Figure 1. Podoplanin-mediated platelet aggregation promotes epithelial-mesenchymal transition in tumour cells.

(a) Immunoblot analysis showing podoplanin expression in UM-UC-5 and H226 but not in A549 cells. TopoIIβ was used as a loading control. (b) UM-UC-5, H226 and A549 cells (5 × 10⁴ cells) were incubated with washed mouse platelets (5 × 10⁷ platelets/200 μl assay) suspended in Tyrode’s buffer containing 2% platelet-poor plasma and 250 μM CaCl₂. Light transmittance of samples was measured to determine the aggregation rate using an aggregometer. (c) Schematic representation of collection of tumour-platelet reactant supernatants. (d,e) Morphological and physiological changes in cells after treatment with or without supernatants of tumor-platelet reactants for 48 h. (d) Images were captured using phase-contrast microscopy (left panels) or immunofluorescence microscopy (right panels) after staining for E-cadherin (green), F-actin (red; phalloidin) and nuclear DNA (blue; Hoechst 33342). Scale bars represent 50 μm. (e) Cellular lysates were immunoblotted with antibodies to N-cadherin, claudin-1, podoplanin (PDPN, clone D2–40) and TopoIIβ.