Figure 3

WNT5A induces a Ca²⁺- and Cdc42-dependent release of soluble mediators. (A) Mewo cells treated with rWNT5A for 3 h were paraffin embedded and stained with hematoxylin & eosin (HE). Scalebars = 10 μm. Arrows indicate differences in the cell cortex. (B) IL-6 Western blot (WB) detecting changes in intracellular IL-6 following cytokine release upon WNT5A treatment for 3 h. (C) Mewo cells treated with DMSO or Actinomycin D for 12 h together with rWnt5 or carrier. The bar graphs show average fold increase as compared to carrier (ctrl) for each time point and treatment indicated. Error bars represent SEM. n = 4. (D) Mewo cells were pre-treated with DMSO, the Ca²⁺-chelator Bapta-AM or PKA inhibitor H89 for 30 min prior to WNT5A treatment for 3 h. The bar graphs show average fold increase as compared to carrier (ctrl) for each time point and treatment indicated. Error bars represent SEM. n = 4. (E) Cdc42 is activated by WNT5A in Mewo cells. Western Blot of a GST-Pull down assay of active Cdc42, upon rWNT5A stimulation for 45 min. Controls are carrier (Ctrl) or rWNT3A for 45 min. (F) Transfection of Mewo cells with DN-Cdc42 or -Rac1 inhibits the WNT5A induced secretion of IL-6. Empty vector used as control. The data shown are ratios of rWNT5A treated (3 h)/untreated cells for each plasmid transfected and normalized against the empty vector control. n = 3. (G) Mewo cells were pre-treated with Tetanus toxin, TeNT, for 30 min prior to WNT5A treatment for 3 h or 24 h. The bar graphs show average fold increase as compared to carrier (ctrl) for each time point and treatment indicated. Error bars represent SD. n = 2. * = p <0.05 ** = p <0.01 by Student’s t-test. IL-6 levels in supernatants were detected using Elisa.