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Effect of EELC on <t>VEGF-A</t> and VEGFR-2 protein expression. HUVECs were treated with EELC for 24 h. The protein levels of VEGF-A in the supernatant of each group and VEGFR-2 from lysed HUVECs were determined by ELISA. Data are the means ± standard deviation of triplicate experiments. *P
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1) Product Images from "Anti-angiogenic effect of Livistona chinensis seed extract in vitro and in vivo"

Article Title: Anti-angiogenic effect of Livistona chinensis seed extract in vitro and in vivo

Journal: Oncology Letters

doi: 10.3892/ol.2017.7075

Effect of EELC on VEGF-A and VEGFR-2 protein expression. HUVECs were treated with EELC for 24 h. The protein levels of VEGF-A in the supernatant of each group and VEGFR-2 from lysed HUVECs were determined by ELISA. Data are the means ± standard deviation of triplicate experiments. *P
Figure Legend Snippet: Effect of EELC on VEGF-A and VEGFR-2 protein expression. HUVECs were treated with EELC for 24 h. The protein levels of VEGF-A in the supernatant of each group and VEGFR-2 from lysed HUVECs were determined by ELISA. Data are the means ± standard deviation of triplicate experiments. *P

Techniques Used: Expressing, Enzyme-linked Immunosorbent Assay, Standard Deviation

Effect of EELC on mRNA expression. The mRNA levels of VEGF-A and VEGFR-2 were detected by reverse transcription-semi-quantitative polymerase chain reaction with an internal control of GAPDH. The treated group data were compared with an untreated control. Data are means ± standard deviation of triplicate experiments. *P
Figure Legend Snippet: Effect of EELC on mRNA expression. The mRNA levels of VEGF-A and VEGFR-2 were detected by reverse transcription-semi-quantitative polymerase chain reaction with an internal control of GAPDH. The treated group data were compared with an untreated control. Data are means ± standard deviation of triplicate experiments. *P

Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Standard Deviation

2) Product Images from "Disrupting VEGF-A paracrine and autocrine loops by targeting SHP-1 suppresses triple negative breast cancer metastasis"

Article Title: Disrupting VEGF-A paracrine and autocrine loops by targeting SHP-1 suppresses triple negative breast cancer metastasis

Journal: Scientific Reports

doi: 10.1038/srep28888

Regorafenib transcriptionally inhibited VEGF-A expression through decreasing the binding of STAT3 on the promoter of VEGF-A. ( A ) Top , MDA-MB-231 cells were co-transfected with a Renilla control vector and plasmids containing the firefly luciferase gene driven by wild-type or STAT3 binding site-mutated VEGF-A promoter. After transfection for 48 h, cells were treated with regorafenib for 24 h. Promoter activity was analyzed by luciferase assay after regorafenib treatment. Bottom , MDA-MB-231 cells were transfected with vector-control or STAT3-overexpression plasmid for 48 h. After that, cells were further co-transfected with Renilla and wild-type VEGF-A promoter and detect promoter activity as mentioned above. *p
Figure Legend Snippet: Regorafenib transcriptionally inhibited VEGF-A expression through decreasing the binding of STAT3 on the promoter of VEGF-A. ( A ) Top , MDA-MB-231 cells were co-transfected with a Renilla control vector and plasmids containing the firefly luciferase gene driven by wild-type or STAT3 binding site-mutated VEGF-A promoter. After transfection for 48 h, cells were treated with regorafenib for 24 h. Promoter activity was analyzed by luciferase assay after regorafenib treatment. Bottom , MDA-MB-231 cells were transfected with vector-control or STAT3-overexpression plasmid for 48 h. After that, cells were further co-transfected with Renilla and wild-type VEGF-A promoter and detect promoter activity as mentioned above. *p

Techniques Used: Expressing, Binding Assay, Multiple Displacement Amplification, Transfection, Plasmid Preparation, Luciferase, Activity Assay, Over Expression

Regorafenib reduced TNBC cell migration and targeted p-STAT3/VEGF-A signaling. ( A ) Chemical structure of regorafenib. ( B,C ), Transwell assay ( B ) and wound-healing assay ( C ) were performed in TNBC cell lines after regorafenib treatment for 24 h. *p
Figure Legend Snippet: Regorafenib reduced TNBC cell migration and targeted p-STAT3/VEGF-A signaling. ( A ) Chemical structure of regorafenib. ( B,C ), Transwell assay ( B ) and wound-healing assay ( C ) were performed in TNBC cell lines after regorafenib treatment for 24 h. *p

Techniques Used: Migration, Transwell Assay, Wound Healing Assay

Both regorafenib and SC-78 targeted autocrine and paracrine regulation of VEGF-A in TNBCs. ( A ) Cells were treated with SC-78 or regorafenib for 24 h. After that, the medium was replaced with fresh medium and incubated for another 24 h. Then, the medium was collected and ELISA was used to measure the VEGF-A level in the CM harvested from TNBC cells w/wo treatment of SC-78 or regorafenib. *p
Figure Legend Snippet: Both regorafenib and SC-78 targeted autocrine and paracrine regulation of VEGF-A in TNBCs. ( A ) Cells were treated with SC-78 or regorafenib for 24 h. After that, the medium was replaced with fresh medium and incubated for another 24 h. Then, the medium was collected and ELISA was used to measure the VEGF-A level in the CM harvested from TNBC cells w/wo treatment of SC-78 or regorafenib. *p

Techniques Used: Incubation, Enzyme-linked Immunosorbent Assay

SHP-1 is negatively associated with p-STAT3/VEGF-A signaling and metastasis in TNBC cells and clinical samples. ( A ) Protein expression pattern of SHP-1, p-STAT3, and VEGF-A in nine TNBC cell lines were analyzed by western blot. ( B ) The migration abilities of nine TNBC cell lines were analyzed by Transwell assay. DAPI stains the nuclei. BT-20 cells were used as a normalization control. ( C ) The correlation (linear regression model) of VEGF-A ( top ), p-STAT3 ( middle ), and SHP-1 ( bottom ) and migration ability in nine TNBC cell lines. ( D ) TNBC cells from representative two patients with SHP-1, p-STAT3, and VEGF-A staining. (200×) ( E ) Kaplan-Meier graph was prepared to compare DMFS ( left ) and DFS ( right ) in patients with high VEGF-A (H score > 160) or low VEGF-A (H score
Figure Legend Snippet: SHP-1 is negatively associated with p-STAT3/VEGF-A signaling and metastasis in TNBC cells and clinical samples. ( A ) Protein expression pattern of SHP-1, p-STAT3, and VEGF-A in nine TNBC cell lines were analyzed by western blot. ( B ) The migration abilities of nine TNBC cell lines were analyzed by Transwell assay. DAPI stains the nuclei. BT-20 cells were used as a normalization control. ( C ) The correlation (linear regression model) of VEGF-A ( top ), p-STAT3 ( middle ), and SHP-1 ( bottom ) and migration ability in nine TNBC cell lines. ( D ) TNBC cells from representative two patients with SHP-1, p-STAT3, and VEGF-A staining. (200×) ( E ) Kaplan-Meier graph was prepared to compare DMFS ( left ) and DFS ( right ) in patients with high VEGF-A (H score > 160) or low VEGF-A (H score

Techniques Used: Expressing, Western Blot, Migration, Transwell Assay, Staining

3) Product Images from "Enhanced Hypoxia-Inducible Factor (HIF)-1α Stability Induced by 5-Hydroxymethyl-2-Furfural (5-HMF) Contributes to Protection against Hypoxia"

Article Title: Enhanced Hypoxia-Inducible Factor (HIF)-1α Stability Induced by 5-Hydroxymethyl-2-Furfural (5-HMF) Contributes to Protection against Hypoxia

Journal: Molecular Medicine

doi: 10.2119/molmed.2014.00007

The enhanced VEGF mRNA expression by 5-HMF in ODD-Luc mice. (A, B) Effects of 5-HMF on the VEGF mRNA expression in the cortex and in the kidney. The transcriptional activity of HIF-1 was estimated by VEGF mRNA expression. The VEGF mRNA levels were analyzed
Figure Legend Snippet: The enhanced VEGF mRNA expression by 5-HMF in ODD-Luc mice. (A, B) Effects of 5-HMF on the VEGF mRNA expression in the cortex and in the kidney. The transcriptional activity of HIF-1 was estimated by VEGF mRNA expression. The VEGF mRNA levels were analyzed

Techniques Used: Expressing, Mouse Assay, Activity Assay

4) Product Images from "Inhibitory effect of arsenic trioxide on angiogenesis and expression of vascular endothelial growth factor in gastric cancer"

Article Title: Inhibitory effect of arsenic trioxide on angiogenesis and expression of vascular endothelial growth factor in gastric cancer

Journal: World Journal of Gastroenterology : WJG

doi: 10.3748/wjg.v12.i36.5780

Amplification Curve of VEGF mRNA in SGC-7901.
Figure Legend Snippet: Amplification Curve of VEGF mRNA in SGC-7901.

Techniques Used: Amplification

5) Product Images from "Macrophage Migration Inhibitory Factor Is Involved in Ectopic Endometrial Tissue Growth and Peritoneal-Endometrial Tissue Interaction In Vivo: A Plausible Link to Endometriosis Development"

Article Title: Macrophage Migration Inhibitory Factor Is Involved in Ectopic Endometrial Tissue Growth and Peritoneal-Endometrial Tissue Interaction In Vivo: A Plausible Link to Endometriosis Development

Journal: PLoS ONE

doi: 10.1371/journal.pone.0110434

Real-time PCR analysis of the expression of genes mainly involved in cell adhesion and vascularisation of endometriosis-like lesions. Histogram representation of the effect of MIF genetic depletion or antagonism versus controls on VEGF (A, B), COX2 (C, D), BCL2 (E, F), BAX (G, H), ITGAV (I, J) and ITGB3 (K, L) mRNA expression in endometriosis-like lesions by quantitative real time PCR. For each factor, the ratio of mRNA level to GAPDH mRNA was determined. Results were from WT and KOmice (n = 6) with no treatment (controls) and from WT treated with ISO-1 (n = 5). Data are mean ± SEM; *, p
Figure Legend Snippet: Real-time PCR analysis of the expression of genes mainly involved in cell adhesion and vascularisation of endometriosis-like lesions. Histogram representation of the effect of MIF genetic depletion or antagonism versus controls on VEGF (A, B), COX2 (C, D), BCL2 (E, F), BAX (G, H), ITGAV (I, J) and ITGB3 (K, L) mRNA expression in endometriosis-like lesions by quantitative real time PCR. For each factor, the ratio of mRNA level to GAPDH mRNA was determined. Results were from WT and KOmice (n = 6) with no treatment (controls) and from WT treated with ISO-1 (n = 5). Data are mean ± SEM; *, p

Techniques Used: Real-time Polymerase Chain Reaction, Expressing

6) Product Images from "A potential small-molecule synthetic antilymphangiogenic agent norcantharidin inhibits tumor growth and lymphangiogenesis of human colonic adenocarcinomas through blocking VEGF-A,-C,-D/VEGFR-2,-3 “multi-points priming” mechanisms in vitro and in vivo"

Article Title: A potential small-molecule synthetic antilymphangiogenic agent norcantharidin inhibits tumor growth and lymphangiogenesis of human colonic adenocarcinomas through blocking VEGF-A,-C,-D/VEGFR-2,-3 “multi-points priming” mechanisms in vitro and in vivo

Journal: BMC Cancer

doi: 10.1186/s12885-015-1521-5

NCTD inhibits the expression of VEGF-A, VEGF-C, VEGF-D, VEGFR-2 and VEGFR-3 proteins/mRNAs of the in-situ colonic xenografts in vivo. a Western-blotting: the expression of VEGF-A, VEGF-C, VEGF-D, VEGFR-2 and VEGFR-3 proteins in NCTD, Sorafenib, or NCTD + Sorafenib group was significantly downregulated as compared to control group (* P
Figure Legend Snippet: NCTD inhibits the expression of VEGF-A, VEGF-C, VEGF-D, VEGFR-2 and VEGFR-3 proteins/mRNAs of the in-situ colonic xenografts in vivo. a Western-blotting: the expression of VEGF-A, VEGF-C, VEGF-D, VEGFR-2 and VEGFR-3 proteins in NCTD, Sorafenib, or NCTD + Sorafenib group was significantly downregulated as compared to control group (* P

Techniques Used: Expressing, In Situ, In Vivo, Western Blot

The expression of VEGF-A, VEGF-C, VEGF-D, VEGFR-2 and VEGFR-3 proteins/mRNAs of HCACCs and the co-culture system and the effect of NCTD on expression of these proteins/ mRNAs by western blotting ( a and b ) and fluorescent quantitative RT-PCR ( c and d ) in vitro . a and b Protein expression by western blotting: the expression of VEGF-C, VEGF-D and VEGFR-3 proteins of the co-culture system was higher than those of alone HCACC culture (* P = 0.001), but there was no difference on VEGF-A and VEGFR-2 expression between alone HCACC culture and the co-culture system. After treatment with NCTD, mF4-31C1 or NCTD + mF4-31C1, the expression of VEGF-C, VEGF-D and VEGFR-3 proteins of the co-culture system was downregulated significantly as compared to control group (all * P
Figure Legend Snippet: The expression of VEGF-A, VEGF-C, VEGF-D, VEGFR-2 and VEGFR-3 proteins/mRNAs of HCACCs and the co-culture system and the effect of NCTD on expression of these proteins/ mRNAs by western blotting ( a and b ) and fluorescent quantitative RT-PCR ( c and d ) in vitro . a and b Protein expression by western blotting: the expression of VEGF-C, VEGF-D and VEGFR-3 proteins of the co-culture system was higher than those of alone HCACC culture (* P = 0.001), but there was no difference on VEGF-A and VEGFR-2 expression between alone HCACC culture and the co-culture system. After treatment with NCTD, mF4-31C1 or NCTD + mF4-31C1, the expression of VEGF-C, VEGF-D and VEGFR-3 proteins of the co-culture system was downregulated significantly as compared to control group (all * P

Techniques Used: Expressing, Co-Culture Assay, Western Blot, Quantitative RT-PCR, In Vitro

The expression of VEGF-A, VEGF-C, VEGF-D, VEGFR-2 and VEGFR-3 protein products of HCACCs and the co-culture system of each group and the effect of NCTD on expression of these protein products in vitro (S-P staining, magnification × 200). a The expression of VEGF-A, VEGF-C, VEGF-D, VEGFR-2 and VEGFR-3 protein products of HCACCs and the co-culture system of each group. The expression of VEGF-C, VEGF-D and VEGFR-3 protein products (brown staining in cytoplasm) of the co-culture system was higher than those of alone HCACC culture (* P = 0.001); but there is no difference on the expression of VEGF-A and VEGFR-2 protein products between alone HCACC culture and the co-culture system. b The inhibitory effect of NCTD on expression of these protein products of the co-culture system. The expression of VEGF-C, VEGF-D and VEGFR-3 protein products in NCTD, mF4-31C1 or NCTD + mF4-31C1 group was downregulated significantly as compared to control group (* P
Figure Legend Snippet: The expression of VEGF-A, VEGF-C, VEGF-D, VEGFR-2 and VEGFR-3 protein products of HCACCs and the co-culture system of each group and the effect of NCTD on expression of these protein products in vitro (S-P staining, magnification × 200). a The expression of VEGF-A, VEGF-C, VEGF-D, VEGFR-2 and VEGFR-3 protein products of HCACCs and the co-culture system of each group. The expression of VEGF-C, VEGF-D and VEGFR-3 protein products (brown staining in cytoplasm) of the co-culture system was higher than those of alone HCACC culture (* P = 0.001); but there is no difference on the expression of VEGF-A and VEGFR-2 protein products between alone HCACC culture and the co-culture system. b The inhibitory effect of NCTD on expression of these protein products of the co-culture system. The expression of VEGF-C, VEGF-D and VEGFR-3 protein products in NCTD, mF4-31C1 or NCTD + mF4-31C1 group was downregulated significantly as compared to control group (* P

Techniques Used: Expressing, Co-Culture Assay, In Vitro, Staining

7) Product Images from "Regulation of IL-8 gene expression in gliomas by microRNA miR-93"

