Rabbit polyclonal VDR antibody (cat. no. ab3508) was the purchased from Abcam (Cambridge, UK) and rabbit polyclonal p-STAT5 antibody (cat. no. 9351) was obtained from Cell Signaling Technology, Inc. (Denver, MA, USA). Mouse monoclonal STAT5 (cat. no. sc-377069) and p-STAT5 (cat. no. sc-81524) antibodies were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). p-STAT5 was fluorescein isothiocyanate (FITC)-labeled and VDR was tetramethylrhodamine (TRITC)-labeled. In addition, horseradish peroxidase-labeled goat anti-mouse immunoglobulin (Ig)G (cat. no. ZB-2305); horseradish peroxidase-labeled goat anti-rabbit IgG (cat. no. ZB-2301); FITC-labeled goat anti-mouse IgG (cat. no. ZF-0312); TRITC-labeled goat anti-rabbit IgG (cat. no. ZF-0316) were purchased from Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd. (Beijing, China); and mouse monoclonal anti-β-actin (cat. no., ICM001-100) was purchased from Beijing 4A Biotech Co., Ltd. (Beijing, China). 1,25-(OH)₂D₃, lipopolysaccharide (LPS) and F-actin were obtained from Sigma-Aldrich (St. Louis, MO, USA). Recombinant human interleukin (IL)-15 was manufactured by Peprotech (Rocky Hill, NJ, USA).

THP-1 cells have been widely used in investigations of human monocytes and macrophages in vitro (17,18). The THP-1 cell line (TcHu 57) used in this study was purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China).

The THP-1 cells were re-suspended in RPMI-1640 culture medium (Gibco Life Technologies, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (Gibco Life Technologies), penicillin and streptomycin (HyClone Laboratories, Inc., Logan, UT, USA) both at 100 U/ml, and flask-cultured in 5% (v/v) CO₂ at 37°C. Prior to each experiment, the cells were allowed to grow in serum-free medium for 24 h to ensure that all cells were synchronized at the G0 phase.

The synchronized cells were divided into the control, LPS + IL-15 and 1,25-(OH)₂D₃ groups according to their differing treatment. The control group was treated with phosphate-buffered saline (PBS) only. In the LPS + IL-15 group, LPS at 1 µg/ml and IL-15 at 100 ng/ml were added for 4 h of incubation. In the 1,25-(OH)₂D₃ group, the cells were pre-treated with 1,25-(OH)₂D₃ at 1×10⁻⁷ mol/l for 48 h, followed by 4 h of incubation with 1 µg/ml LPS and 100 ng/ml IL-15.

The protein expression of STAT5 and p-STAT5 was observed using western blot analysis. Following treatment according to the grouping method, ice-cold protein extraction buffer (Nanjing KeyGen Biotech Co. Ltd., Nanjing, China), supplemented with 1% protease inhibitor and 1% phosphorylation inhibitor (Nanjing KeyGen Biotech Co. Ltd.), was added for protein extraction. Protein concentrations were determined according to the instructions indicated in the bicinchoninic acid (BCA) protein concentration detection kit (Nanjing KeyGen Biotech Co. Ltd.). The protein sample was mixed with sample-loading buffer (Beyotime Institute of Biotechnology, Shanghai, China) and heated to 100°C for 5 min of loading. Proteins were separated in 6 or 10% Tris-glycine polyacrylamide gradient gels (Beyotime Institute of Biotechnology). The obtained proteins were transferred onto a nitrocellulose membrane (Invitrogen Life Technologies, Carlsbad, CA, USA) and then blocked with Tris-buffered saline-Tween® containing 5% bovine serum albumin (Cell Signaling Technology, Inc.) for 1 h. The membrane was incubated overnight at 4°C with the primary rabbit polyclonal anti-STAT and anti-p-STAT5 antibodies (1:500 dilution) or β-actin (1:5,000 dilution). Following washing, the membrane was incubated at room temperature for 2 h with horseradish peroxidase-labeled goat anti-rabbit secondary antibody (ZSGB-BIO, Beijing, China) (1:3,000 dilution with blocking buffer, Abcam). Protein expression was then detected according to the instructions provided in the chemiluminescent staining reagent kit (Beyotime Institute of Biotechnology). The average pixel density was analyzed with UN-SCAN-IT gel analysis software (Silk Scientific Inc., Orem, UT, USA).

