Fig. 2

Abrogation of primary cilia has distinct effects in proliferating and quiescent myoblasts. C2C12 myoblasts were transfected with siRNAs targeting IFT88 to block ciliogenesis, and were analyzed for effects of knockdown on proliferation and quiescence. Cells transfected with non-targeting siRNA were used as control. a qRT-PCR analysis was used to detect levels of IFT88 mRNA in control and siRNA treated samples 48 h after transfection. Knockdown myoblasts show reduced levels of IFT88 mRNA. Values represented are mean ± s.e.m., N = 3, **p-value = 0.0062. b Western blotting analysis demonstrates efficient knockdown of IFT88 protein levels 48 h after transfection. c Primary cilia were visualized by immunofluorescence labeling of Acetylated tubulin (Ac. tubulin) in IFT88 knockdown myoblasts cultured in quiescence-inducing conditions. Representative immunofluorescence images are shown. A single cilium is present per cell in control cultures, whereas knockdown cells show greatly reduced cilia (accompanied by increased tubulin staining in the cell body).

d Quantification of the reduction of IFT88 protein level shown in (b). Values indicated are mean ± s.e.m., N = 3, ***p-value = 0.0004. e Knockdown of IFT88 caused reduction in frequency of ciliated cells. Values represent mean ± s.e.m., N = 4, ** p-value = 0.034.f Proliferation of cells was analyzed by quantification of Ki67⁺ cells, detected by immuno-staining. Targeting ciliogenesis had little effect in proliferating conditions (MB). Values represent mean ± s.e.m., N = 3, ns = not significant (p-value = 0.959). g When induced to enter quiescence (G0), knockdown cells displayed increased frequency of Ki67⁺ compared with control siRNA-transfected cells. Values represent mean ± s.e.m., N = 5, ** p-value = 0.0015. h Ablation of ciliogenesis suppresses expression of quiescence marker p27. C2C12 myoblasts stably expressing the p27 sensor (mVenus-p27K⁻) were transfected with siRNA targeting IFT88or control siRNA and then cultured in quiescence-inducing conditions. The frequency of p27 (mVenus) positive cells was measured by flow cytometry. Values represent mean ± s.e.m., N = 3, * p-value = 0.0234. i Western blotting revealed the reduction of the quiescence marker, p27 in IFT88KD cells under conditions of G0 arrest. The values are relative protein levels compared to Control, and represent mean of quantification from 2 experiments. j Flow cytometric analysis of IFT88 siRNA-transfected cells cultured in conditions that allow proliferation (MB) does not show a significant shift in cell cycle profile. When placed in conditions that induce quiescence (G0) in control cells, knockdown cells display an increased proportion of cells in G2/M. Representative profiles are shown where the X axis shows the DNA content estimated by florescence intensity of the DNA dye DRAQ5, and the Y axis represents cell numbers as a percentage of maximum (normalized to mode). k Quantification of experiment described in (j) showing shifts in proportion of cells in different cell cycle stages (G1, S, G2/M). Values represent mean ± s.e.m., N = 3, * p-value = 0.043. l Flow cytometric analysis of IFT20 and KIF3A siRNA-transfected cells cultured in conditions that induce quiescence (G0). Cells lacking cilia show an altered cell cycle profile with an increase in proportion of cells in G2/M. Representative profiles are shown where the X axis shows the DNA content estimated by florescence intensity of the DNA dye DRAQ5, and the Y axis represents cell numbers as a percentage of maximum (normalized to mode). m Ablation of ciliogenesis using other IFT targets suppresses expression of quiescence marker p27. C2C12 myoblasts stably expressing the p27 sensor (mVenus-p27K⁻) were transfected with siRNA targeting IFT20 or KIF3A, or control siRNA and then cultured in quiescence-inducing conditions. The frequency of p27 (mVenus) positive cells was measured by flow cytometry. Values represent mean ± s.e.m., N = 3, ** p-value = 0.0001, n.s (p-value = 0.38).