Autophagy

Effect of different proteasome inhibitors on dysferlin expression and on membrane resealing in cultured primary myoblasts. Primary myoblasts from patient 2 harboring a homozygous Arg555Trp DYSF mutation that were treated with the indicated amounts of bortezomib for 24 hours. Western blots of protein extracts were stained with anti-dysferlin antibodies and with anti–a-tubulin antibody as loading control.

Inhibition of PI3K, ERK and mTOR prevents the activation of S6K1 and S6 induced by suppression of PKD1 activity. A549 cells were incubated in the absence (-) or presence of either 5 uM Kb or 5 uM Kb and 20 uM LY294002 or 5 uM Kb and 10 uM BKM120 (as indicated) for 1 h prior to stimulation of cells with 50 nM PMA for 30 min and 1 h.

 

Effects of NVP-BKM120 and KU-55933 and their combination on the DNA damage response. A, HCC1937 cells were treated for 18 hours with NVP-BKM120 at 2.5 μmol/L, KU-55933 at 10 μmol/L, or their combination, subjected to ionizing radiation(IR) with 10 Gy or mock, lysed 6 hours later, and subjected to immunoblotting with antibodies as indicated.

Cells were stimulated with TPA (10 nM) for 15 min in the presence of the indicated concentrations of U0126. Samples were collected and analyzed by Western blot to detect phosphorylated p42/p44 MAPK.

Microscope image (406) magnification of U937 cells after exposure to SM-TNs. a) Untreated U937 cells. b) U937 cells treated with 200 nM, and 35 nM ODS empty shells. c) U937 cells treated with microcrystalline (free) Taxol prepared by sonicating Taxol in 20%H3PO4/0.8% SDS solution. d) U937 cells treated with 200 nM SM-TN. A SM-TN is seen in the middle of the picture. e) U937 cells treated with 35 nM SMTN.The nanowires are too small to see at this magnification, but their effect on the cells is clearly visible. Scale bar = 10 mm.

Loss of DUSP4 function upregulates IL-6 and IL-8 and enhances mammosphere growth. Immunoblot analysis of MDA-231 cells after treatment of 24 hours with 1 umol/L selumetinib (MEKi) or 10 umol/L SP600125 (JNKi). I, MDA-231 mammosphere formation quantitated by GelCount software 7 days after siRNA transfection. Where indicated, selumetinib (MEKi) or SP600125 (JNK1) or the combination was added to the mammosphere cultures.

 

L3.6pl cells at 6,000 cells per well were incubated in MEM with 5% FBS in triplicate in a 96-well culture plate and then treated alone with 5 umol/L BMS-777607, 10 umol/L wortmannin, or with BMS-777607 in combination with individual inhibitors. Polyploidy was examined under BK71 Olympus microscope and photographed 72 hours after treatment.

The inhibition of TGF-β1 signaling pathways suppressed EBV-mediated EMT, and prevented the activation of Syk and Src signaling. The EBV-infected HCECs were treated with 100 nM of the dual TGF-β receptor I and II kinase inhibitor, LY2109761, for 48 hours. The EBV-infected HCECs were cultured with anti-TGF-β1 neutralizing antibody (5 ug/mL) or mouse IgG1 antibody (5 ug/mL) for 48 hours. Photographs were taken at x100 magnification by a digital camera.

granulosa cells (GCs) with 24 h of melatonin (10 μM) treatment were rinsed in PBS, and then exposed to H2O2 (200 μM) for 2 h. The autophagy inhibitor 3-MA (10 mM), or the apoptosis inhibitor Z-VAD-FMK (50 μM) were added 1 h prior to H2O2 incubation. Cell viability was determined using the CCK-8 assay. Data represent mean ± S.E; n = 3 in each group. *P < 0.05 (**P < 0.01) vs. vehicle group at 0 h. # Represents P < 0.05 (## Represents P < 0.01) vs. H2O2-only-treated cells. & Represents P > 0.05 vs. H2O2-only-treated cells. N, not significant, P > 0.05. δ Represents P < 0.05 (δδ Represents P < 0.01) vs. Z-VAD-FMK-treated cells.

Protein expression by western blot analysis of cell lysates from MCF-7, TAM^R-4 and 164^R-7 cells treated for 3 days with vehicle (0.1% DMSO), 1 uM or 1.5 uM WP1130.

Verteporfin treatment inhibits proliferation and induces apoptosis of Tsc1-null cells in vivo. Mice were administered i.p. with vehicle or verteporfin at a dose of 100 mg/kg every other day for 10 d before sacrifice. Mice were sacrificed at 6 wk of age. Three independent experiments were performed and mice in different treatments were pooled for analysis. Percentage of Ki67 and αSMA double-positive cells in α-SMA+ mesenchymal lesions in the indicated kidneys. Immunofluorescence staining and counting were performed on three sagittal sections from different kidney regions for each mouse.

Cells were treated with brefeldin A or manumycin A, and the resulting supernatant was collected after 48 h for exosomal preparation (lanes 1 and 2), or exosomes obtained from C81 cells were trypsin-treated or freeze/thawed (F/T) and then trypsin-treated (lanes 3 and 4). Lanes 5 and 6, input exosome controls from C81 or CEM cells, respectively. Resulting exosomes were assayed for the presence of Tax by Western blotting.

Cytosolic extracts or nuclear extracts were examined by Western blot analysis using Abs against p105/p50, p100/p52 and phospho-p65. Solid arrowhead indicates a non-specific band. A nuclear marker, PARP, and cytosolic marker, b-tubulin, were used to assess the purity of each fraction.

A, HeLa cells were treated with DMSO, Taxol (100 nM for 16 h), or Nocodazole (Noco, 100 ng/ml for 16 h). Total cell lysates were probed with the indicated antibodies against Hippo components on Phos-tag SDS-polyacrylamide gels. O and * mark the non-phosphorylated and phosphorylated proteins, respectively.

a-d) LoVo cells were separately or simultaneously treated with 1 μM purmorphamine and 1 μM thiostrepton for the indicated time. a The Gli1, FoxM1, and CCNB1 protein expression levels were examined by immunoblotting after drug treatment for 48 h. b Cell viability was detected after 6 days using an MTT assay. c LoVo cells treated with indicated drugs were cultured for 2 weeks, and outgrowth colonies were stained with crystal violet. d The matched colony count of (c). Error bars represent the mean and S.D. of three independent experiments. **, p < 0.01.

NCI-H929 EV and miR-137 OE cells were treated with ATM activator Chloroquine Phosphate (CQ), specific ATM inhibitor KU-55933, and ATR inhibitor AZ20 for 12 hr. Immunoblotting showed the expression of p-ATM, p-Chk2, p-BRCA1, p-ATR and p-Chk1.

Mitochondrial depolarization mediated relocation of mitochondrial RC-TPP into lysosomes and the ensuing lysosomal acidity triggered rhodamine fluorescence. RC-TPP-loaded Tom20-GFP+ HeLa cells were treated without or with CCCP (20 μM) in the presence or absence of BFA (200 nM). Colocalization of Tom20-GFP (in green) and mitochondrial RC-TPP (in blue) is shown in cyan. Scale bar: 10 μm.

b EdU incorporation assays indicated PFK15 inhibited the cell proliferation of Cal27 cells. c The quantitative data of the EdU incorporation assays.

Inhibition of autophagy advances LPS-induced accumulation of G-MDSCs in vivo and in vitro. (A-E) C57BL/6 mice were treated with SBI-0206965 (5 μg/g) or vehicle for 2 h followed by LPS challenge (10 μg/g). Percentages of CD11b+Gr-1+ MDSCs (A, C), CD11b+Ly6G+Ly6Clow G-MDSCs (B, D) and CD11b+Ly6G-Ly6Chigh M-MDSCs (B, E) in spleens of these mice were analyzed with FACS at 12, 24 and 36 hours. The data are shown as the means ± SEM (n=5 replicates/group) and are representative of three independent experiments. (F, G) Bone marrow cells were pretreated with SBI-0206965 (1 μg/ml) or vehicle for 2 h and then cultured with GM-CSF (40 ng/ml) and IL-6 (40 ng/ml). Four days later, the percentages of CD11b+Gr-1+ MDSCs (F), CD11b+Ly6G+Ly6Clow G-MDSCs (G) and CD11b+Ly6G- Ly6Chigh M-MDSCs (G) were analyzed with FACS. Data are representative from one out of three biological replicates, each with three technical replicates. Error bars represent S.E.M. * p < 0.05, ** p < 0.01, *** p < 0.001, as determined by ANOVA tests; ns denotes p > 0.05.

Western blot analyses show significant inhibition of autophagy-related gene expression in Spautin-1 treated OCI-AML2 cells after Ara-C treatment.

Exosomal miR-124-3p inhibited neuronal inflammation by suppressing the activity of mTOR signaling. A, B) Immunoblot (A) and quantitative (B) data of p-4E-BP1 and p-P70S6K in neurons after scratch injury and exosomal treatment. Their expression was increased after scratch injury and was suppressed by miR-124-3p–up-regulated exosomes, suggesting that miR-124-3p suppresses the activity of mTOR signaling (n = 6/group). ##P < 0.01 vs. control group; **P < 0.01 vs. injury group. C) Expression levels of inflammatory mediators in the culture medium were detected in the 3 groups of neurons: injured neurons (injury group), injured neurons treated with miR-124-3p–up-regulated exosomes (I+Exo-124), and injured neurons treated with miR-124-3p-up-regulated exosomes and the mTOR activator (MHY1485). Compared with the I+Exo-124 group, the MHY1485 group represented an increased expression on proinflammatory cytokines (TNF-α, IL-1β, and IL-6) and a decreased expression of anti-inflammatory cytokines (IL-10), suggesting that MHY1485 blocks the anti-inflammatory effect of miR-124-3p in injured neurons. Thus, the inhibitory effect of exosomal miR-124-3p on neuronal inflammation was exerted by suppressing the activity of mTOR signaling (n = 6/group). #P < 0.05 vs. injury group; *P < 0.05 vs. I+Exo-124 group.

(c) Functional bile canaliculi or lack thereof in PHHs within MPTCs (12 d of drug treatment) as visualized by transport of fluorescent (green) dye into the canaliculi between PHHs. DMSO-treated MPCC control image is shown to the far right. (d) Neutral lipid (Nile red, green) staining of PHHs within MPTCs (12 d of drug treatment). DMSO-treated MPCC control image is shown to the far right. (e) NR1I2 (PXR) gene expression in drug-treated MPTCs relative to DMSOtreated MPTC controls (12 d of treatment). (f) ABCC2 (MRP2) gene expression in drug-treated MPTCs relative to DMSO-treated MPTC controls (12 d of treatment). (g) IL-6 levels in drug-treated MPTC supernatants (6 d of treatment). In all panels, statistical significance is displayed relative to DMSO-treated MPTCs. *p r 0.05, **p r 0.01, ***p r 0.001, and ****p r 0.0001. Scale bars on images represent 80 mm.

C, SA-beta gal staining results of A549-LKB1 cells treated by trametinib (30 nmol/L), radiotherapy (2 Gy), and HCQ (50 μmol/L). Cells were treated by HCQ and/or trametinib 4 hours prior to radiotherapy. Drugs were washed out 24 hours after radiotherapy. Cells were incubated for additional 48 hours before staining. D, Clonogenic survival assay of A549-LKB1 cells treated with trametinib (30 nmol/L) and HCQ (50 μmol/L).