Article Title: Regulation of IL-8 gene expression in gliomas by microRNA miR-93

Journal: BMC Cancer

doi: 10.1186/s12885-015-1659-1

Effects of the treatments of glioma U251 cells with pre-miR-93 and antagomiR-93. VEGF ( a ) and IL-8 released protein ( b , c ) were quantified by Bio-plex analysis. RNA was isolated from cultures after 48 h in vitro growth and analyzed by RT-qPCR. Internal RT-qPCR control were U6 snRNA and let-7c for miR-93, RPL13A and 18S for IL-8 mRNA. Data are in all cases reported in comparison to U251 cells treated with control scrambled sequences. Results represent the average ± S.D. of at least three independent experiments. * p
Figure Legend Snippet: Effects of the treatments of glioma U251 cells with pre-miR-93 and antagomiR-93. VEGF ( a ) and IL-8 released protein ( b , c ) were quantified by Bio-plex analysis. RNA was isolated from cultures after 48 h in vitro growth and analyzed by RT-qPCR. Internal RT-qPCR control were U6 snRNA and let-7c for miR-93, RPL13A and 18S for IL-8 mRNA. Data are in all cases reported in comparison to U251 cells treated with control scrambled sequences. Results represent the average ± S.D. of at least three independent experiments. * p

Techniques Used: Isolation, In Vitro, Quantitative RT-PCR

Effects on IL-8 mRNA ( a , c ) and IL-8 protein ( b , d ) of the treatments of glioma U251 ( a , b ) and T98G ( c , d ) cells with pre-miR-93 and antagomiR-93. RNA was isolated from cultures after 48 h in vitro growth and analyzed by RT-qPCR. Internal RT-qPCR control was RPL13A. Released IL-8 protein was quantified by Bio-plex analysis. Data are in all cases reported in comparison to U251 and T98G cells treated with control sequences. Results represent the average ± S.D. of three independent experiments. * p
Figure Legend Snippet: Effects on IL-8 mRNA ( a , c ) and IL-8 protein ( b , d ) of the treatments of glioma U251 ( a , b ) and T98G ( c , d ) cells with pre-miR-93 and antagomiR-93. RNA was isolated from cultures after 48 h in vitro growth and analyzed by RT-qPCR. Internal RT-qPCR control was RPL13A. Released IL-8 protein was quantified by Bio-plex analysis. Data are in all cases reported in comparison to U251 and T98G cells treated with control sequences. Results represent the average ± S.D. of three independent experiments. * p

Techniques Used: Isolation, In Vitro, Quantitative RT-PCR

Expression of IL-8 and VEGF mRNA in glioblastoma. VEGF mRNA ( a , c , e ) and IL-8 mRNA ( b , d , f ) by mRNA in situ hybridization are shown in separate 5 μm serial tissue sections from glioblastoma specimens at different magnifications ( a , b : x2; c , d : x10; e , f : x32) by peroxidase staining. Nuclei are counterstained with hematoxylin. Positive (GAPDH mRNA) and negative (DAPB mRNA) controls are reported ( g , h : x20 magnification). Squared areas in panels A and B indicate the detail reported in panels c and d , respectively
Figure Legend Snippet: Expression of IL-8 and VEGF mRNA in glioblastoma. VEGF mRNA ( a , c , e ) and IL-8 mRNA ( b , d , f ) by mRNA in situ hybridization are shown in separate 5 μm serial tissue sections from glioblastoma specimens at different magnifications ( a , b : x2; c , d : x10; e , f : x32) by peroxidase staining. Nuclei are counterstained with hematoxylin. Positive (GAPDH mRNA) and negative (DAPB mRNA) controls are reported ( g , h : x20 magnification). Squared areas in panels A and B indicate the detail reported in panels c and d , respectively

Techniques Used: Expressing, In Situ Hybridization, Staining

Expression of VEGF, IL-8 and miR-93 in Low-Grade Gliomas (LGGs) and High-Grade Gliomas (HGGs). VEGF mRNA ( a ) and IL-8 mRNA ( b ) levels relative to GAPDH were measured by RT-qPCR with TaqMan probes on RNAs isolated from FFPE sections of 6 LGG and 10 HGG and normalized to healthy brain reference RNA. Fold changes (FC) of expression over healthy brain reference RNA are reported. In the same LGGs and HGGs miR-93 was quantified ( c ) and normalized to healthy brain reference RNA. For panels a – c : dashed line: mean; solid line: median; grey box includes values from 5th to 95th centiles, vertical lines range from min to max values, excluding outliers which are represented by single dots. The data obtained in each glioma specimen are reported in the right side of the panels. d Relationship between VEGF mRNA and IL-8 mRNA in the same LGG (filled circles) and HGG (open circles) samples analyzed and reported in a – c . Regression straight line showing direct correlation was drawn by the least square method Sigmaplot. Inset reports the same graph expanded
Figure Legend Snippet: Expression of VEGF, IL-8 and miR-93 in Low-Grade Gliomas (LGGs) and High-Grade Gliomas (HGGs). VEGF mRNA ( a ) and IL-8 mRNA ( b ) levels relative to GAPDH were measured by RT-qPCR with TaqMan probes on RNAs isolated from FFPE sections of 6 LGG and 10 HGG and normalized to healthy brain reference RNA. Fold changes (FC) of expression over healthy brain reference RNA are reported. In the same LGGs and HGGs miR-93 was quantified ( c ) and normalized to healthy brain reference RNA. For panels a – c : dashed line: mean; solid line: median; grey box includes values from 5th to 95th centiles, vertical lines range from min to max values, excluding outliers which are represented by single dots. The data obtained in each glioma specimen are reported in the right side of the panels. d Relationship between VEGF mRNA and IL-8 mRNA in the same LGG (filled circles) and HGG (open circles) samples analyzed and reported in a – c . Regression straight line showing direct correlation was drawn by the least square method Sigmaplot. Inset reports the same graph expanded

Techniques Used: Expressing, Quantitative RT-PCR, Isolation, Formalin-fixed Paraffin-Embedded

Interactions of miR-93 with IL-8 mRNA and VEGF mRNA. Predicted secondary structure of the 3′UTR regions of VEGF mRNA ( a ) and IL-8 mRNA ( b ) based on the UCSC genome browser ( http://genome.ucsc.edu ), and of miR-93 by UNAFold Web Server ( http://mfold.rna.albany.edu ). Magnification is also shown of the central portion of 3′UTR IL-8 ( b ) and VEGF ( a ) mRNAs and pointing out the possible interaction between the 3′UTR target strands and the seed region of the lowest energy miR-93 potential stem loops
Figure Legend Snippet: Interactions of miR-93 with IL-8 mRNA and VEGF mRNA. Predicted secondary structure of the 3′UTR regions of VEGF mRNA ( a ) and IL-8 mRNA ( b ) based on the UCSC genome browser ( http://genome.ucsc.edu ), and of miR-93 by UNAFold Web Server ( http://mfold.rna.albany.edu ). Magnification is also shown of the central portion of 3′UTR IL-8 ( b ) and VEGF ( a ) mRNAs and pointing out the possible interaction between the 3′UTR target strands and the seed region of the lowest energy miR-93 potential stem loops

Techniques Used:

Correlations among the expression of miR-93, VEGF and IL-8 mRNAs in HGGs. Regression analysis between VEGF mRNA ( a ) and IL-8 mRNA ( b ) was performed as function of miR-93. Regression straight line showing inverse correlation was drawn by the least square method with Sigmaplot software. The graphical correlations among miR-93, VEGF and IL-8 mRNAs is represented in 3D plot ( c ). All data are reported as fold changes (FC) over healthy brain reference RNA
Figure Legend Snippet: Correlations among the expression of miR-93, VEGF and IL-8 mRNAs in HGGs. Regression analysis between VEGF mRNA ( a ) and IL-8 mRNA ( b ) was performed as function of miR-93. Regression straight line showing inverse correlation was drawn by the least square method with Sigmaplot software. The graphical correlations among miR-93, VEGF and IL-8 mRNAs is represented in 3D plot ( c ). All data are reported as fold changes (FC) over healthy brain reference RNA

Techniques Used: Expressing, Software

8) Product Images from "Dynamic Perfusion Culture of Human Outgrowth Endothelial Progenitor Cells on Demineralized Bone Matrix In Vitro"

Article Title: Dynamic Perfusion Culture of Human Outgrowth Endothelial Progenitor Cells on Demineralized Bone Matrix In Vitro

Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

doi: 10.12659/MSM.897884

Expression of CD34, VE-cadherin, and VEGF protein under static culture and dynamic perfusion culture. (* p
Figure Legend Snippet: Expression of CD34, VE-cadherin, and VEGF protein under static culture and dynamic perfusion culture. (* p

Techniques Used: Expressing

The influence of dynamic perfusion culture on CD34/VE-cadherin/VEGF mRNA expression (* p
Figure Legend Snippet: The influence of dynamic perfusion culture on CD34/VE-cadherin/VEGF mRNA expression (* p

Techniques Used: Expressing

9) Product Images from "The cAMP pathway regulates mRNA decay through phosphorylation of the RNA-binding protein TIS11b/BRF1"

Article Title: The cAMP pathway regulates mRNA decay through phosphorylation of the RNA-binding protein TIS11b/BRF1

Journal: Molecular Biology of the Cell

doi: 10.1091/mbc.E16-06-0379

ACTH induces a cAMP-dependent expression and phosphorylation of TIS11b. (A) BAC cells were preincubated in the absence or presence of H89 (5 μM) for 30 min before addition of 10 nM of ACTH for the indicated periods of time. TIS11b and VEGF protein levels of whole-cell extracts (20 μg) were analyzed by Western blot. The blot was subsequently probed with an anti–β-actin to assess equal loading of samples. (B–D) Quantification of TIS11b, VEGF mRNA, and protein levels from independent experiments ( n = 5, means ± SEM). Protein-level values were normalized to actin and are expressed as percentage of control values at time 0 (unstimulated cells). VEGF mRNA levels were measured by quantitative PCR and normalized to hypoxanthine-guanine phosphoribosyltransferase (HPRT). (E) Time-course of TIS11b phosphorylation in BAC cells stimulated with 10 nM of ACTH in the presence of [ 32 P]orthophosphate and in the presence or absence of H89. TIS11b was immunoprecipitated (IP) from cell extracts, resolved by SDS–PAGE, and then visualized by autoradiography. One representative experiment of four is shown. (F) Quantification of phospho-TIS11b/total TIS11b ratio in ACTH-stimulated BAC cells ( n = 4, means ± SEM). (G) Phosphorylation of recombinant TIS11b by the catalytic subunit of PKA. Purified GST-TIS11b fusion protein was produced as described previously ( Ciais et al. , 2004 ). Increasing doses of GST-TIS11b were subjected to in vitro phosphorylation as described in Materials and Methods . Protein extract from Escherichia coli (30 μg) transformed with empty pGEX vector was used as control in the phosphorylation assay (first lane, 0 μg).
Figure Legend Snippet: ACTH induces a cAMP-dependent expression and phosphorylation of TIS11b. (A) BAC cells were preincubated in the absence or presence of H89 (5 μM) for 30 min before addition of 10 nM of ACTH for the indicated periods of time. TIS11b and VEGF protein levels of whole-cell extracts (20 μg) were analyzed by Western blot. The blot was subsequently probed with an anti–β-actin to assess equal loading of samples. (B–D) Quantification of TIS11b, VEGF mRNA, and protein levels from independent experiments ( n = 5, means ± SEM). Protein-level values were normalized to actin and are expressed as percentage of control values at time 0 (unstimulated cells). VEGF mRNA levels were measured by quantitative PCR and normalized to hypoxanthine-guanine phosphoribosyltransferase (HPRT). (E) Time-course of TIS11b phosphorylation in BAC cells stimulated with 10 nM of ACTH in the presence of [ 32 P]orthophosphate and in the presence or absence of H89. TIS11b was immunoprecipitated (IP) from cell extracts, resolved by SDS–PAGE, and then visualized by autoradiography. One representative experiment of four is shown. (F) Quantification of phospho-TIS11b/total TIS11b ratio in ACTH-stimulated BAC cells ( n = 4, means ± SEM). (G) Phosphorylation of recombinant TIS11b by the catalytic subunit of PKA. Purified GST-TIS11b fusion protein was produced as described previously ( Ciais et al. , 2004 ). Increasing doses of GST-TIS11b were subjected to in vitro phosphorylation as described in Materials and Methods . Protein extract from Escherichia coli (30 μg) transformed with empty pGEX vector was used as control in the phosphorylation assay (first lane, 0 μg).

Techniques Used: Expressing, BAC Assay, Western Blot, Real-time Polymerase Chain Reaction, Immunoprecipitation, SDS Page, Autoradiography, Recombinant, Purification, Produced, In Vitro, Transformation Assay, Plasmid Preparation, Phosphorylation Assay

The phosphomimetic TIS11b-S334D mutant is more potent than WT TIS11b in decreasing VEGF 3′ UTR–driven luciferase activity and endogenous VEGF mRNA steady-state levels. (A) COS7 cells were cotransfected with pLuc 3′ UTR and pTarget plasmids encoding WT TIS11b, TIS11b-S54A, TIS11b-S54D, TIS11b-S334A, or TIS11b-S334D mutants. Firefly/ Renilla luciferase activities of cell lysates were measured as described in Materials and Methods . Results are expressed as relative light units of firefly luciferase activity over relative light units of Renilla luciferase activity and are represented as a percentage of the luciferase activity in control cells transfected with empty pTarget plasmid. Transfections were performed in triplicate, and values are means ± SEM from seven independent experiments. The lower panel is a representative Western blot analysis of overexpressed TIS11b proteins showing that equivalent amounts of overexpressed TIS11b were recovered. (B) Northern blot (NB) analysis of endogenous VEGF mRNA in COS7 cells transfected as in A. (C) Western blot (WB) analysis of TIS11b protein expression levels in the COS7 cells used for the Northern experiment shown in B. (D) Quantification of VEGF mRNA steady-state levels in four independent experiments (means ± SEM). Asterisks: significantly different from WT with * p
Figure Legend Snippet: The phosphomimetic TIS11b-S334D mutant is more potent than WT TIS11b in decreasing VEGF 3′ UTR–driven luciferase activity and endogenous VEGF mRNA steady-state levels. (A) COS7 cells were cotransfected with pLuc 3′ UTR and pTarget plasmids encoding WT TIS11b, TIS11b-S54A, TIS11b-S54D, TIS11b-S334A, or TIS11b-S334D mutants. Firefly/ Renilla luciferase activities of cell lysates were measured as described in Materials and Methods . Results are expressed as relative light units of firefly luciferase activity over relative light units of Renilla luciferase activity and are represented as a percentage of the luciferase activity in control cells transfected with empty pTarget plasmid. Transfections were performed in triplicate, and values are means ± SEM from seven independent experiments. The lower panel is a representative Western blot analysis of overexpressed TIS11b proteins showing that equivalent amounts of overexpressed TIS11b were recovered. (B) Northern blot (NB) analysis of endogenous VEGF mRNA in COS7 cells transfected as in A. (C) Western blot (WB) analysis of TIS11b protein expression levels in the COS7 cells used for the Northern experiment shown in B. (D) Quantification of VEGF mRNA steady-state levels in four independent experiments (means ± SEM). Asterisks: significantly different from WT with * p

Techniques Used: Mutagenesis, Luciferase, Activity Assay, Transfection, Plasmid Preparation, Western Blot, Northern Blot, Expressing

VEGF mRNA is more efficiently destabilized by the phosphomimetic TIS11b S334D mutant. (A) COS7 cells were transfected in 12-well plates with pTarget empty plasmid (Vector) or plasmids encoding WT or mutant TIS11b. AT 48 h after transfection, the transcription inhibitor DRB (10 μg/ml) was added, and total RNA was extracted at the indicated time points and analyzed by Northern blot. The membrane was hybridized to a radiolabeled VEGF 3′ UTR probe and rehybridized to 18S RNA probe for loading control. (B) VEGF mRNA levels were normalized to 18s RNA levels and plotted as a percentage of the initial value against time using nonlinear regression to a first-order exponential decay model. Shown are the mRNA decay rates from three pooled independent experiments. (C) VEGF mRNA half-lives were calculated and compared with the half-life of the transcript in the presence of WT TIS11b. p Values and 95% CIs were determined using an F -test.
Figure Legend Snippet: VEGF mRNA is more efficiently destabilized by the phosphomimetic TIS11b S334D mutant. (A) COS7 cells were transfected in 12-well plates with pTarget empty plasmid (Vector) or plasmids encoding WT or mutant TIS11b. AT 48 h after transfection, the transcription inhibitor DRB (10 μg/ml) was added, and total RNA was extracted at the indicated time points and analyzed by Northern blot. The membrane was hybridized to a radiolabeled VEGF 3′ UTR probe and rehybridized to 18S RNA probe for loading control. (B) VEGF mRNA levels were normalized to 18s RNA levels and plotted as a percentage of the initial value against time using nonlinear regression to a first-order exponential decay model. Shown are the mRNA decay rates from three pooled independent experiments. (C) VEGF mRNA half-lives were calculated and compared with the half-life of the transcript in the presence of WT TIS11b. p Values and 95% CIs were determined using an F -test.

