Fig 2: Neddylation is differentially regulated during C2C12 differentiation. (A–C) Western blot of the indicated proteins (A), and quantifications of NAE1, UBC12, and neddylated Cullins (N8-CULs) (B); quantifications of MYOD1, MYOGENIN, and MYOSIN (skeletal, fast) (MyHC) (C) in proliferative C2C12 cells cultured in growth media (i.e., day 0, D0) and for the indicated days after differentiation induction (D1 to D5). Proteins were quantified and normalized to ACTB. Data were presented as fold changes with D0 cells set at 1. N8-Conj.: NEDD8-conjugated. Arrows indicate neddylated CULs and CUL1. (D) Representative immunofluorescence images of C2C12 cells stained with NEDD8 (green) in growth media (D0) and for 3 and 6 days in differentiation media (D3, D6). Nuclei were counterstained with Hoechst 33342. Scale bars: 50 μm. ** p < 0.005 versus D0. One-way ANOVA. Three independent experiments.

Fig 3: Neddylation inhibition dysregulates SIRT1 intracellular localization. (A) Representative immunofluorescence images of Ctrl and NAE1-KO (KO1, KO2) C2C12 cells at 3 days in differentiation media. Cells were stained with SIRT1 (red), and nuclei were labeled with Hoechst 33342 (blue). Scale bar: 20 μm. (B) Western blots of whole cell lysates (WCL), cytosol, and nucleus fractions of Ctrl and NAE1-KO (KO1 and KO2) C2C12 cells at 3 days in differentiation media. GAPDH and LMNB1 were used as cytosol and nucleus marker proteins, respectively. (C) Quantifications of nuclear fraction of SIRT1 as normalized to LMNB1 with Ctrl set as 1. * p < 0.05 vs. Ctrl. One-way ANOVA. Three independent experiments.

Fig 4: Neddylation inhibition mediated by a specific NAE1 inhibitor MLN4924 blocks C2C12 myotube formation. (A,B) Western blot analysis of neddylated Cullins (N8-CULs), CUL1, and muscle differentiation markers in vehicle (Veh)-treated and MLN4924 (MLN, 0.5 μM)-treated C2C12 for 6 days (D6) in differentiation media. Proteins were quantified and normalized to TUBA1A. Arrow indicates neddylated CUL1. Data were presented as fold changes with Veh-treated cells set at 1. * p < 0.05; ** p < 0.005 vs. Veh. Unpaired t tests. (C) Representative bright-field and immunofluorescence images of Veh-treated and MLN-treated C2C12 at D6 of differentiation. Cells were stained with MyHC (red), and nuclei were counterstained with DAPI (blue). Scale bar: 200 μm. (D) Representative bright-field images of Veh- and MLN-treated C2C12 cells at D6 in differentiation media with insulin. MLN at indicated doses was only treated for the first 2 days of differentiation. Scale bar: 200 μm. Three independent experiments.

Fig 5: NAE1 and Ube2M levels are elevated in OS tumor tissuesLevels of NAE1 and Ube2M in de-paraffinized sections of OS tumor tissues and of normal bone tissues were examined by immunohistological staining. Representative NAE1 staining in OS tumor tissue a. and in normal bone tissue b. are shown. Representative Ube2M staining in OS tumor tissue c. and in normal bone tissue d. are shown.