The mouse NIH3T3 fibroblast cell line (Nanjing Branch Bai Biological Technology Co., Ltd., Nanjing, China) was placed into 25-cm² flasks pretreated with fetal bovine serum (FBS; Gibco, Thermo Fisher Scientific, Inc., Waltham, MA, USA), and incubated horizontally for 1 h and then vertically for 3 h in an atmosphere of 5% CO₂ at 37°C. The cells were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 5.5 mM glucose, 10% FBS and 1% penicillin-streptomycin. Medium was replaced every three days. The cultured cells were digested and passaged with 0.25% trypsin (Gibco; Thermo Fisher Scientific, Inc.) when cell confluence reached ~80%. Cells passaged 3–6 times were used in the following experiments.

To overexpress FGF7 in NIH3T3 cells, FGF7 open reading frame sequences (NM_008008.4, NCBI) were synthesized and N-terminally fused with FLAG-tag coding sequences (Sangon Biotech Co., Ltd., Shanghai, China). To transduce NIH3T3 cells with lentivirus (Beijing Omega Bio-Technology Co., Ltd, Beijing, China), NIH3T3 cells were seeded at 2×10⁵/well into 24-well plates. Following overnight culture, 3, 9 or 12 μl lentivirus (10⁸/ml) was added to the wells in the presence of 4 mg/ml Polybrene^® (Sigma-Aldrich, St. Louis, MO, USA). The plates were then centrifuged at 800 × g at room temperature for 1 h and returned to culture in DMEM. Transduced cells, and mock-treated NIH3T3 cells, were analyzed 24 h later by a confocal microscope (Olympus, Japan) to detect expression of the reporter green fluorescent protein (GFP), and then >20 single transfected cells expressing GFP were collected and maintained for 5–6 generations to increased the cell number. The dose of 12 μl lentivirus was selected for subsequent experiments.

Cell migration was determined using the wound healing scratch assay. Cells were seeded into 6-well plates (10³/plate) and grown overnight at 37°C. Confluent cells were cultured in DMEM containing 0.5% FBS and 5 μg/ml mitomycin-C for 24 h at 37°C, and then wounded by a 1-mm linear scratch from a sterile pipette tip. Images of the wounded cell monolayers were captured at 0, 12 and 24 h following wounding using an inverted light microscope (model IX70; Olympus Corporation, Tokyo, Japan) equipped with a charge-coupled device camera (CoolSNAP HQ; Nippon Roper K.K., Chiba, Japan) and controlled by MetaMorph^® software version 7 (Universal Imaging, Inc., Bedford Hills, NY, USA). All experiments were performed in the presence of 5 mg/ml of mitomycin-C to inhibit cell proliferation. Cells were treated prior to wound healing with 100 ng/ml FGF2, FGF10 or FGF21 (Sigma-Aldrich) for 1 h, or transduced to overexpress FGF7 as previously described, and wound healing was measured at 12 and 24 h. A total of 20 cells/experiment at the edge of wound region were randomly selected from the wound area. At 12 and 24 h following wounding, the distance between the 20 selected cells and the wound edge at 0 h was measured using Image J Fiji software (National Institutes of Health, Bethesda, MD, USA).

The cells were lysed in an ice-cold lysis solution [7 M urea, 2 M thiourea, 2% 3-([3-Cholamidopropyl] dimethylammonio)-1-propanesulfonate, 40 mM Trizma^® base, 40 mM dithiothreitol, 1% protease inhibitor] for 10 min. Following centrifugation at 15,000 × g for 15 min at 4°C, the protein concentration in the supernatant was measured by the Bradford protein assay (Bio-Rad Laboratories, Inc., Hercules, CA, USA) according to the manufacturer's protocol. The proteins (20 μg) were loaded onto a 10% SDS-PAGE gel, separated by electrophoresis at 100 V for 2 h and transferred onto Immobilon-P Transfer Membranes (Merck Millipore, Tokyo, Japan). The membranes were blocked with Tris-buffered saline containing 5% skim milk and 0.05% Tween-20 for 1 h and then probed with primary antibodies at 4°C overnight. Anti-GAPDH (mouse monoclonal; dilution, 1:2,500; catalog no., mAbcam 9484; Abcam, Cambridge, UK), anti-phospho-stress-activated protein kinase (SAPK)/c-Jun N-terminal kinase (JNK) phosphorylated at Thr183/Tyr185 (rabbit monoclonal; dilution, 1:1,000; catalog no., 4668; Cell Signaling Technology, Inc., Danvers, MA, USA), anti-JNK (rabbit monoclonal; dilution, 1:1,000; catalog no., ab179461) and anti-FLAG (mouse monoclonal; dilution, 1:2,000; catalog no., ab49763; Abcam) were used as primary antibodies. The membranes were then incubated for 1 h with an anti-mouse (dilution, 1:2,000; ab131368; Abcam) or anti-rabbit horseradish peroxidase-conjugated secondary antibody (dilution, 1:2,000; catalog no., 7074; Cell Signaling Technology, Inc.), and visualized using an electrochemiluminescence kit (GE Healthcare Life Sciences, Chalfont, UK). Images of western blots were captured using an ImageQuant LAS 4000 Mini (GE Healthcare Life Sciences).