Techniques Used: Mutagenesis, Transfection, Plasmid Preparation, Northern Blot

10) Product Images from "Mesenteric Adipose-derived Stromal Cells From Crohn’s Disease Patients Induce Protective Effects in Colonic Epithelial Cells and Mice With Colitis"

Article Title: Mesenteric Adipose-derived Stromal Cells From Crohn’s Disease Patients Induce Protective Effects in Colonic Epithelial Cells and Mice With Colitis

Journal: Cellular and Molecular Gastroenterology and Hepatology

doi: 10.1016/j.jcmgh.2018.02.001

Mice treated with daily injections of lactoferrin (LTF) displayed decreased severity of colitis as compared with mice treated with vehicle or conditioned media from control (Ctrl) patients (n = 8/group). ( A ) Mice with active DSS colitis (4% w/v) received i.c. injections of vehicle or human apolactoferrin daily for 7 days. Clinical scores ( B ) were decreased and colon length was increased ( C ) in mice treated with LTF at days 6 and 7. ( D ) Quantification of Ki-67 immunohistochemistry using pixel-based densitometry (n = 3/group). ( E ) Representative images from Ki-67 immunoreactive sections. Scale bar = 100 μm; arrows indicate immunoreactive intestinal epithelial cells. ( F and G ) p70s6K and Akt phosphorylation in NCM460 cells, as assessed by Western blot (n = 3). * P
Figure Legend Snippet: Mice treated with daily injections of lactoferrin (LTF) displayed decreased severity of colitis as compared with mice treated with vehicle or conditioned media from control (Ctrl) patients (n = 8/group). ( A ) Mice with active DSS colitis (4% w/v) received i.c. injections of vehicle or human apolactoferrin daily for 7 days. Clinical scores ( B ) were decreased and colon length was increased ( C ) in mice treated with LTF at days 6 and 7. ( D ) Quantification of Ki-67 immunohistochemistry using pixel-based densitometry (n = 3/group). ( E ) Representative images from Ki-67 immunoreactive sections. Scale bar = 100 μm; arrows indicate immunoreactive intestinal epithelial cells. ( F and G ) p70s6K and Akt phosphorylation in NCM460 cells, as assessed by Western blot (n = 3). * P

Techniques Used: Mouse Assay, Immunohistochemistry, Western Blot

Heat maps from subprofiling of gene expression microarray data represent relative expression levels of genes coding for extracellular mediators upregulated in CD patient-derived ADSCs compared with Ctrl patients ( A ) and those downregulated in CD patient-derived ADSCs compared with Ctrl patients ( B ). mRNA ( C ) and protein ( D ) lactoferrin ( LTF) levels are increased in human mesenteric ADSCs and conditioned media from CD patients, respectively (n = 6, * P
Figure Legend Snippet: Heat maps from subprofiling of gene expression microarray data represent relative expression levels of genes coding for extracellular mediators upregulated in CD patient-derived ADSCs compared with Ctrl patients ( A ) and those downregulated in CD patient-derived ADSCs compared with Ctrl patients ( B ). mRNA ( C ) and protein ( D ) lactoferrin ( LTF) levels are increased in human mesenteric ADSCs and conditioned media from CD patients, respectively (n = 6, * P

Techniques Used: Expressing, Microarray, Derivative Assay

11) Product Images from "Nanofibrous Spongy Microspheres for the Delivery of Hypoxia-primed Human Dental Pulp Stem Cells to Regenerate Vascularized Dental Pulp"

Article Title: Nanofibrous Spongy Microspheres for the Delivery of Hypoxia-primed Human Dental Pulp Stem Cells to Regenerate Vascularized Dental Pulp

Journal: Acta biomaterialia

doi: 10.1016/j.actbio.2016.01.032

Hypoxia induced VEGF gene expression of hDPSCs on NF-SMS a) Real-time PCR analysis indicated that VEGF mRNA expression level of hDPSCs on S-MS in the hypoxia group was not different from that in the normoxia group at day 1 and day 3, even became lower than in the normaxia group at day 7 and day 10. ** p
Figure Legend Snippet: Hypoxia induced VEGF gene expression of hDPSCs on NF-SMS a) Real-time PCR analysis indicated that VEGF mRNA expression level of hDPSCs on S-MS in the hypoxia group was not different from that in the normoxia group at day 1 and day 3, even became lower than in the normaxia group at day 7 and day 10. ** p

Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Mass Spectrometry

12) Product Images from "Integrin β4 Signaling Promotes Mammary Tumor Cell Adhesion to Brain Microvascular Endothelium by Inducing ErbB2-mediated Secretion of VEGF"

Article Title: Integrin β4 Signaling Promotes Mammary Tumor Cell Adhesion to Brain Microvascular Endothelium by Inducing ErbB2-mediated Secretion of VEGF

Journal: Annals of biomedical engineering

doi: 10.1007/s10439-011-0321-6

(a) VEGF mRNA levels in WT and 1355T cells. Total RNA was isolated from cells under control and Iressa (10 μM) treatment for the indicated times, and VEGF RNA was quantified by Q-PCR. The data are presented as the ratio of VEGF to GAPDH mRNA (mean±SD) obtained from triplicate samples. The VEGF secretion was quantified by ELISA for (b) WT and (c) 1355T cells under control and under the Iressa treatment at the indicated times. Results are the mean±SD of triplicate samples. Similar results were obtained from two independent experiments. *p
Figure Legend Snippet: (a) VEGF mRNA levels in WT and 1355T cells. Total RNA was isolated from cells under control and Iressa (10 μM) treatment for the indicated times, and VEGF RNA was quantified by Q-PCR. The data are presented as the ratio of VEGF to GAPDH mRNA (mean±SD) obtained from triplicate samples. The VEGF secretion was quantified by ELISA for (b) WT and (c) 1355T cells under control and under the Iressa treatment at the indicated times. Results are the mean±SD of triplicate samples. Similar results were obtained from two independent experiments. *p

Techniques Used: Isolation, Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

13) Product Images from "Studies on the anti-angiogenic effect of Marsdenia tenacissima extract in vitro and in vivo"

Article Title: Studies on the anti-angiogenic effect of Marsdenia tenacissima extract in vitro and in vivo

Journal: Oncology Letters

doi: 10.3892/ol.2013.1105

(A) Effect of MTE on the mRNA expression levels of VEGF-A and VEGFR-2. Cells were treated with various concentrations of MTE for 24 h. The mRNA levels of VEGF-A and VEGFR-2 were determined by RT-PCR in HUVECs and HepG2s. GAPDH was used as an internal control. Data are representative of three independent experiments. (B) Effect of MTE on the protein expression levels of VEGF-A and VEGFR-2.(Bi) The protein levels of VEGF-A secreted in cell culture medium were determined by ELISA after HepG2 cells were treated with indicated concentrations of MTE for 24 h. (Bii) The protein levels of VEGF-A secreted in cell culture medium were determined by ELISA after HUVECs cells were treated with indicated concentrations of MTE for 24 h.. (Biii) The protein levels of VEGFR-2 in cell lysates were determined by ELISA after HUVECs cells were treated with indicated concentrations of MTE for 24 h. Data are the mean ± SD (error bars) from at least three independent experiments. * P
Figure Legend Snippet: (A) Effect of MTE on the mRNA expression levels of VEGF-A and VEGFR-2. Cells were treated with various concentrations of MTE for 24 h. The mRNA levels of VEGF-A and VEGFR-2 were determined by RT-PCR in HUVECs and HepG2s. GAPDH was used as an internal control. Data are representative of three independent experiments. (B) Effect of MTE on the protein expression levels of VEGF-A and VEGFR-2.(Bi) The protein levels of VEGF-A secreted in cell culture medium were determined by ELISA after HepG2 cells were treated with indicated concentrations of MTE for 24 h. (Bii) The protein levels of VEGF-A secreted in cell culture medium were determined by ELISA after HUVECs cells were treated with indicated concentrations of MTE for 24 h.. (Biii) The protein levels of VEGFR-2 in cell lysates were determined by ELISA after HUVECs cells were treated with indicated concentrations of MTE for 24 h. Data are the mean ± SD (error bars) from at least three independent experiments. * P

Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Cell Culture, Enzyme-linked Immunosorbent Assay

14) Product Images from "Adipose-Derived Mesenchymal Stem Cells Migrate and Rescue RPE in the Setting of Oxidative Stress"

Article Title: Adipose-Derived Mesenchymal Stem Cells Migrate and Rescue RPE in the Setting of Oxidative Stress

Journal: Stem Cells International

doi: 10.1155/2018/9682856

Early passage ASCs overexpress the neurotropic protein HGF but not VEGF nor the proinflammatory cytokine IL-1 β . ASCs at passage 3 that were cultured in serum-free conditions for 48 hours were compared to the control group of ASCs at passage 5. Both cells and medium were collected and analyzed at mRNA level and at protein level by qRT-PCR and by ELISA, respectively. (a) qRT-PCR analysis of HGF, VEGF, and IL-1 β . (b) ELISA for HGF protein levels. Each experiment was performed a minimum of 3 samples from 3 different patients. Each experiment was performed a minimum of 3 times.
Figure Legend Snippet: Early passage ASCs overexpress the neurotropic protein HGF but not VEGF nor the proinflammatory cytokine IL-1 β . ASCs at passage 3 that were cultured in serum-free conditions for 48 hours were compared to the control group of ASCs at passage 5. Both cells and medium were collected and analyzed at mRNA level and at protein level by qRT-PCR and by ELISA, respectively. (a) qRT-PCR analysis of HGF, VEGF, and IL-1 β . (b) ELISA for HGF protein levels. Each experiment was performed a minimum of 3 samples from 3 different patients. Each experiment was performed a minimum of 3 times.

Techniques Used: Cell Culture, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

15) Product Images from "Vascular Endothelial Growth Factor Accelerates Compensatory Lung Growth by Increasing Alveolar Units"

Article Title: Vascular Endothelial Growth Factor Accelerates Compensatory Lung Growth by Increasing Alveolar Units

Journal: Pediatric research

doi: 10.1038/pr.2018.41

Pulmonary Function Studies There is no difference between the two groups in normalized inspiratory capacity (A). Although not reaching statistical significance, area under the PV loop is higher in the VEGF group on POD 4 (B). There is no difference in pulmonary compliance (C). Data are shown as mean ± SE.
Figure Legend Snippet: Pulmonary Function Studies There is no difference between the two groups in normalized inspiratory capacity (A). Although not reaching statistical significance, area under the PV loop is higher in the VEGF group on POD 4 (B). There is no difference in pulmonary compliance (C). Data are shown as mean ± SE.

Techniques Used:

Immunohistochemistry VE-Cadherin-labeled lung sections reveal no difference in pulmonary vasculature between the two treatment groups (A). On Ki67 staining, cellular proliferation increased with VEGF treatment on POD 2 but reduced on POD 4 (B–D). Data for Ki67 index are shown as mean ± SE.
Figure Legend Snippet: Immunohistochemistry VE-Cadherin-labeled lung sections reveal no difference in pulmonary vasculature between the two treatment groups (A). On Ki67 staining, cellular proliferation increased with VEGF treatment on POD 2 but reduced on POD 4 (B–D). Data for Ki67 index are shown as mean ± SE.

Techniques Used: Immunohistochemistry, Labeling, Staining

Organ Volume and Weight Measurements Mice treated with VEGF demonstrate increased lung volume/body weight ratio on post-operative day (POD) 4 (A). There is no difference between the two groups in liver (B), kidney (C), or spleen weight (D). Data are shown as mean ± SE.
Figure Legend Snippet: Organ Volume and Weight Measurements Mice treated with VEGF demonstrate increased lung volume/body weight ratio on post-operative day (POD) 4 (A). There is no difference between the two groups in liver (B), kidney (C), or spleen weight (D). Data are shown as mean ± SE.

Techniques Used: Mouse Assay

Morphometric Analyses VEGF-treated mice display increased parenchymal volume (A), alveolar volume (B), and septal surface area (D) on POD 4. Alveolar count is higher in the VEGF group on both POD 4 and 10 (C), but there is a decrease in alveolar count in the VEGF group from POD 4 to 10. There is a trend toward significance for higher septal volume on POD 4 and 10 in the VEGF group (E). There is no statistical difference in mean septal thickness between the two groups although the value is higher on POD 10 with VEGF treatment. Data are shown as mean ± SE.
Figure Legend Snippet: Morphometric Analyses VEGF-treated mice display increased parenchymal volume (A), alveolar volume (B), and septal surface area (D) on POD 4. Alveolar count is higher in the VEGF group on both POD 4 and 10 (C), but there is a decrease in alveolar count in the VEGF group from POD 4 to 10. There is a trend toward significance for higher septal volume on POD 4 and 10 in the VEGF group (E). There is no statistical difference in mean septal thickness between the two groups although the value is higher on POD 10 with VEGF treatment. Data are shown as mean ± SE.

Techniques Used: Mouse Assay

Gene and Protein Expression Analyses VEGF-treated lungs on POD 4 display increased mRNA expression of epidermal growth factor (EGF) on quantitative polymerase chain reactions (A). VEGF-treated lungs also display increased VEGFR2 activation on western blot (B C). Although not reaching statistical significance, there is also an increase in EGFR activation (B–C). Data are shown as mean ± SE.
Figure Legend Snippet: Gene and Protein Expression Analyses VEGF-treated lungs on POD 4 display increased mRNA expression of epidermal growth factor (EGF) on quantitative polymerase chain reactions (A). VEGF-treated lungs also display increased VEGFR2 activation on western blot (B C). Although not reaching statistical significance, there is also an increase in EGFR activation (B–C). Data are shown as mean ± SE.