Following treatment, the THP-1 cells were applied onto adhesive polylysine-coated glass slides (Abcam) and fixed in 4% paraformaldehyde at ambient temperature for 20–30 min for immunofluorescence and laser confocal experiments.

Monocytic cytoskeletons were characterized using laser confocal microscopy. The cells were washed in PBS, fixed in 4% paraformaldehyde, and permeabilized with 0.5% Triton X-100 for 10 min. Following PBS washing, they were incubated with 1:20 rhodamine-labeled phalloidin (Sigma-Aldrich). Specific conjugation to F-actin was allowed at 25°C (room temperature) for 40 min. The cells were washed with PBS and then mounted in an anti-photobleaching mounting medium (Santa Cruz Biotechnology, Inc.). Photomicrographs were captured with an Axio LSM 710 laser confocal microscope (Carl Zeiss GmbH, Jena, Germany) at a magnification of ×2,000.

VDR and p-STAT5 proteins were localized using fluorescence microscopy. The slides were washed thrice with PBS. Triton™ X-100 (0.3%) was added for 10 min of membrane permeabilization. Following another three washes with PBS (5–10 min per wash), they were blocked with bovine serum for 30 min.

Primary antibodies (rabbit polyclonal anti-VDR and mouse monoclonal anti-p-STAT5 antibodies, 1:200 dilution) were applied for incubation at 4°C overnight. Following three washes with PBS, the slides were dried in air. The samples were incubated with secondary antibodies (goat anti-mouse secondary antibody and goat anti-rabbit secondary antibody, 1:50 dilution) at room temperature away from light for 1–2 h. Following washing, the samples were incubated with 4′,6-diamidino-2-phenylindole (DAPI) solution at room temperature for 5–10 min. The slides were washed thrice and then covered with coverslips. Observation was performed under an Axio Observer A1 fluorescence microscope (magnification, ×10–40; Carl Zeiss AG, Oberkochen, Germany) and images were captured.

The IL-6 secretion in the cell culture supernatant was detected using an ELISA kit (PeproTech). The procedures were conducted in accordance with the manufacturer's instructions. The samples were measured in duplicate.

The interaction between VDR and p-STAT5 was validated using the co-immunoprecipitation technique.

The cells in the different groups were collected following treatment. After washing twice with pre-cooled PBS, the cells were dissociated in 1 ml pre-cooled nuclear protein extraction solution (Nanjing KeyGen Biotech Co. Ltd.) and then centrifuged at 700 × g for 15 min. The supernatant was collected. Approximately 5 µg TRITC-labeled rabbit anti-VDR polyclonal antibody (1:100 dilution; cat. no. ab3508; Abcam) was added and agitation was performed at 4°C for 4 h. Protein A-Sepharose beads (Pierce Biotechnology, Inc., Rockford, IL, USA) were added, followed by agitation at 4°C for 30 min. The sample was then centrifuged at 700 × g for 15 min. The beads bearing antigen-antibody complexes were collected. Following thrice washing with pre-cooled lysis buffer (Sangon Biotech Co., Ltd., Shanghai, China), the beads were transferred into a fresh electrophoresis tube and loading buffer was added. The suspension was boiled for 4 min, and then SDS-PAGE gradient gel electrophoresis was performed. The proteins were transferred to polyvinylidene fluoride membranes (Sigma-Aldrich) and incubated with the FITC-labeled mouse monoclonal anti-p-STAT5 antibody (1:50 dilution; cat. no. sc-81524; Santa Cruz Biotechnology, Inc.) overnight at 4°C. Following washing, the secondary antibody (goat anti-rabbit and anti-mouse IgG; 1:50 dilutions) was added and incubation proceeded at room temperature for 1 h. The membranes were washed and chromogenic developmental reagent (Beyotime Institute of Biotechnology) was added in the absence of light. Each experiment was repeated thrice.

