Fig. 4

Effect of 2 μM Fer-1 on ROS levels in H/R-induced cardiomyocytes. (A) Cardiomyocyte viability. (B) LDH release. (C) MDA content. (D) ROS fluorescence image, Merge: A combination of ROS (green fluorescence) and nucleus (blue fluorescence). (E) ROS quantitative analysis. (F) SOD content. (G) JC-1 staining showed that JC-1 aggregated in the mitochondria of normal cells emitted red fluorescence, while JC-1 in dead cells emitted green fluorescence due to its presence in the cytoplasm as a monomer. Merge: A combination of aggregates (red fluorescence), monomers (green fluorescence) and nucleus (blue fluorescence). (H) Red/green fluorescence ratio at the MMP level. (I-K) Western blotting revealing the expression levels of ACSL4 and GPX4 in cardiomyocytes (^*P < 0.05 vs. control; ^#P < 0.05 vs. H/R; scale bars, 100 μm; n = 3). Proteins were then transferred onto PVDF membranes. After cutting according to the molecular size, the primary antibody was incubated. Fer-1, ferrostatin-1; ROS, reactive oxygen species; H/R, hypoxia/reoxygenation; LDH, lactate dehydrogenase; MDA, malondialdehyde; SOD, superoxide dismutase; JC-1, 1,1′,3,3′-tetraethyl-5,5′,6,6′-tetrachloroimidacarbocyanine iodide; MMP, mitochondrial membrane potential; ACSL4, Acyl-CoA synthetase long chain family member 4; GPX4, glutathione peroxidase 4