Fig. 2.

Increased myoblast proliferation rate in 5% compared to 20% oxygen culture conditions. For all graphs, 5% oxygen culture conditions are represented by black bars and 20% oxygen culture conditions are represented by white bars. a *With time there was an increase in cell number in proliferating myoblast cultures, reaching significance at 6 days post-seeding (p < 0.05; main effect time). ^†Low oxygen culture conditions enhanced myoblast proliferation, such that cell numbers were greater in 5% compared to 20% oxygen (p < 0.05; main effect oxygen). b Representative images showing greater myoblast proliferation in 5% than 20% oxygen at 4 days post-seeding; red scale bar represents 100 μm. c Percentage of myoblasts in the G1/G0, S (DNA synthesis) and G2/M phase of the cell cycle as measured by BrdU incorporation and FACS analysis. *At 3 days post-seeding, there were more cells in S phase in 5% than 20% oxygen (p < 0.05; paired t-test) and this was associated with a non-significant reduction in the proportion of cells in the G2/M phase. Oxygen had no effect on the proportion of myoblasts in the G1/G0 phase. d Representative FACS graphs, obtained 3 days post-seeding, showing a greater percentage of cells gated in the S phase of the cell cycle at 5% than 20% oxygen. e *At 4 days post-seeding, mRNA levels of the negative cell cycle regulator p21^Cip1 were higher in 20% compared to 5% oxygen (p < 0.05; paired t-test). Cyclin A2, CDK6 and p27^Kip1 gene expression was not significantly affected by cell culture oxygen levels. f In concordance with the gene expression results, qualitative Western blot data indicate that p21^Cip1 protein levels were increased ~1.5-fold in proliferating myoblasts cultured in 20 versus 5% oxygen; whereas CDK6 protein levels were unaffected by oxygen. To support the graphed group data, note the representative lanes from the p21^Cip1 and CDK6 western blots from myoblasts cultured in 5 and 20% oxygen from two different subjects. (please refer to online manuscript for colour images)