miR-20a inhibits autophagic flux and lysosomal proteolytic activity. (a) MCF7 cells transfected with NC or miR-20a were incubated with bafilomycin A1 (Baf A1, 10 nm) for 1 h. The relative amounts of LC3-II versus GAPDH were determined by western blotting and Image J densitometric analysis. (b) MDA-MB-231 and MCF7 cells transfected with NC or miR-20a were cultured in normal medium or EBSS solution for 4 h. Protein expression of OPTN and ACTB were detected by immunoblotting. (c) Representative fluorescent images of MCF7 cells transfected with miRNAs and mCherry-GFP-LC3. Scale bar, 10 μm. (d) Cells with autolysosomes (red dots; mCherry+/GFP−) and autophagosomes (yellow dots; mCherry+/GFP+) were samples from at least 100 cells. (e) MCF7 cells overexpressing NC or miR-20a were incubated with DQ-BSA for 12 h, cultured in normal medium or EBSS for 4 h, the fluorescence images of DQ Red BSA were captured by confocal microscopy. Scale bar, 10 μm. (f) The red fluorescence intensity of DQ-BSA was quantified with Image J. More than 150 cells were analyzed in each group (**P<0.01).