S1P activation of STAT3 is mediated via an S1PR2/RhoGTPase pathway.

<p>SC-derived myoblasts were treated with indicated dose of: A) Rac1 inhibitor, or B) RhoA inhibitor for 24 hours in complete media, and cell lysates were immunoblotted with indicated antibodies. Levels of total STAT3 (STAT3-T) and phosphorylated STAT3 (STAT3-P) were measured and compared to actin loading control. C) SC-derived myoblasts were treated with 10 µM S1PR2 antagonist JTE-013 (R2i) for 20 hours, cells were incubated in fresh media for 5 minutes with or without R2i, and activated Rac1 was pulled down with GST-PAK1 fusion protein and immunoblotted with Rac1 antibody. STAT3 activation (STAT3-P) also shown for this experiment. D) Myoblasts were transfected with GFP, dominant negative Rac1 (Rac1 DN) or wild type RhoA (RhoA WT) plasmid constructs. Forty-eight hours later, cell lysates were immunoblotted to detect total and phosphorylated STAT3, GFP, Rac1 and RhoA, with GAPDH used as a loading control. E) Representative micrographs (10×) of H&E stained 10 µm frozen sections taken from mdx gastrocnemius muscles 5 days post NTX-injury. Groups were treated with vehicle, THI, STAT3 inhibitor WP1066, or THI+WP1066. F) Quantification of regenerating myofibers/mm² (n = 3/group). Scale bar = 50 microns. G) Analysis of SCs by flow cytometry as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037218#pone-0037218-g005" target="_blank"><b>Figure 5F</b></a>. Data are means ± SD, n = 5/group, * indicates p≤0.05.