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. 2019 May 15;14(5):e0216987. doi: 10.1371/journal.pone.0216987

Fig 6. Knockdown of Ninjurin1 impairs myogenic differentiation.

Fig 6

C2C12 myoblasts were transfected with control siRNA (control_siRNA) or siRNA targeting Ninjurin1 (Ninjurin1_siRNA). Differentiation was initiated 24 hours after transfection. (A) RNA was isolated from C2C12 myoblasts and after 7 days of differentiation (MT7). Quantitative RT-PCR analysis of myosin heavy chain (Myh) 1, 2, 4 and 7 is shown. Glyceraldehyde-3 phosphate dehydrogenase (GAPDH) expression was used as reference. Data are presented as mean ± SD. A two-tailed, unpaired Student’s t-test was used to calculate the P values. * P < 0.05, ** P < 0.01, *** P < 0.001. n = 3. (B) Western blots of proteins isolated from C2C12 myoblasts, and for 3, 5 and 7 days differentiated myotubes, as indicated (MT3, MT5, MT7), with anti-Ninjurin1, slow myosin and anti-fast myosin antibody (as indicated). GAPDH was used as loading control. The Ninjurin1 isoforms at 16kDa (Ninjurin1-16) and 24kDa (Ninjurin1-24) are differentially expressed, therefore the membranes underwent short and long exposure, indicated by short exp. and long exp., respectively. The 16kDa (Ninjurin1-16) and 24kDa (Ninjurin1-24) Ninjurin1 isoforms are indicated. (C) Microscopy pictures of differentiated C2C12 myocytes at day 3 (MT3) and 5 (MT5) of differentiation following transfection with control siRNA (control_siRNA) and siRNA targeting Ninjurin1 (Ninjurin1_siRNA), respectively. Scale bar, 500 μm. (D) Western blots of proteins isolated from C2C12 myoblasts, and 7 days differentiated myotubes, treated with cycloheximide (CHX) and MG132 for different time points, as indicated, with anti-Ninjurin1, Myogenin and Herp antibody (as indicated). GAPDH was used as loading control.