Western blot
Standard western blotting was conducted for protein expression assays in hypoxic H9C2 cells transfected with miR-210 mimic, inhibitor and NC. Two days following transfection, proteins were isolated with RIPA lysis buffer containing 1 mg protease inhibitors (Applygen Technologies Inc., Beijing, China). The protein contents were quantified using the Bicinchoninic Acid (BCA) Protein Assay Kit (CoWin Biotech Co., Ltd., Beijing, China). Primary antibodies against light chain 3 (LC3; ab48394), p62 (ab56416), Caspase8ap2 (ab4052), cleaved caspase 8 (ab25901), cleaved caspase 3 (ab13847), Beclin-1 (ab62557), and the internal controls β-actin and GAPDH were from Abcam (Cambridge, UK). Furthermore, secondary antibodies labelled with horseradish peroxidase were incubated with membranes for 1 h at 37°C. Samples were then transferred to a polyvinylidene fluoride (PVDF) microporous membrane (Millipore, Boston, USA). Blots were imaged by a FluorChem E imager (Cell Biosciences, San Jose, USA). The grey value of each band was determined with ImageJ version 1.51.