Fig. 5
bCAFs rely on MCL-1 for their survival. a BH3 profiling. Values indicate the percentage of cytochrome c loss and are representative of one experiment. b NHLF, NF, and CAFs cells were treated with A-1210477 (2.5 or 5 µM) or ABT-737 (1, 5, or 10 µM) for 48 h in DMEM containing 0.5% FBS, apoptosis was measured by Annexin-V flow cytometry. c Top: NHLF pre-treated or not by TGFβ for 24 h were treated with 10 µM ABT-737 or 5 µM A-1210477 for 48 h in DMEM containing 0.5% FBS, apoptosis was measured by Annexin-V flow cytometry. Bottom: MCL-1 proteins expression levels in NHLF + /-TGF-β cells treated by cycloheximide for the indicated time, were evaluated using western-blot analysis. d CAFs were treated with 5 µM A-1210477 + /- ABT-737 1 µM, Wehi-539 1 µM or ABT-199 1 µM for 48 h in DMEM containing 0.5% FBS, apoptosis was measured by Annexin-V flow cytometry. e Percentage of apoptotic cells estimated in ABT-737-treated CAFs cells previously infected with the control vector or the MCL-1 sh-RNA for 72 h. MCL-1 protein expression in CAFs cells infected with the control vector or the MCL-1 sh-RNA was evaluated by western blotting (insert). f Co-culture experiments. ZR-75-1 cells (1/3) and CAFs (2/3) were treated for 48 h with ABT-737 1 µM and A-1210477 5 µM alone or in combination in DMEM 0.5% FBS. MCL-1 protein expression levels in ZR-75-1 cells isolated by fluorescence-activated cell sorting after their co-culture with CAF is shown on the left. Representative FACS profiles of cell death analyzes (using Annexin V-APC as a marker) in co-culture of ZR-75-1 cells and CAFs (marked by anti CD-90 PE) are shown in the middle. Percentage of apoptotic (Annexin-V–APC positive) tumor cells (CD9O negative) and CAFs (CD 90 positive) grown alone or in co-culture and treated with the indicated combination of BH3 mimetic, measured by flow cytometry are shown on the right. Data are means ± SEM from three independent experiments. P-value was determined by two-way ANOVA. *P < 0.05, **P < 0.01,, ****P < 0.0001
