but that impairment of E-cadherin may provide a growth advantage at an earlier stage – such as in non-malignant, low- grade papillary neoplasms of the bladder [50], early-stage carcinomas of the cervix [51], or in tumours where E-cadherin protein is expressed heterogeneously [50,52]. The understanding of homeostatic regulation of normal epithelial tissue regeneration could have profound implications for developing therapies to target key growth promoting pathways. Materials and Methods Ethics Statement NHU cell lines were established from urological specimens obtained with written informed patient consent and approval from the Leeds (East) and York NHS Research Ethics Committees. Cell culture NHU cell lines of finite lifespan were established and maintained in keratinocyte serum-free medium containing bovine pituitary extract, recombinant EGF and supplemented with cholera toxin (KSFMc), using methods that have been described in detail elsewhere [12,14]. NHU cells were used for retrovirus transduction at passages 1 or 2 and subsequent experiments were performed between passages 2-6. Retrovirus packaging PT67 fibroblasts (BD Biosciences, Oxford, UK) were maintained in DR medium, consisting of a 1:1 (v:v) mixture of DMEM and RPMI 1640 (Invitrogen, Paisley, UK), containing 10% fetal bovine serum (FBS, Harlan Sera-Lab, Loughborough, UK). Preparation of retroviral vectors and retrovirus-producing cell lines The dominant-negative H-2Kd-E-cad mutant [26] was the gift of Professor Fiona Watt (CRUK Research Institute, Cambridge, UK). H-2Kd-E-cad cDNA was sub-cloned from pBabe-puro into the XhoI site of the pLXSP retroviral plasmid [53]. Recombinant vectors pLXSP-ECmut and pLXSP-ECrev were derived, contain- ing the H-2Kd-E-cad fragment in the forward and reverse (non- sense) orientations, respectively (pLXSP-ECrev was used as a specificity control). For the preparation of short hairpin RNA (shRNA) to target b-catenin, an siRNA previously reported [25] was used and adapted for delivery using the pSIREN-RetroQ retroviral vector system. Oligonucleotide primers were prepared and cloned into pSIREN-RetroQ according to the manufacturer’s instructions (Clontech, BD Biosciences), giving rise to vector pSIR- b-cat. The pSIR-Con vector, which contained a scrambled shRNA sequence, was used alongside as negative control. Retroviral transduction of NHU cells Retroviral transductions were performed as described previous- ly [53,54]. PT67 fibroblasts were transfected with pLXSP-ECrev, pLXSP-ECmut, pSIR-Con and pSIR-b-cat vectors using Effecte- neTM (Qiagen, Crawley, UK). Stable retrovirus-producing lines PT67-ECrev, PT67-ECmut, PT67-Con-sir and PT67-b-cat-sir were established by selection in culture medium containing 4 mg/mL puromycin (Autogen Bioclear UK Ltd, Calne, UK). Conditioned media from stable retrovirus producers were 0.45 mm membrane-filtered, supplemented with 8 mg/mL polybrene (Sig- maAldrich, Poole, UK) and used to infect NHU cell cultures for 5– 6 hours. The medium was replaced with KSFMc and two days post-transduction, NHU cells were subjected to antibiotic selection using 1.5 mg/mL puromycin. 2–3 independent NHU lines were each transduced with PT67-ECrev, PT67-ECmut, pSIR-Con and pSIR-b-cat virions resulting in the derivation of isogenic NHU- E-Cadherin Growth Regulation Con and NHU-ECmut sub-lines as well as NHU-Con and NHU- b-cat-KD sub-lines. Cell proliferation assays NHU cells and virally-transduced derivatives were cultured in KSFMc containing low (0.09 mM) or physiological (2.0 mM) Ca2+ and proliferation was assessed by cell counting, by colorimetric assay with thiazolyl blue tetrazolium (MTT; SigmaAldrich) and by 3 [ H]-thymidine incorporation. For counting, cells were seeded in 6-well plates at 26104 cells/well and counts performed for a period of 9 days. For MTT assays, cells were seeded in 96-well plates at 46103/well and proliferation was assessed over the indicated time-course in the presence or absence of 5 mM LY294002. Following treatment with MTT reagent (500 mg/mL) (SigmaAldrich), absorbance at 570 nm was measured. For [3H]- thymidine (TdR) incorporation [53], cells were seeded in 96-well plates at 26104/well; TdR precursor (GE Healthcare, Bucks, UK) was added 24 hours later at 0.5 mCi/well and 16 hours post- pulsing, cells were harvested onto filter-mats and TdR uptake was measured by scintillation spectrometry. Flow cytometry H-2Kd expression was analysed using a FITC-conjugated antibody and an isotype control recommended by the manufac- turer (BD Biosciences). Using previously described methods [54,55], NHU cells were labelled with these antibodies and analysed by flow cytometry on the FL-1 (FITC) channel. A minimum of 10,000 cells were acquired on a CyAnTM instrument and protein expression was analysed using SummitH software (DakoCytomation, Ely, UK). Immunoblotting Cell lysates were prepared, resolved on NuPageH 4–12% bis- Tris acrylamide gels in MES or MOPS running buffer (Invitrogen) and transferred onto 0.45 mm PVDF membranes (GE Healthcare, Bucks, UK) as previously [53,56]. Affinity-purified polyclonal antibodies recognising EGFR (cat#2232), phospho-ERK (cat#9101) and phospho-AKT S473 (cat#9271s) were from Cell Signalling (Autogen Bioclear). Monoclonal antibodies against total-ERK (clone#16) and total-AKT (clone#7) were from BD Biosciences, b-actin (clone#AC-15) was from SigmaAldrich and E-cadherin (mouse monoclonal clone #HECD-1) was from R&D systems (Abingdon, UK). The 8E7 monoclonal antibody recog- nising active, unphosphorylated b-catenin was a kind gift of Prof Hans Clevers (Hubrecht Institute, UMC, Utrecht, Netherlands). Membranes were probed with primary antibodies followed by goat anti-rabbit immunoglobulins conjugated to IRDyeH 800 (Tebu- bio, Peterborough, UK) or goat anti-mouse antibody conjugated to Alexa FluorH 680 (Invitrogen). Immunolabelling was visualised and densitometry performed using an Odyssey system (LiCor, Cambridge, UK). Immunofluorescence microscopy NHU cell cultures were fixed with 10% formalin in PBS and permeabilised in PBS containing 0.1% Triton-X and 10% goat serum (SigmaAldrich). Antibodies used were: a- and b-catenin (rabbit, cat#C-2081 and cat#C-2206, respectively; SigmaAl- d drich). Antibodies against H-2K , EGFR, E-cadherin, total/ phospho-ERK and total/phospho-AKT were as above. Following labelling with primary antibody, cells were washed and incubated with goat anti-mouse or anti-rabbit immunoglobulins conjugated with either Alexa FluorH 488 or 594 (Invitrogen). Cell nuclei were stained using 0.1 mg/mL Hoechst 33258 (SigmaAldrich). Labelled PLoS ONE | www.plosone.org 10 October 2010 | Volume 5 | Issue 10 | e13621