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Figure 6


Fig. 6. Suppression of NIP2 expression caused abnormality in spindle formation. (A) Two different amounts of siRNAs specific to NIP2 (siNIP2), Nek2 (siNek2) or non-specific control siRNA (siCTL) were transfected into HeLa cells. Forty-eight hours after transfection, immunoblot analysis was carried out to determine the cellular levels of NIP2 and Nek2 proteins. Acetylated tubulin and β-tubulin were detected as a control. (B) Twenty-four hours after transfection of siCTL or siNIP2, HeLa cells were co-immunostained with antibodies against NIP2 and {gamma}-tubulin. (C) NIP2-suppressed HeLa cells were co-immunostained with antibodies against NIP2 and β-tubulin. The sizes of the cells were measured using Image-pro software and analyzed statistically. For statistical analysis of this and the following experiments, over 300 cells were counted for each group and the experiments were repeated three times. The results were presented as mean±s.e.m. (D) Microtubule re-growth assay. HeLa cells transfected with siCTL or siNIP2 were placed on ice for 30 minutes to disrupt microtubules and transferred immediately to 37°C for microtubule re-growth. Five minutes after the temperature shift, cells were fixed and co-immunostained with antibodies against NIP2 and β-tubulin. (E) Forty-eight hours after transfection with siCTL or siNIP2, U2OS cells were co-immunostained with antibodies against NIP2 (red) and β-tubulin (green). The number of mitotic cells with abnormal spindles was counted. (F) The nuclei of NIP2-suppressed U2OS cells were stained with DAPI and the number of cells with abnormal nuclear morphology was counted. (G) HeLa cells transfected with siCTL or siNIP2 were cultured for 24, 48 or 72 hours and subjected to FACS analyses to determine the DNA content of each cell population. (H) Ninety-six hours after transfection of siCTL or siNIP2, the MTT assay was carried out to determine the number of live cells. The proportion of the live cells was determined by counting the MTT-stained cells in the culture dishes.





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