WST-1 assay to detect the effects of autophagy on hBM-MSC survival. (A) Survival rates of MSC-shRNA3 (autophagy-suppressed MSCs), MSC-Rapa (autophagy-induced MSCs) and the relevant control groups (MSC-shRNA Cont and MSC-Cont) were assayed at daily intervals during a five-day treatment. (B) Both autophagy modulated and control hBM-MSCs were exposed to different H₂O₂ concentrations for 1 hour. The viability of MSC-shRNA 3 was higher than MSC-Rapa and controls (MSC-Cont and MSC-shRNA Cont) at 2-4 mM H₂O₂. (C) After exposing the experimental groups to serum deprivation for different time durations, we found that the suppression of autophagy in MSC-shRNA 3 renders them more resistant to severe SD stress compared to other groups. (D) Following 8 and 24-hour-duration of hypoxia, ATG7-shRNA knockdown protected the hBM-MSCs from cell death compared to other groups. This demonstrated that autophagy knockdown enhances survival of hBM-MSCs under severe persistent stress conditions (Data represented Mean±SD; ^a)P<0.05, ^b)P<0.01 and ^c)P<0.001).