LC3 and DAPI immunofluorescence staining were performed to detect autophagy in VSC4.1 cells treated with 25 mM HD alone, 25 mM HD + 5 μM PIK-III, 5 μM PIK-III and 0.1% DMSO alone (control group). Scale bar: 20 μm

VE-cadherin-induced Akt activation mediates YAP phosphorylation and translocation in ECs. HUVECs were starved for 1h and treated with thrombin (1U) for 1h. Total cell lysates were probed with anti-pAkt, Akt or b-actin antibody. The representative blots of three independent experiments are depicted, and the normalized values for p-Akt are shown. HUVECs were cultured and starved as described as in d and incubated for 8h in complete medium with the Akt inhibitor, MK-2206 (1 uM). pAkt, Akt, pYAP and YAP were detected by western blotting using specific antibodies.

Role of PARP and BER in the synergy between PTX and GMX in A549 cells. A) Cells were pre-treated +/- 1 uM olaparib (2h) then sequentially +/- 150nM PTX (24h) then +/- GMX 12nM (48h). Cells were harvested for (left) NAD+ quantification by LC-MS/MS (mean +/-SD of quadruplicates) or (right) viability by CellTiter-Glo (mean +/-SD of duplicates) B) PAR modification of proteins and γ-H2AX levels were measured in extracts treated as in A) by western blotting.

The regressing tumour microenvironment stimulates the outgrowth, infiltration and metastasis of drug-resistant clones. b, Bioluminescent signal of drug-resistant A375RTGL cells in vemurafenib-sensitive, A375 tumours, treated with vehicle or vemurafenib for 5 days (vehicle, n = 36; vemurafenib, n = 15 tumours). D, day. c, EdU incorporation in A375R-TGL cells in A375/A375R-TGL tumours treated with vehicle or vemurafenib for 4 days, as determined by FACS (vehicle, n = 8; vemurafenib, n = 6 tumours). d, Bioluminescent signal of A375R-TGL tumours alone, treated with vehicle or vemurafenib for 5 days (vehicle, n 5 38; vemurafenib, n = 15 tumours). e, Bioluminescent signal of TGLexpressing drug-resistant cancer cells (A375R, M249R4, PC9 and H2030) in drug-sensitive tumours (Colo800, LOX, UACC62, M249, H3122 and HCC827) treated with vehicle or drugs (vemurafenib, crizotinib and erlotinib) for 5 days (n (from left to right on the graph) = 6, 7, 12, 12, 9, 9, 25, 26, 9, 12, 12, 12, 16 and 11 tumours). f, Spontaneous lung metastasis by A375R cells in mice bearing A375/A375R-TGL tumours treated with vehicle or vemurafenib (10 days), visualized by BLI (n = 4).

Western blot analysis of histone H3 acetylation in the spleen of untreated and vorinostat-treated hNF-E2 tg mice (n = 4 of each genotype).

Cardiomyocytes transduced with or without Ad-Mst1 were treated with ABT-737 (0, 0.1, 1, 10 uM) for 12 hours. Representative immunoblots with antibodies to p62/SQSTM1, LC3 and GAPDH are shown.

The Rho GTPase-JNK pathway is required for the inhibitory effects of vandetanib on Calu-6 cells invasion. Calu-6 cells were incubated for 24 h in the presence or absence of vandetanib (1 or 2 uM), SP600125 (50 or 100 uM), and Y27632 (5 or 10 uM). The morphology of the Calu-6 cells was examined under a light microscope. Scale bar: 50 um.

MDA-MB-231 cells were treated with 10 uM MG132 and incubated under normoxia or hypoxia for 4 h. Endogenous interaction between LATS2 and SIAH2 was analysed by immunoprecipitation.

Three-dimensional responses of MCF7/IGF-1R cells to TAM (1 μM), E2 and IGF-1. Compared to parental MCF7 cells (a), MCF7/IGF-1R cells (b) in three-dimensional (3D) culture formed bigger acini in response to IGF-1 stimulation and displayed significant TAM resistance when treated with TAM (1 μM) + E2 + IGF-1, which was removable by kinase inhibitors BMS-536924, U0126 and BEZ235 (c). Cells (10,000/well) were seeded in 96-well plates. Acini were formed on 100% Matrigel and cultured for 14 days in starving medium containing 2% Matrigel and 5% charcoal/dextran-stripped fetal bovine serum with the treatments as indicated. Concentrations used: TAM (1 μM), E2 (1 nM) and IGF-1 (100 ng/mL). Confocal image original magnification, × 20. Red, rhodamine phalloidin (actin). Blue, Hoechst blue stain. Results are representative of two individual experiments.

 

Inhibition of breast cancer cell growth using sorafenib. MCF-7 breast cancer cells were treated with increasing concentrations of sorafenib for 5 days. Cell number was measured  using a colorimetric growth assay (crystal violet stain) and expressed relative to DMSO treated control cells.

Combinational treatment of kinase inhibitors induces the similar phenotype produced by PP1. All images are lateral view with dorsal to the top and anterior to the left. The combinational treatment of Dasatinib (D) or U0126 (U) with Sunitinib (SU),PTK787 (PTK), or ZM323881 (Z) resulted in the shrinkage of dorsal aorta.

H4-LC3-GFP cells were treated with 1 nM IFNA2 for the indicated periods in the presence of 200 nM rapamycin. Images of the cells were collected using an ArrayScan HCS 4.0 Reader. Representative cells are shown. The average spot intensity in 500 cells from each indicated sample was determined. Data are displayed as means ?SD of the spot intensity per cell (below). RLU, relative leight unit.

(c) Western blot analyses of p-Akt (Ser473) and p-S6RP (Ser235 and Ser236) in two RCT-E565 transplanted tumors treated with vehicle or PF02341066. Samples were isolated 4 h after the last dose from mice treated with PF02341066 for 3 d. (d) Responses of RCT-E565 transplanted tumors in athymic mice to PF02341066 or vehicle. Data are means ±s.e.m. (each group, n = 6). *P < 0.005, **P < 0.001 (Student^,s t test).

Effects of combined treatment with erlotinib and NPS-1034 in HCC827/ER cells with AXL activation. Lysates were immunoprecipitated with an anti-AXL antibody and immunoblotted with antibodies for phosphotyrosine (p-Tyr) and AXL. HCC827/ER cells were treated with erlotinib. E, erlotinib; N, NPS-1034. **, P < 0.001 for the combination of erlotinib plus NPS-1034 versus either the control or drug alone.

Cytoskeleton organisation of 786-O SuR treated with NVP-LDE225 (2.5 uM), everolimus (1 uM), and their combination for 24 h was analysed by confocal microscopy. Actin-based structures were revealed by rhodaminated phalloidin staining (red fluorescence). Localisation of focal adhesion points was obtained by immunofluorescent staining of p-paxillin (green fluorescence). Merged row images show overlapping of p-paxillin and actin signals. Moreover, all captures were shown in transmitted light. Scale bars, 10 um.

Perturbation of EGFR by its ligand EGF and gefitinib (ZD-1839 Iressat; inhibits EGFR) produces opposite responses in the predicted EGFR target genes SOCS2 and NR2E1.

 

Number of foci detected using laser confocal microscopy and fluorescent Fluor 647 anti-H2A.X-phosphorylated (Ser139) antibody. Double-stranded breaks (red) are clearly augmented in cells incubated with 500 nmol/l of ABT-888 and 500 nmol/l of AZD-2281 compared with PBS and 1% dimethyl sulfoxide controls. Image analysis was performed using ImageJ and the ‘analyze particle’ function.

(A) U87 cells were cultured in the presence of DMSO, 1 uM MS-275 alone, 100 ng/ml IFN-λ1 alone, or both for the course of 4 d. Cell numbers were manually determined by hemacytometer counting at the indicated time points. (B, F) Cell proliferation of U87 cells or U87 spheroids in 3D culture with indicated treatment were performed using the WST-1 assay, which measures active cellular metabolism. (C) U87 spheroid formation in 3D culture was photographed at day 14 in culture (representative images are shown; 200x magnification). (D-E) Quantification of the relative sizes and numbers of U87 spheroids in (C). (G) Cell cycle analysis was performed in U87 cells with indicated treatment using propidium iodide staining. Numbers in the histogram show fractions (percent) of sub-G1, N, 2N, and polyploidy from left to right. (H) U87 cells with indicated treatment were stained with Annexin V-FITC and 7-AAD. Numbers indicate the percentage of FITC-positive cells (upper left quadrant). FITC, fluorescein isothiocyanate; 7-AAD, 7-Aminoactinomycin. In all panels, data represent the mean and SEM of at least three experiments.

TGF-β induces mTORC2 activation. ( A ) NMuMG cells were treated with TGF-β for the indicated times before lysis and immunoblotting. (B ) NMuMG cells were treated or not with TGF-β for the indicated times, in the presence or absence of SB431542 or LY294002. Cell lysates were subjected to anti-Rictor immunoprecipitation, and the immunoprecipitates were subjected to in vitro kinase assays using kinase-inactive Akt1 as a substrate, before immunoblotting of the kinase reactions, immunoprecipitates and cell lysates. The left panels are from the same gel, without differential exposure. ( C ) NMuMG cells were treated or not with TGF-β or insulin for the indicated times, in the presence or absence of SB431542 or GDC-0941. The kinase activity of mTORC2 was then assessed as in B. The top panels are from the same gel, without differential exposure. (D) NMuMG cells were treated or not with TGF-β for the indicated times, in the presence or absence of SB431542 or LY294002. Cell lysates were subjected to immunoprecipitation using Rictor antibody, and/or immunoblotted.

ERK phosphorylates FBW7 at T205. PANC-1 cells were pretreated with the proteasome inhibitor MG132 and trametinib, as indicated, overnight before harvest. Endogenous FBW7 phosphorylation status was examined by immunoblot analysis after immunoprecipitates (IP).

STAT3 phosphorylation as determined by phospho flow, mixed lymphocyte reactions containing BALB/c spleen-derived CD4+ T cells co-cultured with or without C57BL/6 BM-derived DC preactivated with 20 ng/mL LPS.

LSD1 and HDAC inhibitors exhibit synergistic growth inhibition. Cells were simultaneously treated with pargyline or HDAC inhibitors for 48 h.

 

THOC5 phospho-Y225 levels were assessed in the populations shown by flow cytometry following 24 h treatment with 5 μM imatinib, 150 nM dasatinib or 5 μM nilotinib. Results are displayed as mean fluorescence intensity (n= 4) ±s.e.m.

