To investigate the role of miR-20a in regulating the autophagy/lysosome pathway, we used lysosome inhibitors to evaluate autophagic flux.31, 32 Immunoblotting revealed that starvation-induced LC3-II turnover was inhibited by bafilomycin A1 (Baf A1) or hydroxychloroquine (HCQ), miR-20a inhibits basal level and starvation-induced LC3-II turnover (Figure 3a and Supplementary Figure S5). Because MCF7 cells express higher amounts of LC3-II and much less visible LC3-I, we performed more assays to precisely measure autophagic flux. SQSTM1/p62 is commonly used to monitor autophagic flux. Because we have shown that miR-20a directly targets SQSTM1, we used OPTN (Optic neuropathy-inducing protein, Optineurin), a most recently identified selective autophagy substrate, to determine autophagic flux.33 Nutrient starvation destabilized endogenous OPTN protein, overexpression of miR-20a caused OPTN accumulation, especially in nutrient-starved cells (Figure 3b). Then, we used the tandem mCherry-GFP-LC3 probe to distinguish autolysosomes (mCherry positive/GFP negative; red dots) and autophagosomes (mCherry positive/GFP positive; yellow dots).34 Nutrient starvation increased red and yellow LC3 dots per cell; miR-20a-transfected cells showed decreased number of autophgosomes and autolysosomes in nutrient-starved cells, indicating that miR-20a inhibits starvation-induced autophagic flux (Figures 3c and d).