Fig. 2: GBP1 and GPB2 induce apoptotic cell death.

a Cell viability assay was performed in HA-GBP2-transfected cells after treating with a solvent vehicle (0.02% DMSO) or Z-VAD-FMK (1 or 10 μΜ). The expression levels of GBP2 were assessed by western blotting. b The effect of GBP2 silencing on the cell viability of K562 cells was determined following the transfection of siRNAs for GBP2 (100 or 200 nM). Efficient knockdown of GBP2 is demonstrated by immunoblot analysis. Different letters denote statistically significant differences (P < 0.01). c K562 cells were transfected with HA-tagged GBP2 or GBP1 expression plasmids (GBP2: 3 µg, GBP1: 1.8 µg) for 24 h following with cell viability assay (bottom). Different letters denote statistically significant differences (P < 0.001). The expression levels of GBP1/2 were assessed by western blotting. d The differences in cellular viability were examined in HA-GBP2- or HA-GBP1-stably overexpressing K562 cells (1 × 10⁴) after 24 h of culture. Asterisks indicate statistically significant differences compared to WT cells (***P < 0.001). Data are shown as mean ± SEM of three independent experiments. e GBP1- and GBP2-induced activations of Caspase 3, 8, and 9 were determined by western blot analyses in GBPs-overexpressed K562 cells. f GBP2-induced mitochondrial membrane permeability (MMP) was assessed by FACS analysis after the incubation of transfected K562 cells with JC-1. Representative (upper panel) and quantified (lower panel) MMP transition in empty or GBP2-transfected cells are shown. Asterisks indicate statistically significant differences (P < 0.05). Data are presented as the mean ± SEM of three independent experiments. g Cytochrome c levels in the cytosolic or mitochondrial fractions were analyzed in GBP2-transfected K562 cells by western blot analyses. The fractionation quality was demonstrated using anti-COX IV and anti-β-actin antibodies. h K562 cells (1 × 10⁴) were cotransfected with GBP2 expression plasmid (30 ng) and indicated MCL-1 plasmids (30 ng) for 24 h, and then cell viability was measured. Different letters denote statistically significant differences (P < 0.05). Data are shown as the mean ± SEM of three independent experiments performed in triplicate. The expression levels of GBP2, MCL-1 were determined by western blot using indicated antibodies. Representative blots are shown. i, j Cell apoptosis was analyzed by flow cytometry in K562 cells cotransfected with indicated plasmids (3 µg, i) or siRNAs (200 nM, j). Representative (upper panel) and quantitative (lower panel) data are shown. The percentages of early apoptosis (Annexin V-positive, PI-negative) cells and late apoptosis cells (both Annexin V- and PI-positive) are indicated. Efficient MCL-1 knockdown was determined by immunoblot analysis. The histogram data are presented as the mean ± SEM of three independent experiments performed in duplicate. Different letters denote statistically significant differences (P < 0.05).