Fig 2: MAT1A protein levels are significantly increased in female Cgl null mice with severely elevated plasma tHcy. Western blotting analysis of hepatic GNMT and MAT1A protein expression levels in male and female WT and Cgl null mice. Values shown represent the mean±s.d., *P<0.05, **P<0.01.

Fig 3: Effect of H2S or Hcy on SP1 activity in HL1 cardiomyocytes. (A) Western blot and densitometric analyses showing the activity of SP1 (p-SP1: SP1) in cardiomyocytes treated with different doses of Na2S or (B) Hcy. (C) Chromatin immunoprecipitation-qPCR assay showing amplification of CSE promoter copy number in cardiomyocytes treated with different doses of Hcy. (D) (i) Electrophoretic mobility shift assay showing WT CSE-SP-1 complex formation incubated with nuclear extracts from cardiomyocytes treated with different doses of Hcy. The super-shifted band represents binding of p-SP1 antibodies to the CSE-SP1 complex. (D) (ii) Quantification of percentage band shift in ‘D (i)’ in presence of p-SP1 antibody with different doses of Hcy. (D) (iii) Electrophoretic mobility shift assay (EMSA) showing no complex formation with mutant CSE-promoter. Values are represented as mean ± SEM. “*” represents statistically significant when compared to the untreated control. p < 0.05 is considered statistically significant. N = 5.

Fig 4: Methionine, folate and cysteine metabolism in mammals. (A) The transsulfuration and cysteine oxidation pathways and, methionine and folate cycles are shown. Betaine-homocysteine S-methyltransferase (BHMT), cystathionine ß-synthase (CBS), cystathionine ?-lyase (CGL), cysteine dioxygenase (CDO), cysteinesulfinate decarboxylase (CSAD), glutamic-oxaloacetic transaminase (GOT), glycine N-methyltransferase (GNMT), methionine adenosyl transferase (MAT1A), methionine synthase (MTR), methylenetetrahydrofolate reductase (MTHFR), S-adenosyl homocysteine hydrolase (SAHH). (B) Cgl null female but not male mice exhibit severely elevated levels of plasma tHcy. Plasma levels of tHcy in wild type (WT) mice plus and minus treatment with the CGL specific inhibitor PPG and untreated male and female Cgl null mice. Values shown represent the mean±s.d., n=6, ***P<0.001.

Fig 5: Exercise training increases biosynthesis of hydrogen sulfide in the heart of obese mice. Exercise training increased the concentration of H2S in mice fed with both a normal and high-fat diet in the left ventricle tissue of the heart (A) but not in plasma (B). Homocysteine, the precursor substrate for H2S production, was unaltered among the four groups (C). Protein expression of the H2S-producing transulfuration enzyme CBS was increased in the left ventricle of HFDEX mice compared to HFD mice (D). Protein expression of another H2S-producing transulfuration enzyme, CSE, was also increased in the left ventricle of HFDEX mice compared to HFD mice (E). The final H2S-producing enzyme, MPST, localized in mitochondria was unchanged between groups (F). Transcriptional activation of H2S-producing enzymes, CBS and CSE, was observed with increased mRNA expression in HFDEX (G, H), however activation of the transcriptional factor SP1 that activates CSE was unchanged (I). All values are expressed as mean ± SEM with dots of different shape representing each animal in a group. One-way ANOVA and Tukey’s post-hoc test were used for statistical analysis.