Alisertib inhibits AURKA and AURKB in a concentration-dependent manner. (a) Alisertib induces G 2 /M delay or genome reduplication. HeLa cells were exposed to buffer or the indicated concentrations of Alisertib. After 24 h, the cells were harvested and analyzed with flow cytometry. The positions of 2N, 4N and 8N DNA contents are indicated. (b) Alisertib delays mitotic exit or induces slippage. HeLa cells stably expressing histone H2B-GFP were exposed to buffer or the indicated concentrations of Alisertib. Individual cells were then tracked for 24 h with time-lapse microscopy. Each horizontal bar represents one cell (n ¼ 50). Key: light gray ¼ interphase; black ¼ mitosis (from DNA condensation to anaphase or mitotic slippage); dark gray ¼ interphase after mitotic slippage; truncated bars ¼ cell death. (c) Different concentrations of Alisertib are involved in delaying mitotic exit and inducing slippage. Live-cell imaging of cells treated with Alisertib was described in panel (b). The duration of mitosis (mean±90% confidence interval) and the percentage of cells that underwent mitotic slippage during the imaging period was quantified. (d) Alisertib promotes apoptosis in a concentration-dependent manner. HeLa cells were incubated with the indicated concentrations of Alisertib for 48 h. The cells were then harvested and analyzed with flow cytometry. (e) Concentration-dependent cytotoxicity of Alisertib. HeLa cells were cultured in the presence of the indicated concentrations of Alisertib for 48 h. The number of live and dead cells was analyzed with trypan blue exclusion assay. (f) Concentration-dependent suppression of long-term survival by Alisertib. HeLa cells were seeded on 60-mm culture plates and grown in the presence of 250 n M or 1 m M of Alisertib. After 24 h, the cells were washed gently and propagated in normal medium for another 10–12 days. Colonies were fixed and stained with crystal violet solution (examples of the plates are shown). Average±s.d. from three independent experiments. (g) Both AURKA and AURKB are inhibited by Alisertib.Mitotic HeLa cells were obtained by exposure to nocodazole for 16 h followed by mechanical shake off. The cells were incubated with the indicated concentrations of Alisertib for 2 h. Lysates were then prepared and activated phospho-AURKAThr288 and AURKBThr232were detected with immunoblotting. The asterisk indicates the position of an AURKB-like protein (the same throughout this study). Uniform loading was confirmed by immunoblotting for actin. In this assay, nocodazole and MG132 (a proteasome inhibitor) were added to prevent the cells from exiting mitosis. Accordingly, the total AURKA and AURKB levels remained constant throughout the experiment. (h) Alisertib prevents activation of AURKA and AURKB. HeLa cells were incubated with the indicated concentrations of Alisertib for 8 h. Nocodazole was then added for another 6 h to trap cells that entered mitosis. Lysates were prepared and analyzed with immunoblotting. Actin analysis was included to assess loading and transfer.

Inhibition of signaling pathway activation in lung tumor cell lines by kinase inhibitors. Lung tumor cells were cultured in 10% FBS until reaching ∼80% confluence and then the cells were starved in serum-free medium for overnight, followed by 4-hour treatment with the inhibitors. Cell lysates were then prepared and used for determination of the pathway activation signals by the CEER assay.

293T cells were transfected with HA-tagged Fbxo45. At 48 h after transfection, cells were treated with AKT inhibitor (CAL-101; 10 uM, 4 h), cell extracts from the cytoplasm or nuclei were subjected to IP with anti-HA resin followed by western blot analysis with indicated antibodies.

mTOR kinase inhibitor AZD8055 activates PI3K accompanied with induction of expression of EGFR, HER2, HER3 and IRS1. Serum-deprived CHO-EGFP-AKT cells were incubated with 50 nM AZD8055 for 24 hr. The EGFP signal was detected using confocal microscopy. The white arrows indicate EGFP-AKT located on cellular membrane.

C and D, in vivo subcutaneous tumor growth curves (C) and tumor weight quantification of intersected subcutaneous tumor tissues (D) of Huh7 cells after stable LHBs expression under saracatinib treatment (25 mg/kg body weight daily for 4 weeks; n =18). *, P < 0.05. E and F,in vivo subcutaneous tumor growth curves (E) and tumor weight quantification of intersected subcutaneous tumor tissues (F) of SK-Hep1 cells after stable LHBs expression under saracatinib treatment (25 mg/kg body weight daily for 4 weeks; n = 18). *, P < 0.05.

In vivo antitumor efficacies of AZD2281 and GX15-070 alone or in combination in a BxPC-3 xenograft model. Tumor specimens were fixed in 10% formalin, embedded in paraffin, and cut into 4 lm-thick slides for H&E, PCNA, and CD34 staining.

SKBR3 cells were treated with FW-04-806 at 10, 20, 40 uM for 24 h; 17AAG was used as a positive control at 1 and 2 uM. Hsp70, Hsp90, and Cdc37 protein level were analyzed with western blotting using relevant antibodies.

Cell viabilities with increasing concentrations of cisplatin (CP) and doxorubicin (DOXO) under normoxic and hypoxic condition for 48 hours were determined by MTT assay. IC50 values are presented as the means ?SDs (n=4) and * denotes p<0.05.

Effect of nilotinib on Bcr-Abl kinase activity in ABCB1- and ABCG2- overexpressing CD34⁺CD38^- cells. K562 parental cells and CD34⁺CD38^- subpopulation isolated from K562 cells were treated with nilotinib at 0.01, 0.1 and 1.0 umol/L for 12 h. Equal amount of protein was loaded for western blot analysis as described in the Experimental section. The experiments were repeated at least three times independently, and a representative experiment is shown.

mTOR inhibitors attenuate ganetespib-driven elevation of HSPs in multiple tumor cell types. A375 melanoma cells were treated with vehicle, ganetespib (25 nmol/L), BEZ235 (500 nmol/L), or temsirolimus (500 nmol/L), either alone or in combination, for 24 hours. The levels of HSP90α, HSP70, HSP27, and GAPDH were determined by immunoblotting.

We treated all of drugs in T47D which has a PI3KCA H1044R mutation with the concentration shown below for 1 hour and performed western blot analysis using antibodies to phospho-AKT(SERINE 472), and total AKT.

 

RNA incorporating drugs induce SG assembly. HeLa cells were treated with the RNA incorporating agents 5-azacytidine (50 uM) and 6-thioguanine (10 uM), or the DNA incorporating agents trifluorothymidine (10 uM) and gemcitabine (100 nM) for 72 h. Subsequently, the cellular localization of the SG marker protein TIAR (green) and the P-body marker protein DCP1 (red) was analyzed. Nuclei were stained with Hoechst. Scale bars represent 20 um.

 

Comparison of MCAS ovarian cancer cells harboring control and CtBP2 knockdown shRNAs for sensitivity to chemotherapeutic agents. For each cell line, the MTT reading of the untreated cells was assigned as 100%. HDAC inhibitors: (a) Trichostatin A; (b) Vorinostat; (c) Belinostat; (d) MGCD0103; (e) valproic acid; and (f ) carboplatin, a non-HDAC inhibitor.

Hepatoma cells 24 h after plating were treated with vehicle (DMSO), regorafenib (REGO, 0.5 µM), PDE5 inhibitor (sildenafil, 2 µM); or the drugs in combination. 24 hours after treatment cells were isolated and viability determined by trypan blue (n=3, SEM). *P 0.05

PAI-1 expression in HUVECs treated with drugs as indicated.(D) senescent HUVECs were treated with SRT1720, culturing for 24, 48 hours. PAI-1 mRNA and protein (E) levels were analyzed using real-time RT–PCR and Western blotting, respectively. The RNA and protein levels were normalized to the internal control β-actin. Data are presented as the mean±SEM of three independent experiments. *P < 0.05 vs. corresponding control. **P < 0.01 vs. corresponding control ***P < 0.001 vs. corresponding control.

A,IC50 of Bosutinib that block ANDV-induced EC permeability. Endothelial cells were ANDV infected, and 3 days postinfection the permeability of cells in response to VEGF addition was determined in the presence or absence of increasing amounts of kinase inhibitor. The effect of inhibitors is presented as the percentage of ANDV-induced permeability of inhibitor-treated monolayers 3 days postinfection and 30 min post-VEGF and FITC-dextran addition. B, VEGFR2-Src inhibitors block ANDV-induced permeability. Endothelial cells were plated on vitronectin-coated Transwell inserts and infected at an MOI of 0.5 in triplicate with ANDV. Three days postinfection, the permeability of ANDV- and mock-infected endothelial cell monolayers was determined as described for Fig. 1 at indicated times in the presence or absence of Bosutinib .

C, Jurkat cells with SB202190 at 1, 5, and 10 μM were tested, and a decreased SRP72 expression was found when using at 10 μM (lanes 8 and 9). D, results were analyzed and RUA illustrated, finding significant results at 10 μM at 240 versus 0 and 120 versus 0 min (p<0.05).

 

Validation of activity and specificity of chemical inhibitors of; ATM, ATR, and DNAPK. H460 cells were treated with 1 uM camptothecin (CPT) or 20 ug/ml bleomycin for 1 h in the presence of the indicated inhibitors: DNAPK-i1—NU7026, DNAPK-i2—NU7441. MSH6,

PTPH1 confers breast cancer cell sensitivity to fulvestrant. E and F, PTPH1 overexpression increases the growth inhibition by fulvestrant. PTPH1 was overexpressed by a Tet-on system or a stable transfection, and resultant cells were incubated with fulvestrant as indicated for about 2 weeks. Colony formed was stained and counted. Results shown are normalized to its own solvent control of Vector and PTPH1-overexpressed cells, respectively (means ± SD; n = 3–5) with insets showing PTPH1 overexpression. *, versus vector or no Tet cells for E and F.

Role of PARP and BER in the synergy between PTX and GMX in A549 cells. Cells were pretreated ±1 umol/L olaparib (2 hours) then sequentially ±150 nmol/L PTX (24 hours), then ± GMX 12 nmol/L (48 hours). PAR modification of proteins and γ-H2AX levels were measured in extracts treated as in A by Western blotting.

Synergistic effect of BMS-777607 with mTOR inhibitors in reduction of CSCs^+24/44/ESA viability. CSCs^+24/44/ESA at 5,000 cells per well with stem cell culture media in triplicate in an ultra-low adhesion plate were treated with 5 umol/L BMS-777607, 1 umol/L AZD8055, 1 umol/L RAD001, and 1 umol/L PP242 alone, or in their different combinations. Cells were cultured for 72 hours. Percentages of polyploid cells were determined by counting 300 cells from two different regions. Results shown here were from one of two experiments with similar results.

Cellular biomarker responses in HT29 cells exposed to various cytotoxic chemotherapeutic agents in combination with the Chk1 inhibitor V158411. HT29 cells were exposed to the combination GI80 of gemcitabine (0.2 uM), camptothecin (0.44 uM), cisplatin (68 uM), oxaliplatin (131 uM), doxorubicin (1.2 uM) or etoposide (59 uM) for 18 hours followed by DMSO (-) or 400 nM V158411 (+) for a further 24 hours. Protein expression was characterized by immunoblotting.

THP-1 cells were treated with cytarabine alone and in combination with ABT-199 for 8 h. Whole cell lysates were extracted and subjected to Western blotting, and probed with anti-γH2AX or -β-actin antibody. Densitometry for γH2AX expression was measured, normalized to β-actin, and graphed as fold change compared to the no drug control. The data are presented as mean ± standard error from at least 3 independent Western blots. * indicates p < 0.05.

