Fig. 7: The BAX TA amplifies the antagonistic effect of BH3 mimetics on PUMA / BCL-xL complexes.
a Dose-response antagonism of YBCL-xL-CBAX, YBCL-xL-CBAX G222I A226I chimeric proteins complexed with either RPUMA, RPUMA-BIDBH3, RtBID, RPUMAdC, as a function of BAX and BAK expression. A-1331852 BH3m antagonism of the indicated complexes is compared between wild-type and baxCRISPR−/− bak1 CRISPR−/− MCF-7 cells. The representation follows the description provided in Fig. 1b. b Evaluation of the interaction between MBCBAX and GBCL-xL expressed with FPUMA or FPUMAdC in HCT116 cells using the BirA in situ proximal biotinylation assay. Input and labeled (i.e., biotinylated) proteins isolated with biotin affinity beads were analyzed by Western blotting for GBCL-xL, FPUMA, and TOM20. All antibodies specifically target the indicated proteins, except FPUMA and FPUMAdC, detected via anti-FLAG antibody. The presented blots are representative of three independent experiments. Uncropped blots are available in the Source Data file. c Single cell apoptosis analysis in FPUMA/GBCL-xL and FPUMAdC/GBCL-xL HCT116 cells with or without expression of MBCBAX, treated with a concentration range 1:4 serial dilution of A-1331852 (0–10 μM). Apoptosis was analyzed over 13 h for AnnexinV staining by live cell imaging using the Incucyte S3 imager. The heatmap summarizes the Log2 ratio of the percentage of cell death in cells expressing MBCBAX compared to the control vector (pLVX). Data are mean of five independent experiments. Significant difference in overall kinetics were assessed using permutation-based two-sided p values, calculated with the comparegrowthcurve function from the statmod R package. *P < 0.05, **P < 0.01, ***P < 0.001.
