Nek5 loss leads to premature centrosome separation in interphase. (a) RT-PCR of Nek5 and GAPDH with RNA collected after 72 h of siRNA treatment of U2OS cells with mock, GAPDH, or Nek5 siRNAs. Three individual Nek5 siRNAs were tested. (b) Staining of Nek5 and γ-tubulin in U2OS cells transfected with Nek5- or GL2-specific siRNAs for 48 h. (c) Quantification of cells treated as in b with centrosomes split by >2 µm. (d) Staining of γ-tubulin and DNA in U2OS cells transfected for 48 h with GFP, GFP-Nek5WT, or GFP-Nek5KD. (e) Quantification of cells with centrosomes separated by >2 µm in cells treated as in d. (f) U2OS cells were transfected with either siRNAs against Nek5 or GL2, or mock treated for 24 h before GFP-Nek5WT and GFP-Nek5KD RNAi sensitive and resistant constructs were transfected into the cells. Cells were harvested 24 h after plasmid transfection and analyzed by Western blotting with Nek5, GFP, and α-tubulin antibodies. (g) Quantification of cells with centrosomes separated by >2 µm in U2OS cells treated as in f. (h) Box and whisker plot of the distance between centrosomes in mock-, GL2-, or Nek5-depleted cells, and either untransfected cells or those expressing GFP, GFP-Nek5WT, GFP-Nek5KD, or GFP-Nek2WT. Bars: (main images) 10 µm; (insets) 1 µm. *, P < 0.05; **, P < 0.01; ****, P < 0.0001. Histograms show mean + SD (error bars); n = 3, 200 cells per experiment. Boxes represent the 25th and 75th percentile, and whiskers the 10th and 90th percentile of centrosome distances measured in 50 cells.