O8010

C2C12 cell culture medium was removed from the 24-well cell culture plate and rinsed with PBS twice and fixed with 4% paraformaldehyde for 30 min. The cells were stained with oil red O (Solarbio, O8010, Beijing, China) for 30 min, rinsed with 60% isopropanol and rinsed three times with PBS [20 (link)]. Finally, samples were visualized using a 100X microscope (Olympus, CKX41SF, Tokyo, Japan) to observe and collect images. For the oil red O staining of mouse muscles, the mouse gastrocnemius muscle was first frozen sectioned. The slices were dipped into oil red dye solution for 8-10 min (covered to avoid light). Then the slices were took out, and stayed for 3 s and then immersed in 60% isopropanol 2 times, 3 s and 5 s respectively. The slices were immersed in pure water and soaked twice, each for 10 s. After that, we took out the slices, stayed for 3 s and then immersed in hematoxylin for 3-5 min. Then microscope inspection, image acquisition and analysis were performed.

MSCs were isolated from human umbilical cords following previously described methods after informed consent was obtained according to institutional guidelines under the approved protocol^{17 (link)}. MSCs were cultured in DMEM/F-12 (#11320033, GIBCO BRL, Grand Island, NY, USA) containing 10% foetal bovine serum (#10099141C, GIBCO BRL) and 1% penicillin/streptomycin (#15240062, GIBCO BRL). MSCs between passages 5–8 were used for subsequent experiments. To confirm the identity of the cells, the phenotypic profile of MSCs was evaluated by flow cytometry analysis using PE-labeled human anti-CD29 (#102216, Biolegend, San Diego, CA, USA), anti-CD44 (#338804, Biolegend), anti-CD90 (#328109, Biolegend), anti-CD11b (#101210, Biolegend) or anti-CD45 antibody (#103106, Biolegend). The morphology was observed by inverted microscopy. MSCs were cultured in MSC osteogenic differentiation medium for differentiation, and differentiated cells were identified by Alizarin Red staining (#G8550, Solarbio, Beijing, China) and Oil Red O staining (#O8010, Solarbio).