Thirty neonatal 1 to 3 day old and three adult male Sprague-Dawley (SD) rats (180 g–220 g) were purchased from the Experimental Animal Center at Nantong University, China. The rats were specific pathogen free, originally from Charles River Laboratories (Wilmington, MA) and bred in Laboratory Animal Research Center at Nantong University. The animals were housed, in polycarbonate cages with corn cob beddings, in a 12-h light/dark schedule with ad libitum access to food and water in a barrier unit. All animal experiments were performed in accordance with the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals and approved by the Administration Committee of Experimental Animals of Nantong University, China (approval No. 20130410–006).
SCs isolation and transfection
Rat SCs were harvested as previously described  with minor modifications. Briefly, the Sprague-Dawley rats (1 to 3 d- old) were sanitized using 75% ethanol prior to decapitation. Then sciatic nerves were harvested and enzymatically dissociated by incubation at 37 °C sequentially with 1% collagenase and 0.125% trypsin for 30 and 10 min, respectively. The mixture was triturated, centrifuged, and resuspended in 10% FBS in DMEM. The cell pellets were plated on poly-L-lysine precoated dishes using the same media. The following day, 10 μM cytosine arabinoside was added and incubated for an additional 48 h to remove fibroblasts. The cell culture was maintained in DMEM supplemented with 10% FBS, 2 μM forskolin (Sigma Aldrich, St. Louis, MO, USA), and 2 ng/ml heregulin (HRG, R&D system, Minneapolis, MN, USA) to stimulate SC proliferation. For additional purification, the cell culture was gently trypsinized, pelleted, and incubated with anti-Thy1.1 antibody (1:1000, Sigma Aldrich, St. Louis, MO, USA; Cat# M7898, RRID: AB_477242; Clone number: TN26) on ice for 2 h, followed by incubation in complement (Sigma Aldrich, St. Louis, MO, USA) for an additional 2 h. All media and supplements were purchased from Gibco-Invitrogen (Carlsbad, CA, USA).
For cell transfection, purified primary SCs were transfected with Cyr61 siRNAs designated as ((Cyr61-siRNA-1 (sequence: GCAGACCCTGTGAATATAA), Cyr61-siRNA-2 (sequence: GGAATGGGTCTGTGATGAA), Cyr61-siRNA-3 (sequence: GCTCCAGTGTGAAGAAATA)) or NTC (Ribobio, Guangzhou, Guangdong, China), using riboFECT CP Transfection kit (Ribobio, Guangzhou, Guangdong, China) following the manufacturer’s instructions.
Nerve tissue preparation
The adult male SD rats were anesthetized intraperitoneally using a mixture of 85 mg/kg trichloroacetaldehyde monohydrate (RichJoint, Shanghai, China), 42 mg/kg magnesium sulfate (Xilong Scientific, Guangzhou, Guangdong, China), and 17 mg/kg sodium pentobarbital (Sigma Aldrich, St. Louis, MO, USA). After anaesthetization, the rats were transcranial perfused sequentially with saline and 4% (v/v) paraformaldehyde in 0.1 M PBS. Then, the sciatic nerve segments at 10 mm above the bifurcation into the tibial and common fibular nerves were harvested for frozen sections.
Enzyme-Linked Immunosorbent Assay (ELISA)
Primary SCs were transfected with NTC or siRNA targeting Cyr61. After transfection, the media was replaced with serum-free medium for an additional 12 h incubation. The media was then harvested and filtered through a 0.22 μm filter (Millipore, Bedford, MA, USA). The protein levels of Cyr61 in the media were measured using a Cyr61 ELISA Kit (Cusbio, Wuhan, Hubei, China) based on the manufacturer’s instructions. Measurement data were summarized from 3 independent experiments, each run in triplicate.
RNA extraction and quantitative real time RT-PCR (qPCR)
Total RNA of each group was extracted using Trizol (Invitrogen, Carlsbad, CA). Reverse transcription was carried out with SuperScript First-Strand Synthesis System (Invitrogen, Carlsbad, CA). Gene products were analyzed using Fast EvaGreen qPCR Master Mix (Biotium, Hayward, CA) and specific primers in StepOne Real-Time PCR System (Applied Biosystems). Reaction components in each well were composed of 2× Fast Eva Green Master Mix, 10 μl; primers, 1 μl each; template, 1 μl; ROX, 2 μl; and H2O, 5 μl. Three step fast cycling protocol was performed. Relative gene expression levels were calculated as ratios of the mRNA levels normalized against those of 18 s mRNA. All the results were expressed as the mean ± SD of three independent experiments. Primer sequences are provided in Additional file 1: Table 1.
