Figure 5

Setdb1 cellular relocalisation upon terminal differentiation is dependent on Wnt3a signalling. (a) Setdb1 localisation changes in C2C12 myoblasts with increased Wnt3a signalling. Indirect IF and confocal microscopy of Setdb1 (green) was conducted in proliferating C2C12 myoblasts (prolif.) and stimulated with Wnt3a protein for 24 h (prolif.+Wnt3a). DNA was stained with DAPI (red). Scale bar=10 μm. (b) Quantification of a minimum of 100 cells described in (a) according to their phenotype. Setdb1 localisation is cytoplasmic (C), homogeneous (H) or mainly nuclear (N). (c) Setdb1 delocalisation is restricted in differentiating C2C12 myoblasts when Wnt signalling is inhibited. Indirect IF of Setdb1 as explained in (a). C2C12 myoblasts were differentiating for 24 h (24 h diff.) and treated with IWP2 (24 h diff.+IWP2), an inhibitor of Wnt production, in parallel for the same period of time. (d) Quantification of a minimum of 100 cells described in (c) according to their phenotype. Setdb1 localisation is cytoplasmic (C), homogeneous (H) or mainly nuclear (N). (e) Wnt3a signalling is sufficient for Setdb1 delocalisation in C2C12 myoblasts. Left panels: Proliferating cells, at 80–90% confluence, were transfected with control siRNA (siCTRL) or Myogenin siRNA (siMyog) and simultaneously switched to differentiation media for 24 h. Additionally, cells were treated with Wnt3a. Indirect IF was performed, as described in (a). Scale bar=2 μm. Right panel: knockdown efficiency of Myogenin was analysed in parallel in whole-cell extracts by WB. α-Tubulin, loading control. (f) Wnt3a signalling is sufficient for Setdb1 delocalisation in primary myoblasts. Proliferating cells were transfected with control (siCTRL) or Myogenin siRNA (siMyog) and concomitantly stimulated by Wnt3a protein (+Wnt3a) for 24 h. Setdb1 localisation was classified as homogeneous (homogen.) or cytoplasmic (cyto.) and the ratio was calculated. A minimum of 100 cells was counted for each condition. (g) Inhibition of Wnt signalling reduces Ankrd1, Ckm and MyHC levels. WB was performed in whole-cell extracts from C2C12 cells, differentiated (diff.) for the indicated time (in h) and simultaneously treated with IWP2. Vinculin; loading control. For (a, c, e and g): Images are representative of a minimum of three independent experiments. For (b and d): Data are presented as mean of a minimum of three independent experiments. For (f): Data are presented as 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. See also Supplementary Figure S5.