Fig 1: Cep57 is a novel kinetochore component in human cells.(a) Three-dimensional structured illumination microscopy (SIM) images of HeLa cells double-immunostained with antibodies against Cep57 (green) and CREST (red). (b) Immunofluorescence of Cep57 (green) and CENP-A (red) in HeLa cells. (c) Immunofluorescence of Cep57 (green) and Mis12 (red) in HeLa cells at metaphase after treatment with MG132 for 1 h. A linescan through the kinetochore pair indicates the co-localization of Cep57 with Mis12. (d) Stimulated emission depletion (STED) images of immunofluorescence of Cep57 (green) and Zwint-1 (red) in RPE1 cells. (e) Immunofluorescence of Cep57 (green) and CREST (red) in HeLa cells at different stages during mitosis. DNA was stained with 4,6-diamidino-2-phenylindole (DAPI, blue). (f) HEK293T cells were co-transfected with Cep57-GFP and Mis12-HA. The cell lysates were immunoprecipitated (IP) and analysed by western blotting (WB) with the indicated antibodies. (g) Binding assays of Mis12 and Cep57. GST-Mis12 (expressed in E. coli and purified) was incubated with Flag-Cep57 (expressed in HEK293T cells and purified). The IP samples with anti-Flag antibody were analysed by WB with anti-Flag and anti-Mis12 antibodies. (h) Schematic of Cep57 truncated mutants. (i) GST pull-down assays of Cep57 and Mis12. Lysates of HEK293T cells overexpressing Mis12-HA were incubated with Glutathione Sepharose 4B beads coated with GST or GST-Cep57N/C. The samples were analysed by WB with anti-HA antibody. GST-tagged proteins were stained with Coomassie blue. (j) In vitro pull-down assays of Mis12 and Cep57 (1–242 amino acids). GST-Mis12 (expressed in E. coli and purified) and MBP-Cep57 (1–242 amino acids) were incubated with Amylose Magnetic beads. The precipitated samples were analysed by WB with anti-GST antibody and Coomassie blue staining. (k) Quantification of kinetochore signals of Cep57 and Mis12 in HeLa cells depleted of Cep57, Mis12 or negative control (NC) by siRNAs. The signal from control siRNA-treated cells was normalized to 1.0. More than 200 kinetochores from 20 cells were measured. The experiment was repeated three times. Data are mean±s.e.m. ****P<0.0001; ***P<0.001; NS, not significant (unpaired two-tailed Student's t-test). Scale bars, 5 μm in (a–e).