FIGURE 2.

Identification of mediators of E2F1-induced apoptosis. We targeted each of the genes recovered by our screening by shRNA to retest for prevention of E2F1-induced apoptosis. Individual shRNAs listed in Table 1 were stably integrated into U2OS cells. A, serum deprived control and shRNA target cells were infected with E2F1 expressing adenovirus and later measured for apoptosis by quantifying cleaved caspase-3 levels. The level of apoptosis induction by E2F1 compared with control in non-shRNA carrying cells was set to 100%. Three of the 10 shRNA lines (GPR45, MSH6, and ADAMTSL3) did not display reduced E2F1-induced apoptosis. The other seven lines, however, recapitulated the findings of the screen and significantly reduced E2F1 induction of apoptosis from 25–55%. Student's t tests were performed and indicate that the reduced apoptosis seen in these seven cell lines was significant (p < 0.05). B, target gene knockdown was measured by qPCR, comparing levels in vector control to levels in shRNA knockdown lines. Results are reported as % remaining expression in target cells and are listed in the order presented in A.