PI3K

Level of phospho-proteins in RTK and PI3K–Akt–mTOR pathways in treated tumours, detected with western blot.

Clonogenic assays of TNBC cell lines grown continuously in increasing concentrations of palbociclib, divided by gene expression subtypes: LAR, luminal androgen receptor; MSL, mesenchymal stem-like; MES, mesenchymal and basal-like. All cell lines are RB1 wild-type except RB1 mutant BT549.

Type I IFNs induce ISG expression in a serine phosphorylation-independent manner. Cells were treated with kinase inhibitors (SB203580, rottlerin, Ly294002, PD98059, and SP600125) before stimulation with IFN-a and IFN-b for 6 hours. Cell lysates were used in western blotting and probed for serine phosphorylation.The relative amounts of pSTAT1 S727 in (up-graph) were quantied and normalized to an untreated control (below-graph).

Invasive migration of RA FLS was analyzed through growth factor–reduced Matrigel-coated transwell inserts in the presence or absence of 1 µM INK007, 5 µM CAL-101, or 0.3 µM IPI-145, or 0.3 µM GDC-0941 inhibitors or DMSO. Cells were allowed to invade through Matrigel toward PDGF-BB (25 ng/ml) containing media for 24 h and were fixed and stained with Hemacolor staining kit.

NLGN3 mRNA expression in SU-pcGBM2 cells after 12 hr exposure to vehicle, 50 nM NLGN3, 100 nM BKM120, or 50 nM NLGN3 + 100 nM BKM120.

(D) IGROV parental, shControl (two independent clones) and ShPKCiota (two independent clones) were treated with 5 μM GDC-0941 and 1.5 μM PI-103 for 24 hours and subjected to western blot analysis with the indicated antibodies. Each experiment was performed in triplicate.

Cells were treated with cisplatin under the following conditions: pretreatment with Nu7026 (Nu7026, 26) or Nu7441 (Nu7441, 41). Before DNA damage, wortmannin was applied at 100 nM for 30 min, Nu7026 at 10 uM for 1 h, Nu7441 at 1 uM for 1 h. Accompanying quantifications of the adjusted nucleolar EU fluorescence are shown. Each condition was normalized to cells treated with DMSO or indicated inhibitors without DNA damaging treatment. Two-way ANOVA was followed by Holm-Sidak's test for the comparison of cells with and without inhibitors. *, **, *** represent P ≤ 0.05, 0.01, 0.001, respectively.

A p110β-selective antagonist rescues increased protein synthesis in synaptic fractions from Fmr1 KO mice and in LCLs from a patient with FXS. (A–C) Treatment of synaptoneurosomes with TGX-221 (1 μmol/L, 30 min) reduces p110β-specific PI3K activity and phosphorylation of the downstream target AKT in both WT and Fmr1 KO SNS, shown by a radioactive PI3K assay and phosphoAkt- specific western blot-ting (A). (B) Quantification of PI3K activity using a competitive ELISA showed a significant reduction in PI3K activity in both genotypes after treatment [ n = 4, 2-way ANOVA, *P (genotype) = 0.0086, * P (treatment) = 0.0087, P (interaction) = 0.7154]. (C) Densitometric quantification of phosphoAkt-specific western blots showed a significant effect of treatment (C, n = 4, 2-way ANOVA, *P (treatment) = 0.0005, P (genotype) =0.372, P (interaction) = 0.4894).

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.

Breast cancer cells were pretreated with 100ng/ml EGF for 20 min and then treated with the indicated concentrations of XL147 for 24 hours.

j,k, Gene expression ofMYOCD (j) and ACTA2 ( SMA, k) after applying inhibitors of key components involved in the DDR2 downstream signalling pathway to HSCs cultured within 3D collagen matrix subjected to stretching (ST) (n=3, one-way ANOVA, **P=0.0036, ****P<0.0001). l,m, Expression of SMA was significantly reduced after treatment with related inhibitors in early-stage FμNs. (n=4, one-way ANOVA, ***P=0.001, ****P<0.0001). PI3K-i: ZSTK474

AN3CA (B), JHUEM2 (D), and MFE296 (H) cells were treated with the indicated doses of BGJ398 and BYL719 alone or in combination for 96 hours, and an SRB assay was subsequently performed.

