Figure 12. Sequence comparison of 5T4 TM domains. Align- ments were performed using the ClustalW2 multiple sequence alignment program (EMBL-EBI) to compare (A), the TM domains of 5T4 across species and (B), the TM domain of human 5T4 with human LRRC4, identical residues shaded black, similar residues shaded grey. doi:10.1371/journal.pone.0009982.g012 developed using tetramethyl benzidine (TMB; Sigma), the colour reaction was stopped with 1 M sulphuric acid and the absorbance read at wavelength 450–650 nM. The isotypes of the monoclonal antibodies were determined using Isotyping kit (ISO-2; Sigma) according to the manufacture’s instructions. Microarray analysis We utilized the associated loss of pluripotency of murine ES cells with the early upregulation of 5T4 expression to search for other changes in gene expression using an Affymetrix approach [18]. The ES cells were grown with or without LIF for 3 days and the disaggregated cells sorted for expression of cell surface 5T4. E14 TG2a cells were investigated with samples showing minimal intra-replicate variance. Data were preprocessed using RMA[60], as implemented in the ‘affy’ BioConductor library[61] and then analysed using LIMMA [62] to identify those probesets found differentially expressed between pluripotent and differentiated samples (FDR threshold = 0.1; log2 fold change threshold = 1). Resultant probesets mapped to gene identifiers using the annotation packages in BioConductor. Chemotaxis assay Chemotaxis was assessed using transwell chambers as previously described for cellular motility assays [16]. Costar Transwell 24- well plates exhibiting 5-mm pore size were used for all motility assays (Cambridge, MA). Briefly, for ES cells migration assays the transwells were immersed in gelatine solution overnight (0.1% in PBS) and rinsed in PBS. Transwells were blocked in fetal calf serum (FCS)-containing ES cell medium or for MEF migration assays FCS containing DMEM for 30 min at 37uC/5% CO2 and washed in PBS. ES cells and MEF were cultured as described above harvested and resuspended in culture medium (ES cells = 16105 cells/ml, MEF = 16104 cells/ml), and 100 ml of this suspension was added to the transwell plates onto a preformed chemotactic gradient (CXCL12, ES cells = 10 ng, MEF = 30 ng; CXCL10, ES cells = 100 ng; CXCL16, ES cells = 10 ng) and 5T4 Regulates CXCR4 Chemotaxis incubated overnight at 37uC/5% CO2. In all experiments there was no evidence of differential plating with varying conditions; chemotaxis was presented as a ratio with or without the chemokine. The transwells were then washed gently in PBS, and cells were removed from within the transwell using a dry cotton bud followed by two washes in PBS. This washing procedure was repeated twice. The transwells were stained with crystal violet for 10 min, washed in water, and allowed to air dry. Cells present on the underside of the transwell (i.e., migrated cells) were counted by microscopy. The number of cells on the bottom of the plate (i.e., cells that had migrated through the pores and become detached from the transwell) was also counted. P values were calculated using unpaired Student’s t test. All chemotactic experiments were performed at least three times with triplicates for each condition. Inhibition studies were performed in the presence of 10 mM Diprotin A, (Sigma) for CD26, 10 mM AMD3100 (Sigma) for CXCR4, 10 mg/well mouse antibody to CXCL12, (R&D systems), or 0.1–10 mg/ml of the monoclonal antibodies specific for m5T4. Specificity of mAb m5T4 mediated inhibition of chemotaxis was validated using m5T4-IgFc fusion protein [58]. ELISA for CXCL12 The concentration of CXCL12 in 3 day conditioned medium from undifferentiated (+LIF) or following 3 days differentiation (-LIF) of WT- and 5T4KO-ES cells was determined by murine CXCL12 specific ELISA (R&D systems). Flow cytometry Cell surface detection of human and murine 5T4 was performed as previously described [15,16]. Briefly, cells were trypsinized, washed twice in PBS and resuspended at 26106 cells/ ml in FACS buffer, (0.1% sodium azide, Sigma; 0.2% bovine serum albumin, Sigma; in PBS). Cells were labeled with antibodies at 4uC for 1 hour using monoclonal antibody (mAb) anti-SSEA-1- PE (phycoerithrin,) or mouse IgM isotype-PE control at 2 mg/ml (Santa Cruz)); rat anti-mDPIV(CD26)-PE, 5 mg/ml (R&D systems) or rat IgG2A isotype control-PE 5 mg/ml (R&D systems); mAbs recognizing m5T4: 9A7 (rat IgG2a [58] 20 mg/ml) and/or B3F1 (mouse IgG2a, 1 mg/ml), B5C9 (mouse IgG1 10 mg/ml); B1C3 (mouse IgG2a 10 mg/ml); P1C9 (mouse IgG2b 1 mg/ml) and P1H10 (mouse IgG2b 10 mg/ml); and their respective isotype controls rat IgG2a, mouse IgG1, mouse IgG2a and mouse IgG2b (eBioscience). Secondary antibodies were PE-donkey anti rat, 1 mg/ml (eBioscience); PE-goat anti-mouse, 1 mg/ml (DAKO). After washing twice in FACS buffer cells were fixed in 300 ml 1% p-formaldehyde in PBS. In situ immunoflurescence was usually performed for CXCR4 detection as the molecules can be sensitive to trypsin treatment. Modulationof 5T4 expression by mAbs 56105 B16m5T4 cells were incubated with either 1 mg/ml P1C9, 10 mg/ml B1C3, 10 mg/ml mouse IgG in DMEM supplemented with 10% FCS or media only at 37uC overnight. Cells were washed twice with FACS buffer and all of the following steps were performed in 0.1% sodium azide on ice. Cells were washed twice with FACS buffer and incubated with either biotinylated B3F1 (2 mg/ml), biotinylated B5C9 (25 mg/ml) or FACS buffer at 4uC for 45 mins. Cells were then washed twice and labeled with streptavidin PE (BD, 1:100) at 4uC for 45 mins. Following two washes, cells were fixed in 1% p-formaldehyde in PBS and analyzed using a Becton Dickinson FACScan. 10,000 events were acquired and the data obtained was analyzed using WinMIDI (version 2.8) software. PLoS ONE | www.plosone.org 17 April 2010 | Volume 5 | Issue 4 | e9982