Figure 4
HECTD3 decreases the caspase-8 recruitment and inhibits its activation. (a) HECTD3 ablation increases the caspase-8 association with DISC in MDA-MB-231 cells treated with Flag-TRAIL. Stable HECTD3 knockdown and control MDA-MB-231 cells were treated with Flag-TRAIL for 30 min before or after collection. DISC formation was examined by immunoprecipitation using anti-Flag M2 beads and WB. (b) HECTD3 inhibits TRAIL-induced caspase-8 cleavage (41/43 and 18 KDa cleaved caspase-8 bands are indicated by arrows) in HEK293T in HECTD3 E3 ligase activity and caspase-8 ubiquitination site-dependent manners. HEK293T cells were transfected with indicated constructs overnight and treated with 50 ng/ml TRAIL for 4 h. HECTD3m (C744, 823A) cannot decrease TRAIL-induced caspase-8 cleavage as WT HECTD3 (lane 6 versus 5). In addition, HECTD3 cannot decrease TRAIL-induced cleavage of caspase-8-K215R (lane 11 versus 5). L, long exposure; S, short exposure. (c) Re-expression of shRNA resistant HECTD3R in endogenous HECTD3-depleted MDA-MB-231 cells decreases TRAIL-induced cleavage of caspase-8 in an E3 ligase activity-dependent manner. Endogenous HECTD3 was silenced by shRNA. Following that, HECTD3R and HECTD3m (C744, 823A)R were stably introduced by lentiviruses.(d) HECTD3R significantly decreases TRAIL (50 ng/ml)-induced loss of cell viability in an E3 ligase activity-dependent manner in MDA-MB-231. Cell viability was measured by performing the SRB assay. Error bars are S.D. from three independent experiments. **P<0.01 (t-test)
