Additional file 1 of KAI1(CD82) is a key molecule to control angiogenesis and switch angiogenic milieu to quiescent state
Creators: name: Lee, Jin-Woo
name: Hur, Jin
name: Kwon, Yoo-Wook
name: Chae, Cheong-Whan
name: Choi, Jae-Il
name: Hwang, Injoo
name: Yun, Ji-Yeon
name: Kang, Jin-A
name: Choi, Young-Eun
name: Kim, Young Hyun
name: Lee, Sang Eun
name: Lee, Cheol
name: Jo, Dong Hyun
name: Seok, Heeyoung
name: Cho, Byong Seung
name: Baek, Sung Hee
name: Kim, Hyo-Soo
Publisher: name: figshare
Year: 2021
Subjects: subject: Biophysics
subject: Biochemistry
subject: Space Science
subject: Medicine
subject: Cell Biology
subject: Molecular Biology
subject: Pharmacology
subject: 69999 Biological Sciences not elsewhere classified
subject: FOS: Biological sciences
subject: Developmental Biology
subject: Cancer
subject: Hematology

description: Additional file 1: Figure EV1. KAI1 was expressed in PC more than EC and plays a role in the anti-angiogenic effect in PC. (A) Left, vessel regression (empty sleeves, BS-1 lectin-negative, collagen IV-positive) in the WT and Kai1−/− mice. Arrowheads, empty sleeves. Scale bar, 50 μm. Right, quantification of the obtained results (Means ± SEM, 4 technical replicates). (B) Confocal imaging in retinal of WT mice using Kai1 (white) antibody. Scale bar, 20 μm. (C) Confocal imaging of Kai1-GFP expression in brain and heart of the Kai1-GFP fusion transgenic mice. Scale bar, 5 μm. (D) FACS plot showing primary ECs (CD45−/CD31+) and primary PCs (CD45−/Pdgfrβ+) in the bone marrow of Kai1-GFP fusion transgenic mice. Left, KAI1 surface expression of human cell lines, HUVECs and HBVP, analyzed with FACS. Right, quantification of the obtained results. MFI (**p &lt; 0.05, mean fluorescence intensity). (F) CD9 expression as an example in MS1 and 10T1/2 cells. Scale bar, 50 μm. (G) Expression of tetraspanins (Cd9, Cd81, Cd151) in ECs (MS1) and PCs (10T1/2) by using quantitative RT-PCR. (H) In vitro matrigel tube formation assay, performed using co-culture spheroids, consisting of ECs (MS1) and PCs (10T1/2), transduced with the indicated adenovirus at 0, 14, and 107 h, and observed using time-lapse microscopy. Scale bar, 50 μm. Figure EV2. KAI1 localizes at the lipid rafts of PCs and binds with zDHHC3 and zDHHC4. (A) Lipid rafts isolated from ECs (MS1) and PCs (10T1/2) using OptiPrep medium (frozen and crosslinks method). Endogenous Kai1 localization is confirmed. Flotillin-1 (Flot-1) and caveolin-1 (Cav-1) was detected lipid raft membrane fraction of MS1 and 10T1/2, respectively. Fraction 1; no cellular protein, Fraction 2; Lipid raft proteins. Fraction 3; Non-raft membrane proteins. Fraction 4 and 5; Cytosolic proteins. (B) Semi-quantitative RT-PCR analysis for zDHHC family member expression in MS1 and 10T1/2 cells. The genes were normalized by Gapdh (Means ± SEM, 3 technical replicates). (C) zDHHC3 and zDHHC4 binding to KAI1, confirmed by co-immunoprecipitation. Figure EV3. Identification of the downstream effect molecule induced by KAI1. (A) Semi-quantitative RT-PCR analysis for mRNA levels of top 17 genes in “negative angiogenic regulators” in WT and Kai1−/− primary PCs. The genes were normalized by Gapdh and the mRNA expression value are as fold change relative to WT (Means ± SEM, 3 or 4 technical replicates). (B) Mouse EC cell line, MS1, cell cycle analyzed with FACS (ns; not significant, *p &lt; 0.01, **p &lt; 0.05, Means ± SEM, 3 technical replicates). (C) LIF, LIFR, and GP130 expression in mouse and human ECs and PCs by using semi-quantitative RT-PCR. Figure EV4. KAI1 induces LIF through Src/p53. (A) Lif and Kai1 mRNA expression in 10T1/2 cells pre-treated with inhibitor of p38, Erk, Pkc, or Src and then transfected with Kai1-O/E using Flag-Kai1 vector. (B) The effects of Kai1 