HSP (HSP90)

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.

Breast cancer (MDA-MB-231), pancreatic cancer (PaTu2), lung cancer (A549), colon cancer HCT-116, and acute myeloid leukemia (SKM1) cell lines were incubated with increasing amounts of PU-H71 and STA-9090 as indicated. Western blot analysis with PRKD2, cleaved PARP, and cleaved caspase-9 antibodies is depicted.

IC50 determinations of compound S1 and S13 on heat shock poteins ATPase activity using ADP fluorescence assay. (A) inhibition of HSP70 (B) inhibition of HSP90 The test compounds were diluted from mother plates (10 mM in 100% (v/v) DMSO) into series of concentration (in 2.0% (v/v) DMSO). AT13387 and 17-DMAG were used as positive controls in each assay. Data were performed in triplicate and analyzed by GraphPad.Prism.

Growth curves of the MPNST cell line S462 treated with two different HSP90 inhibitors (500nM IPI-504 and 500nM BEP800).

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.

Effect of SNX-2112 on E. histolytica morphology. Trophozoites were treated with 3 uM SNX-2112 for 48 h. (A) E. histolytica trophozoites treated with 0.5% DMSO. Arrow indicates live trophozoite. Magnification, x 10. (B) E. histolytica trophozoites treated with 3 uM SNX-2112. Treatment caused complete lysis of trophozoites (arrow). Magnification, x 10. Trophozoites were imaged under phase-contrast microscope.

B, XL888 (1 mmol/L, 144 hours) is effective at blocking the growth and survival of vemurafenib-resistant melanoma cell lines grown as 3D collagen-implanted spheroids. Staining shows cell viability, in which green corresponds to live cells and red: dead cells. Magnification×4.

Effect of pifithrin-μ (the inhibitor of mitochondrial translocation of p53) on MEG3-induced apoptosis in TGF-β1-treated LX-2 cells. The inhibitor pifithrin-μ attenuated cleavage of caspase 3 which blocked apoptosis. The results are expressed as relative expression against control expression without treatment.

(D) Representative images of paraffin-embedded tumor sections were analyzed by active caspase-3 staining. (E) Representative images of paraffin-embedded tumor sections analyzed by TUNEL staining.

Effects of low-level HSP90 inhibition (by ganetespib, 2-5 nM; PU-H71, 40-70 nM) or febrile-range temperature (39 ℃) on HSP70 and HSP90 protein levels in FANCA wild-type cells. High-level HSP90 inhibition (ganetespib, 25 nM; PU-H71, 300 nM) as well as proteotoxic proteasomal inhibition (MG132, 2.5 mM) induced the expression of HSP70. Constitutive (upper band: C) and inducible forms (lower band: I) of HSP70 are indicated. HSP90 levels are shown for comparison.

Representative images of platelets preincubated with FITC-conjugated CD41 before treated with negative control, pitstop 2, dynasore, A23, or apoptozole spreading on immobilized Fg for 10, 30, and 60 minutes, respectively. Images were taken under oil immersion with magnification × 100. Scale bar, 5 μm.

Western blots of lysates from BaF3 JAK2 V617F cells.

Spironolactone (sp.) and triptolide inhibit NER in myeloma cells. RPMI8226 cells were incubated with dimethyl sulfoxide (DMSO), spironolactone (10 μm) or triptolide (1 μm) for 6 h before NER evaluation. The figure represents the persistence of DNA-damage signal 150 min after exposure to UV (AFU: arbitrary fluorescent unit). Figure 4a shows representative merged pictures of DAPI and DDB2 proteo-probe signal (b).

(D) Microglial cultures treated with 300 μM H2O2 alone or 100 μM SNAP plus 300 μM H2O2 for 24 h in the absence or presence of a chemical inhibitor (20 μM VER-155008 or 20 μM SnPP) were subjected to the determination of cell viability (MTT reduction assay). ∗p < 0.05 vs. H2O2 (300 μM) alone. +p < 0.05 vs. SNAP (100 μM) plus H2O2 (300 μM).

Expression of CD24 and CD44 in HSP90 inhibitor‐treated, shRNA‐transduced HCC1937 cells was analyzed by flow cytometry.

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.

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.

SP cells in inhibitor-treated, shRNA-transduced HCC1937 cells analyzed by flow cytometry using Hoechst 33342 dye. Data are shown as mean±SD. ****P<.0001, ***P<.001, **P<.01, *P<.05.

