HGMSCs were cultivated under standard conditions (37 °C, 95% air: 5% CO2 v/v) in α-Minimum Essential Medium Eagle (α-MEM, Cod. M8042, Sigma-Aldrich, St. Luis, MO, USA) supplemented with 10% heat-inactivated Fetal Bovine Serum (FBS, cod. ECS0180L; Euroclone S.p.A., Milan, Italy), 2 mM L-glutamine (cod. G7513, Sigma-Aldrich) and 1% w/v of Penicillin/Streptomycin (cod. P0781, Sigma-Aldrich). For osteogenic differentiation, cells were incubated up to 21 days in α-MEM supplemented with 50 μg/mL of L-ascorbic acid 2-phosphate (Cod. 49,752, Sigma-Aldrich), 100 nM of dexamethasone (Cod. D1756, Sigma-Aldrich), and 10 mM of β-glycerophosphate (Cod. G9422, Sigma-Aldrich) (referred to as ‘differentiation medium’) [30 (link)]. Differentiation medium was replaced twice or thrice a week by adding all previous reagents. Resveratrol (RV, Cod. R5010, Sigma-Aldrich) was added to the standard or differentiation medium as indicated. Where reported, 5 μM spautin-1 (Sp1, Cod. SML0440, Sigma-Aldrich) was added to the culture medium.
HGMSCs were seeded on 35 mm Petri dishes at 80.000 cells per dish or on sterile glass coverslips at 10.000 cells per dish for western blot and immunofluorescence analysis, respectively. For histochemical staining with Alizarin Red Staining, the cells were cultured on 24-well plates at 20.000 cells per well and let adhere 24 h before treatments.
HGMSCs were seeded on 35 mm Petri dishes at 80.000 cells per dish or on sterile glass coverslips at 10.000 cells per dish for western blot and immunofluorescence analysis, respectively. For histochemical staining with Alizarin Red Staining, the cells were cultured on 24-well plates at 20.000 cells per well and let adhere 24 h before treatments.