Nitrotetrazolium blue chloride

SOD1 enzymatic activity was determined using in-gel zymography as previously described^{57 (link)}. Briefly, lumbar spinal cord PBS-soluble fractions (2.5 μg) and 100 ng of purified recombinant human SOD1^WT protein was separated in 8% native PAGE gels. Subsequently, gels were incubated in 5 mM nitrotetrazolium blue chloride (Sigma-Aldrich) for 20 min with gentle agitation, before incubation in developer solution (10 mM tetramethylethylenediamine and 30 µM riboflavin) for 15 min. Gels were developed by exposure to fluorescent light until sufficient contrast between the achromatic zones and background was achieved and images captured using a calibrated densitometer (GS-900; Bio-Rad, Australia). SOD1 activity was quantified as absorbance of the clear bands using ImageJ software (version 1.48, https://imagej.nih.gov/ij/). Each sample was run in duplicate. To account for equal protein between samples, SOD1 activity was normalised to InstantBlue Coomassie stain (Sigma-Aldrich) from samples run on an adjacent gel.

Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), and Dulbecco’s phosphate buffered saline with calcium and magnesium (PBS) were obtained from Gibco (Invitrogen, Poisley, UK). The LDH Cytotoxicity Detection Kit was purchased from Roche (Roche Diagnostics, Indianapolis, IN, USA), while crystal violet from Fluka (Basel, Switzerland). Methanol, potassium hydroxide, dimethyl sulfoxide (DMSO), formaldehyde, and phosphoric acid were obtained from POCh (Gliwice, Poland). Resazurin, propidium iodide (PI), sulfanilamide, N-(1-naphthyl) ethylenediamine dihydrochloride, 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA), nitrotetrazolium blue chloride (NBT), and RNAse were purchased from Sigma-Aldrich (St. Louis, MO, USA). NIST1648a was obtained from the National Institute of Standards and Technology (Gaithersburg, MD, USA). Culture dishes were purchased from Corning (New York, NY, USA) and Nunc (Roskilde, Denmark).

A549 cells were obtained from ATCC. For soft agar colony formation assays, A549 stable cell lines expressing WT LKB1, LKB1-KD (K78I), or H174R mutants were suspended in F12 culture medium containing 10% FBS and 0.3% agarose. Cells were seeded at 2000 per well onto a layer of F12 culture medium containing 10% FBS and 0.8% agarose in 12-well plates, allowed for proliferation for 14 days, and stained with 0.5 mg/mL nitrotetrazolium blue chloride (Sigma-Aldrich) for 24 hr. Colonies were visualized by light microscopy (4×) and counted. Results were expressed as mean values from triplicate wells.

For tissue analysis, mice were sacrificed after 15 weeks of treatment. Two hours after the last treatment, mice were euthanized by isofluorane inhalation. After euthanasia, tissues were weighted and flash-frozen in isopentane precooled in liquid nitrogen, then stored at −80 °C until further processing. Gastrocnemius muscles were embedded in optimal cutting temperature (OCT) compound (Tissue Tek, Sakura, Torrance, CA), and cross sections (10 μm-thick) were cut with a cryostat (CM1850 UV, Leica Microsystem). Cryosections of gastrocnemius were stained for nicotinamide adenine dinucleotide (NADH). For NADH, sections were air-dried and incubated in NADH solution [0.2 M TRIS-HCL, Nitrotetrazolium Blue chloride (Sigma), β-nicotinamide adenine dinucleotide, (Sigma)] at 37 °C for 40 minutes. Sections were briefly washed in 30%, 60%, 90%, 60%, and 30% acetone solutions. Sections were then washed in water, dried, and mounted with aqueous mounting medium [Gelatin (Serva), sodium azide (Merck), and glycerin]. Images were taken using a Nikon Eclipse 90i upright microscope, the analyses were performed using ImageJ software, and pixel number was converted to μm². The number of fibers and mean cross-sectional area (CSA) distribution were evaluated by a blinded investigator from 3–5 randomly selected areas of 3 sections of each muscle for each genotype.

To further characterize the virulence factors of Gardnerella clinical isolates, we investigated the presence and expression of the sialidase gene. The presence of the sialidase gene in clinical isolates of Gardnerella was identified by PCR using specific primers (Supplementary Table 1). In addition, The Gardnerella cultures were diluted to an OD₆₀₀ value of 0.8 with 50 mM MES buffer (pH 5.5). The reaction system contained 2.2 mM NBT (Nitrotetrazolium Blue chloride, Sigma-Aldrich, St. Louis, MO), 146 mM sucrose, 10.5 mM MgCl₂, 6.3 mM BCIN (5-Bromo-4-chloro-3-indolyl α-D-N-acetylneuraminic acid sodium salt, Sigma-Aldrich, St. Louis, MO), with 0.5% sulfonyl 440 added to avoid bubbles. The mixture was then incubated at 37°C for 20 min and 100 μL of the incubated mixture was added to the wells of black polystyrene microplates (Nunc, Thermo Fisher Scientific). The plates were sealed with an optically clear seal, and BCIN hydrolysis was monitored by measuring the fluorescence at a wavelength of 616 nm using a SynergyH4 hybrid multi-mode microplate reader (Biotek, Winooski, VT, USA) (Zhang and Rochefort, 2013 (link); Janulaitiene et al., 2018 (link)). The fluorescence of each supernatant was analyzed in triplicate.

Dihydrochloride (AAPH), 2,2,-Azinobis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), 2,2′-azobis-2-methyl-propanimidamide, 2,2-Diphenyl-1-picrylhydrazyl (DPPH), 2,4,6-Tris(2-pyridyl)-s-triazine (TPTZ), catechin hydrate, nitrotetrazolium blue chloride, phosphate-buffered saline (PBS), and Trolox were acquired from Sigma-Aldrich (St. Louis, MO, USA). Acetic acid and dimethyl sulfoxide (DMSO) were purchased from RCI Labscan (Bangkok, Thailand). Sodium carbonate (Na₂CO₃), aluminum chloride (AlCl₃), sodium hydroxide (NaOH), potassium persulfate (K₂S₂O₈), ethylenediaminetetraAcetic acid (EDTA), iron (III) chloride hexahydrate (ferric chloride), and sodium nitrite (NaNO₂) were purchased from KAMAUS (New South Wales, Australia). Gallic acid was purchased from Acros Organics (Geel, Belgium). Solvents, including methanol, ethyl acetate, and ethanol, were purchased from J. T. Baker (Radnor, PA, USA). Other agents utilized were analytical-grade substances that were bought from commercial sources. During sample preparation, dilution, and pre-analytical rinses, deionized water was utilized.