C2orf62 and TTC17 act on ciliogenesis by modulating actin polymerization.

<p>Ciliogenesis was induced by serum starvation in hTERT-RPE1 cells transfected with combinations of siRNA and plasmids as indicated <b>(A-C)</b> Cells transiently expressing EGFP or C2orf62 were transfected with siRNA against C2orf62, MAPRE1 or TTC17 (50 µM), with both siRNA against C2orf62 and TTC17 (25 µM each), or with a control siRNA. Cilia were visualized by immunofluorescence using anti α-acetylated tubulin (red) and nuclei by DAPI (blue). (<b>A</b>) Cells transfected with siRNA against MAPRE1, C2orf62 or TTC17 have less cilia than cells transfected with control siRNA. Scale bars, 25 µm. (<b>B</b>) The bar graph shows the percentages of ciliated cells obtained in a minimum of 3 independent experiments with 100 cells counted per condition in each experiment. C2orf62, TTC17 and MAPRE1 siRNA significantly reduce cilia numbers, and effects of C2orf62 and TTC17 siRNA are synergic. Effect of C2orf62 siRNA can be rescued by C2orf62 overexpression (C2orf62 siRNA/Ctrl siRNA<b>**</b>P = 0.0026; MAPRE1 siRNA /Ctrl siRNA <b>**</b>P = 0.0034; TTC17 siRNA/Ctrl siRNA ***P = 0.0005; ½C2orf62 + ½TTC17 siRNA/Ctrl siRNA <b>***</b>P = 0.0004; C2orf62 + C2orf62 siRNA/EGFP + C2orf62 siRNA <b>*</b>P = 0.035; ½C2orf62 + ½TTC17 siRNA/C2orf62 siRNA *P = 0.0127). <b>(C)</b> C2orf62 siRNA does not affect cilia length. (<b>D, E</b>) Cells transfected with 50 µM siRNA against C2orf62 or TTC17 or with a control siRNA were serum-starved in the presence or absence of 500 nM cytochalasin D. Cilia were visualized by immunofluorescence using anti-acetylated α-tubulin antibody (green), F-actin by phalloidin (red) and nuclei by DAPI (blue). Scale bars, 25 µm. (<b>D</b>) Both F-actin polymerization and the reduction in cilia numbers induced by siRNA are reversed by cytochalasin D. (<b>E</b>) The bar graph shows the percentages of ciliated cells obtained in each condition. siRNA against C2orf62 or TTC17 significantly reduce the number of cilia in DMSO-treated cells (C2orf62 siRNA/Ctrl siRNA <b>*</b>P = 0.032; TTC17 siRNA/Ctrl siRNA <b>*</b>P = 0.031). Cells treated with Cytochalasin D exhibit more cilia than cells treated with vehicle (DMSO) in all conditions (C2orf62 siRNA/C2orf62 siRNA + CytD <b>*</b>P = 0.018; C2orf62 siRNA/C2orf62 siRNA + CytD <b>*</b>P = 0.019; Ctrl siRNA/Ctrl siRNA + CytD P = 0–016). In the presence of Cytochalasin D, siRNA against C2orf62 or TTC17 do not affect the number of cilia. See also effects of C2orf62 siRNA on PANC-1 cells in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086476#pone.0086476.s004" target="_blank">Fig. S4B</a>.