Peptide Synthesis
Peptides were synthesized by solid-phase peptide synthesis using Wang resin (Sigma-Aldrich, St. Louis, MO, USA). The peptides were labeled in dimethylformamide (DMF) with five molar equivalents of dye (FPR-675; BioActs, Incheon, South Korea) at the amine of the N-terminal glycine while the resin and protecting group were still intact. The peptides were combined with the TAT peptide at the amine of the C-terminal using a GG linker. After cleavage, the compounds were refined by high-performance liquid chromatography (HPLC) and freeze-dried to obtain fluorophore-labeled peptides.
Cell Culture
Human cervical cancer HeLa cells were obtained from the Department of Biological Sciences of Konkuk University (Seoul, Korea). HeLa cells were grown in Dulbecco’s Modified Eagle medium (DMEM; Welgene, South Korea) containing 10% fetal bovine serum (Biowest, Nuaill´e, France) and 1% penicillin-streptomycin solution (Sigma-Aldrich) at 37°C in a 5% CO2 incubator.
MTT assay
MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2 tetrazolium bromide] was purchased from Sigma–Aldrich (98% purity). HeLa cells were seeded in 24-well plates at a density of 1 × 105 cells per well and incubated for 24 h. HeLa cells were treated with the TAT-S3 probe at different concentrations and incubated for 24 h in complete media. Cells treated with 750 μM H₂O₂ for 1 h were used as a positive control. MTT solution (0.5 mg/ml) was added to each well and incubated for 1 h, then DMSO was added to each well and incubated for 5 min. Absorbance was measured at 570 nm on a microplate reader.
DNA binding assay of the TAT-S3 peptide probe and S3 peptide probe
A DNA-binding assay was conducted using a 5’-end labeled DNA oligonucleotide duplex containing either a single AP site (AP-39mer) or a single 8-oxoG residue (8-oxoG-39mer). The DNA-binding assay was performed in reaction buffer (30 mM KCl, 30 mM HEPES, pH 7.4, and 0.01% Triton X-100) with a 2 μM concentration of the TAT-S3 and S3 probes. The radio-labeled 39-mer oligonucleotide duplex (7 pmol) was immediately added to the TAT-S3 and S3 probes. After incubation at 37°C for 3 h, reactions were terminated using 6× DNA loading dye (Bio Basic Inc., Canada). Samples were loaded on a 10% nondenaturing polyacrylamide gel in 1× TBE buffer (450 mM tris, 450 mM boric acid, 1 mM EDTA, pH 8.0). After electrophoresis, gels were vacuum dried and suctioned.
Confocal fluorescence microscopy
Digital images were obtained with a super-Resolution confocal Laser scanning microscope LSM 800 (Carl Zeiss). HeLa cells were seeded onto a confocal dish at a density of 2 × 10⁵ cells. HeLa cells were treated with the TAT-S3 probe at 100 nM in complete media. After 24 h, the cells were treated with 500 μM H₂O₂. After 1 h, the cells were triple washed with DPBS. HeLa cells were stained with Hoechst (1:5000) for 10 min. After washing 3 times with DPBS, Mitotracker Green FM (Invitrogen) was added at a final concentration 20 nM and incubated for 15 min. The cells were washed 3 times with DPBS. A blue pseudocolor was applied to visualize the nuclear stain, while a green pseudocolor was applied to visualize the mitochondrial stain, and a red pseudocolor was applied to visualize the localization of the TAT-S3 probe within cells.
Cell fractionation
HeLa cells (1 × 105 cells) were plated on a 60-mm2 cell culture dish. The cells were treated with 2 μM TAT-S3 probe and incubated for 24 h. Hoechst was added at a final dilution of 1:5000. The cells were washed 3 times with PBS, then collected and lysed via sonicator. The lysate was centrifuged at 300 ×g for 5 min. The supernatant was cell free extract, and the pellet was resuspended with 200 μl of PBS and centrifuged at 600 ×g for 10 min. This supernatant contained nuclei, and the pellet was resuspended with 200 μl of PBS and centrifuged at 16,000 ×g for 30 min. The final pellet was mitochondria, and this was resuspended with 200 μl of PBS.
