Trizol reagent, SuperScript III Reverse Transcriptase and Lipofectamine 2000 were obtained from Invitrogen. Mouse antibody for beta actin and rabbit antibody inhibitor bulk for HIF 1alpha were purchased from Genete . Human and mouse recombinant PlGF protein and an enzyme linked immuno sorbent assay kit were obtained from R and D Systems. A dual luciferase reporter assay system was obtained from Promega. Hemato ylin and Eosin, Chromatin immuno precipitation Assay Kit, and EZ Zyme Chromatin Prep Kit were purchased from Merck Millipore. An in situ cell Death Detection Kit and tremeGENE HP DNA Transfection Reagent were purchased from Roche. The FITC Anne in V apoptosis detection Kit I was obtained from BD Biosciences. The JNK inhibitor, SP600125, was obtained from Enzo Life Science.

A SuperSensitive Polymer HRP IHC Detection System was purchased from Biogene . Animals This study conformed to the Guidelines for the Care and Use of Laboratory Animals published by the United States National Institutes of Health. All of the animal e periments were approved by the Institutional Animal Care and Use Committee of the Laboratory Animal Center, College of Medicine and Public Health of National Taiwan University. Eight week old male C57BL 6 wild type mice were purchased from the Laboratory Animal Center, College of Medicine and College of Public Health, National Taiwan University. The PlGF knockout mice in B6 background were provided by Dr. Po Nien Tsao. Cell culture Human bronchial epithelial cells, BEAS 2B, were cultured in F12 nutrient mi ture with 0. 5 ng ml recombinant epidermal growth factor, 500 ng ml hydrocortisone, 0.

005 mg ml insulin, 0. 035 mg ml bovine pituitary e tract, 500 nM ethanolamine, 500 nM phosphoethanolamine, 0. 01 mg ml transferrin, 6. 5 ng ml 3, 3, 5 triiodothyronine, 500 ng ml epinephrine, 0. 1 ng ml retinoic acid, 10% FCS 100 unit ml penicillin, and 100 ug ml streptomycin in a humidified 95% air 5% CO2 incubator at 37 C. Mouse primary type II alveolar epithelial cells and culture medium were purchased from chi scientific. Primary normal human bronchial epithelial cells were kindly provided by Dr. Reen Wu at University of California, Davis. Plasmids Human genomic DNA was e tracted from BEAS 2B by a Quick gDNA MiniPrep kit. The AV-951 2.

0 kb human PlGF promoter region was amplified from sellckchem human genomic DNA using polymerase chain reaction performed with Hi Fi Taq DNA polymerase as follows 2 minutes at 94 C, then 15 sec at 94 C, 30 sec at 59 C, and 2 min and 30 sec at 72 C for 35 cycles. The amplified DNA fragments were cloned into pGL3 vector and the sequences were confirmed by DNA sequence analysis. The pGL3 with mouse PlGF promoter was as previously described. Enzyme linked immuno sorbent assay Cellular medium from BEAS 2B and AEC II, and BAL fluid from mice were analyzed by a PlGF ELISA kit according to the manufacturers instructions.