Ampkα sirna

HUVECs were transfected with indicated siRNA or scrambled siRNA (Guangzhou RiboBio, China, siNO5815122147) using a HiPerFect transfection reagent (Qiagen, 301705) according to the manufacturer's protocol. AMPKα siRNA was from Santa Cruz Biotechnology, CA, USA. The sequences are as follows: Caveolin-1 siRNA sense (5′-CGAGAAGCAAGUGUACGACdTdT-3), Caveolin-1 siRNA antisense (5′-GUCGUACACUUGCUUCUCGdTdT-3′), ATG5 siRNA sense (5′-GGAACAUCACAGUACAUUUdTdT-3′), and ATG5 siRNA antisense (5′-AAAUGUACUGUGAUGUUCCdTdT-3′).

AMPKα siRNA (45313, Santa Cruz Biotechnology) was transfected into cells with the Lipofectamine® RNAiMAX transfection kit (13778150, Invitrogen) according to the manufacturer's protocol. Briefly, podocytes were cultured in 6-well plates for 24 h. RNAiMAX transfection reagent and AMPKα siRNA were added to the reaction mixture. Then, the podocytes were cultured with serum-free medium and reaction mixture for 24 h, and the medium was changed for 6–8 h. After the transfection was completed, the podocytes were treated for 48 h with various mediums. The protein level of AMPKα was measured by western blotting analysis to confirm transfection success. Podocytes were collected 24 h after transfection for the following experiments.