Male C57/BL6J mice, aged 3 months and weighing 2835 g, were obtained from the Laboratory Animal Centre of Wenzhou Medical University (Wenzhou, China). The mice were housed at 22°C and 50% humidity, with a 12 h light/dark cycle. Mice had free access to food and water. Approval was given for the use of mice in the present study by the Ethics Committee of Wenzhou Medical University (Wenzhou, China). All mice were anesthetized via intraperitoneal injection of 3% sodium pentobarbital (45 mg/kg) and their dorsal areas were completely depilated using sodium sulfide (8.0%; w/v; both SigmaAldrich) prior to tissue extraction during surgery. Total RNA was extracted from liver, heart, kidney and skin tissues of 9 mice, which were randomly divided into three groups (3 mice/group) for biological duplication (the nature of each group was the same, and simply served as a replication), using TRIzol^® Reagent (Invitrogen; Thermo Fisher Scientific, Inc.). Total RNA (1 μg) was reverse-transcribed using a GoScript Reverse Transcription kit (Promega Corporation, Madison, WI, USA) according to the manufacturer's instructions. PCR was performed using the SapphireAmp^® Fast PCR Master Mix (Takara Biotechnology Co., Ltd., Dalian, China) on a T100 thermal cycler (BioRad Laboratories, Inc.), with the following cycling conditions: 95°C for 5 min, followed by 35 cycles at 94°C for 30 sec and 58°C for 30 sec, 72°C for 30 sec, elongation at 72°C for 5 min and maintenance at 10°C. Gene expression levels were quantified by the ΔΔCq method as described previously (17). mRNA levels were normalized against those of GAPDH using Image J2x version 2.1.4.7 software and the 2^−ΔΔCq method (18). Primers used for RT-qPCR are listed in Table I.

FGF sequences used for similarity searches were collected from the National Center for Biotechnology Information website (http://www.ncbi.nlm.nih.gov/gene/?term=FGF). ClustalW (http://align.genome.jp) was used to perform a homologous sequence alignment of the amino acid sequences of FGF family proteins using default settings. Based on the results of sequence alignment, an unrooted phylogenetic tree of the FGF gene family was constructed using Mega software version 6.0 (http://www.megasoftware.net). The neighbor-joining method was applied to construct a phylogenetic tree (19), in which Poisson correction, pairwise deletion and bootstrapping (1000 replicates; random seeds) served as default values to evaluate the reliability of the tree.

Statistical analyses were performed in GraphPad Prism version 5 (GraphPad Software, Inc., La Jolla, CA, USA). Data are presented as the mean ± standard error. Comparisons between groups were performed using Student's t-test. P<0.05 was considered to indicate a statistically significant difference.

The expression patterns of 23 FGF members in mouse tissues was dissected by analyzing their transcript levels in liver, heart, kidney and skin tissue samples from 9 C57/BL6J male mice. RT-qPCR results revealed that FGF1, 6, 7, 9, 10, 12, 13, 16 and 18 were highly expressed in the heart (Fig. 1A). In the liver, expression of FGF1, 5, 6, 7, 8, 11, 12, 18 and 21 were relatively high, with FGF1 having the greatest expression (Fig. 1B). Notably, the expression of FGFs was relatively less complex in skin compared with other tissues. A total of four FGFs, FGF2, 7, 10 and 21 were significantly highly expressed in the skin (Fig. 1C). In the kidney, levels of FGF1, 5, 7, 8, 9, 10, 11, 12, 16 and 18 were relatively high (Fig. 1D). FGF2/bFGF is widely known for its efficacy in wound healing (7,20), and as the profile of FGFs is relatively simple in skin tissue, a phylogenetic tree was generated to understand the associations between the four FGF proteins. The data revealed that FGF7 and FGF10 belong to the same sub-group, while FGF2 shares a sub-group with FGF1. FGF21 is on a separate branch of the tree (Fig. 2), indicating that the four FGFs are not highly correlated.

FGF2, 7, 10 and 21 are highly expressed in skin tissue (Fig. 1C). Therefore, the function of these FGFs in wound healing was examined. Wound healing involves multiple steps, including cell proliferation and migration. To investigate the effect of FGFs on the cell migration process, mouse NIH3T3 foreskin fibroblast cells were grown in a low glucose medium (5.5 mM) containing 5 mg/ml of mitomycin-C, which prevents cell proliferation, for 24 h at 37°C prior to treatment with FGFs. As presented in Fig. 3, the cells treated with commercially purified FGF2 (P=0.023), 10 (P=0.027) or 21 (P=0.018) at 100 ng/ml exhibited accelerated cell migration 24 h following scratching as compared with control cells. JNK is activated via phosphorylation, and is important for cell migration in wound healing (16); therefore, JNK-phosphorylation was examined following FGF-treatment of NIH3T3 cells for 1 h. The data revealed that treatment with the three FGFs (FGF2, 10 and 21) increased p-JNK levels, while the total JNK level remained unchanged (Fig. 4).

As commercially produced FGF7 is not available, FGF7-FLAG fusion protein was overexpressed in NIH3T3 cells via a lentivirus system. Transduction efficiency was analyzed by monitoring GFP expression, and FGF7 expression was evaluated by western blotting using an anti-FLAG antibody (Fig. 5). In addition, FGF7 transcripts were analyzed in transduced and non-transduced NIH3T3 cells. RT-qPCR revealed that FGF7 levels were much higher in transduced, (FGF7 OX) compared with non-transduced, cells (Fig. 6A). Furthermore, cell migration was evaluated in FGF7 OX and non-transduced cells. However, FGF7 overexpression did not significantly alter the speed of cell migration (Fig. 6B and C; P=0.094). These results indicate that FGF2, 10 and 21 are highly expressed in skin tissue, and are involved in the fibroblast cell migration process.