*2.1. Transfection with Specific siRNAs Targeting COX-1 or -2 Significantly Reduce the Expression Levels of COXs*

Two siRNAs specific for each COX were transfected into primary myoblasts. Forty-eight hours after transfection, the total RNA was collected for quantitative RT-PCR to determine the changes in COX expression level. For each gene, both siRNAs efficiently decreased gene expression (Figure 1A,B). Since the siRNA-2 of COX-1 and -2 had higher levels of knockdown efficiencies, resulting in 97.2% and 79.3% downregulation of COX-1 and -2, respectively, compared with negative control (NC), they were used for all remaining experiments. In addition, the protein levels of COX-1 and -2 were shown around 55% reduction at 48 h post transfection with COX-1 or -2 siRNA (Figure 1C–F). Completed Western blot images are shown in supplementary Figure S1.

After 48 h transfection with COX-1 or -2 siRNA, significant morphological changes were observed in myotubes (Figure 1G). Quantified myogenic differentiation data showed that fusion index was reduced from 79.6% (NC) to 49% (COX-1 siRNA) and 45.4% (COX-2 siRNA), respectively (Figure 1H).

**Figure 1.** Verification of the high efficiency of COX-1 and COX-2 siRNA knockdown. (**A**) Knockdown efficiency of siRNAs targeting COX-1; (**B**) knockdown efficiency of siRNAs targeting COX-2; (**C**) COX-1 Western blot results after siRNA transfection for 48 h; (**D**) quantification of COX-1 Western blot results using ImageJ; (**E**) COX-2 Western blot results after siRNA transfection for 48 h; (**F**) quantification of COX-2 Western blot results using ImageJ; and (**G**) both COX-1 and COX-2 siRNA transfections inhibit primary myoblast myogenic differentiation. Morphological phenotypes observed after transfections with siRNAs. a: Negative control; b: COX-1 siRNA; and c: COX-2 siRNA. (**H**) Treatments with siRNAs significantly reduces fusion index. *n* = 3–4, \*\* *p* < 0.01 compared with NC.
