Fig. 1
Effect of S1P pre-conditioning on hypoxia-impaired differentiation: 0.2 × 105/cm2 C2C12 cells were seeded in each group, maintained in DMEM-HG media and induced to differentiate at 90–95% confluence by switching to DM (DMEM HG with 2% HS). a S1P (1 μM) and VC pretreated differentiating C2C12 cells under normoxia (N) and 0.5% hypoxia (H) at various time points (2D, 4D, 6D and 8D) were stained with calcein-AM (100 nM) and imaged by an inverted fluorescence microscope at ×10 magnification. Scale bar 100 µm. The groups were: 2NVC, 2NS1P, 2HVC, 2HS1P, 4NVC, 4NS1P, 4HVC, 4HS1P, 6NVC, 6NS1P, 6HVC, 6HS1P, 8NVC, 8NS1P, 8HVC, 8HS1P. b MHC (molecular weight: 220 kDa) expression was determined in cell lysate by standard Western blotting protocol. c Densitometric analysis of MHC Western blot normalized against the loading control (β-actin). d Calcein-AM fluorescence signal intensity, an indicator of cell viability, was measured using a multimode plate reader (FLUOstar Omega), and average values relative to 0D were reported. e Cell damage marker, extracellular LDH activity, was estimated in cell culture supernatant by the previously described method. Data are reported as mean ± SD from three independent experiments carried out in triplicate. Statistical analysis was performed using the one-way ANOVA/post hoc Bonferroni’s analysis method. Image analysis and densitometry was performed using ImageJ software. Statistical significance is denoted by NVC vs. HVC ($p < 0.05, *p ≤ 0.005) and HVC vs. HS1P (@p < 0.05, #p ≤ 0.005) at respective time points