*2.2. The Changes in Levels of Lipid Mediators after Knocking Down COX-1 or -2 Are not Limited to PGs and Thromboxane B2 (TXB2)*

To investigate the mechanisms responsible for the effect of COXs in skeletal muscle myogenesis, we first used our new lipidomics method to directly quantify 14 LMs selected from our preliminary studies, mostly AA metabolites through COX and other enzymes in cell differentiation medium (DM). Compared with blank medium, after differentiation for 72 h, the levels of PGE2, PGF2α, 6-keto-PGF1<sup>α</sup> (stable metabolite of PGI2), DHA, and OEA in the medium increased significantly, suggesting that these LMs were released from myocytes/myotubes. We then further analyzed the effect of COXs on their production. Knocking down COX-1 using siRNA significantly reduced the levels of PGE2 and PGF2<sup>α</sup> compared with NC, but had no significant effect on the levels of 6-keto-PGF1α. At the same time, knocking down COX-2 also showed a similar impact on PGE2 levels, but the effect was significantly less than knocking down COX-1. In addition to changes in LMs in AA pathway, COX-1 knockdown significantly reduced the concentration of DHA and OEA in the DM after 72 h (Figure 2). These data demonstrate that the functions of COXs are not limited to regulating the production of PGs from AA. The whole list of LMs identified in these experiments, including LMs with lower levels after 72 h differentiation compared with blank medium (LMs could be consumed by myocytes/myotubes during differentiation), is summarized in supplementary Figure S2.

**Figure 2.** COX-1 and -2 knockdown reduces the levels of key lipid mediators released by primary muscle cells. (**A**) Absolute quantification of lipid mediators (LMs) released in differentiation medium (DM) from primary mouse myocytes/myotubes during differentiation; (**B**) ratio of LMs released in DM at 72 h post-transfection comparing COX-1 siRNA or COX-2 siRNA treatment with NC transfection. *n* = 3, \* *p* < 0.05 and \*\* *p* < 0.01 compared with NC; # *p* < 0.05 compared with COX-1 siRNA.
