Journal: Nature Communications

Article Title: SARI inhibits angiogenesis and tumour growth of human colon cancer through directly targeting ceruloplasmin

doi: 10.1038/ncomms11996

Figure Lengend Snippet: ( a ) Human angiogenesis array analysis of the conditional medium from SW480-controland SW480-SARI cells. ( b ) Summary of the relative signal intensity of indicated cytokines. ( c ) VEGF exacted from SW480 and HCT116 cells was quantified by ELISA ( n =3; ** P <0.01; Student's t -test). ( d ) Western blot showing expression of the SARI protein and that SARI inhibits cellular VEGF expression in SW480 and HCT116 cells in vitro . ( e ) Staining for SARI (red) and VEGF (green) in SW480-control, SW480-SARI, HCT116-control and HCT116-SARI cells; DAPI staining for the nucleus. Scale bar, 50 μm. ( f , g ) Endothelial tube formation estimation following the incubation of HUVECs with conditioned medium from SW480-control, SW480-SARIcancer cells with or without shVEGF-442, or shVEGF-901 transfection. The number of branches was quantified ( g ; n =5; P <0.01; NS, no significant difference; Student's t -test). Scale bar, 50 μm. ( h , i ) Representative photomicrographs of HUVECs that have invaded through Matrigel chambers after incubation with conditioned medium for 72 h from SW480-control, SW480-SARI cancer cells with or without shVEGF-442, or shVEGF-901 transfection. Scale bar, 50 μm. ( i ) Quantification of HUVECs that have invaded through Matrigel chambers ( n =5; P <0.01; NS, no significant difference; Student's t -test). Ctrl, control.

Article Snippet: The supernatant was used to measure the total levels of several cytokines using the human VEGF ELISA kit (NeoBioscience, Shenzhen, China), PDGF-BB ELISA kit (Boster, Wuhan, China), bFGF ELISA kit (NeoBioscience), VEGF-C ELISA kit (Boster), PIGF ELISA kit (NeoBioscience) and VEGF-D ELISA kit (NeoBioscience) according to the manufacturer's instructions.

Techniques: Enzyme-linked Immunosorbent Assay, Western Blot, Expressing, In Vitro, Staining, Control, Incubation, Transfection

Journal: Nature Communications

Article Title: SARI inhibits angiogenesis and tumour growth of human colon cancer through directly targeting ceruloplasmin

doi: 10.1038/ncomms11996

Figure Lengend Snippet: ( a ) Co-immunoprecipitation products after adding anti-SARI antibody were separated by SDS–PAGE, and the differential band was analysed by MS. ( b ) Co-immunoprecipitation analysis of SW480-control and SW480-SARI cell lysates using anti-SARI and anti-Cp antibody. ( c ) Staining for Cp (green) and SARI (red) in SW480-control and SW480-SARI cells; DAPI staining for the nucleus. Scale bar, 50 μm. ( d ) Immunoblots of SARI and Cp expression in SW480 and HCT116-blank, control and SARI cells. GAPDH was used as a loading control. ( e ) Western blotting detection of Cp expression of collected nuclear and cytoplasmic proteins. Histone H3 was used as the nucleus loading control and β-actin as the cytoplasm loading control. ( f ) Immunoblots of Cp and VEGF expression in SW480-control and SW480-SARI cells with or without MG132 treatment. GAPDH was used as the loading control. ( g ) ELISA detection of VEGF expression in SW480-control and SW480-SARI cells with or without MG132 treatment. ( n =4; ** P <0.01; Student's t -test) ( h ) Measurement of Cp in cell lysates harvested at 0, 2, 4, 6, 8 and 10 h after the addition of cycloheximide (CHX) to arrest protein synthesis. ( i ) Densitometry analysis of Cp expression in multiple western blots after CHD addition ( n =3; ** P <0.01; Student's t -test). ( j ) Immunoblots of Cp expression in HCT15, HCT15-shNC, HCT15-shSARI-1068 and HCT15-shSARI-554 cells. GAPDH was used as a loading control. ( k ) Co-immunoprecipitation analysis using anti-HA and anti-CP antibodies of total cell lysates collected from SW480 cells after transfection with HA-SARI (encoding 1–79 amino acids), HA-SARI (encoding 80–274 amino acids) and HA-SARI (encoding 1–274 amino acids). Immunoblots of HA and Cp expression. ( l ) Immunoblots of HA and Cp expression in SW480 cells after transfection with pVax, HA-SARI (encoding 1–79 amino acids), HA-SARI (encoding 80–274 amino acids) and HA-SARI (encoding 1–274 amino acids). GAPDH was used as a loading control. ( m ) ELISA detection of VEGF expression in the conditional medium from the SW480 cells after transfection with pVax, HA-SARI (encoding 1–79 amino acids), HA-SARI (encoding 80–274 amino acids) and HA-SARI (encoding 1–274 amino acids) with or without TM (4.0 μM) treatment. ( n =3; ** P <0.01; analysis of variance analysis). IB, immuoblot.

