Figure 2. 5T4 restores CXCL12 dependent chemotaxis in differentiating 5T4KO-ES cells. (A). Undifferentiated 5T4KO-ES cells forced to express 5T4 following infection with RAd-m5T4, (multiplicity of infection = 30, dark grey columns) show no CXCL12 dependent chemotaxis comparable to undifferentiated WT-ES cells (black columns), mock (white columns), or RAd-eGFP (light grey columns) infection. (B), Differentiating 5T4KO ES cells with 5T4 expression restored by RAd-m5T4, (multiplicity of infection = 30, dark grey columns) show CXCL12 chemotaxis comparable to differentiating WT-ES cells (black columns) 5T4 Regulates CXCR4 Chemotaxis but not following mock (white columns), or RAd-eGFP (light grey columns) infection. (C), Differentiating 5T4KO-ES cells chemotactic response to CXCL12 is also restored following infection with a recombinant adenovirus encoding human 5T4 (multiplicity of infection = 30, black columns). doi:10.1371/journal.pone.0009982.g002 cytoskeleton, Golgi and microtubule disruption was monitored by immunofluorescence with flurochrome conjugated phalloidin, NBD C6 ceramide and antibodies against b-tubulin respectively (not shown). Untreated primary MEF exhibit cell surface expression of both 5T4 and CXCR4 with considerable co- localization (Figure 7). After cytochalasin D treatment, there was no reduction in the CXCR4 cell surface expression or co- localization of 5T4 and possibly an increase compared to untreated controls. Brefeldin A reduced levels of cell surface expression of both antigens and all residual CXCR4 or 5T4 labeling was co-localized at cell surface. One hour after brefeldin A washout, increased cell surface expression of both antigens with marked cell surface co-localization was observed. Following nocodazole treatment there was some intracellular accumulation but no cell surface detection of CXCR4. 5T4 remained detectable at the cell surface albeit at a diminished level and no co- localization with CXCR4 was visible. One hour after nocodazole washout both antigens were detectable at the cell surface with marked co-localization. Clearly, detection of plasma membrane co-localized 5T4/CXCR4 molecules is dependent on microtu- bules and the molecules are not obligatorily associated at the Golgi. Disruption of the Golgi or the actin cytoskeleton per se does not disrupt all 5T4/CXCR4 co-localization at the plasma membrane. It appears that CXCR4 and 5T4 molecules can form a stable interaction at the cell surface facilitating the biological response to CXCL12. Inhibition of CXCL12 chemotaxis by monoclonal antibodies recognizing m5T4 in mouse embryonic cells 5T4 molecules play a role in stabilizing plasma membrane expression of CXCR4 receptors most likely through interaction of their transmembrane domains. It is possible that the binding of antibodies recognizing the extracellular domains of 5T4 molecules might influence 5T4-CXCR4 interactions through modulation of cell surface expression or altering conformation. We investigated the ability of several different monoclonal antibodies to m5T4 to influence the chemotactic response of differentiating ES cells or MEF to CXCL12. Five different monoclonal antibodies recogniz- ing distinct epitopes in the proximal and distal LRR domains of m5T4 were available (Figure 8A). Each antibody showed different affinity in a m5T4 specific ELISA (Figure 8B) or B16m5T4 FACS titration (Figure 8C), decreasing in the order B3F1, P1C9, B5C9, P1H10 and B1C3. All detect the extracellular domain of m5T4-Fc by western blotting except for B1C3 (Figure 8D). Importantly, the chemotactic migration towards CXCL12 exhibited by differenti- ating WT-ES cells was abolished in the presence of mAb B1C3 but not P1C9 or P1H10 while B3F1 and B5C9 showed less but still significant inhibition of the chemotactic response (Figure 9A). Figure 9B shows titration of antibody inhibition of differentiating ES cell chemotaxis for the mAbs; the IC50 for the maximally inhibitory mAb B1C3 was 0.36 mg, 60.11 and for B3F1 and B5C9 was 2.2 mg60.8 and 6.8 mg61.5 respectively. The inhibi- tion of chemotaxis by the mAb B1C3 was prevented by the presence of m5T4-Fc (not shown). Similar results were exhibited by primary WT MEF for four of the monoclonal antibodies tested (Figure 9C). Thus, the chemotactic response of both differentiated ES cells and MEF can be blocked by some but not all antibodies recognizing distinct parts or epitopes of m5T4 molecules. PLoS ONE | www.plosone.org 5 April 2010 | Volume 5 | Issue 4 | e9982