Techniques Used: Expressing, Activation Assay, Western Blot

16) Product Images from "EFFECT OF RHO-KINASE INHIBITOR, Y27632, ON PORCINE CORNEAL ENDOTHELIAL CELL CULTURE, INFLAMMATION AND IMMUNE REGULATION"

Article Title: EFFECT OF RHO-KINASE INHIBITOR, Y27632, ON PORCINE CORNEAL ENDOTHELIAL CELL CULTURE, INFLAMMATION AND IMMUNE REGULATION

Journal: Ocular immunology and inflammation

doi: 10.3109/09273948.2015.1056534

Suppression of inflammatory chemokines from pCECs (A) MCP-1 and VEGF mRNA levels in pCECs with/without 10μM Y27632 were measured by real-time PCR after hTNF-α activation. Although there were increases in MCP-1 and VEGF mRNA in pCECs after activation, Y27632 significantly reduced MCP-1 and VEGF mRNA expression after 6h of activation (43% and 37% reduction respectively, *p
Figure Legend Snippet: Suppression of inflammatory chemokines from pCECs (A) MCP-1 and VEGF mRNA levels in pCECs with/without 10μM Y27632 were measured by real-time PCR after hTNF-α activation. Although there were increases in MCP-1 and VEGF mRNA in pCECs after activation, Y27632 significantly reduced MCP-1 and VEGF mRNA expression after 6h of activation (43% and 37% reduction respectively, *p

Techniques Used: Real-time Polymerase Chain Reaction, Activation Assay, Expressing

Phosphorylation of NF-κB p65 in pCECs after stimulation with hTNF-α (A) Determination of phosphorylated NF-κB p65 in pCECs by flow cytometry. pCECs were cultured with/without Y27632, and then stimulated with hTNF-α for 5 minutes. Black dotted lines show staining with isotype control. Shaded areas show staining with anti-phosphorylation of NF-κB p65 antibody. Data are representative of results of three independent experiments. (B) There was no significant difference in relative MFI and % phosphorylated NF-κB p65 positive cells between pCECs cultured with/without Y27632. Data expressed as the mean ± SD (n=3). (ns = not significant vs. without Y27632)
Figure Legend Snippet: Phosphorylation of NF-κB p65 in pCECs after stimulation with hTNF-α (A) Determination of phosphorylated NF-κB p65 in pCECs by flow cytometry. pCECs were cultured with/without Y27632, and then stimulated with hTNF-α for 5 minutes. Black dotted lines show staining with isotype control. Shaded areas show staining with anti-phosphorylation of NF-κB p65 antibody. Data are representative of results of three independent experiments. (B) There was no significant difference in relative MFI and % phosphorylated NF-κB p65 positive cells between pCECs cultured with/without Y27632. Data expressed as the mean ± SD (n=3). (ns = not significant vs. without Y27632)

Techniques Used: Flow Cytometry, Cytometry, Cell Culture, Staining

17) Product Images from "EFFECT OF RHO-KINASE INHIBITOR, Y27632, ON PORCINE CORNEAL ENDOTHELIAL CELL CULTURE, INFLAMMATION AND IMMUNE REGULATION"

Article Title: EFFECT OF RHO-KINASE INHIBITOR, Y27632, ON PORCINE CORNEAL ENDOTHELIAL CELL CULTURE, INFLAMMATION AND IMMUNE REGULATION

Journal: Ocular immunology and inflammation

doi: 10.3109/09273948.2015.1056534

Suppression of inflammatory chemokines from pCECs (A) MCP-1 and VEGF mRNA levels in pCECs with/without 10μM Y27632 were measured by real-time PCR after hTNF-α activation. Although there were increases in MCP-1 and VEGF mRNA in pCECs after activation, Y27632 significantly reduced MCP-1 and VEGF mRNA expression after 6h of activation (43% and 37% reduction respectively, *p
Figure Legend Snippet: Suppression of inflammatory chemokines from pCECs (A) MCP-1 and VEGF mRNA levels in pCECs with/without 10μM Y27632 were measured by real-time PCR after hTNF-α activation. Although there were increases in MCP-1 and VEGF mRNA in pCECs after activation, Y27632 significantly reduced MCP-1 and VEGF mRNA expression after 6h of activation (43% and 37% reduction respectively, *p

Techniques Used: Real-time Polymerase Chain Reaction, Activation Assay, Expressing

18) Product Images from "EFFECT OF RHO-KINASE INHIBITOR, Y27632, ON PORCINE CORNEAL ENDOTHELIAL CELL CULTURE, INFLAMMATION AND IMMUNE REGULATION"

Article Title: EFFECT OF RHO-KINASE INHIBITOR, Y27632, ON PORCINE CORNEAL ENDOTHELIAL CELL CULTURE, INFLAMMATION AND IMMUNE REGULATION

Journal: Ocular immunology and inflammation

doi: 10.3109/09273948.2015.1056534

Suppression of inflammatory cytokines from human whole blood Inflammatory cytokines, IFN-γ and TNF-α, were measured by intracellular staining from human whole blood after activation with PMA-Ionomycin in the presence or absence of Y27632 at several concentrations. In the presence of Y27632, cytokine levels from (A) CD4 + and (B) CD8 + T cells were significantly decreased. Data expressed as mean ± SD (*p
Figure Legend Snippet: Suppression of inflammatory cytokines from human whole blood Inflammatory cytokines, IFN-γ and TNF-α, were measured by intracellular staining from human whole blood after activation with PMA-Ionomycin in the presence or absence of Y27632 at several concentrations. In the presence of Y27632, cytokine levels from (A) CD4 + and (B) CD8 + T cells were significantly decreased. Data expressed as mean ± SD (*p

Techniques Used: Staining, Activation Assay

Potential application of a Rho kinase inhibitor to corneal xenotransplantation Y27632 enables cultured pCEC-based therapy for (i) in vitro pCEC culture (e.g., enhanced pCEC proliferation, reduced apoptosis), (ii) in vivo graft function (e.g., reproliferation of the pCECs after transplantation), and (iii) in vivo immune regulation (e.g., prevention of graft rejection).
Figure Legend Snippet: Potential application of a Rho kinase inhibitor to corneal xenotransplantation Y27632 enables cultured pCEC-based therapy for (i) in vitro pCEC culture (e.g., enhanced pCEC proliferation, reduced apoptosis), (ii) in vivo graft function (e.g., reproliferation of the pCECs after transplantation), and (iii) in vivo immune regulation (e.g., prevention of graft rejection).

Techniques Used: Cell Culture, In Vitro, In Vivo, Transplantation Assay

Enhanced proliferation resulted in accelerated healing of cultured pCECs with Y27632 in vitro (A) Representative figures of in vitro healing in pCECs +/− 10μM Y27632. A linear defect was created with a pipette tip in confluent pCECs. The healing process was observed at several time-points (0, 8, 16, and 24h) under the microscope. Compared to control, pCECs cultured with Y27632 demonstrated accelerated healing. (B) The defect was significantly reduced in the Y27632 group at all time-points (87% vs. 72%, 59% vs. 41%, 42% vs. 23%, respectively, as a ratio of the width of the initial defect, *p
Figure Legend Snippet: Enhanced proliferation resulted in accelerated healing of cultured pCECs with Y27632 in vitro (A) Representative figures of in vitro healing in pCECs +/− 10μM Y27632. A linear defect was created with a pipette tip in confluent pCECs. The healing process was observed at several time-points (0, 8, 16, and 24h) under the microscope. Compared to control, pCECs cultured with Y27632 demonstrated accelerated healing. (B) The defect was significantly reduced in the Y27632 group at all time-points (87% vs. 72%, 59% vs. 41%, 42% vs. 23%, respectively, as a ratio of the width of the initial defect, *p

Techniques Used: Cell Culture, In Vitro, Transferring, Microscopy

Suppression of inflammatory chemokines from pCECs (A) MCP-1 and VEGF mRNA levels in pCECs with/without 10μM Y27632 were measured by real-time PCR after hTNF-α activation. Although there were increases in MCP-1 and VEGF mRNA in pCECs after activation, Y27632 significantly reduced MCP-1 and VEGF mRNA expression after 6h of activation (43% and 37% reduction respectively, *p
Figure Legend Snippet: Suppression of inflammatory chemokines from pCECs (A) MCP-1 and VEGF mRNA levels in pCECs with/without 10μM Y27632 were measured by real-time PCR after hTNF-α activation. Although there were increases in MCP-1 and VEGF mRNA in pCECs after activation, Y27632 significantly reduced MCP-1 and VEGF mRNA expression after 6h of activation (43% and 37% reduction respectively, *p

Techniques Used: Real-time Polymerase Chain Reaction, Activation Assay, Expressing

Phosphorylation of NF-κB p65 in pCECs after stimulation with hTNF-α (A) Determination of phosphorylated NF-κB p65 in pCECs by flow cytometry. pCECs were cultured with/without Y27632, and then stimulated with hTNF-α for 5 minutes. Black dotted lines show staining with isotype control. Shaded areas show staining with anti-phosphorylation of NF-κB p65 antibody. Data are representative of results of three independent experiments. (B) There was no significant difference in relative MFI and % phosphorylated NF-κB p65 positive cells between pCECs cultured with/without Y27632. Data expressed as the mean ± SD (n=3). (ns = not significant vs. without Y27632)
Figure Legend Snippet: Phosphorylation of NF-κB p65 in pCECs after stimulation with hTNF-α (A) Determination of phosphorylated NF-κB p65 in pCECs by flow cytometry. pCECs were cultured with/without Y27632, and then stimulated with hTNF-α for 5 minutes. Black dotted lines show staining with isotype control. Shaded areas show staining with anti-phosphorylation of NF-κB p65 antibody. Data are representative of results of three independent experiments. (B) There was no significant difference in relative MFI and % phosphorylated NF-κB p65 positive cells between pCECs cultured with/without Y27632. Data expressed as the mean ± SD (n=3). (ns = not significant vs. without Y27632)

Techniques Used: Flow Cytometry, Cytometry, Cell Culture, Staining

Y27632 significantly suppresses proliferation of hPBMCs (A) RhoA (upstream of Rho kinase) was up-regulated in hPBMCs activated by PHA. Phosphorylated myosin phosphatase target subunit 1 (p-MYPT1, 130kDa), which is downstream of Rho kinase was also up-regulated by PHA stimulation, and dose-dependently down-regulated by 10 and 50μM of Y27632. (B) Y27632 significantly reduced proliferation of hPBMCs stimulated with PHA after 72h in a dose-dependent manner (*p
Figure Legend Snippet: Y27632 significantly suppresses proliferation of hPBMCs (A) RhoA (upstream of Rho kinase) was up-regulated in hPBMCs activated by PHA. Phosphorylated myosin phosphatase target subunit 1 (p-MYPT1, 130kDa), which is downstream of Rho kinase was also up-regulated by PHA stimulation, and dose-dependently down-regulated by 10 and 50μM of Y27632. (B) Y27632 significantly reduced proliferation of hPBMCs stimulated with PHA after 72h in a dose-dependent manner (*p

Techniques Used:

Enhanced cell proliferation and Inhibition of apoptosis/necrosis of pCECs in the presence of the Rho kinase inhibitor Y27632 (A) Representative figures of cultured pCECs after being seeded on the plate with/without Rho kinase inhibitor. pCECs were cultured +/− 10μM Y27632, and observed at several time-points (16, 24, 40, and 64h) under the microscope. Compared to control cells, pCECs cultured with Y27632 enhanced initial attachment to the plates, proliferation, and maintained a polygonal-shaped phenotype for 64h. (B) The proliferative capacity of pCECs +/− 10μM Y27632 was compared by direct cell counting for 15 days. Cell numbers were significantly higher at all time-points (2-fold on days 3, 6, 9, 15, and 3-fold on day 12, *p
Figure Legend Snippet: Enhanced cell proliferation and Inhibition of apoptosis/necrosis of pCECs in the presence of the Rho kinase inhibitor Y27632 (A) Representative figures of cultured pCECs after being seeded on the plate with/without Rho kinase inhibitor. pCECs were cultured +/− 10μM Y27632, and observed at several time-points (16, 24, 40, and 64h) under the microscope. Compared to control cells, pCECs cultured with Y27632 enhanced initial attachment to the plates, proliferation, and maintained a polygonal-shaped phenotype for 64h. (B) The proliferative capacity of pCECs +/− 10μM Y27632 was compared by direct cell counting for 15 days. Cell numbers were significantly higher at all time-points (2-fold on days 3, 6, 9, 15, and 3-fold on day 12, *p

Techniques Used: Inhibition, Cell Culture, Microscopy, Cell Counting

19) Product Images from "mi R‐15a/15b Cluster Modulates Survival of Mesenchymal Stem Cells to Improve Its Therapeutic Efficacy of Myocardial Infarction"

Article Title: mi R‐15a/15b Cluster Modulates Survival of Mesenchymal Stem Cells to Improve Its Therapeutic Efficacy of Myocardial Infarction

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

doi: 10.1161/JAHA.118.010157

Vascular endothelial growth factor receptor 2 ( VEGFR ‐2) and Bcl‐2 were validated as a target of miR‐15a/15b.  A , The 3′‐untranslated region ( UTR)  of  VEGFR ‐2 harbored a potential targeting site of miR‐15a/15b, which was conserved among mouse by bioinformatics analysis.  C  and  E , Luciferase assay was performed to show that overexpression of miR‐15a/15b in  HEK  293T cells could significantly suppress the luciferase activity of a reporter fused with 3′‐UTR of  VEGFR 2  mRNA .  HEK  293T cells were transfected with a  pMIR ‐ VEGFR 2‐3′‐ UTR  or  pMIR ‐ VEGFR 2‐m3′‐ UTR . Meanwhile, the cells were cotransfected with an miR‐15a/15b mimics or mimics negative control ( NC ). Compared with the mimics  NC , the miR‐15a/15b mimics could reduce luciferase activity containing a wild‐type miR‐15a/15b binding site ( C  and  E ) but not a mutant binding site ( D  and  F ).  B , 3′‐ UTR  of Bcl‐2 harbored a potential targeting site of miR‐15a/15b, which was conserved among mouse by bioinformatics analysis.  G  and  I , Luciferase assay was performed to show that overexpression of miR‐15a/15b in  HEK  293T cells could significantly suppress the luciferase activity of a reporter fused with 3′‐UTR of Bcl‐2  mRNA .  HEK  293T cells were transfected with a  pMIR ‐Bcl‐2‐3′‐ UTR  or  pMIR ‐Bcl‐2‐m3′‐ UTR . Meanwhile, the cells were cotransfected with an miR‐15a/15b mimics or mimics  NC . Compared with the mimics control, the 15a/15b mimics could reduce luciferase activity containing a wild‐type miR‐15a/15b binding site ( G  and  I ) but not a mutant binding site ( H  and  J ). ** P
Figure Legend Snippet: Vascular endothelial growth factor receptor 2 ( VEGFR ‐2) and Bcl‐2 were validated as a target of miR‐15a/15b. A , The 3′‐untranslated region ( UTR) of VEGFR ‐2 harbored a potential targeting site of miR‐15a/15b, which was conserved among mouse by bioinformatics analysis. C and E , Luciferase assay was performed to show that overexpression of miR‐15a/15b in HEK 293T cells could significantly suppress the luciferase activity of a reporter fused with 3′‐UTR of VEGFR 2 mRNA . HEK 293T cells were transfected with a pMIR ‐ VEGFR 2‐3′‐ UTR or pMIR ‐ VEGFR 2‐m3′‐ UTR . Meanwhile, the cells were cotransfected with an miR‐15a/15b mimics or mimics negative control ( NC ). Compared with the mimics NC , the miR‐15a/15b mimics could reduce luciferase activity containing a wild‐type miR‐15a/15b binding site ( C and E ) but not a mutant binding site ( D and F ). B , 3′‐ UTR of Bcl‐2 harbored a potential targeting site of miR‐15a/15b, which was conserved among mouse by bioinformatics analysis. G and I , Luciferase assay was performed to show that overexpression of miR‐15a/15b in HEK 293T cells could significantly suppress the luciferase activity of a reporter fused with 3′‐UTR of Bcl‐2 mRNA . HEK 293T cells were transfected with a pMIR ‐Bcl‐2‐3′‐ UTR or pMIR ‐Bcl‐2‐m3′‐ UTR . Meanwhile, the cells were cotransfected with an miR‐15a/15b mimics or mimics NC . Compared with the mimics control, the 15a/15b mimics could reduce luciferase activity containing a wild‐type miR‐15a/15b binding site ( G and I ) but not a mutant binding site ( H and J ). ** P

Techniques Used: Luciferase, Over Expression, Activity Assay, Transfection, Negative Control, Binding Assay, Mutagenesis

20) Product Images from "Apelin Affects the Progression of Osteoarthritis by Regulating VEGF-Dependent Angiogenesis and miR-150-5p Expression in Human Synovial Fibroblasts"

Article Title: Apelin Affects the Progression of Osteoarthritis by Regulating VEGF-Dependent Angiogenesis and miR-150-5p Expression in Human Synovial Fibroblasts

Journal: Cells

doi: 10.3390/cells9030594

Src and Akt phosphorylation are involved in APLN-induced VEGF synthesis. ( A , B ) OASFs were pretreated with Src/Akt inhibitors for 30 min or Src/Akt siRNAs for 24 h, then incubated with APLN for 24 h. ( A ) VEGF mRNA levels were examined using RT-qPCR ( n = 4), and ( B ) excreted VEGF protein levels were quantified using ELISA assay ( n = 5). ( C , D ) After pretreatment with Src/Akt inhibitors or Src/Akt siRNA and incubation with APLN, the culture medium was collected and subjected to EPC ( C ) tube formation ( n = 5) and ( D ) migration assays ( n = 5). ( E ) OASFs were incubated with APLN, and the extent of Src/Akt phosphorylation was examined using Western blot analysis ( n = 3). ( F ) OASFs pretreated with a FAK inhibitor before APLN incubation were assessed for the extent of Src/Akt phosphorylation, while OASFs pretreated with a Src inhibitor before APLN incubation were assessed for Akt phosphorylation ( n = 3). * p
Figure Legend Snippet: Src and Akt phosphorylation are involved in APLN-induced VEGF synthesis. ( A , B ) OASFs were pretreated with Src/Akt inhibitors for 30 min or Src/Akt siRNAs for 24 h, then incubated with APLN for 24 h. ( A ) VEGF mRNA levels were examined using RT-qPCR ( n = 4), and ( B ) excreted VEGF protein levels were quantified using ELISA assay ( n = 5). ( C , D ) After pretreatment with Src/Akt inhibitors or Src/Akt siRNA and incubation with APLN, the culture medium was collected and subjected to EPC ( C ) tube formation ( n = 5) and ( D ) migration assays ( n = 5). ( E ) OASFs were incubated with APLN, and the extent of Src/Akt phosphorylation was examined using Western blot analysis ( n = 3). ( F ) OASFs pretreated with a FAK inhibitor before APLN incubation were assessed for the extent of Src/Akt phosphorylation, while OASFs pretreated with a Src inhibitor before APLN incubation were assessed for Akt phosphorylation ( n = 3). * p