(C) In vivo treatment of Tg:Pomc-Pttg;Pomc-eGFP embryos with small-molecule CDK inhibitors (50 μM) or 0.2% DMSO as control from 18 to 40 hpf. One hundred to one hundred fifty embryos were treated with each compound. Representative images of live embryos are shown with gross morphology (Right) and pituitary Pomc-GFP-positive cells at higher magnification (Left) at 40 hpf. Embryos exposed to flavopiridol developed early developmental defect before pituitary POMC cell ontogeny occurs. (D) Relative expression of pituitary Pomc-eGFP fluorescence analyzed using Volocity 5.2 software (Improvision; mean ±SE of relative expression, n = 7). (E) R-roscovitine specifically suppresses expansion of pituitary POMC cells overexpressing zPttg from 18 to 48 hpf. Double transgenic Tg:Pomc-Pttg;Prl-RFP embryos were generated by breeding Tg:Pomc-Pttg fish with a previously generated PRL-RFP transgenic line, in which RFP was targeted to pituitary lactotrophs by a zebrafish Prolactin promoter (34). Representative fluorescent microscopy of pituitary POMC-eGFP (a and b) and PRL-RFP (c and d) expression in live Tg:Pomc-Pttg; Pomc-eGFP and Tg:Pomc-Pttg;Prl-RFP embryos treated with 0.2% DMSO (a and c) or 50 μM R-roscovitine (b and d). (F) Relative expression of pituitary POMC-eGFP or PRL-RFP fluorescence were analyzed (mean ±SE of relative expression; n = 10). Results represent one of three similar experiments;*P < 0.02 and **P < 0.000005. (Scale bar, 50 μm.)

Aberrantly activated PI3K/AKT pathway mediates lapatinib resistance in SK-BR-3-LR cells. (A and B) After drug treatment, phosphorylation of HER2, EGFR, AKT, and ERK1/2 was determined by Western blotting using specific antibodies.

C57BL/6 mice were implanted in the striatum with citrine-GL26-Cherry-HMGB1, which were stably transfected to express the YFP citrine and HMGB1 fused to red fluorescent protein cherry. Fourteen days later, they were treated with saline, Ad-TK+Ad-Flt3L, or Ad-TK+Ad-Flt3L+TMZ (temozolomide). Five days after treatment, the cellular location of cherry-HMGB1 in these cells was assessed by confocal microscopy. Arrows, tumor cells (green) with cytoplasmic HMGB1 (red).

Effect of FK506 on cytosolic calcium homeostasis induced by TG and thrombin in human platelets. (A-B) Human platelets were suspended in a Ca 2+-free medium (100 µM of EGTA was added as indicated by arrowhead), and preincubated for 5 min at 37oC in the absence (solid black traces) or presence of increased concentrations (0.01-100 µM) of FK506 (light-doted and dark solid-grey traces, respectively). Cells were then stimulated with TG (200 nM; A) or Thr (0.1 U/mL; B) and 3 min later 300 μM of CaCl2 was added to extracellular medium to visualize calcium entry.

Cropped immunoblot analyses for downstream effector proteins of the MAPK and PI3K/AKT/mTOR signaling pathways for NRASQ61 mutant lung carcinoma and neuroblastoma cell lines. Dual pathway inhibition can be achieved by combining metformin and trametinib, as evidenced by the abolishment of p-ERK and p-S6.

The ROCK inhibitors fasudil and Y27632 prevented SCP2 cell bone metastasis in nude mice (n = 10 per group). Shown are BLI images of bone metastases, IHC analyses of SMAD3 C-tail phosphorylation and PTHLH, osteoclast TRAP staining, and BLI quantitation.

Immunocytochemical staining of SW620 (metastatic) cells after treatment with 10 uM oxaliplatin (F) or 10 uM ginsenosides 20(S)-Rg 3 (G) and negative staining (H). Cells demonstrated differential expression of histone H4.

The BET protein inhibitor JQ1 reduces c-Myc expression and attenuates primary MCC cell proliferation. A, decreased c-Myc expression in MCC-3 and MCC-5 treated with JQ1 (800 nmol/L) for 72 hours by qRT-PCR and immunoblotting. The mRNA expression of target genes was normalized to that of MRPS2 and a value of 1.0 was assigned to the mRNA expression of target genes in the control group (means+SEM; **, P < 0.01 vs. control); b-actin was used as a loading control for immunoblotting.

Effects of DPN, LY500307, Raloxifene and Tamoxifen on cell viability in BSO-treated FRDA fibroblasts. BSO concentration was 1 mM and all steroid concentrations were 100 nM. Depicted are mean ± SD for n= 8 per group. * indicated p<0.05 versus BSO alone-treated cells.

Huh-7.5 cells were treated with increasing concentration of Torin 1, a known autophagy inducer, and the autophagy response was assessed by measuring LC3II and p62 levels.

R-2HG treatment or chemotherapeutic treatment, especially their combinations, decrease MYC levels in leukemic cells (using MONOMAC-6 as a representative). AZA:Azacitidine.

Primary myoblasts from patient 2 harboring a homozygous Arg555Trp DYSF mutation that were treated with the indicated amounts of MLN2238 for 24 hours.

Lysates of HCT116p53KO cells were harvested 24 hs after treatment with different GSK3 inhibitors and GSK3A/B activation/inactivation checked by western blot: a mix of pSer21-GSK3A and pSer9-GSK3B antibodies and antibody cross-reacting with both pTyr279-GSK3A and pTyr216-GSK3B were used to assess the specificity of the inhibitor for GSK3A. BIO: 6-bromoindirubin-3'-oxime, TWS: TWS119, SB2: SB216763, SB4: SB415286.

Cellular senescence and SIRT1 phosphorylation was monitored in PAECs (P2) incubated in DMEM containing HDL (50 mg/L), LDL (50 mg/L), or resveratrol (100 μM) for 24 hours.

Dexamethasone and largazole cooperate to suppress invasion and to restore E-cadherin localization to the cell peripher y. ( a) Phase contrast micrographs showing morphological changes in MDA-MB-231 cells induced by E-cadherin expression combined with 100 nM dexamethasone and 10 nM largazole treatments. Insets show the cells at higher magnification. (b ) Fluorescence (E-Cad-GFP) or immunofluorescence microscopy (g -catenin (g-Cat.)) of 231/E-Cad-GFP cells treated for 72 h with vehicle (Control), 100 n M dexamethasone, 10 nM largazole or 100 nM dexamethasone + 10 nM largazole (Dex. + Larg.). (c ) Invasion assays were per formed with the indicated cell lines treated for 72 h with or without 100 nM dexamethasone + 10 nM largazole using modified Boyden chambers impregnated with matrigel. The results are presented as the average number of cells that invaded through the membrane per field s.d. of five randomly chosen fields, and are representative of three independently per formed experiments.

Viability and CI vs Fa after 24-h exposure to cytarabine alone or in combination with ABT-199 in Riva, U2932 and VavP-Bcl2/c-MYC murine tumor cells. Viability shown at 500 nM (500 ng/ml for cytarabine; quadruplicates±s.e.m.).

Statin-Related Inhibition of Dehydroepiandrosterone Sulfate (DHEAS) Uptake by SLCO2B1 in Prostate Cancer (PC) Cells. B, Uptake of DHEAS in PC cells with 2.5 µM DHEAS and different concentrations of statins when incubated for 60 minutes. Statistical analysis was performed by comparing each condition with the DHEAS 2.5 µM and no statin state except when indicated. C, Uptake of DHEAS in PC cells before (scrambled short hairpin RNA) and after (short hairpin RNA 2B1) SLCO2B1 is knocked down when incubated with 2.5 μM DHEAS and 100 μM atorvastatin for 10 and 60 minutes. Statistical analysis was performed by comparing each condition with scrambled short hairpin RNA after 10 minutes with DHEAS except when indicated. P = .02 for the comparison between scrambled short hairpin RNA with 10 vs 60 minutes of DHEAS incubation for LNCaP and .01 for 22RV1. Other P values are indicated in the figure. Bars indicate means and error bars indicate standard deviation.

RNA incorporating drugs induce SG assembly. HeLa cells were treated with the RNA incorporating agents 5-azacytidine (50 uM) and 6-thioguanine (10 uM), or the DNA incorporating agents trifluorothymidine (10 uM) and gemcitabine (100 nM) for 72 h. Subsequently, the cellular localization of the SG marker protein TIAR (green) and the P-body marker protein DCP1 (red) was analyzed. Nuclei were stained with Hoechst. Scale bars represent 20 um.

After treated with PF-4708671 (PF, 10 uM) and 4EGI-1 (50 uM) for 48 h, GRP78 and ATF4 in QBC939, RBE and HCCC-9810 cells were analyzed using western blot.

U2OS cells were plated in six-well plates using complete medium. The next day the cells were washed four times with NaCl/P_i before maintaining them for 6 h in serum- and glucose-free DMEM supplemented as indicated in the absence or presence of 0.1 uM Torin 2 for the last 1 h. The cells were control- treated, treated with 1 ug/mL insulin or treated with 1 mM H2O2 for 15 min. Thereafter, cell lysates were prepared and western blotting was performed using the indicated antibodies.

The effects of H89 on bTSH-induced FASN downregulation in 3T3-L1 adipocytes. Differentiated 3T3-L1 adipocytes were pretreated with either 20 µmol/L H89 or vehicle for 1 h, then treated with 0.2 µM bTSH for 8 h. Levels of FASN, pCREB, pERK1/2 and pJNK were determined by Western blotting.

Phenotypic effect of Genetic or Pharmacologic Compromise of the 477 Hsp70-StiA-Hsp90 Complex. The impact of each genetic modification on radial growth, conidiation, and response to various stress conditions was assessed after inoculation of a suspension of 104 conidia on glucose minimal medium (GMM) agar plates and incubation at 37ºC for 5 days.

AMPK inhibition by compound C(Dorsomorphin) inhibited autophagy activation and neuroprotection induced by IPC in PC12 cells. (A) Compound C (Comp C) abolished IPC induced neuroprotection in PC12 cells. Cells were incubated with compound C 5 μM 60 min before the onset of IPC. Twelve hours after IPC, the cells were subjected to OGD for 10 h and cell viability was examined with CCK-8 kit. (B) Compound C reduced LC3II/LC3I ratio. The cells were incubated with compound C 5 μM 60 min before the onset of IPC. Then the cells were harvested 12 h after IPC and subjected to Western blot analysis. Bar represents mean ± SD, n = 3. *P < 0.05, ***P < 0.001 compared with the control group; $$$ P < 0.001 compared with the OGD group; %%% P < 0.001 compared with the IPC + OGD group; # P < 0.05 compared with the IPC group.

Total and phosphorylated Akt were detected by western blot in protein lysates of liver, inguinal WAT and gastrocnemius muscle from mice that were treated with an insulin bolus after an overnight fast.

Effect of metabolic inhibitors on SRC-1 protein levels. A549 cells grown in glucose-containing medium were treated with NAM or FK866 at the indicated concentration.

A, HAECs were per-incubated with PFTα (3 μmol/L) for 60 min followed by transfected with siB-myb or siNC in the presence of PFTα (3 μmol/L) for 7 d. The expression levels of B-myb, p-p53, p53 and p21 proteins were analysed by western blotting. GAPDH was used as a loading control. A typical group of blots is shown from one of three independent experiments.

