Nuclear Exclusion of Forkhead Box O and Elk1 and Activation of Nuclear Factor-B Are Required for C2C12-RasV12C40 Myoblast Differentiation

Supplemental Data

Supplemental Data

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• Supplemental Figure S1 - Functional characterization of H-Ras mutants. A, COS1 cells were transiently cotransfected with 1μg of p42MAPK in pCEFL-KZ-HA and 1 μg of each H-Ras, corresponding to the hyperactive form (V12) or the effector dominant mutant (V12C40) in pCEFL-KZ-AU5. As negative control cells were transfected with the vector (pCEFL-KZ-AU5). All cells were serum-starved for 18 h and then analyzed, as positive control cells transfected with the vector were stimulated with 100 ng/ml EGF for 10 min. The histogram represents p42MAPK relative activity, expressed as fold induction of P32-MBP normalized with respect to control cells and are means ± SEM (n=4). B, COS1 cells were transiently cotransfected with 1 μg of AKT in pCEFL-KZ-HA and 1 μg of each H-Ras mutants. As negative control the cells were transfected with the vector (pCEFL-KZ-AU5). All cells were serum-starved for 18 h and then analyzed, as positive control cells transfected with the vector were stimulated with EGF for 10 min. The histogram represents AKT relative activity, expressed as fold induction of P32-H2B normalized with respect to control cells and are means ± SEM (n=4). C, COS1 cells were transiently transfected with 1 μg of each H-Ras mutants and the vector as negative control. All cells were serum-starved for 18 h and then analyzed, as positive control cells transfected with the vector were stimulated with EGF for 10 min. Ral A-GTP was recovered from cell lysates by binding to immobilized GST-Ral-BD and detected by immunoblotting with anti-Ral A monoclonal antibody. The expression levels of the transfected AU5-Ras constructs and the endogenous Ral A were detected by immunoblotting of the cell extracts with the corresponding anti-AU5 and anti-Ral A monoclonal antibody, respectively. Results presented correspond to a representative experiment, and similar results were obtained in three additional, separated experiments. Histograms represent the Ral A-GTP levels normalized (by GelWoks Analyses) to Ral A levels and correspond to means ± SEM (n=4). D) NIH-3T3 fibroblasts were transfected with 50 ng of each H-Ras mutants and vector. After 14 days the dishes were stained with Giemsa to score the transformed foci. All plasmids DNAs produced similar number of marker-selectable colonies. Results presented correspond to a representative experiment, and similar results were obtained in three additional, separated experiments performed in duplicate.
• Supplemental Figure S2 - Study of myogenic differentiation in a second clone from C2C12 myoblasts that overexpress different H-ras mutants. Myoblasts were cultured as described in Fig. 2. A, Phase-contrast images of the cells for detection of multinucleated myotubes. Magnification, X10. B, At different days of culture (0-4) cells were lysed and total protein was submitted to SDS-PAGE and immunodetected with anti- (MHC, Caveolin-3, p21^CIP or PCNA) antibodies, with anti-phospho- (AKT and p38MAPK) antibodies, and with anti-phospho- and anti-total- p44/p42MAPK. β-actin expression was used for protein loading control. Representative experiments out of two are shown.