Journal: Oncotarget

Article Title: The truncated somatostatin receptor sst5TMD4 stimulates the angiogenic process and is associated to lymphatic metastasis and disease-free survival in breast cancer patients

doi: 10.18632/oncotarget.11076

Figure Lengend Snippet: A. Software-driven functional analysis of genes whose expression is altered by the presence of sst5TMD4 in MCF-7 cells by gene expression microarray (green = inhibition, red = overexpression). B. User-driven supervised functional analysis of genes whose expression is altered by the presence of sst5TMD4 in MCF-7 cells by gene expression microarray (green = inhibition, red = overexpression). C. Examples of sst5TMD4-induced gene expression changes validated by additional qPCR in transfected cell lines. D. Changes in the expression of angiogenesis-related genes (VEGF, EGF, Ang1, Ang2, HIF1a and HIF1b) measured by qPCR in MCF-7 cells stably transfected with sst5TMD4 or pCDNA3.1 empty vector (mock). E. Levels of secreted VEGF in MCF-7 cells stably transfected with sst5TMD4 and mock controls measured by ELISA. F. Percentage and representative images of mammospheres formed from MCF-7 cells stably transfected with sst5TMD4 and the respective mock controls. Data represent mean ± SEM of n=3-6 independent experiments. Asterisks (*, p<0.05; **, p<0.01; ***, p<0.001) indicate significant differences between sst5TMD4- and mock-transfected MCF-7 cells.

Article Snippet: Membranes were blocked with 5% non-fat dry milk in Tris-buffered saline/0.05% Tween 20 and incubated with the rabbit polyclonal antisera against human sst5TMD4 previously described [ , ] or the specific goat anti-human VEGF antibody (AB-293-NA; R&D systems, Minneapolis, MN, USA) and the appropriate secondary antibodies (anti-rabbit or anti-goat IgG-HRP; Santa Cruz Biotechnology, Dallas, TX, USA).

Techniques: Software, Functional Assay, Expressing, Gene Expression, Microarray, Inhibition, Over Expression, Transfection, Stable Transfection, Plasmid Preparation, Enzyme-linked Immunosorbent Assay

Journal: Oncotarget

Article Title: The truncated somatostatin receptor sst5TMD4 stimulates the angiogenic process and is associated to lymphatic metastasis and disease-free survival in breast cancer patients

doi: 10.18632/oncotarget.11076

Figure Lengend Snippet: A. sst5TMD4 protein expression by western blotting in mock- and sst5TMD4-MCF-7 xenografted tumors. B. Expression levels of EGF and VEGF in xenografts derived from mock- and sst5TMD4-MCF-7 cells measured by qPCR. C. and D. VEGF protein expression by western blotting and IHF in mock- and sst5TMD4-MCF-7 xenografted tumors. E. Representative images (x20) and quantification of straight blood vessels in xenografts derived from mock- and sst5TMD4-MCF-7 cells. Data represent mean ± SEM of n=4-5 samples. Asterisks (*, p<0.05) indicate significant differences between sst5TMD4- and mock-transfected tumors.

Article Snippet: Membranes were blocked with 5% non-fat dry milk in Tris-buffered saline/0.05% Tween 20 and incubated with the rabbit polyclonal antisera against human sst5TMD4 previously described [ , ] or the specific goat anti-human VEGF antibody (AB-293-NA; R&D systems, Minneapolis, MN, USA) and the appropriate secondary antibodies (anti-rabbit or anti-goat IgG-HRP; Santa Cruz Biotechnology, Dallas, TX, USA).

