Oxidative DNA Damage

All samples were assessed using a microplate reader spectrophotometer (InfiniteM200, Tecan, Austria). All the determinations were duplicated, and the interassay coefficient of variation was in the range indicated by the kit's manufacturer.
The malondialdehyde (MDA) levels were analysed spectrophotometrically using the modified thiobarbituric acid-reactive substance method to determine the amount of lipid peroxidation in plasma. The measurement of thiobarbituric acid-reactive substances (TBARS) by a commercial assay kit (Cayman Chemical, USA) allows a rapid photometric detection at 535 nm of the thiobarbituric acid malondialdehyde (TBAMDA) adduct, as previously reported [7 (link)]. A linear calibration curve was computed from pure MDA-containing reactions.
The protein carbonyl (PC) content, an index of protein oxidation, was determined utilizing a commercial kit (Cayman Chemical, USA) through the reaction of 2,4-dinitrophenylhydrazine (DNPH) and carbonyls. This reaction forms a Schiff base producing the correspondent hydrazone. The latter was analysed by spectrophotometry, reading the absorbance signal in the 360–385 nm range. Values were normalized to the total protein concentration in the final pellet (absorbance reading at 280 nm) to consider protein loss during the washing steps.
8-OH-2-deoxyguanosine (8-OH-dG), established as a marker of oxidative DNA damage, was assessed by using a commercially available enzyme immune assay EIA kit (Cayman Chemical, USA). The EIA employs an anti-mouse IgG-coated plate and a tracer consisting of an 8-OH-dG-enzyme conjugate, while the sample 8-OH-dG concentration was determined using an 8-OH-dG standard curve. Meanwhile, samples and standards were read at a wavelength of 412 nm.
Nitrite (NO₂⁻)+nitrate (NO₃⁻) (NOx) level determination was performed by the spectrophotometric method to Griess reagent, utilizing a commercial colorimetric assay kit (Cayman Chemical, USA).
Nitric oxide synthase (iNOS) expression was assessed by using a commercial assay EIA kit (cat no. EH0556; FineTest, Wuhan China). This assay was based on sandwich enzyme-linked immune-sorbent assay technology and carried out according to the manufacturer's instructions, while NOS2/iNOS protein synthesis was determined using a standard curve. Samples and standards were read at a wavelength of 450 nm.
Interleukin-6, interleukin-1β, and interleukin-10 (IL-6, IL-1β, and IL-10, respectively) levels were determined by using commercially available enzyme immune assay kits (R&D Systems, USA; Cayman Chemical, USA; and BioVendor, Czech Republic, respectively) following the manufacturer's instruction. The assays are based on a double-antibody sandwich technique. The signal was spectrophotometrically measured.

Sequencing reads were mapped by BWA with a maximum of six mismatches and no gap46 (link). Amplicons with the same tag were collected to generate a read cluster. Since each read cluster was originated from the same template, true mutations were called only if the mutations occurred in 90% of the reads within a read cluster. We acknowledged that this error-correction approach would only correct errors that occured during the deep sequencing process but not those that were introduced during the reverse transcription process. Read clusters with a size below three reads were filtered out. Read clusters were further conflated into “error-free” reads. Average coverages in terms of “error-free” reads were 177028 per nucleotide in the plasmid mutant library, 112355 per nucleotide in replicate 1 of passaged viral mutant library, and 161773 per nucleotide in replicate 2 of passaged viral mutant library (Fig. S1A). Relative fitness index (RF index) for individual point mutations was computed by: For all the downstream analysis, only point mutations covered with ≥30 tag-conflated reads (“error-free” reads) in the plasmid library were included. This arbitrary cutoff filtered out mutants with low statistical confidence, which is ~16% of all possible point mutations (Fig. S1B). In addition, all C → A and G → T mutations are not included in the reported dataset due to an observed DNA oxidative damage during library preparation47 (link). The RF index presented in Table S1 was calculated by averaging all RF indices available for a given amino acid substitution.

The human hOGG1, the primary enzyme for the repair of 8-oxoGua, was used to assess the extent of oxidative modification to the DNA bases [22 (link), 23 (link)]. The enzyme nicks the DNA strand at the 8-oxoGua sites, producing single-strand breaks (SSBs) which can be easily detected by the alkaline comet assay.
The comet assay was performed under alkaline conditions essentially according to the procedure of Singh et al. [24 (link)] with modifications [25 ] as described previously [26 (link)]. A freshly prepared suspension of cells in 0.75% LMP agarose dissolved in PBS was spread onto microscope slides precoated with 0.5% NMP agarose. The cells were then lysed for 1 h at 4°C in a buffer consisting of 2.5 M NaCl, 100 mM EDTA, 1% Triton X-100, 10 mM Tris, pH 10. After lysis, the slides were placed in an electrophoresis unit, the DNA was allowed to unwind for 20 min in the electrophoretic solution consisting of 300 mM NaOH, 1 mM EDTA, pH >13. Electrophoresis was conducted at 4°C (the temperature of the running buffer did not exceed 12°C) for 20 min at an electric field strength of 0.73 V/cm (290 mA).
The slides were then washed in water, drained and stained with 2 μg/ml DAPI and covered with cover slips. To prevent additional DNA damage, all the steps described above were conducted under dimmed light or in the dark. The slides were observed at ×200 magnification using an Eclipse fluorescence microscope (Nikon, Tokyo, Japan) attached to a COHU 4910 video camera (Cohu, Inc., San Diego, CA, USA) equipped with a UV-1 filter block consisting of an excitation filter (359 nm) and barrier filter (461 nm) and connected to a personal-computer-based image analysis system, Lucia-Comet v. 4.51 (Laboratory Imaging, Praha, Czech Republic). A hundred images was randomly selected from each sample and the comet tail DNA (% tail DNA) was measured. Each experiment was repeated three times. % tail DNA is positively correlated with the level of DNA breakage or/and alkali labile sites and is negatively correlated with the level of DNA crosslinks in the alkaline version of the comet assay [17 (link)]. In the pH 12.1 and neutral version, it is positively correlated with strand breaks and DSBs, respectively. The mean value of the % tail DNA in a particular sample was taken as an index of the DNA damage in this sample.
After incubation with HEMA/Bis-GMA and cell lysis the slides from the comet assay were washed three times in the enzyme buffer containing 40 mM HEPES–KOH, 0.1 M KCl, 0.5 mM EDTA, 0.2 mg/ml bovine serum albumin, pH 8.0 for 5 min each time and drained. The agarose on slides was covered with 30 μl of the enzyme buffer either with or without hOGG1 at 1 μg/ml, sealed with a cover glass and incubated for 10 min at 37°C [27 (link)]. The slides were processed as described in “Cells and treatment” section. To check the ability of the enzyme to recognize the DNA oxidative damage, we exposed HGF to 20 μM hydrogen peroxide for 10 min on ice (positive control). We compared the values obtained for the hOGG1 enzyme with the control containing only enzyme buffer.

