Catalase from bovine liver

dSTORM imaging was performed using an Elyra PS.1 microscope (Carl Zeiss Microscopy) equipped with a Plan-Apochromat 100×/1.46 oil objective and a liquid cooled EMCCD camera (Andor Technology). Imaging was carried out in MEA imaging buffer as previously described (36 (link)). In short, fresh stock solutions (1 M cysteamine in 360 mM HCl, 10% glucose in PBS, 70 mg/ml glucose oxidase in PBS, and 20 mg/ml catalase in PBS) were prepared the day before imaging and stored at 4°C and mixed directly before imaging to final concentrations of 0.124 M cysteamine (Sigma), 44.8 mM HCl, 8.6% glucose, 1.08 mg/ml glucose oxidase from Aspergillus niger (Sigma), and 0.0773 mg/ml catalase from bovine liver (Sigma) in PBS. Imaging was performed in 12.8 × 12.8-μm areas in an inclined total internal reflection fluorescence microscope mode (37 (link)). Single molecule fluorescence detection on the EMCCD camera was acquired with 100 × 100-nm pixel size, 20-ms Exposure time, and 100 Gain. 20,000 image frames were acquired for each channel. Both channels were imaged sequentially in 500 frame sequences and the appropriate filters and lasers for each dye were used (642 nm for Alexa Fluor 647 and 488 nm for Atto 488). The images were analyzed with the ImageJ plugin SMLocalizer (38 (link)).

Hydrogen peroxide (H₂O₂) has been used to study the effect of ROS produced during viral infection on nodavirus replication. A 30% stock solution of H₂O₂ (Perhydrol; Merck, Darmstadt, Germany) was sterilized by filtration through a 0.22 µm-pore filter and kept in a dark sealed container. The viral suspension (10⁴ TCID₅₀/mL) was treated with the H₂O₂ solution at a final concentration of 3% and incubated for 5 or 24 h at 4 °C. To stop the reaction and remove residual H₂O₂, the viral suspensions were treated twice with 12.5 U/mL of catalase from bovine liver (Sigma-Aldrich) for 10 min at room temperature. Viral suspensions without H₂O₂ treatment were incubated under the same conditions and served as a control for the infection, and aliquots of H₂O₂-catalase treated medium were also included as a control for the treatment. Then, SSN-1 cells seeded in 24-well plates were inoculated with these suspensions (4 wells per condition) and incubated at 25 °C for 72 h. After this period, viral replication was analysed by qPCR.

Sodium percarbonate (SPO) (20–30% H₂O₂), calcium peroxide (CPO) (75% purity), fibrinogen from bovine plasma (type I-S, 65–85% protein; ≥75% of protein was clottable), hyaluronic acid sodium salt (HA) from Streptococcus equi, glycerol, gelatin from porcine skin (gel strength: ~175 g Bloom, type A), thrombin from bovine plasma, aprotinin from bovine lung, and catalase from bovine liver (2000–5000 U/mg) were purchased from Sigma Aldrich (St. Louis, MO, USA).

Ethanediamine (EDA, 99%), formaldehyde (30–40%), ethyl alcohol (99.7%), 3-aminophenol (99%), ethylenediamine tetraacetic acid disodium salt (EDTA·2Na, 99%), H₂SO₄ (95–98%), HCl (36–38%), NH₃·H₂O (25–28%), and BaCl₂·2H₂O (99.5%) were purchased from Sinopharm Chemical Reagent (Shanghai, China). NaOH (96%) and Na₂SO₄ (99%) were purchased from Shanghai Dahe Chemicals (Shanghai, China). NaH₂PO₄·2H₂O (99%), and Na₂HPO₄·12H₂O (99%) were purchased from Chinasun Specialty Product (Changshu, China). Tetraethoxysilane (TEOS, 99%), catalase (from bovine liver, 2000–5000 units/mg protein), 3,3′,5,5′-tetramethylbenzidine (TMB, 98%), and peroxidase from horseradish (HRP, ~200 units/mg) were purchased from Sigma–Aldrich (St. Louis, MO, USA). All chemicals were used without further purification.

Glucose oxidase (GOx), sodium L-ascorbate (L-ASC), and catalase from bovine liver were obtained from Sigma Aldrich (St Louis, MO, USA), the reagents were dissolved in sterile distilled water. Hydrogen peroxide (H₂O₂) was dissolved in appropriate medium. Adenanthin (ADNT) was purchased from Faces Biochemical Co. (Wuhan, China) and dissolved in DMSO.

Catalase activity was determined by the rate of hydrogen peroxide absorbance decrease at 240 nm [30 (link)]. The reaction mixture contained 50 mM potassium phosphate buffer (pH 7.0) and an aliquot of the sample. The reaction started with adding hydrogen peroxide (final concentration of 10 mM), and absorbance was monitored at 25 °C for 5 min. Catalase from bovine liver (Sigma-Aldrich, St. Louis, MO, USA) was used as a standard.