Representative confocal microscopy images of oocytes with DZNep or GSK343 treatment. The oocytes presented with a typical barrel-shaped spindle and well-aligned chromosomes on the metaphase plate not only in DZNep or GSK343 treated group but also in the control group. Spindle was recognized by α-tubulin (green) and DNA was recognized by PI (Propidium iodide, red). Scale bar = 4 μm.

Nutlin-3a preserved p53 expression without influencing high glucose (HG)-induced podocyte impairment. A-D: cultured podocytes were pre-treated by nutlin-3a for 2 hrs before subjected to HG treatment. Western blotting gel documents (A) and summarized data (B) showing the expression of p53 and MDM2 in podocytes under HG exposure for 24 hrs. n = 4. Western blotting gel documents (C) and summarized data (D) showing the expression of Desmin in podocytes under HG exposure for 24 hrs. n = 3. *P < 0.05 vs. Ctrl, #P < 0.05 vs. Vehl + HG. Ctrl: control; Vehl: vehicle; nutlin-3a: nutlin-3a treatment.

Whole-cell lysates from NRAS- or BRAF-mutant melanoma cells treated with encorafenib or/and binimetinib or DMSO as a control for 24 hours were subjected to Western blot analysis to detect pERK, ERK, and b-actin. The experiment shown is a representative of three independent experiments.

Cell survival curves of A549 cells treated with increasing doses of A-674563, PHA-848125, and H89 2HCl for 72 hours. Cells were incubated with cell proliferation reagent WST-1 for 2 hours and absorbance was read at 450nm. Optical density was then normalized to a 1% DMSO control. The data is presented as the percentage of cell survival relative to the DMSO control ± SEM of three independent trials. Statistical significance was determined with multiple T-tests using the Holm-Sidak method without assuming a consistent SD and is represented by *p<0.05.

Involvement of EV linc-VLDLR in tumor cell responses to chemotherapy. Cells were incubated with sorafenib, camptothecin, or doxorubicin. EVs were obtained after 24 hours, and qRT-PCR was performed for linc-VLDLR. The bars represent the mean ?SEM of the increase in cell viability from 3 independent studies. *, P < 0.05.

Effects of some confirmed hits on IRF7 transcription level in response to IFN-α2a treatment (1 h) in SH-SY5Y cells. Data represent mean expression fold±SEM relative to GAPDH, measured from three independent experiments, each in triplicates. *P<0.05, **P<0.01, and ***P<0.001 compared to IFN-α2a treated cells.

Immunocytochemistry demonstrates NR4A1 nuclear location at control (non-chymase treated) in HL-1 cells (a). Chymase (2.5 ug/ml) treatment for 2 h induces NR4A1 (red) cytoplasmic translocation in HL-1 cells as well as myosin (green) disruption (panel b). Active chymase enters HL-1 cells and is prevented by Dynasore (c and d). There marked entry into HL-1 cell nuclei and cytoplasm after treatment with chymase (5 ug/ml) for 2 h that is prevented by pre-treatment with Dynasore. Lack of co-staining with caveolin 3 (green) demonstrates that chymase is not transported via caveolae. Dynasore prevents transferrin uptake in HL-1 cells.

For MTT assays, cells (2,000 ~ 5,000 cells/well) were subcultured into 96-well plates according to their growth properties. Cell proliferation was assayed at 72 hr after treatment of CHIR-99021 by adding 20 μl of 5 mg/ml 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) solution per 100 μl of growth medium. After incubating for 3-4 h at 37°C, the media were removed and 150 µl/well of MTT solvent (either absolute DMSO or isopropanol containing 4 mM HCl and 0.1% Nonidet-40) was added to dissolve the formazan. The absorbance of each well was measured by ELx808 (BioTek, Winooski, VT) or Wallac Victor2 (Perkin-Elmer Life Sciences, Boston, MA) Microplate Reader. Viable cells are presented as percent of control, vehicle-treated cells. 

Apoptosis evaluation of UACC62R, SK-MEL-28 R and R3 cells treated with Gant61 (10 μM) for 48 h by flow cytometry detection of Annexin V staining.

Drugs across therapeutic indications induce lipid formation in hiPS-CM. Lipid accumulation was detected in cardiac cells using the LipidTox plate-based fluorescent assay on the Thermo Scientific CellInsight High Content platform. A) Ten drugs increased lipid levels in hiPS-CM following 48 h treatment. The lowest drug dose that induced a N1.5-fold increase in lipid formation is shown. B) Representative images (20×) from the assay are shown to the right. All drugs had >55% cell viability at 48 h at these tested concentrations. C) Of these 10 drugs, 8 significantly increased lipid accumulation following only 24 h treatment (images not shown). All drugs had >80% cell viability at 24 h at these drug doses. The graphs represent the mean fold-change of the lowest concentration of drug that significantly induced lipid formation >1.5-fold more than vehicle control. *P<0.05, **P<0.01, and ***P<0.0001.

Low-micromolar amounts of loperamide inhibit MERS-CoV-induced cytopathology. Huh7 cells in 96-well plates were infected with MERS-CoV isolate EMC/2012 (MOI, 0.005) in the presence of 0 to 8 μM LPM (C). Cells were incubated for 2 days, and cell viability was monitored using an MTS assay. In addition, the potential toxicity of compound treatment only was monitored in parallel mock-infected Huh7 cell cultures. Graphs show the results (averages and standard deviations [SD]) of a representative experiment that was performed in quadruplicate. All experiments were repeated at least twice. For each compound, the calculated EC50, CC50, and SI values are given.

Dexamethasone and largazole cooperate to suppress invasion and to restore E-cadherin localization to the cell peripher y. ( a) Phase contrast micrographs showing morphological changes in MDA-MB-231 cells induced by E-cadherin expression combined with 100 nM dexamethasone and 10 nM largazole treatments. Insets show the cells at higher magnification. (b ) Fluorescence (E-Cad-GFP) or immunofluorescence microscopy (g -catenin (g-Cat.)) of 231/E-Cad-GFP cells treated for 72 h with vehicle (Control), 100 n M dexamethasone, 10 nM largazole or 100 nM dexamethasone + 10 nM largazole (Dex. + Larg.). (c ) Invasion assays were per formed with the indicated cell lines treated for 72 h with or without 100 nM dexamethasone + 10 nM largazole using modified Boyden chambers impregnated with matrigel. The results are presented as the average number of cells that invaded through the membrane per field s.d. of five randomly chosen fields, and are representative of three independently per formed experiments.

Inhibitory curves and IC50 values for the reference compound flubendazole (B) against VEGFR2.

Assessment of the role of Ca2+ channels during RSV replication. HEp-2 cells were infected with RSV strain long at an MOI of 0.1, and treated with calcium channel blockers nifedipine, nimodipine, and tetrandrine (A), at indicated concentrations. Supernatants were collected at 48 h postinfection and viral titers were determined by immunoplaque assay. Grey bars represent cytotoxicity of the compounds. The data presented were obtained from two independent experiments. Error bars represent the standard deviations from two independent experiments. NS, no differences at a significance level of 0.05.

Ectopic expression of a RNAi-resistant SMARCE1 cDNA resensitizes SMARCE1-knockdown cells to MET inhibition in MET-amplified NSCLC cells. The above-described cells were grown in the absence or presence of 300 nM Crizotinib, 150 nM EMD1214063, or 150 nM PHA665752. Cells were then fixed, stained and photographed after 12 days (untreated) or 28 days (treated).

(C) Western blot analysis of mature TGF-β1, FN and Col-I protein levels in sham, sham+ PF-543, UUO and UUO +PF-543 groups at 7 days (n =3). (average ± SEM; ANOVA; *P < 0.05, **P < 0.01 versus sham group. #P < 0.05 versus UUO group). (D) Periodic acid-Schiff staning showed that tubular atrophy was evident in the obstructed kidneys at UUO 7 days after PF-543 treatment (n =6). (E) Masson's trichrome staining showed that matrix accumulation increased in the obstructed kidneys at UUO 7 days after PF-543 treatment(n = 6). (original magnification, ×400, scale bar =50 μm).

Sunitinib decreases FLT-3 and RET phosphor ylation but increases ERK phosphorylation in a time-dependent manner. H295R and SW13 cells were treated with sunitinib (10 nM) for various time points as indi-cated. Cell lysates were prepared and phospho-FLT-3, RET, and ERK levels were monitored by Western Blot-ting. Re-probing against FLT-3, RET, and ERK was done to ensure equal protein loading.

Effects of combined treatment with erlotinib and NPS-1034 in HCC827/ER cells with AXL activation. Lysates were immunoprecipitated with an anti-AXL antibody and immunoblotted with antibodies for phosphotyrosine (p-Tyr) and AXL. HCC827/ER cells were treated with erlotinib. E, erlotinib; N, NPS-1034. **, P < 0.001 for the combination of erlotinib plus NPS-1034 versus either the control or drug alone.

Apoptotic sensitivity of K562 resistant (E) cells exposed to A1331852 (10 nM) for 4 h was restored following pharmacological inhibition of glutamine uptake or metabolism with GPNA (5 mM) for 48 h, CB-839 (10 μM) for 72 h, azaserine (25 μM) for 16 h and AOA (500 μM) for 24 h but not with EGCG (50 μM) for 24 h. Western blots confirmed the knockdown efficiency of the different siRNAs. ***P<0.001, **P<0.01; Error bars = Mean ± SEM (n=3).

Western blot was performed to analyze p65 signaling and the expression of Bcl2 and cleaved Caspase 3. GAPDH served as the loading control

FGF21 activates myogenic and aerobic myofiber-associated genes expression via AMPK pathway. The activator (acadesine) or inhibitor (dorsomorphin) of AMPK pathway were used to treat the pcDNA3.1-21 or control transfected C2C12 myoblasts. For this experiment, four groups were set up: FGF21-ACA (pcDNA3.1-21‡acadesine), Control-ACA (control‡acadesine), FGF21-DOR (pcDNA3.1-21‡dorsomorphin), and Control-DOR(control‡dorsomorphin). The qRT-PCR was performed to detect the genes expression of FGF21 (A), AMPK (B), Sirt 1, Myoglobin(C), Desmin (D), MEF2c (E), a-actin (F). (G) The C2C12 myoblasts were transiently transfected with pCDNA3.1-21 or pCDNA3.1, Western blot showed FGF21 activated AMPK signal via FGF21-Sirt1-AMPK. For the phosphorylated AMPK (right), the intensity of band was normalized total AMPK, and then normalized by control. (H and I) FGF21, as well as AMPK activator acadesine (ACA), increased phosphorylation of AMPM, and myogenic genes expression, especially MyHC I, which was activated by ACA (FGF21‡ACA) and suppressed by AMPK inhibitor DOR(FGF21‡DOR). The data are presented as mean±SD (*P<0.05, **P<0.01, and P<0.001), n=ˆ3.

E, Analysis of BrCSC viability upon treatment with tamoxifen (tam) and fulvestrant (fulv) at the indicated doses. The experiments were performed in triplicates. ** P<0.01.

Inhibition of GSK3 activity induces an autophagic response in human pancreatic cancer cells. PANC1 cells were treated for 24 h with DMSO, CHIR99021 (CHIR; 5 uM) or the mTOR inhibitor Torin1 (250 nM). Autophagosome (punctate LC3B) (D) and lysosome (E) labeling was visualized with a Zeiss LSM700 confocal microscope. Images were acquired with a 20x (D) or 63x (E) objective. Nuclei were stained with DAPI. Scale bars, 20 祄.