overexpression sup. and PP2 (Src inhibitor) on angiogenesis using tube formation assay (***p &lt; 0.001, Means ± SEM, 3 technical replicates). (C) The effects of Kai1 overexpression sup. and PP2 (Src inhibitor) on angiogenesis using migration assay (ns; not significant, ***p &lt; 0.001, Means ± SEM, 3 technical replicates). (D) Protein level of p53 and Pbx1 by Kai1 O/E in 10T1/2 cells. The quantification of the obtained results (*p &lt; 0.01, Means ± SEM, 3 technical replicates). Figure EV5. KAI1 inhibits VEGFR2 and PDGFRβ signaling through direct binding to VEGF-A and PDGF-BB. (A) Protein-protein interactions between rhKAI1 and rhVEGF-R2 observed using surface plasmon resonance (BLItz system), which suggests no binding between KAI1 and VEGF-R2. (B) The effects of KAI1 on angiogenesis using tube formation. (C) The effects of KAI1 on angiogenesis using migration. rhVEGF-A (40 ng/ml), rhKAI1 (800 ng/ml) were used. ns; not significant, *p &lt; 0.01, **p &lt; 0.05, ***p &lt; 0.001. Figure EV6. The expression of KAI1 proteins in human prostate cancer and melanomas. (A, D) Representative immunohistochemistry staining for KAI1/CD82 protein expression in normal/malignant prostate cancer tissue and normal skin/melanomas. Scale bar, 50 μm. (B, E) Representative fluorescence images of the immunostained normal prostate and skin. (C, F) Representative fluorescence images of the immunostained malignant prostate cancer tissue and melanomas. Scale bar, 8 μm. DIC (Differential interference contrast). (G) Representative immunohistochemistry staining for KAI1/CD82 protein expression in normal/tumor pancreatic tissue. Scale bar, 100 μm. Figure EV7. Identification of the role of B16 proliferation by Kai1. WST1 assay measuring B16 cell proliferation rate after Kai1 overexpression .Figure EV8. The anti-angiogenic effect through Lif induction in vivo by Kai1. (A) Immunofluorescence image after transducing the Kai1-Flag vector into the mouse TA muscle and detected co-localized Kai1 and Lif. (B) Quantification of the obtained results (Means ± SEM, 4 technical replicates). **p &lt; 0.05, Scale bar, 100 μm. Figure EV9. Therapeutic potential of KAI1 and characteristics of KAI1 peptide. (A) Venn diagram showing the design of peptide which interact with VEGF and PDGF, we screened the consensus amino acid sequences of VEGF- and PDGF-receptor but exclusive with FGF-receptor sequence in KAI1 large extracellular loop. (B) Schematic figure showing the KAI 1 peptide and characteristics. Figure EV10. Wild-type peptide of KAI1 interact with VEGF and PDGF. Mutant peptide does not interact with them. (A) Peptide-protein interactions between KAI1 peptide and VEGF observed using surface plasmon resonance (BLItz system), which suggests binding between KAI1 WT peptide and VEGF but no binding between mutant peptide and VEGF. (B) Peptide-protein interactions between KAI1 peptide and PDGF observed using BLItz system, which suggests binding between KAI1 WT peptide and PDGF but no binding between mutant peptide and PDGF. Figure EV11. Comparative analysis of exosome miRNAs in the aorta of WT and Kai1 knockout mice. Left, Comparison of expression pattern of miR-181a in exosomes of WT and Kai1 knockout mice. Right, Comparison of the expression pattern of miR-107 in exosomes of WT and Kai1 knockout mice. *p &lt; 0.01. Expanded View movie 1. KAI1 in PCs has anti-angiogenic effects. In vitro matrigel tube formation assay, performed using co-culture spheroids, consisting of ECs (MS1) and PCs (10T1/2), transduced with the indicated adenovirus at 0, 14, and 107 h, and observed using time-lapse microscopy. (A) Mock virus transduced MS1 and 10T1/2 co-culture spheroids. (B) Kai1-O/E MS1 and 10T1/2 co-culture spheroids. Video sprouting from the spheroid. Scale bar, 50 μm.