Modulation of c-FLIP levels by enforcing expression of ectopic c-FLIP ( A and B ) or siRNA-mediated downregulation of c-FLIP ( C and D ) regulates cell sensitivity to GGTI-298-induced apoptosis. A and B, The indicated transfectants were treated with the given concentrations of GGTI-298 (A) or 15 μM GGTI-298 (B) for 48 h and then subjected to estimation of cell number using the SRB assay (A) or detection of apoptosis by Annexin V staining (B). C and D, H226 cells seeded in a 6-well plate (C) or a 96-well (D) plate were transfected with control (Ctrl) or c-FLIP siRNA. Twenty-four hours later, the cells were exposed to 30 μM (C) or the indicated concentrations (D) of GGTI-298. After 24 h, the cells were subjected to preparation of whole-cell protein lysates and subsequent detection of the indicated proteins using Western blotting (C) or to estimation of cell number by the SRB assay (D). Data in A and D are means of four replicate determinations. Bars, ± SDs. CF, cleaved form.