Functional evaluation of Bcl2 and BAX mutations. (A) ABT-199 was unable to bind F101C and F101L Bcl2 mutant proteins. (B) Bcl2 mutations had no impact on Bcl2 binding to Bim in both ABT-199–sensitive and –resistant lymphomas. Immunoprecipitation experiments were performed in duplicate. (C) Ectopic expression of WT BAX (but not G179E) in HBL2R cells restored ABT-199 sensibility. Impact of ABT-199 on cell viability (left). Induction of apoptosis with and without ABT-199 treatment (right). Experiments were performed in triplicate. (D) IF analysis showing that ABT-199 induced BAX translocation from cytoplasm to mitochondria in HBL2 but not in HBL2R cells, as shown by colocalization of BAX and mitotraker (Mito). Experiments were performed in duplicate: a representative example is shown. Quantification values of colocalization BAX and mitotraker IF images are provided in the main text. DAPI, 4,6 diamidino-2-phenylindole. (E) Subcellular localization of BAX performed by cell fractionation experiments showed that mutant BAX remained cytoplasmic after ABT-199 treatment. In addition, BAX G179E mutation prevented release of cytochrome C to cytoplasm. Experiments were performed in duplicate.