and VEGF and that it could ultimately be involved in the regulation of PCV. Further studies evaluating the influence of HTRA1 on vascular endothelial cell and protein-protein interac- tions are needed. Materials and Methods Construction and identification of the HTRA1 expression plasmid The primers targeting the human HTRA1 gene were synthe- sized based on a cDNA library (Genechem,Shanghai, China), and the sequences were as follows: a) HTRA1-AgeI-F 59- GAGGATCCCCGGGTACCGGTCGCCACCATGCAGAT- CCCGCGCGCCGCTCTTCTC-39, and HTRA1-AgeI-R 59- AGTCCATGGTGGCGACCGGTGGGTCAATTTCTTCGG- GAATC-39. These primers contain the complement bases, the AgeI restriction site and a partial sequence of the 39 end of the target gene, which is used for isolating the target gene by polymerase chain reaction (PCR). b) HTRA1-SEQF 59- CTCTCCGGCCCTCGCCCTGTC-39. This primer was located in the coding sequence of the HTRA1 gene and used in colony PCR to identify positive transformants. c) EGFP-N-R 59- CGTCGCCGTCCAGCTCGACCAG-39. This primer was used in PCR to identify transformants and in sequencing. Each of these fragments was amplified, digested with AgeI and then ligated into the pGC-FU-3FLAG vector for 15 min at 23uC and 42uC. The recombinant was transformed into competent Escherichia coli cells treated with calcium chloride and incubated for 16 h at 37uC. PCR conditions were as follows: 94uC for 5 min, 30 cycles of 94uC for 30 s, 55uC for 30 s, 72uC for 2 min and finally 72uC for 10 min. Positive clones, as confirmed by PCR, were chosen for sequencing. The plasmids with the correct sequence were transfected into 293T cells, and the expression of the confluency protein was observed under a fluorescence microscope. Western blotting was applied to detect the expression of the confluency protein. The virus titer was determined by quantitative real-time PCR. Cell culture and lentivirus transduction A rhesus monkey choroid-retina endothelial cell line (RF/6A) and human umbilical vein endothelial cells (HUVECs) were obtained from the American Tissue Culture Collection(Manassas, VA) and the cell bank of the Chinese Academy of Science (Shanghai, China), respectively. The cells were cultured in Dulbecco’s Modified Eagle Medium/F12 (Gibco, Grand Island, NY) with 10% fetal bovine serum (FBS) at 37uC in 5% CO2 and 95% humidity. When the RF/6A cells and HUVEC cells were approximately 50% confluent in fresh serum-free medium, they were transiently transduced with control lentivirus or HTRA1- overexpression lentivirus at MOIs (multiplicities of infection) of 100 and 10, respectively. The cells were further cultured in DMEM with 10% FBS after infecting for 4 hours and then selected using 200 mm/ml puromycin. The stable overexpression lines were established when more than 90% of the transduced cells were found to strongly express GFP under fluorescent microscopy. The RF/6A cells and HUVEC cells were both divided into 3 groups: control(C), the lentiviral vector control group (NC), and the HTRA1-transfected group (HTRA1). RNA isolation and real-time RT-PCR The total RNA was isolated from RF/6A cells and HUVEC cells using TRIzol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. RNA extract (2 ml) was reverse- transcribed into cDNA in a total reaction volume of 20 ml using a Overexpression of HTRA1 in RF/6A Cells and HUVECs RevertAidTM First Strand cDNA Synthesis Kit (Fermentas, Burlington, Canada). Real-time quantitative PCR was performed using IQ Supermix (Bio-Rad, Hercules, CA), with 20-ml reaction mixtures containing 2 ml cDNA, 7.2 ml sterilized water, 10 ml SYBR Green Real-time PCR Master Mix (TaKaRa, Japan), and 0.8 ml of primer (10 mM). The HTRA1, VEGF and fibronectin amplification signals were normalized to b-actin expression and evaluated using the equation: fold change = 22DDct. The primer sequences used in this study were as follows: HTRA1, forward primer 59-GGGAAAGCACCCTGAACAT-39, reverse primer 59- CCAGAGTCCTCATCCGTCAT-39; VEGF, forward primer 59- AGTTCCACCACCAAACATGC-39, reverse primer 59- TGAAGGGACACAACGACACA-39; fibronectin, forward prim- er 59-GGGAGCCTCGAAGAGC-39, reverse primer 59-AA- CAAGTACAAACCAACGCA-39; and b-actin, forward primer 59-CTTAGTTGCGTTACACCCTT-39, reverse primer 59- CCTTCACCGTTCCAGTTT-39. Laminin, forward primer 59- TAACTGGTGGCAAAGTCC-39, reverse primer 59-AGA- GAACGCTCCAAAATC-39; MMP-2, forward primer 59- AACTTCTTCCCTCGCAAG-39, reverse primer 59- TGTCTGCCTCTCCATCAT-39; PDGFR, forward primer 59- TCAGACAGAAGAGAATGAGC-39, reverse primer 59- GTCTCGGGATCAGTTGTG-39. Protein extraction and western blot analysis RF/6A cells and HUVEC cells were washed three times with ice-cold phosphate-buffered saline (PBS, 4uC, pH 7.4) for 5 min at room temperature and prepared using a protein extraction kit and a protease inhibitor kit (Pierce, Rockford, IL). The supernatant was collected and the protein content of each lysate was determined using a BCA Protein Assay Kit (Tianlai Shengwu Jishu, Tianlai, China) according to the manufacturer’s instruc- tions. Equal amounts (20 mg) of protein were electrophoresed on a 10% sodium dodecyl sulfate (SDS) polyacrylamide gel and transferred onto a 0.22 mm PVDF membrane (Millipore). The primary antibodies used to probe the membranes included anti- HTRA1 (1:300; Abcam, Cambridge, UK, Cat No. ab38610), anti-Fibronectin (1:1800; Abcam, Cambridge, UK, Cat No. ab2413), anti-laminin (1:600; Abcam, Cambridge, UK, Cat No. ab11575) and anti-b-actin (1:3000; Boster, Wuhan, China). The membranes were washed and incubated with peroxidase- conjugated secondary antibodies (1:5000; Boster, China). En- hanced chemiluminescence Western blotting detection reagents (Pierce) were used to detect HTRA1, fibronectin and laminin protein levels. Measurement using an enzyme-linked immunosorbent assay The RF/6A and HUVEC cells (untransfected and stably transfected cells) were seeded in 24-well plates. Next, the culture supernatants were separately collected at 12, 24, 48 ,72 h and 96 h and were centrifuged to eliminate cellular fragments. The concentration of the secreted VEGF and MMP-2 in the media was determined with commercially available enzyme-linked immunosorbent assay (ELISA) kits (Boster, Wuhan, China) according to the manufacturer’s protocol. All samples were assayed in triplicate and the mean values were calculated and expressed as pg VEGF or MMP-2/105 cells. In vitro cell proliferation assay To determine the role of pGC-FU-3FLAG-HTRA1 transfec- tion in the proliferation of RF/6A cells and HUVECs, a 3-[4,5- dimethylthiazol-2-yl]-2,5-diphenyltetrazoliumbromide (MTT; PLOS ONE | www.plosone.org 2 October 2012 | Volume 7 | Issue 10 | e46115