RK1 (cat. no. 42754) and LY294002 (cat. no. L9908) were purchased from Sigma-Aldrich (Merck KGaA) and the purity was ≥95 and ≥98%, respectively. Additional reagents used in the present study were commercially available and of analytical purity.

The PIG1 immortalized human melanocyte cell line was purchased from The Cell Bank of Type Culture Collection of The Chinese Academy of Sciences, and cultured in Medium 254 (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% FBS (Gibco; Thermo Fisher Scientific, Inc.) and 1% penicillin-streptomycin (Beijing Solarbio Science & Technology Co., Ltd.) at 37°C in a humidified atmosphere with 5% CO₂.

Cell viability was determined using a Cell Counting Kit-8 (CCK-8; Dojindo, Molecular Technologies, Inc.) assay. The PIG1 cell line was seeded into 96-well plates at a density of 7×10³/well. When 60–70% confluence was reached, the cells were pretreated with different concentrations of RK1 (0, 0.05, 0.1, 0.2, 0.4, 0.8 or 1.6 mM) for 2 h, and then treated with 1.0 mM H₂O₂ for 24 h at 37°C with 5% CO₂. Subsequently, 10 µl CCK-8 solution was added to each well and cultured for 90 min. The absorbance was measured using a microplate reader (Molecular Devices, LLC) at 450 nm. Cell viability was calculated according to the following formula: Viability rate (%) = (absorbance of test sample-absorbance of blank)/(absorbance of control-absorbance of blank) ×100. In addition, following treatment with RK1 (0.1,0.2 and 0.4 mM) for 2 h, and treatment with 1.0 mM H₂O₂ for 24 h at 37°C with 5% CO₂, cell morphology was observed using an inverted light microscope (Olympus Corporation).

Cell apoptosis was detected using an Annexin V-fluorescein isothiocyanate (FITC)-PI apoptosis detection kit (BD Biosciences). Briefly, PIG1 cells were seeded into 6-well plates at a density of 3×10⁵/well and cultured until they had reached 60–70% confluence. The cells were exposed to 1 mM H₂O₂ for 24 h after pretreatment with different concentrations of RK1 (0, 0.1, 0.2 or 0.4 mM) or LY294002 (10 µM) for 2 h at 37°C with 5% CO₂. Then, the collected PIG1 cells were resuspended in 100 µl binding buffer (1×10⁵ cells), stained with 5 µl Annexin V-FITC and 5 µl PI, and incubated at room temperature (20–25°C) for 15 min in the dark. The cell apoptosis rate (early + late apoptosis) was detected using a FACSCalibur flow cytometer and CellQuest software (version 3.3; BD Biosciences) within 1 h.

The cells from each group were disrupted using an ultrasonic cell disruptor (3 times; each for 3 sec with an interval of 1 sec on ice) and the lysate was centrifuged at 4°C at 16,000 x g for 5 min to obtain the total protein. The activity levels of SOD, CAT and GSH-Px were determined using a SOD (cat. no. A001-3-2), CAT (cat. no. A007-1-1), GSH-Px (cat. no. A005-1-2) analysis kit (Nanjing Jiancheng Bioengineering Institute) according to the manufacturer's instructions.

Total protein from PIG1 cells in each group was extracted using RIPA lysis buffer. Nuclear and plasma proteins were extracted using the Nuclear Protein Extraction Kit (cat. no. R0050; Beijing Solarbio Science & Technology Co., Ltd.). Protein concentrations were determined using a BCA kit (cat. no. AR0146; Wuhan Boster Biological Technology, Ltd). Protein samples (20 µg) were separated via SDS-PAGE on 10% gels, and then transferred onto PVDF membranes. The membranes were blocked with 5% skimmed milk in TBS-Tween-20 (0.05%) for 1 h at room temperature, followed by incubation at 4°C overnight with primary antibodies against Bax (cat. no. ab53154; 1:500), caspase-3 (cat. no. ab4051; 1:500), Bcl-2 (cat. no. ab196495; 1:500), phosphorylated (p)-AKT (cat. no. ab38449; 1:500), AKT (cat. no. ab18785; 1:500), Nrf2 (cat. no. ab137550; 1:500), HO-1 (cat. no. ab13243; 1:2,000), lamin A/C (cat. no. ab227176; 1:1,000), tubulin (cat. no. ab6130; 1:2,000) and β-actin (cat. no. ab8227; 1:1,000) (all from Abcam). Subsequently, the membranes were incubated with HRP-conjugated goat anti-rabbit immunoglobulin G secondary antibody (cat. no. BA1054; Wuhan Boster Biological Technology, Ltd.) at 1:5,000 dilution for 1 h at room temperature. The chemiluminescent signals were developed using an enhanced chemiluminescence kit (MilliporeSigma). The images of the proteins were collected and the intensity was analyzed using ImageJ software (version 2.0; National Institutes of Health). β-actin was used as a whole cell internal reference, lamin A/C was used as a nuclear internal reference and tubulin was used as a cytosolic internal reference.

