DUOXA1 inhibits myogenesis through a mechanism involving DUOX1 and ASK1. Small interfering RNAs targeting DUOX1 (DUOX1 siRNA), ASK1 (ASK1 siRNA) or a scrambled control (CON siRNA) were introduced into myoblasts using nucleofection. After 24 hrs, samples were subjected to adenoviral vectors containing GFP-DUOXA1 (DUOXA1) or GFP. (A) Quantitative RT-PCR was performed on samples harvested on day 1. Data was normalized to GAPDH and DUOXA1 values are presented relative to corresponding GFP controls. Reductions in the levels of myogenin and MyHC mRNA associated with DUOXA1 overexpression are alleviated upon knockdown of DUOX1 or ASK1. (B) Images demonstrating the ability of ASK1 siRNA to rescue the DUOXA1 phenotype. GFP is visualized in samples. Scale bars: 500 μm. (C) Graphical representation of cell counts. Confocal immunofluorescence was performed on samples harvested at day 1, and the number of cells expressing Myogenin and MyHC were counted. The number of MyHC+ cells was reduced in samples subjected to CON siRNA and DUOXA1 overexperssion compared to CON siRNA and GFP controls. This was alleviated by ASK1 knockdown. Counts are represented relative to their corresponding controls (e.g. DUOX1si-DUOXA1 relative to DUOX1si-GFP). The large number of Annexin-V+ cells witnessed upon DUOXA1 overexpression and defects in cell fusion can be reversed with siRNAs targeting DUOX1 and/or ASK1. (D-F) To determine whether DUOX1 or ASK1 knockdown alone would have an effect on differentiation, we subjected samples to DUOX1 siRNA or ASK1 siRNA and a suitable control (CON siRNA). DUOX1 knockdown enhances fusion (D), but not Myogenin or MyHC mRNA (E) or protein levels (F). ASK1 knockdown had no effect on differentiation. *Significantly different from samples infected with the corresponding GFP control (P < 0.05. # Significantly different from samples nucleofected with a scrambled control (CON siRNA) and infected with a DUOXA1 construct (CONsi-DUOXA1, P < 0.05).