Fig. 4

CIB1 depletion upregulates TRAIL-R2 expression Death receptors TRAIL-R1/R2 levels in MDA-436 cells were measured at Days 2, 3, and 4 post-infection with either control or CIB1 shRNA. a FACS analysis shows upregulation of cell surface levels of TRAIL-R2, but not TRAIL-R1, starting at 2, 3 and 4 days post-infection (2nd and 3rd row) with CIB1 shRNA (red) relative to control (black). b Mean ± SD (n = 3) of fluorescence intensities of CIB1 shRNA-infected MDA-436 cells normalized to control cells shown in a. c Representative western blot (n = 3) showing increased whole cell expression of TRAIL-R2, but not TRAIL-R1, in CIB1-depleted MDA-436 cells at 3 and 4 days post-infection with CIB1 shRNA. d TRAIL-R2 neutralizing antibody blocks TRAIL-induced cell death in CIB1-depleted cells. Control or CIB1-depleted MDA-436 cells were pre-treated with either IgG control or TRAIL-R2 neutralizing antibody (TRAIL-R2 Fc, 1 ng/ml) for 24 h before addition of vehicle (water) or 10 ng/ml recombinant TRAIL ligand for 48 h (n = 3). Cell death was quantified as in Fig. 1 (**P < 0.01). e Blocking TRAIL-R2 inhibits TRAIL-induced PARP cleavage and caspase-8 activation in CIB1-depleted MDA-436 cells. Representative Western blot of PARP, TRAIL-R2, cleaved caspase-8, CIB1, and GAPDH (loading control) in treatment groups shown in d) (n = 3)