Reduction of evodiamine-(Evo)-induced protective autophagy by silencing the TRPV1 gene. (a) Cells were transfected with control or TRPV1 siRNA using the lipofectamine RNAimax reagent for 48 h, and the level of TRPV1 protein was measured by an immunoblot assay using anti-TRPV1 and anti-GAPDH anti-bodies to monitor the efficiency of the siRNA. GAPDH was used as an internal control to normalize the amount of proteins applied in each lane. (b) The elevation of cytosolic calcium induced by Evo was reduced by knockdown of TRPV1. Cells with or without knockdown of TRPV1 were treated with Evo for 8 h and then analyzed by Fluo-3 AM staining using flow cytometry. After transfection, cells were treated with Evo for another 24 or 48 h, trypsinized, and collected to determine percentages of autophagy (c) and apoptosis (d), respectively, using acridine orange staining and annexin V/PI staining. , compared to the respective control, by Student’s -test.
