AOH and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich, Inc. (St. Louis, MO, USA). Anti-phosphorylated (p-)p38MAPK [rabbit polyclonal immunoglobulin G (IgG); catalog no., sc-17852-R], anti-DNA polβ (goat polyclonal IgG; catalog no., sc-5927), anti-β-actin (mouse monoclonal; catalog no., sc-47778) and anti-α-tubulin (mouse monoclonal; catalog no., sc-23948) primary antibodies and goat anti-rabbit IgG-horseradish peroxidase (HRP) (catalog no., sc-2004), rabbit anti-mouse IgG-HRP (catalog no., sc-358914) and donkey anti-goat IgG-HRP (sc-2020) secondary antibodies were obtained from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Anti-p-ATF2 (rabbit polyclonal; catalog no., CST9225) was obtained from Cell Signaling Technology, Inc. (Danvers, MA, USA). RPMI-1640 and fetal bovine serum (FBS) was purchased from Thermo Fisher Scientific, Inc. (Waltham, MA, USA). SB203580 and the Enhanced Chemiluminescence (ECL) Plus Detection kit were purchased from Beyotime Institute of Biotechnology (Haimen, China). Reagents for protein concentration analysis were obtained from Bio-Rad Laboratories, Inc. (Hercules, CA, USA).

The NIH3T3 cell line was obtained from China Center for Type Culture Collection (Wuhan, China). The cells were cultured in RPMI-1640 with 10% FBS, 100 units (U)/ml penicillin and 100 µg/ml streptomycin at 37°C and 5% CO₂ in a humidified incubator.

To detect the DNA damage of NIH3T3 cells induced by various concentrations of AOH, an alkaline comet assay was performed. Cells (2×10⁶) were cultured in 10-cm dishes for ~48 h. When the cells reached 80% confluence, they were treated with 2, 10 or 20 µM AOH or DMSO for 2 h. Fully frosted slides were then coated with 1% normal melting agarose at 45°C as a base layer, and were solidificated at 4°C for 10 min. The cells were suspended in 0.8% low melting agarose at 37°C. This suspension was pipetted over the base layer, which was covered with a cover slip immediately. The slides were placed at 4°C for 10 min, and then the cover slips were removed. The slides were kept in the lysis buffer (pH 10) at 4°C for 1 h and transferred into electrophoretic running buffer (pH 13) for unwinding for 30 min. Electrophoresis was performed in the same buffer for 20 min at 0.8 v/cm. Subsequently, the slides were washed with neutralizing buffer and stained with ethidium bromide (20 µg/ml) for 5 min, and washed with cold saline. Finally, the glycerine buffer was placed on the slides in a dropwise manner and cover slips were placed on top. The slides were visualized using a fluorescence microscope (Olympus, Tokyo, Japan).

Cells (2×10⁶) were cultured in 10-cm dishes for ~48 h. When the cells reached 80% confluence, they were treated with various concentrations (2, 10 or 20 µM) of AOH, cells were lysed in radioimmunoprecipitation assay buffer [50 mm Tris/HCl, pH 7.5, 150 mM NaCl, 1 mM ethylenediaminetetraacetic acid, 1 mM Na₃VO₄, 0.1% sodium dodecyl sulfate (SDS), 1% (v/v) NP-40, 1 mM NaF, containing complete protease and phosphatase inhibitor cocktail (Roche Diagnostics, Indianapolis, IN, USA)], and supernatants were collected by centrifugation at 21,341 × g for 20 min. Cell lysate (50 µg) was separated on a 10% SDS-polyacrylamide gel and transferred to a polyvinylidene fluoride membrane. After blocking with 5% nonfat dried milk for 1 h, the membrane was incubated with a primary antibody at 4°C overnight (anti-p-p38MAPK, 1:200 dilution; anti-p-ATF2, 1:1,000 dilution; anti-DNA polβ, 1:200 dilution; anti-α-tubulin and anti-β-actin, 1:1,000 dilution). IgG-HRP secondary antibodies were incubated for 2 h at room temperature. Protein bands were visualized using the ECL Plus Detection kit. The bands were subjected to densitometry for quantitative analysis using Quantity One software (Bio-Rad Laboratories, Inc.).

The pLKO.1, pCMV-dR8.2 and pCMV-VSVG vectors were kindly provided by Dr Zigang Dong (The Hormel Institute, Austin, MN, USA). The lentivirus-based shRNA-expressing vectors was a pLKO.1 back bone with the human U6 promoter driving RNA polymerase III transcription. The puromycin resistance marker encoded in pLKO.1 allows for convenient stable selection. The shRNA sequence of p38MAPK (5′-ATACATTCGGCTGACATAATT-3′) was cloned into the lentiviral shRNA-expressing vectors at the AgeI and EcoRI restriction sites (New England Biolabs Ltd., Beijing, China), and were confirmed by direct DNA sequencing (Genewiz Inc., Beijing, China).

