FIGURE 1:

EACC inhibits autophagic flux. (A) HeLa cells were either left untreated or treated with BafA1 (100 nM) or EACC (2.5–25 µM) for 2 h in starvation conditions. Samples were collected and immunoblotted for anti-LC3 and anti–β-actin antibodies. (B) Relative levels of LC3-II:β-actin in untreated vs. treated samples were quantitated for three independent experiments. **, P < 0.01; *, P < 0.05; ns = nonsignificant (two-way ANOVA, replicate means compared with Bonferroni posttest). (C) HeLa cells were either left untreated or pretreated with BafA1 (100 nM) in basal or starvation conditions for 1 h in order to block the autophagic flux. This was followed by treatment with EACC (10 µM) for 2 h. Samples were collected and immunoblotted for anti-LC3 and anti–β-actin antibodies. (D) Relative levels of LC3-II:β-actin in untreated vs. treated samples were quantitated for three independent experiments. ns = nonsignificant. Statistical significance was analyzed by Student’s unpaired t test. (E) HeLa cells transfected with tandem-tagged ptfLC3 (mRFP-GFP-LC3) construct were either left untreated or treated with BafA1 (100 nM) or EACC (2.5–25 µM) for 2 h in starvation conditions. Scale = 10 µm. (F) The autophagosomes (RFP⁺/GFP⁺ structures) and autolysosomes (RFP⁺/GFP⁻ structures) per cell were counted using the cell counter plug-in of ImageJ software. Data shown represent the number of autophagosomes (RFP⁺/GFP⁺) and autolysosomes (RFP⁺/GFP⁻) as compared with control of a minimum of 45 cells from three independent experiments plotted as mean ± SEM. Statistical significance was analyzed by Student’s unpaired t test. ***, P < 0.001; **, P < 0.01; *, P < 0.05; ns = nonsignificant. (G) Immunostaining with anti-SQSTM1/p62 antibody in RFP-LC3 transfected HeLa cells treated with EACC (10 µM) for 2 h in starvation conditions. Scale = 15 µm. (H) Graph showing the mean intensity of colocalization between p62 and RFP-LC3 in control vs. EACC-treated group. Mean intensity of colocalization was measured using colocalization and analyze plug-ins of ImageJ software. Data shown here represents a minimum of 60 cells from three independent experiments plotted as mean ± SEM. Statistical significance was analyzed by Student’s unpaired t test. *, P < 0.05.