Fig. 5
CDK16 regulates cell proliferation by phosphorylating PRC1 and modulating spindle formation. A Spindle formation analysis of control and CDK16-KD MDA-MB-231 cells. Cells were immunostained with antibodies for pericentrin (red), alpha-tubulin (green), followed by DAPI (blue) staining. Shown are representative immunofluorescence images of control cells in distinct cell cycle phases and CDK16-depleted cells stuck in prophase (left, scale bar: 10 μm), and statistics of cells with spindle formation defects (right). B MDA-MB-231 cells were synchronized in M phase by nocodazole followed by release back into the cell cycle, and the cell cycle phases were monitored by FACS (left). CDK16 and p-PRC1 (T481) expression was detected by immunoblot analysis at indicated time points (right). C Immunoblot analysis for total PRC1 and p-PRC1 (T481) expression of MDA-MB-231 cells when CDK16 was knocked down. D qPCR analysis of PRC1-targeted cell cycle and apoptosis related genes in MDA-MB-231 cells when CDK16 was knocked down. E Subcellular localization analysis of PRC1 in control and CDK16-KD MDA-MB-231 cells. Cells were immunostained with antibodies for PRC1 (purple), alpha-tubulin (green), followed by DAPI (blue) staining (scale bar: 10 μm). F Immunoblot analysis for PRC1 distribution in distinct subcellular fractions (Cyt: cytoplasm, Nuc: nucleus) of MDA-MB-231 cells when CDK16 was knocked down. G qPCR analysis to verify the knockdown of CDK16 and the overexpression of PRC1T481D or PRC1T481A mutant in MDA-MB-231 cells. H 2D-colony formation assay of MDA-MB-231 cells stably expressing control vector, PRC1T481D or PRC1T481A mutant followed with transfection of scramble-shRNA or CDK16-shRNA. Shown are representative images (left) and quantification of colonies (right). Data are presented as mean ± SD and p values were obtained by two-tailed Student’s t-test. All *p < 0.05, ** p < 0.01, ***p < 0.001, **** p < 0.0001, ns, not significant