Journal: Nature cancer

Article Title: A small-molecule allosteric inhibitor of BAX protects against doxorubicin-induced cardiomyopathy

doi: 10.1038/s43018-020-0039-1

Figure Lengend Snippet: a, Chemical structure of BAI1. b, Microscale thermophoretic analysis of BAI1 binding BAX. ΔFnorm, normalized change in fluorescence. Representative of three independent experiments. c, Lack of an effect of BAI1 (0.1 μM) on early conformational changes in BAX activation in staurosporine (STS)-treated MEFs as assessed by exposure of 6A7 epitope. Three independent experiments each with three independent immunoprecipitations per group. One-way ANOVA, *P = 0.0141, NS, P > 0.05. d, BAI1 inhibits BAX insertion into the OMM. Only noninserted BAX can be recovered from isolated mitochondria by treatment with strong alkali (n = 4 independent experiments). One-way ANOVA, *P = 0.0301, ***P = 0.0002. e,f, Inhibition of BAX mitochondrial translocation by BAI1. e, Representative images of immunofluorescence for BAX translocation in WT MEFs. f, Quantification of percentage of cells with BAX puncta (n = 4, 11, 9, 11 and 12 images). Data are representative of five independent experiments. One-way ANOVA, *P = 0.0237, ****P < 0.0001. g, BAI1 (10 μM) inhibits BAX-dependent cytochrome c release as assessed by ELISA. Mitochondria isolated from BAX/BAK DKO MEFs were incubated with recombinant BAX and tBID (n = 3 independent mitochondrial isolates per group). Representative of three independent experiments. One-way ANOVA, **P = 0.0016, ##P = 0.0074. Data in c,d,f,g are presented as mean ± s.e.m. Unprocessed images of blots are provided as source data.

Article Snippet: Isolated mitochondria (40 μg) from BAX/BAK DKO MEFs were incubated with 25 nM recombinant human BAX, 10 nM recombinant human tBID (R&D Systems, 882-b8–050) with or without 10 μM BAI1 at 37 °C.

Techniques: Binding Assay, Fluorescence, Activation Assay, Isolation, Inhibition, Translocation Assay, Immunofluorescence, Enzyme-linked Immunosorbent Assay, Incubation, Recombinant