Journal: Journal of Translational Medicine

Article Title: ANGPTL4 accelerates ovarian serous cystadenocarcinoma carcinogenesis and angiogenesis in the tumor microenvironment by activating the JAK2/STAT3 pathway and interacting with ESM1

doi: 10.1186/s12967-023-04819-8

Figure Lengend Snippet: The expression, potential functions, diagnostic value and prognostic value of ARGs in OC patients. A The mRNA expression of ARGs in OC samples compared to normal ovary samples based on the TCGA database. B The diagnostic value of ARGs for OC patients. C The PPI network for ARGs. D The correlation among these ARGs in OC patients. E GO and F KEGG enrichment analysis for these ARGs. G ANGPTL4 protein expression in OC and normal ovaries based on the HPA database. H The prognostic value of ANGPTL4 for OC patients based on the TCGA database. I The expression of ANGPTL4 in normal ovary samples and benign and malignant ovarian disease samples. J GSEA of ANGPTL4 based on the TCGA database. K Immune infiltration analysis for OC patients according to ANGPTL4 expression. L The expression of ANGPTL4 in multiple OC cell lines. M The expression of ESM1 and ANGPTL4 in multiple OC cell lines by Western blot. *P < 0.05, **P < 0.01, ***P < 0.001

Article Snippet: According to the requirements of Beijing Genomics institution (BGI), the samples of Hey-A8 cells overexpressing ANGPTL4 and transfected with vector were broken and lysed, and RNA was purified and collected.

Techniques: Expressing, Diagnostic Assay, Western Blot

Journal: Journal of Translational Medicine

Article Title: ANGPTL4 accelerates ovarian serous cystadenocarcinoma carcinogenesis and angiogenesis in the tumor microenvironment by activating the JAK2/STAT3 pathway and interacting with ESM1

doi: 10.1186/s12967-023-04819-8

Figure Lengend Snippet: The expression of ANGPTL4 in benign and malignant ovarian diseases based on IHC staining

Article Snippet: According to the requirements of Beijing Genomics institution (BGI), the samples of Hey-A8 cells overexpressing ANGPTL4 and transfected with vector were broken and lysed, and RNA was purified and collected.

Techniques: Expressing

Journal: Journal of Translational Medicine

Article Title: ANGPTL4 accelerates ovarian serous cystadenocarcinoma carcinogenesis and angiogenesis in the tumor microenvironment by activating the JAK2/STAT3 pathway and interacting with ESM1

doi: 10.1186/s12967-023-04819-8

Figure Lengend Snippet: The expression of ANGPTL4 in ovarian cancer based on TCGA database

Article Snippet: According to the requirements of Beijing Genomics institution (BGI), the samples of Hey-A8 cells overexpressing ANGPTL4 and transfected with vector were broken and lysed, and RNA was purified and collected.

Techniques: Expressing

Journal: Journal of Translational Medicine

Article Title: ANGPTL4 accelerates ovarian serous cystadenocarcinoma carcinogenesis and angiogenesis in the tumor microenvironment by activating the JAK2/STAT3 pathway and interacting with ESM1

doi: 10.1186/s12967-023-04819-8

Figure Lengend Snippet: The effects of the ANGPTL4/JAK2/STAT3 pathway on OC proliferation, migration and invasion. A The protein levels of ANGPTL4, p-JAK2, JAK2, p-STAT3, STAT3, Vimentin, PCNA and hypoxia-inducible factor alpha (HIF-α) were detected in the NC-SKOV3, Vector-SKOV3, ANGPTL4 KD-SKOV3, ANGPTL4 KD-SKOV3 with Colivelin, NC-HeyA8, Vector-HeyA8, ANGPTL4 OE-HeyA8, and ANGPTL4 OE-HeyA8 with AG490 groups by Western blotting. B A workflow for transfecting cells, collecting conditioned medium, and conducting analyses on OC cells and HUVECs. The effect of ANGPTL4, ANGPTL4 + AG490, ANGPTL4 KD, and ANGPTL4 KD + Colivelin on Hey-A8 and SKOV3 cell proliferation ability via MTT ( C ) and EdU ( D ) analysis. The migration and invasion abilities of ANGPTL4, ANGPTL4 + AG490, ANGPTL4 KD, and ANGPTL4 KD + Colivelin were measured by wound healing ( E ) and Transwell invasion assays ( F ) in Hey-A8 and SKOV3 cells, respectively. *P < 0.05, **P < 0.01, ***P < 0.001

Article Snippet: According to the requirements of Beijing Genomics institution (BGI), the samples of Hey-A8 cells overexpressing ANGPTL4 and transfected with vector were broken and lysed, and RNA was purified and collected.

