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

Journal: Journal of the American Chemical Society

Article Title: OptoChaperoneA Biohybrid Tool for Regulating Protein Condensates in Cells and In Vitro

doi: 10.1021/jacs.6c04074

Figure Lengend Snippet: Effect of OptoChaperone on cell viability under heat treatment. (a) Schematic of cell viability assay. HeLa cells electroporated with OptoChaperone were seeded 96-well plates. After adherence, cells were irradiated for 5 min with UV (OFF) or blue (ON) light to switch the state of OptoChaperone immediately before heat treatment. Cells were then subjected to 120 min of heat treatment at 43 °C, followed by a 48 h incubation at 37 °C to assess delayed cytotoxicity. Cell viability was measured using a water-soluble tetrazolium 8 (WST-8) assay after 4 h of incubation with the reagent. (b) Quantitative analysis of cell viability after light irradiation and heat treatment. Viability values were normalized to those of HeLa cells without OptoChaperone and without heat treatment (set as 100%). The plots show each data points, and the mean ± s.d. ( N = 3). Statistical significance was determined by one-way ANOVA followed by Tukey’s HSD (n.s., not significant; * p < 0.05).

Article Snippet: Subsequently, cells were incubated at 37 °C and 5% CO 2 for 48 h to allow cell viability phenotypes to manifest, with fresh DMEM introduced after 24 h. After the 48 h incubation, 100 μL of DMEM (prewarmed to 37 °C) containing 10% v/v WST-8 reagent (Cat No. 260-96162; Kishida Chemical Co., Osaka, Japan) was added to each well.

Techniques: Viability Assay, Irradiation, Incubation