Analysis of ROS production

The level of intracellular total ROS was assessed by 2′,7′-dichlorofluorescin diacetate (DCFH-DA) using a Reactive Oxygen Species Assay kit (Beyotime Institute of Biotechnology). Briefly, H9c2 cells were seeded in 6-well plates at 2×10⁵ cells/well and treated as aforementioned when the cells had grown to 80% confluence. DCFH-DA was diluted to 10 μM in serum-free DMEM and added into the medium (1 ml/well), and the cells were incubated at 37°C away from light for 20 min. Subsequently, the cells were washed with PBS thrice to remove the residual DCFH-DA and examined in at ≥3 fields per sample under a laser scanning confocal microscope (magnification, ×100). The density of ROS fluorescence was examined by ImageJ software 1.26 (National Institutes of Health).