Techniques: Migration, Plasmid Preparation, Western Blot

Journal: Journal of Translational Medicine

Article Title: ANGPTL4 accelerates ovarian serous cystadenocarcinoma carcinogenesis and angiogenesis in the tumor microenvironment by activating the JAK2/STAT3 pathway and interacting with ESM1

doi: 10.1186/s12967-023-04819-8

Figure Lengend Snippet: ANGPTL4 promotes OC growth and angiogenesis by activating the JAK2/STAT3 pathway in vivo. A Morphological observation and HE and IHC staining of xenografts in the NC-SKOV3, Vector-SKOV3, ANGPTL4 KD-SKOV3, ANGPTL4 KD-SKOV3 with Colivelin, NC-HeyA8, Vector-HeyA8, ANGPTL4 OE-HeyA8, and ANGPTL4 OE-HeyA8 with AG490 groups. B The tumor weight and volume and the IHC staining score of xenografts in the NC-SKOV3, Vector-SKOV3, ANGPTL4 KD-SKOV3, ANGPTL4 KD-SKOV3 with Colivelin, NC-HeyA8, Vector-HeyA8, ANGPTL4 OE-HeyA8, and ANGPTL4 OE-HeyA8 with AG490 groups. C The proliferation and tube formation of HUVECs cultured with the CM of HeyA8 cells were tested by EdU staining and tube formation assays. The chick chorioallantoic membrane assay measured the angiogenesis ability of HeyA8 CM in vivo. Angiogenesis ability of HeyA8 cells in a zebrafish model. The experimental groups were NC, Vector, ANGPTL4 OE and ANGPTL4 + AG490. D The proliferation and tube formation of HUVECs cultured with the CM of SKOV3 cells were tested by EdU staining and tube formation assays. The chick chorioallantoic membrane assay measured the angiogenesis ability of SKOV3 CM in vivo. Angiogenesis ability of SKOV3 cells in the zebrafish model. The experimental groups were NC, Vector, ANGPTL4 KD and ANGPTL4 KD + Colivelin. *P < 0.05, **P < 0.01, ***P < 0.001

Article Snippet: According to the requirements of Beijing Genomics institution (BGI), the samples of Hey-A8 cells overexpressing ANGPTL4 and transfected with vector were broken and lysed, and RNA was purified and collected.

Techniques: In Vivo, Immunohistochemistry, Plasmid Preparation, Cell Culture, Staining, Chick Chorioallantoic Membrane Assay

Journal: Journal of Translational Medicine

Article Title: ANGPTL4 accelerates ovarian serous cystadenocarcinoma carcinogenesis and angiogenesis in the tumor microenvironment by activating the JAK2/STAT3 pathway and interacting with ESM1

doi: 10.1186/s12967-023-04819-8

Figure Lengend Snippet: ANGPTL4 interacted with ESM1. A The correlation expression of ESM1 and ANGPTL4 in OC samples based on TCGA database. B IF staining for ANGPTL4 and ESM1 in OC tissue samples, SKOV3 cell lines, HeyA8 cell lines and HUVECs. C The interaction of ANGPTL4 and ESM1 in HeyA8 cells by Co-IP analysis. D GST pull-down analysis for GST-ANGPTL4 and His-ESM1. E Molecular docking for ANGPTL4 and ESM1 by HADDOCK.

Article Snippet: According to the requirements of Beijing Genomics institution (BGI), the samples of Hey-A8 cells overexpressing ANGPTL4 and transfected with vector were broken and lysed, and RNA was purified and collected.

Techniques: Expressing, Staining, Co-Immunoprecipitation Assay

Journal: Journal of Translational Medicine

Article Title: ANGPTL4 accelerates ovarian serous cystadenocarcinoma carcinogenesis and angiogenesis in the tumor microenvironment by activating the JAK2/STAT3 pathway and interacting with ESM1

doi: 10.1186/s12967-023-04819-8

Figure Lengend Snippet: ESM1 promotes ANGPTL4 to combine with LPL by accelerating proliferation, invasion and lipid accumulation in OC cells. A Molecular docking for ANGPTL4, LPL and ESM1 by HADDOCK. B Co-IP showed the effects of ESM1 on the interaction between ANGPTL4 and LPL in HeyA8 and SKOV3 cells. The effect of vector, ANGPTL4, ANGPTL4 + ESM1, ANGPTL4 KD, and ANGPTL4 KD + ESM1 on Hey-A8 and SKOV3 cell lipid levels and proliferation ability via oil red O staining ( C ), MTT ( D ) and EdU ( E ) analysis. The migration and invasion abilities were measured by wound healing ( F ) and Transwell assays ( G ) in Hey-A8 and SKOV3 cells transfected with vector, ANGPTL4, ANGPTL4 + ESM1, ANGPTL4 KD, and ANGPTL4 KD + ESM1. *P < 0.05, **P < 0.01, ***P < 0.001

