for the TEAD4 transcript. The GAPDH housekeeping gene served as the loading control. Cell Proliferation Assay Cell proliferation was determined using a standard assay (CyQuant NF Cell Proliferation Assay Kit, Molecular Probes, Eugene OR). The TEAD4216 cDNA insert was cloned into the pLenti7.3 vector (Invitrogen, Carlsbad, CA) and lentivirus (LV- TEAD4216) was prepared according to the manufacturer’s protocol. The LV-TEAD4216 virus was used to transduce 293T cells in wells of a 6-well plate, and transduced cells were FAC sorted using the emerald GFP encoded by the vector. LV-GFP was used as a control. LV-TEAD4216 positive cells were seeded at 105 cells in 80 ml of media into each well of a 96-well culture plate (N = 8). Cells were incubated for 3 to 4 days at 37uC with 5% CO2 in a humidified chamber. Media was carefully aspirated from wells without disturbing the cells. Exactly 50 ml of Cyquant NF staining solution was added to each well, and wells were incubated at 37uC with 5% CO2 for 30 min to 1 hour. The plate was read in a Victor3 plate reader (Perkin Elmer, Waltham, MA). Statistical significance was determined by a paired Student’s t-test. Each experiment was performed in octuplicate and each proliferation assay was performed at least twice. Immunostaining of Eyes with Neovascular AMD Three globes from 3 patients diagnosed with AMD were obtained post-mortem, fixed in 10% neutral buffered formalin and embedded in paraffin; these individuals were aged 77, 80 and 85 years at time of death. The Oregon Health & Science University Institutional Review Board provided a determination that this study did not constitute human subjects research. Ocular cross- sections were cut 5 microns thick, and mounted on Superfrost Plus glass slides (Fisher Scientific, Pittsburgh, PA) and heated overnight at 37uC, followed by 1 hour at 60uC, to promote firm adhesion. TEAD4 was visualized in tissue sections, according to a previously published indirect immunostaining method [58], using rabbit polyclonal anti-TEAD4 antibody (made to order by Genemed Synthesis, San Antonio, TX, see Methods S1 for details of custom made antibody) or, as negative control, rabbit IgG (Vector Laboratories, Burlingame, CA), at a concentration of 11 ug/mL. Antigen retrieval was performed by immersing the slides in 10 mM citrate buffer (pH 6.0) and boiling for 10 minutes in a microwave oven. To visualize antibody complexes, sections were incubated with Fast Red (Biogenex, San Ramon, CA), as described by the manufacturer. Subsequently the sections were counterstained with hematoxylin. Immunostaining was interpreted by comparison with hematoxylin and eosin-stained sections. Supporting Information Figure S1 Human TEAD4 is differentially spliced within retinal, choroidal and iris derived vascular endothelial cells under hypoxia/normoxia. Agarose gel electrophoresis showing RT-PCR from RNA isolated from primary cultures of REC, CEC and IECs after culture under normoxic or hypoxic conditions. Donor #1 (pooled REC, CEC and IEC cells) gave products for TEAD4434 (1305 bp band), TEAD4311 (936 bp band), TEAD4216 (651 bp band) and TEAD4148 (447 bp band) under hypoxic conditions, and under normoxic conditions only the TEAD4216 product was amplified. TEAD4216 was present in REC, CEC and IEC but interestingly expression levels differed The TEAD4216 Isoform Inhibits VEGF Expression between each EC type and between normoxic and hypoxic conditions. (TIF) Figure S2 Western blot for TEAD4 on nonhuman primate ocular vascular endothelial cell proteins recog- nizes TEAD4434 and other isoforms. Two different com- mercially available antibodies (ARP33426 and ARP38276, Aviva) recognize the full length TEAD4 (,50 kD) in monkey retinal- choroidal ECs (RF/6A). No significant difference in TEAD4 expression was observed when normoxic (N) samples were compared to hypoxia (H) treated samples. Other TEAD4 isoforms appear to also be recognized at 30 KD, 26 kD and 24 kD. (TIF) Figure S3 Band shift (EMSA) assay for TEAD4 isoforms on Sp-I sequences found within the VEGF promoter. The enhancer isoforms TEAD4434, TEAD4216 and TEAD4148 bound to the SpI sequences present within the human VEGF promoter. The repressor isoform TEAD4216, although lacking one of the 3 helixes present within the TEA DNA binding domain, is still able to bind the Sp-I sequences within the VEGF promoter. (TIF) Figure S4 TEAD4216 can competitively inhibit enhancer mediated VEGF promoter activity. Increasing concentra- tions of a plasmid containing TEAD4216, inhibited the enhancer properties of TEAD4148. (TIF) Figure S5 Expressed protein from transfected TEAD4 isoforms is stable over time. Protein from cells was isolated at various time points (24 to 96 hours) after transfection with pcDNA expression vector containing a TEAD4 isoform. Western blot was performed with a custom made TEAD4 antibody (Genemed Synthesis). (TIF) Figure S6 TEAD4216 requires an NLS to enhance VEGF promoter activity. A reporter assay using the full length human VEGF promoter F1–R3 and chimeric TEAD4 isoforms, TEAD4216 with a NLS and TEAD4148 without an NLS showed that the NLS domain was crucial for enhancers to function and that loss of the NLS converts a potent enhancer (TEAD4148) into an inhibitor. The chimeric TEAD4216+NLS isoform significantly enhanced promoter activity (p,0.01, n = 3). (TIF) Methods S1 A description of electrophoretic mobility shift assays, culture of RF/6A cells, antibodies used and TEAD4 chimeric constructs. (DOC) Acknowledgments We thank Dr. James T. Rosenbaum for generously providing primary human ocular endothelial cell RNA. Author Contributions Conceived and designed the experiments: BA JTS. Performed the experiments: BA TJM JBK DB YZ KW RW A. Stout HI KR MWH YP A. Stempel. Analyzed the data: BA TJM MWH DJW JRS JTS A. Stempel. Contributed reagents/materials/analysis tools: MLK. Wrote the paper: BA TJM JRS JTS. PLoS ONE | www.plosone.org 10 June 2012 | Volume 7 | Issue 6 | e31260