Hydrogen peroxide solution h2o2

Antibodies against ZO-1 (Abcam, ab190085; dilution 1:1,000), 5hmC (Active Motif, #39769, dilution 1:1,000), GFAP (Abcam, ab7260, dilution 1:1000), NeuN (Abcam, ab177487, dilution 1:1000), CD68 (Abcam, ab237988, 1:1000) and Claudin-5 (Abcam, ab131259, dilution 1:500) were used for immunohistochemistry staining. Antibody against 5hmC (Active Motif, #39769, dilution 1:5,000) was used for dot-blotting. Antibody against ZO-1 (Proteintech, 22601-1-AP, dilution 1:1000) and TET2 (Proteintech, 21207-1-AP, dilution 1:1000) were used for western-blotting.
Hydrogen peroxide solution (H₂O₂) (Sigma, 323381), N-acetyl cysteine (NAC) (Sigma, A7250), and DAPI (Sigma, D9542) were commercially obtained.
Fluorescein isothiocyanate (FITC)-conjugated dextran (40 kDa) and FITC-conjugated dextran 10 kDa were purchased from Thermo Fisher Scientific Inc. (Waltham, MA USA).

Indanyloxyacetic acid-94 (IAA-94) was purchased from Sigma (MO, USA). Anti-ICAM-1 and VCAM-1 antibodies were obtained from ImmunoWay Biotechnologies (California, USA). Anti-CLIC1 antibody was acquired from Santa Cruz Biotechnologies (sc-81873, CA, USA). Anti-Na⁺/K⁺-ATPase antibody was acquired from SanYing Biotechnology (14418-1-AP, Wuhan, China). Goat Anti-Mouse IgG (CW0102) and Goat Anti-Rabbit IgG (CW0103) were acquired from CWBIO (Beijing, China). FITC Goat Anti-Mouse IgG was acquired from EarthOx (E031210-01, CA, USA). Hydrogen peroxide solution (H₂O₂) was provided by Sigma. Chemicals and reagents of analytical grade were obtained from Sigma, TOYOBO, or Invitrogen.

Sodium hydroxide (NaOH), citric acid (CH₂COOH₂), chitosan powder (90% DD, degree of deacetylation), ethanol (CH₃CH₂OH), 2,2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH), ethylene diamine-tetraacetic acid (EDTA), bovine serum albumin (BSA), agarose, iron (III) chloride (FeCl₃), hydrogen peroxide solution (H₂O₂), and L-ascorbic acid were purchased from Sigma-Aldrich (St. Louis, MO, USA), and LB broth, Petri dish, and 96-well polystyrene plates from (Thermo Scientific™. Waltham, MA, USA) Moringa was purchased from the local supermarket.

Reactive oxygen species was measured using the carboxy derivative of fluorescein, CM-H2DCFDA (Life Technologies), according to the protocol provided by the manufacturer with the following modification: briefly, an overnight-grown C. difficile culture was refreshed to OD₆₀₀ of approximately 0.8 in BHIS broth. Moreover, 198 μL of the bacterial suspension was incubated with 2 μL of stock CM-H2DCFDA anaerobically at 37°C for 30 min. Cells were then treated with 1× MBC of lauric acid for 10 min, and fluorescence was then measured using the FlexStation^® 3 Multi-Mode Microplate Reader (Molecular Devices) at an excitation wavelength of 488 nm and an emission wavelength of 535 nm. The following controls were included: bacterial suspensions in BHIS broth containing 1% DMSO as the negative control; bacterial suspensions in BHIS broth containing 0.0035% hydrogen peroxide solution (H₂O₂; Sigma–Aldrich) and 10 mM tert-butyl hydroperoxide solution (TBHP; Sigma–Aldrich) as the positive control. The results are representative of three independent experiments.

The wild-type strain and the msh2Δ, mlh1Δ, and pms1Δ mutants were streaked for single colonies on YPD medium or YPD medium-nourseothricin (100 µg/ml) plates and incubated at 28°C. Single colonies were restreaked every 7 days for 2 months (approximately 600 generations). Three independent strains were generated for the wild-type strain and each mutant (Table S2). Strains were then cultured overnight using YPD medium, 10-fold serially diluted, and plated onto YPD medium with or without the following stress agents: 0.25 mM paraquat dichloride hydrate (paraquat) (Sigma-Aldrich; catalog no. 36541), 5 mM hydrogen peroxide solution (H₂O₂) (Sigma-Aldrich; catalog no. 216763), 1.5 mg/ml calcofluor white, and 5 mg/ml Congo red. Plates were incubated at 28°C for 2 days. One set of plates was incubated at 37°C. Strains were also plated on l-DOPA (l-3,4-dihydroxyphenylalanine) medium to assess melanization.

Antibodies against LDHA (Cell Signaling Technology, Danvers, MA, USA, 3582, 1:2500), DOT1L (Cell Signaling Technology, 77087, 1:1000), H3K79me2 (Cell Signaling Technology, 5427, with 1:1000), BCAT1 (Cell Signaling Technology, Cat.88785, 1:1000), Tubulin (Santa Cruz, Santa Cruz, CA, USA, Cat. sc-5286, 1:2000), Lamin B1 (Cell Signaling Technology, Cat.13435, 1:2000), β-actin (Santa Cruz, Cat. sc-047778, 1:2000), and GAPDH (Santa Cruz, Cat.sc-47724, 1:2000) were used for Western blotting.
Antibodies against LDHA (Cell Signaling Technology, 3582, 1:500), BCAT1 (Cell Signaling Technology, Cat.88785, 1:500), GCLM (Proteintech, Rosemont, IL, USA, Cat.14241, 1:500), and TXNRD1 (Proteintech, Cat.11117, 1:500) were used for immunohistochemistry staining (IHC).
Hydrogen peroxide solution (H₂O₂) (Sigma, St. Louis, MO, USA, Cat.323381), N-acetyl cysteine (NAC) (Sigma, Cat.A7250), DAPI (Sigma, Cat.D9542), sodium 2-hydroxybutyrate (Santa Cruz, Cat. sc-258161), sulfasalazine (SSA) (Sigma, Cat.S0087), and EPZ004777 (Selleck, Washington, DC, USA, Cat.S7353) were commercially obtained.