Figure 4. Crosstalk between ROS and autophagy.

A. Logarithmically growing Capan-2 and BxPC-3 cells were treated with autophagy inhibitors (25μM CQ or 1mM 3-MA) for 24h. Level of intracellular ROS was determined by CellROX^® deep red reagent (Invitrogen, NY) following manufacturer's instructions. Fold change in intracellular ROS was calculated relative to untreated samples. Statistical significance between groups was determined using students t-test and p values less than 0.05 was considered significant. B. Capan-2 and BxPC-3 cells were treated with Nx as described in legends figure 2 in the presence or absence of autophagy inhibitors (25μM CQ or 1mM 3-MA) for 24h for measuring intracellular levels of ROS. Level of intracellular ROS was determined by CellROX^® deep red reagent (Invitrogen, NY) following manufacturer's instructions. Fold change in intracellular ROS was calculated relative to untreated samples. Statistical significance between groups was determined using students t-test and p values less than 0.05 was considered significant with *indicating p<0.05; **; p<0.01; and *** p<0.005). C. Protein levels of p62 and LC3 in Capan-2 and BxPC-3 cells treated with Nx in the presence and absence of 5 mM NAC for 24h were determined by immunoblot analysis. Quantification data normalized to β-actin were shown below the blot. Representative blot from multiple experiments is shown. D. Effect of knocking down Atg5 and 7 on levels of p62 and LC3 and intracellular ROS in Capan-2 and BxPC-3 cells: Levels of Atg5, 7, p62 and LC3 were determined by immunoblot analysis of Capan-2 or BxPC-3 cells stably expressing Atg5 or Atg7 shRNA. E. Intracellular ROS production was measured in Capan-2 or BxPC-3 cells stably expressing Atg5 or Atg7-specific shRNA or scrambled shRNA using CellROX^® deep red reagent (Invitrogen, Carlsbad, CA; E). Statistical significance between groups was determined using students t-test and p values less than 0.05 was considered significant (*p<0.05 and ***p<0.005).