Fig 1: Changes in endogenous CSE–H2S system in HHcy-induced atherosclerosis. (a) CSE immunohistochemical staining in aortic root local plaque (n = 5 for each of three groups, bar = 20 µm). Insert images in the left corner are overall perspective pictures. Changes in aortic mRNA expression of CSE (b), 3-MST (c), and CBS (d) (n = 5 for each of three groups). H2S release from liver tissues was measured by methylene blue assay (n = 5 for each of three groups) (e). Liver expression of CSE and CBS protein (f) and CSE (g) and CBS mRNA (h) (n = 5 to 6 for each of three groups)
Fig 2: Changes in binding to L-homocysteine associated with CSE sulfhydration or nitrosylation. (a) Microscale thermophoresis assay of CSE binding to L-homocysteine after changes in CSE sulfhydration or nitrosylation (n = 4). (b) Biotin switch assay of CSE sulfhydration by NaHS or nitrosylation by GSNO, with mutated CSE cysteine residues: M1 (C252S + C255S), M2 (C307S + C310S), and M3 (C252S + C255S + C307S + C310S) (n = 5). (c) Changes in CSE binding activity with mutated sulfhydration/nitrosylation CSE sites (n = 4) with NaHS treatment and (d) GSNO treatment (n = 5)
Fig 3: CSE sulfhydration and nitrosylation changes on exposure to HHcy. (a) Detection by biotin switch assay in HepG2 cells with different doses of homocysteine (n = 5) and (b) in liver tissue of atherosclerosis mice with HHcy (n = 6 for each of three groups)
Fig 4: HERPUD1 downregulates GSH by reducing the protein level of GSS.A, B GSH and Fe2+ content in liver cancer cells with or without HERPUD1 after adding erastin, RSL3, CA. C The ferroptosis-related metabolic axis from glucose to GSH. D mRNA levels of GCLC, GSS, CBS, CTH, and SHMT2 were analyzed by qPCR in Bel-7402 and Bel-7404 cells with or without HERPUD1. E Protein levels of GCLC, GSS, CBS, CTH, and SHMT2 were analyzed by immunoblotting in Bel-7402 and Bel-7404 cells with or without HERPUD1. F The relative protein level of GCLC, GSS, CBS, CTH, and SHMT2 are normalized according to the level of GAPDH calculated by ImageJ software. The data were analyzed using Student’s t test and shown as the mean ± SD from three biological replicates. *P < 0.05; **P < 0.01; ***P < 0.001 indicates statistical significance.
Fig 5: RRM2 upregulates GSH by sustaining GSS. a The ferroptosis-related metabolic axis from glucose to GSH. b–g The levels of glycine (b), glutamate (c), cysteine (d), cystathionine (e), serine (f) and glucose (g) were measured in HepG2 and SMMC-7721 cells with or without ectopically expression or knocked down of RRM2. h, i mRNA levels of CBS, CTH, SHMT2, GSS and GPX4 were analyzed by qPCR in HepG2 (h) and SMMC-7721 cells (i) administered the indicated treatment. j, k Protein levels of CBS, CTH, SHMT2, GSS and GPX4 were analyzed by immunoblotting in HepG2 and SMMC-7721 cells (j). The level of RRM2 was normalized to that of GAPDH, and the normalized level of RRM2 in the untreated group was arbitrarily set to 100% (k). (l) GSH levels were measured in WT and GSS-/- HepG2 and SMMC-7721 cells with or without RRM2 overexpression or knockdown, as indicated. The data are shown as the mean ± SD from three biological replicates. **P < 0.01 indicates statistical significance. Data from b-i and k-l were analyzed using one-way ANOVA
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