Figure 1

E3 ubiquitin ligases Siah2 and POSH can regulate caspase-8 activity. a: (left) MTT viability and (right) annexin V apoptosis analysis of DU145 cells treated with Velcade (50 nM), GST-TRAIL (100 ng/mL) or the combination for 24 and 16 hours, respectively. b: Flow chart illustrating the experimental design of the E3 ubiquitin ligase screen utilized in this study. c: Representative screening results showing 90 out of 239 E3 ligases examined in the siRNA screen. DU145 cells were plated/reverse-transfected for 72 hours and then treated with TRAIL (50 ng/ml) for 16 hours. Caspase-8 activity was quantified using a luminescent reporter. Values were normalized by subtracting out the background levels of caspase-8. The black bar indicates treatment with TRAIL and Velcade. Gray bars denote each TRAIL treated and siRNA targeted E3. 'No TRAIL' indicates no treatment. d: Assessment of Siah2 and POSH silencing in DU145 cells using qRT-PCR 24 hours post-transfection. All data are represented as the mean +/- the standard deviation. *P < 0.05, **P < 0.01, ***P < 0.001 using a two-tailed Student's t-test.