HL-60 cells, obtained from The Cancer Research Institute, Xiangya Medical College, Central South University (Changsha, China), were maintained in L-glutamine-containing RPMI 1640 medium (HyClone; GE Healthcare Life Sciences, Logan, UT, USA), supplemented with 10% fetal bovine serum (Zhejiang Tianhang Biotechnology Co., Ltd., Hangzhou, China) and without antibiotics. The cells were incubated at 37°C in 5% CO₂ for 2 days before they were harvested. DADS (purity 80%; the remaining 20% being diallyl trisulfide and diallyl sulfide) was purchased from Sigma-Aldrich; Merck Millipore (Darmstadt, Germany), dissolved in Tween 80 at 8 mg/ml and stored at −20°C. HL-60 cells (2×10⁶ cells/ml) in the logarithmic growth phase were placed in fresh medium prior to use in experiments. DADS was diluted to 1.25, 2.5, 5 and 10 mg/l with culture medium and added to cell cultures. Following 4, 8 or 12 h DADS treatment, cells were collected for analysis.

HL-60 cells (2×10⁶ cells/ml) in the logarithmic growth phase were harvested and washed with ice-cold phosphate-buffered saline (PBS). Nuclear and cytoplasmic proteins were isolated from cells using NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher Scientific, Inc., Waltham, MA, USA) according to the manufacturer's instructions. Briefly, cells were transferred to a 1.5 ml microcentrifuge tube and pelleted by centrifugation at 500 × g for 5 min, 4°C before a pipette was used to remove the supernatant. Ice-cold Cytoplasmic Extraction Reagent (CER) I was subsequently added to the cell pellet, and the tube was vortexed vigorously for 10 sec before incubating on the ice for 20 min. Ice-cold CER II, was added to the solution, and the tube was vortexed for 5 sec and then centrifuged for 10 min, 4°C at maximum speed in a microcentrifuge (160,000 × g). The resulting supernatant was the cytoplasmic extract, and the insoluble fraction, containing the cell nuclei, was dissolved in ice-cold Nuclear Extraction Reagent. The sample was subsequently vortexed for 15 sec and then incubated on ice for 10 min. This step was repeated 4 times over the course of 40 min and was followed by centrifugation at maximum speed (160,000 × g) in a microcentrifuge for 30 min at 4°C, to obtain the nuclear protein extract.

Mitochondrial proteins were isolated using a Cell Mitochondria Isolation kit (Beyotime Institute of Biotechnology, Haimen, China) according to the manufacturer's instructions. Briefly, cells (2×10⁶ cells/ml) in the logarithmic growth phase were harvested and washed twice with ice-cold PBS. Cells were incubated with cell lysis buffer (1 ml cell lysis buffer/2×10⁷ cells) for 10 min at 4°C, before they were homogenized with a glass homogenizer. The cell lysate was centrifuged at 600 × g for 10 min at 4°C to remove any remaining whole cells, and the supernatant was further centrifuged at 11,000 × g for 10 min at 4°C. The supernatant was removed and the remaining mitochondrial pellet was resuspended in mitochondrial lysis buffer at 4°C, and centrifuged at 24,000 × g for 15 min at 4°C to remove the cell nuclei. Mitochondrial extracts were stored at −80°C until required. The concentration of cytoplasmic, nuclear and mitochondrial protein extracts was determined using a bicinchoninic acid assay kit (Pierce; Thermo Fisher Scientific, Inc.).

Sample protein extracts were denatured by boiling at 100^ºC for 5 min in 5X SDS-PAGE Sample Loading buffer (Beyotime Institute of Biotechnology), at a ratio of 4:1. Proteins were then separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes by electroblotting. Membranes were blocked using 5% non-fat dry milk in tris-buffered saline with 0.1% Tween 20 (TBST) buffer for 2 h at room temperature. Membranes were subsequently incubated with mouse monoclonal anti-DJ-1 (dilution, 1:3,000; catalog no. 05–828; EMD Millipore, Billerica, MA, USA), mouse anti-β-actin (dilution, 1:2,000; catalog no. A5441; Sigma-Aldrich; Merck Millipore), rabbit polyclonal anti-TATA-box binding protein (TBP; dilution, 1:2,000; catalog no. 22246-1-AP; ProteinTech Group, Inc., Chicago, IL, USA), or rabbit polyclonal anti-voltage-dependent anion channel 1 (VDAC1; dilution, 1:1,000; catalog no. 10866-1-AP; ProteinTech Group, Inc.) primary antibodies overnight at 4°C. β-actin, VDAC1 and TBP were used to normalize protein loading in cytoplasmic, mitochondrial and nuclear protein extracts, respectively. Membranes were then washed with TBST and incubated for 1 h with horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG, (dilution, 1:2,000; catalog no. CW0102M; Beijing ComWin Biotech Co., Ltd., Beijing, China) or HRP-conjugated goat anti-rabbit IgG, (dilution, 1:1,000; catalog no. CW0103M; Beijing ComWin Biotech Co., Ltd.). After washing the membranes in TBST, antibody binding was detected using Ponceau S staining solution (Beyotime Institute of Biotechnology). Densitometry was performed using AlphaImager software version 3.3.0 (ProteinSimple Bioscience and Technology Co., Ltd., Shanghai, China), and target protein expression levels were normalized to the reference protein expression levels.

