Fig. 6: Dependence of Eltrombopag binding and subsequent apoptosis on BAK α4/α6/α7 groove.

a, b After BAKΔTM R156A (a) or R156E (b) and indicated concentrations of Eltro were incubated in CHAPS buffer for 1 h at 25 °C, the mixtures were separated by FPLC on a Superdex75 size exclusion column and absorbance at 280 nm was plotted as a function of elution volume. Labels on top indicate size markers. c After BAKΔTM WT and different BAKΔTM mutants were analyzed by the BAK oligomerization assay shown in a, b, the percentage of BAK oligomers were calculated. d After recombinant BAKΔTM WT, BAKΔTM R156A or R156E was labeled at cysteine using maleimide, the dissociation constants (K_D) was evaluated by MST analysis using increasing concentrations of Eltro. A summary of data from three independent experiments is shown. e, f. Liposome permeabilization assay was performed in the presence of 50 nM BAKΔTM WT, BAKΔTM R156A, or BAKΔTM R156E and the indicated concentrations of Eltro or BIM BH3 peptide as a positive control. A representative experiment (e) and a summary from three independent experiments (f) are shown. g Bak^−/−Bax^−/− MEFs were transduced with WT BAK or BAK R156E. After 2 weeks of selection, a pool of cells was subjected to western blot. h, i Bak^−/−Bax^−/− MEFs reconstituted with WT BAK or BAK R156E were treated with the indicated Eltro concentrations for 24 h and analyzed by Annexin V/PI staining (h) or sub-G1 analysis (i). c, d, f, h, i Differences between BAK mutants and WT were assessed by ANOVA. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; and ns, not significant.