Techniques: Expressing, Western Blot, Derivative Assay, Transfection

Journal: Oncotarget

Article Title: The truncated somatostatin receptor sst5TMD4 stimulates the angiogenic process and is associated to lymphatic metastasis and disease-free survival in breast cancer patients

doi: 10.18632/oncotarget.11076

Figure Lengend Snippet: A. Expression levels of VEGF, ANG1 and CD34 according to sst5TMD4 expression in the battery of 117 grade 3 infiltrating ductal breast carcinoma samples. Data represent mean ± SEM. B. Correlation between sst5TMD4 expression and the expression of VEGF, ANG1 and CD34 in breast carcinoma samples. C. Association between the presence of sst5TMD4 and lymphatic and distant metastasis in breast carcinoma samples. Graphs, obtained from a frequency table, show the distribution of 117 grade 3 ductal breast carcinoma with low or high sst5TMD4 expression according to lymphatic and distant metastasis. D. Kaplan-Meier plots showing the association between increased sst5TMD4 and disease-free survival (DFS) in breast carcinoma series. Significant correlation was studied using a Chi-square and Long-rank-p-value methods. Asterisks (*, p<0.05; **, p<0.01; ***, p<0.001) indicate significant differences between samples with low and high sst5TMD4 expression.

Article Snippet: Membranes were blocked with 5% non-fat dry milk in Tris-buffered saline/0.05% Tween 20 and incubated with the rabbit polyclonal antisera against human sst5TMD4 previously described [ , ] or the specific goat anti-human VEGF antibody (AB-293-NA; R&D systems, Minneapolis, MN, USA) and the appropriate secondary antibodies (anti-rabbit or anti-goat IgG-HRP; Santa Cruz Biotechnology, Dallas, TX, USA).

Techniques: Expressing, Battery

Journal: Odontology

Article Title: Targeting hepatocyte growth factor in epithelial–stromal interactions in an in vitro experimental model of human periodontitis

doi: 10.1007/s10266-021-00625-0

Figure Lengend Snippet: Effects of recombinant HGF or HGF neutralizing antibody on collagen gel degradation. a Effect of HGF (25 or 50 ng/mL) on collagen gel degradation. Representative gels are shown. Scale bar: 1 mm. b Effect of HGF neutralizing antibody on collagen gel degradation. Three-dimensional co-culture gels were treated with control IgG (normal goat 20 µg/mL) or different concentrations of HGF neutralizing antibody (2, 10 and 20 µg/mL). Representative gels are shown. c Three-dimensional co-culture gels containing gingival epithelial cells and PAFs were assessed with regard to collagen gel degradation. The residual collagen gel content was quantified ( n = 6). HGF neutralizing antibody was used at the concentration of 10 µg/mL

Article Snippet: Collagen gels were treated with the indicated concentrations of recombinant HGF (#100-39, Peprotech, NJ, USA) or HGF neutralizing antibody (AB-294-NA, R&D Systems, Minneapolis, MN, USA).

Techniques: Recombinant, Co-Culture Assay, Control, Concentration Assay

Journal: Odontology

Article Title: Targeting hepatocyte growth factor in epithelial–stromal interactions in an in vitro experimental model of human periodontitis

doi: 10.1007/s10266-021-00625-0

Figure Lengend Snippet: Impact of HGF neutralizing antibody on vacuolization in collagen gels. Three-dimensional co-culture gels containing PAFs derived from periodontitis patient #5 and #7 were treated with control IgG or HGF neutralizing antibody (10 µg/mL). Collagen gels were paraffin-embedded and sections were stained with hematoxylin–eosin. Treatment with HGF neutralizing antibody resulted in a reduction of vacuole numbers. Arrows point to vacuoles surrounding PAFs observed in the control gel. The numbers of vacuoles decreased under the HGF neutralizing antibody treatment (10 µg/mL). Scale bar: 50 μm

Article Snippet: Collagen gels were treated with the indicated concentrations of recombinant HGF (#100-39, Peprotech, NJ, USA) or HGF neutralizing antibody (AB-294-NA, R&D Systems, Minneapolis, MN, USA).

