N2a mouse neuroblast cells (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) were cultured in high-Dulbecco's modified Eagle's medium/OPTI-Minimal Essential Medium (1:1; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) containing 5% (v/v) fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.) in a humidified atmosphere of 5% CO₂ at 37°C. Cells were passaged by trypsinization and seeded at ~10⁵ cells/ml. When cells reached 60–80% confluence, the culture medium was replaced with serum-free medium for 12–24 h. Cells were initially treated with 20, 50, 100, 300, 600 and 1,000 µM H₂O₂ for 12 h to assess the effect of different doses of H₂O₂ on N2a cell viability. The results of this treatment indicated that 600 uM H₂O₂ was the median lethal dose. Therefore, 600 µM H₂O₂ was used for subsequent experiments. Subsequently, cells were pretreated with 100 µM H₂O₂ for 90 min followed by 12 h recovery and subsequent exposure to the median lethal dose of 600 µM H₂O₂ for 12 h. To evaluate the involvement of ATM in preconditioning-induced protection, additional experiments were performed. N2a cells were treated with 10 µM ATM-specific inhibitor Ku55933 (Sigma-Aldrich; Merck KGaA) for 30 min or transfected with ATM small interfering RNA (siRNA) for 36 h prior to H₂O₂ preconditioning. Following H₂O₂ preconditioning, these cells were subjected to the lethal dose of 600 µM H₂O₂.

An MTT assay was used to determine cell viability. N2a cells were seeded at a density of 1×10⁴ cells/well in a 96-well culture plate. At the end of each experiment, 10 µl MTT (0.5 mg/ml) was added to the cell medium and incubated for 4 h at 37°C. Following incubation, MTT solutions were removed, dimethyl sulfoxide was added, and the absorbance at 490 nm was measured using a microplate reader. Data are expressed as a percentage of the control, which was considered to be 100% viable.

N2a cells were transfected with 50 nM ATM siRNA or Scramble control siRNA using Lipofectamine 2000^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. The siRNA sequences utilized targeted the following mouse ATM coding sequence: 5′-GCTTGAGGCTGATCCATATTC-3′. To determine the effect of siRNA transfection, the N2a cells were collected and lysed with lysis buffer [50 mM NaCl, 10 mM Tris-base, 1 mM EDTA, 2 mM sodium orthovanadate (Na₃VO₄), 1 mM NaF, 1 mM phenylmethyl-sulfonyl fluoride, 1% sodium dodecyl sulfate (SDS)] at 95°C for 10 min for western blot analysis 48 h following transfection.

Total cellular RNA was isolated using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) and cDNA was generated from 1 µg total RNA using the M-MLV reverse transcription kit (Promega Corporation, Madison, WI, USA). Quantification of gene copies was performed using the ABI 7300 Real-Time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.) with the Power SYBR^® Green PCR Master Mix kit (Promega Corporation). The primer sequences used were as follows: Forward, 5′-GCACACGGATTGCTCAAGGA-3′ and reverse, 5′-GCCCATTCGGAATATGGATCAG-3′ for ATM (14); and forward, 5′-CAATGACCCCTTCATTGA-3′ and reverse, 5′-GACAAGCTTCCCGTTCTCAG-3′ for GAPDH (15). The following thermocycling conditions were used: An initial predenaturation step at 95°C for 10 min, followed by 40 cycles of denaturation at 95°C for 15 sec and annealing at 60°C for 60 sec. All amplification reactions for each sample were repeated in at least triplicate, and the relative expression values were normalized to those of GAPDH using the 2^−ΔΔCq method (16).

