Ap live staining

To quantify hMSCs osteogenic differentiation, alkaline phosphatase enzyme activities were evaluated and measured by using two ALP staining assays, AP live staining (ThermoFisher) and ALP staining kit (for fixed cells, Sigma-Aldrich). For fixed cells, the staining solution was first prepared by mixing Fast Red Violet solution, Naphthol AS-BI phosphate solution and water at a ratio of 2:1:1. Next, hMSCs were fixed using 4% cold- Paraformaldehyde (PFA) for 2 min which enable the maintenance of the ALP activation. After fixation, the PFA was aspirated without wash. The staining solution was then added to the fixed cells for 15 min under room temperature and protected from light. The cells were then washed three times with 1x PBS, 15 min each time, before taking images. For AP live staining, hMSCs were stained using AP live stain at the concentration of 10X stock solution for 30 min according to manufacturers’ instructions. After staining, hMSCs were washed twice using basal medium. Images were captured after 30 min of staining. For F-actin staining, hMSCs were first fixed with 4% PFA solution for 10 min before being permeabilized and blocked with the PBST solution (PBS + 0.5% Triton + 1% BSA) for 1 h. After wash with 1x PBS three times, hMSCs were incubated with phalloidin (1:30) for 1 h at room temperature. The cells were then washed three times using 1x PBS, before imaging.