ATP Synthase control transcript levels increase in early differentiation, decrease around D7, and then increase again at D12. This late spike is most likely due to cells obtaining their contractile functions around this time. Surprisingly, fibronectin treatment showed down-regulation on the expression levels compared to the control sample, although there could be spikes during days that were not tested. Gelatin results are interesting due to very high expression levels during D0. Although this was apparent in both trials, an explanation is difficult to arrive at. This could simply be a result of mitochondrial biogenesis being upregulated during gelatin-coated differentiation, or perhaps a more abstract reason which would need to be tested further.

The overall change in MyoD gene expression on both coated plates and on control. Fibronectin protein coating shows a steady increase in MyoD gene expression until day 4. In contrast, both control and gelatin plates display an increase in MyoD gene expression on day 0 (D0) and then a substantial drop (more than 50%) for control and less than 50% drop for gelatin plates on day 4 for MyoD gene expression. All data were normalized to the reference gene, GAPDH. Myoblast was used as a calibrator for each corresponding plate. n = 2 for Myoblast (MB) and D0; n=1 for day 4 (D4).

(TOP)The change in M-cadherin expression from day 0 to 4 saw gelatin coated plates having a higher degree of expression relative to the myoblast stage. In contrast, fibronectin plates saw a dip in expression levels before rising but still being lower than both control and gelatin plates. (BOTTOM) However when the relative expression is calibrated to control myoblast plates, it can be seen that the expression of M-cadherin on fibronectin coated plates sees a nearly 30-fold increase in expression versus control and gelatin plates. This could potentially indicate that peak M-cadherin expression occurs earlier in fibronectin plates than in gelatin or control plates. All data points were normalized to the reference gene, GAPDH. n=2 for Myoblast and D0; n=1 for D4 and D7.

Interestingly, it appears as if fibronectin enhances mitochondrial biogenesis, while 2% gelatin inhibits relative mitochondrial biogenesis (DNM1L) genetic transcription is directly correlated with this process). Expected trends of increased prevalence of mitochondrial biogenesis during myoblast stages qPCR data has been normalized to the relative expression of GAPDH (reference gene) for all samples; r-squared < 0.01, data values calculated using the Pfaffle Method, where respective primer efficiencies (E) are E (GAPDH) ~87%, E (Drp-1) ~70.8%. Error bars are representative of relative standard deviation values; all qPCR experiments were conducted in triplicate.