NOMO is required for myogenesis and depends on the Ig 1/10/11 interface. (A) Cartoon representation of C2C12 myogenesis, whereby myoblasts exit the cell cycle to differentiate into myocytes, which then fuse to form multinucleated myotubes expressing Myhc. (B) Fixed myotubes stained for Myhc and DAPI (DNA) 3 days after differentiation induction; left scale bars, 50 μm; right scale bars, 10 μm. (C) Electron micrograph of cells treated with either non-targeting (Ctrl) or Nomo siRNA (siNomo) during myoblast differentiation. (D) Representative images of the differentiation assay performed as in A, with myoblasts stably expressing siRNA-resistant F-NOMOr (WT) or F-NOMOr4-Mut (4-Mut) constructs under Nomo knockdown. (E) Myogenesis quantification of conditions shown in B and C, scored by the mean fraction of 3+ nuclei in Myhc-expressing myotubes (fusion index). N = 21 frames, 2,788 nuclei (Ctrl), N = 21 frames, 2,677 nuclei (siNomo), N = 16 frames, 1,957 nuclei (siNomo, WT rescue), and N = 18 frames, 2,457 nuclei (siNomo, 4-Mut rescue) from n = 3 biological replicates. Statistical analyses were performed using an ordinary one-way ANOVA test; ****P < 0.0001, **P < 0.01, and *P < 0.05. Source numerical data are available in source data.
Figure 7.

NOMO is required for myogenesis and depends on the Ig 1/10/11 interface. (A) Cartoon representation of C2C12 myogenesis, whereby myoblasts exit the cell cycle to differentiate into myocytes, which then fuse to form multinucleated myotubes expressing Myhc. (B) Fixed myotubes stained for Myhc and DAPI (DNA) 3 days after differentiation induction; left scale bars, 50 μm; right scale bars, 10 μm. (C) Electron micrograph of cells treated with either non-targeting (Ctrl) or Nomo siRNA (siNomo) during myoblast differentiation. (D) Representative images of the differentiation assay performed as in A, with myoblasts stably expressing siRNA-resistant F-NOMOr (WT) or F-NOMOr4-Mut (4-Mut) constructs under Nomo knockdown. (E) Myogenesis quantification of conditions shown in B and C, scored by the mean fraction of 3+ nuclei in Myhc-expressing myotubes (fusion index). N = 21 frames, 2,788 nuclei (Ctrl), N = 21 frames, 2,677 nuclei (siNomo), N = 16 frames, 1,957 nuclei (siNomo, WT rescue), and N = 18 frames, 2,457 nuclei (siNomo, 4-Mut rescue) from n = 3 biological replicates. Statistical analyses were performed using an ordinary one-way ANOVA test; ****P < 0.0001, **P < 0.01, and *P < 0.05. Source numerical data are available in source data.

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