*2.6. Western Immunoblotting*

Protein content of Tf receptor 1 (TFR1), ZIP8, ZIP14, and 8-oxoguanine DNA glycosylase (OGG1), and levels of 3-nitrotyrosine (3-NT) were measured in muscle samples by Western immunoblotting. Whole-tissue extracts were prepared as described elsewhere [24]. Briefly, 50 μg proteins were separated on 12%–15% polyacrylamide gels (Bio-Rad Laboratories, Hercules, CA, USA), transferred onto polyvinylidene difluoride membranes (Bio-Rad Laboratories), and blocked for 1 h in 5% milk in Tris-buffered saline Tween (Bio-Rad Laboratories). Blots were probed with commercially available primary antibodies for OGG1 (1:2500, Abcam, Cambridge, MA, USA; #ab63942), TFR1 (1:1000, Cell Signaling Technology, Beverly, MA, USA; #13113), ZIP14 (1:1000, Sigma–Aldrich, St. Louis, MO, USA; #HPA016508), and 3-NT (1:1000, Cell Signaling Technology; #9691S). A custom-made polyclonal rabbit primary antibody was used for detecting ZIP8 (1:1000). The antibody was raised to a peptide [(NH2) FGNDNFGPQEKT (COOH)] selected from the full-length sequence [33] designed by Dr. Tolunay Beker Aydemir (University of Florida, Gainesville, FL, USA) who also performed the purification [34]. To allow affinity purification, a cysteine residue was added to the N terminus for coupling to the carrier protein and for conjugation to Sulfolink (Pierce, Rockford, IL, USA). The antibody was prepared in rabbit as previously described [35]. Anti-rabbit secondary antibody conjugated with horseradish peroxidase (1:10000, Cell Signaling Technology; #7074) was used to enable subsequent protein detection. Protein bands were visualised with SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Scientific) using a ChemiDoc XRS imager (Bio-Rad Laboratories). Spot density of the target bands was normalised to the amount of protein loaded in each lane, as determined by densitometric analysis of the corresponding Ponceau S-stained membranes [36]. Bands were quantified using Image Lab 6.0 software (Bio-Rad Laboratories) according to the "Total Lane Protein" setting.

#### *2.7. Quantification of Mitochondrial DNA (mtDNA) Content*

Genomic DNA was purified from muscle samples using a Wizard Genomic DNA Purification Kit according to the manufacturer's instructions (Promega, Madison, WI, USA). Briefly, 10–20 mg of muscle tissue were homogenised in 1 mL of nuclei cell lysis solution using a hard tissue disposable probe (Omni international, Kennesaw, GA, USA) on a PowerGen 500 homogenator (Thermo Fisher Scientific). Total DNA quantification was carried out on a NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific) and integrity was verified by gel electrophoresis on 0.8% agarose gel in 1× Tris-borate-EDTA (TBE) (90 mM Tris-borate pH 7.4, 90 mM boric acid, 2.5 mM EDTA). Determination of mtDNA content was performed with the Human Mitochondrial DNA Monitoring Primer Kit (Takara Bio, Mountain View, CA, USA) using real-time polymerase chain reaction (RT-PCR). Amplification reactions were run on a CFX96 Touch™ Real-Time PCR Detection System (Bio-Rad Laboratories). Primers included in the kit specifically amplified mitochondrial genes corresponding to mitochondrial NADH dehydrogenase subunit 1 and 5 (ND1, ND5) and nuclear genes corresponding to solute carrier organic

anion transporter family, member 2b1 (SLCO2B1), and serpin family A member 1 (SERPINA1) [37]. Melting curve analysis, non-template control reactions, and gel electrophoresis of PCR products were used to check amplification specificity of each experiment. Each sample was analysed in triplicate in 20 μL final volume. The reaction mixture consisted of 1× Terra qPCR Direct SYBR Premix (Takara Bio), 0.2 μM forward and reverse primers, and 10 ng of genomic DNA template. Amplification proceeded for 40 cycles. Quantification of relative mtDNA content was accomplished according to the Pfa ffl mathematical model [38]. Di fferences in threshold cycle values for the ND1/SLCO2B1 pair ( ΔCt1 = Ct for SLCO2B1 – Ct for ND1) and the ND5/SERPINA1 pair ( ΔCt2 = Ct for SERPINA1 – Ct for ND5) were calculated, and the average of 2ΔCt for the values of ΔCt1 and ΔCt2 was used as a measure of relative mtDNA abundance.

#### *2.8. Analysis of mtDNA Damage*

Quantitative RT-PCR was used to assess mtDNA damage according to the method described by Furda et al. [39] with minor adjustments. Briefly, 225 ng of purified total DNA was digested with PvuII Restriction enzyme (New England Biolabs, Ipswich, UK). Fifteen ng of digested DNA were used to amplify a 8.9-kb mtDNA fragment (accession number: J01415; 5 sense position: 5999; 5 antisense position: 14841) [39] with a TaKaRa LA Taq ® DNA Polymerase with GC Bu ffer (Takara Bio) and a 221-bp mtDNA fragment (accession number: J01415; 5 sense position: 14620; 5 antisense position: 14841) [39] with a DreamTaq DNA Polymerase (Thermo Fisher Scientific). Amplification was carried out using a CFX96 Touch ™ PCR Detection System (Bio-Rad Laboratories) as described by Furda et al. [39]. Each sample was analysed in triplicate in 20 μL final volume. The reaction mixture for the 8.9-kb mtDNA fragment consisted of 1× GC Bu ffer I, 2U TaKaRa LA Taq ® DNA Polymerase (Takara Bio), 0.2 mM dNTPs, and 0.4 μM forward and reverse primers. The reaction mixture for the 221-bp mtDNA fragment included 1× DreamTaq Bu ffer (Thermo Fisher Scientific), 0.2 mM dNTPs, and 0.4 μM forward and reverse primers. Prior to quantification, amplification products of the 8.9-kb and the 221-bp fragments were electrophoresed on 0.8% agarose and 1.5% agarose gels, respectively, to check for PCR product specificity. Amplicons were quantified by Pico-Green (Thermo Fisher Scientific) using a Synergy HT multidetection microplate reader (BioTek) with excitation and emission wavelengths at 485 and 530 nm, respectively. Data obtained from the 221-bp mtDNA fragment were used to normalise results of the 8.9-kb fragment amplification. The number of mtDNA lesions was calculated using the equation: D = [1 − 2−(Δ8.9-kb − Δ221-bp)] × 10,000 bp/8900 bp [40].

## *2.9. Statistical Analysis*

The normal distribution of data was ascertained through the Kolmogorov–Smirnov test. Comparisons for normally distributed continuous variables were performed by one-way analysis of variance (ANOVA) followed by Tukeys post-hoc test when applicable. The non-parametric tests Mann–Whitney U and Kruskal–Wallis H (with Dunns post-hoc test as appropriate) were applied to assess di fferences for non-normally distributed continuous data. Di fferences in categorical variables among groups were determined via χ2 statistics. Correlations between variables were explored via Pearsons or Spearmans tests as appropriate. All analyses were performed using the GraphPrism 5.03 software (GraphPad Software, Inc., San Diego, CA, USA), with statistical significance set at *p* < 0.05.