Wound healing assays using DMSO vehicle or 5 µM AS-605240. Resealing of the ‘wounded’ monolayer was examined after 48 h.

A549 cells were treated with DMSO or PIK-75 (200 nM) for 1 h and subsequently stimulated with izTRAIL for 24 h. Long-term survival was visualized after 7 days by crystal violet staining. One of two independent experiments is shown.

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.

(C) Western blot analyses of pAkt in MMTV-MT tumor derived cells treated with various PI3K inhibitors (in micromolar) as indicated following growth factor starvation. (D) Western blot analyses of pAkt in MMTV-NeuT tumor-derived cells treated with various PI3K inhibitors (in micromolar) as indicated. (*) P < 0.01; (**) P < 0.001 (Student’s t-test).

The TMZ/XL765 combination decreases serum GH and PRL of mice bearing GH3 xenografts. A and B, After treatment with DMSO, TMZ, XL765, or the XL765/TMZ combination, the blood of the nude mice xenograft with GH3 tumor was collected and serum rat-GH and rat-PRL were measured by IRMA (TMZ/XL765 combination vs TMZ or XL765 alone: ***P <.001).

After starved in serum-free medium for 24h,A549 cells incubated with the indicated concentrations of PIK-93 for 3h,followed by 20-minute stimolation of 100ng/ml EGF.

A, Effects of AUY922 and GSK458 alone on pathways downstream of KRAS. Western blotting of EGFR, pEGFR, pMEK, MEK, pAKT, AKT, pERK, ERK, HSP70, cleaved (c)PARP, and pRSK levels in two PI3K inhibitor-resistant NSCLC cell lines following treatment with each drug. β-Actin was included as a loading control.

3D cultures expressing control (pQCXIP GFP) or K-Ras V12 (pQCXIP GFP K-Ras V12) were continuously treated with 5, 10, or 20 uM of the MEK inhibitor U0126 or 2.0 uM of the PI3K inhibitor PIK-90. At day 10, these structures were fixed and stained for aPKC and DNA. Representative images are shown. Bar, 50 um.

Collagen-embedded melanoma spheroids were treated for 72 h with SGI-1776 [5 μM] or AZD6482 [10 μM] as single agents or in combination before staining for live (green) and dead (red) cells. Experiments were conducted in triplicate and representative images are shown. Scale bar represents 150 microns.

BMDMs from WT animals were treated with different concentrations of PI3K inhibitors (500 nmol/L PF4691502, PI-103, BKM120 and 25 μmol/L SF1126) followed by hypoxia for 4 hours for Western blots. These macrophages were either used for lysate preparation (nuclear extracts for HIFα or WCE for pAKT and AKT) and Western blot analysis.

Inhibition of PI3Kγ or PI3Kδ significantly decreased the migration of JVM3 cells. JVM3 cells were cultured in medium or stimulated for 24 h with CD40L/IL-4 and then incubated with the PI3Kγ-specific inhibitor CZC24832 (2 µM), the PI3Kδ-specific inhibitor idelalisib (1 µM), dual PI3Kδ/γ inhibitor duvelisib (1 µM), or the pan-PI3K inhibitor GDC0980 (1 µM) and subjected to a transwell migration assay. DMSO served as the vehicle control for the inhibitors, while SDF1α (100 ng/ml) served as the chemoattractant (n = 7).