Western blotting showing increased unconjugated SUMO1 levels in Notch1 ΔE cells treated with 10 uM DAPT for 3 days. Tubulin was used as a loading control.

for HXO-RB44 cells, IC50 values for VCR were changed in the different SG600 treatment groups. The results are representative of three independent  experiments and of f our replicates in each experiment.

Western blot analysis of Acetylated Histone and Histone. 0-10μM sodium valproate was added.

 

Columns depict relative densitometric values that were obtained by comparing with (β-actin. Presenilin 1, Nicastrin, and Pen-2 were increased with the 3-MA treatment (*P<0.001) while the other component of the γ-secretase complex (APH-1 and Presenilin 2) has no difference, compared with the STF-62247 treatment. Moreover, Bace1 and ADAM17 respectively had no difference between 3-MA and STF-62247 treatments. Both autophagy inhibitor and inducer activated α-, β-, and γ-secretases (*P<0.005). Abbreviations: Aβ, beta-amyloid; ADAM17, a disintegrin and metalloprotease 17; APH-1, anterior pharynx-defective 1; APP, amyloid protein precursor; Bace1, beta-site APP cleavage enzyme; 3-MA, 3-methyladenine; Con, control, LC3, microtubule-associated protein 1A/1B-light chain 3.

C. Lysosomal and endosomal inhibition selectively targets Tsc2−/− MEFs. Proliferation of Tsc2+/+ and Tsc2−/− MEFs treated with vehicle control (DMSO), CQ (5uM), Vps34 inhibitor (SAR405; 1uM) or the combination for 72 hours (crystal violet staining). D. Confirmation of selective growth inhibition using VPS34-IN1 inhibitor. Proliferation of Tsc2+/+ and Tsc2−/− MEFs treated with vehicle control (DMSO), CQ (5uM), VPS34-IN1 (500nM) or the combination for 72 hours (crystal violet staining). Two-sample t-test was carried out between CQ and SAR405 and each drug alone. Results are representative of three independent experiments. Bar graph represent means ±SD.

Effect of different proteasome inhibitors on dysferlin expression and on membrane resealing in cultured primary myoblasts. Primary myoblasts from patient 2 harboring a homozygous Arg555Trp DYSF mutation that were treated with the indicated amounts of bortezomib for 24 hours. Western blots of protein extracts were stained with anti-dysferlin antibodies and with anti–a-tubulin antibody as loading control.

Inhibition of PI3K, ERK and mTOR prevents the activation of S6K1 and S6 induced by suppression of PKD1 activity. A549 cells were incubated in the absence (-) or presence of either 5 uM Kb or 5 uM Kb and 20 uM LY294002 or 5 uM Kb and 10 uM BKM120 (as indicated) for 1 h prior to stimulation of cells with 50 nM PMA for 30 min and 1 h.

 

Effects of NVP-BKM120 and KU-55933 and their combination on the DNA damage response. A, HCC1937 cells were treated for 18 hours with NVP-BKM120 at 2.5 μmol/L, KU-55933 at 10 μmol/L, or their combination, subjected to ionizing radiation(IR) with 10 Gy or mock, lysed 6 hours later, and subjected to immunoblotting with antibodies as indicated.

Cells were stimulated with TPA (10 nM) for 15 min in the presence of the indicated concentrations of U0126. Samples were collected and analyzed by Western blot to detect phosphorylated p42/p44 MAPK.

Microscope image (406) magnification of U937 cells after exposure to SM-TNs. a) Untreated U937 cells. b) U937 cells treated with 200 nM, and 35 nM ODS empty shells. c) U937 cells treated with microcrystalline (free) Taxol prepared by sonicating Taxol in 20%H3PO4/0.8% SDS solution. d) U937 cells treated with 200 nM SM-TN. A SM-TN is seen in the middle of the picture. e) U937 cells treated with 35 nM SMTN.The nanowires are too small to see at this magnification, but their effect on the cells is clearly visible. Scale bar = 10 mm.

Loss of DUSP4 function upregulates IL-6 and IL-8 and enhances mammosphere growth. Immunoblot analysis of MDA-231 cells after treatment of 24 hours with 1 umol/L selumetinib (MEKi) or 10 umol/L SP600125 (JNKi). I, MDA-231 mammosphere formation quantitated by GelCount software 7 days after siRNA transfection. Where indicated, selumetinib (MEKi) or SP600125 (JNK1) or the combination was added to the mammosphere cultures.

 

L3.6pl cells at 6,000 cells per well were incubated in MEM with 5% FBS in triplicate in a 96-well culture plate and then treated alone with 5 umol/L BMS-777607, 10 umol/L wortmannin, or with BMS-777607 in combination with individual inhibitors. Polyploidy was examined under BK71 Olympus microscope and photographed 72 hours after treatment.

The inhibition of TGF-β1 signaling pathways suppressed EBV-mediated EMT, and prevented the activation of Syk and Src signaling. The EBV-infected HCECs were treated with 100 nM of the dual TGF-β receptor I and II kinase inhibitor, LY2109761, for 48 hours. The EBV-infected HCECs were cultured with anti-TGF-β1 neutralizing antibody (5 ug/mL) or mouse IgG1 antibody (5 ug/mL) for 48 hours. Photographs were taken at x100 magnification by a digital camera.

granulosa cells (GCs) with 24 h of melatonin (10 μM) treatment were rinsed in PBS, and then exposed to H2O2 (200 μM) for 2 h. The autophagy inhibitor 3-MA (10 mM), or the apoptosis inhibitor Z-VAD-FMK (50 μM) were added 1 h prior to H2O2 incubation. Cell viability was determined using the CCK-8 assay. Data represent mean ± S.E; n = 3 in each group. *P < 0.05 (**P < 0.01) vs. vehicle group at 0 h. # Represents P < 0.05 (## Represents P < 0.01) vs. H2O2-only-treated cells. & Represents P > 0.05 vs. H2O2-only-treated cells. N, not significant, P > 0.05. δ Represents P < 0.05 (δδ Represents P < 0.01) vs. Z-VAD-FMK-treated cells.

Protein expression by western blot analysis of cell lysates from MCF-7, TAM^R-4 and 164^R-7 cells treated for 3 days with vehicle (0.1% DMSO), 1 uM or 1.5 uM WP1130.

Verteporfin treatment inhibits proliferation and induces apoptosis of Tsc1-null cells in vivo. Mice were administered i.p. with vehicle or verteporfin at a dose of 100 mg/kg every other day for 10 d before sacrifice. Mice were sacrificed at 6 wk of age. Three independent experiments were performed and mice in different treatments were pooled for analysis. Percentage of Ki67 and αSMA double-positive cells in α-SMA+ mesenchymal lesions in the indicated kidneys. Immunofluorescence staining and counting were performed on three sagittal sections from different kidney regions for each mouse.

Cells were treated with brefeldin A or manumycin A, and the resulting supernatant was collected after 48 h for exosomal preparation (lanes 1 and 2), or exosomes obtained from C81 cells were trypsin-treated or freeze/thawed (F/T) and then trypsin-treated (lanes 3 and 4). Lanes 5 and 6, input exosome controls from C81 or CEM cells, respectively. Resulting exosomes were assayed for the presence of Tax by Western blotting.

Cytosolic extracts or nuclear extracts were examined by Western blot analysis using Abs against p105/p50, p100/p52 and phospho-p65. Solid arrowhead indicates a non-specific band. A nuclear marker, PARP, and cytosolic marker, b-tubulin, were used to assess the purity of each fraction.

A, HeLa cells were treated with DMSO, Taxol (100 nM for 16 h), or Nocodazole (Noco, 100 ng/ml for 16 h). Total cell lysates were probed with the indicated antibodies against Hippo components on Phos-tag SDS-polyacrylamide gels. O and * mark the non-phosphorylated and phosphorylated proteins, respectively.

a-d) LoVo cells were separately or simultaneously treated with 1 μM purmorphamine and 1 μM thiostrepton for the indicated time. a The Gli1, FoxM1, and CCNB1 protein expression levels were examined by immunoblotting after drug treatment for 48 h. b Cell viability was detected after 6 days using an MTT assay. c LoVo cells treated with indicated drugs were cultured for 2 weeks, and outgrowth colonies were stained with crystal violet. d The matched colony count of (c). Error bars represent the mean and S.D. of three independent experiments. **, p < 0.01.

NCI-H929 EV and miR-137 OE cells were treated with ATM activator Chloroquine Phosphate (CQ), specific ATM inhibitor KU-55933, and ATR inhibitor AZ20 for 12 hr. Immunoblotting showed the expression of p-ATM, p-Chk2, p-BRCA1, p-ATR and p-Chk1.

Mitochondrial depolarization mediated relocation of mitochondrial RC-TPP into lysosomes and the ensuing lysosomal acidity triggered rhodamine fluorescence. RC-TPP-loaded Tom20-GFP+ HeLa cells were treated without or with CCCP (20 μM) in the presence or absence of BFA (200 nM). Colocalization of Tom20-GFP (in green) and mitochondrial RC-TPP (in blue) is shown in cyan. Scale bar: 10 μm.

b EdU incorporation assays indicated PFK15 inhibited the cell proliferation of Cal27 cells. c The quantitative data of the EdU incorporation assays.

Inhibition of autophagy advances LPS-induced accumulation of G-MDSCs in vivo and in vitro. (A-E) C57BL/6 mice were treated with SBI-0206965 (5 μg/g) or vehicle for 2 h followed by LPS challenge (10 μg/g). Percentages of CD11b+Gr-1+ MDSCs (A, C), CD11b+Ly6G+Ly6Clow G-MDSCs (B, D) and CD11b+Ly6G-Ly6Chigh M-MDSCs (B, E) in spleens of these mice were analyzed with FACS at 12, 24 and 36 hours. The data are shown as the means ± SEM (n=5 replicates/group) and are representative of three independent experiments. (F, G) Bone marrow cells were pretreated with SBI-0206965 (1 μg/ml) or vehicle for 2 h and then cultured with GM-CSF (40 ng/ml) and IL-6 (40 ng/ml). Four days later, the percentages of CD11b+Gr-1+ MDSCs (F), CD11b+Ly6G+Ly6Clow G-MDSCs (G) and CD11b+Ly6G- Ly6Chigh M-MDSCs (G) were analyzed with FACS. Data are representative from one out of three biological replicates, each with three technical replicates. Error bars represent S.E.M. * p < 0.05, ** p < 0.01, *** p < 0.001, as determined by ANOVA tests; ns denotes p > 0.05.

Western blot analyses show significant inhibition of autophagy-related gene expression in Spautin-1 treated OCI-AML2 cells after Ara-C treatment.

