Antibodies and reagents. Monoclonal antibodies (Abs) against MyoD (sc-32758), Mth1 (sc-271082) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-RAS (ab 52939) and anti-pY20 (ab10321) monoclonal Abs were from Abcam (Cambridge, U.K.). Monoclonal Abs against RhoA (76B9) (#2117), ERK (#4695S) and p-JNK(#9255), as well as polyclonal Abs against Cdc42 (#2462), Rac (#2465) p38 (#9212), pERK (#9101S), p-p38 (#9211S), and JNK (#9252), were purchased from Cell Signaling Technology (Danvers, MA, USA). Monoclonal Abs against GST (HT601-01), GFP (HT801-01), and GAPDH (HC301-01) were from TransGen Biotech (Beijing, China). The polyclonal Ab against Ogg1 (BS6826) was from Bioworld Technology (Minnesota, USA). The monoclonal Ab against MHC (F20) was from DSHB (Developmental Studies Hybridoma Bank). Anti-8-oxoG (MOG100P) Ab was from JaICA (Shizuoka, Japan). 8-Oxo-guanine (8-oxoG) was from (Cayman Chemical, Ann Arbor, MI, USA). Guanine (G) (G104274) and 8-hydroxyguanosine (8-oxodG) (H303620) were from Aladdin (Shanghai, China). Recombinant human OGG1 (NBP1-45318) and human MTH1 (NBP1-49309) were purchased from Novas biologicals (Littleton, USA). Y27632 (RhoA inhibitor), Kobe0065 (Ras inhibitor) and EHop-016 (Rac1 inhibitor) were purchaded from Selleck (Shanghai, China).
Plasmids
Eukaryotic expressional plasmid pCS2(+)-Flag-MyoD were kindly provided by Dr. Stephen Tapscott (FHCRC, Seattle, WA). To generate the Myog-promoter luciferase reporter plasmid, the mouse myogenin promoter (-1348 ~ -65 nt, containing multiple MyoD transcription factor motifs predicted by JASPAR CORE 2018 vertebrates) was cloned from the C2C12 genome and inserted into reporter vector pGL4.2 (Promega, Madison, WI) using restriction enzyme sites KpnI and BglII. GST-MyoD, GFP-MyoD, GFP-MyoD (Y156F/V). Mutant derivatives of mice MyoD were created by changing the amino acid at position 156 from tyrosine to phenylalanine/ valine (Y156F/V) using a Fast Mutagenesis System kit according to the manufacturer’s instructions (TRANS, FM111-01).
Cell Culture and treatment. CH310T1/2 fibroblasts and C2C12 myoblasts were cultured in growth medium (Dulbecco’s modified Eagle’s medium with L-glutamine (400 mM), in 10% fetal bovine serum and 1% penicillin/streptomycin at 37°C in a 5% CO2 atmosphere. To initiate myogenic differentiation of C2C12 myoblasts, 10% fetal bovine serum was replaced with 2% horse serum (Gibco). When the cells were treated with 8-oxo-guanine (8-oxoG) or guanine (G) , the reagents were dissolved as recommended by the manufacturer (Cayman Chemical, Ann Arbor, MI). Briefly, a 10 mM stock solution was prepared in 100 mM NaOH (pH 12), and the working concentration is 10 µM. Stock solutions were used immediately or stored at room temperature in dark for a maximum of 1 week35.
Quantitative real-time PCR (qRT-PCR). To detect the mRNA levels of Total RNA was extracted using Trizol (Takara, Japan), and reverse-transcribed by using Prime Script 1st strand cDNA synthesis kit (Takara). Real-time PCR was performed by using the Prime Script RT Master Mix (Takara) and TB Green Premix Ex Taq II (Takara). The gene expression was calculated by ∆∆Ct method. The primer sequences were shown in Supplementary Table S1.
