Fig. 2.

S1PR3-null satellite cells have increased proliferation. (A) EDL myofibres were isolated from age-matched wild-type or S1PR3-null mice and immunostained for Pax7 to identify quiescent satellite cells. There was no difference in the number of quiescent satellite cells per EDL myofibre in 6–8 week old S1PR3-null or wildtype mice. (B) and (C) Satellite cells associated with a myofibre from S1PR3^−/− and control mice were cultured for 72 h and co-immunostained for Pax7 (green) and Ki67 (red), with DAPI counterstaining, to assess proliferation and self-renewal. A significantly lower proportion of satellite cells lacking S1PR3 exited the cell cycle to adopt the self-renewal phenotype compared to wild type cells. (D) Satellite cells associated with a myofibre from S1PR3^−/− and control mice were cultured in medium to promote satellite cell proliferation and samples pulsed with EdU for 4 h and fixed at 24 h intervals. More S1PR3-null satellite cells had incorporated EdU after 48 and 72 h, compared to controls. (E) and (F) Plated expanded satellite cell-derived myoblasts were cultured in serum-rich medium and pulsed for 2 h with EdU before fixation. Co-staining for EdU and DAPI confirmed that more satellite cells from S1PR3-null mice incorporated EdU compared to control cells. Values are mean±SEM from three mice (n=3) where an asterisk denotes significantly different from wild-type controls (p<0.05) using a two-tailed T-test.