Figure 4: Cep57 depletion accelerates anaphase onset and leads to chromosome lagging.

(a) Time-lapse images of RPE1 cells co-transfected with H2B-GFP and negative control (NC)- or Cep57-siRNA for 60 h. NEBD, nuclear envelope breakdown; M, metaphase; A, anaphase. The numbers indicate the time (minutes) after NEBD. Arrows, misaligned chromosomes and lagging chromosomes. Scale bar, 5 μm. (b,c) Quantification of time from NEBD to anaphase onset of HeLa (b) and RPE1 cells (c) co-transfected with H2B-GFP and NC- or Cep57-siRNA for 60 h. Scatter plots show data from three independent experiments (red lines indicate the average). Data are mean±s.e.m. **P<0.01 (unpaired two-tailed Student’s t-test). (d) Mitotic progression from NEBD to anaphase onset in HeLa cells transfected with H2B-RFP and NC- or Cep57-siRNA for 60 h. The cells were selected at random. (e) Immunostaining of γ-tubulin (green) in HeLa cells with NC-siRNA, Cep57-siRNA or Cep57-siRNA and siRNA-resistant Cep57 (resCep57) for 60 h. DNA was stained with 4,6-diamidino-2-phenylindole (DAPI, blue). Scale bar, 5 μm. Arrows, lagging chromosomes. (f,g) Percentages of anaphase HeLa (f) and RPE1 cells (g) after transfection with NC-siRNA, Cep57-siRNA or Cep57-siRNA and siRNA-resistant Cep57 (resCep57) for 60 h. The experiments were repeated three times. Data are mean±s.e.m. **P<0.01; *P<0.05 (unpaired two-tailed Student’s t-test); n>200 cells per experiment. (h) Western blots show endogenous Mis12 depletion in HeLa cells after twice transfection of siRNA for 48 h, and rescue by expressing siRNA-resistant GFP-tagged wild-type (WT) or mutant L132G Mis12 (resMis12). (i,j) Percentages of anaphase HeLa (i) and RPE1 cells (j) after twice transfection of Mis12 siRNA and rescue by expressing siRNA-resistant GFP-tagged wild-type (WT) or mutant L132G Mis12 (resMis12). The experiments were repeated three times. Data are mean±s.e.m. **P<0.01; *P<0.05; NS, not significant (unpaired two-tailed Student’s t-test); n>200 cells per experiment.