Techniques: Co-Culture Assay, Derivative Assay, Control, Staining

Journal: Odontology

Article Title: Targeting hepatocyte growth factor in epithelial–stromal interactions in an in vitro experimental model of human periodontitis

doi: 10.1007/s10266-021-00625-0

Figure Lengend Snippet: Gene expression profiling of collagen gels after anti-HGF treatment. RNA was extracted from collagen gel co-cultures of PAFs and gingival epithelial cells treated with HGF neutralizing antibody treatment (10 µg/mL) or control IgG. Venn diagram illustrates the a downregulated or b upregulated transcripts in three independent collagen gels containing PAFs derived from three individuals (periodontitis patient #6, #8, and #9). Numbers of transcripts are indicated. Annotated genes that were concordant in three independent experiments are highlighted

Article Snippet: Collagen gels were treated with the indicated concentrations of recombinant HGF (#100-39, Peprotech, NJ, USA) or HGF neutralizing antibody (AB-294-NA, R&D Systems, Minneapolis, MN, USA).

Techniques: Gene Expression, Control, Derivative Assay

Journal: Odontology

Article Title: Targeting hepatocyte growth factor in epithelial–stromal interactions in an in vitro experimental model of human periodontitis

doi: 10.1007/s10266-021-00625-0

Figure Lengend Snippet: Gene expression changes after anti-HGF treatment for selected genes by quantitative real-time PCR. Gene expression changes in four different co-cultures models treated with HGF neutralizing antibody (10 µg/mL) or control IgG. Collagen gels-containing PAFs derived from four individuals (periodontitis patient #1, #5, #8 and #9) were evaluated. The mRNA levels of BOC (BOC Cell Adhesion Associated, Oncogene Regulated), LAMA3 (Laminin Subunit Alpha 3) and WFDC5 (WAP Four-Disulfide Core Domain 5) were normalized to the expression of the house-keeping gene GAPDH. PAFs #8 and #9 were also used for microarray analysis

Article Snippet: Collagen gels were treated with the indicated concentrations of recombinant HGF (#100-39, Peprotech, NJ, USA) or HGF neutralizing antibody (AB-294-NA, R&D Systems, Minneapolis, MN, USA).

Techniques: Gene Expression, Real-time Polymerase Chain Reaction, Control, Derivative Assay, Expressing, Microarray

Journal: Angiogenesis

Article Title: ADAMTS3 activity is mandatory for embryonic lymphangiogenesis and regulates placental angiogenesis

doi: 10.1007/s10456-015-9488-z

Figure Lengend Snippet: Normal angiogenesis but absence of lymphatics in Adamts3 − / − embryos. Superficial sagittal sections in the skin of wild type ( Adamts3 + / + ) ( a , b ) and Adamts3 − / − ( d ) E14.5 embryos were stained using antibodies specific to prox-1 ( a ) or Lyve-1 ( b , d ). While many lymphatics were identified in Adamts3 + / + skin, they were never observed in Adamts3 − / − . V vein, L lymphatic, M macrophage. Full-thickness whole mounts of Adamts3 + / + ( c ) and Adamts3 − / − ( e ) dorsal skin were immunostained to identify blood vessels (CD31, in red ) and lymphatics (VEGF-R3, in green ). Lympatics were observed only in Adamts3 + / + skin (see also Fig S3 for individual pictures and additional data). Sections at the border between the thoracic cavity and the rib cage ( f – j ) were also stained with H&E ( f , g , i , j ) or using an anti-prox-1 antibody ( h ). Enlarged views of the boxed regions in f , i are shown in g , j , respectively. Prox-1 staining ( h ) of the section following the section in f is also provided. Nerve ( N ), artery ( A ) and vein ( V ) are seen in Adamts3 + / + and Adamts3 − / − . Lymphatics ( L ) are often seen in Adamts3 + / + but never in Adamts3 − / −

Article Snippet: Blood and lymphatic vessels were identified using, respectively, anti-CD31 (Dia310, Dianova, Germany) or antibodies against LYVE1 (07-538, Upstate, USA), Prox1 (AF2777, R&D Systems) and VEGF-R3 (AF743, R&D Systems).

