Recombinant SDF-1 protein was constructed as described previously (17). Briefly, the human SDF-1 sequence was amplified by PCR (Perkin Elmer, Inc.) using primers flanked with restriction sites for XhoI and KpnI (forward, 5′-XhoI-AACCTCGAGAGCGAT GGCAAACCGGTG-3′; reverse, 5′-KpnI-GTTGGTACCTTA TTTGTTCAGCGCT-3′). The thermocycling conditions consisted of 30 cycles at 94°C for 1 min, 55°C for 1 min and 72°C for 1 min. Following digestion with XhoI and KpnI at 37°C for 60 min, ligation with the pBAD/HisA vector at 20°C for 4 h was performed to produce the pBAD/HisA-SDF-1 construct.

Ligation mixture was spread in Luria-Bertani medium (LPS Solution Co., Ltd.) containing 100 µg/ml ampicillin after heat-shock at 42°C for 1 min. Following bacterial transformation, TOP10 E. coli cells (Invitrogen; Thermo Fisher Scientific, Inc.) were cultured in Luria-Bertani medium containing 100 µg/ml ampicillin overnight at 37°C. Once the optical density value at 600 nm reached 0.6, bacterial cells were incubated with 0.1% (w/v) L-arabinose at 20°C for 6 h. Cells were centrifuged at 6,000 × g at for 10 min at 4°C, then lysed (50 mM NaH₂PO₄, 300 mM NaCl, 10 mM imidazole, pH 8.0). After centrifugation at 14,000 × g at 4°C for 30 min, the soluble protein was purified from the supernatant by affinity to a nickel-nitrilotriacetic acid resin (Invitrogen; Thermo Fisher Scientific, Inc.). The purified recombinant SDF-1 protein was analyzed via 10% SDS-PAGE followed either by Coomassie Brilliant Blue staining or western blotting. The protein concentrations were measured using the Bradford method (Bio-Rad Laboratories, Inc.). The purity of SDF-1 protein was determined by Coomassie Brilliant Blue staining at room temperature for 30 min, and the molecular size was confirmed by western blotting. For western blotting, SDF-1 protein (50 µg/lane) was electrophoresed via SDS-PAGE on a 10% gel, and subsequently transferred to nitrocellulose membrane. Following which, membranes were blocked with 1% BSA at room temperature for 1 h, washed and then incubated with the mouse anti-His antibody (1:1,000; cat. no. sc-8036; Santa Cruz Biotechnology, Inc.) at 4°C overnight. The blotted membrane was washed with Tris-buffered saline with 0.5% Tween-20 and incubated with HRP-conjugated anti-mouse secondary IgG (1:2,000; cat. no. sc-2371; Santa Cruz Biotechnology, Inc.) at room temperature for 2 h. Bands were detected using ECL detection reagent (Immobilon^® ECL Ultra Western HRP Substrate; EMD Millipore).

The PC-12 rat pheochromocytoma cell line was purchased from American Type Culture Collection and cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco; Thermo Fisher Scientific, Inc.) containing 10% heat-inactivated fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.) and 5% penicillin-streptomycin solution in a 5% CO₂ atmosphere at 37°C.

Cell viability was evaluated using an MTT assay. PC-12 cells were seeded at a density of 1×10⁴ cells/well and incubated for 24 h at 37°C with 5% CO₂. The cells were washed three times with PBS, then incubated for 24 h at 37°C with 5% CO₂ in the presence of 0.5, 1 and 5 µg/ml recombinant SDF-1 protein. Untreated cells were also used as a control. Subsequently, 500 µl MTT was added to each well and incubated at 37°C for 4 h. Then, the media was removed and dissolved with DMSO. Optical density at 540 nm (OD₅₄₀) was read using a microplate reader. Cell proliferation activity was similarly evaluated. PC-12 cells (1×10³ cells/well) were incubated for 3 and 5 days with various concentration of SDF-1 protein. Cell proliferation was also measured by reading at OD₅₄₀.

Cell migration was evaluated using an in vitro wound healing assay (18). PC-12 cells were seeded at a density of 1×10⁶ cells/well to create a confluent monolayer. Following overnight serum starvation at 37°C with 5% CO₂, a scratch was made using a 200 µl pipette tip. Cells were incubated for 6 days in the presence of 5 µg/ml. Untreated cells were used as a negative control. Cell migration was evaluated at day 0, 2, 4 and 6 (magnification, ×10) under a CKX41 light microscope (Olympus Corporation).

PC-12 cells were seeded at a density of 1×10³ cells/well and incubated with 5 µg/ml SDF-1 protein for 5 days at 37°C with 5% CO₂. Untreated cells were also used as a negative control. On the fifth day, neurite number and length were examined under a CKX41 light microscope (magnification, ×10). Three images per well were captured in each three experiments. To evaluate neurite number, cell images were captured and the neurite number in the field was counted. Neurite number was normalized with the control and expressed as a percentage. To evaluate neurite length, neurite with 1.5 times longer than the cell body were scored.

