Figure 6

WNT5A induces release of exosomes that contain solube mediators. (A) Electronmicroscopic picture of isolated exosome fraction from supernatant of Mewo cells treated with 0.2 μg/ml rWNT5A for 3 h. (B) Western blot of isolated exosomes from supernatant of Mewo cell stimulated with 0.2 μg/ml rWNT5A, carrier or 0.1 ng/ml rWNT3A for 3 h. GAPDH (upper), CD63 (center) and Rab5b (lower). (C) Quantitative Exosome Elisa (ExoELISA) of isolated exosome fraction from Mewo cells transfected with pcDNA3 or CA-Cdc42. The bar graphs show the relative increase of exosomes produced in CA-Cdc42 transfected as compared to pcDNA3 transfected Mewo cells. Error bars represent SEM. n = 5. * = p <0.05 by Student’s t-test. (D) IL-6 or (E) MMP2 levels as measured by Elisa of frozen exosome fractions isolated from supernatant of Mewo cells that were treated with 0.2 μg/ml rWNT5A, carrier or 0.1 ng/ml rWNT3A for 3 h. The low protein yield in the isolated exosome fractions might explain the higher relative background level in the control as compared to IL-6 measured in supernatant. The bar graphs show average fold increase as compared to carrier (ctrl) for each treatment indicated. Error bars represent SD. n = 3. * = p <0.05, ** = p <0.01 by Student’s t-test.