## 3 Results
### The Effect of Hypoxia on Myogenic Relative Markers Expression  
Having demonstrated that hypoxia enhances cell viability even under low cell numbers, in order to examine whether hypoxia modulates the myogenic differentiation, the expression of Myo, myogenin, NOS1, VEGF, mTOR and MHC7 was quantified after 3 h, 72 h, and 7 days of differentiation. The data analysis revealed that MyoD expression was upregulated at 72 h under normoxia (_p_ < 0.05) compared to both the non-induced cells in basal medium (_p_ < 0.05) and matched condition after 3 h (Figure 5a). Additionally, the quantification of the differentiation markers showed an upregulated myogenic expression at day 7 (_p_ < 0.05) compared to non-induced cells for both experimental groups regardless of the effect of hypoxia (Figure 5b). The data analysis demonstrated an upregulated NOS1 at day 7 under normoxia (_p_ < 0.01) compared to matched conditions at 3 h and 72 h. Moreover, the data showed an upregulated NOS1 expression at day 7 under normoxia (_p_ < 0.01) compared to the cells cultivated under hypoxia and non-induced cells at the same time point (Figure 5c). It is well established that ischemic conditions stimulate an alternative vascularization, and thus we examined whether in vitro hypoxia induction modulates angiogenesis. Indeed, the data analysis exhibited an upregulated VEGF expression after 72 h under hypoxia compared to non-induced cells in basal medium (_p_ < 0.05). Additionally, enhanced VEGF expression was observed (_p_ < 0.05) when comparing 72 h with matched conditions after 3 h (Figure 5d). To examine whether hypoxia modulates the late myogenic markers relative to myotube maturation, the relative expression of mTOR, MHC7, and MHC1 was quantified. The data analysis showed that mTOR expression was upregulated after 72 h under normoxia (_p_ < 0.05) compared to both the non-induced cells in the basal medium and the cells cultivated under hypoxia. Similarly, mTOR expression was upregulated after 72 h under normoxia (_p_ < 0.05) compared to the matched condition after 3 h (Figure 5e). The data analysis revealed a late upregulation of MHC7 expression after day 7 under hypoxia (_p_ < 0.05) compared to either non-induced cells in basal medium or the early time point after 3 h (Figure 5f). Surprisingly, the expression of HIF1\(\alpha\) showed a discrepancy between 3 h, 72 h, and day 7 for cells cultivated under normoxia and hypoxia that was not statistically identified.  
Figure 4: Effect of low oxygen concentration on the physiological parameters of muscle precursors after 3h, 24 h, and 72 h under normoxic (NX) and hypoxic conditions (HX). (**a**) Semi-quantitative analysis of cell viability (\(n\) = 6) at 570 nm absorbance using the MTT assay. (**b**) Semi-quantitative analysis of total cell protein contents indicative of cell number (\(n\) = 6) at 565 nm absorbance using SRB assay. (**c**) Percentage of MyoD-positive cells after 24 h. (**d**) Percentage of myogenin-positive cells after 72 h. (**e**–**h**) Immunofluorescence shows myogenin-positive cells (red) after 72 h and (**e**,**f**) myosin heavy chain-positive myotubes after 7 days (**g**,**h**) under NX and HX conditions. (**i**,**j**) Colony formation unit assay for C2C12 cultivated under normoxic and hypoxic conditions in a growth medium for 7 days (\(n\) = 6). (**i**) Number of colonies (\(n\)) at 1 \(\times\) 10\({}^{2}\) cell density. (**j**) Average colony size (\(\mu\)m\({}^{2}\)) at 1 \(\times\) 10\({}^{2}\) cell density. Data are presented as means \(\pm\) SEM. * = \(p\) < 0.05, ** = \(p\) < 0.01, *** = \(p\) < 0.001. DAPI was used as a nuclear stain (blue). Scale bar = 10 \(\mu\)M.