Figure 4.

Increased expression of GODZ sensitizes tumor cells to TRAIL or DR4. (a) Ectopic expression of GODZ increases TRAIL-mediated apoptosis. HeLa cells were transiently co-transfected with pEGFP (Clontech) and either pcDNA3-HA (Ctrl) or pGODZ-HA for 24 h, and then left untreated (MOCK) or treated with TRAIL (100 ng/ml) for the indicated times. Cell death rates were measured by counting the number of both GFP- and EtHD-positive cells among total GFP-positive cells after staining with EtHD. Values indicate mean±S.D. (n=3). (b) Overexpression of GODZ sensitizes HeLa cells to TRAIL, but not to other insults. HeLa cells were co-transfected with pEGFP (Clontech) and either pcDNA3-HA (Ctrl) or pGODZ-HA for 24 h, and then treated with TRAIL (100 ng/ml) or TNF-α (30 ng/ml) with CHX (5 μg/ml) for 3 h, 25 μM etopo or 2 μg/ml tuni. for 24 h, or 125 ng/ml doxo for 24 h. Cell death ratios were measured as described in (a). Bars indicate mean±S.D. (n=3). P values were estimated using t-test versus control. (c) Enhanced activation of caspases by GODZ. HeLa cells transiently transfected with pcDNA3-HA (Ctrl) or pGODZ-HA for 24 h were treated with TRAIL (100 ng/ml) for the indicated times. Western blotting was performed with the indicated antibodies. (d) Effects of GODZ deletions on TRAIL sensitivity. HeLa cells were transiently co-transfected with pEGFP (Clontech) and one of GODZ deletion mutants for 24 h, and then exposed to TRAIL (100 ng/ml) for additional 3 h. Cell death ratios were measured as described in (b). Bars indicate mean±S.D. (n=3). P-values were estimated using t-test versus control (^*P<0.05; ^**P<0.01). (e and f) Distinct sensitivity of DR4 or DR5 knockdown cells to GODZ and TRAIL. HeLa cells were stably transfected with control pshRNA (Ctrl), pDR5 shRNA (shDR5) (e), or pDR4 shRNA (shDR4) (f), and their expression levels were examined by western blotting using anti-DR4, anti-DR5, and anti-β-actin antibodies (inserts). HeLa stable cells were then transfected with pEGFP (Clontech), and either pcDNA3-HA (Ctrl) or pGODZ-HA (GODZ) for 24 h and then left untreated (MOCK) or exposed to TRAIL (100 ng/ml) for 3 h. Cell death ratios were measured as described in (b). Bars indicate mean±S.D. (n=3). P-values were estimated using t-test versus control