We examined whether knockdown of miR-20a targets phenocopy the effect of miR-20a on DNA damage and ROS. Immunoblotting revealed that depletion of ATG16L1, BECN1 or SQSTM1 moderately reduced LC3-II levels; DQ Red BSA assay showed that both siRNAs significantly reduced EBSS-induced lysosomal proteolytic activity, indicating that these siRNAs can efficiently block autophagy and lysosomal activity (Figure 5g). Knockdown of ATG16L1, BECN1 or SQSTM1 increased expression of γH2AX and intracellular ROS levels in nutrient-starved cells (Figures 5d–f). These results indicate that defective autophagy increases ROS and activates the DNA damage response.7