Figure 3: Cep57 contributes to activation of the spindle assembly checkpoint.
From: Cep57 is a Mis12-interacting kinetochore protein involved in kinetochore targeting of Mad1–Mad2
(a,b) HeLa cells with 100 nM nocodazole treatment after co-transfection with H2B-RFP and negative control (NC)-, Cep57-, Mad1- or Mad2-siRNA for 48 h. (a) Time-lapse images of HeLa cells. The numbers indicate the time (minutes) after entry into mitosis. Arrowhead, multiple nuclei. Scale bar, 5 μm. (b) Quantification of mitotic duration of HeLa cells from a. Scatter plots show data from three independent experiments. (c) Quantification of mitotic duration of live HeLa cells with 80 nM taxol treatment after co-transfection with H2B-RFP and negative control (NC)-, Cep57-, Mad1- or Mad2-siRNA for 48 h. Scatter plots show data from three independent experiments. (d) Diagram of experimental design. The siRNA-transfected HeLa cells were synchronized by double-thymidine block, and released into nocodazole. (e–g) HeLa cells were treated as in d. (e) After 12 h nocodazole treatment, mitotic cell lysates were used to perform immunoprecipitation (IP) and western blotting assays with the indicated antibodies. WCL, whole-cell lysate. (f) Western blots of the indicated proteins in HeLa cells after the indicated nocodazole (Noc.) treatment time. (g) Quantification of the protein levels of securin and cyclin B1 from (f). The experiment was repeated three times. For b,c and g, data are mean±s.e.m. ****P<0.0001; ***P<0.001; **P<0.01; *P<0.05; NS, not significant (unpaired two-tailed Student’s t-test). DIC, differential interference contrast.
