Fig. 3

RSPO3 loss modestly decreases proliferation but significantly increases invasion. DU145 or 22RV1 cells were transiently transfected with control (siCtrl) or RSPO3-specific siRNA (siRSPO3). All figures shown are representative of 3 independent experiments. a Matrigel transwell invasion assay with RSPO3 knockdown. Graphs are shown with means, standard deviations, and statistical significance (left panel), and representative pictures are given with a scale bar = 20 μm, magnification: ×60. (**p < 0.01; n = 3 independent experiments). b Matrigel transwell invasion assays with RSPO3 overexpression. DU145 or 22RV1 cells were transfected with an empty vector (DU145-ctrl and 22RV1-ctrl) or a vector encoding a gene cassette for overexpression of RSPO3 (DU145-RSPO3 and 22RV1-RSPO3). Graphs are shown with means, standard deviations, and statistical significance (left), as well as representative images (right). Scale bar = 20 μm, magnification: ×60. (**p < 0.01; ***p < 0.001; n = 3 independent experiments). c Western blot for EMT markers. DU145-ctrl and DU145-RSPO3 cells were lysed and western blotting performed for the EMT markers indicated. d The chick chorioallantoic membrane assay for ex ovo extravasation/metastasis. 22RV1 cells were transduced with lentiviral particles carrying a vector encoding for control (22RV1.shCtrl) or RSPO3-specific (22RV1.shRSPO3) shRNA. Following selection of a colony of stable transductants, CAM assay was performed as described. Extravasation efficiency is shown with means, standard deviation, and individual measurements (dots/triangles). (**p < 0.001; n = 3 independent experiments.)