Immunocytochemical analysis of DJ-1 expression in HL-60 cell specimens was performed using a DAB Detection Kit (Fuzhou Maixin Biotech, Co., Ltd., Fuzhou, China). Cells (20 µl/coverslip; 1×10⁶ cells/l) were first seeded on coverslips and fixed with 95% ethanol for 30 min at room temperature. Endogenous peroxidase activity was inhibited by incubating cells in 0.3% (v/v) hydrogen peroxide in methanol for 10 min, followed by washing three times with PBS for 5 min each time. Samples were then blocked with 10% (v/v) normal goat serum diluted in PBS for 30 min, before incubating with the anti-DJ-1 antibody [diluted, 1:200 in PBS containing 3% (wt/vol) bovine serum albumin; catalog no. 05–828; EMD Millipore] overnight at 4°C. After washing three times with PBS for 5 min each time, cells were incubated with a biotinylated goat anti-mouse IgG antibody (dilution, 1:100; catalog no. bs-0296G-Bio; BIOSS, Beijing, China) for 15 min at room temperature, followed by three additional 5-min wash steps with PBS. Cells were subsequently incubated with streptavidin-avidin-HRP for 20 min at room temperature, and then washed with PBS as described above. Samples were visualized by incubating cells in 3,3′-diaminobenzidine at room temperature for 2 min. After counterstaining with hematoxylin for 30 sec and rinsing with tap water, samples were immediately dehydrated by sequential immersion in gradient ethanol solutions. Images were obtained using a light microscope (Olympus Corporation, Tokyo, Japan).

Immunofluorescence analysis of DJ-1 expression in HL-60 cells was achieved by first seeding cells (20 µl/coverslip; 1×10⁶ cells/l) on coverslips and fixing with 4% paraformaldehyde for 20 min at room temperature. Samples were then blocked in PBS containing 0.2% Triton X-100 and 5% normal goat serum for 1 h at room temperature. Cells were subsequently incubated with mouse monoclonal anti-DJ-1 (diluted, 1:200 in PBS containing 0.2% Triton X-100 and 1% normal goat serum; catalog no. 05–828; EMD Millipore) at 4°C overnight. After washing three times with PBS, cells were incubated with a Texas Red-conjugated goat anti-mouse IgG (dilution, 1:50; catalog no. SA00005-1; ProteinTech Group, Inc.) secondary antibody at room temperature for 1 h. Nuclei were stained with 4′,6-diamidino-2-phenylindole for 5 min at room temperature, before the cells were washed twice with PBS and analyzed using an EVOS FL Auto fluorescence microscope (Thermo Fisher Scientific, Inc.).

Experiments were repeated a minimum of three times. Data are presented as the mean ± standard deviation. Differences between treatment groups were analyzed with the Student's t-test using GraphPad Prism software version 5.01 (GraphPad Software, Inc., La Jolla, CA, USA). P<0.05 was considered to indicate a statistically significant difference.

As shown in Fig. 1, western blot analysis demonstrated that the protein expression levels of cytoplasmic DJ-1 in HL-60 cells treated with DADS (1.25 mg/l for 8 h) were significantly reduced (P=0.013), while the nuclear expression levels of DJ-1 were significantly increased (P=0.032).

As shown in Fig. 2, western blot analysis indicated that the protein expression levels of DJ-1 in the mitochondria of HL-60 cells following treatment with by DADS (1.25 mg/l) were not significantly different at 4, 8 and 12 h when compared with untreated controls (P=0.178). By contrast, exposure of HL-60 cells to 5 and 10 mg/l DADS for 8 h was associated with a significant decrease in mitochondrial DJ-1 protein expression levels (P=0.029; Fig. 3).

As presented in Fig. 4A, immunocytochemical analysis demonstrated that DJ-1 protein expression was detected primarily in the cytoplasm, and low levels were detected in the nucleus. By contrast, following exposure of HL-60 cells to 1.25 mg/l DADS for 8 h, an increase in the expression of DJ-1 protein was observed in the nucleus (Fig. 4B).

As demonstrated in Fig. 4, DJ-1 expression was observed primarily in the cytoplasm, with low levels observed in the nucleus. In order to confirm the observed increase in DJ-1 expression in the nucleus following exposure of cells to DADS, the intracellular distribution DJ-1 in HL-60 cells was analyzed by immunofluorescence. As shown in Fig. 5, an increase in the number of DJ-1-positive nuclei were observed in HL-60 cells following exposure to DADS (1.25 mg/l) for 8 h (Fig. 5F), when compared with untreated controls (Fig. 5C). These results provide additional evidence demonstrating that DJ-1 translocated from the cytoplasm to the nucleus following DADS treatment.