Confirmation of autophagy suppression via ATG7 gene silencing using shRNA. (A) Fluorescence microscopic observation 48 hours after transfection of hBM-MSCs with ATG7 shRNA clone 3 containing a GFP vector. Intrinsic GFP fluorescence and green cytoplasm in MSC-shRNA 3 confirmed the transfection. (B) RT-PCR analysis of transfected hBM-MSCs for selection of the most suppressive vector. Following transfection of hBM-MSCs with ATG7 shRNA suppression vectors, ATG7 expression was analyzed by RT-PCR. ATG7 was effectively downregulated in MSC-shRNA 3 cells. shRNA clone 3 was selected as a proper vector for autophagy suppression in hBM-MSCs. Expression of β-actin was also evaluated for normalization. (C) qPCR of ATG7 demonstrated that ATG7 was expressed at a very low level in MSC-shRNA 3 compared with MSC-shRNA Cont. (Data represented Mean±SD, ^a)P<0.001).