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: Stem cells international

Article Title: Aucubin Impeded Preosteoclast Fusion and Enhanced CD31 hi EMCN hi Vessel Angiogenesis in Ovariectomized Mice.

doi: 10.1155/2022/5226771

Figure Lengend Snippet: Figure 2: Aucubin represses osteoclast formation, promotes PDGF-BB secretion, and promotes type H vessel formation in OVX mice. (a) TRAP staining of the femora to calculate the numbers of osteoclasts and preosteoclasts of bone surface. Scale bar: 200 μm. (b) Quantification of OCs and POCs per bone surface of different groups. BS: bone surface. (c) Quantification of PDGF-BB levels in bone marrow was detected by ELISA and analyzed. (d) Type H vessels were detected by CD31hiEmcnhidouble IF. Scale bar: 100 μm. (e) Type H vessels were quantitative analysis in each group. BM: bone marrow; GP: growth plate. n = 5 per group. (f) Representative images of IF staining of OCN, Scale bar: 50 μm. (g) The quantification of OCN+cell surfaces/bone surfaces. TB: trabecular bone; BM: bone marrow. ∗P < 0:05 compared to the sham group; #P < 0:05 compared to the OVX group.

Article Snippet: PDGF-BB ELISA Kit was obtained from Cusabio (Wuhan, China).

Techniques: Staining, Enzyme-linked Immunosorbent Assay