Neurite morphology was examined under a CLSM. Cells were treated as aforementioned, and subsequently fixed with 10% formalin for 30 min and stained with Alexa Fluor 488 conjugated phalloidin and DAPI for 30 min (Invitrogen; Thermo Fisher Scientific, Inc.). Then, neurite morphology was observed under an LSM700 confocal microscope (magnification, ×10; Carl Zeiss AG).

Neurite morphology was also examined under SEM. Cells were fixed with 2.5% glutaraldehyde at room temperature for 30 min, and serially dehydrated with ethanol solution (50, 70, 90, 95 and 100%) for 10 min each. Specimens were then treated with hexamethyldisilazane (Sigma-Aldrich; Merck KGaA) and coated with platinum (magnification, ×250).

PC-12 cells were seeded at density of 1×10³ cells/well and incubated for 5 days at 37°C with 5% CO₂. Total RNA was extracted using an Easy-spin Total RNA Extraction kit (Intron Biotechnology, Inc.). Then, cDNA was synthesized at 50°C for 50 min and 85°C for 5 min using SuperScript^® IV-First Strand Synthesis System (Invitrogen; Thermo Fisher Scientific, Inc.). The expression of the NF gene was quantified by RT-qPCR. The reaction mixture contained 0.1 µm of each primer, 10 µl of 2X SYBR-Green PCR Master Mix (Applied Biosystems; Thermo Fisher Scientific, Inc), including AmpliTaq Gold™ DNA Polymerase with Buffer, dNTPs mix, SYBR-Green I dye, Rox dye and 10 mm MgCl₂, and 1 µl template cDNA. PCR was conducted with activation (94°C for 10 min), followed by 40 cycles of denaturation (94°C for 15 sec), and annealing and extension (60°C for 1 min). Primer sequences are listed in Table I. The Cq value for each gene was normalized to GAPDH using the formula: dCq = Cq_(GAPDH) - Cq_(NF) (19).

All experiments were conducted three times. Data are expressed as the mean ± SD. Date was analyzed using Student's t-test or one-way ANOVA followed by Duncan's multiple range test. Statistical analysis was carried out using GraphPad Prism 7 software (GraphPad Software, Inc.). P<0.05 was considered to indicate a statistically significant difference.

Recombinant SDF-1 protein was constructed and purified from E. coli. The yield for purified SDF-1 protein from a 1 L culture was 0.7 mg/ml. The molecular weight of the purified recombinant SDF-1 protein was ~15 kDa (Fig. 1).

Cell viability was measured to evaluate the potential cytotoxic effects of the recombinant SDF-1 protein on PC-12 cells. PC-12 cells incubated with SDF-1 concentrations ranging from 0.5, 1 and 5 µg/ml retained >95% cell viability, compared with untreated cells (Fig. 2A).

PC-12 cell proliferation was measured after incubation for 3 or 5 days in the presence of 0.5, 1 or 5 µg/ml SDF-1. At both timepoints, cell proliferation significantly increased in a dose-dependent manner (Fig. 2B). In particular, cell proliferation in the presence of 5 µg/ml SDF-1 increased ~1.5-fold. Based on cell viability and proliferation findings, SDF-1 was used at a concentration of 5 µg/ml in subsequent experiments.

Cell migration in PC-12 cells in the presence of SDF-1 was assessed using an in vitro scratch assay over a 6-day time course. At day 2, SDF-1 induced PC-12 cell migration from the edge of the scratch at 2 days, compared with the initial time point (Fig. 3). However, at day 6, cell migration in the presence SDF-1 was similar to the control. These results confirmed the chemoattractant effect of SDF-1 in PC-12 cells.

Neurite number and length in PC-12 cells were determined from microscopy images (Fig. 4). In all examined microscopic images, SDF-1 induced neurite outgrowth in PC-12 cells (Fig. 4A-C). This stimulating effect of SDF-1protein on neurite outgrowth was observed in images obtained from an optical microscope, as well as CLSM and SEM. In addition, SDF-1 significantly increased neurite number in PC-12 cells 8.97-fold, compared with the control (Fig. 4D). Moreover, SDF-1 also significantly increased the average neurite length in the SDF-1-treated cells, compared with the control (230.56±47.91 vs. 101.39±37.74 µm, respectively; Fig. 4E). Therefore, SDF-1 stimulates neurite outgrowth in PC-12 cells. Collectively, these results suggest that recombinant SDF-1 protein might be utilized for nerve regeneration by inducing neurite outgrowth.

NF gene expression levels were significantly upregulated in PC-12 cells treated with 5 µg/ml SDF-1 protein, compared with control cells (Fig. 5). These results suggested that SDF-1 might influence neurite outgrowth in part by upregulating the NF gene.