Genomic DNA competition assay with ARP/MX and the TAT-S3 peptide probe
Genomic DNA was extracted using a genomic DNA extraction kit (Bioneer). Solutions of aldehyde reaction probe (ARP)/methoxyamine (MX) and the TAT-S3 probe were prepared in H₂O, varying the ARP/MX between 0-10 μM while maintaining the TAT-S3 probe at 10 μM. The ratios of ARP/MX:TAT-S3 probe used in this study were: 0.002, 0.01, 0.02, 0.1, 0.2, and 0.5. The following samples were prepared in triplicate: 10 μl ARP or MX/TAT-S3 solution was added to 5 μl genomic DNA (100 ㎍/ml). Samples were incubated at 37°C for 24 h in the dark. Tris-EDTA buffer (85 μl, pH 7.0; Sigma) and 1 μl of glycogen (Sigma) were added to the samples, then 10 μl of 3 M sodium acetate was added to the samples. Ice-cold ethanol (300 μl) was added, and the DNA was purified by ethanol precipitation. The pellet was washed 3 times with 70% ethanol. DNA was dissolved in 100 μl H₂O. Samples were added to a 96 well black plate (Corning) and analyzed at 685 nm excitation and 709 nm emission.
Flow cytometry
HeLa cells were seeded as 3 × 105 cells/60-mm2 dish and cultured. The cells were treated with the TAT-S3 probe (100 nM) for 24 h, then the cells were treated with the indicated concentration of H₂O₂ for 1 h. After washing 3 times with DPBS, the cells were treated with 20 nM Mitotracker Green FM for 15 min, then washed 3 times with DPBS. The cells were collected with a scrapper and the cell lysate (100 μl) was analyzed via flow cytometry (CytoFLEX, Beckman Coulter).
ATP assay
ATP assays were performed according to the manufacturer’s protocol (Promega, ATP assay kit) and detection was performed with a luminometer (Veritas™). HeLa cells were seeded at 3 × 105 cells/60-mm2 dish. The cells were treated with the TAT-S3 probe and incubated 24 h, then collected and lysed with 0.5% trichloroacetic acid solution (TCA, Sigma). The lysate received pH 8.0 Tris-EDTA buffer (Sigma), then 100 μl of cell lysate was analyzed for luciferase activity using a luminometer.
Measurement of anti-oxidant effects by confocal microscopy
HeLa cells were seeded at 2 × 10⁵ cells/confocal dish, then treated with the TAT-S3 probe at 100 nM. After 24 h, the cells were treated with 0.5 µg/ml Mitoquinone (MitoQ, Biovison), a mitochondria-targeted anti-oxidant. After 1 h, the cells were washed 3 times with DPBS, then treated with 20 nM Mitotracker Green FM for 15 min. The cells were washed 3 times with DPBS, then green pseudocolor was applied to visualize mitochondrial staining while red pseudocolor was applied to visualize the localization of the TAT-S3 probe within cells.
Measurement of OGG1 inhibitor effects by confocal microscopy
HeLa cells were seeded at 2 × 10⁵ cells/confocal dish and treated with the TAT-S3 probe (100 nM) alone or in combination with O8 (Sigma), an inhibitor of OGG1. After 24 h, the cells were washed 3 times with DPBS then treated with 20 nM Mitotracker Green FM for 15 min. The cells were washed 3 times with DPBS and a green pseudocolor was applied to visualize mitochondrial staining, while a red pseudocolor was applied to visualize the localization of the TAT-S3 probe within cells.
Maintenance of zebrafish
Zebrafish was purchased from a commercial dealer (Seoul aquarium, Seoul, Korea). Zebrafish were maintained and bred according to the methods described by Kim et al., 2014 (57). All animal experiments were approved by the Jeju National University Animal Care and Use Committee (2016-0052).
Measurement of antioxidant effect in zebrafish embryos
From approximately 7-9 hours post-fertilization (hpf), 15 embryos were transferred to individual wells of 12-well plate containing 1.8 mL embryo media. The embryos were treated with 0.5 µM MitoQ. After 1 h, 10 mM H2O2 was added to the embryo exposed with MitoQ for up to 72 hpf. Then, 72 hpf of zebrafish larva was transferred to one well of 96-well plate, treated with 100 nM the TAT-S3 probe and incubated for 24 h in the dark at 28.5 ± 0.5 °C. The zebrafish larvae were rinsed 3 times with fresh embryo media. After anesthesia on 0.03% MS-222, the image of stained larvae was observed and photographed under the microscope (Gen 5 version 3.03, BioTek, Winooski, Vermont, U.S.). A fluorescence intensity of larvae was quantified using the image J program.
Statistical analysis
The values in this study are representative of at least three independent experiments. All results are shown as the means ± S.D. Statistical analysis of the data between experimental groups was performed using the Student’s t-test. P values less than 0.05 were considered statistically significant.