miR-30b inhibits autophagy to alleviate hepatic ischemia-reperfusion injury via decreasing the Atg12-Atg5 conjugate

Figure 3 miR-30b inhibits autophagy by sequestering Atg12 in AML12 cells. A and B: The predicted miR-30b binding site on the Atg12 mRNA 3′-untranslated region (UTR) is shown, and a luciferase reporter assay was performed to determine the effects of miR-30b on the Atg12 mRNA 3′-UTR; C: Western blot was used to detect expression of Atg12 and LC3 in AML12 cells treated with miR-30b mimics or inhibitor; D: Confocal immunofluorescence of AML12 cells demonstrated increased numbers of GFP-RFP-LC3 dots in the IR group; when autophagy is induced, both GFP and RFP are expressed as yellow dots (autophagosomes). Red dots represent autolysosomes as the GFP degrades in an acid environment. Scale bars = 20 μm; E: TEM images of AML12 cells after ischemia followed by reperfusion at 12 h. TEM images show representative examples of autophagosomes (red arrows). Scale bars = 1.0 μm. And the data were quantified by counting the number of autophagosomes per cross-sectioned cell; ^aP < 0.05, ^bP < 0.01, ^cP < 0.001 vs miR-NC group. Every experiment was repeated three times.