Figure 4

Siah2 functions down stream of procaspase-8 processing. a: Siah2 E3 activity is required to maintain apoptosis resistance. PPC-1 cells stably expressing wtSiah2, rmSiah2 or empty vector (inset) were treated with TRAIL (100 ng/mL) for 16 hours and stained for annexin V. b: Siah2 acts downstream of caspase-8 processing. (left) Caspase-8 Western blot on PPC-1 cell extracts transfected with Siah2 or non-targeting siRNA and TRAIL treated from 0 to 16 hours. GAPDH serves as loading control. (right) Caspase-8 activity determined on the 16 hr timepoints used in (B). c: PC-3 cells were transfected with siRNA to target Siah2, POSH, or a non-targeting control. After 48 hours cells were plated to a 24-well dish. After 72 hours, cells were treated with staurosporine (100 nM for 6 hours), doxorubicin (1 μM for 30 hours), thapsigargin (1 μM for 24 hours) or TRAIL (100 ng/mL for 16 hours) and annexin V stained to assess apoptosis. All values indicate the mean percent annexin V positive cells +/- the standard deviation. d: Western blot analysis of sub-cellular fractionation from PPC-1 cells stably expressing rmSiah2-myc (top) or rmPOSH-myc (bottom) and treated +/- TRAIL (100 ng/mL). Mito, mitochondria; cyto, cytoplasm; micro, microsomal; PDI, protein disulfide isomerase. e: Representative confocal images of PPC-1 cells stably expressing rmSiah2. Siah2 and proteasome localization was visualized by incubation with anti-myc and anti-20S antibodies. *P < 0.05, **P < 0.01.