Fig. 3. ASPA-mediated catabolism is responsible for NAA effects in mytubes.

A Bioinformatic analysis of ASPA expression in EDL and soleus muscles. Data are expressed as Log₂(fold change) ± SD (*p < 0.05 EDL vs soleus). B Bioinformatic analysis of GO of transcripts that correlate with ASPA expression in muscle. C RT-qPCR analysis of ASPA expression in C2C12 cells. ACTB was used as a reference control. Data are shown as fold change ± SD of n = 3 independent experiments (* p < 0.05 vs CTRL). Western blot analysis of ASPA levels on D C2C12 myotubes treated with NAA for 48 h and on E differentiated ASPA KO C2C12 cells. The Western blots reported are representative of three independent experiments that gave similar results. F Diameter measurement of C2C12 ASPA KO cells treated with NAA. Data are expressed as mean ± SD of n = 3 independent experiments (*p < 0.05 vs CTRL). G Representative images of C2C12 ASPA KO myotubes after staining with Oil Red O. Scale bars, 50 µm. (n = 3). H The ratio of MFI of cells untreated and treated with ATGListatin. Data are expressed as mean ± SD of n = 3 independent experiments (***p < 0.001 vs untreated). I Evaluation of extracellular lactate content in C2C12 ASPA KO cells. Lactate concentration was normalized on total proteins and data are expressed as mean ± SD of n = 3 independent experiments (*p < 0.05 vs CTRL Cas9).