Figure 1

Setdb1 is required for accurate adult skeletal muscle stem cell amplification and negatively regulates terminal differentiation. (a) Setdb1 increases during muscle satellite cells (MuSCs) activation. Single myofibres were isolated from extensor digitorum longus (EDL) muscles of C57BL/6N mice. Myofibres were directly fixed after isolation (upper, T=0 h) or cultured in floating conditions for 24 h (lower, T=24 h). Setdb1 (red) and Pax7 (green) proteins were revealed by indirect immunofluorescence (IF). DNA was labelled with Hoechst (blue). A representative picture of an MuSC is shown. Scale bar=5 μm. (b) Setdb1 knockdown in MuSCs. Freshly isolated EDL mouse single myofibres were transfected with control (siCTRL) or Setdb1 siRNA (siSetdb1) 2 h after isolation and cultured for 48 h post-transfection. IF in MuSCs was done as in (a). Scale bar=10 μm. (c) Setdb1 knockdown reduces MuSCs amplification. Quantification of MuSCs progeny (Myogenin⁺ or Pax7⁺) on cultured myofibres transfected with control (siCTRL) or Setdb1 siRNA (siSetdb1) 72 h after transfection. (d) Setdb1 knockdown decreases MuSCs self-renewal. Myofibres were transfected as described in (b). Quantification (in %) of MuSC descendants at the surface of cultured myofibres 72 h post-transfection. The proportion of committed cells (Pax7⁻/MyoD⁺), proliferating cells (Pax7⁺/MyoD⁺) and self-renewing cells (Pax7⁺/MyoD⁻) in myofibres transfected with control (siCTRL) or Setdb1 siRNA (siSetdb1) is presented. (e) Setdb1 limits MuSCs differentiation. EDL single myofibres were cultured for 72 h following transfection with control (siCTRL) or Setdb1 siRNA (siSetdb1). For detection of proliferating and differentiating MuSCs indirect IF was performed to detect Pax7 (green) and Myogenin (red), respectively. DNA was labelled with Hoechst (blue). Representative myogenic cell clusters are shown. Scale bar=20 μm. See Supplementary Figure S1A for images with lower magnification. (f) Setdb1 knockdown increases the proportion of differentiating MuSCs. % of Myogenin⁺ MuSCs in myofibres transfected with control (siCTRL) or Setdb1 siRNA (siSetdb1) as described in (d). (g) Setdb1 protein levels decrease during terminal differentiation of C2C12 myoblasts. Western blot (WB) analysis of Setdb1, Myosin Heavy Chain (MyHC), Creatine Kinase Muscle (Ckm) and Myogenin in whole-cell extracts from proliferating C2C12 myoblasts (prolif.) and after 24, 48 and 96 h of differentiation (diff.). Vinculin served as a loading control for Setdb1 and Myogenin and α-Tubulin for Ckm and MyHC. A typical experiment in shown. * Shifted Setdb1 signals. Images are representative of a minimum of three independent experiments. For Setdb1 signal quantification see Supplementary Figure S1D. (h) Setdb1 knockdown promotes differentiation and fusion of myotubes. Proliferating C2C12 myoblasts, at 80–90% confluence, were transfected with control (siCTRL) or Setdb1 (siSetdb1) siRNA and simultaneously switched to differentiation media for 72 h. Cellular Ckm was revealed by indirect IF (green) and DNA with DAPI (red). Images are representative of a minimum of three independent experiments. Relative 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. Scale bar=20 μm. See Supplementary Figure S1A for additional images. (i) Setdb1 knockdown increases Ckm mRNA levels. C2C12 cells were transfected with control (siCTRL) or Setdb1 siRNA (siSetdb1) and differentiated as described in (h). Relative mRNA expression of Setdb1 and Ckm were represented as fold change relative to siCTRL and normalised to Cyclophilin A (CycloA) and TATA-box-binding protein (TBP). Data are presented as mean±s.e.m. of three independent experiments. For significance Student paired t-test was applied. *P-values <0.05 and are considered significant. **P-values <0.01. For (a, b, e): all images are representative of a minimum of three independent experiments using three different mice. For (c,d, f): data are presented as mean±s.e.m. of three independent experiments using three different mice. For each mouse at least 30 fibres were counted. For significance Student paired t-test was applied. *P-values <0.05 and are considered significant. See also Supplementary Figure S1.