Evaluation of autophagy induction and inhibition in the Huh7.5 cell line through flow cytometry

Abstract

Background: Autophagy is a physiologic process in which double membrane vesicles engulf damaged proteins and organelles for delivering them to lysosomein order to degrade and recycle them via lysosomal digestion. Beclin1 is one of the basic proteins involved in the initial step of autophagosome formation. In the current study, the effect of exogenous Beclin1 to induce autophagy and the effect of 3MA to inhibit of autophagy was assessed in Huh7.5 cells as an in vitro models of hepatocellular carcinoma. Material and methods: The Recombinant pcDNA-Beclin1was transfected into Huh7.5 cells. Also, the cell treated with 3MA. Next, the autophagy induction and inhibition was conducted via LC3 staining as a main autophagy marker using flow cytometry. Results: The result of this study suggest that the over expression of exogenous Beclin1 in Huh7.5 cells elevated the autophagosome formation as shown by intracellular autophagosomal marker LC3-II staining for about 32.32 % and   3MA decreased  it up to2% in compared with control cells in which the  stained LC3-II was12.08. Conclusion:  Recombinant beclin1 may be used as a potential autophagy inducer agent and 3-methyl-Adenin inhibits autophagy formation in Huh7.5 cell. The staining autophagy formation marker LC3-II with specific antibody is a reliable method to measure autophagy activation via flow cytometry.