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2.6.1

DNA extraction from tissue

Approximately 1 gram of frozen tissue sample was incubated in lysis buffer (1M Tris, 0.5M EDTA, 10% SDS, 5M NaCl, pH 8.0) and 20 mg/mL proteinase K (Roche, Mannheim, Germany) overnight at 55℃ with constant agitation. Samples were centrifuged at 200xg for 10 minutes at room temperature, and the supernatants in each sample were transferred into new tubes. 100 μL of Protein precipitation solution (catalog number: A975A, Promega Corporation, WI, USA) was added to samples and centrifuged at 14,000xg for 10 minutes. Supernatants were briefly chilled on ice followed by centrifugation at 14,000xg for 5 minutes, with pellets discarded. Immediately after, ice-cold isopropanol (500 μL) was added to supernatants, mixed by inversion five times and incubated at -20℃ for 30 minutes.

All samples were centrifuged at 14,000xg for 15 minutes, and pellets were washed with ice-cold 70% ethanol. After, samples were centrifuged at 14,000xg for 10 minutes again and washed pellets using ice-cold 70%. Resulting pellets were air-dried at room temperature and suspended in 20 μL of ddH2O. Sample concentration were measured using

NanoDrop One/OneC (ThermoFisher Scientific, MA, USA), and stored in -20℃ until further use. Only samples with an A260/280 ratio between 1.8 to 1.9 were used in future

experiments.

2.6.2

8-oxo-2’-deoxyguanosine ELISA

Nuclear ROS-adducts 8-oxo-2’-deoxyguanosine (8-oxo-dG) were detected using a commercially available ELISA kit (catalog number: 4380-096-K, Trevigen, MD, USA). DNA was extracted from frozen tissue as described in section 2.5.1. Samples were diluted in nuclease-free ddH2O to 200 μg/mL, and 100X cations (catalog number: 4380-096-05)

was added for a final 1X concentration. 0.8 μL of DNase I (catalog number: 4380-096-06, 5 U/uL) and alkaline phosphatase (catalog number: 4386-096-07) were added to each sample and incubated at 37℃ for 1 hour separately. Digested samples were aliquoted and

were serially diluted with assay diluent (catalog number: 4380-096-02) to construct a standard curve (200 nM, 100 nM, 50 nM, 25 nM, 12.5 nM, 6.25 nM, and 3.13 nM). 25 μL of standards, blank and DNA samples were pipetted in triplicates into a 96-well plate pre- coated with 8-oxo-dG, with 25 μL of anti-8-OHdG monoclonal antibody (catalog number: 4380-096-03, diluted 1:250-fold in assay diluent) added to each well. The plate was covered with a film sealer and incubated for 1 hour at 25℃. All wells were washed with 4

changes of 1xPBST (137 mM NaCl, 12 mM phosphate, 2.7 mM KCl, pH 7.4, 0.1% Tween- 20) for 30 seconds, with excess liquid aspirated. Goat-anti-mouse IgG-HRP conjugate (catalog number: 4380-096-04, diluted 1:500-fold in assay diluent) was added to all wells, and incubated at 25℃ for 1 hour. The plate was then washed with 4 changes of 1xPBST

with excessive liquid aspirated. 50 μL of room-temperature TACS-Sapphire colorimetric substrate (catalog number: 4822-096-08) was added to all wells, and incubated for 15 minutes at 25℃ in the dark. The reactions were stopped by adding 50 μL of 5% phosphoric

acid to all wells, and absorbance was read immediately at 450 nm.

The average absorbance for all standards, samples and blanks were calculated, with the blank average subtracted from standard and sample averages to determine relative absorbance. The log of 8-oxo-dG standard concentration (ng/mL) was plotted against the relative absorbance to derive a standard curve, and 8-oxo-dG concentration in samples was calculated and interpolated from the standard curve.

2.6.3

Cyclobutane pyrimidine dimer ELISA

Protocol was carried at as described by the manufacturer (catalog number: STA-322, Cell Biolabs). Briefly, genomic DNA (gDNA) was extracted from tissue samples as described in section 2.7.1, and incubated at 95℃ for 10 minutes to denature double-stranded DNA

(dsDNA) into single-stranded DNA (ssDNA). Samples were briefly chilled on ice, and diluted to 4 μg/mL in ice-cold 1xTE buffer (10 mM Tris, pH 8.0, 1mM EDTA). Similarly, CPD-DNA standards and reduced DNA provided in the commercial kit were denatured into ssDNA via incubation at 95℃ for 10 minutes. Standards were serially diluted to create

a standard curve (0, 1.56, 3.13, 6.25, 12.5, 25, 50, 100 ng/mL) in ice-cold TE buffer. Samples and CPD-DNA standards were added to a 96-well plate, with an equal volume of

DNA binding solution added to each well and mixed thoroughly via pipetting. The plate was incubated at room temperature overnight with constant agitation. Solutions were aspirated and wells were washed twice with 1xPBS, with excess fluid removed by blotting. Assay diluent was added to each well to block non-specific binding at room temperature for 1 hour. After assay diluent was removed from well, anti-CPD antibody (catalog number: 232202, Cell Biolabs) were diluted 1:1000 in 1xTE buffer and added to each well and incubated at room temperature for 1 hour with constant agitation. All wells were washed five times with wash buffer and blotted to remove excess fluid, followed by incubation with secondary antibody-HRP conjugate (diluted 1:1000 in 1xTE buffer, catalog number:10902, Cell Biolabs) at room temperature for 1 hour on an orbital shaker. Plate was washed five times in wash buffer and excess fluid was removed. Substrate solution warmed to room temperature was incubated in each well for 20 minutes, stop solution was added to each well to stop the enzyme reaction. Absorbance of all wells was read using a microplate reader at 450 nm immediately after stop solution addition. Reduced DNA standard was used as an absorbance blank, and all DNA samples were assayed in triplicates. The average absorbance for all standards, samples and blanks was calculated, with the blank average subtracted from standard and sample averages to determine relative absorbance. The CPD-DNA standard concentration (ng/mL) was plotted against the relative absorbance at OD450nm to derive a standard curve. The CPD concentration in

extracted DNA was calculated and interpolated using the standard curve.