Cells were lysed with lysis buffer [50 mM NaCl, 10 mM Tris-base, 1 mM EDTA, 2 mM Na₃VO₄, 1 mM NaF, 1 mM phenylmethyl-sulfonyl fluoride, 1% SDS], and the protein content of the lysates was measured using the bicinchoninic acid assay. Subsequently, 40 µg/lane protein was separated by 7.0% SDS-polyacrylamide gel electrophoresis and electrophoretically transferred to a nitrocellulose membrane. The membranes were blocked with 5% bovine serum albumin (Beyotime Institute of Biotechnology, Haimen, China) in TBS containing 1% Tween 20 at room temperature for 1 h, and incubated with ATM (mouse monoclonal antibody; dilution, 1:1,000; cat. no. sc-47739; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), phosphorylated (p)-ATM antibodies (mouse monoclonal antibody; dilution, 1:500; cat. no. sc-73615; Santa Cruz Biotechnology, Inc.) and β-actin (rabbit polyclonal antibody; dilution, 1:1,000; cat. no. sc-130656; Santa Cruz Biotechnology, Inc.) at 4°C overnight, followed by incubation with a horseradish peroxidase-conjugated secondary antibody (either goat anti-rabbit; dilution, 1:1,000; cat. no. A0208. Or goat anti-mouse; dilution, 1:1,000; cat. no. A0216; Beyotime Institute of Biotechnology) for 1 h at room temperature. The immunostaining was visualized by enhanced chemiluminescence (Beyotime Institute of Biotechnology). The blots were scanned, and the pixel count and intensity of each band was quantified using the Scion Image software (version 4.2.3.2; Scion Corporation, Frederick, MD, USA). The results were normalized to β-actin expression.

Flow cytometry was performed as described in a previous study by Tang et al (1). Briefly, treated N2a cells (2×10⁶) were collected and centrifuged at 5,000 × g at 4°C for 10 min. The cell pellet was resuspended in cold PBS and fixed using 70% ethanol at 4°C for 1 h. The cells were then centrifuged at 5,000 × g for 10 min, and resuspended in PBS. DNase-free RNaseA (100 µl, 200 µg/ml) was added to the cells and incubated at 37°C for 10 min. Cells were subsequently incubated with propidium iodide (PI) at a final concentration of 100 mg/l, filtered and incubated in the dark at room temperature for 10 min prior to flow cytometric analysis. The PI fluorescence of individual nuclei was measured using a flow cytometer (Beckman-Coulter, Inc., Brea, CA, USA). DNA labeling data were analyzed using CellQuest v.3.0 sampling software (BD Biosciences, Franklin, NJ, USA) for flow cytometry.

Caspase-3 activity was measured using a colorimetric CaspACE kit (Promega Corporation) according to the manufacturer's protocol. Cells were lysed using the kit lysis buffer (Promega Corporation) and centrifuged for 5 min at 5,000 × g and 4°C. The supernatant was used for the measurement of caspase-3 activity.

DNA was extracted from N2a cells with the DNA Extractor kit (Wako Pure Chemical Industries, Ltd., Osaka, Japan) according to the manufacturer's protocol. The extracted DNA was digested with 8 units nuclease P1 (Cell Biolabs, Inc., San Diego, CA, USA) for 2 h at 37°C in a final concentration of 20 mM sodium acetate (pH 5.2), followed by treatment of 6 units alkaline phosphatase for 1 h at 37°C in a final concentration of 100 mM Tris (pH 7.5). The reaction mixture was centrifuged for 5 min at 6,000 × g and 4°C, and the supernatant was used for the 8-OHdG Quantitation ELISA assay (catalog no. STA-320; Cell Biolabs, Inc.), according to the manufacturer's protocol.

SPSS statistical software was used for statistical analysis (version 18.0; SPSS, Inc., Chicago, IL, USA). The data are expressed as the mean ± standard error of at least 3 replicate experiments. Comparisons among multiple groups were performed using one-way analysis of variance followed by the Student-Newman-Keuls post hoc test. P<0.05 was considered to indicate a statistically significant difference.