Ox-mtDNA for treatment was synthesized from mtDNA extracted using the Mitochondrial Extraction Kit according to the manufacture’s protocol (Active Motif, Carlsbad CA, USA) and amplified by ND1 primers (ND1 Forward: 5′-CCCTAAAACCCGCCACATCT-3′; ND1 Reverse: 5′-GAGCGATGGTGAGAGCTAAGGT-3′) with the addition of oxidized guanosine to the master mix and mtDNA amplified, as described in [13 (link)] (Supplemental Figure S1). The pyroptotic TLR4 signaling pathway was activated by incubation with LPS, ATP, and nigericin (LAN) [23 (link)]. Caspase-Glo^® 1 Inflammasome and LDH-Glo^® Cytotoxicity assays (Promega Corporation, Madison, WI, USA) were used to assess inflammasome activation following manufacturer’s protocols. Ox-mtDNA levels were quantified using the DNA/RNA Oxidative Damage (High Sensitivity) ELISA Kit (Cayman Chemical Company, Ann Arbor, MI, USA) [19 (link)].

The effect of IPC on skeletal muscle damage due to ischemia-reperfusion injury was studied using immunohistochemical staining of neovascular factors (VEGF), oxidative DNA damage (8-hydroxyguanosine (8-OHdG)), and inflammatory markers (cyclooxygenase 2 (COX-2)). In all groups, femoral sections were prepared, and sections obtained from the femoral proximal medial diaphysis of each group were deparaffinized with xylene and ethanol. For antigen retrieval, the sections were autoclaved at 121 °C for 15 min. Using 0.3% H₂O₂, endogenous peroxidase was eliminated, blocking was performed using a mouse or goat normal serum, and the primary antibody was made to react. As the primary antibody, anti-Bax rabbit polyclonal antibody (Abcam, Cambridge, UK), anti-VEGF rabbit polyclonal antibody (Proteintech, Rosemont, IL, USA), anti-8-OHdG mouse monoclonal antibody (Abcam), and COX-2 rabbit polyclonal antibody (Proteintech) were used at 1.25, 5.0, 20.0, and 5.0 µg/mL, respectively. The reaction time was overnight at 4 °C in a cool dark room. After reacting with the primary antibody, the secondary antibody (biotin) reacted with an enzymatic agent (streptavidin). After 5 min immersion in DAB to allow for color development, the nuclei were stained. Photographs were taken using a BX53 microscope (Olympus, Tokyo, Japan) and a DP71 camera (Olympus).
VEGF and COX-2 were randomly extracted from their respective muscle tissues with 10 fields of view (400 times) and quantified using ImageJ. In 8-OHdG, cell nuclei expressed by 8-OHdG were extracted from 10 fields of view per sample, and the IPC (+) group was compared with the IPC (−) group.

As a marker of lipid peroxidation, MDA was measured from the cell culture media with a specific colorimetric test. According to the protocol, 300 μL freshly prepared thiobarbituric acid (TBA) stock solution was mixed with 100 μL cell culture media. Solutions were incubated at 95 °C for 1 h, followed by 10 min cooling on ice. The absorbance of the samples was read at 532 nm with a Multiscan GO 3.2 reader.
Protein damage caused by oxidative stress was examined with a Protein Carbonyl ELISA Kit (Cat. No. MBS2600294, MyBioSource, San Diego, CA, USA) by measuring the protein carbonyl content of the cell lysate. To monitor the oxidative DNA damage, 8-OHdG concentration was assayed from the cell lysate with a specific 8-OHdG ELISA kit (Cat. No. MBS808265, MyBioSource, San Diego, CA, USA). The measurements were carried out according to instructions of the manufacturer’s protocol. The absorbances of the samples were read at 450 nm by a Multiscan GO 3.2 reader.
As a marker of endoplasmic reticulum stress, a chaperone protein—GRP78—was measured from cell lysate with a feline-specific ELISA kit (Cat. No. MBS072358, MyBioSource, San Diego, CA, USA), based on the instructions of the manufacturer’s protocol. The absorbance was read at 450 nm via a Multiscan GO 3.2 reader.