Article Snippet: According to the requirements of Beijing Genomics institution (BGI), the samples of Hey-A8 cells overexpressing ANGPTL4 and transfected with vector were broken and lysed, and RNA was purified and collected.

Techniques: Co-Immunoprecipitation Assay, Plasmid Preparation, Staining, Migration, Transfection

Journal: Journal of Translational Medicine

Article Title: ANGPTL4 accelerates ovarian serous cystadenocarcinoma carcinogenesis and angiogenesis in the tumor microenvironment by activating the JAK2/STAT3 pathway and interacting with ESM1

doi: 10.1186/s12967-023-04819-8

Figure Lengend Snippet: ESM1 inhibits ANGPTL4 binding to integrin α5β1 and VE-cadherin, which represses HUVEC proliferation and migration to induce vascular permeability in the tumor microenvironment. A Co-IP showed that ESM1 inhibited ANGPTL4 binding to α5β1 and VE-cadherin in HUVECs cultured with HeyA8 and SKOV3 cells, respectively. The effect of vector, ANGPTL4, ANGPTL4 + ESM1, ANGPTL4 KD, and ANGPTL4 KD + ESM1 on HUVECs cultured with Hey-A8 and SKOV3 cell lipid levels and proliferation ability via oil red O staining ( B ), MTT ( C ) and EdU ( D ) analysis. The migration ability was measured by wound healing ( F ) and Transwell assays ( G ) in HUVECs cultured with Hey-A8 and SKOV3 cells transfected with vector, ANGPTL4, ANGPTL4 + ESM1, ANGPTL4 KD, and ANGPTL4 KD + ESM1. *P < 0.05, **P < 0.01, ***P < 0.001

Article Snippet: According to the requirements of Beijing Genomics institution (BGI), the samples of Hey-A8 cells overexpressing ANGPTL4 and transfected with vector were broken and lysed, and RNA was purified and collected.

Techniques: Binding Assay, Migration, Permeability, Co-Immunoprecipitation Assay, Cell Culture, Plasmid Preparation, Staining, Transfection

Journal: Journal of Translational Medicine

Article Title: ANGPTL4 accelerates ovarian serous cystadenocarcinoma carcinogenesis and angiogenesis in the tumor microenvironment by activating the JAK2/STAT3 pathway and interacting with ESM1

doi: 10.1186/s12967-023-04819-8

Figure Lengend Snippet: A model of the molecular mechanism by which ANGPTL4 promotes OC development and progression. A OC is a type of tumor with a rich blood supply. B There are many angiogenic factors in the tumor microenvironment of OC. C A variety of cells in the tumor microenvironment can take up these angiogenesis factors in a variety of ways to produce different pathophysiological effects. D ANGPTL4 can promote the proliferation and migration of OC cells by activating the JAK-STAT3 pathway, and ESM1 can bind ANGPTL4 and stabilize the binding of ANGPTL4 to LPL, thus promoting lipid accumulation and accelerating the proliferation and migration of OC cells. ANGPTL4 can also promote endothelial cell proliferation and migration through the JAK-STAT3 signaling pathway. ESM1 can inhibit the binding of ANGPTL4 to integrin α5β1 to increase vascular stability. Moreover, ESM1 promotes angiogenesis in the ovarian cancer microenvironment by binding to ANGPTL4 to inhibit its destruction of VE-cadherin-mediated tight junctions. E The molecular mechanism might be attributed to the interaction of ESM1 and ANGPTL4.

Article Snippet: According to the requirements of Beijing Genomics institution (BGI), the samples of Hey-A8 cells overexpressing ANGPTL4 and transfected with vector were broken and lysed, and RNA was purified and collected.

