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Nübel et al, Fig. 1 - Carcinogenesis

Nübel et al, Fig. 1 - Carcinogenesis


MATERIALS AND METHODS Materials Lovastatin was purchased from Calbiochem-Novabiochem (Bad Soden, Germany). Oligonucleotides originate from Sigma-Aldrich Fine Chemicals (Taufkirchen, Germany). Clostridium difficile toxin A (ToxA) was a gift from I. Just (Institute of Toxicology, Hannover, Germany). 9-cis retinoic acid and all-trans retinoic acid were purchased from ICN Biomedicals GmbH (Eschwege, Germany). Anti-E-selectin, anti-P-selectin and ERK2 antibodies used in this study were purchased from Santa Cruz Biotechnology Inc. (Heidelberg, Germany), antibody directed against ICAM-1 was from R&D Systems GmbH (Wiesbaden-Nordenstadt, Germany) POD-conjugated anti-mouse antibody used for ELISAanalyses was obtained from Promega Corporation (Mannheim, Germany). Phospho-specific IκBα antibody was purchased from New England Biolabs GmbH (Frankfurt am Main, Germany). For treatment of cells with ionizing radiation (i.e. γ-rays), a 60 Co source (Atomic Energy of Cananda Ltd.) was used. The Titan One Tube RT-PCR Kit originates from Roche Diagnostics GmbH (Mannheim, Germany). For cloning of the E-selectin promoter PowerScript DNA Polymerase was used (PAN TM Biotech GmbH, Aidenbach, Germany). Luciferase activity was determined by use of the Luciferase assay system (Promega Corporation, Mannheim, Germany). CDNAs and epression vectors for dominant negative Rho GTPases (N17Rac and N19RhoB) are described elswhere [32]. Dominant-negative RhoA (N19Rho) originates from A. Hall (University College London, UK). The cell line EA.hy-926 was obtained from Cora-Jean S. Edgell (University of North Carolina, USA). Human umbilical vein endothelial cells (HUVEC) were purchased from Cambrex (BioWhittaker Europe, Verviers, Belgium). Cell culture conditions EA.hy-926 cells and human colon carcinoma cells (DLD1) (originating from the American Type Culture Collection (ATCC)) were grown in Dulbeccos medium containing 10% heat inactivated fetal bovine serum (FBS). EA.hy-926 culture medium additionally contains 1% HAT media supplement. Human umbilical vein endothelial cells (HUVEC) were cultured using endothelial cell growth media system (EGM-2) from Cambrex (BioWhittaker Europe,Verviers, Belgium) containing 2% fetal calf serum (FCS) according to the manufacturer´s protocol. HUVEC were used in the fourth to sixth passage for experiments. Western blot analysis To detect activation of NF-κB signaling pathway, phosphorylation of the inhibitory molecule IκBα was measured by Western blot analysis using phospho-specific IκBα antibody (detecting IκBα phosphorylated on Ser32). 15 min after irradiation, cells were harvested and 30 µg of protein was separated by polyacrylamide gel electrophoresis (10 % denaturing gels). After transfer of the proteins to nitrocellulose, membrane was blocked (5 % dry milk in PBS/0.1 % tween, 2h, RT), before overnight incubation with phospho-specific antibody (1:1000) according to the manufacturers protocoll. After washing and incubation with peroxidase- coupled secondary anti-rabbit antibody, phosphorylated IκBα was visualized by chemiluminescence. ELISA analysis For quantitation of E-selectin protein expression an ELISA-based method was used. To this end, 4x10 4 endothelial cells were seeded into 96-well microtitre plates and grown overnight. Before pretreatment and stimulation of the cells the medium was replaced. About 5h after stimulation, plates were stored on ice for 10 min. Afterwards the cell layer was washed twice 4 Downloaded from by guest on December 18, 2012