The PIG1 cell line was seeded into 12-well plates containing single layer glass slides and cultured until it reached 60–70% confluence. The cells were then pretreated with 0.4 mM RK1 or 10 µM LY294002 for 2 h, and with 1 mM H₂O₂ for 24 h at 37°C with 5% CO₂. Following treatment, the PIG1 cell line was fixed with 4% formaldehyde solution for 20 min at room temperature, permeabilized with 0.1% Triton X-100/PBS for 5 min at room temperature, blocked with 10% newborn calf serum (cat. no. 22012-8612; Zhejiang Tianhang Biotechnology Co., Ltd.) for 30 min at room temperature and incubated with an anti-Nrf2 primary antibody (cat. no. ab137550; 1:500; Abcam) overnight at 4°C. The slides were washed twice with PBS/0.1% Tween-20 and incubated with Alexa Fluor^® 488 goat anti-rabbit IgG (green color; cat. no. ab150077; 1:1,000; Abcam) for 1 h at room temperature. DAPI was used to stain the cell nuclei (blue) for 15 min at room temperature. The protein expression of Nrf2 was observed using an Olympus confocal microscope (Olympus FV300; Olympus Corporation).

SPSS v20.0 software (IBM Corp.) was used for statistical analysis. All data are presented as the mean ± standard deviation from three separate experiments. Differences among multiple groups were compared by one-way analysis of variance with Tukey's post hoc test. P<0.05 was considered to indicate a statistically significant difference.

To identify the cytotoxic concentration of RK1, the effects of RK1 on PIG1 cell viability were assessed using a CCK-8 assay. As shown in Fig. 1A, compared with that in the control group, there were no significant changes in cell viability at doses of RK1 between 0.05 and 0.4 mM. Nevertheless, RK1 at a concentration of 0.8 and 1.6 mM significantly suppressed cell viability. Therefore, 0.4 mM RK1 was used as the highest concentration in the subsequent experiments. To investigate the protective effect of RK1 on the PIG1 cell line, the cells were treated with RK1 (0, 0.1, 0.2 and 0.4 mM) for 2 h followed by exposure to 1.0 mM H₂O₂ for 24 h. It was previously found that 1 mM H₂O₂ was the optimal dose for inducing a sufficient cytotoxic effect on melanocytes (24,25). As shown in Fig. 1B and C, treatment with 1.0 mM H₂O₂ for 24 h significantly inhibited cell viability, and the dendrites of the melanocytes were also shortened compared with that in the control cells. However, pretreatment with RK1 (0.2 and 0.4 mM) for 2 h significantly increased cell viability and decreased the amount of cell shrinkage compared with that in cells treated with H₂O₂. Taken together, these data suggested that RK1 could effectively reduce H₂O₂-induced human melanocyte death.