Techniques: Migration, Binding Assay

Journal: Journal of Translational Medicine

Article Title: ANGPTL4 accelerates ovarian serous cystadenocarcinoma carcinogenesis and angiogenesis in the tumor microenvironment by activating the JAK2/STAT3 pathway and interacting with ESM1

doi: 10.1186/s12967-023-04819-8

Figure Lengend Snippet: The expression of ANGPTL4 in ovarian cancer based on HPA database

Article Snippet: According to the requirements of Beijing Genomics institution (BGI), the samples of Hey-A8 cells overexpressing ANGPTL4 and transfected with vector were broken and lysed, and RNA was purified and collected.

Techniques: Expressing

Journal: Drug Design, Development and Therapy

Article Title: Anti-Tumor Xanthones from Garcinia nujiangensis Suppress Proliferation, and Induce Apoptosis via PARP, PI3K/AKT/mTOR, and MAPK/ERK Signaling Pathways in Human Ovarian Cancers Cells

doi: 10.2147/DDDT.S258811

Figure Lengend Snippet: ( A and B ) The cell viability of human OC cells HEY and ES2 under the treatment of 1, 2 , and 3 at different concentration for 24 and 48 hours, respectively. ( C ) The cell viability of bronchial epithelial cell line 16HBE under the treatment of 1, 2 , and 3 at different concentration for 48 hours, respectively. Data are presented as mean ±SEM of three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001, versus the control group.

Article Snippet: Human OC cell lines HEY and ES2, and human bronchial epithelioid cell lines 16HBE were purchased from Beyotime Institute of Biotechnology (Shanghai, China).

Techniques: Concentration Assay, Control

Journal: Communications Biology

Article Title: Hypoxia-induced inhibin promotes tumor growth and vascular permeability in ovarian cancers

doi: 10.1038/s42003-022-03495-6

Figure Lengend Snippet: a Relative qRT-PCR analysis of (i) INHA and (ii) VEGFA mRNA expression normalized to levels in 20% O 2 in HEY and OV90 cells exposed to indicated oxygen concentration for 24 hrs. Mean ± SEM, ( n = 3). * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001, One-way ANOVA followed by Tukey’s multiple comparison. (iii) Western blot of HIF-1α stabilization at indicated oxygen concentrations in HEY (left) and OV90 (right). b Relative qRT-PCR analysis of (i) INHA and (ii) VEGFA mRNA expression normalized to corresponding levels in normoxia in indicated cells grown under hypoxia (0.2%) or normoxia (17–21%) for 24 h except for OVCAR5 (12 h). Mean ± SEM, n of independent trials for PA1 = 3, OVCAR5 n = 7, HEY n = 3, OV90 n = 3, * p < 0.05; ** p < 0.01, unpaired t -test. (iii) Western blot of HIF-1α stabilization in indicated cell lines. c Total inhibin ELISA (inhibin A/B, inhibinα) of conditioned media collected from OV90 and HEY cells grown in normoxia or after 24 h exposure to hypoxia (0.2% O 2 ). Mean ± SEM, ( n = 3). ** p < 0.01, unpaired t -test. d (i) Western blot of HIF-1α levels in HEY and OV90 following exposure to hypoxia (0.2% O 2 ) for 24 h and after indicated reoxygenation times. (ii) Relative qRT-PCR analysis of INHA expression in HEY and OV90 cells following exposure to hypoxia (0.2% O 2 ) and indicated reoxygenation time normalized to corresponding levels in normoxia. Mean ± SEM, ( n = 3). * p < 0.05; ** p < 0.01, One-way ANOVA followed by Tukey’s multiple comparison.

Article Snippet: 3 × 10 6 HEY cells either exposed to normoxia or hypoxia (0.2% O 2 ) for 24 h were subcutaneously injected into right flank of 6-week old Ncr Nude mice (Taconic).

Techniques: Quantitative RT-PCR, Expressing, Concentration Assay, Comparison, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Communications Biology

Article Title: Hypoxia-induced inhibin promotes tumor growth and vascular permeability in ovarian cancers

