primary antibodies against vegf c  (Santa Cruz Biotechnology)

Journal: Breast Cancer Research : BCR

Article Title: GPER mediates the angiocrine actions induced by IGF1 through the HIF-1α/VEGF pathway in the breast tumor microenvironment

doi: 10.1186/s13058-017-0923-5

Figure Lengend Snippet: GPER correlates with IGF1R and CD34 expression in breast tumor samples. Data showing angiocrine-related genes across the 17 study integrated Affymetrix ( a - c ) and METABRIC ( d - f ) datasets of 2999 and 1973 breast cancer patients, respectively. In the heatmaps, ranked from left to right GPER expression correlates with IGF1R and the angiogenic marker CD34. Colors are log2 mean-centered values; red indicates high, and green indicates low. Tumours in which GPER is called ‘present’ (GPER+) using the MAS5 detection calls are indicated in grey. GPER G-protein estrogen receptor, HIF-1 hypoxia inducible factor-1, IGF1 insulin-like growth factor 1, VEGF vascular endothelial growth factor

Article Snippet: Then cells were fixed in 4% paraformaldehyde, permeabilized with 0.2% Triton X-100, washed three times with PBS and incubated overnight with a mouse primary antibody against VEGF (C-1) (Santa Cruz Biotechnology).

Techniques: Expressing, Marker

Journal: Breast Cancer Research : BCR

Article Title: GPER mediates the angiocrine actions induced by IGF1 through the HIF-1α/VEGF pathway in the breast tumor microenvironment

doi: 10.1186/s13058-017-0923-5

Figure Lengend Snippet: IGF1 induces the expression of HIF-1α, GPER and VEGF . mRNA expression of HIF-1α, GPER and VEGF in CAFs ( a ) and SKBR3 ( b ) cells treated for 8 hours with 100 ng/mL, as evaluated by real-time PCR. Values are normalized to the 18S expression and shown as fold changes of mRNA expression induced by IGF1 compared to cells treated with vehicle. Data are mean ± SEM of three independent experiments performed in triplicate. c Evaluation of luciferase activity in SKBR3 cells infected with a HRE reporter construct (SKBR3-HRE-luc) and treated for 18 hours with IGF1 (100 ng/mL). The luciferase activities were normalized to the protein content, evaluated in parallel plate by SRB (sulforhodamine B) assay. The transactivation of a VEGF (pVEGF) ( d ) and a GPER (pGPER) ( e ) promoter construct is observed in SKBR3 cells treated with 100 ng/mL IGF1 for 18 hours. Luciferase activity was normalized to the internal transfection control. Results are expressed as the % change of normalized RLU values relative to vehicle-treated cells. Data shown are the mean ± SEM of two independent experiments performed in triplicate. Representative immunoblots showing the increase of HIF-1α and GPER protein expression in CAFs ( f ) and SKBR3 cells ( g ) treated with 100 ng/mL IGF1 for 8 hours. The upregulation of GPER protein expression observed treating CAFs ( h ) and SKBR3 cells ( i ) for 8 hours with 100 ng/mL IGF1 is abrogated by silencing HIF-1α. β-actin serves as loading control. j The transactivation of a GPER promoter construct (pGPER) detected in SKBR3 cells treated with 100 ng/mL IGF1 for 18 hours is abrogated by HIF-1α silencing. (*), (○), p < 0.05; (**), (○○), (●●) p < 0.01; (***), (●●●) p < 0.001. CAFs cancer-associated fibroblasts, GPER G-protein estrogen receptor, HIF-1 hypoxia inducible factor-1, IGF1 insulin-like growth factor 1, VEGF vascular endothelial growth factor

Article Snippet: Then cells were fixed in 4% paraformaldehyde, permeabilized with 0.2% Triton X-100, washed three times with PBS and incubated overnight with a mouse primary antibody against VEGF (C-1) (Santa Cruz Biotechnology).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Luciferase, Activity Assay, Infection, Construct, Sulforhodamine B Assay, Transfection, Control, Western Blot