To determine the protective effect of RK1 against H₂O₂-induced apoptosis, the PIG1 cell line was pretreated with various concentrations of RK1 (0.1, 0.2 and 0.4 mM) for 2 h, then treated with or without 1.0 mM H₂O₂ for 24 h. As shown in Fig. 2A and B, the number of apoptotic cells was significantly increased in the H₂O₂ group compared with that in the control group; however, RK1 significantly reversed these effects in a dose-dependent manner. In addition, the western blot analysis results revealed that H₂O₂ treatment resulted in a significant increase in the anti-apoptotic protein expression levels of caspase-3 and Bax, and a decrease in the expression of Bcl-2 protein compared with that in the control group. However, treatment with RK1 (0.2 and 0.4 mM) significantly decreased caspase-3 and Bax protein expression levels, and increased Bcl-2 expression levels compared with that in H₂O₂-treated cells (Fig. 2C-F). These data indicated that RK1 could protect human melanocytes from H₂O₂-induced apoptosis.

To investigate whether RK1 protects melanocytes against H₂O₂-induced OS, the PIG1 cell line was pretreated with different concentrations of RK1 for 2 h, followed by incubation with 1.0 mM H₂O₂ for 24 h, then the activity levels of SOD, CAT and GSH-Px were determined. As shown in Fig. 3A-C, the activity levels of SOD, CAT and GSH-Px were significantly decreased in the H₂O₂ group compared with that in the control group, whereas RK1 significantly reversed this effect. These results indicated that RK1 could protect melanocytes against H₂O₂-induced oxidative damage.

The effect of RK1 on the AKT/Nrf2/HO-1 signaling pathway was further evaluated in the PIG1 cell line. The results showed that H₂O₂ treatment significantly decreased the protein expression levels of HO-1, the ratio of p-AKT to AKT, and Nrf2 nuclear to cytosolic translocation in the PIG1 cell line (Fig. 4A-D). In addition, compared with that in the H₂O₂ group, the protein expression levels of HO-1, the ratio of p-AKT to AKT, and Nrf2 nuclear to cytosolic translocation were increased in the RK1 group (Fig. 4A-D). The Nrf2 translocation in the PIG1 cell line following treatment with RK1 (0.4 mM) was subsequently investigated using immunofluorescence staining. As expected, RK1 promoted nuclear translocation of Nrf2 (Fig. 4E). These results indicated that H₂O₂ treatment inhibited the activation of the AKT/Nrf2/HO-1 signaling pathway and promoted Nrf2 translocation from the nucleus to the cytoplasm, while RK1 pretreatment could reverse this effect.

To clarify whether the PI3K/AKT signaling pathway is involved in the protective effects of RK1 in the PIG1 cell line against H₂O₂-induced damage, the PI3K inhibitor LY294002 was used to treat the melanocytes. As shown in Fig. 5A, pretreatment with LY294002 significantly decreased the effects of RK1 on the protein expression levels of p-AKT, Nrf2 and HO-1. In addition, LY294002 also reversed the nuclear translocation of Nrf2 induced by RK1 (Fig. 5C). The findings suggested that the protective effect of RK1 against H₂O₂-induced downregulation of the Nrf2/HO-1 signaling pathway and Nrf2 nuclear translocation was mediated by the PI3K/AKT signaling pathway in the PIG1 cell line.

The effect of LY294002 on the cell viability of the PIG1 cell line was subsequently investigated. As shown in Fig. 5B, LY294002 significantly reduced the protective effect of RK1 on the cell viability of the PIG1 cell line. Then, the effect of LY294002 on apoptosis in the PIG1 cell line exposed to H₂O₂ was investigated. As shown in Fig. 5D and E, RK1 treatment significantly decreased the apoptotic rate of the PIG1 cell line exposed to H₂O₂, while the pretreatment of LY294002 significantly inhibited the protective effect of RK1 on the PIG1 cell line. These results demonstrated that pretreatment with RK1 increased the viability of the PIG1 cell line and resulted in a significant decrease of apoptotic cells via the PI3K/AKT signaling pathway.

To verify whether RK1 had a protective effect on H₂O₂-induced OS via the PI3K/AKT signaling pathway, the PIG1 cell line was treated with LY294002. As shown in Fig. 6A-C, treatment with RK1 significantly increased the activity level of SOD, CAT and GSH-Px in the PIG1 cell line exposed to H₂O₂, whereas LY294002 could significantly reverse this effect. These data indicated that RK1 alleviated the oxidative damage of the PIG1 cell line induced by H₂O₂ via the PI3K/AKT signaling pathway.