Measurement data were presented as mean ± standard error of the mean. Statistical analysis was carried out using SPSS software (version 15.0; SPSS Inc., Chicago, IL, USA) using a t-test for comparisons between groups and one-factor analysis of variance for comparisons among groups. Differences of P<0.05 were considered to be statistically significant.

To assess the response of l,25-(OH)₂D₃ pretreated monocytes to LPS + IL-15, the protein expression of STAT5 and p-STAT5 was observed using western blot analysis. The results are shown in Fig. 1.

In the LPS + IL-15 group, the cells exhibited a significantly higher level of p-STAT5 expression compared with that in the control group (0.481±0.16 vs. 0.086±0.024; P=0.016). In the 1,25-(OH)₂D₃ group, however, p-STAT5 expression did not show a significant difference compared with that in the control group (0.092±0.028 vs. 0.086±0.024; P>0.05). No significant differences in the level of STAT5 expression were observed among the three groups (the expression levels in the control, LPS + IL-15 and 1,25-(OH)₂D₃ groups were 0.580±0.098, 0.594±0.086 and 0.568±0.105, respectively; P>0.05).

Monocytic cytoskeletons in the different groups were characterized using laser confocal microscopy. The results are shown in Fig. 2. In the control group, THP-1 cytoskeletons were primarily distributed at the periphery of the cytoplasm (Fig. 2A). In the LPS + IL-15 group, the cell morphology altered, the actins were remodeled, the actin bands at the cytoplasmic periphery disappeared and the actin masses noticeably shrank. In addition, the lysis of microfilaments was observed in certain cells (Fig. 2B). In the 1,25-(OH)₂D₃ group, the pretreatment with 1,25-(OH)₂D₃ partially prevented the effects caused by LPS + IL-15 (Fig. 2C).

To detect the interaction between VDR and p-STAT5, co-localization was performed using immunofluorescence. The cells were grouped and treated according to the method described previously. Prior to stimulation with LPS + IL-15, STAT5 was expressed in the cytoplasm. Subsequent to LPS + IL-15 stimulation, STAT5 was phosphorylated and expressed in the nucleus. In all three groups, VDR was mostly expressed in the nucleus with a small amount expressed in the membrane. p-STAT5 was almost undetectable in the control group (Fig. 3A). Compared with the control group, the LPS + IL-15 group exhibited a greater amount of nuclear p-STAT5 and some co-expressed VDR + p-STAT5 complexes (Fig. 3B; indicated in yellow). The pretreatment with 1,25-(OH)₂D₃ markedly enhanced the nuclear expression levels of VDR and p-STAT5; their co-localization was more noticeable than that in the LPS + IL-15 group (stained in yellow in Fig. 3C).

As shown in Fig. 4, the IL-6 level in the LPS + IL-15 group was significantly higher compared with that in the control and 1,25-(OH)₂D₃ groups (53.122±17.756 vs. 0.063±0.006 and 13.472±5.056 pg/ml, respectively; both P<0.01).

The possible interaction between VDR and p-STAT5 was further tested using the co-immunoprecipitation method. VDR and proteins interacting with p-STAT5 were precipitated from the cell extracts, and western blot analysis was performed (Fig. 5). In the control group, when LPS and IL-15 were absent, p-STAT5 was not observed. In the LPS + IL-15 group, p-STAT5 became noticeable in VDR-containing protein complexes. In the 1,25-(OH)₂D₃ group, the association between VDR and p-STAT5 became more evident compared with that in the LPS + IL-15 group. These data, as well as the immunofluorescence results, provided evidence of the interaction between VDR and p-STAT5. Each experiment was repeated three times and yielded a consistent result.