Transfection with siRNA. The transfection of siRNA into C2C12 cells was performed in six-well plates. The first round of transfection with siOgg1, siMth1, siMyoD or control was performed 12 h prior the culture medium was changed to differentiation medium. Cells were transfected again 48 h after the first round of transfection and the expreiments were performed 2 days later. For all transfections, Lipofectamine 3000 (Invitrogen) was used following the nanufacturer’s protocol. SiRNA duplexes (100 pmol) was used for each well of a six-well plate, and the siRNA sequences are shown in Supplementary Table S2.
Immunofluorescence staining. C2C12 cells were cultured on coverslips in 24 wells plates and exposed to vehicle/G/8-oxoG for 4 days (one does per day). The coverslips were washed once with PBS (in PBS for 5 min), and then incubated in 0.25% Triton X-100 in PBS for 10 min at room temperature, and after another two washes, blocked in 3% FBS for 2 h. The coverslips were then incubated with anti-MHC antibody (1:200) at 4 °C overnight, followed by FITC-conjugated goat anti-mouse fluorescent secondary antibody (1:200) for 1 h. After three times of washing with PBS, cells were incubatd with DAPI (D1306, Invitrogen) (1:8000) for 5 min to visualize cell nuclei.
Differentiation Index. The differentiation index indicating the efficiency of C2C12 differentiation was determined by calculating the number of MHC+ cells in each microscopic field as described previously84.
Luciferase reporter assays. CH310T1/2 cells were seeded in the 24-well plates overnight in the absence of antibiotics. The cells were then transfected myogenin promoter-driven firefly luciferase reporter plasmid (or control vector (pGL3-Control)) and Renilla luciferase reporter plasmid (an internal control, Promega), as well as the expressional plasmid Flag-MyoD, using Lipofectamine 2000 transfection reagent (Invitrogen). Cells were challenged with or without 8-oxoG/G, and 6 h later, lysed in 100 µl passive lysis buffer, and then the extracts (20 µl) were used for luciferase activity using a Dual Luciferase Reporter Assay kit (Promega). To signify the the rapid transcriptional activation of myogenin, RNA was extracted after the cell were challenged with or without 8-oxoG/G for 30 min. Firefly luciferase mRNA levels were measured by real-time PCR and calibrated to that of Renilla. Primers used were shown in Supplementary Table S1.
Subcellular Fractionation and Western Blot. Protein C2C12 cells were lysed in lysis buffer85 for 30 min on ice . Lysates were centrifuged at 12,000 g for 20 min at 4 °C, and the supernatants were taken as the whole-cell extract (WE). Cytoplasmic and nuclear fractions were prepared using the CelLytic NuCLEAR Extraction kit (Sigma, NXTRACT) following the manufacturer's guidance. Briefly, cells were lysed with cytosolic lysis buffer for 20 min, and then centrifuged (11,000 g, 1 min, 4 °C), and supernatants were collected (CE). The pellets were washed twice with cytosolic lysis buffer and lysed with extraction buffer. Nuclear lysates were clarified by centrifugation (21,000 g, 5 min, 4 °C), and the supernatants were collected (NE). Then, 20 mg of protein from each sample was was mixed with 2 × SDS sample buffer, resolved by SDS–polyacrylamide gel, and then transferred on nitrocellulose (NC) membranes. Each NC membrane blot was sealed with 5% skimmed milk powder for 1 h at room temperature and incubated with primary antibodies overnight at 4°C. After three washes in TBST (Tris-buffered saline containing Tween 20), the membranes were incubated with HRP-conjugated secondary antibody for 1 h and detected by freshly made ECL (enhanced chemiluminescence) solution. The primary antibodies were as follow: anti-MHC (1:200), anti-Flag (1:3000), anti-RAS (1:1000), anti-RhoA (1:1000), anti-Cdc42 (1:1000), anti-Rac (1:1000), anti-ERK (1:3000), anti-p38 (1:3000), anti-pERK (1:3000), anti-pp38 (1:3000), anti-JNK (1:1000), anti-pJNK(1:1000) anti-pTyr (1:500), anti-MyoD (1:1000), anti-Ogg1 (1:1000), anti-Mth1 (1:500), anti-GST (1:3000), anti-GFP (1:3000), anti-GAPDH (1:3000).
