FIGURE 4.

Growth inhibiting effects of Roc1, Ddb1, and Cul4a silencing on ovarian cancer cell in vitro. A and B, Western blot (A) and quantitative RT-PCR (B) results for Roc1 and Roc2 RNAi depletion efficiency. OV2008 cells were transfected with control siRNA (siCON), siRoc1, or siRoc2. Total RNAs and proteins were isolated 48 h after transfection. C, effects of Roc1/2 RNAi depletion on ovarian cancer cell growth. OV2008 cells were seeded in 96-well plates (3,000 cells/well) at 48 h after transfection, and then assessed by MTT assay. D and E, Roc1 or Roc1/2 double silencing inhibited ovarian cancer cell proliferation. Cells were counted at 72 h after transfection. F and G, Roc1/2 depletion induced G₂-M cell cycle arrest in ovarian cancer cells. Cells were transfected with siCON, siRoc1, siRoc2, or siRoc1/2 for 72 h and then subjected to PI staining and FACS analysis to determine cell cycle profiles. H and I, Roc1/2 depletion inhibited colony formation of OV2008 cells. *, p < 0.01; **, p < 0.001. J, Western blot results for the levels of pH2AX, pCHK1, p27, and CDT1 after RNAi depletion of Roc1, Roc2, and Roc1/2 in OV2008 cells. K, quantitative RT-PCR results for Cul4a and Cul4b RNAi depletion efficiency. L, Western blot results for Ddb1 RNAi depletion efficiency. M, effects of Ddb1, Cul4a, and Cul4b depletion on ovarian cancer cell proliferation as assessed by MTT assay. N and O, effects of Ddb1, Cul4a, and Cul4b depletion on colony formation by OV2008 cells. P, immunofluorescent staining results for increased caspase 3 cleavage after RNAi depletion of Ddb1 and Cul4a.