The murine monocyte/macrophage cell line, RAW264.7, was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and routinely cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS) (both from Gibco-BRL, Carlsbad, CA, USA) at 37°C in a humidified air atmosphere and 5% CO₂. The RAW264.7 cells were plated at 5×10⁵ cells/well into 6-well plates and then treated with 1.5 ml of medium.

A siRNA expression vector was constructed by introducing synthetic double-stranded oligonucleotides as follows: Kif4 sense, 5′-GCUGAAGUUUAGGCAAUTT-3′ and antisense, 5′-AUUGCCUAAACUUCUCAGCTT-3′; negative control (NC) sense, 5′-UUCUCCGAACGUGUCACGUTT-3′ and antisense, 5′-ACGUGACACGUUCGGAGAATT-3′ synthesized by GeneChem, Co., Ltd. (Shanghai, China) and green fluorescent FAM-labeled negative control siRNA (Shanghai GenePharma Co., Ltd., Shanghai, China) was used to detect the transfection efficiency. The RAW264.7 cells at 70% confluency were transfected with the Kif4 siRNA (si-Kif4) or NC siRNA (si-NC) or FAM labeled NC siRNA (6 µl) premixed with Lipofectamine™ 2000 (Invitrogen Life Technologies, Carlsbad, CA, USA) (4.5 µl) in Opti-MEM in 6-well plates. The mock-transfected cells were transfected only with Lipo fectamine™ 2000 and were included as a control. After 6 h, the cells were placed in fresh complete medium. In order to confirm whether siRNA was able to transfect into cells, the cells transfected with FAM labeled NC siRNA were observed under an inverted fluorescence microscope (serial no. 321463; Leica, Wetzlar, Germany). Cells were observed and photographed at ×100 magnification under light field and fluorescence (520 nm), respectively. The transfection efficiency was determined by the positive rate of FAM green fluorescence from 5 different fields. To examine the effects of si-Kif4 on macrophage morphology, RAW 264.7 cells were examined using an inverted microscope (serial no. 321463; Leica). After being transfected with the si-NC or si-Kif4 for 48 h, RAW264.7 cells were observed under light field at ×200 magnification. Mouse insulin-like growth factor-1 (IGF-1) (100 ng/ml) was used to treat the cells for 36 h in si-NC and si-Kif4 groups, after 48-h transfection with si-NC or si-Kif4. IGF-1 was purchased from ProSpec-Tany TechnoGene, Ltd. (Ness Ziona, Israel).

The RAW264.7 cells were collected and washed with cold phosphate-buffered saline (PBS). The cells were then incubated with monoclonal antibodies against CD11c (M10118-02B) and CD80 (M10801-09B) (both from Tianjin Sungene Biotech Co., Ltd., Tianjin, China) for 20 min in the dark. The cells were washed with cold PBS again and analyzed with a flow cytometer (FACSCalibur; Becton-Dickinson, Mountain View, CA, USA). A minimum of 10,000 events/sample were collected for analysis.

Total RNA was extracted from the cells using an RNeasy Mini kit (Qiagen, Dusseldorf, Germany) in accordance with the RNeasy Mini Handbook. RNA was reverse transcribed using the ReverTra Ace qPCR RT kit (Toyobo Co., Ltd., Osaka, Japan). PCR analysis was performed with cDNA and the following primers: mouse VEGFR1 forward, 5′-CTTTCTCAAGTGCAGAGGGG-3′ and reverse, 5′-TCATGTGCACAAGTTTGGGT-3′; mouse VEGF-A forward, 5′-GGGACCCCTTCGTCCTCTC-3′ and reverse, 5′-GTCTCCTGGGGACAGAATTAGTG-3′; and mouse glyceraldehyde 3-phosphate dehydrogenase (GAPDH) forward, 5′-CCCACTAACATCAAATGGGG-3′ and reverse, 5′-CCTTCCACAATGCCAAAGTT-3′.

The cDNA sample amplification was carried out using SYBR PremixEx Taq™ II (Tli RNaseH Plus; Takara, Dalian, China). The RT-PCR conditions were as follows: 3 sec at 95°C, followed by 40 cycles consisting of denaturing at 95°C for 5 sec, annealing at 60°C for 10 sec, and extending at 72°C for 15 sec. Relative quantification values were calculated using the ΔΔCqmethod.

