Fig. 3: BAK is an essential mediator of GBP1- and GBP2-induced cell death, and GBP2 competitively inhibits MCL-1-BAK interaction.

a, b The effects of GBPs on cell viability were determined in MEF cells of WT, Bak^−/−, Bax^−/−, and Bax^−/−Bak^−/−. Asterisks indicate statistically significant values compared with those of WT cells (***P < 0.001). Data are presented as the mean ± SEM of three independent experiments performed in triplicate. Cell lysates were subjected to western blot to confirm similar ectopic expression of GBP2 and GBP1 in all cell lines. c The effects of BAK or BAX knockdown on GBP2-induced cell death activity was determined in K562 cells. Efficient silencing of BAK and BAX was confirmed by western blot analyses. Different letters denote statistically significant differences (P < 0.01). Data are presented as the mean ± SEM of three independent experiments performed in triplicate. d, e Effects of GBP2 overexpression (d) or silencing (e) on the complex formation between MCL-1 and BAK were evaluated by immunoprecipitation following transfection of K562 cells with HA-GBP2 (0, 3, or 6 μg; d) or with siRNAs of GBP2 (200 nM). Representative blots and quantified data are presented. Asterisks indicate statistically significant differences (*P < 0.05). Data are presented as mean ± SEM of three independent experiments. f, g The interaction between MCL-1 and BAK were evaluated in two independent GBP2 KO cell lines (f) or GBP2- or GBP1-stably overexpressing cell lines (g). Representative blots (left panel) and quantified data of bound BAK protein to MCL-1 (top on the right) and BAK protein level in total lysates (bottom on the right) are presented. The data are shown as mean ± SEM from three independent experiments. Asterisks indicate statistically significant differences (*P < 0.05; **P < 0.01; ***P < 0.005). Values denoted with different letters are statistically significant ones (*P < 0.05). h Changes in the levels of the BCL-2 family proteins after GBP2 knockdown (200 nM of siRNAs) or GBP2 overexpression (3 µg of plasmid) were determined by western blot analyses in HL-60 cells. Both representative blots and quantified data are presented. Data presented are the mean ± SEM of three independent experiments (*P < 0.05; **P < 0.01). i Changes in BAK mRNA levels were quantified by real-time PCR in K562 and HL-60 cells after transfection with siRNAs in GBP2 (200 nM) or GBP2 plasmid (3 µg) for 24 h. Data presented are mean ± SEM of three independent experiments performed in triplicate (*P < 0.05; **P < 0.01; ***P < 0.001).