Fig 1: Expression of H2S-producing and -catabolizing enzymes in GYY4137-treated cells. A Representative Western blot images of adipocytes treated with different concentration of GYY4137 and corresponding densitometry analysis of B 3-MST, C CBS, D CSE, E ETHE1, and F TST in differentiated adipocytes (day 7). ß-Actin was used as loading control. Data refer to mean values of N = 5 independent experiments ± SEM. *p < 0.05 and **p < 0.01 show significant differences compared to control values in the absence of GYY417 treatment
Fig 2: DS is associated with the upregulation of CBS and 3-MST enzymes in a brain-region-specific manner. Following the cognitive assessment, animals were euthanized and tissue from the prefrontal cortex (PFC), hippocampus (HIP), entorhinal cortex (ETC), and the basal forebrain (BF) brain regions were collected for protein extraction. Protein samples were processed for immunoblotting detection of the H2S producing enzymes cystathionine ß-synthase (CBS) (A, B, C), 3-mercaptopyruvate sulfurtransferase (3MST) (A, D), and cystathionine ?-lyase (CSE) (A, E). (B): Quantification of the proteolytic, 45 kDa isoform of CBS. The expression of ß-actin served as loading control for our analysis. Data, expressed as mean ± SEM of 5 animals per experimental condition, were analyzed by two-way ANOVA followed by post hoc Bonferroni multiple comparison t-tests at each brain region of interest. *p=0.05 or **p=0.01 shows significant differences between saline-treated DS rats and saline-treated WT rats; #p = 0.05 or ##p = 0.01 shows significant effect of AOAA treatment on the expression of various H2S-producing enzymes in Dup(Rno20)Yah rats.
Fig 3: NW improved CSE/H2S production. L-Cysteine and pyridoxal phosphate and indicated concentration of NW are added to tissue homogenates. H2S production in the heart (A), aorta (B), kidney (C), and liver (D) was measured, N = 4. SD rats were subcutaneously injected with NW 4.4 mg/kg/day for 1 week. H2S production in the heart and kidney was detected (E), N = 5. In primary adipocytes, the effect of 100 µM NW treatment on H2S production was detected (F), N = 5. In cultured HepG2 cells, the effect of different concentrations of NW on the CSE protein level was detected. The left panel is representing graph, and the right panel is statistical graph (G), N = 3. **p < 0.01.
Fig 4: Growth Hormone Signaling Inhibits Hepatic H2S Production In vivo(A–C) Hepatic CGL and CBS mRNA expression (n = 3/group) (A), protein expression (n = 3/group) (B), and H2S production capacity (n = 3/group) (C) in male and female growth hormone receptor knockout (GHRKO) mice. The asterisk indicates the significance of the difference between genotypes within sex; *p < 0.05.(D) Liver H2S production capacity (n = 8/group) in WT mice treated for 2 weeks with recombinant IGF-1, GH, or saline vehicle as indicated. The asterisk indicates the significance of the difference between GH and saline treatment; *p < 0.05.(E and F) Liver H2S production capacity in male and female IRS-1 WT or KO mice (n = 4–11/group) (E) and in male FGF21 WT or overexpressing (OE) mice (n = 6/group) (F). The asterisk indicates the significance of the difference between genotypes within sex; *p < 0.05. Error bars are ± SEM. See also Figure S2.
Fig 5: Effect of pharmacological inhibition of CBS/CSE on the expression of various H2S-producing and -metabolizing enzymes in AdeCIN TS and AdeCIN TSK organoids. (A,B) Western blot analysis of H2S-producing and H2S-metabolizing enzymes in AdeCIN TS in the presence of increasing concentrations of AOAA for 48 h. (C,D) Western blot analysis of H2S-producing and -metabolizing enzymes in AdeCIN TSK in the presence of increasing concentrations of AOAA for 48 h. Data represent mean ± SEM of at least 4 independent experiments. CBS refers to the 45 kDa, truncated form, which was the CBS isoform predominantly present in these organoids. * p < 0.05, ** p < 0.01 compared to control.
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