Figure 6A Chemical structures of Hcy and its structural analogue acetyl Hcy thioether (AHT) are shown. The analogous parts are circled with dashed lines. B MARS- or MARS2-catalysed HTL production from Hcy (20 μM) was carried out in vitro in the presence of different AHT concentrations. The amount of HTL produced in each reaction was determined by thin layer chromatography. C MARS and MARS2 activities were determined (n=4) in the presence of the indicated AHT concentrations in the reactions. AHT IC50 values were determined for MARS and MARS2 with 20 μM Hcy. Data are presented as the mean ± SEM and were compared using an unpaired Student's t test. nsnot significant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. One-way ANOVA with Dunnett's correction was used for multiple comparisons. D Relative HTL levels were determined (n=4) for NE4C cells treated with various AHT concentrations for 4 h. Data are presented as the mean ± SEM and were compared using an unpaired Student's t test. nsnot significant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. One-way ANOVA with Dunnett's correction was used for multiple comparisons. E AHT (71 or 110 μg/kg per day) was injected into C57BL/6 mice for 6 days. Plasma HTL levels were detected by LC-MS. Data are presented as the mean ± SEM and were compared using an unpaired Student's t test. nsnot significant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. One-way ANOVA with Dunnett's correction was used for multiple comparisons. F Hcy levels were compared in NE4C or H9C2 cells without and with 100 μM AHT treatments. Data are presented as the mean ± SEM and were compared using an unpaired Student's t test. nsnot significant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. One-way ANOVA with Dunnett's correction was used for multiple comparisons. NE4C cells were cultured in DMEM with or without 20 μM Hcy and supplemented with the indicated AHT levels; N-homocysteinylation levels were detected by western blot. NE4C cells were cultured in DMEM with or without 20 μM Hcy and supplemented with the indicated AHT levels; Band intensities were quantified relative to the untreated group using Image J (H). Data are presented as the mean ± SEM and were compared using an unpaired Student's t test. nsnot significant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. One-way ANOVA with Dunnett's correction was used for multiple comparisons. I Cellular superoxide levels were determined (n=4) for NE4C cells treated with various AHT concentrations for 4 h. Superoxide levels were normalized to those of untreated NE4C cells. Data are presented as the mean ± SEM and were compared using an unpaired Student's t test. nsnot significant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. One-way ANOVA with Dunnett's correction was used for multiple comparisons. J Apoptotic rates were determined in the absence or presence of 20 μM Hcy in the culture media in control and 70 μM AHT-treated NE4C cells (n=4). Data are presented as the mean ± SEM and were compared using an unpaired Student's t test. nsnot significant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. One-way ANOVA with Dunnett's correction was used for multiple comparisons. K Levels of β-catenin in untreated, Hcy-, or HTL-treated NE4C cells were detected (n=3) with the presence or absence of 70 μM AHT in the culture media. Mean β-catenin levels in untreated NE4C cells were set at 100%. Data are presented as the mean ± SEM and were compared using an unpaired Student's t test. nsnot significant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. One-way ANOVA with Dunnett's correction was used for multiple comparisons.