Chromatin Immunoprecipitation assay. C2C12 cells were grown to a final count of 5-10×107 cells for each sample. Cells were cross-linked in 1% formaldehyde at room temperature for 5 min and then added 125 nM Glysine. After washing with PBS, the cells were resuspended and lysed in lysis buffers and sonicated to solubilize and shear the crosslinked DNA.. Sonicated chromatin was diluted to a final SDS concentration of 0.1% and aliquots 500 µL for each sample. DNA-protein complexes (near 25 µg of genomic DNA fragment per sample) were immunoprecipitated using 2 µg of MyoD Ab at 4°C overnight, then incubated with 30 µL of magnetic beads for 3 hours. The precipitates were washed three times, decrosslinked, and subjected to qPCR following the manufacturer's instructions for the Simple ChIP Enzymatic Chromatin IP Kit (cell signaling technology, US, #9003). The primer sequences were shown in Supplementary Table S3.
Expression and Purification of the Recombinant Proteins. GST and GST-MyoD proteins were induced in BL21(DE3) of Escherichia coli. The protein expression was achieved by adding 1 mM IPTG (isopropyl-b-D-thiogalactopyranoside) and 40 µM ZnSO4 at 37°C overnight. The ulture was centrifuged at 12000 rpm/min for 5 min, and the pellet was resuspended in chilled lysates buffer (20 mM HEPES, 120 mM NaCl, 10% Glycerin, 2 mM EDTA, protease inhibitor cocktail) and homogenized by sonication. The lysate was centrifuged at 14000 rpm for 20 min at 4°C. The supernatant was then applied to glutathione-Sepharose 4B beads (GE Healthcare). The protein-loaded 4B beads were washed with 5 volumes of PBS and 5 volumes of TEN100 (20 mM Tirs-HCl (pH 7.5), 0.1 mM EDTA (pH 8.0), 100 mM NaCl) and NETN (20 mM Tirs-HCl (pH 7.5), 0.1 mM EDTA (pH 8.0), 100 mM NaCl, 0.5% NP-40). Free GST and GST-MyoD proteins were purified and eluted in 100 µL buffer (50 mM Tris-HCl (pH 8.0), 100 mM KCl and 40 mM glutathione, and protease inhibitor cocktail), and used for EMSA.
Electrophoretic Mobility Shift Assay (EMSA). The EMSA analysis was performed using the LightShift chemiluminescent EMSA kit (ThermoFisher Scientific, 20148). GST-fused Recombinant MyoD (20, 10, or 5 nM) or nuclear extracts (1 µg) were mixed with 10 fmol of Biotin-labeled and canonical E box-containing oligonucleotide probe in a binding buffer (20 mM HEPEs (pH 7.6), 1.5 mM MgCl2, 10 mM KCl, 1 mM EDTA, 1 mM dithiothreitol, 1 mg/ml BSA) with 1µg poly(dI-dC) in a total volume of 10 µL. After incubation at room temperature for 20 min, the entire reaction was loaded on a 6% non-denaturing poly acrylamide-Tris-borate-EDTA (TBE) gel and run at 100 V for 2 h at 4°C. The sequence information of the probe was shown in Supplementary Table S4.
Small GTPase activity assay. To measure the level of the activity of small GTPases (Ras, Rac1/Cdc42 and RhoA), C2C12 cells were treated with vehicle or 10 µM 8-oxoG for 0, 3, 6, 9, 12 min. After treatment, cells were immediately harvested on ice in lysis buffer containing 25 mM Tris-Cl (pH 7.4), 150 mM NaCl, 50 mM MgCl2, 1 mM NaF, 1 mM of NaVO3, 1% IGEPAL, and 1 mM protease inhibitors. Lysates were vortexed, incubated on ice for 5 min and then clarified by centrifugation at 15,000 rpm for 15 min at 4°C, and 500 µg of protein was used in a pulldown assay with GST-Raf1-RBD (for Ras), GST-Pak1-RBD (for Rac1/Cdc42), or GST-Rhotekin-RBD (for RhoA) beads for 1 h with rotation at 4°C. The protein-coated beads were washed thrice with lysis buffer and then suspended in 2× loading buffer. The resulting pulldown was then subjected to western blotting to measure the activity Ras, Rac1/Cdc42 and RhoA by using anti-Ras, anti-Rac1/Cdc42 and anti-Rho antibodies.
