Fig. 6

Nedd4-1 ablation results in decreased expansion and differentiation of myogenic progenitors in vitro. A Representative images of primary myoblasts colonies by phase contrast microscopy from SC-N4wt and SC-N4KO mice, maintained in proliferation conditions for 48 h. Quantification shows the average number of cells per colony (n = 3, **P < 0.01). Scale bar = 100 μm. B Primary myoblasts isolated from SC-N4wt and SC-N4KO mice and maintained in proliferation conditions were labeled with EdU for 6 h prior to fixation. EdU (magenta) incorporation and PAX7 (green) or MYOD (red) expression were analyzed by IF. Quantification shows the average number of myogenic EdU(+) cells, normalized by the total number of cells, in each condition; n = 3, *P < 0.05. Scale bar = 100 μm. C Single myofiber-associated SCs were transfected upon isolation with control (siScramble) or Nedd4-1 targeting siRNA (siNedd4-1) for 24 h prior to fixation and IF. Transfection was monitored by co-transfection with a FAM-labeled non-targeting siRNA (FAM; green). SYNDECAN-4 (Sdc-4; blue), NEDD4-1 (red, upper panel), and MYOD (red, lower panel) were detected by IF. Quantification shows the percentage of MYOD(+)/SDC-4(+)/FAM(+) cells in each condition. n = 3. D Primary myoblasts obtained and treated as in (I) were maintained in differentiating culture conditions for 72 h. NEDD4-1 and MYOGENIN (Myog) expression were analyzed by IF (green and red, respectively). Nuclei were marked with DAPI staining. Dotted lines trace the periphery of multinucleated cells identified by phase contrast. Quantification shows the average myotube diameter in each condition. n = 3, **P < 0.01