Fig 2: CSE/H2S pathway mediated ISO-induced heart dysfunction. A, Western blots and summarized data showing CSE protein levels in the indicated hearts of mice injected by vehicle or ISO for 4 weeks. n = 6. B, H2S concentrations in the indicated hearts of mice injected by vehicle or ISO for 4 weeks. n = 8. C-D, Left ventricular ejection fraction (EF) (C) and left ventricular fraction shortening (FS) were measured by echocardiography (D). n = 8–9. E-F, Left Ventricular internal dimension diastole (E) and left ventricular end-diastolic volume (F) were measured by echocardiography. n = 8. G, Plasma BNP levels in mice after 4 weeks' vehicle or ISO administration. n = 8. H, Quantification of heart weight (HW) over tibia length (TL) ratio in mice injected by vehicle or ISO for 4 weeks. n = 8. I-J, Representative images of HE images (I) and masson trichrome staining images (J) in indicated hearts of mice after 4 weeks' vehicle or ISO administration. Scale bar = 200 µm. K–N, Western blotting images (K) and quantification of Bcl-2 (L), Bax (M) and cleaved caspase 9 (N) protein levels in the indicated hearts of mice after 4 weeks' vehicle or ISO administration. n = 6. *: p < 0.05, **: p < 0.01.

Fig 3: Western blot images showing the expression levels of various key Hcy metabolizing enzymes in normal thigh muscles: Methylenetetrahydrofolate reductase (MTHFR), Cystathionine ß-synthase (CBS) and Cystathionine ?-lyase (CSE). We also measured the levels of another key H2S producing enzyme 3-mercaptopyruvate sulfur transferase (3MST) in conjunction with CBS and CSE in the thigh muscles. GAPDH was used as a loading control. Liver tissue lysate from the wild type mouse was used as a positive control.

Fig 4: PDAC GLSi proteomics identifies up- and downregulated pathways.(a,b) Volcano plots illustrate statistically significant protein abundance differences in MPDAC-4 cells treated with CB-839 for 24 h (a) or 72 h (b). Volcano plots display the –log10(P value) versus the log2 of the relative protein abundance of (a) mean CB-839 24 h treatment or (b) mean CB-839 72 h treatment to mean control (DMSO). Red circles represent the top 5% upregulated proteins with a P value<0.05, blue circles represent the 5% most downregulated proteins and a P value <0.05. The remainder of proteins are represented as gray circles. Data are from three independent plates from a representative experiment of three experiments. (c) Principal component analysis of the CB-839 treated proteomes represented in a two-dimensional space. (d) Enrichment map of gene set enrichment analysis (GSEA) of CB-839-72 h versus DMSO, FDR<0.01, co-efficient overlap (CO) >0.25, node size is related to number of components identified within a gene set and the width of the line is proportional to the overlap between related gene sets. GSEA terms associated with upregulated (red) and downregulated (blue) proteins are coloured accordingly and grouped into nodes with associated terms. (e) Proteins in the oxidative stress response protein network are upregulated in response to CB-839 treatment. Graphs represent mean of Log2(CB-839 treated sample/control) from MPDAC-4 Experiments 1–3 of select proteins in oxidative stress response pathways (n=9, three independent plates for each Experiment 1–3). Error bars represent±s.e.m. Upregulated (red), downregulated (blue), no relative change (grey). Abbreviations: ALDH1L2: Aldehyde Dehydrogenase 1 Family Member L2, GSR: Glutathione reductase, mitochondrial, GLS: Glutaminase, GLS2: Glutaminase 2, CTH: Cystathionine gamma-lyase, GCLC: Glutamate-cysteine ligase catalytic subunit, GCLM: Glutamate-cysteine ligase regulatory subunit, GPX1: Glutathione peroxidase 1, GSTT3: Glutathione S-transferase Theta 3, MTX: Methotrexate, BSO: L-Buthionine-S,R-sulfoximine.

Fig 5: Schematic model showing the mechanism of CSE/H2S regulated Drp1 activity in heart failure. During heart failure, CSE/H2S pathway was inhibited, whereas iNOS/NO pathways was potentiated. Although Drp1 S-sulfhydration and S-nitrosylation were competitively modified at the same cysteine, the effect of these two modifications on Drp1 activity was reverse. Aberrant activation of Drp1 translocated to mitochondria outer membrane and interacted with VDAC1 to cause mitochondria hyperfission and dysfunction.