The PI3K/AKT pathway significantly contributes to the ligand-driven HER2 signaling activities detected by CELx HSF tests. a and b SKBr3 cells were seeded in sensor plates and then treated with a serial titration of the PI3K/AKT pathway inhibitor GSK1059615 (0 nM to 810 nM) two hours prior to maximal stimulation with NRG1b (800 pM) (a) or EGF (600 pM) (b). CELx curves are displayed using Delta CI values to demonstrate the relative signals to the time point (arrow) when the stimulus (EGF or NRG1b) was added. Dose–response curves of GSK1059615 inhibition on NRG1b and EGF-driven HER2 signals are shown in the insets

Inhibition of PI3Kγ or PI3Kδ significantly decreased the migration of JVM3 cells. JVM3 cells were cultured in medium or stimulated for 24 h with CD40L/IL-4 and then incubated with the PI3Kγ-specific inhibitor CZC24832 (2 µM), the PI3Kδ-specific inhibitor idelalisib (1 µM), dual PI3Kδ/γ inhibitor duvelisib (1 µM), or the pan-PI3K inhibitor GDC0980 (1 µM) and subjected to a transwell migration assay. DMSO served as the vehicle control for the inhibitors, while SDF1α (100 ng/ml) served as the chemoattractant (n = 7).

PI3K inhibitors promote apoptosis in checkpoint-defective cell lines. Two checkpoint-functional (A2058, D28) and three defective (HT144, D20, SKMel13) melanoma cell lines growth as tumour spheres as in Figure 4B were either untreated or treated with 5 uM PF-05212384 for 72 h, harvested and immunoblotted for pAkt Ser473.

(E) Phosphorylation of CREB affected by different concentrations of TG100-115 determined by using immunoblotting

Effects of AS252424 on KL-induced cPLA 2 phosphorylation in BMMCs. BMMCs were pre-incubated with AS252424 for 1 h followed by stimulation with KL for 15 min. The phosphorylation of cPLA 2 was evaluated via immunoblot analysis. Results are representative of three independent experiments.*P<0.05, **P<0.01, ***P<0.001 and ### P<0.001, compared with BMMCs with KL induction but without pretreatment by AS252424.

IGROV1-R10 cells were treated with BGT266(250nM) for 8 h.The effect of BGT226 on PI3K/Akt/mTOR pathway activation (A) and on expression of Mcl-1 and Bim (B).

Representative Oil Red O staining of lipid-filled mature adipocytes on day 7 for uninduced cells (a), adipocyteinduced hMSCs exposed to the vehicle control (b) or CUDC-907-treated cells (500 nM) (c). Nile red staining (d and e) on day 7 of post-adipocytic induction in hMSCs and after exposure to CUDC-907. Images were captured at ×20 magnification using the FLoid Cell Imaging Station. The level of Nile red staining was quantified using the Molecular Devices M5 Microplate Reader (f). Data are presented as mean ± S.E (n = 16) from three independent experiments, ***P <0.0005.

After starved in serum-free medium for 24 h,A549 cells incubated with the indicated concentrations of PIK-294 for 3 h,followed by 20-minute stimolation of 100ng/ml EGF.

Numbers of AID on-target (Igh and Igk loci) and off-target hot spots from WT and AID−/− B cells. Data pooled from at least n = 3 biological replicates

Cells were treated as indicated. Medium and drug were refreshed every 2 to 3 days, and protein lysates were analyzed as above.

(A) Total cell lysates were immunoblotted with the indicated antibodies. β-actin served as the loading control.

Validation study for vacuolin-1 and YM201636. (A) HeLa cells were treated with 3 lM vacuolin-1 or YM201636 in the presence or absence of the lysosomal protease inhibitor E64d (10 μg/mL) and pepstatin A (10 μg/mL). After 24 h of treatment, cell lysates (10 μg) were separated by 10% polyacrylamide gel electrophoresis, and LC3 was detected via immunoblotting.

Effect of selected compounds on MLC phosphorylation induced by 2-AG. Washed platelets (1.0 x 10⁹ platelets/mL) were preincubated at 37 ℃ for 10 min with saline or 20 uM LY294002 (L), 10 uM CH5132799 (C), 10 uM TGX221 (T), 10 uM MK2206 (M), 20 uM Y27632 (Y), 1 uM PIK-75 (P), 1 U/mL apyrase (AP), 5 uM AS252424 (AS), 1 uM IC87114 (IC) and then stimulated for 30 s with 10 uM 2-AG. At the end of incubation suitable aliquots were immunoblotted with anti-p-MLC (thr18/ser19) as detailed in Methods. Blot is representative of four independent experiments.

