Figure 3

The newly identified Setdb1 target Ankrd1 is crucial for myoblast terminal differentiation. (a) Downregulation of Ankrd1 increases MuSC amplification in single myofibres. Control siRNA (siCTRL) or Ankrd1 siRNA (siAnkrd1) were transfected directly after isolation and cultured in suspension for 72 h. Indirect IF was conducted to visualise Pax7 (green) and Myogenin (red) in MuSCs. DNA was detected with Hoechst (blue). Scale bar=5 μm. (b) Quantification of MuSCs progeny (Pax7⁺ or Myogenin⁺) in myofibres transfected with control siRNA (siCTRL) or Ankrd1 siRNA (siAnkrd1). (c) Quantification of differentiating MuSCs (Myogenin+) in myofibres transfected with control siRNA (siCTRL) or Ankrd1 siRNA (siAnkrd1). (d) Ankrd1 knockdown impairs myotube formation. Proliferating C2C12 myoblasts, at 80–90% confluence, were transfected with control siRNA (siCTRL) or Ankrd1 siRNA (siAnkrd1) and simultaneously switched to differentiation media for 72 h post-transfection. Cellular Ckm was stained by indirect IF (green). DNA was co-stained with DAPI (red). Scale bar=20 μm. Fusion index (number of nuclei in myotubes divided by total number of nuclei) is indicated (in blue) for each condition. A minimum of 300 nuclei was counted. Data are presented as mean±s.e.m. of three independent experiments. P-values are indicated. (e) Ankrd1 downregulation reduces Ckm mRNA levels. Proliferating C2C12 myoblasts were transfected and cultured as described in (d). Relative mRNA expression levels of Ankrd1 and Ckm were evaluated by qPCR. Data are represented as fold change relative to siCTRL and normalised to CycloA and TBP. (f) Proliferating C2C12 myoblasts were transfected and differentiated as described in (d). Apoptotic cells were stained by performing the TdT-mediated dUTP-biotin nick-end labelling (TUNEL) reaction. Cells containing a signal inside the nucleus were considered as apoptotic. A minimum of 400 cells was counted. For (a, d): Images are representative of at least three independent experiments. For (b, c): Presented data are mean±s.e.m. of four independent experiments using four different mice. For each experiment at least 25 fibres were evaluated. For significance Student’s paired t-test was applied. *P-values <0.05 are considered significant. For (e, f): Presented data are mean±s.e.m. of a minimum of three independent experiments. For significance Student’s paired t-test was applied. *P-values <0.05 are considered significant.