Fig. 6

Immunophenotype of breast cancer cells, from patients with Luminal B subtype, used in all experiments. Cells are shown under phase contrast microscopy and indirect immunofluorescence for vimentin, cytokeratin, Ecadherin, N-cadherin, PAI-1, claudin 1, phalloidin, and DAPI (blue, for nuclei). (a) Phase contrast–confluent culture of tumor cells with EMT (epithelialmesenchymal- transition) after 3 days. (b-e) Analysis of epithelial and mesenchymal markers by confocal microscopy; cytokeratin, vimentin, Ecadherin, and N-cadherin, respectively. (f and g) Representative flow cytometry histograms of PAI-1 and claudin 1 on tumor cells with EMT. The histogram on the left represents a control staining using an isotype-matched control antibody. (h) Zymography for MMP-9, in conditioned medium, in EMT and epithelial cells isolated from women with breast cancer. (i) Phase contrast— epithelial cells confluent culture after 2 days. (j and k) Analysis of epithelial marker cytokeratin. We observed the cytoskeletal organization pattern when using phalloidin. (l) The expression of cat K is up-regulated in co-cultured breast cancer cells and platelets. Cat K was assessed by immunoblotting. (m) Breast cancer cells in epithelial-mesenchimal transition co-cultured with platelets and activated by cat K showed up-regultation of SHH, PTHrP, OPN, and TGF-ß expression, and increased phospho-Src. The graph represents the densitometric analyses from the immunoblotting results. The results are represented as band intensities in arbitrary units relative to the respective total loading control (ß-actin). (n) The flow cytometry results are reported as percentages of CD44 human-specific antibody in breast cancer cells, and P-selectin in human platelets activated by cat K after exposure to breast cancer cells. See also Fig. 2