FIG. 5.

Knock-down of cFLIP sensitizes hESC to TRAIL-induced apoptosis. Stable cell lines were established following transduction of the hESC line CCTL14 with a lentivirus expressing nontargeting, nonsilencing shRNA (NS shRNA) or shRNA targeting cFLIP mRNA (shFLIP1). (A) Western blot showing a decrease in cFLIP levels in shFLIP1-transduced cells compared with NS shRNA-transduced cells. Alpha tubulin was used as a loading control. (B) NS shRNA and shFLIP1 cells were left untreated (control) or treated with 200 ng/mL TRAIL. The morphology of colonies and cell detachment were observed by light microscopy after 6 h of treatment. (C) Nontransduced (WT), NS shRNA-transduced, and shFLIP1-transduced cells were left untreated (control) or treated with 200 ng/mL TRAIL for 6 h. Induction of apoptosis was determined as in Figure 3B. Each bar represents the mean of n=3 experiments, error bars show±SEM. (D) Western blot analysis of the activation of the extrinsic apoptotic pathway in NS shRNA and shFLIP1-transduced cells after 30 min and 1, 2, 3, and 6 h of treatment with 200 ng/mL TRAIL. Alpha tubulin was used as a loading control.