with ice-cold PBS/1% BSA. Cells were incubated with anti-E-selectin antibody for 90 min (1:50 in PBS/1%BSA). After washing (3x), POD-conjugated secondary antibody (1:1000 in PBS/1%BSA) was added for 60 min. All steps were performed on ice. Upon addition of pnitrophenyl phosphate (pNPP) solution enzymatic reaction was allowed to proceed for 30 min at room temperature before optical densitiy (E 405 nm) was quantitated using ELISA plate reader. Specific binding of the antibody was calculated after substracting non-specific binding of IgG 2a-isotype(= Δ405 nm). At least three independent experiments (each performed in triplicate) were used for statistical interpretation of the data (mean value ± sd) . RNA expression analysis (RT-PCR) Total RNA was isolated by use of RNA Purification Kit (Sigma-Aldrich Chemie GmbH, Germany). 1µg of purified total RNA was used for RT-PCR analysis using the Titan One Tube RT-PCR System (Roche Diagnostics GmbH, Mannheim, Germany). Reverse transcription was performed at 50°C for 30min. For PCR-reaction 25 cycles were performed (denaturation: 95°C, 1min; annealing: 60°C, 2min; polymerization: 68°C, 2min). PCR products were separated onto agarose gels and DNA was visualized by ethidium bromide staining. Primers for E-selectin RNA amplification were designed according to the E-selectin mRNA sequence published (NCBI Nucleotide GenBank (accession number: NM-000450) (5'primer: 5´-TCTCTCAGCTCTCACTTTG-3', 3´-primer: 5'-TTCTTCTTGCTGCACCTCT-3'). E-selectin PCR product is 383 bp in length. Primers for GAPDH amplification (392 bp PCR product) were as follows: 5´-primer: 5´-GTCTTCACCACCATGGAGAAGGCT-3´ and 3´primer: 5´-CATGCCAGTGAGCTTCCCGTTCA-3´) Cloning of the E-selectin promoter and reporter gene analysis Total genomic DNA was isolated from EA.hy-926 cells using the DNeasy TM -Tissue Kit (Qiagen GmbH, Hilden, Germany). PCR primers for cloning of the human E-selectin promoter were designed according to the E-selectin gene sequence published (Montgomery et al., PNAS USA 88 (1991)) (5'-primer: 5´- AGGCTGGTCTTGAACTCCCG-3'; 3´- primer: 5'-GACTTCAAGAGTTCTTTTCACCC-3'). After amplification by touch down PCR (30 cycles) from 66°C down to 54°C, the 1.24 kb E-selectin promoter fragment was cloned into XhoI/HindIII site of pGL3-Basic Vector (Promega Corporation, Mannheim, Germany). Succesful cloning was confirmed by sequencing. To examine transcriptional activation of the E-selectin promoter or of NF-κB minimal promoter construct (3xNF-κB-luciferase [33] subconfluent EA.hy-926 cells or HUVEC were transiently transfected with 2 µg of the corresponding construct. In case of EA.hy-926 cells, transfection was done by use of the Effectene method (Qiagen GmbH, Hilden, Germany), transfection of HUVEC was performed using ESCORT TM III Transfection Reagent (Sigma-Aldrich Fine Chemicals, Taufkirchen, Germany). 8 hours after transfection, cells were pretreated or not and stimulated after a further incubation period of 16h. About 12h after exposure, cells were harvested and luciferase activity was determined according to the manufacturers protocoll (Promega Corporation, Mannheim, Germany). Determination of protein concentration of cell extracts was done by the method of Bradford [34]. Relative luciferase activity of treated cells was related to that of untreated control which was set to 1.0. Analysis of cell adhesion The adhesion of human colon carcinoma cells (DLD1) to a confluent monolayer of endothelial cells (HUVEC) was analyzed upon fluorometrical labeling of the tumor cells by calcein AM (excitation wavelength: 495 nm; emission at 517 nm) basically according to the Vybrant TM cell adhesion assay (Molecular Probes, Europe, Leiden, The Netherlands). Shortly, colon carcinoma cells were labeled by incubation with 5 µM of calcein AM (Sigma Aldrich 5 Downloaded from by guest on December 18, 2012

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