Fig 1: The effects of PEDF on extravasation and inhibition of EndoMT. (A) Immunoblot of mouse spleen (Sp), kidney (Kd), liver (Lv), and lung (Lu) lysates by anti-PEDF receptor molecules. PNPLA2/PEDF-R was expressed in liver and lung. Laminin receptor/67lr (lamR) was expressed in the kidney and the lung (arrow). The lamR in liver sample was truncated (open arrow). The endogenous PEDF was not significantly different among these organs. (B) The cells extravasated in the lung of tail intravenous injected U2OS+mCherry (above). The cells extravasated in the lung of U2OS+PEDF (+mCherry) injected mouse. The mCherry-positive cells were observed everywhere in the lung (below). (C) After two weeks of the intracardiac injection of MG63Neo or MG63+PEDF, Evans Blue (EB) dye was injected from the tail vein two hours before the sacrifice. EB dye was visualized as the red fluorescence, and when the vascular permeability was increased, the EB dye remained in the organs (See Materials & Methods). In MG63+PEDF-injected mouse lung, the large puncta of EB remained (right) while the small vein was stained in control (left). (D) The quantitative analyses of Milles assay shown in (C). The remained EB dyes were extracted from each dissected organ by the heated formamide and quantified by OD600. (E) The schematic illustration of the double-sided cell culture on the atelocollagen membrane. The mouse kidney endothelial cell line, TKD2 cells expressing GFP (TKD2-GFP) were cultured on the one side of the chemically cross-linked atelocollagen membrane, and the appropriate size of 143B cell cluster was transferred on the other side of the membrane. We could see the localized effects of PEDF because only secretory molecules could go across this cross-linked membrane (See Material and Methods). (F, G) The Z-stack images were taken under the confocal microscopy. The nucleus of the cells (blue), TKD2-GFP (green), osteosarcomas (red), and ß-catenin immunofluorescence (IF) (white) images were merged. (F) TKD2 layer was disturbed by 143B+PEDF. The optical section of TKD2 side (above). The projection image of cancer side (below). (G) ß-catenin IF of TKD2 layer. ß-catenin staining was disappeared from the region where the opposite side were occupied by the cancer cells. (H) The schematic diagram of the wound healing assay of TKD2-derived cell layers. TKD2 cell layers were formed inside of the silicon molds. The cancer cells were cultured around the mold, and time-lapse images were captured after removing the mold. (I) Western blot analyses of TKD2-laminin receptor knockout cells (lamRKO). The PEDF or its receptor expressions were not changed. (J, K) [TKD2 migrations with Bkid (red)], [TKD2 with Bkid+PEDFmiR (pink)], [TKD2-lamRKO cell migrations with Bkid (blue)] and [TKD2-lamRKO with Bkid+PEDFmiR (dark blue)], [TKD2 with Bkid-lamRKO(purple)] and [lamRKO-TKD2 with Bkid-lamRKO (green)]. (K) The graph indicated the cell free area at 14 hours after the start. PEDF knockdown in Bkid cells or lamR knockout in TKD2 accelerated the endothelial cell migration. NS, Not significant. The symbol **** means 4 digit below the decimal point, enough significant difference according to the result of t-test.