Fig. 6: BCL-XL inhibition triggers MCL-1 dependence, and resistance to WEHI-539 is overcome by combination treatment with S63845.

ONS76 (A) and UW228 (C) cells were treated with PBS or 1 µM WEHI-539 for 18 h followed by BH3 profiling. Bars represent the mean ± SEM of N = 3 independent experiments carried out in triplicate, normalised to DMSO (0% mitochondrial depolarisation) and FCCP (100% mitochondrial depolarisation). Data were analysed using Two-Way ANOVA followed by Sidak’s test for multiple comparisons, whereby **p < 0.01, *p < 0.05. Cell viability of ONS76 (B) and UW228 (D) was measured following 48 h treatment with increasing concentrations of WEHI-539 combined with the indicated concentrations of S63845, using the WST-1 viability assay. Bars represent the mean of N = 3 independent experiments carried out in duplicate ±SEM. Data were analysed using Two-Way ANOVA followed by Tukey’s test for multiple comparisons, whereby *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Combination index values were calculated based on the above data to assess synergistic effects between WEHI-539 and S63845 where <1 indicates synergy between the two agents, while <0.8 indicates strong synergy. ONS76 (E) and UW228 (F) cells were treated for 48 h with 1 µM of the indicated BH3 mimetics either singly or in combination, and Annexin-V/PI staining followed by flow cytometry analysis was used to determine the proportion of apoptotic cells. Data are expressed as mean of N = 3 independent experiments ± SEM. Data were analysed using One-Way ANOVA with Tukey’s multiple comparison test, whereby *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. G ONS76 cells were treated as indicated for 48 h and the colony forming capacity of surviving cells was assessed by clonogenic assay. 14 days post-treatment, colonies were stained with crystal violet (left) and the number of colonies counted in ImageJ (right). Data are expressed as the mean of N = 3 independent experiments ± SEM. Data were analysed using One-Way ANOVA with Tukey’s multiple comparison test, whereby ***p < 0.001, **p < 0.01. ONS76 (H) and UW228 (I) cells were treated with either DMSO, 1 µM of the indicated BH3 mimetics, or 1 µM of the indicated BH3 mimetics following a 30 min pre-treatment with 30 µM Q-VD-OPh. Cells were treated for 0, 1, 2, 3 and 6 h, and Annexin-V/PI staining followed by flow cytometry analysis was used to determine the proportion of apoptotic cells. Data are expressed as mean of N = 3 independent experiments ± SEM, and were analysed Two-Way ANOVA with Tukey’s multiple comparison test, whereby ****p < 0.0001. The break in the x axis indicates a shortened axis.