Quantitative PCR, (qPCR) For each RNA sample, 2 mg of total RNA was reverse transcribed according to manufacturer’s instructions, (Promega). Murine primers were designed using Primer Express 2.0 (read 5’ to 3’; forward-F, reverse-R): m5T4-F: agctcttcggtaccctcgtc, m5T4-R: gttgcggttcacgcactta, mCD26-F: ggcaatttgtaaaaatgggatt, mCD26-R: aggttacataccctccatatgacc, mCXCL12-F: tccaaattccccagcaga, mCXCL12-R: ctgaacccatcgctgcttagac, mCXCR4-F: caggacctgtggccaagttctt, mCXCR4-R: agctgaggatcacggctagctt. SybrGreen qPCR reactions were performed in MicroAmp optical 384-well reaction plates, (Applied Biosystems). Amplifica- tions were carried out using a 7900 ABI Prism thermocycler, (Applied Biosystems) and amplification analyzed with SDS 2.1 software, (Applied Biosystems). Melt curves (derivative of fluores- cence intensity versus temperature) were made and inspected to ensure that only one peak, indicating one amplicon product, was produced. Amplification efficiencies for each primer pair were determined by constructing standard dilution curves (mean Ct of triplicate reactions plotted against cDNA mass), with cDNA inputs of 1 ng, 5 ng, 10 ng, 50 ng and 100 ng per reaction and calculating r2 values and gradients of linear regression lines. For testing of relative gene expression, 10 ng cDNA was used per reaction SDS-Polyacrylamide Gel Electrophoresis (PAGE) and Western Blotting Samples were prepared in non-reducing PAGE loading buffer (Thermo Fisher), heated to 100uC for 3 minutes and loaded on to a preformed 10% or 4-15% gradient gel (BioRad) and run in Lamelli buffer (25 mM Tris-base, 192 mM glycine, 0.1% SDS). Western transfer to PVDF membranes used a BioRad Mini- PROTEAN Tetra cell system. For western probing, primary antibody concentrations were: Polyclonal rabbit anti-CXCR4, (1 mg/ml) (Abcam) and appropriate concentrations of the different mouse 5T4 specific monoclonal antibodies. For the detection of phosphorylated and/or intracellular proteins cells were lysed in M- PER supplemented with protease and phosphatase inhibitor cocktails, (Thermo Fisher). The compounds PD98059 (50 mM), LY294002 (50 mM) (both Cell Signaling Technology) or AMD3100 (10 mM) were applied to cells for 1 hour prior to CXCL12 (12.5 nM) or Phorbol 12-Myristate 13-Acetate, (PMA) (50 nM) (Sigma) stimulation in order to inhibit MEK1, PI3 kinase or CXCR4 respectively. Primary antibodies used as per suppliers instructions were ERK1/2, phospho-ERK1/2 (Thr202/Tyr204), (Cell Signalling Technology). Following secondary antibody labeling using appropriate HRP conjugates, (AbSerotec) hybrid- izing bands were detected using SuperSignal West Dura, (Thermo Fischer). Immunofluorescence in situ These studies were performed using methods described previously [63], with cells grown on 24 well glass bottomed plates, (Iwaki, supplied through Jencons) coated with 0.1% gelatine. Primary antibodies used were rabbit anti-CXCR4, (5 mg/ml) (Abcam), rat-anti-DPIV (5 mg/ml), (R&D), mAb anti-m5T4, 9A7 (20 mg/ml), B5C9 or B3F1 (5 mg/ml) or appropriate isotype controls. Secondary detection was performed by incubation for 1 hour at 4uC with species or IgG sub-class specific Alexa Fluor conjugated secondary antibodies (Invitrogen) (6 mg/ml) as appro- 5T4 Regulates CXCR4 Chemotaxis priate for multiple antigen detection. Labeling of the endoplasmic reticulum and Golgi apparatus was performed using C6-Ceramide or Endotracker, (Invitrogen, Molecular Probes) as per manufac- turer’s instructions. Labeling of lipid rafts was performed using cholera toxin subunit B conjugated to FITC, 1 mg/ml (Sigma) for 30 minutes at room temperature after secondary antibody labeling. F-Actin was labeled using Phaloidin-633 Alexa Fluor (Invitrogen). Inhibition of the Golgi, actin cytoskeleton and microtubule network was performed by overnight incubation of MEF with optimized concentrations of 3.57 mM Brefeldin FA, 985 nM Cytochalasin D or 332 nM Nocodazole respectively. Cells were fixed following washout at 0, 30 minutes and 1 hour and processed as above for immunofluorescence. Cytoskeleton disruption following 24 hours treatment with cytochalasin D was achieved as no polymerized actin filaments were detectable (data not shown). Likewise, Golgi disruption following brefeldin A treatment was confirmed by immunofluorescence detection of sphingolipids using BODIPY labeled NBD C6 ceramide (data not shown). Microtubule disruption following nocodazole treatment was confirmed by immunofluorescence detection using an antibody against b-tubulin (data not shown). In some experiments, MEF were seeded and treated with 332 nM nocodazole for 18 hours and following washout, cells were incubated in growth medium or growth medium with mAb B1C3 and P1C9 (10 mg/ ml) or mIgG (10 mg/ml) for 3 or 6 hours. Cells were imaged on a Zeiss Axiovert 200 M with a plan-fluar 6100 1.45NA objective lens and a Roper Cascade EMCCD 512B camera. Illumination was achieved using a 300W Xenon system, (Sutter) which presented the system as an even field of illumination in addition to the appropriate neutral density and Schott filters to modulate the light source. Wavelength selection was achieved using external filter wheels, (Applied Scientific Instrumentation) and the ET-Sedat set, (Chroma). Data sets were captured with an axial resolution of 100 nm using the MS-2000 stage (Applied Scientific Instrumentation) and a lateral resolution of 0.1645 microns per pixel. The system was full controlled and automated via the FRAP-AI software, (MAG Biosystems/Metamorph). All of the data sets were deconvolved using Huygens (Scientific Volume Imaging) after which visualization and analysis was carried out using Imaris, (Bitplane). Deconvolved images were assessed utilizing the ImarisColoc software (Bitplane) in manual mode. A 2D scatter plot showing intensity pairs in the image was thresholded to include only co-localized points in the three dimensional volume. This data was then extracted to a separate channel containing three dimensional co-localized points only. For each in situ immunofluorescence investigation, a minimum of 50 cells per experimental condition were examined in at least duplicate. In addition a more detailed quantitative analysis (as above) of the patterns of expression (e.g. intracellular versus membrane) was performed on between 10–25 cells and represen- tative images are presented. 5T4 constructs A series of 5T4 constructs were built for this study and cloned into pCMVa, and the retroviral vector SFb91 [64] containing a cDNA cassette eGFP under the transcriptional control of an IRES, (Clonetech). Chimeric constructs of mouse 5T4/CD44 molecules with reciprocally exchanged TM and cytoplasmic domains were engineered. CD44 molecules are 80–95 kDa transmembrane glycoproteins expressed on a variety of normal cells as well as some tumors where particular spliced forms have been associated with increased metastasis [65]. E1/E3 replication deficient recombinant adenoviral vectors were constructed by cloning of the m5T4 or h5T4 cDNA into the adenoviral shuttle vector PLoS ONE | www.plosone.org 18 April 2010 | Volume 5 | Issue 4 | e9982