Fig. 3.

Myogenic progression in S1PR3-null satellite cells is relatively normal. EDL myofibres and their associated satellite cells were isolated from either age-matched wild-type (S1PR3^+/+) or S1PR3-null (S1PR3^−/−) mice and cultured in proliferation medium for 72 h. (A)–(C) Samples were taken at 24 h intervals, fixed and co-immunostained for Pax7, MyoD and Myogenin, which showed a general increase in each population when S1PR3 was absent, consistent with enhanced proliferation. (D) When expressed as a ratio though, the proportions of cells expressing Pax7, MyoD or myogenin were similar between wildtype and S1PR3-null mice. (E) and (F) Expanded satellite cell-derived myoblasts were also plated at high confluency and then cultured in differentiation medium for 48 h before immunostaining for Myosin Heavy Chain (MyHC) and counterstaining with DAPI. Counting the number of nuclei within MyHC+ve myotubes (>2 nuclei) to determine the fusion index revealed that more satellite cells from S1PR3-null mice had differentiated and fused than from control mice ((E), quantified (F)). Values are expressed as mean±SEM from multiple samples from at least three mice (n=3/4) where an asterisk denotes significantly different (p<0.05) from wild-type controls using a two-tailed T-test.