Techniques Used: Incubation, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Migration, Western Blot

21) Product Images from "Effects of vascular endothelial growth factor and epidermal growth factor on biological properties of gastric cancer cells"

Article Title: Effects of vascular endothelial growth factor and epidermal growth factor on biological properties of gastric cancer cells

Journal: Archives of Medical Science : AMS

doi: 10.5114/aoms.2019.88443

mRNA expression of VEGF and EGF in cancer tissues and survival curves. A – mRNA levels of VEGF and EGF in cancer tissues from the patients with positive exfoliative cytology or negative exfoliative cytology. B – The quantification of band density (mean ± SD). mRNA levels of VEGF and EGF were normalized to the β-actin mRNA level. * P
Figure Legend Snippet: mRNA expression of VEGF and EGF in cancer tissues and survival curves. A – mRNA levels of VEGF and EGF in cancer tissues from the patients with positive exfoliative cytology or negative exfoliative cytology. B – The quantification of band density (mean ± SD). mRNA levels of VEGF and EGF were normalized to the β-actin mRNA level. * P

Techniques Used: Expressing

mRNA expression of VEGF and EGF in cancer cells with or without Endostar or Erbitux treatment. A – mRNA levels of VEGF, EGF, EGFR, VEGFR1 and VEGFR2 in colon cancer cells: MGC803, HGC27, BGC823 and SGC7901. B – VEGF, VEGFR1 and VEGFR2 in MGC803 cancer cells treated with Endostar. C – VEGF, EGFR and EGF in MGC803 cancer cells treated with Erbitux. Quantification of band density is shown. VEGF and EGF mRNA levels were normalized to the β-actin mRNA level
Figure Legend Snippet: mRNA expression of VEGF and EGF in cancer cells with or without Endostar or Erbitux treatment. A – mRNA levels of VEGF, EGF, EGFR, VEGFR1 and VEGFR2 in colon cancer cells: MGC803, HGC27, BGC823 and SGC7901. B – VEGF, VEGFR1 and VEGFR2 in MGC803 cancer cells treated with Endostar. C – VEGF, EGFR and EGF in MGC803 cancer cells treated with Erbitux. Quantification of band density is shown. VEGF and EGF mRNA levels were normalized to the β-actin mRNA level

Techniques Used: Expressing

22) Product Images from "Granulocyte-macrophage colony-stimulating factor-transfected bone marrow stromal cells for the treatment of ischemic stroke ☆"

Article Title: Granulocyte-macrophage colony-stimulating factor-transfected bone marrow stromal cells for the treatment of ischemic stroke ☆

Journal: Neural Regeneration Research

doi: 10.3969/j.issn.1673-5374.2012.16.003

BMSCs-GM-CSF increased VEGF mRNA levels at 48 hours after MCAO. (a) VEGF mRNA was measured by reverse transcription-PCR before MCAO (baseline), as well as at 24 hours after MCAO (pre-injection) and 48 hours after MCAO. (b) Bar graph shows VEGF mRNA levels at different time points. VEGF mRNA expression is the percentage of VEGF mRNA to GAPDH mRNA/baseline. Baseline value was standardized to 1. Data are expressed as mean ± SD. a P
Figure Legend Snippet: BMSCs-GM-CSF increased VEGF mRNA levels at 48 hours after MCAO. (a) VEGF mRNA was measured by reverse transcription-PCR before MCAO (baseline), as well as at 24 hours after MCAO (pre-injection) and 48 hours after MCAO. (b) Bar graph shows VEGF mRNA levels at different time points. VEGF mRNA expression is the percentage of VEGF mRNA to GAPDH mRNA/baseline. Baseline value was standardized to 1. Data are expressed as mean ± SD. a P

Techniques Used: Polymerase Chain Reaction, Injection, Expressing

23) Product Images from "Defect of Adaptation to Hypoxia in Patients With COPD Due to Reduction of Histone Deacetylase 7"

Article Title: Defect of Adaptation to Hypoxia in Patients With COPD Due to Reduction of Histone Deacetylase 7

Journal: Chest

doi: 10.1378/chest.11-1536

VEGF messenger RNA (mRNA) is correlated with HIF-1α protein. A, VEGF mRNA in lung tissue was quantified using real-time quantitative polymerase chain reaction (PCR). B, Correlation between FEV 1 and VEGF mRNA expression in the lung tissue. C, PBMCs were incubated in the hypoxic chamber (1% oxygen, 5% CO 2 , and 94% N 2 ) for 24 h, and then VEGF mRNA was quantified using quantitative real-time PCR. D, VEGF mRNA correlated with HIF-1α protein expression. E, PBMCs were incubated under hypoxia (1% oxygen, 5% CO 2 , and 94% N 2 ) for 24 h, and then IL-8 in the supernatants was measured. VEGF mRNA values were normalized by GNB2L1 mRNA. HIF-1α values were normalized using lamin A. * P
Figure Legend Snippet: VEGF messenger RNA (mRNA) is correlated with HIF-1α protein. A, VEGF mRNA in lung tissue was quantified using real-time quantitative polymerase chain reaction (PCR). B, Correlation between FEV 1 and VEGF mRNA expression in the lung tissue. C, PBMCs were incubated in the hypoxic chamber (1% oxygen, 5% CO 2 , and 94% N 2 ) for 24 h, and then VEGF mRNA was quantified using quantitative real-time PCR. D, VEGF mRNA correlated with HIF-1α protein expression. E, PBMCs were incubated under hypoxia (1% oxygen, 5% CO 2 , and 94% N 2 ) for 24 h, and then IL-8 in the supernatants was measured. VEGF mRNA values were normalized by GNB2L1 mRNA. HIF-1α values were normalized using lamin A. * P

Techniques Used: Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, Expressing, Incubation

HDAC7 reduction by RNA interference or cigarette smoke extract affected HIF-1α nuclear translocation and VEGF promoter binding in A549 cells. siRNA of HDAC7 was transfected to A549, and then cells were incubated under hypoxia (0.1% oxygen, 5% CO 2 ) for 24 h. A, HIF-1α nuclear translocation was evaluated. B, HIF-1α binding to the VEGF promoter under treatment with CoCl 2 (50 μM) (mimic hypoxia) was also analyzed by chromatin immunoprecipitation (ChIP) assay. The ratio of the cycle threshold (Ct) value of HIF-1α ChIP product to input (total cells) is shown (** P
Figure Legend Snippet: HDAC7 reduction by RNA interference or cigarette smoke extract affected HIF-1α nuclear translocation and VEGF promoter binding in A549 cells. siRNA of HDAC7 was transfected to A549, and then cells were incubated under hypoxia (0.1% oxygen, 5% CO 2 ) for 24 h. A, HIF-1α nuclear translocation was evaluated. B, HIF-1α binding to the VEGF promoter under treatment with CoCl 2 (50 μM) (mimic hypoxia) was also analyzed by chromatin immunoprecipitation (ChIP) assay. The ratio of the cycle threshold (Ct) value of HIF-1α ChIP product to input (total cells) is shown (** P

Techniques Used: Translocation Assay, Binding Assay, Transfection, Incubation, Chromatin Immunoprecipitation

24) Product Images from "Could an endoneurial endothelial crosstalk between Wnt/β-catenin and Sonic Hedgehog pathways underlie the early disruption of the infraorbital blood–nerve barrier following chronic constriction injury?"

Article Title: Could an endoneurial endothelial crosstalk between Wnt/β-catenin and Sonic Hedgehog pathways underlie the early disruption of the infraorbital blood–nerve barrier following chronic constriction injury?

Journal: Molecular Pain

doi: 10.1177/1744806917727625

In vitro, in the human cerebral microvascular endothelial cell line (hCMEC/D3), modulation of Wnt/β-catenin pathway induces significant expression changes in the VE-cadherin/Fzd-7/β-catenin complex mRNAs levels. (a) Presence of functional Frizzled-7 within the hCMEC/D3 endothelial cells was assessed using immunofluorescent labeling of Frizzled-7, β-catenin, and TO-PRO observed with confocal microscopy imaging. Paraformaldehyde-fixed cells were incubated with anti-Frizzled-7 antibodies (green), anti-β-catenin (cell membrane surrogate marker, red), and TO-PRO (nuclear stain, blue) (Scale bar = 20 µm). (b) Changes in Frizzled-7, β-catenin, and VE-cadherin mRNAs expression levels were assessed in vitro following stimulation with either Wnt agonist I (5 μM; Wnt pathway agonist) or PKF 118-310 (2 μM; Wnt pathway antagonist). Data are presented as relative quantification (R.Q.) in arbitrary units (A.U.) corresponding to the ratio of specific mRNA over RPS18 mRNA. Each bar corresponds to the mean ± SEM of n = 4 experiments for each condition; * p
Figure Legend Snippet: In vitro, in the human cerebral microvascular endothelial cell line (hCMEC/D3), modulation of Wnt/β-catenin pathway induces significant expression changes in the VE-cadherin/Fzd-7/β-catenin complex mRNAs levels. (a) Presence of functional Frizzled-7 within the hCMEC/D3 endothelial cells was assessed using immunofluorescent labeling of Frizzled-7, β-catenin, and TO-PRO observed with confocal microscopy imaging. Paraformaldehyde-fixed cells were incubated with anti-Frizzled-7 antibodies (green), anti-β-catenin (cell membrane surrogate marker, red), and TO-PRO (nuclear stain, blue) (Scale bar = 20 µm). (b) Changes in Frizzled-7, β-catenin, and VE-cadherin mRNAs expression levels were assessed in vitro following stimulation with either Wnt agonist I (5 μM; Wnt pathway agonist) or PKF 118-310 (2 μM; Wnt pathway antagonist). Data are presented as relative quantification (R.Q.) in arbitrary units (A.U.) corresponding to the ratio of specific mRNA over RPS18 mRNA. Each bar corresponds to the mean ± SEM of n = 4 experiments for each condition; * p

Techniques Used: In Vitro, Expressing, Functional Assay, Labeling, Confocal Microscopy, Imaging, Incubation, Marker, Staining

Chronic constriction injury of the infraorbital nerve (IoN-CCI) induces early and transient alterations in the Fzd-7/β-catenin/VE-cadherin AJ complex within endoneurial endothelial cells in association with a transient increased production of VEGF-A protein in the infraorbital nerve. (a) IoN-CCI induces early and transient significant downregulation of Fzd-7 and VE-cadherin mRNAs and early upregulation followed by transient downregulation of β-catenin mRNA expression levels. Changes over time of Fzd-7, β-catenin, and VE-cadherin mRNA levels were assessed in the IoN of sham- or CCI-injured animals using semi-quantitative RT-PCR analyses. Data are presented as relative quantification (R.Q.) in arbitrary units (A.U.) corresponding to the ratio of specific mRNA over RPS18 mRNA. Each bar corresponds to the mean ± SEM of n = 5–8 animals for each time point (post-injury); * p
Figure Legend Snippet: Chronic constriction injury of the infraorbital nerve (IoN-CCI) induces early and transient alterations in the Fzd-7/β-catenin/VE-cadherin AJ complex within endoneurial endothelial cells in association with a transient increased production of VEGF-A protein in the infraorbital nerve. (a) IoN-CCI induces early and transient significant downregulation of Fzd-7 and VE-cadherin mRNAs and early upregulation followed by transient downregulation of β-catenin mRNA expression levels. Changes over time of Fzd-7, β-catenin, and VE-cadherin mRNA levels were assessed in the IoN of sham- or CCI-injured animals using semi-quantitative RT-PCR analyses. Data are presented as relative quantification (R.Q.) in arbitrary units (A.U.) corresponding to the ratio of specific mRNA over RPS18 mRNA. Each bar corresponds to the mean ± SEM of n = 5–8 animals for each time point (post-injury); * p

Techniques Used: Expressing, Quantitative RT-PCR

In vivo , at 3 h post-injury, inhibition of Wnt/β-catenin signaling could not prevent the molecular and vascular alterations following infraorbital nerve chronic constriction injury (IoN-CCI). (a to d) Changes in mRNAs expression levels of TJ proteins Claudin-1, Claudin-5, Occludin (a), inflammatory markers TLR2 and CD11b (b), Hedgehog pathway markers Patched-1 and Gli-1 (c), and Fzd-7/ β-catenin/VE-cadherin AJ complex proteins (d) were assessed using semi-quantitative RT-PCR following either perineural injections (used as a control condition), or infraorbital nerve chronic constriction injury (CCI) following either PKF 118-310 (50 μM; Wnt pathway antagonist) or NaCl 0.9% injections (three injections, spaced 6 h apart, starting 24 h before injury), as compared to noninjured IoN of naïve rats (serving as baseline values for mRNA levels comparisons). Data are presented as relative quantification (R.Q.) in arbitrary units (A.U.) corresponding to the ratio of specific mRNA over RPS18 mRNA. Each bar corresponds to the mean ± SEM of n = 5–8 animals for each time point (post-injury); * p
Figure Legend Snippet: In vivo , at 3 h post-injury, inhibition of Wnt/β-catenin signaling could not prevent the molecular and vascular alterations following infraorbital nerve chronic constriction injury (IoN-CCI). (a to d) Changes in mRNAs expression levels of TJ proteins Claudin-1, Claudin-5, Occludin (a), inflammatory markers TLR2 and CD11b (b), Hedgehog pathway markers Patched-1 and Gli-1 (c), and Fzd-7/ β-catenin/VE-cadherin AJ complex proteins (d) were assessed using semi-quantitative RT-PCR following either perineural injections (used as a control condition), or infraorbital nerve chronic constriction injury (CCI) following either PKF 118-310 (50 μM; Wnt pathway antagonist) or NaCl 0.9% injections (three injections, spaced 6 h apart, starting 24 h before injury), as compared to noninjured IoN of naïve rats (serving as baseline values for mRNA levels comparisons). Data are presented as relative quantification (R.Q.) in arbitrary units (A.U.) corresponding to the ratio of specific mRNA over RPS18 mRNA. Each bar corresponds to the mean ± SEM of n = 5–8 animals for each time point (post-injury); * p

Techniques Used: In Vivo, Inhibition, Expressing, Quantitative RT-PCR

In vivo, at 3 h post-injury, CCI of the infraorbital nerve elicited a significant increase in hypoxia marker HIF-1α mRNA expression levels as compared to sham-injured animals. Changes in HIF-1α mRNA levels were assessed in the IoN of CCI-injured animals compared to sham-injured controls, using semi-quantitative RT-PCR analyses. Data are presented as relative quantification (R.Q.) in arbitrary units (A.U.) corresponding to the ratio of specific mRNA over RPS18 mRNA. Each bar corresponds to the mean ± SEM of n = 5–6 animals for each condition; * p
Figure Legend Snippet: In vivo, at 3 h post-injury, CCI of the infraorbital nerve elicited a significant increase in hypoxia marker HIF-1α mRNA expression levels as compared to sham-injured animals. Changes in HIF-1α mRNA levels were assessed in the IoN of CCI-injured animals compared to sham-injured controls, using semi-quantitative RT-PCR analyses. Data are presented as relative quantification (R.Q.) in arbitrary units (A.U.) corresponding to the ratio of specific mRNA over RPS18 mRNA. Each bar corresponds to the mean ± SEM of n = 5–6 animals for each condition; * p

