Fig. 6

Treating monocytes with Wnt-3a induces cell migration and CCL2 production. Naïve monocytes were plated in the top chamber of a transwell and exposed to different conditioned media (CM). Cells that crossed the transwell were collected from the bottom well and counted by flow cytometry. A Schematic illustration of experimental design. Naïve monocytes were exposed to Wnt-3a/control CM obtained from L-cells for 2 h (I). Naïve monocytes were exposed to Wnt-3a/control CM obtained from monocytes treated with Wnt-3a/control for 8 h (II). Treated monocytes (as in II) were washed and transferred to fresh RPMI medium for an additional 1 h. CM was collected and used to treat naïve monocytes in the transwell assay (III). B Flow cytometry counts of cells crossing the transwell. *P-value = 0.0307, ***P-value = 0.0003 and **P-value = 0.0045 for paired t-tests. Y-axis represents the absolute cell count. C ELISA assays of CCL2 concentration in media used for the indicated experiments. Protein concentrations were calculated by regression from a standard curve. D Flow cytometry counts of cells crossing the transwell. Y-axis represents the absolute cell count. X-axis represents the media used as in I and II of the experimental design, with or without 2 μg/ml CCL2 inhibitor, marked as circles and squares, respectively. The white and black shapes represent each donor. FC – average fold change of the marked pairs. E A representative blot of media collected from the top and bottom chambers of the different experiments (as indicated), at the end of the transwell incubation period. Media were centrifuged to remove cells and analyzed by western blot using a specific anti-Wnt-3a antibody. C – control/control-treated media, W3 – Wnt-3a/Wnt-3a-treated media