Roche, Molecular Biochemicals, Mannheim, Germany) assay was used to measure cell proliferation over 4 consecutive days. RF/6a cells and HUVECs, both divided into three groups, were plated at a density of 16104 cells per well in 96-well culture plates. After a 24-h incubation, MTT was added, and the cells were incubated for an additional 4 h. Formazan crystals were then dissolved by the addition of dimethyl sulfoxide (DMSO, 100 ml/well). The absorbance at 570 nm was measured using an ELISA plate reader (Dynatech Medica, Guernsey, UK). Each group was measured using a modified MTT assay at 24, 48, 72 and 96 h, and the experiment was performed at least three times. Flow cytometry Each group of cells was seeded in 6-well plates for 48 h. The cells were detached using ethylene diamine tetraacetic acid (EDTA), washed in ice-cold PBS (4uC), and treated with the BD CycletestTM Plus DNA Reagent Kit (Becton Dickinson) according to the manufacturer’s protocol. Samples were analyzed using a FACSCalibur flow cytometer (Becton Dickinson, Franklin Lakes, NJ). Three samples were used in one experiment and each experiment was repeated. Cell migration A migration assay was performed as described previously [21]. Briefly, 26104 cells were placed in the upper chamber in a final volume of 200 ml of serum-free medium, and 10% FBS then was added to the bottom chamber for a final volume of 600 ml. All migration assays were conducted for 4 h at 37uC. At the end of the assay, the cells were fixed in 95% ethanol and stained with hematein for 25 min. The remaining cells were wiped away with a cotton swab, and the membrane was imaged. The numbers of cells from five random fields of view were counted. Tube formation A tube formation assay was performed to investigate the effect of pGC-FU-3FLAG-HTRA1 transfection on RF/6A cells and HUVECs as described previously [22]. Aliquots (150 ml) of matrigel solution were poured into the 48-well plates (repeated twice), and the plates were incubated at 37uC for 1 h in a 5% CO2 incubator to form a matrigel. Three groups of RF/6A cells and HUVECs were seeded on the matrigel and cultured in DMEM medium for 14 h. Five random fields of the networks in the matrigel were counted and photographed under a microscope. Results Identification of recombinant plasmid pGC-FU-3FLAG- HTRA1 The positive clones were identified after PCR amplification; the size of the PCR product was 1486 bp. The detected sequence was identical to the known HTRA1 sequence in GenBank (Gene ID 5654). A western blot revealed a 55-kDa band in cell extracts, which was in accordance with the expected size of an HTRA1– Flag confluency protein (53 kDa+2 kDa = 55 kDa). These results indicate that the HTRA1 recombinant plasmid was successfully expressed in RF/6A and HUVEC cells and suggested that these cells were successfully transduced with lentivirus. Overexpression of HTRA1 by stable transfection of pGC- FU-3FLAG-HTRA1 Total mRNA and protein extracts were prepared from untransfected control (UT), lentiviral vector control (NC) and HTRA1-transfected (HTRA1) RF/6A cells and HUVECs. The Overexpression of HTRA1 in RF/6A Cells and HUVECs HTRA1 mRNA and protein expression levels in these cells were determined by real-time RT-PCR and Western blot assays, respectively. Real-time RT-PCR demonstrated that the mRNA levels of HTRA1 protein expression in the transfectants containing pGC-FU-3FLAG-HTRA1 were increased 2-fold and 3-fold, respectively, compared with untreated control RF/6A cells and HUVECs, which was consistent with the increase of HTRA1 protein expression (p,0.01; Figure 1A, Figure 1B). There was no significant difference between the cells transfected with the control lentiviral vector and the untransfected cells (p.0.05). These results indicate the stable transfection of pGC-FU-3FLAG-HTRA1 up- regulated HTRA1 expression in both RF/6A cells and HUVECs. Effect of HTRA1 on fibronectin, laminin , VEGF and MMP- 2 protein expression Fibronectin plays an important role in maintaining dynamic equilibrium in the extracellular matrix and is associated with vascular development [23,24]. We investigated whether the overexpression of HTRA1 induced by pGC-FU-3FLAG-HTRA1 could affect fibronectin and angiogenesis-associated genes, such as VEGF. The levels of fibronectin mRNA and protein in the UT, NC, and HTRA1-transfected cells were determined by real-time RT-PCR and western blot. When compared with the control groups, HTRA1 transfection led to a significant 54% decrease (p,0.01) and a 72% (p,0.01) decrease in fibronectin mRNA levels in RF/6A cells and HUVECs, respectively. The results were consistent with the decrease of fibronectin protein expression (p,0.01; Figure 1C,Figure 1B). We also analyzed the levels of VEGF mRNA in the cells and secreted VEGF protein in the conditioning media by real-time RT-PCR and ELISA assays. There were no significant differences between HTRA1-transfected groups and control groups of RF/6A cells and HUVECs in either mRNA (Figure 1D) or protein levels of VEGF secretion in the cells or in the culture medium (P.0.05; Figure 1E, Figure 1F), with the exception of a slight decrease of protein level in cultured HUVEC supernatants collected after by 48 h of incubation (P,0.05). In addition, the mRNA level of laminin, PDGFR and MMP-2 had no significant differences between groups in RF/6A cells (Figure 2A, Figure 2B and Figure 2C). The protein levels of laminin and MMP-2 showed no significant changes in HTRA1- transfected groups and control groups of RF/6A cells (Figure 2D, Figure 2E). HTRA1 regulates RF/6A cells and HUVECs proliferation, cell cycle and cell migration To determine the effect of HTRA1-induced RF/6A and HUVEC proliferation, the metabolic activities of UT, NC and HTRA1 cells were quantified using the MTT assay. RF/6A cell proliferation was reduced by 7% at 48 h compared with NC cells (P.0.05) and the reduction peaked at 17% on the fourth day (p,0.01). HUVEC proliferation was reduced by 10% at 48 h compared with NC cells (P,0.05) and the reduction peaked at 23% on the fourth day (p,0.01) (Figure 3). Next, flow cytometric analysis was applied to analyze the effect of HTRA1 on RF/6A cells and HUVEC cycles. As shown in Figure 4, the percentage of RF/6A cells in G0/G1 phase increased from 46.27% (NC) to 58.83% (HTRA1), and the S-phase cells decreased from 42.29% (NC) to 34.17% (HTRA1). The percent- age of HUVECs in G0/G1 phase increased from 35.97% (NC) to 65.32% (HTRA1) and the S-phase cells decreased from 57.10% (NC) to 28.61% (HTRA1). The difference between HTRA1- transfected RF/6A cells and NC RF/6A cells in G2/M phase was slight (11.44% versus 5.66%, p,0.05), whereas the difference PLOS ONE | www.plosone.org 3 October 2012 | Volume 7 | Issue 10 | e46115