BMC Cancer 2005, 5:3 http://www.biomedcentral.com/1471-2407/5/3 Biotech) according to the manufacturer's instructions. EST sequences corresponding to the coding sequence of the genes-of-interest were identified by BLAST [26] and obtained from the I.M.A.G.E. consortium through the UK Human Genome Mapping Project Resource Centre (Hinx- ton, UK). ESTs bearing the following I.M.A.G.E. cloneIDs were used: Autotaxin – 533819; CD14 – 2936787; Ecm1 717050; Hig2 – 367488; Ramp3 – 615797, HIG2 4366895). Following overnight hybridisation with the labelled probe, the membranes were washed twice in 1X SSC, 0.1% (w/v) SDS at room temperature for 20 mins, and twice in 0.2X SSC, 0.1% (w/v) SDS at 68°C for 10 mins and exposed to film at -80°C for 48 hours. Bound probe was quantitated using a phosphorimager (Molecu- lar Dynamics). Western blotting Cell monolayers were rinsed twice with ice-cold Phos- phate Buffered Saline and total cell lysates were prepared by scraping cells into a minimal volume of 50 mM Tris. HCl pH 7.5, 150 mM NaCl, 0.5% NP40 and Complete protease inhibitor cocktail (Roche). Aliquots containing 80 µg protein from each sample were analysed by SDS- PAGE [27], and transferred electrophoretically to a PVDF membrane. Mouse monoclonal antibodies were used to detect tTA (Clontech), β-catenin (Transduction Laborato- ries) and EGFP (Santa Cruz Biotechnology). Samples of conditioned medium were concentrated 12-fold using Microcon YM-10 centrifugal filter units (Millipore) prior to analysis. Construction of plasmids A BgIII fragment containing the lacZ cDNA was excised from the CMV-lacZ construct (a gift of Trevor Dale) and sub-cloned into BamHI digested RevTRE to make RevTRE- lacZ. A plasmid containing a myc-tagged ∆Nβ-catenin was obtained from Hans Clevers. The myc-tagged ∆Nβ-catenin was excised with KpnI and NotI and the ends were blunted, and subcloned into HpaI digested RevTRE to make RevTRE-∆Nβ-catenin. The mouse Hig2 open reading frame was amplified by PCR from I.M.A.G.E. cDNA clone 367488 using the primers TTTACTAGTAGGAGCT- GGGCACCGTCGCC and TTTTACCGGTGCCTGCACTC- CTCGGGATGGATGG. The PCR product was digested with AgeI and SpeI and subcloned into the AgeI and NheI sites in pEGFP-C1 (Clontech) to make the Hig2-EGFP fusion gene. Site directed mutagenesis was carried out by the method of Sawano and Miyawaki (2000) [28]. The primer TGCTGAACCTCGAGGAGCTGGGCATCATG was used to make the Hig2-EGFP(Y8V9/D8D9) mutant. Transient transfections Transient transfections were performed using Lipo- fectamine (Invitrogen) according to the manufacturer's instructions. Briefly, 1.5 × 105 cells were plated in 3.5 cm wells on the day prior to transfection. Each well was trans- fected with a total of 0.9 µg DNA under serum-free condi- tions for six hours, after which the cells were washed and incubated for a further 48 hours before assaying expression. β-galactosidase activity assay For the tetracycline dose response curve, 5000 HC11-lacZ cells for each condition, were cultured in triplicate in 96 well plates for 72 hours, and beta-galactosidase activity was determined as previously described [24]. Results Generation of HC11-lacZ and HC11-∆Nβ-catenin cell lines Stable cell lines were generated in which either ∆Nβ-cat- enin or β-galactosidase was expressed in a tetracycline dependent manner. These cell lines were established using a novel autoregulatory system in which the expression level of the tetracycline transactivator (tTA) protein is minimised during routine culture and is induced upon withdrawal of tetracycline with concomitant upregulation of the transgene-of-interest [24]. This strategy helps to minimise deleterious effects due to tTA toxicity. A dose-response analysis for the HC11-lacZ cell line is shown in Figure 1A. β-galactosidase expression is effec- tively repressed at tetracycline concentrations in excess of 20 ng/ml and is strongly induced in the absence of tetra- cycline. The N-terminal truncation mutant of β-catenin can be detected by western blotting by both its myc- epitope tag and an anti-β-catenin antibody (Figure 1B). tTA expression is detectable only in the absence of tetracy- cline demonstrating the autoregulatory nature of this system. Microarray analysis Transgene expression was induced in HC11-lacZ and HC11-∆Nβ-catenin cells by withdrawal of tetracycline for 72 hours. Total RNA was isolated, from which mRNA was purified. cDNAs were labelled and hybridized to an 8962 element Incyte mouse GEM1 cDNA microarray (Incyte Genomics, Palo Alto, CA). These data are provided as sup- plementary material (See Additional file 1). Among those genes upregulated were two genes shown by other work- ers to be transcriptional targets of this pathway – Fibronec- tin [29] and Autotaxin [30] (data not shown) – suggesting that our model of Wnt/β-catenin signalling deregulation results in the activation of a set of target genes which over- laps, at least partially, with pathway targets in other cell lines. The microarray experiment described here was per- formed only once but differential expression was repeat- edly validated by northern blotting from independent samples for the genes discussed here. Page 3 of 9 (page number not for citation purposes)