Exosomal miR-124-3p inhibited neuronal inflammation by suppressing the activity of mTOR signaling. A, B) Immunoblot (A) and quantitative (B) data of p-4E-BP1 and p-P70S6K in neurons after scratch injury and exosomal treatment. Their expression was increased after scratch injury and was suppressed by miR-124-3p–up-regulated exosomes, suggesting that miR-124-3p suppresses the activity of mTOR signaling (n = 6/group). ##P < 0.01 vs. control group; **P < 0.01 vs. injury group. C) Expression levels of inflammatory mediators in the culture medium were detected in the 3 groups of neurons: injured neurons (injury group), injured neurons treated with miR-124-3p–up-regulated exosomes (I+Exo-124), and injured neurons treated with miR-124-3p-up-regulated exosomes and the mTOR activator (MHY1485). Compared with the I+Exo-124 group, the MHY1485 group represented an increased expression on proinflammatory cytokines (TNF-α, IL-1β, and IL-6) and a decreased expression of anti-inflammatory cytokines (IL-10), suggesting that MHY1485 blocks the anti-inflammatory effect of miR-124-3p in injured neurons. Thus, the inhibitory effect of exosomal miR-124-3p on neuronal inflammation was exerted by suppressing the activity of mTOR signaling (n = 6/group). #P < 0.05 vs. injury group; *P < 0.05 vs. I+Exo-124 group.

(c) Functional bile canaliculi or lack thereof in PHHs within MPTCs (12 d of drug treatment) as visualized by transport of fluorescent (green) dye into the canaliculi between PHHs. DMSO-treated MPCC control image is shown to the far right. (d) Neutral lipid (Nile red, green) staining of PHHs within MPTCs (12 d of drug treatment). DMSO-treated MPCC control image is shown to the far right. (e) NR1I2 (PXR) gene expression in drug-treated MPTCs relative to DMSOtreated MPTC controls (12 d of treatment). (f) ABCC2 (MRP2) gene expression in drug-treated MPTCs relative to DMSO-treated MPTC controls (12 d of treatment). (g) IL-6 levels in drug-treated MPTC supernatants (6 d of treatment). In all panels, statistical significance is displayed relative to DMSO-treated MPTCs. *p r 0.05, **p r 0.01, ***p r 0.001, and ****p r 0.0001. Scale bars on images represent 80 mm.

C, SA-beta gal staining results of A549-LKB1 cells treated by trametinib (30 nmol/L), radiotherapy (2 Gy), and HCQ (50 μmol/L). Cells were treated by HCQ and/or trametinib 4 hours prior to radiotherapy. Drugs were washed out 24 hours after radiotherapy. Cells were incubated for additional 48 hours before staining. D, Clonogenic survival assay of A549-LKB1 cells treated with trametinib (30 nmol/L) and HCQ (50 μmol/L).

LC3 and DAPI immunofluorescence staining were performed to detect autophagy in VSC4.1 cells treated with 25 mM HD alone, 25 mM HD + 5 μM PIK-III, 5 μM PIK-III and 0.1% DMSO alone (control group). Scale bar: 20 μm

VE-cadherin-induced Akt activation mediates YAP phosphorylation and translocation in ECs. HUVECs were starved for 1h and treated with thrombin (1U) for 1h. Total cell lysates were probed with anti-pAkt, Akt or b-actin antibody. The representative blots of three independent experiments are depicted, and the normalized values for p-Akt are shown. HUVECs were cultured and starved as described as in d and incubated for 8h in complete medium with the Akt inhibitor, MK-2206 (1 uM). pAkt, Akt, pYAP and YAP were detected by western blotting using specific antibodies.

Role of PARP and BER in the synergy between PTX and GMX in A549 cells. A) Cells were pre-treated +/- 1 uM olaparib (2h) then sequentially +/- 150nM PTX (24h) then +/- GMX 12nM (48h). Cells were harvested for (left) NAD+ quantification by LC-MS/MS (mean +/-SD of quadruplicates) or (right) viability by CellTiter-Glo (mean +/-SD of duplicates) B) PAR modification of proteins and γ-H2AX levels were measured in extracts treated as in A) by western blotting.

The regressing tumour microenvironment stimulates the outgrowth, infiltration and metastasis of drug-resistant clones. b, Bioluminescent signal of drug-resistant A375RTGL cells in vemurafenib-sensitive, A375 tumours, treated with vehicle or vemurafenib for 5 days (vehicle, n = 36; vemurafenib, n = 15 tumours). D, day. c, EdU incorporation in A375R-TGL cells in A375/A375R-TGL tumours treated with vehicle or vemurafenib for 4 days, as determined by FACS (vehicle, n = 8; vemurafenib, n = 6 tumours). d, Bioluminescent signal of A375R-TGL tumours alone, treated with vehicle or vemurafenib for 5 days (vehicle, n 5 38; vemurafenib, n = 15 tumours). e, Bioluminescent signal of TGLexpressing drug-resistant cancer cells (A375R, M249R4, PC9 and H2030) in drug-sensitive tumours (Colo800, LOX, UACC62, M249, H3122 and HCC827) treated with vehicle or drugs (vemurafenib, crizotinib and erlotinib) for 5 days (n (from left to right on the graph) = 6, 7, 12, 12, 9, 9, 25, 26, 9, 12, 12, 12, 16 and 11 tumours). f, Spontaneous lung metastasis by A375R cells in mice bearing A375/A375R-TGL tumours treated with vehicle or vemurafenib (10 days), visualized by BLI (n = 4).

Western blot analysis of histone H3 acetylation in the spleen of untreated and vorinostat-treated hNF-E2 tg mice (n = 4 of each genotype).

Cardiomyocytes transduced with or without Ad-Mst1 were treated with ABT-737 (0, 0.1, 1, 10 uM) for 12 hours. Representative immunoblots with antibodies to p62/SQSTM1, LC3 and GAPDH are shown.

The Rho GTPase-JNK pathway is required for the inhibitory effects of vandetanib on Calu-6 cells invasion. Calu-6 cells were incubated for 24 h in the presence or absence of vandetanib (1 or 2 uM), SP600125 (50 or 100 uM), and Y27632 (5 or 10 uM). The morphology of the Calu-6 cells was examined under a light microscope. Scale bar: 50 um.

MDA-MB-231 cells were treated with 10 uM MG132 and incubated under normoxia or hypoxia for 4 h. Endogenous interaction between LATS2 and SIAH2 was analysed by immunoprecipitation.

Three-dimensional responses of MCF7/IGF-1R cells to TAM (1 μM), E2 and IGF-1. Compared to parental MCF7 cells (a), MCF7/IGF-1R cells (b) in three-dimensional (3D) culture formed bigger acini in response to IGF-1 stimulation and displayed significant TAM resistance when treated with TAM (1 μM) + E2 + IGF-1, which was removable by kinase inhibitors BMS-536924, U0126 and BEZ235 (c). Cells (10,000/well) were seeded in 96-well plates. Acini were formed on 100% Matrigel and cultured for 14 days in starving medium containing 2% Matrigel and 5% charcoal/dextran-stripped fetal bovine serum with the treatments as indicated. Concentrations used: TAM (1 μM), E2 (1 nM) and IGF-1 (100 ng/mL). Confocal image original magnification, × 20. Red, rhodamine phalloidin (actin). Blue, Hoechst blue stain. Results are representative of two individual experiments.

 

Inhibition of breast cancer cell growth using sorafenib. MCF-7 breast cancer cells were treated with increasing concentrations of sorafenib for 5 days. Cell number was measured  using a colorimetric growth assay (crystal violet stain) and expressed relative to DMSO treated control cells.

Combinational treatment of kinase inhibitors induces the similar phenotype produced by PP1. All images are lateral view with dorsal to the top and anterior to the left. The combinational treatment of Dasatinib (D) or U0126 (U) with Sunitinib (SU),PTK787 (PTK), or ZM323881 (Z) resulted in the shrinkage of dorsal aorta.

H4-LC3-GFP cells were treated with 1 nM IFNA2 for the indicated periods in the presence of 200 nM rapamycin. Images of the cells were collected using an ArrayScan HCS 4.0 Reader. Representative cells are shown. The average spot intensity in 500 cells from each indicated sample was determined. Data are displayed as means ?SD of the spot intensity per cell (below). RLU, relative leight unit.

(c) Western blot analyses of p-Akt (Ser473) and p-S6RP (Ser235 and Ser236) in two RCT-E565 transplanted tumors treated with vehicle or PF02341066. Samples were isolated 4 h after the last dose from mice treated with PF02341066 for 3 d. (d) Responses of RCT-E565 transplanted tumors in athymic mice to PF02341066 or vehicle. Data are means ±s.e.m. (each group, n = 6). *P < 0.005, **P < 0.001 (Student^,s t test).

Effects of combined treatment with erlotinib and NPS-1034 in HCC827/ER cells with AXL activation. Lysates were immunoprecipitated with an anti-AXL antibody and immunoblotted with antibodies for phosphotyrosine (p-Tyr) and AXL. HCC827/ER cells were treated with erlotinib. E, erlotinib; N, NPS-1034. **, P < 0.001 for the combination of erlotinib plus NPS-1034 versus either the control or drug alone.

Cytoskeleton organisation of 786-O SuR treated with NVP-LDE225 (2.5 uM), everolimus (1 uM), and their combination for 24 h was analysed by confocal microscopy. Actin-based structures were revealed by rhodaminated phalloidin staining (red fluorescence). Localisation of focal adhesion points was obtained by immunofluorescent staining of p-paxillin (green fluorescence). Merged row images show overlapping of p-paxillin and actin signals. Moreover, all captures were shown in transmitted light. Scale bars, 10 um.

Perturbation of EGFR by its ligand EGF and gefitinib (ZD-1839 Iressat; inhibits EGFR) produces opposite responses in the predicted EGFR target genes SOCS2 and NR2E1.

 

Number of foci detected using laser confocal microscopy and fluorescent Fluor 647 anti-H2A.X-phosphorylated (Ser139) antibody. Double-stranded breaks (red) are clearly augmented in cells incubated with 500 nmol/l of ABT-888 and 500 nmol/l of AZD-2281 compared with PBS and 1% dimethyl sulfoxide controls. Image analysis was performed using ImageJ and the ‘analyze particle’ function.

(A) U87 cells were cultured in the presence of DMSO, 1 uM MS-275 alone, 100 ng/ml IFN-λ1 alone, or both for the course of 4 d. Cell numbers were manually determined by hemacytometer counting at the indicated time points. (B, F) Cell proliferation of U87 cells or U87 spheroids in 3D culture with indicated treatment were performed using the WST-1 assay, which measures active cellular metabolism. (C) U87 spheroid formation in 3D culture was photographed at day 14 in culture (representative images are shown; 200x magnification). (D-E) Quantification of the relative sizes and numbers of U87 spheroids in (C). (G) Cell cycle analysis was performed in U87 cells with indicated treatment using propidium iodide staining. Numbers in the histogram show fractions (percent) of sub-G1, N, 2N, and polyploidy from left to right. (H) U87 cells with indicated treatment were stained with Annexin V-FITC and 7-AAD. Numbers indicate the percentage of FITC-positive cells (upper left quadrant). FITC, fluorescein isothiocyanate; 7-AAD, 7-Aminoactinomycin. In all panels, data represent the mean and SEM of at least three experiments.