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.

Breast cancer (MDA-MB-231), pancreatic cancer (PaTu2), lung cancer (A549), colon cancer HCT-116, and acute myeloid leukemia (SKM1) cell lines were incubated with increasing amounts of PU-H71 and STA-9090 as indicated. Western blot analysis with PRKD2, cleaved PARP, and cleaved caspase-9 antibodies is depicted.

IC50 determinations of compound S1 and S13 on heat shock poteins ATPase activity using ADP fluorescence assay. (A) inhibition of HSP70 (B) inhibition of HSP90 The test compounds were diluted from mother plates (10 mM in 100% (v/v) DMSO) into series of concentration (in 2.0% (v/v) DMSO). AT13387 and 17-DMAG were used as positive controls in each assay. Data were performed in triplicate and analyzed by GraphPad.Prism.

Growth curves of the MPNST cell line S462 treated with two different HSP90 inhibitors (500nM IPI-504 and 500nM BEP800).

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.

Effect of SNX-2112 on E. histolytica morphology. Trophozoites were treated with 3 uM SNX-2112 for 48 h. (A) E. histolytica trophozoites treated with 0.5% DMSO. Arrow indicates live trophozoite. Magnification, x 10. (B) E. histolytica trophozoites treated with 3 uM SNX-2112. Treatment caused complete lysis of trophozoites (arrow). Magnification, x 10. Trophozoites were imaged under phase-contrast microscope.

B, XL888 (1 mmol/L, 144 hours) is effective at blocking the growth and survival of vemurafenib-resistant melanoma cell lines grown as 3D collagen-implanted spheroids. Staining shows cell viability, in which green corresponds to live cells and red: dead cells. Magnification×4.

Effect of pifithrin-μ (the inhibitor of mitochondrial translocation of p53) on MEG3-induced apoptosis in TGF-β1-treated LX-2 cells. The inhibitor pifithrin-μ attenuated cleavage of caspase 3 which blocked apoptosis. The results are expressed as relative expression against control expression without treatment.

(D) Representative images of paraffin-embedded tumor sections were analyzed by active caspase-3 staining. (E) Representative images of paraffin-embedded tumor sections analyzed by TUNEL staining.

Effects of low-level HSP90 inhibition (by ganetespib, 2-5 nM; PU-H71, 40-70 nM) or febrile-range temperature (39 ℃) on HSP70 and HSP90 protein levels in FANCA wild-type cells. High-level HSP90 inhibition (ganetespib, 25 nM; PU-H71, 300 nM) as well as proteotoxic proteasomal inhibition (MG132, 2.5 mM) induced the expression of HSP70. Constitutive (upper band: C) and inducible forms (lower band: I) of HSP70 are indicated. HSP90 levels are shown for comparison.

Representative images of platelets preincubated with FITC-conjugated CD41 before treated with negative control, pitstop 2, dynasore, A23, or apoptozole spreading on immobilized Fg for 10, 30, and 60 minutes, respectively. Images were taken under oil immersion with magnification × 100. Scale bar, 5 μm.

Western blots of lysates from BaF3 JAK2 V617F cells.

Spironolactone (sp.) and triptolide inhibit NER in myeloma cells. RPMI8226 cells were incubated with dimethyl sulfoxide (DMSO), spironolactone (10 μm) or triptolide (1 μm) for 6 h before NER evaluation. The figure represents the persistence of DNA-damage signal 150 min after exposure to UV (AFU: arbitrary fluorescent unit). Figure 4a shows representative merged pictures of DAPI and DDB2 proteo-probe signal (b).

(D) Microglial cultures treated with 300 μM H2O2 alone or 100 μM SNAP plus 300 μM H2O2 for 24 h in the absence or presence of a chemical inhibitor (20 μM VER-155008 or 20 μM SnPP) were subjected to the determination of cell viability (MTT reduction assay). ∗p < 0.05 vs. H2O2 (300 μM) alone. +p < 0.05 vs. SNAP (100 μM) plus H2O2 (300 μM).

Expression of CD24 and CD44 in HSP90 inhibitor‐treated, shRNA‐transduced HCC1937 cells was analyzed by flow cytometry.

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.

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.

SP cells in inhibitor-treated, shRNA-transduced HCC1937 cells analyzed by flow cytometry using Hoechst 33342 dye. Data are shown as mean±SD. ****P<.0001, ***P<.001, **P<.01, *P<.05.