Stempro osteogenesis differentiation kit medium

hMSC were seeded at 5 × 10³ cells/cm² in α-MEM. After 24 h, cells were approximately 80% confluent and osteogenic differentiation was initiated by switching to commercially available Stempro^® Osteogenesis Differentiation kit medium (Invitrogen) or osteogenic differentiation medium consisting of α-MEM, 15% FBS, 100 μM ascorbic acid (Scientific Lab Supplies, UK), 2.5 mM β-glycerophosphate (Calbiochem) and 100 nM dexamethasone (Sigma). For experiments on undifferentiated hMSC, cells were grown in α-MEM supplemented with 10% heat-inactivated foetal bovine serum (FBS, Sigma), 1000 U/mL penicillin, and 10 mg/mL streptomycin (Sigma). Medium was changed every 3–4 days, and cells were used until passage 8 only. VCP/p97 and the proteasome were inhibited with DBeQ (Biovision) or bortezomib (Calbiochem). Tunicamycin was purchased from Sigma.