Journal: Redox Biology

Article Title: Formation of a reducing microenvironment and regulation of protein supersulfidation by gut microbial supersulfides

doi: 10.1016/j.redox.2026.104123

Figure Lengend Snippet: Gut bacterial supersulfides are linked to fecal reducing capacity in the distal gut (A–C) Mice were treated with an antibiotic cocktail containing 1 mg/mL ampicillin and 0.5 mg/mL vancomycin in drinking water for 2 weeks (Abx, antibiotic treatment). (A, B) Fecal supersulfides, related sulfur metabolites, and cystine were quantified by LC-ESI-MS/MS. (C) The reducing capacity of fecal supernatants was evaluated using WST-8 (absorbance at 460 nm). (D) The effect of N-ethylmaleimide (NEM) on the reducing capacity of fecal supernatants was evaluated using WST-8. (E, F) Mice were fed a high-cystine diet for 2 weeks. (E) Fecal sulfur metabolites were quantified by LC-ESI-MS/MS. (F) The reducing capacity of fecal supernatants was analyzed using WST-8. Data are expressed as the means ± SEM. Statistical significance was assessed using unpaired Student's t -test in (A-C, E and F) or paired Student's t -test in (D). ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ns, not significant.

Article Snippet: Next, 190 μL of bacterial lysate were mixed with 10 μL of WST-8 reagent (Tokyo Chemical Industry Co., Ltd., Tokyo, Japan) and incubated for 2 h at room temperature.

Techniques: Tandem Mass Spectroscopy

Journal: Redox Biology

Article Title: Formation of a reducing microenvironment and regulation of protein supersulfidation by gut microbial supersulfides

doi: 10.1016/j.redox.2026.104123

Figure Lengend Snippet: Supersulfides shape the extracellular redox environment and oxidative stress resistance (A) Each bacterial strain was incubated with cystine. Cell pellets and culture supernatants were collected to obtain intracellular and extracellular fractions, respectively. Heatmap colors represent fold changes of the absorbance values in the cystine-supplemented group relative to the vehicle control. (B) Levels of supersulfides in intracellular and extracellular fractions after incubation with cystine were quantified using LC-ESI-MS/MS. The extracellular-to-intracellular ratio of supersulfides was then calculated and expressed as a relative value compared with the vehicle control. (C, D) Bacterial culture supernatants with or without cystine were treated with NEM, and thiol levels and reducing capacity were analyzed using DTNB (C) and WST-8 (D). (E) Bacterial culture supernatants with or without cystine were treated with H 2 O 2 , and residual H 2 O 2 was quantified. Data was normalized to the optical density at 600 nm (OD 600 ). (F) Intracellular oxidative stress levels in each bacterial strain after H 2 O 2 exposure were analyzed by flow cytometry. For the cystine-supplemented group, bacteria were pre-cultured in the presence of cystine prior to H 2 O 2 treatment. Data are expressed as the means ± SEM. Statistical significance was assessed using unpaired Student's t -test in (B) and (E) or one-way ANOVA followed by Holm–Šidák multiple comparisons test in (C, D) and (F). ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001.

Article Snippet: Next, 190 μL of bacterial lysate were mixed with 10 μL of WST-8 reagent (Tokyo Chemical Industry Co., Ltd., Tokyo, Japan) and incubated for 2 h at room temperature.

Techniques: Incubation, Control, Tandem Mass Spectroscopy, Flow Cytometry, Bacteria, Cell Culture