FIG 2.

AZT inhibits autophagic flux. (A) C2C12 cells stably expressing LC3-GFP were treated with AZT (8.6 µM), 3TC (8.7 µM), 3MA (0.2 mM), nocodazole-vinblastine (N/V; 2 µM), or CQ (50 µM) for 24 h. (A and B) Flow cytometry analysis of autophagic flux in C2C12 cells expressing the LC3-GFP fusion protein. Data are presented as the percent change from the value for the untreated control. C2C12 cells were treated with AZT (42.8 µM) for 72 h or PP242 (5 µM) for 4 h in the presence or absence of 5 µM ACH for the last 3 h. The data in panels A and B are presented as the mean ± SD and are representative of those from at least three independent experiments with three replicates. ***, P < 0.001. MFI, mean fluorescent intensity. (C) Western blot analyses of the conversion of LC3-I to LC3-II with and without ammonium chloride (ACH) blockage of autolysosomal degradation using whole-cell lysate and anti-LC3 and β-actin antibodies (top) with densitometry analysis (bottom). A representative blot from three experiments is shown.