Figure 4

DUOXA1 knockdown decreases H ₂ O ₂  production and enhances differentiation. Primary myoblasts were subjected to nucleofection using shRNA constructs targeting DUOXA1 (DUOXA1 shRNA) or luciferase (CON shRNA). Twenty-four hours later, growth medium was replaced with differentiation medium, and samples were harvested 48 hours after the initiation of differentiation (day 2). (A) Quantitative RT-PCR was performed on representative samples subjected to DUOXA1 shRNA and CON shRNA. Data was normalized to GAPDH and DUOXA1 shRNA values are presented relative to CON shRNA cells. (B) Confocal immunofluorescence and flow cytometry were used to demonstrate the ability of the DUOXA1 shRNA constructs to reduce DUOXA1 protein levels. Scale bars: 20 μm. (C) Amplex red was used to illustrate that DUOXA1 knockdown reduces the amount of H₂O₂ released by the cells. Levels of H₂O₂ were corrected using cell counts, and the amount of H₂O₂ produced by DUOXA1 shRNA cells are expressed relative to CON shRNA samples. (D) Quantitative RT-PCR was used to confirm significantly higher levels of myogenin, but not MyoD or MyHC, in DUOXA1 shRNA samples. (E-G) Immunostaining on differentiating samples suggests that the number of Myogenin⁺ cells is higher in DUOXA1 shRNA (E) samples than in controls (F). Scale bars: 200 μm. (G) Graphical representation of some of the data in E &F. The number of MyoD⁺ cells was not different between groups. (H) At day 2, enhanced fusion was associated with DUOXA1 knockdown. (I) Annexin staining on samples subjected to DUOXA1 shRNA or CON shRNA suggest that DUOXA1 knockdown results in significantly reduced apoptosis. Values represent means for myoblasts isolated from 3–5 mice SE. * Significantly different from CON shRNA (P < 0.05).