Interphase Cep164 levels are affected in Nek2-overexpressing breast cell lines. (A) Immunofluorescence analysis of Cep164 centrosomal levels in RPE1, MCF10A, MCF10AT, and MCFCA1 interphase cells. DNA was stained with DAPI, and γ-tubulin labels centrosomes. Percentage of interphase cells with high and low Cep164 levels was quantified. Representative images are shown to the left. Bar graphs represent mean ± SD. n > 900 cells per cell type in five independent experiments. Scale bar, 5 µm. (B) Percentage of cells with low Cep164 centrosomal levels in interphase in control and Nek2 siRNA-treated cells. Cells were analyzed as in A. Bar graphs represent mean ± SD. n > 300 cells per cell type and condition in four independent experiments for MCF10A and MCF10AT and three experiments for MCF10CA1. (C) Correlative analysis of Cep164 and centrosomal Nek2 levels. The box/dot plots show Cep164 fluorescent intensity (int.) measured at the centrosome in interphase cells with high or low Nek2 centrosomal signals (as shown in D). The graphs show one representative experiment. Dots represent individual cells, boxes show interquartile range, lines inside the box represent the median, and whiskers show minimum and maximum values excluding outliers. n = 80 cells per sample. Similar results were obtained in five independent experiments. (D) Pictures show representative images for Nek2-positive and low/negative cells. Specific antibodies against Nek2, Cep164, and γ-tubulin were used. Scale bar, 10 µm. A.U., arbitrary units; n.s., not significant. Significance probability values are: n.s., P > 0.05; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001.