Fig. 5

SHCBP1 knockdown causes multipolar spindle formation and delays mitotic exit in tumour cells. A The percentage of mitotic cells counted in HeLa, A549 and HBE cells transfected with control or SHCBP1 siRNA after release from RO3306 block (9μΜ) for indicated time. Immunofluorescence staining for α-tubulin (red) and γ-tubulin (green) followed by confocal fluorescence imaging and cell counting were performed at each time point. Three different fields at each time point were randomly selected for counting at least 500 cells within each field. Data are shown as mean ± SD (n = 3) and analyzed by the unpaired Student’s t test. ***, P < 0.001; ****, P < 0.0001; ns = not significant. B The percentage of mitotic cells prior to anaphase in total M-phase cells was analyzed from the same experiment in (A). Three different fields at each time point were randomly selected for counting at least 200 mitotic cells within each field. Data are shown as mean ± SD (n = 3) and analyzed by the unpaired Student’s t test. **, P < 0.01; ***, P < 0.001. C Representative immunofluorescence images of α-tubulin (red) and γ-tubulin (green) staining after 0.5 h release from RO3306 arrest (9 μM) in HeLa and HBE cells transfected with siCtrl or siSHCBP1. White arrows indicate the cells with multipolar spindle in M phase. Scale bars, 20 μm. D The percentage of cells with multipolar spindle in M phase over total mitotic cells in the same experiment from (C) were analyzed. Three different fields were randomly selected for counting at least 200 mitotic cells within each field. Data are shown as mean ± SD (n = 3) and analyzed by the unpaired Student’s t test. ***, P < 0.001. E Representative immunofluorescence images of multipolar spindle formation throughout M phase in HeLa cells transfected with SHCBP1 siRNA compared to siCtrl cells with normal spindle pole formation throughout M phase. α-tubulin (red), γ-tubulin (green), and Hoechst (blue) were co-stained in cells. Scale bars, 5 μm. F, G Western blot images (F) and mRNA expression (G) analysis of changes in NEK7, ZW10, cyclin B1, PLK1, XLF and γH2AX in A549, NCI-H460, HeLa and NCI-H1299 cells transfected with SHCBP1 siRNA compared to controls. GAPDH was used as internal reference. Data are shown as mean ± SD (n = 3) and analyzed by the unpaired Student’s t test. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001