DUOXA1 overexpression in primary myoblasts inhibits differentiation. Primary myoblasts were infected with adenoviral vectors containing pCMV5-GFP (GFP), or pCMV5-DUOXA1-GFP (GFP-DUOXA1). (A) Cells were subjected to adenoviral constructs and, 24 hours later, growth medium (GM) was replaced with differentiation medium (DM - designated as day 0). mRNA was harvested 24 hours after the initiation of differentiation (day 1). Quantitative RT-PCR was performed on representative samples from DUOXA1 overexpressing cells and GFP controls. Data was normalized to GAPDH and GFP-DUOXA1 values are presented relative to GFP control cells. MyoD, myogenin and MyHC mRNA levels are all significantly reduced in samples overexpressing DUOXA1. (B-H) Immunostaining on samples harvested on day 2 suggests the number of GFP+/myogenin+ and GFP+/MyHC+ cells are clearly reduced in DUOXA1 overexpressing cells (E, G, H) compared to GFP controls (D, F, H). Numbers of GFP+/MyoD+ cells are not different between samples (B, C, H). Counterstaining with DAPI is provided as a nuclear marker. Scale bars: 100 μm. (H) Graphical representation of the data shown in B-G. GFP-DUOXA1 counts are represented relative to GFP controls. (I) The ability of cells overexpressing DUOXA1 to fuse is also impaired compared to GFP control cells collected at day 2. (J) In order to assess the amount of H2O2 released from the cells, the amplex red reagent was used to demonstrate that DUOXA1 overexpression results in significant increases in H2O2 in the culture medium. Levels of H2O2 were corrected using cell counts and the amount of H2O2 in DUOXA1 overexpressing cells are expressed relative to GFP controls. Values represent means from myoblasts isolated from 3–5 mice ± SE. * Significantly different from GFP control (P < 0.05).