Fig. 7. KIFC1-S26 phosphorylation induces chromosomal instability.

a Representative images showing lagging chromosomes induced by centrosome clustering in the MDA-MB-231 stable cell lines. The cells were pretreated with VE-822 for 1 h, and then treated with etoposide (2 μM) for another 15 h. Spindle and DNA were co-stained with DAPI and α-tubulin. In quantitative analysis, anaphase cells with lagging chromosomes induced by centrosome clustering were compared with all anaphase cells. For each experimental condition, 100–110 cells were counted, and three independent experiments were performed. Scale bar, 10 μm. Two-tailed t test p values (from left to right): p = 0.0115, 0.0030, and 0.0038. b Establishment of KIFC1 WT, S26A, or S26D mutant stable cell lines from MCF-10A cells. Cell lysates were immunoblotted with antibodies against KIFC1 or β-actin (as the internal standard). The Western blot images are representative of two independent experiments with similar results. (c, d) Histogram showing the percentage of >2 centrosomes per cell (centrosome amplification) (c), pseudo-bipolar mitosis (centrosome clustering), and multipolar mitosis (non-efficient centrosome clustering) (d) in the indicated stable cell lines in response to etoposide (5 μM) for 15 h (c, d) or 48 h (c). c Two-tailed t test p values: p = 0.00044, 0.0137, 0.0033, 0.0033, and 0.0023. (d) Two-tailed t test p values: p = 0.0097, and 0.0099. (e, f) The stable cell lines were treated with etoposide (0.1 μM) for 30 generations and then were used to assess the rate of chromosomal instability (CIN). For each experimental condition, 100–120 cells were counted, and three independent experiments were performed. Scale bar, 10 μm. e Representative metaphase plates containing different chromosome numbers with quantitative analysis of chromosome numbers. For each experimental condition, 100–164 cells were counted, and three independent experiments were performed. Two-tailed t test p values (from left to right): p = 0.0278, 0.0078, 0.0098, 0.0079, and 0.0056. f Representative image of the centromeric DNA of chromosomes 3 and 7 obtained by fluorescence in situ hybridization (FISH) analysis with quantitative analysis of chromosome numbers. For each experimental condition, 100–112 cells were counted, and three independent experiments were performed. Two-tailed t test p values (from left to right): p = 0.0027, 0.0037, 0.0086, and 0.0084. Statistical data presented in this figure show mean values ± SD of three times of independent experiments. *p < 0.05; **p < 0.01. Source data are provided as a Source Data file.