Western blot analysis
Total proteins from SCs were extracted using the M-PER cell protein extraction reagent (Pierce, Rockford, IL, USA). Extracted proteins were quantified using the Fast Silver Stain Kit (Beyotime, Haimen, Jiangsu Province, China). Twenty microgram of total protein were loaded onto a 12% (w/v) SDS-PAGE, electrophoresed, and transferred to a PVDF membrane (Millipore, Bedford, MA). After blocking for 1 h with 5% (w/v) non-fat dry milk in TBS-T (0.05% (v/v) Tween 20 in Tris-buffered saline), the membrane was incubated with specific primary antibodies diluted in blocking buffer overnight at 4 °C. The rabbit polyclonal antibody to Cyr61 (1:500, Abcam, Cambridge, MA, USA; Cat# ab24448, RRID: AB_2088724) and rabbit monoclonal antibody to c-Jun (1:2000, Abcam, Cambridge, MA, USA; Abcam Cat# ab40766, RRID: AB_731602, Clone number: EP693Y) were used. Afterward, the membranes were washed with TBS-T and then incubated with HRP conjugated secondary antibody diluted in blocking buffer (1:5000, Abcam, Cambridge, MA, USA) at RT for 2 h. Immunoreactive bands were visualized using enhanced chemiluminescence (Beyotime, Haimen, Jiangsu Province, China). Densitometry analysis was performed using the Image J software (http://imagej.nih.gov/ij/).
Cells were plated on poly-L-lysine pre-coated coverslips and cultured overnight. They were then fixed in 4% paraformaldehyde for 30 min at room temperature (RT). Sciatic nerve segments from adult rats were dissected, fixed in 4% paraformaldehyde for 24 h, dehydrated in 30% sucrose at 4 °C, then cut and mounted onto microscope slides. Cells and sciatic nerve sections were blocked for 2 h at 37 °C. Cells were incubated with the following antibodies overnight at 4 °C: mouse monoclonal or rabbit polyclonal antibody to S100β (1:100, Abcam, Cambridge, MA, USA; Cat# ab14849, RRID: AB_301508, Clone number: 4B3; Cat# ab52642, RRID: AB_882426), rabbit polyclonal antibody to Cyr61 (1:200, Abcam, Cambridge, MA, USA; Cat# ab24448, RRID: AB_2088724) or mouse monoclonal antibody to αvβ3 (1:200, R&D system, Minneapolis, MN, USA; Cat# MAB3050, RRID: AB_2128187; Clone number: #23C6). After washing, the cells and sciatic nerve sections were incubated with FITC-conjugated rabbit anti-mouse IgG and Cy3-conjugated donkey anti-rabbit IgG (1:400, Abcam, Cambridge, MA; Cat# ab6724, RRID: AB_955315; Cat# ab97075, RRID: AB_10679955) for 2 h at RT. Nuclei were counterstained with Hoechst 33342 dye (5 μg/mL, Sigma Aldrich, St. Louis, MO, USA). Fluorescence was visualized under a TCS SP5 confocal microscope (Leica Microsystems, Wetzlar, Germany).
Cell proliferation analysis
SC proliferation was assessed after siRNA transfection, or exposure to recombinant Cyr61 with/without αvβ3 neutralizing antibody (R&D system, Minneapolis, MN, USA; Cat# MAB3050, RRID: AB_2128187; Clone number: #23C6) and mouse IgG1 isotype control (R & D, Minneapolis, MN, USA; Cat# MAB002, RRID: AB_357344; Clone number:11711) was used as a control for neutralizing antibody. Cell Counting Kit8 (CCK-8) (Biyuntian Company, Jiangsu Province, China) was then used. Briefly, an equal number of cells were plated onto a 96-well plate. Cells in each treatment group were cultured for 0-60 h. Then, 10 μL of CCK-8 solution was added to each well and incubated at 37 °C for an additional 2 h. Optical density (OD) was determined at a wavelength of 450 nm.
Cell migration analysis
SC migration was monitored after siRNA transfection, or exposure to recombinant Cyr61 with/without neutralizing antibody and mouse IgG1 isotype control was used as a control for neutralizing antibody. The transwell migration assay was used as previously described . After transfection, 2 × 104 cells in serum-free DMEM were plated onto the upper chamber of each transwell with 8 μm pore size (Costar, Corning, Inc., NY). The lower chamber was supplemented with 800 μL of complete media (DMEM+ 10%FBS) or complete media with Cyr61 or complete media with Cyr61 and αvβ3 neutralizing antibodies. Cells were incubated for 24 h at 37 °C in 5% CO2. Non-migrating cells were removed from the upper surface of the membrane using a cotton swab. Cells on the lower side of the membrane were stained with crystal violet, and migration was quantified by counting cells from four microscope fields. Each treatment condition was run in triplicate.
Data were expressed as mean ± SEM, and statistical analysis was performed using GraphPad Prism Software (GraphPad Software, LaJolla, CA). Comparisons between two groups were performed using the Student’s t-test. Differences of p < 0.05 were considered statistically significant.