Microscale thermophoresis (MST). The MST experiments were performed using a Monolith NT.115 instrument (NanoTemper) as described previously48. For each binding experiment recombinant Human OGG1 / MTH1 was diltued to 50 nM in MST buffer (PBS with 3 mM DTT, 0.05% Tween-20, and 0.2% Prionex). A series of 16 tubes with 8-oxoG/G/8-dG were prepared in MST buffer, producing 8-oxoG/G/8-dG ligand xonxentrations ranging from 120 nM to 4 pM. The reaction was mixed by pipetting, incubated for 30 min at room temperture followed by centrifugation at 10,000 × g for 10 min. Capillary forces were used to load the samples into Monolith NT.115 Premium Capillaries (NanoTemper Technologies). Measurements were performed using a Monolith NT.115Pico instrument (NanoTemper Technologies) at an ambient temperature of 25°C. Instrument parameters were adjusted to 20% LED power, medium MST power and MST on-time of 10 s. An initial fluorescence scan was performed across the capillaries to determine the sample quality and afterwards 16 subsequent thermophoresis measurements were performed. The Kd values were calculated using MO. Affinity Analysis software, (NanoTemper Technologies). The data presented here was repeated at three times confirming the results.
Isolation and identification of muscle satellite cells. Mouse satellite cells were isolated as previously described48. Briefly, tibialis anterior muscles were dissected and minced into fine pieces, and then transferred into the digestion buffer (0.5% collagenase typeII, 1% dispase in PBS) and incubated for 40 min at 37°C with regularly shaking during the incubation. Digestion was terminated by addition of 20% FBS. Samples were centrifuged at 500×g for 10 min. The pellets (containing satellite cells and fibroblast cells) were suspended in complete medium (Ham’s F-10 with 10% FBS). After four rounds of pre-plating, cells were plated in 24 wells plates. After removing the non-adhered cells and debris 24 later, the cells were cultured in the DMEM as used for proliferating for 3 days, and were passed into differentiation conditions (DMEM with 2% horse serum) to day 3. To identify the myoblast lineage of the culture, cells were washed with PBS, and fixed with 4% formaldehyde/PBS at room temperature for 15min. After washing three times with PBS, cells were treated with 0.25% Triton X-100/PBS (containing 5 mM EDTA and 2% FBS) for 5 min and washing for three times, followed by incubation with anti-MyoD (1:200) at 4 °C overnihgt. After eliminating the excessive primary antibodies with PBS, cells were incubated with TRITC-conjugated secondary antibodies (1:200) for 1 h. After washing three times in PBS, specimens were labeled with DAPI for 5 min. Finally, cells were washed twice with PBS for 5 min and fluorescence-labeled cells were observed under the fluorescence microscope. The images were visualized and captured by an Eclipse Ti2 microscope (Nikon Instruments Inc., Melville, NY).
Mice. Mice were bred and cohoused, four to six mice per cage, in a specific pathogen-free facility with a standard 12-hr alternate light/dark cycle at an ambient temperature of 22 ± 2°C and 30–70% humidity at the Animal Research Center of Northeast Normal University (Changchun, China). Health status of mice was determined via daily observation by technicians supported by veterinary care. All mouse experiments were conducted in accordance with the protocols for animal use, treatment, and euthanasia approved by the local ethics committee (reference number: AP2019085, the institutional animal care and use committee (IACUC) of the Northeast Normal University). Ogg1 KO mice were kindly provided by Dr. Yusaku Nakabeppu (Division of Neurofunctional Genomics, Department of Immunobiology and Neuroscience, Medical Institute of Bioregulation, Kyushu University, Fukuoka, Japan )86.
