FIGURE 3.

Knockdown of FAS induces activation of caspase-8, expression of DDIT4, suppression of eIF4E phosphorylation, and reduction in the anti-apoptotic protein FLIP. A, MDA-MB-435 cells were cotransfected with 25 nm FAS duplex 9 siRNA together with 25 nm non-silencing control duplex 1 or DDIT4 duplex 9–11 siRNAs. Caspase-8 cleavage was measured 96 h after transfection by Western blotting. Transfection with 50 nm non-silencing control siRNA was used to indicate the basal level of caspase-8 cleavage occurring in response to siRNA transfection. B, the effect of non-silencing control siRNA or siRNA targeting FAS, ACC-α, or ACL (25 nm; left panel) and orlistat (0–50 μm; right panel) on phosphorylation and the expression levels of the translation protein eIF4E (p-eIF4E and eIF4E) were measured by Western blotting in MDA-MB-435 cells 96 h after transfection. C, MDA-MB-435 cells were transfected with non-silencing control or FAS siRNA (25 nm) and analyzed 48–96 h post-transfection for changes in the expression of the anti-apoptotic protein FLIP. Actin was used as a loading control.