Hnmpa

Cells were plated in muscle growth media (fetal bovine serum 0.05 mL/mL, fetuin 50 μg/mL, epidermal growth factor 10 ng/mL, basic fibroblast growth factor 1 ng/mL, insulin 10 μg/mL, and dexamethasone 0.4 μg/mL) for 48 hours before changing to skeletal muscle differentiation media, which is a proprietary serum-free medium containing 10 μg/mL insulin (PromoCell) for 14 days. PMSCs were treated every 3 days with 200 nM of IGF-IR inhibitor PPP, 25 μM of AKT inhibitor LY294002, 10 μM of MEK1/2 inhibitor U0126, or 10 μM of IR inhibitor HNMPA (Santa Cruz Biotechnology) under muscle differentiation conditions. Treatment concentrations for LY294002, U0126, and HNMPA were determined by a dose-response experiment using PMSCs in muscle differentiation media (Supplementary Figure 1). For IGFBP-6 supplementation with the inhibitors, recombinant human IGFBP-6 (ProSpec) was added to the media (375 ng/mL) every 3 days at the time of media change. The dose of IGFBP-6 was based on our previous studies [26 (link), 31 (link)].

Recombinant human neuritin was purchased from Pepro Tech (Rocky Hill, NJ). Poly-l-lysine, triethanolamine, tetrodotoxin, CsA, ascomycin (FK520), and CMZ were obtained from Sigma. FBS, DMEM, and antibiotic-antimycotic solution were purchased from Invitrogen, Thermo Fisher Scientific. HNMPA was purchased from Santa Cruz Biotechnology (Dallas, TX).