Techniques Used: In Vivo, Marker, Expressing, Quantitative RT-PCR

In vitro, in the hCMEC/D3 cell line, activation of Wnt/β-catenin pathway downregulates the mRNAs of key Hedgehog pathway readouts and mediates the molecular changes observed following TLR4 stimulation. (a) Changes in Gli-1 or Patched-1 mRNAs expression levels were assessed in vitro following stimulation with either Wnt agonist I (5 μM; Wnt pathway agonist) or PKF 118-310 (2 μM; Wnt pathway antagonist). Data are presented as relative quantification (R.Q.) in arbitrary units (A.U.) corresponding to the ratio of specific mRNA over RPS18 mRNA. Each bar corresponds to the mean ± SEM of n = 4 experiments for each condition; * p
Figure Legend Snippet: In vitro, in the hCMEC/D3 cell line, activation of Wnt/β-catenin pathway downregulates the mRNAs of key Hedgehog pathway readouts and mediates the molecular changes observed following TLR4 stimulation. (a) Changes in Gli-1 or Patched-1 mRNAs expression levels were assessed in vitro following stimulation with either Wnt agonist I (5 μM; Wnt pathway agonist) or PKF 118-310 (2 μM; Wnt pathway antagonist). Data are presented as relative quantification (R.Q.) in arbitrary units (A.U.) corresponding to the ratio of specific mRNA over RPS18 mRNA. Each bar corresponds to the mean ± SEM of n = 4 experiments for each condition; * p

Techniques Used: In Vitro, Activation Assay, Expressing

In vivo, at 3 h post-injury, CCI of the infraorbital nerve elicited a significant increase in chemokine marker CCL2 mRNA expression levels as compared to sham-injured animals. Changes in CCL2 mRNA levels were assessed in the IoN of CCI-injured animals compared to sham-injured controls, using semi-quantitative RT-PCR analyses. Data are presented as relative quantification (R.Q.) in arbitrary units (A.U.) corresponding to the ratio of specific mRNA over RPS18 mRNA. Each bar corresponds to the mean ± SEM of n = 5–6 animals for each condition; * p
Figure Legend Snippet: In vivo, at 3 h post-injury, CCI of the infraorbital nerve elicited a significant increase in chemokine marker CCL2 mRNA expression levels as compared to sham-injured animals. Changes in CCL2 mRNA levels were assessed in the IoN of CCI-injured animals compared to sham-injured controls, using semi-quantitative RT-PCR analyses. Data are presented as relative quantification (R.Q.) in arbitrary units (A.U.) corresponding to the ratio of specific mRNA over RPS18 mRNA. Each bar corresponds to the mean ± SEM of n = 5–6 animals for each condition; * p

Techniques Used: In Vivo, Marker, Expressing, Quantitative RT-PCR

25) Product Images from "Hypoxia potentiates the cytotoxic effect of piperlongumine in pheochromocytoma models"

Article Title: Hypoxia potentiates the cytotoxic effect of piperlongumine in pheochromocytoma models

Journal: Oncotarget

doi: 10.18632/oncotarget.9643

Piperlongumine activates apoptosis and necroptosis, and inhibits cell migration and invasion A. RIP1-IP was performed on cell lysates from MPC cells treated with the indicated concentrations of PL at 21% and 1% O 2 for 24 hours and probed for RIP1 (top lane) and RIP3 (bottom lane). A representative image (n=3) is shown. B. MTT cells were treated with indicated concentrations of PL at 21% and 1% O 2 for 24 hours. Total cell lysates were analyzed by Western blot for cleaved PARP, cleaved caspase 3 and caspase 3. β-tubulin was used as a loading control. The representative image (n=3) is shown. C. 1.5 × 10 5 MPC cells were plated in the upper part of transwell chambers and allowed to migrate for 24 hours in the presence of 0, 1, and 5μM PL. The box and whiskers graph represents data from three independent experiments. D. 1.5 × 10 5 MPC cells were plated in the upper part of matrigel-coated transwell chambers and allowed to migrate for 24 hours in the presence of 0, 1, and 5μM PL. The box and whiskers graph represents data from three independent experiments. E. MPC cells were treated with indicated concentrations of PL for 24 hours. mRNA expression levels of Twist1, Vegfa, Mmp9, and Nanog were assessed by quantitative real-time PCR. The target gene transcript levels were normalized to Actb . A graph represents data from three independent experiments as mean +/− SEM. *P
Figure Legend Snippet: Piperlongumine activates apoptosis and necroptosis, and inhibits cell migration and invasion A. RIP1-IP was performed on cell lysates from MPC cells treated with the indicated concentrations of PL at 21% and 1% O 2 for 24 hours and probed for RIP1 (top lane) and RIP3 (bottom lane). A representative image (n=3) is shown. B. MTT cells were treated with indicated concentrations of PL at 21% and 1% O 2 for 24 hours. Total cell lysates were analyzed by Western blot for cleaved PARP, cleaved caspase 3 and caspase 3. β-tubulin was used as a loading control. The representative image (n=3) is shown. C. 1.5 × 10 5 MPC cells were plated in the upper part of transwell chambers and allowed to migrate for 24 hours in the presence of 0, 1, and 5μM PL. The box and whiskers graph represents data from three independent experiments. D. 1.5 × 10 5 MPC cells were plated in the upper part of matrigel-coated transwell chambers and allowed to migrate for 24 hours in the presence of 0, 1, and 5μM PL. The box and whiskers graph represents data from three independent experiments. E. MPC cells were treated with indicated concentrations of PL for 24 hours. mRNA expression levels of Twist1, Vegfa, Mmp9, and Nanog were assessed by quantitative real-time PCR. The target gene transcript levels were normalized to Actb . A graph represents data from three independent experiments as mean +/− SEM. *P

Techniques Used: Migration, MTT Assay, Western Blot, Expressing, Real-time Polymerase Chain Reaction

Piperlongumine inhibits tumor growth and metastases formation and decreases expression levels of EMT and angiogenesis markers in vivo A. Nude female mice bearing MTT-Luc tumors were treated with 24 mg/kg/day PL (n=9) for 28 days. The control group was treated with vehicle (n=10). Tumor growth was assessed once a week. The graph shows a significant inhibition of tumor growth at all time points of treatment in the treated group (green) compared to control (red) animals. The graph represents data from 9 (treated) and 10 (vehicle) mice assessed at specific time points as mean +/− SEM. Statistical analysis was performed by Mann Whitney, U-test, at each time point. B. Representative tumors from control mice compared to the tumors from animals treated with PL. Scale bar: 1cm. C. Lungs were resected and subjected to bioluminescence imaging. The presence of lung metastases in treated mice was 46% lower than in control group. D. mRNA expression levels of Pou5f1, Twist1, Vegfa, Mmp9, and Nanog in tumors from both treated and control groups were assessed by quantitative real-time PCR. The target gene transcript levels were normalized to Actb . The box and whiskers graphs represent data from control (n=10) and treated (n=9) groups. *P
Figure Legend Snippet: Piperlongumine inhibits tumor growth and metastases formation and decreases expression levels of EMT and angiogenesis markers in vivo A. Nude female mice bearing MTT-Luc tumors were treated with 24 mg/kg/day PL (n=9) for 28 days. The control group was treated with vehicle (n=10). Tumor growth was assessed once a week. The graph shows a significant inhibition of tumor growth at all time points of treatment in the treated group (green) compared to control (red) animals. The graph represents data from 9 (treated) and 10 (vehicle) mice assessed at specific time points as mean +/− SEM. Statistical analysis was performed by Mann Whitney, U-test, at each time point. B. Representative tumors from control mice compared to the tumors from animals treated with PL. Scale bar: 1cm. C. Lungs were resected and subjected to bioluminescence imaging. The presence of lung metastases in treated mice was 46% lower than in control group. D. mRNA expression levels of Pou5f1, Twist1, Vegfa, Mmp9, and Nanog in tumors from both treated and control groups were assessed by quantitative real-time PCR. The target gene transcript levels were normalized to Actb . The box and whiskers graphs represent data from control (n=10) and treated (n=9) groups. *P

Techniques Used: Expressing, In Vivo, Mouse Assay, MTT Assay, Inhibition, MANN-WHITNEY, Imaging, Real-time Polymerase Chain Reaction

26) Product Images from "Prognostic significance of Cytokeratin 20-positive lymph node vascular endothelial growth factor A mRNA and chromodomain helicase DNA binding protein 4 in pN0 colorectal cancer patients"

Article Title: Prognostic significance of Cytokeratin 20-positive lymph node vascular endothelial growth factor A mRNA and chromodomain helicase DNA binding protein 4 in pN0 colorectal cancer patients

Journal: Oncotarget

doi: 10.18632/oncotarget.23424

Kaplan-Meier curves of recurrence for the 2nd cohort of 47 CK20+ pN0 CRC patients after follow-up for 60 months based on VEGF-A mRNA copy numbers
Figure Legend Snippet: Kaplan-Meier curves of recurrence for the 2nd cohort of 47 CK20+ pN0 CRC patients after follow-up for 60 months based on VEGF-A mRNA copy numbers

Techniques Used:

( A ) VEGF-A mRNA and ( B ) TCF20 mRNA copy numbers per 0.05 μg total RNA in CK20+ and CK20- LNs from the 2nd cohort of pN0 CRC patients. The median in each group of subjects was indicated by a black horizontal line.
Figure Legend Snippet: ( A ) VEGF-A mRNA and ( B ) TCF20 mRNA copy numbers per 0.05 μg total RNA in CK20+ and CK20- LNs from the 2nd cohort of pN0 CRC patients. The median in each group of subjects was indicated by a black horizontal line.

Techniques Used:

27) Product Images from "Ultrasound-assisted zsiRNA delivery via arginine-grafted bioreducible polymer and microbubbles targeting VEGF for ovarian cancer treatment"

Article Title: Ultrasound-assisted zsiRNA delivery via arginine-grafted bioreducible polymer and microbubbles targeting VEGF for ovarian cancer treatment

Journal: Journal of controlled release : official journal of the Controlled Release Society

doi: 10.1016/j.jconrel.2014.03.025

Optimization of MB:cell ratio for VEGF protein knockdown. Transfection efficiency of VEGF-siRNA using polyplexes (ABP-siRNA), SAM complexes (MB:cell ratio 500 and 1000) and US. VEGF ELISA and MTT assay in A2780 cell line treated with US 1 MHz, 0.5 Watt/cm 2 and 50% duty. (A) VEGF concentration after transfection of A2780 cells in serum containing media after US exposure with 100 nM siRNA targeting VEGF, complexed in SAM or polyplexes. (B) Cell viability of (A) was determined by MTT assay and expressed as relative cell viability compared to the control. Data represent mean ± SD and significance tested (P
Figure Legend Snippet: Optimization of MB:cell ratio for VEGF protein knockdown. Transfection efficiency of VEGF-siRNA using polyplexes (ABP-siRNA), SAM complexes (MB:cell ratio 500 and 1000) and US. VEGF ELISA and MTT assay in A2780 cell line treated with US 1 MHz, 0.5 Watt/cm 2 and 50% duty. (A) VEGF concentration after transfection of A2780 cells in serum containing media after US exposure with 100 nM siRNA targeting VEGF, complexed in SAM or polyplexes. (B) Cell viability of (A) was determined by MTT assay and expressed as relative cell viability compared to the control. Data represent mean ± SD and significance tested (P

Techniques Used: Transfection, Enzyme-linked Immunosorbent Assay, MTT Assay, Concentration Assay

VEGF knockdown using SAM complexes. Transfection efficiency of VEGF-siRNA using polyplexes (ABP-VEGF-siRNA, PEI-VEGF-siRNA), SAM-VEGF complexes (MB:cell ratio 500), naked VEGF-siRNA, SAM-Luc (siRNA negative control, MB:cell ratio 500) and US. VEGF ELISA and MTT assay in A2780 cell line treated with US 1 MHz, 0.5 Watt/cm 2 and 50% duty. (A) VEGF concentration after transfection of A2780 cells in serum containing media after US exposure with 100 nM siRNA targeting VEGF or luciferase (SAM-Luc). (B) Cell viability of (A) was determined by MTT assay and expressed as relative cell viability compared to the control. Data represent mean ± SD and significance tested (P
Figure Legend Snippet: VEGF knockdown using SAM complexes. Transfection efficiency of VEGF-siRNA using polyplexes (ABP-VEGF-siRNA, PEI-VEGF-siRNA), SAM-VEGF complexes (MB:cell ratio 500), naked VEGF-siRNA, SAM-Luc (siRNA negative control, MB:cell ratio 500) and US. VEGF ELISA and MTT assay in A2780 cell line treated with US 1 MHz, 0.5 Watt/cm 2 and 50% duty. (A) VEGF concentration after transfection of A2780 cells in serum containing media after US exposure with 100 nM siRNA targeting VEGF or luciferase (SAM-Luc). (B) Cell viability of (A) was determined by MTT assay and expressed as relative cell viability compared to the control. Data represent mean ± SD and significance tested (P

Techniques Used: Transfection, Negative Control, Enzyme-linked Immunosorbent Assay, MTT Assay, Concentration Assay, Luciferase

28) Product Images from "Oral squamous carcinoma cells promote macrophage polarization in an MIF-dependent manner"

Article Title: Oral squamous carcinoma cells promote macrophage polarization in an MIF-dependent manner

Journal: QJM: An International Journal of Medicine

doi: 10.1093/qjmed/hcy163

MIF shRNA expression in RAW 264.7 macrophages phenocopies 4-IPP. mRNA expression by qRT-PCR in RAW 264.7 macrophages transduced with non-sense (control) or anti-MIF shRNA; co-cultured with SCCVII cells. ( A ) Arginase 1; ( B ) VEGF-A; ( C ) COX-2; ( D ) iNOS; ( E ) CD206. RQ, relative quantity (relative to the reference sample transduced with control shRNA). ns, non-significant: P > 0.05; * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001; **** P ≤ 0.000.1.
Figure Legend Snippet: MIF shRNA expression in RAW 264.7 macrophages phenocopies 4-IPP. mRNA expression by qRT-PCR in RAW 264.7 macrophages transduced with non-sense (control) or anti-MIF shRNA; co-cultured with SCCVII cells. ( A ) Arginase 1; ( B ) VEGF-A; ( C ) COX-2; ( D ) iNOS; ( E ) CD206. RQ, relative quantity (relative to the reference sample transduced with control shRNA). ns, non-significant: P > 0.05; * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001; **** P ≤ 0.000.1.

Techniques Used: shRNA, Expressing, Quantitative RT-PCR, Transduction, Cell Culture

SCCVII co-culture induces M2 marker expression in RAW 264.7 cell line and inhibition by 4-IPP. mRNA expression by qRT-PCR in RAW 264.7 macrophages treated with vehicle control (0.1% DMSO) or 4-IPP; co-cultured with different densities of SCCVII cells (0.3 = 0.3×10 5 cells/insert; 1.0 = 1.0×10 5 cells/insert). ( A ) Arginase 1; ( B ) VEGF-A; ( C ) COX-2; ( D ) iNOS; ( E ) CD206. RQ, relative quantity (relative to the reference sample treated with vehicle control DMSO). ns, non-significant: P > 0.05; * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001; **** P ≤ 0.000.1.
Figure Legend Snippet: SCCVII co-culture induces M2 marker expression in RAW 264.7 cell line and inhibition by 4-IPP. mRNA expression by qRT-PCR in RAW 264.7 macrophages treated with vehicle control (0.1% DMSO) or 4-IPP; co-cultured with different densities of SCCVII cells (0.3 = 0.3×10 5 cells/insert; 1.0 = 1.0×10 5 cells/insert). ( A ) Arginase 1; ( B ) VEGF-A; ( C ) COX-2; ( D ) iNOS; ( E ) CD206. RQ, relative quantity (relative to the reference sample treated with vehicle control DMSO). ns, non-significant: P > 0.05; * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001; **** P ≤ 0.000.1.

Techniques Used: Co-Culture Assay, Marker, Expressing, Inhibition, Quantitative RT-PCR, Cell Culture

4-IPP treatment of macrophages—but not SCCVII cells—reduces co-culture-induced M2 marker expression. mRNA expression by qRT-PCR in RAW 264.7 macrophages treated with vehicle control (0.1% DMSO) or 4-IPP; co-cultured with SCCVII cells pre-treated with vehicle control (DMSO) or 4-IPP. ( A ) Arginase 1; ( B ) VEGF-A; ( C ) COX-2; ( D ) iNOS; ( E ) CD206. RQ, relative quantity (relative to the reference sample treated with vehicle control DMSO). ns, non-significant: P > 0.05; * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001; **** P ≤ 0.000.1.
Figure Legend Snippet: 4-IPP treatment of macrophages—but not SCCVII cells—reduces co-culture-induced M2 marker expression. mRNA expression by qRT-PCR in RAW 264.7 macrophages treated with vehicle control (0.1% DMSO) or 4-IPP; co-cultured with SCCVII cells pre-treated with vehicle control (DMSO) or 4-IPP. ( A ) Arginase 1; ( B ) VEGF-A; ( C ) COX-2; ( D ) iNOS; ( E ) CD206. RQ, relative quantity (relative to the reference sample treated with vehicle control DMSO). ns, non-significant: P > 0.05; * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001; **** P ≤ 0.000.1.

