*4.7. Intracellular Ca2*<sup>+</sup> *Measurements*

Intracellular Ca2<sup>+</sup> measurements were performed as previously described [58]. A Photon Technology International (PTI) imaging system was used to measure intracellular Ca2<sup>+</sup> homeostasis. Myotubes imaged were loaded with 2 μM Fura-2 AM (Thermo Fisher Scientific) for 30 min at 37 ◦C, followed by RT de-esterification for 15 min. Only cells that had an initial ratio below 1.0, indicating healthy and not Ca2<sup>+</sup>-overloaded myotubes, were selected for application of 20 mM caffeine (Thermo Fisher Scientific) with a perfusion system (Bioscience Tools, San Diego, CA, USA). Ratiometric analysis (350/375 nm excitation ratio; 510 nm emission) was performed using software PTI EasyRatioPro 2 (HORIBA, Edison, NJ, USA). These experiments were repeated 5 times and at least 6 myotubes were tested on each experiment.

#### *4.8. Statistical Analysis*

Three to five independent replicates were performed for each experiment, except for the customized gene array study. One-way ANOVA with post hoc Tukey's test was performed for data analysis. Results were expressed as mean ± SD. Differences were considered significant at *p* < 0.05.

#### **5. Conclusions**

In conclusion, by using lipidomic analysis, our data have provided new insights regarding the functions of COXs in skeletal muscle. COX-1 may play a major role in the step from myoblast to fusion in myogenic differentiation. Its effect could also be related with the alteration in AA/LOX pathway. Further studies on the lipid mediators from AA/COX pathway, and the interactions between COXs

and LOXs will advance the knowledge of COX and related lipid signaling in skeletal muscle and other tissues, which could benefit the development of new treatments for inflammation related diseases in skeletal muscle and other tissues.

#### **Supplementary Materials:** Supplementary materials can be found at http://www.mdpi.com/1422-0067/20/18/ 4326/s1.

**Author Contributions:** For Conceptualization, C.M. and M.B.; methodology, C.M., Z.W., and M.B.; validation, C.M., Z.W. and M.B.; formal analysis, C.M., Z.W. and M.B.; investigation, C.M. and Z.W.; resources, L.B. and M.B.; data curation, C.M. and Z.W.; writing—original draft preparation, C.M. and Z.W.; writing—review and editing, L.B. and M.B.; supervision, M.B.; project administration, M.B.; funding acquisition, L.B. and M.B.

**Funding:** This work was supported by NIH-National Institutes of Aging PO1 AG039355 (L.B., M.B.), and the George W. and Hazel M. Jay and Evanston Research Endowments (M.B.). C.M. and Z.W. were partially supported by grants from National Institutes of Aging R01AG056504 and R01 AG060341 (M.B.) and the National Institutes of Diabetes, Digestive, and Kidney Diseases (M.B.).

**Acknowledgments:** We thank Yating Du for her assistance in the experiments, and we are grateful for instrumentation support from Shimadzu Scientific Instruments, Inc.

**Conflicts of Interest:** The authors declare no conflict of interest.
