Confirmation of autophagy induction with rapamycin. (A) Western blot analysis of 24 hour-rapamycin-treated hBM-MSCs exposed to 10, 200, and 500 nM rapamycin. LC3-I induction and conversion of LC3-I to LC3-II were determined by western blot analysis. Expression of LC3-II and its significantly increased level compared to LC3-I in MSCs-500 nM Rapa indicated induction of autophagy in these cells. β-actin served as the loading control. (B) Microscopic observation of transfected hBM-MSCs before and after rapamycin treatment. hBM-MSCs were transfected with GFP-LC3 containing a GFP vector (MSC-LC3), and 48 hours later, MSC-LC3 was treated with 500 nM rapamycin for 24 hours (MSC-LC3-Rapa). Fluorescence microscopic observations indicated that MSC-LC3-Rapa manifested green dots (punctate signal) (B.I), which confirmed effective autophagy induction in MSC-LC3-Rapa compared to MSC-LC3 (B.II). Autophagy was observed in MSC-shRNA Cont-Rapa cells, which were transfected with a negative control shRNA vector and treated with rapamycin (shiny green dots) (B.III). Very few green dots were detected in MSC-shRNA 3-Rapa cells, which were treated with rapamycin after suppression of autophagy (B.IV). (C) Western blot analysis of shRNA 3-transfected hBM-MSCs before and after rapamycin treatment. hBM-MSCs were treated with 500 nM rapamycin for 24 hours following transfection with ATG7-shRNA clone 3 (ATG7 suppressive vector) and the negative control shRNA, and subjected to western blot analysis.