Figure 4.
BAX expression governs the response to MCL1 BH3-mimetic S63845. (A) BAX silencing inhibited S63845-induced cell death. XG7 TP53+/+ no. 1 and TP53−/− no. 1 were transiently transfected with siCont, siBAX, siBAK1, or both, before treatment with increasing doses of S63845. The graph represents the mean ± SD of 3 independent experiments. Statistical analyses were performed using the unpaired t test. (B) CRISPR/Cas9-mediated BAX, but not BAK1, inactivation inhibited response to S63845 in NCI-H929 cells. (Left) TP53+/+ no. 1, TP53−/− no. 2, BAX−/− (n = 3), and BAK1−/− (n = 3) clones were treated with increasing doses of S63845 for 24 hours and cell death was determined using flow cytometry. The graph represents the mean ± SD of 3 experiments. (Right) The graph represents the LD50 values of each clone (mean ± SD). Statistical analysis was performed using the Mann-Whitney U test. (C) BAX, but not BAK1, expression correlated with sensitivity to S63845 in NCI-H929 and XG7 clones. Expression of BAX and BAK1 (mean DGE-seq values) were plotted against LD50 S63845 values. Correlation was assessed using the Spearman test. (D) BAX, but not BAK1, expression correlated with sensitivity to S63845 in HMCLs. Expression of BAX and BAK1 (microarray) in 30 HMCLs was plotted against LD50 S63845 value. Correlation was assessed using the Spearman test. (E) CRISPR/Cas9-mediated TP53 inactivation increased MCL1/BAK and decreased MCL1/BAX complexes. For immunoprecipitation (IP) assays, cells were lysed in 1% digitonin-containing buffer and lysates were precleared with protein A conjugated to sepharose beads. For MCL1 IP assay, 700 μg of protein lysate was incubated overnight with an agarose-conjugated monoclonal antibody anti-MCL1 from Santa Cruz Biotechnology. After immunoblotting, MCL1, BAK and BAX levels were determined in the immunoprecipitated fraction and BAX/MCL1 and BAK/MCL1 ratios were calculated to evaluate the abundance of both complexes. One experiment out of 3 is represented. The graph represents the quantities of BAX and BAK bound to MCL1 in TP53+/+ and TP53−/− NCI-H929 and XG7 cells (3 independent experiments). Statistical analyses were performed using the Mann-Whitney U test. ∗∗∗∗P < .0001; ∗∗∗P < .001; ∗∗P < .01; ∗P < .05.

BAX expression governs the response to MCL1 BH3-mimetic S63845. (A) BAX silencing inhibited S63845-induced cell death. XG7 TP53+/+ no. 1 and TP53−/− no. 1 were transiently transfected with siCont, siBAX, siBAK1, or both, before treatment with increasing doses of S63845. The graph represents the mean ± SD of 3 independent experiments. Statistical analyses were performed using the unpaired t test. (B) CRISPR/Cas9-mediated BAX, but not BAK1, inactivation inhibited response to S63845 in NCI-H929 cells. (Left) TP53+/+ no. 1, TP53−/− no. 2, BAX−/− (n = 3), and BAK1−/− (n = 3) clones were treated with increasing doses of S63845 for 24 hours and cell death was determined using flow cytometry. The graph represents the mean ± SD of 3 experiments. (Right) The graph represents the LD50 values of each clone (mean ± SD). Statistical analysis was performed using the Mann-Whitney U test. (C) BAX, but not BAK1, expression correlated with sensitivity to S63845 in NCI-H929 and XG7 clones. Expression of BAX and BAK1 (mean DGE-seq values) were plotted against LD50 S63845 values. Correlation was assessed using the Spearman test. (D) BAX, but not BAK1, expression correlated with sensitivity to S63845 in HMCLs. Expression of BAX and BAK1 (microarray) in 30 HMCLs was plotted against LD50 S63845 value. Correlation was assessed using the Spearman test. (E) CRISPR/Cas9-mediated TP53 inactivation increased MCL1/BAK and decreased MCL1/BAX complexes. For immunoprecipitation (IP) assays, cells were lysed in 1% digitonin-containing buffer and lysates were precleared with protein A conjugated to sepharose beads. For MCL1 IP assay, 700 μg of protein lysate was incubated overnight with an agarose-conjugated monoclonal antibody anti-MCL1 from Santa Cruz Biotechnology. After immunoblotting, MCL1, BAK and BAX levels were determined in the immunoprecipitated fraction and BAX/MCL1 and BAK/MCL1 ratios were calculated to evaluate the abundance of both complexes. One experiment out of 3 is represented. The graph represents the quantities of BAX and BAK bound to MCL1 in TP53+/+ and TP53−/− NCI-H929 and XG7 cells (3 independent experiments). Statistical analyses were performed using the Mann-Whitney U test. ∗∗∗∗P < .0001; ∗∗∗P < .001; ∗∗P < .01; ∗P < .05.

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