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. 2021 Sep 1;22(17):9509. doi: 10.3390/ijms22179509

Figure 3.

Figure 3

Neddylation inhibition mediated by knockout of NAE1 inhibits C2C12 differentiation. C2C12 cells were infected with lentiviruses expressing gRNAs (gRNA1 and gRNA2) against murine Nae1, respectively, to generate bulk cultures of cells with NAE1 knockout (KO1 and KO2). Cells infected with lentiviruses expressing no gRNA were used as controls (Ctrl). (A) Western blot analysis of NAE1, neddylated Cullins (N8-CULs), CUL1, myogenic transcription factors, and muscle differentiation markers during the differentiation. Arrow indicates neddylated CUL1. (B) Quantifications of protein expressions as normalized to TUBA1A at D6. Data were presented as fold changes with Ctrl set at 1. (C) qRT-PCR analysis of Myog gene expression at D0, D3, and D6 of myoblast differentiation. (D) Representative bright-field and immunofluorescence images at 6 days in differentiation medium. Cells were stained with MyHC (red), and nuclei were counterstained with DAPI (blue). Scale bar: 200 μm. * p < 0.05; ** p < 0.01 vs. Ctrl. One-way ANOVA. Three independent experiments.