Article Snippet: The supernatant was used to measure the total levels of several cytokines using the human VEGF ELISA kit (NeoBioscience, Shenzhen, China), PDGF-BB ELISA kit (Boster, Wuhan, China), bFGF ELISA kit (NeoBioscience), VEGF-C ELISA kit (Boster), PIGF ELISA kit (NeoBioscience) and VEGF-D ELISA kit (NeoBioscience) according to the manufacturer's instructions.

Techniques: Immunoprecipitation, SDS Page, Control, Staining, Western Blot, Expressing, Enzyme-linked Immunosorbent Assay, Transfection

Journal: Nature Communications

Article Title: SARI inhibits angiogenesis and tumour growth of human colon cancer through directly targeting ceruloplasmin

doi: 10.1038/ncomms11996

Figure Lengend Snippet: ( a ) Immunoblots of SARI and HIF-1α expression in SW480 and HCT116 cells after stable expression of SARI. HCT116-control and HCT116-SARI cells were incubated under 1% O 2 for 48 h or treated with DMOG for 24 h. β-Actin was used as a loading control. ( b ) Immunoblots of Cp, HIF-1α and VEGF expression in control and SARI cells with or without PX-478 (45 μM) or Tm (4.0 μM) treatment under normoxia and hypoxia. GAPDH was used as the loading control. ( c ) Immunoblots of Cp, HIF-1α and VEGF expression in control and SARI cells after transfection with or without p-Cp plasmid under normoxia and hypoxia. GAPDH was used as the loading control. ( d ) Expression of VEGF exacted by SW480-control and SW480-SARI cells with or without Tm (4.0 μM) and PX-478 (45 μM) treatment, after transfection with or without Cp expression plasmid ( n =3; ** P <0.01, NS, no significant difference; Student's t -test). ( e ) Endothelial tube formation estimation following the incubation of HUVECs with conditioned medium from SW480-control and SW480-SARI cells with or without Tm treatment (4.0 μM) and PX-478 (45 μM), after transfection with or without Cp expression plasmid. The number of branches were quantified ( n =5; ** P <0.01; NS, no significant difference; Student's t -test). Scale bar, 50 μm. ( f ) Representative photomicrographs of HUVECs that have invaded through Matrigel chambers after incubation with conditioned medium from SW480-control and SW480-SARI cells with or without Tm treatment (4.0 μM) and PX-478 (45 μM), after transfection with or without Cp expression plasmid. Quantification of HUVECs that have invaded through Matrigel chambers ( n =5; ** P <0.01; NS, no significant difference; Student's t -test). ( g ) Immunoblots of Cp and HIF-1α expression in HCT15, HCT15-shNC, HCT15-shSARI-1068 and HCT15-shSARI-554 cells with or without Tm (4.0 μM) treatment. GAPDH was used as a loading control. ( h ) Expression of VEGF exacted by HCT15, HCT15-shNC, HCT15-shSARI-1068 and HCT15-shSARI-554 cells with or without Tm treatment (4.0 μM) was measured by ELISA ( n =3; ** P <0.01; ## P <0.01; analysis of variance analysis).

Article Snippet: The supernatant was used to measure the total levels of several cytokines using the human VEGF ELISA kit (NeoBioscience, Shenzhen, China), PDGF-BB ELISA kit (Boster, Wuhan, China), bFGF ELISA kit (NeoBioscience), VEGF-C ELISA kit (Boster), PIGF ELISA kit (NeoBioscience) and VEGF-D ELISA kit (NeoBioscience) according to the manufacturer's instructions.

Techniques: Western Blot, Expressing, Control, Incubation, Transfection, Plasmid Preparation, Enzyme-linked Immunosorbent Assay

Journal: Nature Communications

Article Title: SARI inhibits angiogenesis and tumour growth of human colon cancer through directly targeting ceruloplasmin

