Newly activated satellite cells and primary myoblasts express DUOXA1. (A) Scheme of myogenesis indicating common markers for precursor cells (Pax7), myoblast commitment (Myf5, MyoD), early differentiation (myogenin) and late differentiation (Myosin heavy chain - MyHC). Myofibres were isolated and incubated with 10 μM bromodeoxyuridine (BrdU). Samples were cultured for 24 hours, upon which time they were harvested, fixed in 2% paraformaldehyde, and immunostained for DUOXA1 (green) and BrdU (red). Results show evidence of activated DUOXA1+/BrdU+ satellite cells. Scale bar: 20 μm. (B) Myofibre and primary myoblast cultures obtained from the extensor digitorum longus muscles of adult mice demonstrate DUOXA1 expression in newly-activated satellite cells, primary myoblasts and differentiated myotubes. Primary myoblasts which have migrated away from the parent fibre also show extensive cytoplasmic and nuclear staining of DUOXA1 (green) in cells that co-express DUOX1 (red). The nuclei of differentiated myotubes are generally devoid of DUOXA1. White arrows indicate alterations in the localization of DUOXA1 in single cells compared to that in fused myotubes. Counterstaining with DAPI is provided as a nuclear marker. Scale bars: 50 μm. Inset scale bars: 20 μm. (C) The expression of DUOXA1 and DUOX1 (along with Myf5, MyoD, myogenin & MyHC) in myoblast (MB) and myotube (MT) samples was analyzed by quantitative reverse transcription (qRT)-PCR. The levels of mRNA were normalized to GAPDH and presented as values relative to expression at day 0 (d0). (D) DUOXA1 levels in MB and myocyte samples (MC - which had not undergone fusion) were also analyzed by flow cytometry. The normal arrow and broken arrow heads in MC samples indicate populations of cells that have either downregulated or upregulated DUOXA1 expression respectively. (E) Markers of myogenesis were also analyzed by Western blot. * Significantly different from d0 (P < 0.05). mRNA R.L. indicates mRNA relative level.