Exercise training. Experimental animals include: 6-8 weeks male wistar rats (weight 180-200 g), 6-8weeks male C57BI/6 (WT) mice and Ogg1 KO mice (weight 20-25 g). Generally, the animals were divided into 3 groups: sendentary, running and running follwed by rest. Armstrong training programs46 was modifies and utilized in the present study. Briefly, animals were acclimated with the treadmill apparatus (JD-PT, JiDe Shanghai, China) 1 week prior to the formal experiment: 5 to 10 min of running on the platform at a speed of 5 to 10 m/min with a slope of zero. Thereafter, the slope angle of treadmill was set to 16°, and all Rat/mice were trained using the following exercise program: 8-min running and 2-min rest with total 6 cycles (60 min). To determine the role of OGG1 in myogenic gene activation in response to exercise, after the last bout of training (40-min), the TA muscles of wild type and Ogg1 KO mice were collected, and the expression of MyoD and Myogenin was measured by RT-qPCR. Ras activation was detected by pull down assay and MEK activation was examined by western blotting.
Muscle damage and regeneration. For muscle injury, Six-to-eight week male C57BI/6 (WT) mice and Ogg1 KO mice (weight 20-25 g) were anesthetized with inhalation of 1-2% isoflurane (Vedco), and treated with 25 µl of 10 μM (in pyrogen-free saline) cardiotoxin VII4 (CTX, Sigma-Aldrich) via intramuscular injection into the anterior compartments of both hindlimbs. Mice (n = 5 mice/time point) were sacrificed on day 1, 3, 7, 9 and 11 after CTX-induced injury. Tibialis anterior muscles were collected and mRNA levels of MyoD and myogenin of samples were measured. To detect the role of 8-oxoG in muscle regeneration, after CTX-induced injury, 20 µL of vehicle (1 mM NaOH in saline) and 8-oxoG (100 µM) were injected intramuscularly into the left and right tibialis anterior of these mice, respectively, on day 1, 2, 3, 5 and 7. Tibialis anterior muscles were collected on day 3 and 7, mRNA levels of MyoD and myogenin of samples were measured; or in other experiments, tibialis anterior muscles were collected on day 7, placed in 10% neutral buffered formalin (NBF) for histological examination to confirm the extent of muscle regeneration.
Hematoxylin and Eosin (H&E) staining and Immunohistochemistry. H&E staining was performed according to the method described previously87. Skeletal muscle samples were immersion-fixed in 4% paraformaldehyde and embedded with paraffin, sectioned (5 µm). H&E staining was processed to show the morphology of regenerated myofibers. Randomly, five fields of each sample were captured at a magnification of 10× using an Olympus AX70 microscope (Olympus Optical, Japan). The average cross-sectional area analysis was achieved by using Image J software (1.4.3 V, US National Institutes of Health). To detect the generation 8-oxoG and its release from myofiber nuclei, anti-8-oxoG immuno-histochemical staining was performed. The level of 8-oxoG was determined by mean optical density (MOD), MOD = (integrated optical density (IOD)/area (sum)). The IOD of 8-oxoG and sum area were calculated by using Image-Pro Plus version 6.0.
Statistical analysis. Fluorescence quantification and normalization were performed using ImageJ software. All data are representative of three independent experiments and shown as mean ± standard deviaton (SD). Differences between two groups were analyzed by two-tailed Student’s t-test paired (figures 7d, 7e, 7f, 7g for TA muscles from the same mouse with different treatment) or unpaired (figures 6f、7i、7j). One-way analysis of variance (ANOVA) was performed for all experiments that require a comparison between three or more groups within one categorical variable followed by a Sidak’s post hoc test (figure 6c) or Tukey’s post hock test (including figures 1c、1g、2a-f、2g、2h、2n、4a、7b). To test the effect of two independent variables on a dependent variable, two-way ANOVA followed by Tukey’s post hock test (figures 1d、1e 、3d) or Sidak’s post hock test (figure 6g) was used. In all analyses, significance was accepted with P < 0.05 and analyses were performed using GraphPad Prism 7.00 software (GraphPad Software Inc., CA).