Fig. 2

MMA-mediated ROS contributes to cardiomyocytes injury. In vitro, AC16 cells were treated with MMA (17.5 mM) for 3 h or H₂O₂ (600 μM) for 6 h and then used for further analyses. a, b ROS production was observed by DHE and DCFH-DA staining (n = 3/group) (Scale bar: 50 μm). c Flow cytometry was used to detect DCFH-DA fluorescence intensity under MMA treatment. d MMA increased the percentage of DHE-positive cells detected by flow cytometry (n = 4/group). e, f GSH and MDA were measured by ELISA kit in AC16 cells under MMA or H₂O₂ treatment (n = 3–4/group). g-i NOX2 and NOX4 expression in AC16 cells were analyzed by immunofluorescence staining (n = 3–5/group) (Scale bar: 100 μm). j, k Proteins were isolated from AC16 cells, and the levels of NOX2 and NOX4 were analyzed through western blot (n = 3–5/group). Data are expressed as the mean ± standard deviation. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 and n.s, not significant