FIGURE 3:

EACC does not affect early autophagic events. (A) HeLa cells were either left untreated or treated with BafA1 (100 nM) or EACC (10 µM) for 2 h in starvation conditions. Samples were collected and immunoblotted with anti–phospho-P70S6K (T389), anti-P70S6K, anti–phospho-4EBP1, and anti-4EBP1 antibodies. (B) Samples collected after EACC treatment were immunoblotted with anti–phospho-ULK1 (S757), anti-ATG14, anti-ATG5, anti-ATG16L1, and anti–β-actin antibodies. (C) HeLa cells cotransfected with mCherry-DFCP1, GFP-LC3, and HA-ATG14 were either left untreated or treated with EACC and immunostained with anti-HA antibody. Scale = 15 µm, 1 µm. (D) Graph showing the percent of LC3 puncta colocalizing with DFCP1 and ATG14. This population represents immature autophagosomes. The colocalized dots were counted and plotted as in Figure 2B. Data shown here represent a minimum of 50 cells from three independent experiments plotted as mean ± SEM. Statistical significance was analyzed by Student’s unpaired t test. ns = nonsignificant. (E) GFP-LC3 transfected HeLa cells were treated with EACC (10 µM) for 2 h in starvation conditions and immunostained with anti-WIPI2 antibody. Scale = 15 µm. (F) Graph showing the percent of LC3 puncta colocalizing with WIPI2. This population represents omegasomes. The analysis was done similarly as in D. Data shown here represent a minimum of 45 cells from three independent experiments plotted as mean ± SEM. Statistical significance was analyzed by Student’s unpaired t test. ns = nonsignificant.