The effect of different concentrations of H₂O₂ on N2a cell viability was evaluated by MTT assay (Fig. 1). Treatment with 20–100 µM H₂O₂ did not significantly affect N2a cell viability; however, concentrations of 300, 600 and 1,000 µM significantly decreased N2a cell viability compared with the control, to 78.5±6.5 (P<0.05), 44.2±3.5 (P<0.01) and 11.4±1.4% (P<0.001), respectively. In addition, an MTT assay demonstrated that preconditioning cells with 50 or 100 µM H₂O₂ attenuated the reduction of N2a cell viability induced by 600 µM H₂O₂, compared with the non-preconditioned group (P<0.05 and P<0.01, respectively; Fig. 2), with 100 µM H₂O₂ most effective. Preconditioning with 20 µM H₂O₂ failed to significantly attenuate the reduction in cell viability induced by treatment with 600 µM H₂O₂ (P>0.05; Fig. 2). Therefore, 100 µM H₂O₂ was selected for preconditioning in subsequent experiments.

Following exposure of N2a cells to 600 µM H₂O₂ for 12 h, the percentage of apoptotic N2a cells increased significantly compared with the control (62.8±5.2 vs. 6.5±0.5%; P<0.01; Fig. 3). Preconditioning with 100 µM H₂O₂ for 90 min did not significantly alter the apoptotic rate compared with the control (9.5±0.89 vs. 6.5±0.5%; n=5; P>0.05; Fig. 3); however, subsequent 600 µM H₂O₂-induced apoptosis was significantly inhibited following preconditioning compared with the non-preconditioned group (33.8±3.1 vs. 62.8±5.2%, respectively; n=5; P<0.01; Fig. 3).

The caspase 3 protein is a member of the cysteine-aspartic acid protease (caspase) family (17). Sequential activation of caspases serves a central role in the execution-phase of cell apoptosis, thus caspase-3 activity is a marker of cell apoptosis (18). Consistent with the results of flow cytometric analysis, caspase-3 activity was significantly decreased in N2a cells preconditioned with 100 µM H₂O₂ and exposed to 600 µM H₂O₂ compared with the non-preconditioned group (P<0.01; Fig. 4).

8-OHdG is a marker of oxidative stress (19). The present study observed that 8-OHdG content was additionally significantly decreased in N2a cells preconditioned with 100 µM H₂O₂ and exposed to 600 µMH₂O₂ compared with the non-preconditioned group (P<0.01; Fig. 5).

The effect of 100 µM H₂O₂ preconditioning on ATM mRNA and protein expression levels was determined. Preconditioning of N2a cells with 100 µM H₂O₂ for 90 min significantly increased p-ATM protein expression levels compared with the control (P<0.01; Fig. 6). Preconditioning with 100 µM H₂O₂ for 90 min, then 12 h later, increased the expression of ATM mRNA and protein when compared with the control (P<0.01; Fig. 7A and B).

To determine the involvement of ATM in H₂O₂ preconditioning, RNA interference (RNAi) with siRNA, and treatment with an ATM inhibitor, was performed. siRNA-mediated knockdown of ATM resulted in reduction of ATM protein expression compared with the untransfected control and scramble control groups (P<0.01; Fig. 8). When N2a cells were incubated with 10 µmol/l Ku55933 for 36 h or transfected with 50 nM control siRNA, the percentage of apoptotic cells was 8.0±0.68 and 11.1±0.96%, respectively, and were not significantly different compared with the control group (6.5±0.5%; P>0.05; Fig. 3). However, the anti-apoptotic effect of preconditioning with 100 µM H₂O₂ was decreased by pretreatment with the ATM inhibitor Ku55933 or silencing of ATM with RNAi compared with the preconditioned group (P<0.01 and P<0.01, respectively; Fig. 3). In addition, the decreased caspase-3 activity observed following preconditioning with 100 µM H₂O₂ was inhibited by the pretreatment of cells with the ATM inhibitor Ku55933 or silencing of ATM with RNAi compared with the preconditioned group (P<0.01 and P<0.01, respectively; Fig. 4) and the decrease in 8-OHdG content observed following preconditioning with 100 µM H₂O₂ was also inhibited by pretreatment with the ATM inhibitor Ku55933 or silencing of ATM with RNAi compared with the preconditioned group (P<0.01 and P<0.01, respectively; Fig. 5).