Next, we determined whether knockdown of endogenous miR-20a would affect autophagic flux and lysosomal proteolytic activity. In comparison, LNA-20a-transfected cells showed measurable lower levels of OPTN in control and starved cells (Figure 4c). LNA-20a also increased the numbers of autophagosomes and autolysosomes (Figure 4d and Supplementary Figure S6). We used DQ Red BSA to measure proteolytic activity; LNA-20a-transfected cells showed higher levels of fluorescence intensity compared with LNA-NC-transfected cells (Figures 4e and f). These results demonstrate that endogenous miR-20a negatively regulates autophagic flux and lysosomal proteolytic activity.