(D) Box plots showing the results of primary MM cell treatment (n = 22; in co-culture with BMSCs) with PI3K inhibitors (10 umol/l each). BYL-719 exerted the strongest anti-myeloma effects of TGX-221, CAY10505 or CAL-101. The mean survival value is indicated by a horizontal bar and the end of the whiskers show the values of the most sensitive and most resistant sample, respectively. (E) Treatment of PBMCs (n = 5) for 5 d with PI3K isoform-specific inhibitors (10 umol/l each).

After starved in serum-free medium for 24 h,A549 cells incubated with the indicated concentrations of PIK-293 for 3 h,followed by 20-minute stimolation of 100ng/ml EGF.

The compounds BAY80-6946 and TG100713, which are respectively an alpha/beta- and a pan-isoforms inhibitors, demonstrated a very good ability to block Jurkat E6.1 proliferation with an IC50 slightly higher than 1 mM.

HCT116 cells were treated with Go 6983 (0.75 μM) or VS-5584 (0.6 μM) alone or in combination with cobimetinib (0.15 μM) for 48 h. Then, cell viability was evaluated by MTT.

(c) MM primary cells were treated with idelalisib (1 μm), CZC24832 (1 μm) and duvelisib (1 μm) for 72 h and then assessed for apoptosis by PI/Annexin V staining (n=4).

b, d Serum-deprived MG-63 (b) and U20S cells (d) were pre-treated with 1 nmol/L HS-173 (PI3Kα inhibitor), 10 nmol/L TGX-221 (PI3Kβ inhibitor), 10 nmol/L CZC24832 (PI3Kγ inhibitor) and 10 nmol/L CAL-101 (PI3Kδ inhibitor) for 1 h, then were incubated with 100 ng/mL Wnt5a and harvested at 30 min after the start of Wnt5a treatment. Data were presented as mean ± SD of 3 determinations. The relative RhoA activity was normalized to the average value of each inhibitor-untreated group

After starved in serum-free medium for 24h,A549 cells incubated with the indicated concentrations of Quercetin for 3h,followed by 20-minute stimolation of 100ng/ml EGF.

Confirmation of selective growth inhibition using VPS34-IN1 inhibitor. Proliferation of Tsc2+/+ and Tsc2-/- MEFs treated with vehicle control (DMSO), CQ (5 uM), VPS34-IN1 (500 nM) 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.

HL60, HL60/ADR, K562 and K562/A02 cells pretreated with various concentrations of Voxtalisib (0, 0.625, 1.25, 2.5, 5 and 10 μM) for 48 h were harvested for western blot. The levels of cyclin D1, p27, and p-pRb in the nuclei were determined.

The structure and formula weight of LY3023414 were presented (A). Established human glioma cells (U251MG and A172 lines), primary human astrocytes (“Astrocytes”) or primary human glioma cells [three lines, “Glioma (L1/2/3)”], were either left untreated (“Ctrl”) or treated with LY3023414 at 1-1000 nM, cells were further cultured for indicated time; Cell survival was tested (B, C, and E) (n=5). Cell proliferation was also tested by the BrdU ELISA assay (D and F) (n=5). *p < 0.05 vs. “Ctrl”. Experiments in this figure were repeated three times.

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.

RT-112 and RT-112 (CisPt-R) cells were treated with cisplatin, SAR405, and a combination of both in absence and presence of QVD (10 µM). Again, the concentrations used were the IC50 or half IC50 values for RT-112 (CisPt-R) and RT-112, respectively. After 48 hours, the cells were lysed, and cleared cellular lysates were subjected to SDS-PAGE and immunoblotting for PARP, caspase-3 (Casp3), and Actin. One representative immunoblot is shown.

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

I. OMI index decreases in BT474 organoids treated with single and combination therapies at 24 hr. J. OMI index of BT474 organoids treated for 72hr. Red bars denote p<0.05 for treated organoids vs. control. H:trastuzumab (anti-HER2); P:paclitaxel (chemotherapy); XL147 (X).