doi: 10.1038/ncomms11996

Figure Lengend Snippet: ( a ) Staining for VEGF (red) revealed less VEGF-positive cells in SARI than in controlSW480Matrigel plugs, Scale bar, 50 μm; VEGF-positive (%; VEGF positive per total cells; n =5; ** P <0.01; Student's t -test). ( b ) Staining for VEGF (red) revealed less VEGF-positive cells in SARI than in control HCT116 tumours ( d ), Scale bar, 100 μm; VEGF positive (%;VEGF positive per total cells; n =5; ** P <0.01; Student's t -test). ( c ) Staining for Cp revealed less Cp-positive cells in SARI than in control HCT116 tumours, Scale bar, 100 μm; Cp positive (%;Cp positive per total cells; n =5; ** P <0.01; Student's t -test). ( d ) Staining for HIF-1α (red)-positive cells in SARI and control HCT116 tumours, Scale bar, 100 μm; HIF-1α positive (%; HIF-1α positive per total cells; n =5; ** P <0.01; Student's t -test). ( e ) Immunoblots of VEGF, Cp and HIF-1a expression in SW480-control and SW480-SARI tumours. GAPDH was used as the loading control. ( f ) ELISA detection of VEGF expression in SW480-control and SW480-SARI tumours ( n =3; ** P <0.01; Student's t -test). ( g ) Staining for CD31, VEGF, Cp and HIF-1α in colonic tumours of AOM/DSS induction. Analysis of the number of microvessels, VEGF-positive cells, Cp-positive cells and HIF-1α-positive cells ( n =4; ** P <0.01; Student's t -test). Scale bar, 100 μm. ( h ) Immunoblots of VEGF, Cp and HIF-1α expression in colonic tumours of SARI −/− and SARI WT mice after AOM/DSS induction. GAPDH was used as the loading control. ( i ) ELISA detection of VEGF expression in colonic tumours of SARI −/− and SARI WT mice after AOM/DSS induction. ( n =3; ** P <0.01; Student's t -test) ( j ) Nuclear and cytoplasmic proteins were collected from the colonic tumours of SARI −/− and SARI WT mice after AOM/DSS induction. Western blotting was performed to detect Cp expression. Histone H3 was used as the nuclear loading control and β-actin as the cytoplasmic loading control.

Article Snippet: The supernatant was used to measure the total levels of several cytokines using the human VEGF ELISA kit (NeoBioscience, Shenzhen, China), PDGF-BB ELISA kit (Boster, Wuhan, China), bFGF ELISA kit (NeoBioscience), VEGF-C ELISA kit (Boster), PIGF ELISA kit (NeoBioscience) and VEGF-D ELISA kit (NeoBioscience) according to the manufacturer's instructions.

Techniques: Staining, Control, Western Blot, Expressing, Enzyme-linked Immunosorbent Assay

Journal: Thrombosis and Haemostasis

Article Title: Katacine Is a New Ligand of CLEC-2 that Acts as a Platelet Agonist

doi: 10.1055/a-1772-1069

Figure Lengend Snippet: H igh-throughput screening based on CLEC-2 and podoplanin interaction. ( A ) Schematic representation of ALPHA screen based on podoplanin and CLEC-2 interaction. ALPHA screen assay is based on the proximity of the donor and acceptor beads mediated by the protein–protein interaction. After laser excitation, podoplanin-conjugated donor beads transform ambient oxygen to singlet oxygen, which is absorbed by CLEC-2-conjugated nickel acceptor beads, then ALPHA signal is emitted and detected by the plate reader. ( B-i ) Scatter plot showing the effect of each compound on the ALPHA signal values emitted by CLEC-2 and podoplanin interaction; dots below the cut off ( red line ) represent compounds that significantly decreased signal and potential hits. ( B-ii ) Scatter plot showing TruHits assay to identify potential false-positive compounds on the screening; dots over the cut-off indicate potential hits. ( C ) The dose–response curves of potential hits and IC50 of each potential compound (i–vi). C-i and C-ii show the dose–response curves of the two most potent compounds, further evaluated them in functional assays. shows a chemical description of the compounds tested.

Article Snippet: Katacine was purchased from TargetMol (Boston, Massachusetts, United States), and recombinant human podoplanin-rFc and recombinant human CLEC-2–6xHis (Ser55-Pro229) were expressed in house HEK293T cells.

Techniques: Functional Assay

Journal: Thrombosis and Haemostasis

Article Title: Katacine Is a New Ligand of CLEC-2 that Acts as a Platelet Agonist

doi: 10.1055/a-1772-1069

Figure Lengend Snippet: Katacine binds to the same binding site as rhodocytin based on molecular binding site prediction. Molecular docking has been performed using AutoDock vina and ADT for protein preparation. Grid boxes for binding prediction have covered the extracellular domain of CLEC-2, using the crystal structure of human CLEC-2 (PDB: 2C6U). Katacine can bind to a positive charge surface rich of arginine in the CLEC-2 extracellular domain (Arg-107, Arg-118, Arg-152, Arg-157) previously reported as the binding site for podoplanin and rhodocytin (enclosed in a red circle ), with a predicted binding energy of −6.6 kcal/mol. ADT, AutoDock tools.

Article Snippet: Katacine was purchased from TargetMol (Boston, Massachusetts, United States), and recombinant human podoplanin-rFc and recombinant human CLEC-2–6xHis (Ser55-Pro229) were expressed in house HEK293T cells.

Techniques: Binding Assay