Journal: Breast Cancer Research : BCR

Article Title: GPER mediates the angiocrine actions induced by IGF1 through the HIF-1α/VEGF pathway in the breast tumor microenvironment

doi: 10.1186/s13058-017-0923-5

Figure Lengend Snippet: ERK1/2 and AKT signaling pathways are involved in the upregulation of VEGF expression induced by IGF1. The upregulation of HIF-1α and GPER protein expression observed treating CAFs ( a ) and SKBR3 cells ( b ) with 100 ng/mL IGF1 for 8 hours is abolished in the presence of 10 μM IGF1R inhibitor AG1024 (AG), 10 μM MEK inhibitor PD98059 (PD) and 100 nM PI3K inhibitor Wortmannin (WM). The activation of ERK1/2 and AKT (Ser 473) is prevented in CAFs ( c , e ) and SKBR3 cells ( d , f ) treated for 60 minutes with 100 ng/mL IGF1, alone and in combination with AG (10 μM), PD (10 μM) and WM (100 nM). ERK2, AKT and β-actin serve as loading control, as indicated. g VEGF protein expression in CAFs treated with 100 ng/mL IGF1 for 8 hours, alone and in combination with AG (10 μM), PD (10 μM) and WM (100 nM), as evidenced by immunfluoerscence experiment. VEGF accumulation is shown by the green signal , nuclei are stained by DAPI ( blue signal ), bar scale 100 μM. Results shown are representative of two independent experiments. Evaluation of luciferase activity in SKBR3 cells infected with a HRE reporter construct (SKBR3-HRE-luc) ( h ), and in SKBR3 cells transiently transfected with a GPER (pGPER) ( i ) or a VEGF (pVEGF) promoter construct ( j ) and treated with 100 ng/mL IGF1 for 18 h in the presence of AG, PD and WM. Luciferase activity was normalized to the internal transfection control. Results are expressed as the % change of normalized RLU values relative to vehicle-treated cells. Each data point represents the mean ± SEM of two independent experiments performed in triplicate. (**) p < 0.01; (***) p < 0.001. CAFs cancer-associated fibroblasts, GPER G-protein estrogen receptor, HIF-1 hypoxia inducible factor-1, IGF1 insulin-like growth factor 1

Article Snippet: Then cells were fixed in 4% paraformaldehyde, permeabilized with 0.2% Triton X-100, washed three times with PBS and incubated overnight with a mouse primary antibody against VEGF (C-1) (Santa Cruz Biotechnology).

Techniques: Protein-Protein interactions, Expressing, Activation Assay, Control, Staining, Luciferase, Activity Assay, Infection, Construct, Transfection

Journal: Breast Cancer Research : BCR

Article Title: GPER mediates the angiocrine actions induced by IGF1 through the HIF-1α/VEGF pathway in the breast tumor microenvironment

doi: 10.1186/s13058-017-0923-5

Figure Lengend Snippet: HIF-1α and GPER are involved in VEGF protein increase induced by IGF1. a Evaluation of VEGF protein expression by immunofluorescence experiment in CAFs transfected for 24 hours with control shRNA ( panels 1–4 ) or shHIF-1α ( panels 5–8 ) and treated with 100 ng/mL IGF1 for 8 hours, as indicated. b The transactivation of a VEGF (pVEGF) promoter plasmid observed in SKBR3 cells treated with 100 ng/mL IGF1 for 18 hours is abrogated silencing the expression of HIF-1α. c Evaluation of VEGF protein expression by immunofluorescence experiment in CAFs transfected for 24 hours with control shRNA ( panels 1–4 ) or shGPER ( panels 5–8 ) and treated with 100 ng/mL IGF1 for 8 hours, as indicated. In immunofluorescence experiments, VEGF accumulation is evidenced by the green signal, nuclei are stained by DAPI ( blue signal ), bar scale 100 μM. Images shown are representative of two independent experiments. d The transactivation of a VEGF (pVEGF) promoter plasmid observed in SKBR3 cells treated with 100 ng/mL IGF1 for 18 hours is abrogated silencing the expression of GPER. In luciferase assays, luciferase activity was normalized to the internal transfection control. Results are expressed as the % change of normalized RLU values relative to vehicle-treated cells. Each data point represents the mean ± SEM of two independent experiments performed in triplicate. (*) p < 0.05, p < 0.01 (**). CAFs cancer-associated fibroblasts, GPER G-protein estrogen receptor, HIF-1 hypoxia inducible factor-1, IGF1 insulin-like growth factor 1, VEGF vascular endothelial growth factor

