STEM CELLS – Wiley Online Library

Pax7 stability is regulated by the proteasome during myogenesis. (A): Proteasome inhibition results in myogenin (Myog) and Pax7 coexpression (arrows) in adult mouse primary myoblasts (pMbs). Syndecan-4 (Sdc-4) expression was used as lineage marker. Scale bar=10 m. Right panel: quantification (three separate experiments; [meanSEM]) of percentage of Myog(+)/Pax7(+) cells from the total Sdc-4(+) population; Student's t-test; *, p <0.005. (B): Western blot analysis of Pax7 and myogenin expression in 48 hours cultured pMbs treated as in (A). (C): Pax7 levels are significantly increased upon proteasome inhibition during differentiation commitment in C2C12 myoblasts. Myogenin (Myog): differentiation marker, tubulin (Tub): loading control. Right panel: quantification of Pax7 fold change (meanSEM) of three separate experiments. One-way analysis of variance (ANOVA); *, p<0.005. (D): Epoxomicin-mediated proteasome inhibition recapitulates Pax7 accumulation in differentiating C2C12 myoblasts. MG132 effect is shown as positive control. Right panel: quantification of Pax7 fold change (meanSEM) of three separate experiments for 1 M epoxomicin and 25 M MG132 treatments. One-way ANOVA; * and **p<0.005. (E): Proteasome inhibition results in Pax7 nuclear accumulation. Pax7 expression was determined by Western blot in cytoplasmic and nuclear fractions: (tubulin: cytoplasmic marker; histone deacetylase 2 [HDAC2]: nuclear marker), Right panel: quantification of Pax7 protein levels normalized to HDAC2 (meanSEM) of three separate experiments. Student's t-test; *, p<0.005. MG132 was used at 25 M in (B), (C), and (E). Abbreviations: DMSO, dimethyl sulfoxide; HDAC2, histone deacetylase 2.

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