Figure 2.

miR-101 overexpression inhibits autophagy. (A) miR-101 overexpression inhibits eGFP–LC3 translocation. MCF-7 eGFP–LC3 cells were transfected with indicated miRNAs or siRNAs and fixed 72 h post-transfection. Percentage of eGFP–LC3 puncta-positive cells was quantified by automated image acquisition and analysis using a threshold of >5 dots/cell. Data are shown as the mean±s.d. of five replicates and is representative of three independent experiments. ^*P<0.05, ^**P<0.005. (B) Representative images from the quantification shown in (A). Scale bars represent 20 μM. (C) miR-101 overexpression inhibits autophagic flux. MCF-7 RLuc–LC3^WT and RLuc–LC3^G120A cells were reverse transfected with indicated miRNAs or siRNAs and 42 h later 50 μM etoposide was added. Luciferase activity was measured at 42, 54 and 66 h after transfection. Data are shown as the mean±s.d. of three replicates and are representative of three independent experiments. ^*P<0.05, ^**P<0.005. (D) miR-101 overexpression leads to accumulation of p62 protein. Western blot analysis 72 h after transfection with miR-101 or scramble control. p62 bands were quantified relative to the vinculin loading control using ImageJ software and the relative quantifications are shown. The data are representative of three independent experiments.