Fig. 5

CCL5 secreted by aPKCι-induced mesenchymal-like CCA cells mediates the chemotactic migration and activation of macrophages. A. Cytokine array of the CM of mesenchymal-like TFK-1. The Euclidean average summarizing the relative signal intensity of the indicated cytokines is presented. B. qRT-PCR and ELISA showing the relative levels of CCL5 in mesenchymal-like or epithelial-like CCA cells treated without or with the NF-κB inhibitor PDTC. C. Luciferase reporter analysis of the NF-κB-mediated activation of CCL5 transcription. TFK-1 cells were transfected with luciferase reporter plasmids containing wild-type (CCL5-WT-Luc), NF-κB mutant (CCL5-ΔkB-Luc), or NF-κB response elements (κB-Luc, active control) in the absence or presence of TGF-β or TNF-α. D. Migration assay in CD14⁺ monocytes stimulated with or without CM from TFK-1 cells transfected with an empty vector (negative control, NC-CM), CM from mesenchymal-like TFK-1 cells (transfected with aPKCι-cDNA) alone or with anti-CCL5 neutralizing antibody, aPKCι-siRNA, PDTC, or recombinant human CCL5 treatment. Scale bar, 200 μm. E. Flow cytometry for expression of CD80/CD206 in macrophages treated with CM from mesenchymal-like TFK-1 cells in the presence or absence of control IgG, a CCL5 neutralizing antibody, PDTC, or recombinant human CCL5. The CM of TFK-1 cells transfected with empty vector was used as a negative control