The cells were collected and lysed in ice-cold radioimmunoprecipitation assay (RIPA) lysis buffer containing 1 mM phenylmethanesulfonyl fluoride (Solarbio, Beijing, China). The supernatant were collected following centrifugation at 10,000 rpm for 15 min at 4°C. The protein concentration was determined using a Micro BCA™ Protein Assay kit (Pierce, Rockford, IL, USA). Equal amounts of protein were loaded, separated by SDS-PAGE and transferred onto PVDF membranes (Invitrogen Life Technologies). For 2 h, the membranes were blocked with 5% (w/v) fat-free milk dissolved in Tris-buffered saline containing 0.05% Tween-20 (TBST). The membranes were then hybridized to a mouse anti-AKT antibody (1:1,000 #9272), a phosphorylated Akt (p-Akt) antibody (1:1,000; #9271) or GAPDH antibody (1:2,000; #97166) (all from Cell Signaling Technology, Inc., Boston, MA, USA) at room temperature for 2 h. The membranes were washed in TBST and then incubated with HRP-conjugated secondary antibody (1:2,000; ZDR-5307; ZSGB-BIO, Beijing, China). Blots were visualized using an ECL chemiluminescent kit (Millipore, Billerica, MA, USA). The bands were processed and analyzed using the FluorChem system (Alpha Innotech, Cell Biosciences Inc., Santa Clara, CA, USA).

The concentrations of mouse sVEGFR1 and VEGF-A in the cell culture medium were detected using ELISA kits (R&D Systems, Inc., Minneapolis, MN, USA), according to the manufacturer's instructions. Briefly, 50 µl assay diluent was added to each well and the same volume of serially diluted standards and samples were loaded on the plate followed by incubation for 2 h at room temperature on the shaker at 200 rpm. Excess antibodies were then removed 5 times with wash buffer, and incubated for 2 h with conjugate. The reaction was developed by substrate and the optical density was measured using a spectrophotometer (GeneQuant 100; Biochrom, Holliston, MA, America) at 450 nm (correcting at 540 nm).

The values are expressed as the means ± standard deviation (SD). Data were analyzed using the Student's t-test or by analysis of variance (ANOVA). A P-value <0.05 was considered to indicate a statistically significant difference.

The RAW264.7 cells were transfected with si-NC or FAM si-NC for 6 h, and were observed under a fluorescence microscope. We observed green fluorescence in the RAW264.7 cells. This suggests that the FAM si-NC with green fluorescent tags was successfully transfected into the cells (Fig. 1A).

RNA interference targeting Kif4 was determined by RT-PCR. The reduction of Kif4 mRNA expression by ~60% was observed in the si-Kif4 transfected cells compared to the si-NC- and Lipofectamine 2000 only-transfected cells (Fig. 1B). No morphological changes in the cells were observed at 48 h following transfection (Fig. 1C). In both the si-NC group and si-Kif4 group, RAW264.7 cell bodies were plump, oval and thin at the cell ends. Most of the cells exhibited two or fewer extended dendritic pseudopods. Flow cytometric analysis indicated that the expression of the costimulatory molecules, CD11c and CD80, was not obviously altered (Fig. 1D).

The results of RT-PCR revealed that at 24 h following transfection of the RAW264.7 cells with si-Kif4, VEGF-A mRNA expression was markedly decreased (Fig. 1E). However, at 48 h post-transfection, the level of VEGF-A was increased in the cell supernatant, as shown by ELISA (Fig. 1F), which was inconsistent with the decreased gene level. We hypothesized that this was due to a decrease in the level of sVEGFR1 in the cell supernatant, which is a decoy receptor of VEGF-A, which kept the levels of free VEGF-A from decreasing. Thus, we examined whether Kif4 regulates the levels of VEGFR1 and sVEGFR1 in the RAW264.7 cells.

The results of RT-PCR revealed that at 24 h post-transfection, VEGFR1 mRNA expression was decreased (Fig. 2A). The results of ELISA also revealed that the level of sVEGFR1 was decreased in the cell supernatant following transfection with si-Kif4 (Fig. 2B). In addition, the results of western blot analysis indicated that the protein expression of VEGFR1 was decreased following transfection with si-Kif4 (Fig. 2C).

We then explored the signaling mechanisms involved in the regulatory effects of si-Kif4 on the levels VEGFR1 and sVEGFR1 in RAW264.7 cells. We found that the levels of p-Akt significantly decreased at 48 h post-transfection in the si-Kif4-transfected group compared with the mock- and si-NC-transfected groups (Fig. 3A). IGF-1 is the specific agonist of PI3K/Akt. Thus, we also used IGF-1 to examine its effects on the transfected cells. The results revealed a high level of p-Akt at 30 min after the addition of IGF-1 (100 ng/ml) in the si-Kif4-transfected group; the level remained high at 60 min (Fig. 3B).

Following the addition of IGF-1 (100 ng/ml) for 36 h in the si-NC- and si-Kif4-transfected groups, we detected a significant increase in the VEGFR1 mRNA (Fig. 4A), and VEGFR1 protein levels (Fig. 4B); the levels of sVEGFR1 also increased in the cell supernatant in the si-Kif4-transfected group (Fig. 4C).

Following the addition of IGF-1 (100 ng/ml) for 36 h, we found that VEGF-A mRNA expression was did significantly altered between the si-NC- and si-Kif4-transfected groups (Fig. 5A); however, the expression of VEGF-A in the cell supernatant was decreased in the si-Kif4-transfected group (Fig. 5B).