doi: 10.1038/s42003-022-03495-6

Figure Lengend Snippet: a (i) Western blotting of HIF-1α protein from indicated cells grown in either 2D or under anchorage independence (3D) conditions, vinculin is loading control and (ii) relative qRT-PCR of INHA mRNA expression in OVCA420 and PA1 after 48 h (OVCA420) or 72 h (PA1) of growth under anchorage independence (3D). Mean ± SEM, n = 3. ** p < 0.01; *** p < 0.001, unpaired t -test. b Total inhibin ELISA of ascites fluid from 25 ovarian cancer patients sorted by stage. c (i) Percent hypoxic area in tumors of indicated size range determined by quantitation of pimonidazole staining in tumors (Supplementary Fig. ). Graph represents average hypoxic area of all HEY xenograft tumors sorted by size as <500 mm 3 or >500 mm 3 . Mean ± SEM, n = 4 for <500 mm 3 and n = 7 for >500 mm 3 . * p < 0.05, unpaired t -test. (ii) Relative qRT-PCR of INHA expression in tumors from indicated sizes of HEY cells implanted subcutaneously. Mean ± SEM, n = 8. *** p < 0.001, unpaired t -test. d Correlation analysis between INHA expression and either (i) Buffa or (ii) Winter hypoxia scores from TCGA OVCA (i–ii) or breast (iii) cancer patient data sets from cBioportal measured by RNA-Seq. Correlation analysis was performed by Pearson correlation.

Article Snippet: 3 × 10 6 HEY cells either exposed to normoxia or hypoxia (0.2% O 2 ) for 24 h were subcutaneously injected into right flank of 6-week old Ncr Nude mice (Taconic).

Techniques: Western Blot, Control, Quantitative RT-PCR, Expressing, Enzyme-linked Immunosorbent Assay, Quantitation Assay, Staining, RNA Sequencing

Journal: Communications Biology

Article Title: Hypoxia-induced inhibin promotes tumor growth and vascular permeability in ovarian cancers

doi: 10.1038/s42003-022-03495-6

Figure Lengend Snippet: a (i) Western blot of HIF-1α at indicated time points after treatment with 100 μM CoCl 2 . (ii) Relative qRT-PCR analysis of INHA and VEGF mRNA in OVCAR5 and PA1 cells after indicated time of treatment with 100 μM of CoCl 2 normalized to untreated. Mean ± SEM, ( n = 2). * p < 0.05; ** p < 0.01, One-way ANOVA followed by Tukey’s multiple comparison. b Relative qRT-PCR analysis of INHA and ARNT mRNA in HEY shControl or sh ARNT cell lines after exposure to hypoxia (0.2% O 2 ) for 24 h normalized to corresponding shControl normoxia levels. Mean ± SEM, ( n = 4). n.s.,not significant; * p < 0.05; ** p < 0.01, unpaired t -test. c Representative western blot (above) and relative qRT-PCR analysis of INHA expression (below) from (i) HEY or (ii) OV90 cells transfected with either siScr, siHIF-1α, siHIF-2α, or a combination of siHIF-1/2α and exposed to hypoxia (0.2% O 2 ) for 24 h. Mean ± SEM, ( n = 3) * p < 0.05; *** p < 0.001; **** p < 0.0001, One-way ANOVA followed by Tukey’s multiple comparison test. d Relative qRT-PCR analysis using primers that amplify the proximal HRE region in the INHA promoter (Supplementary Fig. ) after chromatin immunoprecipitation (ChIP) of HIF-1α in OVCAR5 and OV90 cells. ChIP qRT-PCR results were quantified as normalized enrichment over IgG and normalized to normoxia. Mean ± SEM, OVCAR5 ( n = 3), OV90 ( n = 2). n.s.,not significant; * p < 0.05; ** p < 0.01, Two-way ANOVA followed by Fishers LSD test. e Luciferase activity of HEK293 cells transfected with the INHA promoter driven luciferase reporter construct (pGL4.10) and a SV-40 renilla control vector. Cells were either (i) exposed to hypoxia (0.2% O 2 ) or (ii) co-transfected with HIF-1α overexpression plasmid (HIF-1 ODD) and luciferase activity measured and normalized to either normoxia in (i) or PCDNA3.1 in (ii). Mean ± SEM, n = 3 (Hypoxia), n = 2 (HIF-1 ODD) * p < 0.05; ** p < 0.01, unpaired t -test.

Article Snippet: 3 × 10 6 HEY cells either exposed to normoxia or hypoxia (0.2% O 2 ) for 24 h were subcutaneously injected into right flank of 6-week old Ncr Nude mice (Taconic).