Techniques Used: Co-Culture Assay, Marker, Expressing, Quantitative RT-PCR, Cell Culture

29) Product Images from "Multiple Autologous Bone Marrow‐Derived CD271+ Mesenchymal Stem Cell Transplantation Overcomes Drug‐Resistant Epilepsy in Children"

Article Title: Multiple Autologous Bone Marrow‐Derived CD271+ Mesenchymal Stem Cell Transplantation Overcomes Drug‐Resistant Epilepsy in Children

Journal: Stem Cells Translational Medicine

doi: 10.1002/sctm.17-0041

Molecular and functional analysis of BMMSCs. (A): Percentages of positive cells for subsequent surface markers are given in table. (B): Graphical overview of comparative genomic hybridization microarray results. Expression levels of mRNA for neurotrophic (C) , proangiogenic (D) , and tissue remodeling (E) factors in patients' BMMSCs. qRT‐PCR analysis revealed expression of BDNF, NGF, HGF, PDGF‐A, Flt‐1, VEGF‐A, FGF‐1, FGF‐2, ANGPT‐1, and MMP‐2. Slight expression of GDNF, CNTF, and NGF was observed. No expression of MMP‐9 was detected. The results are mean values ± SD (from triplicates) of mRNA expression relative to expression of housekeeping gene—GAPDH. (F): Western blot detection of VEGF in patients' BMMSCs lysates. Both forms of VEGF were identified: dimer (42 kDa) and monomer (21 kDa). The intermediary band is an effect of nonspecific reaction of antibody, observed also in manufacturer's specification. Standardized protein content in samples from different patients was confirmed by GAPDH detection. (G, H): qRT‐PCR analysis of IDO1 and TSG‐6 mRNA expression levels in patients' BMMSCs stimulated with proinflammatory cytokines. (G): Pretreatment of BMMSCs with INF‐γ caused manifestation of IDO1 expression. (H): Moreover, stimulation of BMMSCs with either TNF‐α or Il‐1β revealed elevated expression of TSG‐6. Pronounced effect was observed for Il‐1β. The results are mean values ± SD (from triplicates) of mRNA expression relative to expression of housekeeping gene—GAPDH. As a control in experiments, BMMSCs in stimulation medium only (2% FBS) were utilized. Abbreviations: ANGPT‐1, angiopoietin 1; BDNF, brain‐derived neurotrophic factor; BMMSC, bone marrow mesenchymal stem cell; CNTF, ciliary neurotrophic factor; FBS, fetal bovine serum; FGF, fibroblast growth factor; Flt‐1, vascular endothelial growth factor receptor 1; GAPDH, glyceraldehyde 3‐phosphate dehydrogenase; GDNF, glial‐derived neurotrophic factor; HGF, hepatocyte growth factor; IDO1, indoleamine 2,3‐dioxygenase; Il‐1β, interleukin 1 β; INF‐γ, interferon γ; MMP, metalloproteinase; NGF, nerve growth factor; PDGF, platelet‐derived growth factor; qRT‐PCR, real‐time quantitative reverse transcription polymerase chain reaction; TNF‐α, tumor necrosis factor α; TSG‐6, tumor necrosis factor‐inducible gene 6; VEGF, vascular endothelial growth factor.
Figure Legend Snippet: Molecular and functional analysis of BMMSCs. (A): Percentages of positive cells for subsequent surface markers are given in table. (B): Graphical overview of comparative genomic hybridization microarray results. Expression levels of mRNA for neurotrophic (C) , proangiogenic (D) , and tissue remodeling (E) factors in patients' BMMSCs. qRT‐PCR analysis revealed expression of BDNF, NGF, HGF, PDGF‐A, Flt‐1, VEGF‐A, FGF‐1, FGF‐2, ANGPT‐1, and MMP‐2. Slight expression of GDNF, CNTF, and NGF was observed. No expression of MMP‐9 was detected. The results are mean values ± SD (from triplicates) of mRNA expression relative to expression of housekeeping gene—GAPDH. (F): Western blot detection of VEGF in patients' BMMSCs lysates. Both forms of VEGF were identified: dimer (42 kDa) and monomer (21 kDa). The intermediary band is an effect of nonspecific reaction of antibody, observed also in manufacturer's specification. Standardized protein content in samples from different patients was confirmed by GAPDH detection. (G, H): qRT‐PCR analysis of IDO1 and TSG‐6 mRNA expression levels in patients' BMMSCs stimulated with proinflammatory cytokines. (G): Pretreatment of BMMSCs with INF‐γ caused manifestation of IDO1 expression. (H): Moreover, stimulation of BMMSCs with either TNF‐α or Il‐1β revealed elevated expression of TSG‐6. Pronounced effect was observed for Il‐1β. The results are mean values ± SD (from triplicates) of mRNA expression relative to expression of housekeeping gene—GAPDH. As a control in experiments, BMMSCs in stimulation medium only (2% FBS) were utilized. Abbreviations: ANGPT‐1, angiopoietin 1; BDNF, brain‐derived neurotrophic factor; BMMSC, bone marrow mesenchymal stem cell; CNTF, ciliary neurotrophic factor; FBS, fetal bovine serum; FGF, fibroblast growth factor; Flt‐1, vascular endothelial growth factor receptor 1; GAPDH, glyceraldehyde 3‐phosphate dehydrogenase; GDNF, glial‐derived neurotrophic factor; HGF, hepatocyte growth factor; IDO1, indoleamine 2,3‐dioxygenase; Il‐1β, interleukin 1 β; INF‐γ, interferon γ; MMP, metalloproteinase; NGF, nerve growth factor; PDGF, platelet‐derived growth factor; qRT‐PCR, real‐time quantitative reverse transcription polymerase chain reaction; TNF‐α, tumor necrosis factor α; TSG‐6, tumor necrosis factor‐inducible gene 6; VEGF, vascular endothelial growth factor.

Techniques Used: Functional Assay, Hybridization, Microarray, Expressing, Quantitative RT-PCR, Western Blot, Derivative Assay, Reverse Transcription Polymerase Chain Reaction

30) Product Images from "Adipose-Derived Mesenchymal Stem Cells Migrate and Rescue RPE in the Setting of Oxidative Stress"

Article Title: Adipose-Derived Mesenchymal Stem Cells Migrate and Rescue RPE in the Setting of Oxidative Stress

Journal: Stem Cells International

doi: 10.1155/2018/9682856

Early passage ASCs overexpress the neurotropic protein HGF but not VEGF nor the proinflammatory cytokine IL-1 β . ASCs at passage 3 that were cultured in serum-free conditions for 48 hours were compared to the control group of ASCs at passage 5. Both cells and medium were collected and analyzed at mRNA level and at protein level by qRT-PCR and by ELISA, respectively. (a) qRT-PCR analysis of HGF, VEGF, and IL-1 β . (b) ELISA for HGF protein levels. Each experiment was performed a minimum of 3 samples from 3 different patients. Each experiment was performed a minimum of 3 times.
Figure Legend Snippet: Early passage ASCs overexpress the neurotropic protein HGF but not VEGF nor the proinflammatory cytokine IL-1 β . ASCs at passage 3 that were cultured in serum-free conditions for 48 hours were compared to the control group of ASCs at passage 5. Both cells and medium were collected and analyzed at mRNA level and at protein level by qRT-PCR and by ELISA, respectively. (a) qRT-PCR analysis of HGF, VEGF, and IL-1 β . (b) ELISA for HGF protein levels. Each experiment was performed a minimum of 3 samples from 3 different patients. Each experiment was performed a minimum of 3 times.

Techniques Used: Cell Culture, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

31) Product Images from "Crenigacestat, a selective NOTCH1 inhibitor, reduces intrahepatic cholangiocarcinoma progression by blocking VEGFA/DLL4/MMP13 axis"

Article Title: Crenigacestat, a selective NOTCH1 inhibitor, reduces intrahepatic cholangiocarcinoma progression by blocking VEGFA/DLL4/MMP13 axis

Journal: Cell Death and Differentiation

doi: 10.1038/s41418-020-0505-4

Relationship between microvessel density and NOTCH1, HES1, DLL4, MMP13, and VEGFA levels. Levels of tumor microvessel density (MVD) correlate with mRNA expression of NOTCH1 ( a ), HES1 ( b ), DLL4 ( c ), and MMP13 ( e ), but not with those of VEGFA ( d ), in a collection of human intrahepatic cholangiocarcinoma (iCCA) samples ( n = 42). Linear regression analysis was used. f Representative examples of human iCCA specimens with high and low MVD.
Figure Legend Snippet: Relationship between microvessel density and NOTCH1, HES1, DLL4, MMP13, and VEGFA levels. Levels of tumor microvessel density (MVD) correlate with mRNA expression of NOTCH1 ( a ), HES1 ( b ), DLL4 ( c ), and MMP13 ( e ), but not with those of VEGFA ( d ), in a collection of human intrahepatic cholangiocarcinoma (iCCA) samples ( n = 42). Linear regression analysis was used. f Representative examples of human iCCA specimens with high and low MVD.

Techniques Used: Expressing

LY3039478 inhibits VEGFA, CD31, and DLL4 expression in the iCCA PDX model. a Western blot analysis and semiquantitative evaluation of DLL4, VEGFA, and CD31 expression in PDX mice tissues by densitometry analysis of protein bands reveals a downregulation of DLL4, VEGFA, and CD31 protein expression in PDX mice treated with GSI. The bands were measured compared with the housekeeping GAPDH protein band, for each tissue. Average value of DLL4, VEGFA, and CD31 expression levels among all mouse treated with LY3039478 or vehicle is reported in the graph. P value showed versus vehicle treatment. Tissues PDX mice n = 10 for vehicle treatment in gray, n = 10 for LY3039478 treatment in black. b Representative images with immunofluorescence staining show DLL4 and CD31 downregulation in representative images of PDX tissues treated with LY30349478. DLL4 (green) and CD31 (red) and overlapping staining (yellow) were immunolocalized in PDX tissues. The yellow arrows highlight the detail of the co-localization of DLL4 and CD31 in PDX tissues (#4, #14, #24) not treated with LY339478. DAPI, 4′,6‐diamidino‐2‐phenylindole. c Immunofluorescence staining with MMP13 in red and nucleus in DAPI shown a significantly reduction of MMP13 in iCCA PDX tissues treated with LY3039478. Magnifications: ×20; inset ×60. d Representative images demonstrate a significant ( P
Figure Legend Snippet: LY3039478 inhibits VEGFA, CD31, and DLL4 expression in the iCCA PDX model. a Western blot analysis and semiquantitative evaluation of DLL4, VEGFA, and CD31 expression in PDX mice tissues by densitometry analysis of protein bands reveals a downregulation of DLL4, VEGFA, and CD31 protein expression in PDX mice treated with GSI. The bands were measured compared with the housekeeping GAPDH protein band, for each tissue. Average value of DLL4, VEGFA, and CD31 expression levels among all mouse treated with LY3039478 or vehicle is reported in the graph. P value showed versus vehicle treatment. Tissues PDX mice n = 10 for vehicle treatment in gray, n = 10 for LY3039478 treatment in black. b Representative images with immunofluorescence staining show DLL4 and CD31 downregulation in representative images of PDX tissues treated with LY30349478. DLL4 (green) and CD31 (red) and overlapping staining (yellow) were immunolocalized in PDX tissues. The yellow arrows highlight the detail of the co-localization of DLL4 and CD31 in PDX tissues (#4, #14, #24) not treated with LY339478. DAPI, 4′,6‐diamidino‐2‐phenylindole. c Immunofluorescence staining with MMP13 in red and nucleus in DAPI shown a significantly reduction of MMP13 in iCCA PDX tissues treated with LY3039478. Magnifications: ×20; inset ×60. d Representative images demonstrate a significant ( P

Techniques Used: Expressing, Western Blot, Mouse Assay, Immunofluorescence, Staining

NOTCH1 , HES1 , DLL4 , VEGFA , and MMP13 mRNA expression in iCCA patients. a Analysis of 31 primary tumors from iCCA patients and matched surrounding normal liver tissues downloaded from the GEO database (GSE107943). Mean expression data were expressed in RPKM (Reads Per Kilobase Million). *** P
Figure Legend Snippet: NOTCH1 , HES1 , DLL4 , VEGFA , and MMP13 mRNA expression in iCCA patients. a Analysis of 31 primary tumors from iCCA patients and matched surrounding normal liver tissues downloaded from the GEO database (GSE107943). Mean expression data were expressed in RPKM (Reads Per Kilobase Million). *** P

Techniques Used: Expressing

32) Product Images from "Oleic Acid Increases Synthesis and Secretion of VEGF in Rat Vascular Smooth Muscle Cells: Role of Oxidative Stress and Impairment in Obesity"

Article Title: Oleic Acid Increases Synthesis and Secretion of VEGF in Rat Vascular Smooth Muscle Cells: Role of Oxidative Stress and Impairment in Obesity

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms140918861

Influence of a 24-h incubation with 100 μM oleic acid (on VEGF-A synthesis (Panels A ) and secretion (Panels B ) in VSMC from LZR, without or with a 1-h pre-incubation with inhibitors of PKC (Staurosporine and LY379196).
Figure Legend Snippet: Influence of a 24-h incubation with 100 μM oleic acid (on VEGF-A synthesis (Panels A ) and secretion (Panels B ) in VSMC from LZR, without or with a 1-h pre-incubation with inhibitors of PKC (Staurosporine and LY379196).

Techniques Used: Incubation

Influence of a 6-h incubation with 100 μM oleic acid on VEGF-A synthesis (Panel A ) and secretion (Panel B ) in VSMC from LZR without or with a 24-h pre-incubation with H 2 O 2 . H 2 O 2 completely blunts the above mentioned effects of oleic acid on VEGF-A ( n = 6, p = 0.0001 vs. oleic acid alone).
Figure Legend Snippet: Influence of a 6-h incubation with 100 μM oleic acid on VEGF-A synthesis (Panel A ) and secretion (Panel B ) in VSMC from LZR without or with a 24-h pre-incubation with H 2 O 2 . H 2 O 2 completely blunts the above mentioned effects of oleic acid on VEGF-A ( n = 6, p = 0.0001 vs. oleic acid alone).

Techniques Used: Incubation

Time-dependent (4–24 h of incubation with 100 μM oleic acid) influence of oleic acid on VEGF-A mRNA transcription (Panel A ); protein synthesis (Panel B ) and secretion (Panel C ) in VSMC from LZR.
Figure Legend Snippet: Time-dependent (4–24 h of incubation with 100 μM oleic acid) influence of oleic acid on VEGF-A mRNA transcription (Panel A ); protein synthesis (Panel B ) and secretion (Panel C ) in VSMC from LZR.

Techniques Used: Incubation

Influence of a 24-h incubation with 100 μM oleic acid on VEGF-A: ( A ) mRNA transcription; ( B ) protein synthesis and ( C ) secretion in VSMC from LZR and OZR.
Figure Legend Snippet: Influence of a 24-h incubation with 100 μM oleic acid on VEGF-A: ( A ) mRNA transcription; ( B ) protein synthesis and ( C ) secretion in VSMC from LZR and OZR.

Techniques Used: Incubation

Dose-dependent (24 h of incubation with 50–100 μM oleic acid) influence of oleic acid on VEGF-A mRNA transcription (Panel A ); protein synthesis (Panel B ) and secretion (Panel C ) in VSMC from LZR.
Figure Legend Snippet: Dose-dependent (24 h of incubation with 50–100 μM oleic acid) influence of oleic acid on VEGF-A mRNA transcription (Panel A ); protein synthesis (Panel B ) and secretion (Panel C ) in VSMC from LZR.

