Figure 6

Involvement of RNF183 in TRAIL-induced apoptosis. (a) Effect of RNF183 knockdown on TRAIL-induced caspase activation. RNF183-siRNA was transfected into HEK293 cells stably expressing V5-tagged RNF183. At 30 h after transfection, TRAIL (100 ng/mL) was added and cells were incubated for 2.5, 5, and 7.5 h. Cell lysates were subjected to western blotting with anti-cleaved caspase-3, cleaved caspase-8 antibodies, and anti-caspase-3 antibody (for procaspase-3). The full-length blots are presented in Supplementary Fig. S12. (b,c) Effect of RNF183 overexpression on TRAIL-induced caspase activation. HEK293 cells expressing mock or V5-tagged RNF183 were treated with TRAIL for 5 h. Cells were subjected to immunofluorescence staining with cleaved caspase-3, cleaved caspase-8 antibodies (red), and DAPI (blue). (d,e) Quantitative graph of data from (b,c). Asterisks represent significant differences (n = 3; Student’s t-test, *p < 0.05, **p < 0.01). (f) Apoptotic cell death was evaluated by Annexin V assay. HEK293 cells expressing mock or RNF183 were treated with TRAIL (300 ng/ml) for 3 h. The percentage of apoptotic cells was determined by FITC-annexin-V/propidium iodide (PI) staining.