Techniques: Western Blot, Quantitative RT-PCR, Comparison, Expressing, Transfection, Chromatin Immunoprecipitation, Luciferase, Activity Assay, Construct, Control, Plasmid Preparation, Over Expression

Journal: Communications Biology

Article Title: Hypoxia-induced inhibin promotes tumor growth and vascular permeability in ovarian cancers

doi: 10.1038/s42003-022-03495-6

Figure Lengend Snippet: a (i) Hemoglobin content in Matrigel plugs collected 12 days after subcutaneous injection of HEY conditioned media collected from cells exposed to normoxia or hypoxia for 24 h and mixed with either 2 μg of IgG or anti-inhibin R1 antibody. Mean ± SEM, n = 6 plugs per condition. n.s., not significant; *** p < 0.001, One-way ANOVA followed by Tukey’s multiple comparison test. (ii) Representative images of Matrigel plugs from (i) Scale bar: 2 mm. b Quantitation of HMEC-1 migration through fibronectin coated 8 μm trans-well filter (i–ii) towards conditioned media from OV90 or HEY cells exposed to hypoxia (0.2% O 2 ) with either 2 μg of R1 or PO23 anti-inhibin antibody or IgG as a control, or towards (iii) serum free media containing 1 nM inhibin A or 1 nM VEGFA . Nuclei from three representative fields per filter were counted. Mean ± SD. ** p < 0.01; *** p < 0.001; **** p < 0.0001, One-way ANOVA followed by Tukey’s multiple comparison. c , d Quantitation of endothelial cell permeability by measuring FITC-dextran changes across a HMEC-1 monolayer treated with (i–ii) conditioned media from (i) OV90 or (ii) HEY cells exposed to hypoxia (0.2% O 2 ) with either 2 μg of R1 or PO23 anti-inhibin antibody or IgG as a control, or ( d ) treated with 1 nM inhibin A or 10 μg/mL LPS. Mean ± SEM * p < 0.05; *** p < 0.001; **** p < 0.0001, One-way ANOVA followed by Tukey’s multiple comparison. e HEY trans-endothelial migration (TEM) across HMEC-1 monolayer either treated with inhibin A for 4 h or untreated. (i) Representative transmigrated GFP positive HEY cells and (ii) quantitation of transmigration ( n = 3). * p < 0.05, unpaired t -test. Scale bar: 100 μm.

Article Snippet: 3 × 10 6 HEY cells either exposed to normoxia or hypoxia (0.2% O 2 ) for 24 h were subcutaneously injected into right flank of 6-week old Ncr Nude mice (Taconic).

Techniques: Injection, Comparison, Quantitation Assay, Migration, Control, Permeability, Transmigration Assay

Journal: Communications Biology

Article Title: Hypoxia-induced inhibin promotes tumor growth and vascular permeability in ovarian cancers

doi: 10.1038/s42003-022-03495-6

Figure Lengend Snippet: a Growth curves of subcutaneously implanted HEY shControl or sh INHA tumors exposed to either normoxia or hypoxia (0.2% O 2 ) 24 h prior to injection. 10 mg/kg R1 antibody or vehicle control was intraperitoneally injected three times a week. Data shown as box plots where center line is median, box limits are upper and lower quartile, n = 10 for vehicle and n = 6 for R1 receiving groups. ** p < 0.01; **** p < 0.0001, Two-way ANOVA followed by Tukey’s multiple comparison test. b Fold change of proteins most altered in shControl and sh INHA tumors ( a ) using the (i) human or (ii) mouse angiogenesis proteome array. ( n = 2 tumors per group). c (i) Average tumor volume of HEY shControl or sh INHA subcutaneous tumors used for analysis of vasculature and permeability in ii and iii. Mean ± SEM, n = 4. (ii) Quantitation of extravasated rhodamine-dextran (red) shown as signal per 10x field from tumors in Fig. 7ci (Methods). Mean ± SD. n = 12 fields from 4 tumors. *** p < 0.001, unpaired t -test. (iii) Representative images of rhodamine-dextran (red) extravasation into either shControl or sh INHA subcutaneous tumors from c.i Scale bar:100 μm. d (i–ii) Quantitation of average (i) vessel number and (ii) size in a 10x field using ImageJ (Methods). Mean ± SD. n = 8 which represents averages of 8 fields in four tumors from c.i. (iii) Representative images of CD-31 (red) staining in HEY shControl and sh INHA subcutaneous tumors from Fig. 7ci. Scale bar:100 μm, insets scale bar: 20 μm. * p < 0.05; ** p < 0.01, unpaired t -test.

Article Snippet: 3 × 10 6 HEY cells either exposed to normoxia or hypoxia (0.2% O 2 ) for 24 h were subcutaneously injected into right flank of 6-week old Ncr Nude mice (Taconic).

Techniques: Injection, Control, Comparison, Permeability, Quantitation Assay, Staining