previously described, in the laboratory of Dr James T. Rosen- baum. [11] Total RNA was extracted from these cells when they were 99% pure, as evaluated by morphologic criteria, and at passages 2 to 5. Human 239T cells and ARPE-19 cells were purchased from ATCC and cultured in DMEM with 10% FBS, or serum free, and used for promoter reporter assays. The D407 RPE cell line (a gift from Dr. R. C. Hunt, University of South Carolina, Columbia, SC) were cultured in RPMI media with 10% FBS. Induction of Hypoxia Cells were cultured in either 6 well or 12 well plates after transfection with expression and reporter gene plasmids. Cells were recovered overnight under normoxic conditions, and media was exchanged before placement in the Modulator Incubator Chamber (Billups-Rothenberg, Del Mar, CA) for hypoxic gas flushing. A 1% O2, 5% CO2 and 94% N2 gas mix was flushed through the chamber for exactly 5 minutes. The chamber was sealed and placed in a humidified 37˚C incubator. After an 8 hour incubation, the chamber was flushed again with hypoxic gas for 5 minutes, and resealed and incubated for a further 8 hours. The flush and reincubation sequence was repeated once more before total RNA was isolated. Media was collected for reporter protein analysis 8 hours after the final flush. RNA Extraction and RT-PCR Total RNA was isolated using an RNAqueous kit (Ambion Inc, Austin, TX) and 150 ng of this RNA was used with an oligo-dT primer for first strand synthesis (SuperScript II, Stratagene, La Jolla, CA). The following primers, F1: 59-ttggagggcacggccggca-39 and R1: 59-tcattctttcaccagcctgta-39 was used for second strand PCR amplification of TEAD4 using standard conditions. Ampli- fied products were electrophoresed and visualized on a 1.5% agarose gel and subsequently purified from the gel (Qiaquick Gel Extraction, Qiagen, Valencia, CA) for standard dideoxynucleotide sequencing on an ABI 310 automated sequencer. Reporter Gene Analysis The cDNA for TEAD4 isoforms were directionally cloned into the pcDNA 3.1 expression plasmid (Invitrogen, Carlsbad, CA). Human VEGF 59 proximal promoter fragment (F1–R3) of 1,136 bp containing 54 bp of 5 UTR and 1,082 bp upstream of the transcription start site (TSS) was cloned 59 to the secretable alkaline phosphatase (SEAP) gene within the pSEAP reporter plasmid (Clontech, Mountain View, CA). Truncated VEGF promoter fragment (F2–R3), which lacked the HRE sequence, containing 580 bp sequence 59 to the TSS, was generated by nested amplification from the F1–R3 clone. The F2–R3 fragment was directionally cloned into the promoterless pSEAP vector. All constructs were sequence confirmed on both strands prior to transfection studies. Transfection Assays The Amaxa Nucleofection Device (Amaxa Inc, Gaithersburg, MD), with Amaxa reagents and standard manufacturer’s protocol, was used to transfect plasmid vectors into cells. Briefly, 293T cells were cultured until 80% confluent and subsequently trypsinized. One million cells were resuspended in 100 ml of Nucleofect solution, mixed with 5 ml (containing 2 mg) of total plasmid DNA, and then electroporated (program #A023). Nucleofected cells were immediately resuspended in pre-warmed media and seeded into single wells of a 6 or 12-well plate. Cells were recovered overnight, before media was replaced with exactly 500 ml of fresh media. After 6 to 24 hours incubation, 200 ml of media was The TEAD4216 Isoform Inhibits VEGF Expression carefully removed and 25 ml of this was assayed immediately or stored at 220uC for SEAP analysis. Two separate 25 ml media aliquots were used for SEAP analysis according to manufacturer’s protocol (BD Biosciences, San Jose, CA). The two readings from one well were averaged for comparison to triplicate/quadruplicate repeat wells. Each co-transfection was repeated in triplicate or quadruplicate for a single experiment. Each experiment was repeated independently two more times with separate plasmid preparations (n = 9–12). A representative experiment is presented. Statistical analysis was performed using a Student’s t-test (two- tailed) to compare the triplicate samples in a single experiment. Bonferroni correction for multiple testing was applied and a P,0.01 was considered as significant. For each co-transfection, the copy number of each plasmid was adjusted to be equivalent to the copy number of the largest plasmid used. The pcDNA 3.1 expression plasmid with no insert served as a control. As positive controls, a SV40 promoter pSEAP plasmid and a pGFPmax vector were transfected to ensure efficient and equal transfection efficiencies. The pSEAP plasmid with an SV40 promoter served as a positive control for subsequent SEAP protein analysis. The pGFPmax vector also served as positive control for transfection for each batch of cells allowing visual confirmation of consistent transfection efficiency. Nucleo- fection consistently gave 75–90% transfection efficiency in all experiments. Quantification of Secreted VEGF Protein The level of VEGF165 protein was quantified using a standard ELISA kit (Chemicon, Temecula, CA), according to the manu- facturer’s protocol. Cells were Nucleofected as described above with either 2 mg of pcDNA vector containing the cDNA for TEAD4216 or an equal copy number of the vector with no insert as a control. Cells were recovered overnight, and the media was replaced. The conditioned media was collected 24 hrs later and 2650 ml of media was collected from each well (n = 4) for use in the ELISA. Laser Induced Central Retinal Artery Occlusion (CRAO) All animal experiments were approved by the Institutional Animal Care and Use Committee and handled in accordance with the ARVO statement for the Use of Animals in Ophthalmic and Vision Research. Retinal artery occlusion was induced in the right eye of a cynomolgus primate via Rose bengal-assisted laser occlusion technique (532 nm laser); the left eye served as a control. Color photographs and fluorescein angiography (FA) were taken immediately after the procedure to confirm occlusion after the CRAO was induced. Briefly, cynomolgus primates (Macaca fascicularis, Sierra Biomedical Inc., Whitmore CA) were anesthe- tized by intramuscular injection of a mixture containing ketamine hydrochloride (20 mg/ml) and acepromazine maleate (0.125 mg/ ml). Pupils were dilated (1% Cyclogel, 2.5% Phenylepherine, 0.5% Tropicamide). Eyes were topically sterilized and proparacaine hydrochloride was used as topical anesthesia. Immediately after Rose Bengal (Sigma, St. Louis, Mo, 20 mg/ml in normal saline) was injected into the femoral vein (20 mg/kg), the central retinal artery was irradiated with a 532 nm argon green laser (Iridex Inc., Mountain View, CA) via a stabilized slit-lamp delivery system. Following color photography (Ziess, fundus camera), 0.5 ml of fluorescein was injected and FA was performed to confirm occlusion of the central retinal artery. Retinal, choroidal, and iris tissue were harvested 24 hours after occlusion, and RNA was obtained. Total RNA was isolated from the three tissue groups (Ambion), quantified and followed by semi-quantitative RT-PCR PLoS ONE | www.plosone.org 9 June 2012 | Volume 7 | Issue 6 | e31260