Fig. 5

Nek2 ablation suppresses centrosome clustering and p53LOH. a Colony formation assay showing suppressed proliferation of H/−;ErbB2 cells, as compared to +/+;ErbB2 cells, following treatment with Nek2 inhibitor JH295. b Colony formation assay showing partial suppression of proliferation of H/+;ErbB2 cells, as compared to −/+;ErbB2 cells, following treatment with Nek2 inhibitor JH295. c Growth curve showing suppressed proliferation in H/+;ErbB2 cells following CRISPR/Cas9 Nek2 deletion (Nek2cc9). Inset shows western blot for Nek2 before and after CRISPR/Cas9 Nek2 deletion. α-Tubulin is loading control. d–f Bar graphs showing percent of cells with ≥ 3 centrosomes, with centrosome clustering and with multipolar spindle, respectively, in H/+;ErbB2 cells before and following CRISPR/Cas9 Nek2 deletion. n = 3 independent experiments per genotype. g, h Bar graphs showing percent of cells with ≥ 3 centrosomes and with centrosome clustering, respectively, in ErbB2 mammary epithelial tumor cell lines with different p53 status with and without treatment with Nek2 inhibitor JH295. n = 3 independent experiments per genotype (one cell line per genotype except for p53+/+ and p53 H/− where 2 different cell lines derived from different tumors and result per genotype was averaged). i Analysis of LOH in H/+;ErbB2 cell line. n = 3 independent experiments per treatment. Non-irradiated cells (lanes 1–3) and cells treated with JH295 (lanes 4–6) are showing no LOH. Irradiated cells showing LOH (lanes 7–9). Cells irradiated and treated with JH295 are showing no LOH (lanes 10–12). j Densitometric analysis of band intensity ratio of PCR amplification product shown in i