Techniques: Staining

Journal: Angiogenesis

Article Title: ADAMTS3 activity is mandatory for embryonic lymphangiogenesis and regulates placental angiogenesis

doi: 10.1007/s10456-015-9488-z

Figure Lengend Snippet: Activation of the intermediate form into the fully active form of VEGF-C by ADAMTS3. HEK293 cells were transfected with an empty vector (Ctr) or with an expression vector containing the complete coding sequence of VEGF-C ( lanes 2–5 ). In some conditions, cells were simultaneously transfected with expression vectors for CCBE1 ( 3 , 5 ) and/or ADAMTS3 ( 4 , 5 ). Culture media were collected after 48 h and analyzed by WB. Conversion of the intermediate processed form of VEGF-C (31 kDa) into its mature form (21 kDa) is observed in the presence of CCBE1 alone and is almost complete in the presence of ADAMTS3

Article Snippet: Blood and lymphatic vessels were identified using, respectively, anti-CD31 (Dia310, Dianova, Germany) or antibodies against LYVE1 (07-538, Upstate, USA), Prox1 (AF2777, R&D Systems) and VEGF-R3 (AF743, R&D Systems).

Techniques: Activation Assay, Transfection, Plasmid Preparation, Expressing, Sequencing

Journal: Angiogenesis

Article Title: ADAMTS3 activity is mandatory for embryonic lymphangiogenesis and regulates placental angiogenesis

doi: 10.1007/s10456-015-9488-z

Figure Lengend Snippet: Main functional categories of genes identified by microarray analysis as characterized by a progressive increased or decreased expression correlating with the severity of liver alterations

Article Snippet: Blood and lymphatic vessels were identified using, respectively, anti-CD31 (Dia310, Dianova, Germany) or antibodies against LYVE1 (07-538, Upstate, USA), Prox1 (AF2777, R&D Systems) and VEGF-R3 (AF743, R&D Systems).

Techniques: Functional Assay, Microarray, Expressing, Protein-Protein interactions

Journal: Angiogenesis

Article Title: ADAMTS3 activity is mandatory for embryonic lymphangiogenesis and regulates placental angiogenesis

doi: 10.1007/s10456-015-9488-z

Figure Lengend Snippet: Immunohistologic analysis of placental blood vessels. Transverse sections were performed in E14.5 placenta ( Adamts3 + / + in a , d , g , j ; Adamts3 − / − in b , c , e , f , h , i , k ). All the pictures were taken in the labyrinthine layer where the blood vessels containing embryonic blood are present. After H&E staining ( a – c ), the structure of the Adamts3 − / − placenta seemed grossly normal (compare b to a ), except for the accumulation of nucleated erythroblasts in some vessels ( c ). CD31 staining ( d – f ) showed a normal pattern of blood vessel distribution in Adamts3 − / − placenta but suggested some reduction in vessel diameter ( d – f ). Transverse sections were also performed in the middle of other placenta ( Adamts3 +/+ in g ; two different Adamts3 −/− placenta in h and i ; see also Fig. S10 for additional data). At low magnification, the labyrinthine layer ( L delineated by yellow lines ) is clearly identified by its high blood vessel density. Its surface is significantly reduced in Adamts3 −/− placenta. Immunofluorescence staining ( j , k ) showed that blood vessels are formed by cells with different levels of expression of CD31 ( green ) and VEGF-R3 ( red ). No difference was found between Adamts3 + / + and Adamts3 − / − samples. Bars 50 µm in a – f and 300 µm in g – i . CP chorionic plate, S spongiotrophoblast layer

Article Snippet: Blood and lymphatic vessels were identified using, respectively, anti-CD31 (Dia310, Dianova, Germany) or antibodies against LYVE1 (07-538, Upstate, USA), Prox1 (AF2777, R&D Systems) and VEGF-R3 (AF743, R&D Systems).

Techniques: Staining, Immunofluorescence, Expressing

Journal: BMC Cancer

Article Title: Identification of proteins involved in neural progenitor cell targeting of gliomas

doi: 10.1186/1471-2407-9-206

Figure Lengend Snippet: Expression profiles of selected genes and proteins from microarray analysis, RT-PCR, immunocytochemistry and western blot analysis.

