C2C12 differentiation assay

8000 cells were inoculated in a well of 96 well plate and cultured in growth medium. The next day, medium was replaced with DMEM supplemented with 2% FBS or 5% FBS (myotube differentiation medium) for myotube differentiation. After 4 days, cells were fixed and stained with anti-MyHC antibody (Developmental Studies Hybridoma Bank; cat. no. MF20) and anti-mouse IgG Alexa Fluor 488 or Alexa Fluor 555 (Life Technologies; cat. no. A-11029 or A-21424) as described in [17]. Nuclei were stained with Hoechst33258 (Life Technologies; cat. no. H3569). Myotube index was calculated as percentage of nuclei in MyHC positive myotube that had more than 2 nuclei. For quantification, at least five random fields were imaged with EVOS FL Cell Imaging System (Life Technologies). For osteoblast differentiation, growth medium was replaced with DMEM supplemented with 2% FBS containing 75 ng/ml or 100 ng/ml human recombinant BMP-4 (R&D Systems; cat. no. 314-BP-010) (osteoblast differentiation medium). After 3 days, alkaline phosphatase (ALP) activity was detected with luminescent ALP assay kit (Anaspec; cat. no. AS-72122) following the manufacturer’s protocol. Cells in the 96 well plate were lysed with 50 µl lysis buffer for 3 min under agitation and centrifuged at 2,500 g for 5 min at 4 °C. The supernatant was collected and combined with the same volume of chemiluminescent substrate and incubated for 15 min at 4 °C in the dark. Luminescence was measured using a VICTOR3 plate reader (PerkinElmer) and ALP activity was calculated by using ALP standard. Protein concentration was determined by DC Protein Assay Kit (BIO-RAD; cat. no. 5000112) and ALP activity was normalized against protein concentration.