Fig. 5. KIFC1-S26 phosphorylation promotes centrosome clustering.

a Establishment of Flag-tagged KIFC1-WT, S26A, or S26D mutant stable cell lines from MDA-MB-231 cells. Cell lysates were immunoblotted with antibodies against KIFC1 and β-actin (as internal standard). b KIFC1-S26 phosphorylation stabilizes KIFC1 protein level. Western blot analysis of lysates of indicated cell lines treated with cycloheximide (CHX, 50 μm ml⁻¹). Relative KIFC1 band intensities were quantified using densitometry and presented. c 293T cells transfected with His-ubiquitin and Flag-KIFC1 or indicated Flag-KIFC1 mutant plasmids were treated with etoposide (20 μM) for 6 h. MG132 (25 μM) was added for 3 h prior to lysis. Ubiquitinated proteins were precipitated using Ni-NTA beads. KIFC1 ubiquitination was detected by western blot using anti-Flag antibody. The Western Blot images are representative of 2 independent experiments with similar results (a–c). d–f KIFC1-S26 phosphorylation promotes centrosome clustering. Histogram showing the percentage of >2 centrosomes per cell (centrosome amplification) (d), pseudo-bipolar mitosis (centrosome clustering), and multipolar mitosis (non-efficient centrosome clustering) (e, f) in the indicated stable cell lines in response to etoposide (5 μM) for 15 h (d–f) or 48 h (d). e Representative images showing pseudo-bipolar and multipolar mitosis in indicated cell lines (scale bar, 10 μm). Spindle poles, centrioles, and DNA were co-stained with α-tubulin, centrin, and DAPI. Insets show magnification of the centriole area. f The cells were pretreated with VE-822 (5 μM) for 1 h and then treated with etoposide and VE-822 for another 15 h. d Two-tailed t test p values (from left to right): p = 0.0006, 0.0364, 0.0071, 0.0050, and 0.0441. e Two-tailed t test p values (from left to right): p = 0.0073, 0.0019, 0.0034, 0.1816, and 0.6059. f Two-tailed t test p values (from left to right): p = 0.0088, 0.3163, and 0.4928. Statistical data show mean values ± SD of three independent experiments. NS = not significant, *p < 0.05, **p < 0.01. Source data are provided as a Source Data file.