RPMI-1640 (Gibco, Thermo Fisher Scientific, Inc., Waltham, MA, USA), supplemented with 100 U/ml penicillin and 100 µ/ml streptomycin (Beijing Solarbio Science and Technology Co., Ltd., Beijing, China), fetal bovine serum (Hangzhou Sijiqing Biological Engineering Materials Co., Ltd., Hangzhou, China), SDF-1 (R&D Systems, Inc., Minneapolis, MN, USA), and nitric oxide synthase inhibitor N^G-monomethyl-l-arginine monoacetate salt (L-NMMA, Beyotime Institute of Biotechnology, Haimen, China) were obtained and used without further modification. Total Nitric Oxide assay kit was from the Beyotime Institute of Biotechnology (cat. no. S0021). Anti-NOS2 (Phospho-Tyr151) rabbit polyclonal antibody (NOS2-pY151; cat. no. D151565) and anti-NOS3 (Phospho-Thr494) rabbit polyclonal antibody (NOS3-pY494; cat. no. D151279) were obtained from Sangon Biotech Co., Ltd. (Shanghai, China).

Jurkat cells from Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China) were maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum, 25 ng/ml SDF-1 and 1 mM L-NMMA in a humidified incubator with 5% CO₂ at 37°C. A total of 1 mM L-NMMA was dissolved in double distilled water and cells were pre-treated with L-NMMA for 2 h. Jurkat cells were starved with 0.5% serum overnight in all experiments.

Total nitric oxide production in the culture medium was determined by measuring the concentration of nitrate and nitrite, a stable metabolite of NO, by the modified Griess reaction method (29,30). The procedure was performed according to the manufacturer's protocol of the Total Nitric Oxide assay kit (Beyotime Institute of Biotechnology). RPMI-1640 medium was used as the control for SDF-1.

Cells were stimulated at 37°C for 0, 1, 2, 3 or 4 h and lysed in ice-cold lysis buffer (50 mM Tris pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Nonidet P-40, 2.5 mM sodium pyrophosphate, 1 mM each NaF, Na3VO4, and β-glycerolphosphate, 10 µg/ml aprotinin and leupeptin). After 30 min on ice, lysates were centrifuged at 13,000 × g for 30 min. Protein concentration was determined by the BCA kit (cat. no. P0010) from Beyotime Institute of Biotechnology. A total of 20 µg lysate from each sample were subjected to 10% SDS-PAGE. Proteins were transferred to the nitrocellulose membranes using chilled transfer buffer (25 mM Tris, 192 mM glycerin, and 20% methanol) at 100 V for 1 h. Following protein transfer, nitrocellulose membranes were washed with TBST (20 mM Tris, 500 mM NaCl, 0.05% Tween-20, pH 7.5) for ≥3 times, and were immediately incubated with 3% bovine serum albumin (BSA, cat. no. SW3015) from Beijing Solarbio Science and Technology Co., Ltd., Beijing, China. After the incubation for 1 h at room temperature, the membrane was incubated with primary antibodies NOS2-pY151 and NOS3-pY494 respectively diluted at a ratio of 1:1,000 in TBST at 4°C overnight, washed with TBST 3 times at room temperature, and incubated with HRP-conjugated goat anti-rabbit secondary antibody (cat. no. D110058; Sangon Biotech Co., Ltd., Shanghai, China) diluted at a ratio of 1:1,000 in TBST at 37°C for 1 h. For the detection of the reference gene, mouse monoclonal antibody against actin (cat. no. D190606; Sangon Biotech Co., Ltd., Shanghai, China) and HRP-conjugated goat anti-mouse IgG (cat. no. D110087; Sangon Biotech Co., Ltd., Shanghai, China) were used. Chemiluminescent detection was performed by using ECL plus Western blot reagents (BeyoECL Plus; cat. no. P0018; Beyotime Institute of Biotechnology). Western blot assay was repeated three times, analyzed by image-Pro Plus 6.0 (Media Cybernetics, Inc., Rockville, MD, USA), and expressed as fold increase relative to the basal level in unstimulated cells.

The MTT assay was employed to quantify cell viability. Jurkat cells were washed once with RPMI-1640, and ~3×10⁵ cells were seeded to each sample. The cells were then cultured at 37°C for 6 h with different concentrations of L-NMMA (0, 0.25, 0.5, 1, 2 and 4 mM). At the end of the treatment, MTT reagent (5 mg/ml, 20 µl) was added to each sample and cells were incubated at 37°C for 3 h, DMSO (200 µl) was then added to each sample, in order to dissolve the cell precipitation. A total of 150 µl liquid was transferred to the well of a96-well plate and the optical density value of each well was determined using a plate reader at 570 nm.