Level of phospho-proteins in RTK and PI3K–Akt–mTOR pathways in treated tumours, detected with western blot.

Clonogenic assays of TNBC cell lines grown continuously in increasing concentrations of palbociclib, divided by gene expression subtypes: LAR, luminal androgen receptor; MSL, mesenchymal stem-like; MES, mesenchymal and basal-like. All cell lines are RB1 wild-type except RB1 mutant BT549.

Type I IFNs induce ISG expression in a serine phosphorylation-independent manner. Cells were treated with kinase inhibitors (SB203580, rottlerin, Ly294002, PD98059, and SP600125) before stimulation with IFN-a and IFN-b for 6 hours. Cell lysates were used in western blotting and probed for serine phosphorylation.The relative amounts of pSTAT1 S727 in (up-graph) were quantied and normalized to an untreated control (below-graph).

Invasive migration of RA FLS was analyzed through growth factor–reduced Matrigel-coated transwell inserts in the presence or absence of 1 µM INK007, 5 µM CAL-101, or 0.3 µM IPI-145, or 0.3 µM GDC-0941 inhibitors or DMSO. Cells were allowed to invade through Matrigel toward PDGF-BB (25 ng/ml) containing media for 24 h and were fixed and stained with Hemacolor staining kit.

NLGN3 mRNA expression in SU-pcGBM2 cells after 12 hr exposure to vehicle, 50 nM NLGN3, 100 nM BKM120, or 50 nM NLGN3 + 100 nM BKM120.

(D) IGROV parental, shControl (two independent clones) and ShPKCiota (two independent clones) were treated with 5 μM GDC-0941 and 1.5 μM PI-103 for 24 hours and subjected to western blot analysis with the indicated antibodies. Each experiment was performed in triplicate.

Cells were treated with cisplatin under the following conditions: pretreatment with Nu7026 (Nu7026, 26) or Nu7441 (Nu7441, 41). Before DNA damage, wortmannin was applied at 100 nM for 30 min, Nu7026 at 10 uM for 1 h, Nu7441 at 1 uM for 1 h. Accompanying quantifications of the adjusted nucleolar EU fluorescence are shown. Each condition was normalized to cells treated with DMSO or indicated inhibitors without DNA damaging treatment. Two-way ANOVA was followed by Holm-Sidak's test for the comparison of cells with and without inhibitors. *, **, *** represent P ≤ 0.05, 0.01, 0.001, respectively.

A p110β-selective antagonist rescues increased protein synthesis in synaptic fractions from Fmr1 KO mice and in LCLs from a patient with FXS. (A–C) Treatment of synaptoneurosomes with TGX-221 (1 μmol/L, 30 min) reduces p110β-specific PI3K activity and phosphorylation of the downstream target AKT in both WT and Fmr1 KO SNS, shown by a radioactive PI3K assay and phosphoAkt- specific western blot-ting (A). (B) Quantification of PI3K activity using a competitive ELISA showed a significant reduction in PI3K activity in both genotypes after treatment [ n = 4, 2-way ANOVA, *P (genotype) = 0.0086, * P (treatment) = 0.0087, P (interaction) = 0.7154]. (C) Densitometric quantification of phosphoAkt-specific western blots showed a significant effect of treatment (C, n = 4, 2-way ANOVA, *P (treatment) = 0.0005, P (genotype) =0.372, P (interaction) = 0.4894).

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.

Breast cancer cells were pretreated with 100ng/ml EGF for 20 min and then treated with the indicated concentrations of XL147 for 24 hours.

j,k, Gene expression ofMYOCD (j) and ACTA2 ( SMA, k) after applying inhibitors of key components involved in the DDR2 downstream signalling pathway to HSCs cultured within 3D collagen matrix subjected to stretching (ST) (n=3, one-way ANOVA, **P=0.0036, ****P<0.0001). l,m, Expression of SMA was significantly reduced after treatment with related inhibitors in early-stage FμNs. (n=4, one-way ANOVA, ***P=0.001, ****P<0.0001). PI3K-i: ZSTK474

AN3CA (B), JHUEM2 (D), and MFE296 (H) cells were treated with the indicated doses of BGJ398 and BYL719 alone or in combination for 96 hours, and an SRB assay was subsequently performed.

