Fig 1: Apoptosis induction by BOK is comparable to and independent of BAX and BAK.A Representative images of HCT-AKO cells expressing GFP-BAX, GFP-BOK and GFP 16 h after transfection. Transfected cells and annexin V+ cells appear in green and red respectively. Scale bar; 100 µm. B Effect of GFP-BOK overexpression on cell death in different cell lines (HCT-WT, DKO, AKO and Casp9 KO), measured as percentage of cells with Annexin V from the total transfected cells (Annexin V+/GFP+). C Effect of BAX and BOK overexpression on cell death in HCT AKO, in the presence or absence of ZVAD, measured as fraction of cells with Annexin V normalized to total cell area. D Effect of tBID-GFP and GFP-BOK overexpression on cell death in different cell lines (HCT-WT and DKO) quantified as in B. E Effect of GFP-BAX, GFP-BOK, GFP-BOK G35A, GFP-BOK?C, GFP-BCL-xL and GFP overexpression on cell death in HCT AKO, in the presence or absence of ZVAD and calculated as in (B). ?: untransfected. F Representative images of GFP-BOK and GFP-BOK-xL subcellular localization 16 h after transfection in U2OS BAX-/-/BAK-/- cells. BOK appears depicted in green and mitochondria (labelled with Mitotracker-644) in magenta. Scale bar, 10 µm. G Effect of GFP-BAX, GFP-BOK, GFP-BOK-xL, GFP-BCL-xL and GFP overexpression on cell death in HCT AKO quantified as in B. H Effect of GFP-BAX, GFP-BOK, GFP-BCL-xL and GFP overexpression on mitochondrial depolarization in U2OS DKO cells, measured as a decrease on TMRE signal. ?: untransfected. I Release of cytochrome c from isolated mitochondria from HCT116 cells was assessed by immunoblotting of pellet (mitochondria) and supernatant fractions. Antibody against TOM22 was used as a control. ***P = 0.001, **P = 0.01, *P = 0.05, ns not significant. Experiments were averages of at least three replicates.