[View Larger Version of this Image]

Figure S4. Emi1 knockdown induces rereplication that can be rescued by exogenous Emi1. (A) HeLa cells were transfected with either control or Emi1 siRNA, labeled with BrdU for 1 h before the indicated times, and BrdU incorporation was analyzed by flow cytometry 24, 48, and 72 h after transfection. (B) hTert-RPE cells were transfected with control (siCTR) or Emi1 (siEmi1_1) siRNA, and samples were harvested 24, 48, and 72 h after transfection for immunoblotting. Actin is shown as a loading control. Molecular mass markers (given in kilodaltons) are shown. Representative of two experiments. (C) hTert-RPE cells treated as for B were analyzed by flow cytometry. Note that the appearance of the polyploid cell population at 48 h is partially masked by the expansion of the nontransfected cell population at 72 h, which is consistent with the reappearance of Emi1 levels detected by Western blotting (see Results section). (D) Cells transfected as in B were fixed, and DNA was stained with Hoechst 33342 to analyze the size of the nuclei. The presence at 72 h of both large and normal-size nuclei is consistent with the DNA content measured by flow cytometry and the levels of Emi1 detected by immunoblotting (see Results section). Bars, 20 µm. (E) Representative images of the cells quantified in Fig. 5 D. Bars, 30 µm.