To detect the distribution of F-actin, Jurkat cells were pre-treated with L-NMMA (1 mM) at 37°C for 2 h, and then stimulated with SDF-1 at 37°C for 4 h. At 4 h time, chilled 500 µl PBS was added to cells to rapidly stop the reaction. Jurkat cells were then fixed with 4% paraformaldehyde for 20 min at room temperature. After washing twice with PBS, the Jurkat cells were permeabilized with 0.2% Triton X-100 in PBS (containing 5mM EDTA and 2% FBS) for 10 min. Following washing twice with PBS, the cells were stained with 2×10⁻⁷ M Fluorescein isothiocyanate (FITC)-conjugated phalloidin at room temperature for 20 min, washed three times with PBS, and then observed under a confocal microscope at ×600 magnification.

The amount of F-actin in cells was examined by flow cytometry. Following stimulation with SDF-1, Jurkat cells were fixed with 4% paraformaldehyde at room temperature for 20 min, permeabilized with 0.2% Triton X-100 in PBS (containing 5 mM EDTA and 2% FBS) for 10 min, blocked in 1% BSA (cat. no. SW3015) from Beijing Solarbio Science and Technology Co., Ltd. (Beijing, China) in 0.05% Tween 20 and PBS for 30 min at room temperature, stained with 2×10⁻⁷ M FITC-conjugated phalloidin (cat. no. P5282-.1MG; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) at room temperature for 20 min, and then washed with PBS. Cells were examined by flow cytometry (FACSCalibur; BD Biosciences, Franklin Lakes, NJ, USA), analyzed by FlowJo 7.6 software (TreeStar, Inc., Ashland, OR, USA), and the results were expressed as the relative fluorescence index (RFI) by dividing the fluorescence value of the experimental groups by that of the control group. RPMI-1640 medium was used as the control for SDF-1.

The migration assay was performed using 3.0 µm Transwell inserts in 24-well culture plates. The Jurkat cells were resuspended in serum-free RPMI-1640 at a density of 3×10⁶ cell/ml and 100 µl cell suspensions were pre-treated with L-NMMA (1 mM) at 37°C for 2 h and seeded onto the upper chambers. Next, 500 µl of RPMI-1640 supplemented with SDF-1 was added to the lower chamber. After 4 h incubation at 37°C, the migrated cells were imaged at ×100 magnification under a light microscope, and the relative migration rates were analyzed. RPMI-1640 medium was used as the control for SDF-1.

All experiments were performed in triplicate. The data were expressed as the mean ± standard deviation and were analyzed by one-way analysis of variance with Bonferroni's post-hoc test using the statistical software package SPSS 17.0 (SPSS, Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference.

NO serves a critical role in signal transduction. Therefore, cellular NO levels were analyzed following SDF-1 treatment for 1, 2, 3 or 4 h. NO levels were significantly increased following SDF-1 stimulation for 4 h compared with the control group (Fig. 1A). Additionally, NO synthases 2 and 3 were phosphorylated following SDF-1 treatment (Fig. 1B), corresponding to activities, which catalyze the generation of NO.

To investigate the role of NO in SDF-1-induced cell migration, the NOS inhibitor, L-NMMA was used. The results demonstrated that pretreatment of Jurkat cells with L-NMMA led to the inhibition to cell migration towards SDF-1 (Fig. 2A), which suggested that NO was required for cell migration. Jurkat cells were then treated with different concentrations of L-NMMA and found that the different concentrations of L-NMMA had no significant effect on cell viability (Fig. 2B), suggesting that the inhibitory effect of L-NMMA on cell migration was not due to a reduction in cell viability.

The mechanism by which NO affects SDF-1-induced Jurkat cell migration was further tested by observing and measuring the distribution of the F-actin cytoskeleton, in the presence or absence of L-NMMA when stimulated by SDF-1. The results showed that L-NMMA significantly inhibited the F-actin rearrangement induced by SDF-1 (Fig. 3A) and the F-actin polymerization-induced by SDF-1 (Fig. 3B). NO was required for F-actin rearrangement and polymerization in response to SDF-1. This suggests that SDF-1-induced migration was regulated by NO production and the subsequent F-actin rearrangement and polymerization.