Catalase cat

Gallic acid, Folin-Ciocalteu reagent, catechin, rutin, thiobarbituric acid, catalase (CAT), and malondialdehyde (MDA) were purchased from Sigma–Aldrich Co. (Sigma, St. Louis, USA). Standards (iron (Fe), zinc (Zn), calcium (Ca), potassium (K), magnesium (Mg), manganese (Mn), phosphorus (P), and sodium (Na)) were obtained from Merck (Darmstadt, Germany). Superoxide dismutase (SOD) and glutathione peroxidase (GPx) assay kits were purchased from Randox Laboratory Ltd. Methanol was purchased from PanReac (Barcelona, Spain). All other chemicals and solvents were of analytical grade and were obtained from Sigma–Aldrich (Sigma, St. Louis, USA).

Caspase activity was evaluated using the luminescent kit Caspase-Glo™3/7 Assay (Promega Corporation, Fitchburg, Wisconsin, USA). Morphological membrane changes were detected using Annexin V-EGFP/PI detection kit (Biovision, Mt. View, CA, USA). Viability was measured using the colorimetric CellTiter 96^® Aqueous One Solution Cell Proliferation Assay (Promega). The CellTiter 96^® Aqueous One Solution Reagent contains the tetrazolium compound 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS), and an electron coupling reagent (1-methoxy phenazine methosulfate—PMS). The liquid scintillation cocktail was the Ready-Gel (Beckman Instrument, Fullerton, CA, USA). The proteasome inhibitors PS-341 and MG-132, the necroptosis inhibitor Necrostatin-1 and the pan-caspase inhibitor carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]fluoromethylketone (Z-VAD-fmk) were supplied by Vinci-Biochem (Florence, Italy). The hydrogen peroxide scavenger catalase (CAT) and fludarabine (FLU) were purchased by Sigma-Aldrich. For SDS-PAGE, precasted gels and buffer strips obtained from GE Healthcare were used. Other reagents used were from Merck (Darmstadt, Germany), Carlo Erba (Milano, Italy) and Sigma-Aldrich.

Intracellular ROS was measured using 2′,7′-dichlorodihydrofluorescein diacetate (H₂DCF-DA, Eugene, OR, United States). Briefly, L929 cells were treated with 100 nM CNP for 24 h or 100 μM NAC, 100 UI SOD (Sigma-Aldrich, St. Louis, MO, United States) and 100 UI catalase (CAT, Sigma-Aldrich, St. Louis, MO, United States) for 1 h and irradiated with UVA at a unique dose of 30 J/cm² or with 15 J/cm² followed by an additional dose (15 J/cm²) after 24 h. After different times, cells were incubated with 5 μM H₂DCF-DA for 30 min in the dark at 37°C. Cell-associated fluorescence was detected using a spectrofluorimeter (VICTOR X3, PerkinElmer, United States, λex = 488 nm, λem = 525 nm). The fluorescence percentage was expressed as arbitrary fluorescence units per μg of protein determined by Bradford method (Bio-Rad, CA, United States).

PdCl₂ for the preparation of the PdNPs was purchased from Sigma-Aldrich (St. Louis, MO, USA). Penicillin-streptomycin solution, trypsin-ethylenediaminetetraacetic acid (EDTA) solution, Dulbecco’s modified Eagle’s medium (DMEM, F-12), and 1% antibiotic-antimycotic solution were obtained from Life Technologies (Carlsbad, CA, USA). A fetal bovine serum (FBS) in vitro toxicology assay kit was purchased from Sigma-Aldrich. The reagent kits for the measurement of malondialdehyde (MDA), superoxide dismutase (SOD), and catalase (CAT) were purchased from Sigma-Aldrich.

Four spin traps were selected to cover a large range of lipophilicity and reactivity against the main families of radicals. DEPMPO and EMPO were obtained from Radical Vision (Marseille, France). POBN and PBN were purchased from Enzo-Alexis Biochemicals (Lausanne, Switzerland). Spin traps were dissolved in water, except for PBN which was dissolved in DMSO. Stock solutions (2 M) were stored at -80°C.
Chemicals for the radical generating systems (xanthine, xanthine oxidase, diethylenetriaminepentaacetic acid (DTPA), ferrous ammonium sulfate (Fe (NH₄)₂(SO₄)₂), hydrogen peroxide (H₂O₂), sodium sulfite, sodium dichromate, sodium azide and horseradish peroxidase) were purchased from Sigma Aldrich (St Louis, MO).
Toxic agents were chosen in order to produce radicals representative of the main types commonly observed (O, N, C or S-centered radicals). Menadione bisulfite, hydrogen peroxide, tert-butylhydroperoxide, phenylhydrazine, catalase (CAT) and superoxide dismutase-polyethylene glycol (PEG-SOD) were obtained from Sigma Aldrich (St Louis, MO).

The cell viability after CT infection was also evaluated on cells pre-treated with 100 μM of the pan-caspase inhibitor Z-Vad-fmk (Z-VAD), 100 μM of the necroptosis inhibitor necrostatin-1 (Nec) and 100 U/ml of the hydrogen peroxide scavenger catalase (CAT) (Sigma Aldrich, Missouri, USA).
To evaluate the involvement of caspase-1 during chlamydial infection, the cell viability was also evaluated on cells pre-treated with 100 μM of the selective caspase-1 inhibitor Ac-YVAD-cmk (Sigma Aldrich).
Cells (1 × 10⁴/well) were seeded in a 96-well plate in 100 μL of complete medium and allowed to reach the 60% of confluence. Inhibitors or scavenger were added 3 h before the infection with CT at MOI 3 and plates were centrifuged at 640 × g for 2 h and incubated at 37 °C with 5% CO₂. After a 72 h-incubation, cell viability was evaluated as above described.