Dissecting molecular mechanisms underlying ferroptosis in human umbilical cord mesenchymal stem cells: Role of cystathionine γ-lyase/hydrogen sulfide pathway

Figure 4 Effect of cystathionine γ-lyase overexpression on ferroptosis in human umbilical cord mesenchymal stem cells. A and B: Cell apoptosis was analyzed by Annexin V-fluorescein isothiocyanate (FITC)/propidine iodide staining using flow cytometry analysis (n = 3), and quantified on the basis of apoptosis rate (n = 3); C and D: Iron level was detected by FeRhoNox-1 staining (C) and iron assay kit (D); E and F: Immunofluorescent staining of total reactive oxygen species; G and H: Level of lipid peroxidation detected by flow cytometry analysis after staining with C11-BODIPY; I: Intracellular cystine-FITC levels measured by flow cytometry. Results represent three independent experiments; J: Malonaldehyde level; K: Ratio of reduced glutathione/oxidized glutathione disulfide; L: Mitochondrial morphological changes detected by transmission electron microscopy; M: Western blot analysis of expression of the glutathione-dependent antioxidant enzyme glutathione peroxidase, ferritin heavy chain 1, nuclear receptor coactivator 4, Fe³⁺-bound transferrin receptor 1, 4-hydroxynonenal, and SLC7A11 protein. Glyceraldehyde-3-phosphate dehydrogenase was used for normalization for protein (n = 3). Fer-1: Ferrostatin-1; Annexin V-FITC/PI: Annexin V-fluorescein isothiocyanate/propidine iodide; ROS: Reactive oxygen species; sh-CSE: Short hairpin RNA targeting cystathionine γ-lyase; MDA: Malonaldehyde; GSH/GSSG: Reduced glutathione/oxidized glutathione disulfide; GPX4: Glutathione-dependent antioxidant enzyme glutathione peroxidase; FTH1: Ferritin heavy chain 1; NCOA4: Nuclear receptor coactivator 4; TFRC1: Fe³⁺-bound transferrin receptor 1; 4-HNE: 4-hydroxynonenal; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase. ^aP < 0.05, ^bP < 0.01.