Wound healing assays using DMSO vehicle or 5 µM AS-605240. Resealing of the ‘wounded’ monolayer was examined after 48 h.

A549 cells were treated with DMSO or PIK-75 (200 nM) for 1 h and subsequently stimulated with izTRAIL for 24 h. Long-term survival was visualized after 7 days by crystal violet staining. One of two independent experiments is shown.

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.

(C) Western blot analyses of pAkt in MMTV-MT tumor derived cells treated with various PI3K inhibitors (in micromolar) as indicated following growth factor starvation. (D) Western blot analyses of pAkt in MMTV-NeuT tumor-derived cells treated with various PI3K inhibitors (in micromolar) as indicated. (*) P < 0.01; (**) P < 0.001 (Student’s t-test).

The TMZ/XL765 combination decreases serum GH and PRL of mice bearing GH3 xenografts. A and B, After treatment with DMSO, TMZ, XL765, or the XL765/TMZ combination, the blood of the nude mice xenograft with GH3 tumor was collected and serum rat-GH and rat-PRL were measured by IRMA (TMZ/XL765 combination vs TMZ or XL765 alone: ***P <.001).

After starved in serum-free medium for 24h,A549 cells incubated with the indicated concentrations of PIK-93 for 3h,followed by 20-minute stimolation of 100ng/ml EGF.

A, Effects of AUY922 and GSK458 alone on pathways downstream of KRAS. Western blotting of EGFR, pEGFR, pMEK, MEK, pAKT, AKT, pERK, ERK, HSP70, cleaved (c)PARP, and pRSK levels in two PI3K inhibitor-resistant NSCLC cell lines following treatment with each drug. β-Actin was included as a loading control.

3D cultures expressing control (pQCXIP GFP) or K-Ras V12 (pQCXIP GFP K-Ras V12) were continuously treated with 5, 10, or 20 uM of the MEK inhibitor U0126 or 2.0 uM of the PI3K inhibitor PIK-90. At day 10, these structures were fixed and stained for aPKC and DNA. Representative images are shown. Bar, 50 um.

Collagen-embedded melanoma spheroids were treated for 72 h with SGI-1776 [5 μM] or AZD6482 [10 μM] as single agents or in combination before staining for live (green) and dead (red) cells. Experiments were conducted in triplicate and representative images are shown. Scale bar represents 150 microns.

BMDMs from WT animals were treated with different concentrations of PI3K inhibitors (500 nmol/L PF4691502, PI-103, BKM120 and 25 μmol/L SF1126) followed by hypoxia for 4 hours for Western blots. These macrophages were either used for lysate preparation (nuclear extracts for HIFα or WCE for pAKT and AKT) and Western blot analysis.

Inhibition of PI3Kγ or PI3Kδ significantly decreased the migration of JVM3 cells. JVM3 cells were cultured in medium or stimulated for 24 h with CD40L/IL-4 and then incubated with the PI3Kγ-specific inhibitor CZC24832 (2 µM), the PI3Kδ-specific inhibitor idelalisib (1 µM), dual PI3Kδ/γ inhibitor duvelisib (1 µM), or the pan-PI3K inhibitor GDC0980 (1 µM) and subjected to a transwell migration assay. DMSO served as the vehicle control for the inhibitors, while SDF1α (100 ng/ml) served as the chemoattractant (n = 7).

