Direct injection results in elevated S1P levels which correlate with the activation of receptor 1 in muscle fibers. ( A ) To quantify the elevation of S1P following direct administration, we injected a single dose (same dose as Figure 5) of S1P in left TAs and vehicle in right TAs of uninjured mdx4cv (n = 3, 11-MO) mice. TA muscles were harvested 15 minutes post injection for analysis by LC-MS/MS. Results indicate a significant elevation of S1P following direct injection. ( B ) To visualize the location of S1P following injection, biotinylated-S1P was injected in left TAs versus vehicle in right TAs of uninjured mdx4cv mice (n = 2, 11-MO). Once more, TAs were harvested 15 minutes following injection. Staining with streptavidin conjugated to Alexa Fluor 594 reveals the presence of S1P-biotin around the perimeter of muscle fibers. ( C ) Staining of mdx4cv TAs for S1PR1 and S1PR3 reveals S1PR1 is localized to the perimeter and perinuclear area (arrow) of muscle fibers (left photo). In contrast, staining for S1PR3 was mainly localized to the muscle vasculature (middle photo). Staining in parallel with an IgG isotype control for both antibodies shows the absence of non-specific staining (right graph). ( D ) Staining for S1PR1 in CTX-injured TAs (same tissue from Figure 5) reveals S1PR1 is present at the perimeter and perinuclear area of regenerating eMyHC+ fibers. ( E ) Staining for phosphorylated S1PR1 in the same mdx4cv TAs was more prominent in the perinuclear area of eMyHC+ fibers, indicating the presence of active S1PR1 signaling in regenerating fibers. Scale bars = 50 μm. **P <0.005 by student’s t-test. Error bars represent SEM. CTX, cardiotoxin; eMyHC, embryonic myosin heavy chain; IgG, immunoglobulin G; LC-MS/MS, liquid chromatography-tandem mass spectrometry; MO, month-old; S1P, sphingosine-1-phoshate; S1PR1, S1P receptor 1; S1PR3, S1P receptor 3; SEM, standard error of the mean; TA, tibialis anterior.