Fig. 1

Primary cilia are dynamically regulated and are preferentially present on quiescent myoblasts in vitro and in vivo.a Immuno-detection of primary cilia on C2C12 myoblasts in different cellular states -proliferating (MB), quiescent (G0), and 24 h after reactivation into cell cycle from quiescence (R24). Antibodies against γ-tubulin (γ-tub) mark centrosome and Acetylated tubulin (Ac.Tub) mark cilia; DAPI was used to label DNA. While only a small proportion of MB is ciliated, G0 myoblasts show a higher frequency of ciliation, which is reversed at R24. Lower panel shows magnified views (boxed insets). (Scale bar, 10 μm.). b Quantification of data showed in (a). Values are mean ± s.e.m., N ≥ 3, * p < 0.05. c The length of cilia present on MB, G0, and R24 cultures was measured and the distribution plotted. Cilia elaborated by G0 myoblasts are longer (Average length 2.2 μm) than those in proliferating cultures (Average length 1.8 μm). The numbers of ciliated cells analyzed are MB (40), G0 (30), and R24 (54). d Increased frequency of ciliated cells observed as proliferating myoblasts withdraw into quiescence during a time course of suspension arrest (0–48 h). Values are mean ± s.e.m., N ≥ 3, * p < 0.05, ** p < 0.01, *** p < 0.005. e Rapid loss of cilia during reactivation of G0 cells by re-plating on to an adhesive substratum, and gradual increase in ciliation during cell cycle re-entry. Values are mean ± s.e.m., N = 3, * p < 0.05. f Immunofluorescence analysis of cilia in 5 day differentiated culture of C2C12 cells: cilia are marked with Acetylated tubulin (Ac.Tub), Myogenin marks differentiated cells, DNA is labeled with DAPI (Scale bar, 10 μm). Cilia are absent on multinucleated myotubes (open arrowheads), and preferentially present on mononuclear (unfused) cells that lack Myogenin (closed arrowheads). Zoomed insets are representative of (1) unfused myoblasts (Myogenin⁻ Cilium⁺, and (2) multinucleated myotubes (Myogenin⁺ Cilium⁻). g Quantitative analysis of ciliation during a time course of differentiation (represented by image in f) reveals increased proportion of ciliated cells as myoblasts withdraw into terminal differentiation. Values are mean ± s.e.m., N = 3, * p < 0.05. h Increased proportion of Myogenin⁺ cells during the period where ciliation was analyzed in (g) highlights the time course of increase in Myogenin expression during differentiation. Values are mean ± s.e.m., N = 3, * p < 0.05. i Quantitative analysis of cells that are both ciliated and Myogenin⁺ shows that ciliated cells are largely negative for Myogenin, indicating exclusion from differentiation. Values are mean ± s.e.m., N = 2. j Primary cilia are present on reserve cells in differentiated cultures. 5-day differentiated C2C12 cultures were mildly trypsinised to remove myotubes, enriching the adherent undifferentiated mononuclear reserve cells. Cilia were detected using γ-tubulin (γ tub) and Acetylated tubulin (Ac.Tub). (Scale bar, 10 μm). The graph shows quantification of the data represented in the image, values are mean ± s.e.m. 47% of reserve cells in differentiated cultures are ciliated. k Primary cilia are associated with Pax7⁺ satellite cells (SC) but not with Pax7⁻ myonuclei (MN). Representative immunofluorescence image of a single fiber isolated from mouse EDL muscle and stained for Pax7 to mark satellite cells, and Pericentrin (PCNT) and Acetylated tubulin (Ac.Tub) to mark primary cilia, DAPI marks nuclei (Scale bar, 10 μm). The graph shows the quantification of the data derived from 4 independent staining experiments on single fibers: each experiment used fibers isolated from one mouse and a total of 121 nuclei were evaluated for the presence/absence of an associated cilium.