Article Snippet: Sections were blocked with 5% goat serum (Jackson ImmunoResearch Laboratories Inc. West Grove, P.A, USA) for 20 minutes and incubated with the primary antibody mouse anti-human follistatin (R&D Systems, Minneapolis, MN, USA) for 60 minutes in room temperature (RT).

Techniques: Expressing, Microarray, Immunocytochemistry, Western Blot

Journal: BMC Cancer

Article Title: Identification of proteins involved in neural progenitor cell targeting of gliomas

doi: 10.1186/1471-2407-9-206

Figure Lengend Snippet: Validation of protein expression . Immunocytochemical analysis of protein expression of (a-f) Follistatin, (g-l) CD81, (m-r) AXL, Scale bars indicate 50 μm. (s) total protein expression analysis of the proteins Gas6 and CXCL1 comparing gliomas (N25, N29, N32) and NPC (HiB5, ST14A, RN33B). As well as RT-PCR analysis of activin expression in the tumors.

Article Snippet: Sections were blocked with 5% goat serum (Jackson ImmunoResearch Laboratories Inc. West Grove, P.A, USA) for 20 minutes and incubated with the primary antibody mouse anti-human follistatin (R&D Systems, Minneapolis, MN, USA) for 60 minutes in room temperature (RT).

Techniques: Biomarker Discovery, Expressing, Reverse Transcription Polymerase Chain Reaction

Journal: BMC Cancer

Article Title: Identification of proteins involved in neural progenitor cell targeting of gliomas

doi: 10.1186/1471-2407-9-206

Figure Lengend Snippet: Blocking antibody against the follistatin protein results in an increased tumor proliferation . Co-cultures of tumor N29 (A) and N32 (B) together with irradiated NPC (HiB5/ST14A), were treated by adding an antibody targeting the follistatin protein. Cells were seeded together and allowed to attach before addition of the antibodies. The plates were pulsed with 3 H-thymidine after 3 days in culture and cell division was measured as the amount of incorporated 3 H-thymidine. White bars show the proliferation of NPC + tumor control cultures and black bars show the proliferation after the addition of follistatin antibody. Mean ± stdev: Statistical analysis was made using t-test and Mann Whitney Wilcoxon rank sum test * P < 0.05.

Article Snippet: Sections were blocked with 5% goat serum (Jackson ImmunoResearch Laboratories Inc. West Grove, P.A, USA) for 20 minutes and incubated with the primary antibody mouse anti-human follistatin (R&D Systems, Minneapolis, MN, USA) for 60 minutes in room temperature (RT).

Techniques: Blocking Assay, Irradiation, Control, MANN-WHITNEY

Journal: BMC Cancer

Article Title: Identification of proteins involved in neural progenitor cell targeting of gliomas

doi: 10.1186/1471-2407-9-206

Figure Lengend Snippet: A subpopulation of grade IV human GBM display follistatin expression . Figure 6. Follistatin expression by subsets of tumor cells within the glioblastoma multiforme (GBM) tumor cell bulk. Samples from GBM patients were freeze sectioned and subsequently stained using a follistatin antibody against the human antigen. A) Central tumor sections of patient A. A few selectively follistatin positive cells are seen within the central portions of the tumor. B) Central section from patient B. Follistatin expression is seen in a subset of tumor cells within the central bulk of the tumor. The histological examination of patient A and B tumors displays a heterogeneity within the tumor bulk with both positive and negative tumor cells. As negative control, the primary antibody was omitted, C. Scale bars indicate 50 μm.

Article Snippet: Sections were blocked with 5% goat serum (Jackson ImmunoResearch Laboratories Inc. West Grove, P.A, USA) for 20 minutes and incubated with the primary antibody mouse anti-human follistatin (R&D Systems, Minneapolis, MN, USA) for 60 minutes in room temperature (RT).

Techniques: Expressing, Staining, Negative Control