Fig 1: PUMA is required for the antitumor effects of BRD9 inhibition.A Nude mice were injected s.c. with 5 × 106 WT or PUMA-KO GIST-882 cells. After 7 days, mice were treated with 10 mg/kg GSK602 or the vehicle control for 10 consecutive days. Tumor volume at indicated time points after treatment was calculated and plotted with p values, n = 6 in each group. B Mice weight after treatment. C Mice with WT GIST-882 xenograft tumors were treated with 10 mg/kg GSK602 or the vehicle as in (A) for 4 consecutive days. The level of PUMA in three randomly selected tumors were analyzed by Western blotting. D Paraffin-embedded sections of WT or PUMA-KO tumor tissues from mice treated as in (C) were analyzed by TUNEL staining. Left, representative TUNEL staining pictures; Right, TUNEL-positive cells were counted and plotted, Scale bar: 25 µm. E Tissue sections from (D) were analyzed by active caspase 3 staining. Left, representative staining pictures; Right, active caspase 3-positive cells were counted and plotted, Scale bar: 25 µm. Results were expressed as means ± SD of three independent experiments. *P < 0.05.
Fig 2: BRD9 is overexpressed in GISTs.A BRD9 protein expression in GISTs and adjacent nontumor tissues was examined by IHC. Scale bar: 100 µm. B BRD9 protein expression in GISTs and adjacent nontumor tissues was examined by western blotting. C BRD9 mRNA level in GISTs and adjacent nontumor tissues was examined by real-time PCR. Results were expressed as means ± SD of three independent experiments. *P < 0.05; **P < 0.01.
Fig 3: PUMA is required for BRD9 inhibition-induced apoptosis in GIST.A GIST-882 cells were treated with GSK602 at indicated concentrations for 24 h. Left, PUMA expression was analyzed by western blotting. Right, PUMA mRNA level was analyzed by real-time PCR. B GIST-T1 cells were treated with GSK602 at indicated concentrations for 24 h. Left, PUMA expression was analyzed by western blotting. Right, PUMA mRNA level was analyzed by real-time PCR. C GIST-882 cells were treated with GSK602 at indicated concentrations for 24 h. The expression of indicated Bcl-2 family members was analyzed by western blotting. D GIST-T1 cells were treated with GSK602 at indicated concentrations for 24 h. The expression of PUMA was analyzed by western blotting. E WT and PUMA-KO GIST-882 cells were treated with 0.5 µM GSK602 for 24 h. Apoptosis was analyzed by fragment nuclei assay. F WT and PUMA-KO GIST-T1 cells were treated with 0.5 µM GSK602 for 24 h. Apoptosis was analyzed by fragment nuclei assay. G WT and PUMA-KO GIST-882 or GIST-T1 cells were treated with 0.5 µM GSK602 for 24 h. Indicated protein level was analyzed by western blotting. H Cytosolic fractions isolated from WT and PUMA-KO GIST-882 cells treated with 0.5 µM GSK602 for 24 hr were probed for cytochrome c by western blotting. ß-actin and cytochrome oxidase subunit IV (Cox IV), which are expressed in cytoplasm and mitochondria, respectively, were analyzed as the control for loading and fractionation. I WT and PUMA-KO GIST-882 or GIST-T1 cell lines were treated 0.5 µM GSK602 for 24 h. Cell viability was analyzed by colony formation assay. Results were expressed as means ± SD of three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001.
Fig 4: BRD9 inhibition enhances the antitumor effect of imatinib in vitro.A GIST-882 cells were treated with GSK602 combined with imatinib at indicated concentration for 48 h. Cell viability was analyzed by MTT. B Combination index (CI) and fraction affected of GSK602 and imatinib combining at different concentration in GIST-882 cells treated for 48 h were analyzed by the CompuSyn program (ComboSyn). C GIST-T1 cells were treated with GSK602 combined with imatinib at indicated concentration for 48 h. Cell viability was analyzed by MTT. D Combination index (CI) and fraction affected of GSK602 and imatinib combining at different concentration in GIST-T1 cells treated for 48 h were analyzed by the CompuSyn program (ComboSyn). E Indicated cells were treated with 100 nM GSK602 and 50 nM imatinib for 24 h. The indicated proteins were analyzed by western blotting. F WT and PUMA-KO GIST-882 cells were treated with 100 nM GSK602 and 50 nM imatinib for 24 h. Apoptosis was analyzed by fragment nuclei assay. G WT and PUMA-KO GIST-T1 cells were treated with 100 nM GSK602 and 50 nM imatinib for 24 h. Apoptosis was analyzed by fragment nuclei assay. H WT and PUMA-KO GIST-882 or GIST-T1 cells were treated with the combination of 100 nM GSK602 and 50 nM imatinib for 24 h. Caspase 3 level was analyzed by western blotting. Results were expressed as means ± SD of three independent experiments. ***P < 0.001.
Fig 5: BRD9 inhibition reduced GIST cell proliferation.A Indicated cell lines were transfected with siRNA against BRD9 for 24 h, BRD9 protein level was analyzed by western blotting. B The viability of GIST-882 and GIST-T1 cells transfected with siRNA against BRD9 was determined by the MTT. C The viability of GIST-882 and GIST-T1 cells transfected with siRNA against BRD9 was determined by colony formation assay. D Indicated cell lines were transfected with siRNA against BRD9 for 24 h. Cleaved PARP and caspase 3 level were analyzed by western blotting. E Indicated cell lines were treated increasing concentration of GSK602 for 24 h. Cell viability was analyzed by colony formation assay. F Indicated cell lines were treated increasing concentration of GSK602 for 24 h. ?H2AX level was analyzed by western blotting. G Indicated cell lines were treated increasing concentration of GSK602 for 24 h. Apoptosis was analyzed by fragment nuclei assay. H Indicated cell lines were treated increasing concentration of GSK602 for 24 h. Cleaved PARP and caspase 3 level was analyzed by western blotting. Results were expressed as means ± SD of three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001.
Supplier Page from Abcam for Anti-BRD9 antibody