Effects of various autophagy modulators on the expression of autophagic markers LC3II and p62

Western blotting of autophagic markers LC3II and p62 are widely used for estimating autophagic activity. To compare the regulation of various autophagy modulators on LC3II and p62, HEK293 cells were treated separately with mTOR-dependent autophagy activator rapamycin or -independent autophagy activators trehalose, and autophagy inhibitors including 3-methyladenine(3-MA), bafilomycin A1 or E64d and pepstatin A that inhi-bited the initiation of autophagy, the fusion of autophagosome and lysosome, and the activities of lysosomal enzymes accordingly, and then LC3II and p62 levels were assessed. Western blot results demonstrated that rapamycin enhanced the conversion of LC3I to LC3II, promoted the degradation of p62 simultaneously, while trehalose merely increased the expression of LC3II with no influence on the p62 level. Moreover, inhibition of autophagy commonly led to accumulation of LC3II as well as blockage of p62 degradation in a concentration- and time- dependent manner. These results indicate that obvious differences exist in the regulation of LC3II and p62 by various modulators although both are autophagic markers.