Raptinal bypasses BAX, BAK, and BOK for mitochondrial outer membrane permeabilization and intrinsic apoptosis

a HCT116, PCI-1 and Jurkat J16 cells were challenged with the indicated concentrations of Raptinal for 18？h. Data points and mean？±？SEM from three independent experiments are shown. b HCT116 cells were challenged with Raptinal (10？μM) for the indicated periods of time in the absence and presence of the pan-caspase inhibitor zVAD-fmk (100？μM). After washing and lysis, western blot analyses were performed with antibodies specific for the indicated proteins. Detection of tubulin served as a loading control. c HCT116 cells were treated as in b and subsequently analyzed by flow cytometry for 7-AAD- and annexin-V positivity. For b and c, data shown are representative of two experiments performed. d, e HCT116 cells and caspase-8-deficient variants thereof were challenged with Raptinal (10？μM) for 60？min or 120？min in the presence and absence of the pan-caspase inhibitors zVAD-fmk (100？μM) or QVD-OPh (100？μM). f HCT116 Caspase-8 KO cells were treated with Raptinal (10？μM) for the indicated periods of time in the presence and absence of zVAD-fmk (100？μM). Caspase-3/-7 activity was assessed using the fluorogenic substrate (DEVD)2-R110. For d–f, individual data points of at least two independent experiments are shown. RFU, relative fluorescence unit