Fig 1: Increased fibrosis, ERS and Warburg effect and decreased CSE expression and left atrial function were found in AF patients compared with SR patients. (A) H2S content was determined by Methylene blue method in the LAA of patients with SR or AF (n = 6 in each group). (B) Representative Western blotting and relative densitometry analysis of CSE, CBS, 3-MST, PDK-4, LDHA, PDH, ATF-4, CHOP, caspase-12 and GAPDH in the LAA of patients with SR or AF (n = 6 in each group). (C) Western blotting was used to analyze the protein levels of p-PERK and t-PERK in the LAA of patients with SR or AF (n = 6 in each group). (D) Western blotting was used to analyze the protein levels of p-elf2a and t-elf2a in the LAA of patients with SR or AF (n = 6 in each group). (E) Lactate acid was determined by kit analysis in the LAA of patients with SR or AF (n = 6 in each group). Blue staining with representative Masson staining (scale bar = 50 µm) and quantification of atrial fibrosis (n = 6 in each group, with =40 fields in each group) were used in the (F) AF group and (G) SR group. (H) Western blotting was used to analyze the protein levels of MMP-9 and GAPDH in the LAA of patients with SR or AF (n = 6 in each group). Representative RT-PCR and relative densitometry analysis of (I) collagen Ia and (J) collagen IIIa in the LAA of patients with SR or AF (n = 6 in each group). Left atrial function was measured by color Doppler echocardiography (K) LAESVI, (L) LAEF and (M) LAFI (n = 6 in each group) in the patients with SR or AF during the echocardiographic studies. **p < 0.01 VS AF. A Student’s t-test was used for each of these comparisons between AF and SR groups.
Fig 2: 3-MST inhibition in HCT116 cells induces cyclin D1 downregulation in part via ATF1, CREB and CDK4, which is associated with G1-S phase cell arrest and cell death. A,B: Western blot analysis of Cyclin D1, CDK4 and GSK3ß proteins after treatment of HCT116 cells with HMPSNE, AOAA or their combination for 48 h. Data are shown as mean ± SEM, n = 4, **p < 0.01 compared to control. C: Representative FACS plots showing the Q1 population (early apoptotic cells), Q2 population (late apoptotic cells), Q3 population (necrotic cells) and Q4 population (cells alive). D: Quantification of apoptotic cells, distinguishing the late and the earlier apoptosis ratios, and necrotic cells compared to control. Data are shown as mean ± SEM, n = 5, *p<0.05, **p<0.01 compared to control.
Fig 3: 3-MST inhibition induces BID downregulation and CyR61 upregulation in part via RhoA. Endogenously produced CyR61 acts as a negative regulator of apoptosis in HCT116 cells treated with 3-MST inhibitor. A,B: Western blot analysis of BID, CyR61 and RhoA proteins in HCT116 cells treated with HMPSNE, AOAA or their combination for 48 h. Data are presented as mean ± SEM of at least 5 independent experiments. *p < 0.05, **p < 0.01 compared to control. C: Histogram showing the annexin-positive cell population in HCT116 ShCTR and Sh3 CyR61 after 48 h incubation with HMPSNE. D: Comparison of the apoptotic cell population in HCT116 ShCTR vs. Sh3 CyR61 after treatment of the cells with HMPSNE for 48 h. Data are shown as mean ± SEM, n = 5, **p<0.01 compared to control.
Fig 4: Upregulation of H2S-producing and H2S-metabolizing enzymes occurs during the accumulation of ‘driver’ pathway mutations in human intestinal epithelial cell organoids. Western blot analysis (A) and densitometric quantification (B) of H2S-producing (CBS, 3-MST, CSE) and H2S-metabolizing (TST, ETHE1, SQR) enzymes in NL, A, AT, AKST, AdeCIN TS and AdeCIN TSK organoids after 7 days in culture. Data represent mean ± SEM of at least 5 independent experiments. * p < 0.05, ** p < 0.01 compared to control.
Fig 5: Effect of pharmacological inhibition of 3-MST on the expression of various H2S-producing and H2S-metabolizing enzymes in AdeCIN TS and AdeCIN TSK organoids. (A,B) Western blot analysis of H2S-producing and H2S-metabolizing enzymes in AdeCIN TS organoids in presence of increasing concentrations of HMPSNE. (C,D) Western blot analysis of H2S-producing and H2S-metabolizing enzymes in AdeCIN TSK organoids in presence of increasing concentrations of HMPSNE. Analysis was performed at 48 h. CBS refers to the 45 kDa, truncated form, which was the CBS isoform predominantly present in these organoids. Data represent mean ± SEM of at least 4 independent experiments. * p < 0.05, ** p < 0.01 compared to control.
Supplier Page from Abcam for Anti-MST antibody