Article Snippet: Then cells were fixed in 4% paraformaldehyde, permeabilized with 0.2% Triton X-100, washed three times with PBS and incubated overnight with a mouse primary antibody against VEGF (C-1) (Santa Cruz Biotechnology).

Techniques: Expressing, Immunofluorescence, Transfection, Control, shRNA, Plasmid Preparation, Staining, Luciferase, Activity Assay

Journal: Breast Cancer Research : BCR

Article Title: GPER mediates the angiocrine actions induced by IGF1 through the HIF-1α/VEGF pathway in the breast tumor microenvironment

doi: 10.1186/s13058-017-0923-5

Figure Lengend Snippet: HIF-1α and GPER are involved in the formation of endothelial tubes mediated by VEGF. Tube formation in HUVECs cultured in medium collected from CAFs treated with vehicle or 100 ng/ml IGF1 for 18 hours; CAFs were transfected for 24 hours with control shRNA ( a ), shHIF-1α ( b ) or shGPER ( c ) before adding treatments. c 10 ng/mL VEGF rescues tube formation in HUVECs cultured in conditioned medium from GPER-silenced CAFs, which were treated with 100 ng/mL IGF1 for 18 hours. d Quantification of the number of tubes, observed in HUVECs, as indicated. Data are representative of three independent experiments performed in triplicate. (***) p < 0.001. CAFs cancer-associated fibroblasts, GPER G-protein estrogen receptor, HIF-1 hypoxia inducible factor-1, HUVECs human umbilical vein endothelial cells, IGF1 insulin-like growth factor 1, VEGF vascular endothelial growth factor

Article Snippet: Then cells were fixed in 4% paraformaldehyde, permeabilized with 0.2% Triton X-100, washed three times with PBS and incubated overnight with a mouse primary antibody against VEGF (C-1) (Santa Cruz Biotechnology).

Techniques: Cell Culture, Transfection, Control, shRNA

Journal: Breast Cancer Research : BCR

Article Title: GPER mediates the angiocrine actions induced by IGF1 through the HIF-1α/VEGF pathway in the breast tumor microenvironment

doi: 10.1186/s13058-017-0923-5

Figure Lengend Snippet: VEGF triggers endothelial tube formation via VEGFR2. a Endothelial tube formation is abrogated in HUVECs cultured for 4 hours in medium collected from CAFs treated with 100 ng/mL IGF1 for 18 hours, in the presence of the VEGFR2 inhibitor SU5416 (1 μM). b Quantification of the number of tubes observed in HUVECs, as indicated. Data are representative of three independent experiments performed in triplicate. (***) p < 0.001. CAFs cancer-associated fibroblasts, HUVECs human umbilical vein endothelial cells, IGF1 insulin-like growth factor 1, VEGF vascular endothelial growth factor

Article Snippet: Then cells were fixed in 4% paraformaldehyde, permeabilized with 0.2% Triton X-100, washed three times with PBS and incubated overnight with a mouse primary antibody against VEGF (C-1) (Santa Cruz Biotechnology).

Techniques: Cell Culture