Figure 2

Loss of Haspin leads to longer and more persistent primary cilia. (a,b) shSCR and shHaspin were treated as described in Figure 1. For Haspin inhibition, entry into G0 was achieved by 48 h of serum starvation to induce ciliation. 5-ITu was added after the first 24 h of starvation. Cells were then incubated in the presence of serum to induce cell-cycle entry and cilia resorption. Samples were analyzed by immunofluorescence in G0, 2 h, and 24 h after serum readdition. Cells were fixed and processed for immunofluorescence against γ-tubulin and acetylated tubulin; representative images are shown in (a), and the percentage of cells with or without cilia is shown in (b); error bars represent standard deviation; (c) cells were processed as above to measure cilia length at the end of the incubation in serum-free medium. Representative images are shown; (d) SH-SY5Y cells were differentiated and processed by immunofluorescence to visualize basal bodies (γ-tubulin) and cilia (ARL13B). Representative images are shown; (e) RPE_1-hTERT cells were transfected with GFP or Haspin–Venus encoding plasmids and serum-starved for 48 h. At the end of the starvation, cells were fixed and processed for immunofluorescence to measure cilia length. Graphs in (c–e) show the median cilia length calculated as described in Material and Methods; boxes include 50% of the data points, notch represent confidence interval (median ± 1.58 IQR/sqrt(n)). t-test was applied as a statistical measurement, n.s.; not significant, * p < 0.05, *** p < 0.005. Scale bar in (a): 20 µm, (c,d): 5 µm.