To investigate the role of miR-20a in autophagy, we transfected miR-20a and GFP-LC3 into MDA-MB-231 triple-negative breast cancer cells and cultured cells in Earle's Balanced Salt Solution (EBSS) medium to induce nutrient starvation. miR-20a significantly reduced GFP-LC3 puncta formation in nutrient-starved cells. Immunofluorescence assay with anti-LC3 confirmed these results (Figure 1d and Supplementary Figure S2a). Next, we used two breast cancer cell lines MDA-MB-231 and MCF7 to examine LC3-I to LC3-II conversion. Quantitative real-time PCR showed MDA-MB-231 cells expressed much higher levels of endogenous miR-20a than MCF7 cells, but much lower amounts of miR-20a after transfection, indicating MCF7 cells have much better transfection efficiency than MDA-MB-231 cells (Supplementary Figures S3a and b). miR-20a attenuated LC3-II protein expression in both cells, especially in MCF7 cells presumably because MCF7 cells have higher levels of miR-20a after transfection (Figure 1e).