The PI3K/AKT pathway significantly contributes to the ligand-driven HER2 signaling activities detected by CELx HSF tests. a and b SKBr3 cells were seeded in sensor plates and then treated with a serial titration of the PI3K/AKT pathway inhibitor GSK1059615 (0 nM to 810 nM) two hours prior to maximal stimulation with NRG1b (800 pM) (a) or EGF (600 pM) (b). CELx curves are displayed using Delta CI values to demonstrate the relative signals to the time point (arrow) when the stimulus (EGF or NRG1b) was added. Dose–response curves of GSK1059615 inhibition on NRG1b and EGF-driven HER2 signals are shown in the insets

Inhibition of PI3Kγ or PI3Kδ significantly decreased the migration of JVM3 cells. JVM3 cells were cultured in medium or stimulated for 24 h with CD40L/IL-4 and then incubated with the PI3Kγ-specific inhibitor CZC24832 (2 µM), the PI3Kδ-specific inhibitor idelalisib (1 µM), dual PI3Kδ/γ inhibitor duvelisib (1 µM), or the pan-PI3K inhibitor GDC0980 (1 µM) and subjected to a transwell migration assay. DMSO served as the vehicle control for the inhibitors, while SDF1α (100 ng/ml) served as the chemoattractant (n = 7).

PI3K inhibitors promote apoptosis in checkpoint-defective cell lines. Two checkpoint-functional (A2058, D28) and three defective (HT144, D20, SKMel13) melanoma cell lines growth as tumour spheres as in Figure 4B were either untreated or treated with 5 uM PF-05212384 for 72 h, harvested and immunoblotted for pAkt Ser473.

(E) Phosphorylation of CREB affected by different concentrations of TG100-115 determined by using immunoblotting

Effects of AS252424 on KL-induced cPLA 2 phosphorylation in BMMCs. BMMCs were pre-incubated with AS252424 for 1 h followed by stimulation with KL for 15 min. The phosphorylation of cPLA 2 was evaluated via immunoblot analysis. Results are representative of three independent experiments.*P<0.05, **P<0.01, ***P<0.001 and ### P<0.001, compared with BMMCs with KL induction but without pretreatment by AS252424.

IGROV1-R10 cells were treated with BGT266(250nM) for 8 h.The effect of BGT226 on PI3K/Akt/mTOR pathway activation (A) and on expression of Mcl-1 and Bim (B).

Representative Oil Red O staining of lipid-filled mature adipocytes on day 7 for uninduced cells (a), adipocyteinduced hMSCs exposed to the vehicle control (b) or CUDC-907-treated cells (500 nM) (c). Nile red staining (d and e) on day 7 of post-adipocytic induction in hMSCs and after exposure to CUDC-907. Images were captured at ×20 magnification using the FLoid Cell Imaging Station. The level of Nile red staining was quantified using the Molecular Devices M5 Microplate Reader (f). Data are presented as mean ± S.E (n = 16) from three independent experiments, ***P <0.0005.

After starved in serum-free medium for 24 h,A549 cells incubated with the indicated concentrations of PIK-294 for 3 h,followed by 20-minute stimolation of 100ng/ml EGF.

Numbers of AID on-target (Igh and Igk loci) and off-target hot spots from WT and AID−/− B cells. Data pooled from at least n = 3 biological replicates

Cells were treated as indicated. Medium and drug were refreshed every 2 to 3 days, and protein lysates were analyzed as above.

(A) Total cell lysates were immunoblotted with the indicated antibodies. β-actin served as the loading control.

Validation study for vacuolin-1 and YM201636. (A) HeLa cells were treated with 3 lM vacuolin-1 or YM201636 in the presence or absence of the lysosomal protease inhibitor E64d (10 μg/mL) and pepstatin A (10 μg/mL). After 24 h of treatment, cell lysates (10 μg) were separated by 10% polyacrylamide gel electrophoresis, and LC3 was detected via immunoblotting.

Effect of selected compounds on MLC phosphorylation induced by 2-AG. Washed platelets (1.0 x 10⁹ platelets/mL) were preincubated at 37 ℃ for 10 min with saline or 20 uM LY294002 (L), 10 uM CH5132799 (C), 10 uM TGX221 (T), 10 uM MK2206 (M), 20 uM Y27632 (Y), 1 uM PIK-75 (P), 1 U/mL apyrase (AP), 5 uM AS252424 (AS), 1 uM IC87114 (IC) and then stimulated for 30 s with 10 uM 2-AG. At the end of incubation suitable aliquots were immunoblotted with anti-p-MLC (thr18/ser19) as detailed in Methods. Blot is representative of four independent experiments.