The lentiviral vector containing the p38MAPK-shRNA, a packaging vector (pCMV-dR8.2) and an envelope vector (pCMV-VSVG) were co-transfected into 293T cells using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.). After 24 h and 48 h, the viral supernatants were harvested and added to the NIH3T3 cells. Subsequently, 2 mg/ml puromycin was used to select the stably infected cells.

All results were presented as mean ± standard deviation of the mean. Statistics analysis was performed using analysis of variance in SPSS 11.0 software (SPSS, Inc., Chicago, IL, USA). P<0.05 was considered to indicate statistical significance.

To investigate the DNA toxic effect of AOH on NIH3T3 cells, a comet assay was conducted. NIH3T3 cells were treated with DMSO or 2, 10 or 20 µM AOH for 2 h. The results of single cell gel electrophoresis showed that 10 and 20 µM AOH increased the length and area of comet tails, a phenotype of DNA damage (Fig. 1A). DNA polβ is a polymerase that participates in the repair of DNA strand breaks. Thus, the present study investigated whether or not DNA polβ was activated in DNA damage induced by AOH. NIH3T3 cells were treated with AOH. Western blotting was performed to determine polβ protein level with various doses of AOH treatment. The results showed that the protein level of polβ protein increased in a dose-dependent manner with AOH treatment compared with the control (Fig. 1B).

Western blotting was used to determine the level of p38MAPK phosphorylation after AOH treatment at various doses and time points. Results showed that the activation of p38MAPK increased in a dose-dependent manner (2 µM AOH group, P=0.002 vs. control group; 10 µM AOH group, P<0.001 vs. control group; 20 µM AOH group, P<0.001 vs. control group) (Fig. 2A). The level of p38MAPK phosphorylation was highest when the NIH3T3 cells were treated with 10 µM AOH at 2 h (1 h group, P<0.001 vs. control group; 2 h group, P<0.001 vs. control group; 4 h group, P<0.001 vs. control group) (Fig. 2B). SB203580, an inhibitor of p38MAPK, can effectively block the phosphorylation of p38MAPK. To determine whether AOH induces the phosphorylation of p38MAPK, the NIH3T3 cells were pretreated with 10 µM SB203580 for 1 h, then treated with 10 µM AOH. Western blot analysis showed a significant decrease of p38MAPK phosphorylation in SB203580 pretreatment group compared with the AOH-only treated group (SB203580+AOH group, P=0.013 vs. control group; AOH group, P<0.001 vs. control group) (Fig. 2C).

ATF2 is a transcription factor that can be activated by p38MAPK. Activated p38MAPK can phosphorylate ATF2 in the cell stress response process. To further identify the activation of ATF2 evoked by phosphorylated p38MAPK with AOH treatment, western blotting was performed to determine the level of ATF2 phosphorylation when NIH3T3 cells were treated with various doses of AOH at different time points. The results were consistent with the change of p38MAPK phosphorylation, which revealed that phosphorylation occurred in a dose-dependent manner and that the peak activation of ATF2 occurred at the same time as p38MPAK (in dose groups, 2 µM AOH group, P=0.384 vs. control group; 10 µM AOH group, P=0.001 vs. control group; 20 µM AOH group, P<0.001 vs. control group; in time groups, 1 h group, P<0.001 vs. control group; 2 h group, P<0.001 vs. control group; 4 h group, P=0.001 vs. control group) (Fig. 2A and B). Furthermore, the level of ATF2 phosphorylation decreased in the SB203580 pretreatment group compared with the AOH-only treated group (SB203580+AOH group, P=0.057 vs. control group; AOH group, P<0.001 vs. control group) (Fig. 2C).

Subsequently, the AOH-induced phosphorylation of p38MAPK and ATF2 was assessed for associations with the expression level of polβ. Polβ expression was observed in western blotting assays when cells were pretreated with SB203580, an inhibitor of p38MAPK. The expression level of polβ was found to decrease in the pretreatment group compared with the AOH stimulation group (Fig. 3).

To further verify the role of the p38MAPK-ATF2 pathway in polβ expression, p38MAPK knockdown was performed by lentiviral shRNA transfection. The results of the western blotting showed that p38MAPK knockdown was associated with the reduced phosphorylation of ATF2 and the decreased expression of polβ (Fig. 4).