TGF-β induces mTORC2 activation. ( A ) NMuMG cells were treated with TGF-β for the indicated times before lysis and immunoblotting. (B ) NMuMG cells were treated or not with TGF-β for the indicated times, in the presence or absence of SB431542 or LY294002. Cell lysates were subjected to anti-Rictor immunoprecipitation, and the immunoprecipitates were subjected to in vitro kinase assays using kinase-inactive Akt1 as a substrate, before immunoblotting of the kinase reactions, immunoprecipitates and cell lysates. The left panels are from the same gel, without differential exposure. ( C ) NMuMG cells were treated or not with TGF-β or insulin for the indicated times, in the presence or absence of SB431542 or GDC-0941. The kinase activity of mTORC2 was then assessed as in B. The top panels are from the same gel, without differential exposure. (D) NMuMG cells were treated or not with TGF-β for the indicated times, in the presence or absence of SB431542 or LY294002. Cell lysates were subjected to immunoprecipitation using Rictor antibody, and/or immunoblotted.

ERK phosphorylates FBW7 at T205. PANC-1 cells were pretreated with the proteasome inhibitor MG132 and trametinib, as indicated, overnight before harvest. Endogenous FBW7 phosphorylation status was examined by immunoblot analysis after immunoprecipitates (IP).

STAT3 phosphorylation as determined by phospho flow, mixed lymphocyte reactions containing BALB/c spleen-derived CD4+ T cells co-cultured with or without C57BL/6 BM-derived DC preactivated with 20 ng/mL LPS.

LSD1 and HDAC inhibitors exhibit synergistic growth inhibition. Cells were simultaneously treated with pargyline or HDAC inhibitors for 48 h.

 

THOC5 phospho-Y225 levels were assessed in the populations shown by flow cytometry following 24 h treatment with 5 μM imatinib, 150 nM dasatinib or 5 μM nilotinib. Results are displayed as mean fluorescence intensity (n= 4) ±s.e.m.

Alisertib inhibits AURKA and AURKB in a concentration-dependent manner. (a) Alisertib induces G 2 /M delay or genome reduplication. HeLa cells were exposed to buffer or the indicated concentrations of Alisertib. After 24 h, the cells were harvested and analyzed with flow cytometry. The positions of 2N, 4N and 8N DNA contents are indicated. (b) Alisertib delays mitotic exit or induces slippage. HeLa cells stably expressing histone H2B-GFP were exposed to buffer or the indicated concentrations of Alisertib. Individual cells were then tracked for 24 h with time-lapse microscopy. Each horizontal bar represents one cell (n ¼ 50). Key: light gray ¼ interphase; black ¼ mitosis (from DNA condensation to anaphase or mitotic slippage); dark gray ¼ interphase after mitotic slippage; truncated bars ¼ cell death. (c) Different concentrations of Alisertib are involved in delaying mitotic exit and inducing slippage. Live-cell imaging of cells treated with Alisertib was described in panel (b). The duration of mitosis (mean±90% confidence interval) and the percentage of cells that underwent mitotic slippage during the imaging period was quantified. (d) Alisertib promotes apoptosis in a concentration-dependent manner. HeLa cells were incubated with the indicated concentrations of Alisertib for 48 h. The cells were then harvested and analyzed with flow cytometry. (e) Concentration-dependent cytotoxicity of Alisertib. HeLa cells were cultured in the presence of the indicated concentrations of Alisertib for 48 h. The number of live and dead cells was analyzed with trypan blue exclusion assay. (f) Concentration-dependent suppression of long-term survival by Alisertib. HeLa cells were seeded on 60-mm culture plates and grown in the presence of 250 n M or 1 m M of Alisertib. After 24 h, the cells were washed gently and propagated in normal medium for another 10–12 days. Colonies were fixed and stained with crystal violet solution (examples of the plates are shown). Average±s.d. from three independent experiments. (g) Both AURKA and AURKB are inhibited by Alisertib.Mitotic HeLa cells were obtained by exposure to nocodazole for 16 h followed by mechanical shake off. The cells were incubated with the indicated concentrations of Alisertib for 2 h. Lysates were then prepared and activated phospho-AURKAThr288 and AURKBThr232were detected with immunoblotting. The asterisk indicates the position of an AURKB-like protein (the same throughout this study). Uniform loading was confirmed by immunoblotting for actin. In this assay, nocodazole and MG132 (a proteasome inhibitor) were added to prevent the cells from exiting mitosis. Accordingly, the total AURKA and AURKB levels remained constant throughout the experiment. (h) Alisertib prevents activation of AURKA and AURKB. HeLa cells were incubated with the indicated concentrations of Alisertib for 8 h. Nocodazole was then added for another 6 h to trap cells that entered mitosis. Lysates were prepared and analyzed with immunoblotting. Actin analysis was included to assess loading and transfer.

Inhibition of signaling pathway activation in lung tumor cell lines by kinase inhibitors. Lung tumor cells were cultured in 10% FBS until reaching ∼80% confluence and then the cells were starved in serum-free medium for overnight, followed by 4-hour treatment with the inhibitors. Cell lysates were then prepared and used for determination of the pathway activation signals by the CEER assay.

293T cells were transfected with HA-tagged Fbxo45. At 48 h after transfection, cells were treated with AKT inhibitor (CAL-101; 10 uM, 4 h), cell extracts from the cytoplasm or nuclei were subjected to IP with anti-HA resin followed by western blot analysis with indicated antibodies.

mTOR kinase inhibitor AZD8055 activates PI3K accompanied with induction of expression of EGFR, HER2, HER3 and IRS1. Serum-deprived CHO-EGFP-AKT cells were incubated with 50 nM AZD8055 for 24 hr. The EGFP signal was detected using confocal microscopy. The white arrows indicate EGFP-AKT located on cellular membrane.

C and D, in vivo subcutaneous tumor growth curves (C) and tumor weight quantification of intersected subcutaneous tumor tissues (D) of Huh7 cells after stable LHBs expression under saracatinib treatment (25 mg/kg body weight daily for 4 weeks; n =18). *, P < 0.05. E and F,in vivo subcutaneous tumor growth curves (E) and tumor weight quantification of intersected subcutaneous tumor tissues (F) of SK-Hep1 cells after stable LHBs expression under saracatinib treatment (25 mg/kg body weight daily for 4 weeks; n = 18). *, P < 0.05.

In vivo antitumor efficacies of AZD2281 and GX15-070 alone or in combination in a BxPC-3 xenograft model. Tumor specimens were fixed in 10% formalin, embedded in paraffin, and cut into 4 lm-thick slides for H&E, PCNA, and CD34 staining.

SKBR3 cells were treated with FW-04-806 at 10, 20, 40 uM for 24 h; 17AAG was used as a positive control at 1 and 2 uM. Hsp70, Hsp90, and Cdc37 protein level were analyzed with western blotting using relevant antibodies.

Cell viabilities with increasing concentrations of cisplatin (CP) and doxorubicin (DOXO) under normoxic and hypoxic condition for 48 hours were determined by MTT assay. IC50 values are presented as the means ?SDs (n=4) and * denotes p<0.05.

Effect of nilotinib on Bcr-Abl kinase activity in ABCB1- and ABCG2- overexpressing CD34⁺CD38^- cells. K562 parental cells and CD34⁺CD38^- subpopulation isolated from K562 cells were treated with nilotinib at 0.01, 0.1 and 1.0 umol/L for 12 h. Equal amount of protein was loaded for western blot analysis as described in the Experimental section. The experiments were repeated at least three times independently, and a representative experiment is shown.

mTOR inhibitors attenuate ganetespib-driven elevation of HSPs in multiple tumor cell types. A375 melanoma cells were treated with vehicle, ganetespib (25 nmol/L), BEZ235 (500 nmol/L), or temsirolimus (500 nmol/L), either alone or in combination, for 24 hours. The levels of HSP90α, HSP70, HSP27, and GAPDH were determined by immunoblotting.

We treated all of drugs in T47D which has a PI3KCA H1044R mutation with the concentration shown below for 1 hour and performed western blot analysis using antibodies to phospho-AKT(SERINE 472), and total AKT.

 

RNA incorporating drugs induce SG assembly. HeLa cells were treated with the RNA incorporating agents 5-azacytidine (50 uM) and 6-thioguanine (10 uM), or the DNA incorporating agents trifluorothymidine (10 uM) and gemcitabine (100 nM) for 72 h. Subsequently, the cellular localization of the SG marker protein TIAR (green) and the P-body marker protein DCP1 (red) was analyzed. Nuclei were stained with Hoechst. Scale bars represent 20 um.

 

Comparison of MCAS ovarian cancer cells harboring control and CtBP2 knockdown shRNAs for sensitivity to chemotherapeutic agents. For each cell line, the MTT reading of the untreated cells was assigned as 100%. HDAC inhibitors: (a) Trichostatin A; (b) Vorinostat; (c) Belinostat; (d) MGCD0103; (e) valproic acid; and (f ) carboplatin, a non-HDAC inhibitor.

Hepatoma cells 24 h after plating were treated with vehicle (DMSO), regorafenib (REGO, 0.5 µM), PDE5 inhibitor (sildenafil, 2 µM); or the drugs in combination. 24 hours after treatment cells were isolated and viability determined by trypan blue (n=3, SEM). *P 0.05

PAI-1 expression in HUVECs treated with drugs as indicated.(D) senescent HUVECs were treated with SRT1720, culturing for 24, 48 hours. PAI-1 mRNA and protein (E) levels were analyzed using real-time RT–PCR and Western blotting, respectively. The RNA and protein levels were normalized to the internal control β-actin. Data are presented as the mean±SEM of three independent experiments. *P < 0.05 vs. corresponding control. **P < 0.01 vs. corresponding control ***P < 0.001 vs. corresponding control.

A,IC50 of Bosutinib that block ANDV-induced EC permeability. Endothelial cells were ANDV infected, and 3 days postinfection the permeability of cells in response to VEGF addition was determined in the presence or absence of increasing amounts of kinase inhibitor. The effect of inhibitors is presented as the percentage of ANDV-induced permeability of inhibitor-treated monolayers 3 days postinfection and 30 min post-VEGF and FITC-dextran addition. B, VEGFR2-Src inhibitors block ANDV-induced permeability. Endothelial cells were plated on vitronectin-coated Transwell inserts and infected at an MOI of 0.5 in triplicate with ANDV. Three days postinfection, the permeability of ANDV- and mock-infected endothelial cell monolayers was determined as described for Fig. 1 at indicated times in the presence or absence of Bosutinib .

C, Jurkat cells with SB202190 at 1, 5, and 10 μM were tested, and a decreased SRP72 expression was found when using at 10 μM (lanes 8 and 9). D, results were analyzed and RUA illustrated, finding significant results at 10 μM at 240 versus 0 and 120 versus 0 min (p<0.05).

 

Validation of activity and specificity of chemical inhibitors of; ATM, ATR, and DNAPK. H460 cells were treated with 1 uM camptothecin (CPT) or 20 ug/ml bleomycin for 1 h in the presence of the indicated inhibitors: DNAPK-i1—NU7026, DNAPK-i2—NU7441. MSH6,

PTPH1 confers breast cancer cell sensitivity to fulvestrant. E and F, PTPH1 overexpression increases the growth inhibition by fulvestrant. PTPH1 was overexpressed by a Tet-on system or a stable transfection, and resultant cells were incubated with fulvestrant as indicated for about 2 weeks. Colony formed was stained and counted. Results shown are normalized to its own solvent control of Vector and PTPH1-overexpressed cells, respectively (means ± SD; n = 3–5) with insets showing PTPH1 overexpression. *, versus vector or no Tet cells for E and F.