(D) Box plots showing the results of primary MM cell treatment (n = 22; in co-culture with BMSCs) with PI3K inhibitors (10 umol/l each). BYL-719 exerted the strongest anti-myeloma effects of TGX-221, CAY10505 or CAL-101. The mean survival value is indicated by a horizontal bar and the end of the whiskers show the values of the most sensitive and most resistant sample, respectively. (E) Treatment of PBMCs (n = 5) for 5 d with PI3K isoform-specific inhibitors (10 umol/l each).

After starved in serum-free medium for 24 h,A549 cells incubated with the indicated concentrations of PIK-293 for 3 h,followed by 20-minute stimolation of 100ng/ml EGF.

The compounds BAY80-6946 and TG100713, which are respectively an alpha/beta- and a pan-isoforms inhibitors, demonstrated a very good ability to block Jurkat E6.1 proliferation with an IC50 slightly higher than 1 mM.

HCT116 cells were treated with Go 6983 (0.75 μM) or VS-5584 (0.6 μM) alone or in combination with cobimetinib (0.15 μM) for 48 h. Then, cell viability was evaluated by MTT.

(c) MM primary cells were treated with idelalisib (1 μm), CZC24832 (1 μm) and duvelisib (1 μm) for 72 h and then assessed for apoptosis by PI/Annexin V staining (n=4).

b, d Serum-deprived MG-63 (b) and U20S cells (d) were pre-treated with 1 nmol/L HS-173 (PI3Kα inhibitor), 10 nmol/L TGX-221 (PI3Kβ inhibitor), 10 nmol/L CZC24832 (PI3Kγ inhibitor) and 10 nmol/L CAL-101 (PI3Kδ inhibitor) for 1 h, then were incubated with 100 ng/mL Wnt5a and harvested at 30 min after the start of Wnt5a treatment. Data were presented as mean ± SD of 3 determinations. The relative RhoA activity was normalized to the average value of each inhibitor-untreated group

After starved in serum-free medium for 24h,A549 cells incubated with the indicated concentrations of Quercetin for 3h,followed by 20-minute stimolation of 100ng/ml EGF.

Confirmation of selective growth inhibition using VPS34-IN1 inhibitor. Proliferation of Tsc2+/+ and Tsc2-/- MEFs treated with vehicle control (DMSO), CQ (5 uM), VPS34-IN1 (500 nM) 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.

HL60, HL60/ADR, K562 and K562/A02 cells pretreated with various concentrations of Voxtalisib (0, 0.625, 1.25, 2.5, 5 and 10 μM) for 48 h were harvested for western blot. The levels of cyclin D1, p27, and p-pRb in the nuclei were determined.

The structure and formula weight of LY3023414 were presented (A). Established human glioma cells (U251MG and A172 lines), primary human astrocytes (“Astrocytes”) or primary human glioma cells [three lines, “Glioma (L1/2/3)”], were either left untreated (“Ctrl”) or treated with LY3023414 at 1-1000 nM, cells were further cultured for indicated time; Cell survival was tested (B, C, and E) (n=5). Cell proliferation was also tested by the BrdU ELISA assay (D and F) (n=5). *p < 0.05 vs. “Ctrl”. Experiments in this figure were repeated three times.

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.

RT-112 and RT-112 (CisPt-R) cells were treated with cisplatin, SAR405, and a combination of both in absence and presence of QVD (10 µM). Again, the concentrations used were the IC50 or half IC50 values for RT-112 (CisPt-R) and RT-112, respectively. After 48 hours, the cells were lysed, and cleared cellular lysates were subjected to SDS-PAGE and immunoblotting for PARP, caspase-3 (Casp3), and Actin. One representative immunoblot is shown.

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

I. OMI index decreases in BT474 organoids treated with single and combination therapies at 24 hr. J. OMI index of BT474 organoids treated for 72hr. Red bars denote p<0.05 for treated organoids vs. control. H:trastuzumab (anti-HER2); P:paclitaxel (chemotherapy); XL147 (X).