Figure 5: Cep57 depletion reduces kinetochore anchoring of Mad1–Mad2.

(a–d) Depletion of the indicated proteins in HeLa cells by siRNAs for 60 h followed by treatment with nocodazole and MG132 for 1 h. NC, negative control. Immunostaining of Cep57 (green), Mad1 (purple) and CREST (red, a); and Cep57 (green), Mad2 (purple) and CREST (red, c). DNA was stained with 4,6-diamidino-2-phenylindole (DAPI, blue). Scale bars, 5 μm. Cep57 and Mad1 (b) signals; and Cep57 and Mad2 (d) signals in cells from a and c, respectively, were normalized against CREST. The experiment was repeated three times. (e) Immunostaining of Mad1 (green) and CREST (red) in HeLa cells transfected with the indicated siRNAs and vectors for 60 h followed by nocodazole and MG132 treatment for 1 h. ResCep57, siRNA-resistant Cep57. DNA was stained with DAPI (blue). Scale bar, 5 μm. (f) Mad1 signal in cells from e was normalized against CREST. The experiment was repeated three times. (g) Immunostaining of Cep57 (green) and CENP-A (red) in HeLa cells transfected with the indicated shRNAs. DNA was stained with DAPI (blue). Scale bar, 5 μm. (h) Quantification and normalization of the kinetochore signal of Cep57 from g. The experiment was repeated three times. (i) Immunostaining of Mad1 (green) and CENP-A (red) in HeLa cells transfected with NC- or Cep57-siRNA for 60 h followed by treatment with 80 nM taxol and MG132 for 4 h. DNA was stained with DAPI (blue). Scale bar, 5 μm. (j) Quantification and normalization of the kinetochore signal of Mad1 from i. The experiment was repeated three times. For b,d,f,h and j, >200 kinetochores from 20 cells were measured per experiment. Data are mean±s.e.m. ****P<0.0001; ***P<0.001; **P<0.01; NS, not significant (unpaired two-tailed Student’s t-test).