Fig 2: Loss-of-function analyses of PEDF in Bkid cells. (A) PEDF micro RNA (miR) expressing vector was transfected into Bkid cells. Immunoblot by anti-PEDF antibody showed the reduction of PEDF proteins by miRs. (B) The renal and the hepatic lesions of the results of the intracardiac injections of Bkid cells or Bkid+PEDFmiR cells. The adrenal metastasis was observed. Also, by Bkid cell injections, the renal metastasis was observed frequently (left, 12-positive/12), while no renal nodule was observed in Bkid+PEDFmiR injected mouse (right, 17-negative/18). The ruler is 1mm square. (C) The hepatic metastases of both cells were not inhibited with or without PEDF. (D) The section Hematoxylin/Eosin (H&E) staining of the similar samples to (B). The adrenal hypertrophy was observed both in Bkid- and Bkid+PEDFmiR-injected mouse organs. The renal metastatic lesion (met) was seen as the lighter color. The scale is 2 mm. (E) The hepatic metastasis. Both tumors had the cavity. (F) PEDF-immunohistochemistry (IHC) of the section (D). PEDF staining was observed inside of the adrenal gland cystic vesicle of Bkid tumors. The overall staining level of PEDF in Bkid tumor was higher than Bkid+PEDFmiR cells. The scale is 1 mm. (G) IHC for PEDF on paraffin sections of collagen gel-embedded 143B and 143B+PEDF cells. (H-L) The renal section IHC by various antibodies. (H) Anti-SPARC (osteonectin)-IHC indicates that the lesions are derived from the injected osteosarcoma. In the section of kidney from Bkid-injected mouse, there is SPARC-positive tumor both in kidney and adrenal gland sides. On the right, the kidney from Bkid+PEDFmiR-injected mouse had SPARC-positive cells only in adrenal gland. The scale indicates 100 µm. (I–L) The scale indicates 20 µm. (I) The higher magnified picture of the tissue surrounded glomerulus. There is SPARC-positive cells were seen around the glomerulus of Bkid-injected mouse (arrow), while no signal was seen in Bkid+PEDFmiR-injected mouse, (J) PEDF-IHC. PEDF was accumulated in the podocytes around the glomerulus. (K) 67kDa laminin receptor (lamR)-IHC. lamR is a potential PEDF receptor. Arrows indicated lamR signals in the podocytes around and the vein in the glomerulus. (L) CD31-IHC in glomerulus. (M, N) The liver section IHCs with CD31 antibody. The vascular formation was observed. (M) No closed blood vessel was seen in Bkid tumor (above). Near the tumor-liver boundary in Bkid sample (below). (N) The blood vessels could be seen as the closed oval form in Bkid+PEDFmiR tumor (above). Tumor-liver boundary of Bkid+PEDFmiR (below). There were a lot of the closed blood vessels (arrowheads). (O) The closed shapes of CD31-staining were counted only in the tumor regions, and the results were normalized by CD31-positive area. PEDF expression severely disturbed the formation of blood vessels (p=0.015). ad, adrenal gland; kid, kidney; met, metastatic lesion; liv, liver.

Fig 3: PEDF-lamR downstream in TKD2 endothelial cells. (A) The phosphorylation of Akt in TKD2 cells were stimulated by Bkid supernatant and inhibited by wortmanin (WMN) treatment. The numbers showed the ratio of phosphorylated Akt to Akt. (B) The qPCR of the same sample as (A). Lysyl-oxidase like 1(Loxl1) was increased by Bkid stimulation and blocked by WMN. Conversely, SNAI3 (Snail3) expression was downregulated by Bkid and increased by Bkid+WMN, while Loxl2 and Snail2 were not affected by such stimulations. MMP2 were regulated similarly to SNAI3. (C) The gene expressions of PEDF-overexpressing, SNAI3-overexpressing and SNAI3+PEDF-coexpressing TKD2. Endothelial-to-mesenchymal Transition (EndoMT) markers were tested. Tcf12, S100A4 (fibroblastic), ACTA2 (fibroblastic) markers indicated that SNAI3 overexpression promoted the EndoMT, and SNAI3 function was blocked by PEDF-coexpression. SM-22a (smooth muscle protein) was not inhibited by PEDF. The known EMT marker, Metadherin (MTDH) was also upregulated by SNAI3 overexpression. The endogenous SNAI3 was not significantly changed by the overexpression. (D) TKD2+Loxl1 cell line was established. The layer formation of TKD2 with Bkid+PEDF-KD was promoted, however, TKD2+Loxl1 with Bkid+PEDF-KD was inhibited as same as that of TKD2+Bkid. (E, E’) The co-immunoprecipitation between 3xFLAG-Loxl1 and EGFP-ETV1 (negative control of EGFP-tag) and EGFP-SNAI3. FLAG-tagged bovine alkaline phosphatase (3xFLAG-BAP) was negative control of FLAG-tag. Loxl1 pulled down SNAI3 while Loxl1 lacking Lox domain (Loxl1?L) could not bind to SNAI3 same way as full-length Loxl1, thus, the center of binding between SNAI3 and Loxl1 is the Lox domain of Loxl1. (E’) The co-expression of lamR protein did not affect on the binding between Loxl1 and SNAI3. (F) The binding between HaloTag-fusion protein of laminin receptor (Halo-lamR) and 3xFLAG-Loxl1 was tested with or without Bkid stimulations. Loxl1 could bind to lamR (lane 2), while Bkid stimulation released Loxl1 from Halo-lamR (lane 5). Non-specific binding was slightly increased after adding Bkid proteins (lane 4).

Fig 4: PEDF did not inhibit the proliferation but inhibited the migration. (A–D) Cell numbers over time. (E–H) The decrease ratio of the cell free area by wound healing. (A) The growth curves of 143B and 143B+PEDF cells. PEDF overexpression slightly increased the cell proliferation, but the differences were not significant. (B) The cell numbers of MG63+MOC vector (MG63Neo) and MG63+PEDF in cell culture. MG63+PEDF grow slightly faster than MG63Neo. (C) The cell numbers of U2OS+mCherry vector and U2OS+PEDF in cell culture. Likely U2OS+PEDF grow faster than that of U2OS+mCherry. (D) The comparisons among Bkid, Bkid+PEDFmiR, and Bkid lamR knockout cells. The proliferations were not significantly changed by loss-of-function. (E) Wound healing abilities of 143B and 143B+PEDF cells. PEDF overexpression decreased cell motility of 143B. (F) Wound healing of MG63Neo and MG63+PEDF. MG63+PEDF delayed the healing. (G) Wound healing of U2OS+mCherry and U2OS+PEDF. Same as other cells, U2OS+PEDF significantly delayed the healing. (H) The wound healing phenotype of Bkid was cancelled by the knockdown of PEDF-lamR pathway. (I) Western blot of the effect of gene knockdown and gene knockout cell lysates. LamR knockout and PEDF knock down did not affect on the other’s expression each other.