Techniques Used: Incubation

Influence of a 24-h incubation with 100 μM oleic acid on VEGF-A synthesis (Panels A ) and secretion (Panels B ) in VSMC from LZR, without or with a 1-h pre-incubation with inhibitors of: (i) NADPH oxidase (Apocynin); (ii) mitochondrial electron transport chain complex (Rotenone). All the inhibitors impair the oleic acid effects ( n = 6, p = 0.04–0.02 vs. oleic acid alone).
Figure Legend Snippet: Influence of a 24-h incubation with 100 μM oleic acid on VEGF-A synthesis (Panels A ) and secretion (Panels B ) in VSMC from LZR, without or with a 1-h pre-incubation with inhibitors of: (i) NADPH oxidase (Apocynin); (ii) mitochondrial electron transport chain complex (Rotenone). All the inhibitors impair the oleic acid effects ( n = 6, p = 0.04–0.02 vs. oleic acid alone).

Techniques Used: Incubation

Influence of a 24-h incubation with 100 μM oleic acid on VEGF-A synthesis (Panels A ) and secretion (Panels B ) in VSMC from LZR, without or with a 1-h pre-incubation with inhibitors of: (i) Akt (LY294002); (ii) mTOR (Rapamycin); (iii) MEK (PD98059). All the inhibitors impair the oleic acid effects ( n = 6, p = 0.04–0.02 vs. oleic acid alone).
Figure Legend Snippet: Influence of a 24-h incubation with 100 μM oleic acid on VEGF-A synthesis (Panels A ) and secretion (Panels B ) in VSMC from LZR, without or with a 1-h pre-incubation with inhibitors of: (i) Akt (LY294002); (ii) mTOR (Rapamycin); (iii) MEK (PD98059). All the inhibitors impair the oleic acid effects ( n = 6, p = 0.04–0.02 vs. oleic acid alone).

Techniques Used: Incubation

33) Product Images from "Studies on the anti-angiogenic effect of Marsdenia tenacissima extract in vitro and in vivo"

Article Title: Studies on the anti-angiogenic effect of Marsdenia tenacissima extract in vitro and in vivo

Journal: Oncology Letters

doi: 10.3892/ol.2013.1105

(A) Effect of MTE on the mRNA expression levels of VEGF-A and VEGFR-2. Cells were treated with various concentrations of MTE for 24 h. The mRNA levels of VEGF-A and VEGFR-2 were determined by RT-PCR in HUVECs and HepG2s. GAPDH was used as an internal control. Data are representative of three independent experiments. (B) Effect of MTE on the protein expression levels of VEGF-A and VEGFR-2.(Bi) The protein levels of VEGF-A secreted in cell culture medium were determined by ELISA after HepG2 cells were treated with indicated concentrations of MTE for 24 h. (Bii) The protein levels of VEGF-A secreted in cell culture medium were determined by ELISA after HUVECs cells were treated with indicated concentrations of MTE for 24 h.. (Biii) The protein levels of VEGFR-2 in cell lysates were determined by ELISA after HUVECs cells were treated with indicated concentrations of MTE for 24 h. Data are the mean ± SD (error bars) from at least three independent experiments. * P
Figure Legend Snippet: (A) Effect of MTE on the mRNA expression levels of VEGF-A and VEGFR-2. Cells were treated with various concentrations of MTE for 24 h. The mRNA levels of VEGF-A and VEGFR-2 were determined by RT-PCR in HUVECs and HepG2s. GAPDH was used as an internal control. Data are representative of three independent experiments. (B) Effect of MTE on the protein expression levels of VEGF-A and VEGFR-2.(Bi) The protein levels of VEGF-A secreted in cell culture medium were determined by ELISA after HepG2 cells were treated with indicated concentrations of MTE for 24 h. (Bii) The protein levels of VEGF-A secreted in cell culture medium were determined by ELISA after HUVECs cells were treated with indicated concentrations of MTE for 24 h.. (Biii) The protein levels of VEGFR-2 in cell lysates were determined by ELISA after HUVECs cells were treated with indicated concentrations of MTE for 24 h. Data are the mean ± SD (error bars) from at least three independent experiments. * P

Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Cell Culture, Enzyme-linked Immunosorbent Assay

34) Product Images from "Spica prunellae promotes cancer cell apoptosis, inhibits cell proliferation and tumor angiogenesis in a mouse model of colorectal cancer via suppression of stat3 pathway"

Article Title: Spica prunellae promotes cancer cell apoptosis, inhibits cell proliferation and tumor angiogenesis in a mouse model of colorectal cancer via suppression of stat3 pathway

Journal: BMC Complementary and Alternative Medicine

doi: 10.1186/1472-6882-13-144

Effect of EESP on the expression of Bcl-2, Bax, Cyclin D1, CDK4, VEGF-A and VEGFR-2 in CRC mice. ( A ) The mRNA levels in tumor tissues from control and EESP-treated group were determined by RT-PCR. GAPDH was used as an internal control. The data of densitometric analysis were normalized to the mean mRNA expression of untreated control (100%). ( B-D ) The protein expression of Bcl-2, Bax, Cyclin D1, CDK4, VEGF-A and VEGFR-2 in tumor tissues was analyzed via immunohistochemical assay. The photographs are representative images taken at a magnification of 400 ×. Quantification of immunohistochemical assay was represented as percentage of positively-stained cells. Data shown are averages with S.D. (error bars) from 9 individual mouse in each group. *P
Figure Legend Snippet: Effect of EESP on the expression of Bcl-2, Bax, Cyclin D1, CDK4, VEGF-A and VEGFR-2 in CRC mice. ( A ) The mRNA levels in tumor tissues from control and EESP-treated group were determined by RT-PCR. GAPDH was used as an internal control. The data of densitometric analysis were normalized to the mean mRNA expression of untreated control (100%). ( B-D ) The protein expression of Bcl-2, Bax, Cyclin D1, CDK4, VEGF-A and VEGFR-2 in tumor tissues was analyzed via immunohistochemical assay. The photographs are representative images taken at a magnification of 400 ×. Quantification of immunohistochemical assay was represented as percentage of positively-stained cells. Data shown are averages with S.D. (error bars) from 9 individual mouse in each group. *P

Techniques Used: Expressing, Mouse Assay, Reverse Transcription Polymerase Chain Reaction, Immunohistochemistry, Staining

35) Product Images from "Effects of Synergistic Massage and Physical Exercise on the Expression of Angiogenic Markers in Rat Tendons"

Article Title: Effects of Synergistic Massage and Physical Exercise on the Expression of Angiogenic Markers in Rat Tendons

Journal: BioMed Research International

doi: 10.1155/2014/878095

Bands corresponding to VEGF-A 21kDA isoform obtained from tendons sampled in the fifth week of the experiment from the massage (M), premassage (PM), and control (C) groups (a). Optical density (OD) of the 21 kDa VEGF-A protein expression in individual study groups. Significant differences were detected between the M and C study groups. **** P
Figure Legend Snippet: Bands corresponding to VEGF-A 21kDA isoform obtained from tendons sampled in the fifth week of the experiment from the massage (M), premassage (PM), and control (C) groups (a). Optical density (OD) of the 21 kDa VEGF-A protein expression in individual study groups. Significant differences were detected between the M and C study groups. **** P

Techniques Used: Expressing

H E staining. A microphotograph of a rat tendon presenting nuclei of tendon cells (arrows) between parallel plates of collagen fibers. Note: no tissue lesions or inflammatory cells are visible in any of the sampled tendons, regardless of the duration or the experimental group (a). Blood vessels with erythrocytes in the lumen (indicated by arrows) are present in the resected rat tendons (b). IHC staining. In the peritendinous tissues VEGF-A expression was observed in fibroblastic-like cells (indicated by arrow) (c). CD34 expression was noted in blood vessel endothelium (indicated by arrow) (d).
Figure Legend Snippet: H E staining. A microphotograph of a rat tendon presenting nuclei of tendon cells (arrows) between parallel plates of collagen fibers. Note: no tissue lesions or inflammatory cells are visible in any of the sampled tendons, regardless of the duration or the experimental group (a). Blood vessels with erythrocytes in the lumen (indicated by arrows) are present in the resected rat tendons (b). IHC staining. In the peritendinous tissues VEGF-A expression was observed in fibroblastic-like cells (indicated by arrow) (c). CD34 expression was noted in blood vessel endothelium (indicated by arrow) (d).

Techniques Used: Staining, Immunohistochemistry, Expressing

Differentiated mRNA expression of VEGF-A (a), CD34 (b), and FGF-2 (c) in the massage (M), premassage (PM), and control (C) groups. Significant differences were noted between the M and C and between the PM and C groups. * P
Figure Legend Snippet: Differentiated mRNA expression of VEGF-A (a), CD34 (b), and FGF-2 (c) in the massage (M), premassage (PM), and control (C) groups. Significant differences were noted between the M and C and between the PM and C groups. * P

Techniques Used: Expressing

36) Product Images from "Angiogenic factor-driven inflammation promotes extravasation of human proangiogenic monocytes to tumours"

Article Title: Angiogenic factor-driven inflammation promotes extravasation of human proangiogenic monocytes to tumours

Journal: Nature Communications

doi: 10.1038/s41467-017-02610-0

CX3CL1 prevents HPMo activation by chemokines required for monocyte homing. a Role of chemokines in HPMo transmigration promoted by angiogenic factors. Human monocytes were treated with PBS or pertussis toxin (PTX) at 1 µg/ml before applying them under flow to HUVECs prestimulated with TNF or TNF + VEGF-A. n = 3 experiments, data are mean ± s.d., two-way ANOVA with Holm-Sidak adjustment. b Kinetics of calcium flux in monocyte induced by sequential stimulations with chemokines analyzed by Fura2 QBT ratiometric assay. Black arrows indicate the time of chemokine addition. The chemokines were tested at 50 ng/ml. c Effect of blocking CX3CR1 on calcium flux induced by sequential stimulation of monocytes by chemokines. Monocytes were treated with either rabbit IgG or anti-CX3CR1 at 50 µg/ml prior to sequential activation. Data were reproduced in three experiments. d Analysis of the effect of CX3CR1 blockade on transmigration of monocyte subpopulations through HUVECs activated by TNF or TNF + VEGF. n = 3 experiments, data are mean ± s.d., two-way ANOVA with Holm-Sidak adjustment. e , f Analysis of the influence of CX3CR1 blockade on extravasation of adoptively transferred human monocytes to mouse peritoneum in thioglycolate +Tnf− ( e ) and thioglycolate +TnfVegf-a- ( f ) induced peritonitis. Peritonitis was induced as described in the schematic in Supplementary Fig. 11 . Isolated monocytes were treated with either IgG or anti-CX3CR1 antibody at 50 µg/ml (with/without PTX) prior to mouse injection, n = 6 mice per group pooled from two experiments, mean ± s.d., ANOVA. g Effect of CX3CR1 blockade on HPMo recruitment to SKBR7 tumour xenografts. Human monocyte recruitment to the xenografts was performed as described in Fig. 1 with human monocytes treated with the antibodies at 50 µg/ml (with/without PTX) before mouse injection. Left: Percentage of HPMo within recruited human monocytes, n = 9 mice per group injected with monocytes from 3 donors. Data are mean ± s.d., * p -value
Figure Legend Snippet: CX3CL1 prevents HPMo activation by chemokines required for monocyte homing. a Role of chemokines in HPMo transmigration promoted by angiogenic factors. Human monocytes were treated with PBS or pertussis toxin (PTX) at 1 µg/ml before applying them under flow to HUVECs prestimulated with TNF or TNF + VEGF-A. n = 3 experiments, data are mean ± s.d., two-way ANOVA with Holm-Sidak adjustment. b Kinetics of calcium flux in monocyte induced by sequential stimulations with chemokines analyzed by Fura2 QBT ratiometric assay. Black arrows indicate the time of chemokine addition. The chemokines were tested at 50 ng/ml. c Effect of blocking CX3CR1 on calcium flux induced by sequential stimulation of monocytes by chemokines. Monocytes were treated with either rabbit IgG or anti-CX3CR1 at 50 µg/ml prior to sequential activation. Data were reproduced in three experiments. d Analysis of the effect of CX3CR1 blockade on transmigration of monocyte subpopulations through HUVECs activated by TNF or TNF + VEGF. n = 3 experiments, data are mean ± s.d., two-way ANOVA with Holm-Sidak adjustment. e , f Analysis of the influence of CX3CR1 blockade on extravasation of adoptively transferred human monocytes to mouse peritoneum in thioglycolate +Tnf− ( e ) and thioglycolate +TnfVegf-a- ( f ) induced peritonitis. Peritonitis was induced as described in the schematic in Supplementary Fig. 11 . Isolated monocytes were treated with either IgG or anti-CX3CR1 antibody at 50 µg/ml (with/without PTX) prior to mouse injection, n = 6 mice per group pooled from two experiments, mean ± s.d., ANOVA. g Effect of CX3CR1 blockade on HPMo recruitment to SKBR7 tumour xenografts. Human monocyte recruitment to the xenografts was performed as described in Fig. 1 with human monocytes treated with the antibodies at 50 µg/ml (with/without PTX) before mouse injection. Left: Percentage of HPMo within recruited human monocytes, n = 9 mice per group injected with monocytes from 3 donors. Data are mean ± s.d., * p -value

Techniques Used: Activation Assay, Transmigration Assay, Flow Cytometry, Blocking Assay, Isolation, Injection, Mouse Assay

Human proangiogenic monocytes transmigrate in angiogenic factor-driven inflammation. a Time-lapse imaging of proangiogenic monocyte transmigration assay under flow through endothelium activated with a combination of TNF and VEGF-A. Scale bar = 25 µm. Green and blue arrows indicate two transmigrating proangiogenic monocytes. b Analysis of proangiogenic and non-angiogenic monocyte adhesion in ADIn (TNF + VEGF-A or TNF + FGF2) versus conventional (TNF alone) inflammation under flow. c Analysis of proangiogenic and non-angiogenic monocyte transmigration in ADIn versus conventional inflammation under flow. The data are presented as mean ± s.d., * p -value
Figure Legend Snippet: Human proangiogenic monocytes transmigrate in angiogenic factor-driven inflammation. a Time-lapse imaging of proangiogenic monocyte transmigration assay under flow through endothelium activated with a combination of TNF and VEGF-A. Scale bar = 25 µm. Green and blue arrows indicate two transmigrating proangiogenic monocytes. b Analysis of proangiogenic and non-angiogenic monocyte adhesion in ADIn (TNF + VEGF-A or TNF + FGF2) versus conventional (TNF alone) inflammation under flow. c Analysis of proangiogenic and non-angiogenic monocyte transmigration in ADIn versus conventional inflammation under flow. The data are presented as mean ± s.d., * p -value

Techniques Used: Imaging, Transmigration Assay, Flow Cytometry

VEGF-A promotes GATA3-mediated alteration of CX3CL1 expression and HPMo homing. a Heat map of the influence of inflammatory conditions on transcription factor binding to the promoter of CX3CL1 in HUVECs. HUVECs were stimulated with inflammatory regimens, then chromatin immunoprecipitation (ChIP) was performed with antibodies against indicated transcription factors and immunoprecipitated DNA fragments in parallel with total input DNA were analysed. Analysis was done by qPCR for the presence of the CX3CL1 promoter. The result is presented as percentage of input DNA. The data represent individual mean values of experimental replicates (3 experiments #1 to #3). b Analysis of the effect of inflammatory conditions on the binding of candidate transcription factors to the CX3CL1 promoter from a . Data are presented as mean ± s.d. ( n = 3 experiments). c Analysis of the binding of Nfκb and Gata3 to Cx3cl1 promoter in tumour associated endothelial cells. DLD1 and SKBR7 xenograft-bearing mice were treated either with the mixture of DC101 and bevacizumab (D/B mix) or control IgG for 24 h. Mice were killed, and tumours collected to isolate endothelial cells for ChIP analysis of Nfκb and Gata3. Data are presented as mean ± s.d. of experimental replicates ( n = 3). d Analysis of GATA3 downregulation in HUVEC with specific siRNA by western blotting and quantification. e Effect of GATA3 downregulation on CX3CL1 protein expression induced by TNF and TNF + IFNγ in HUVECs. f Quantification of GATA3 expression in e . g Role of GATA3 in HPMo transmigration induced by angiogenic factors-driven inflammation. The data are presented as mean ± s.d. of three independent experiments. * p
Figure Legend Snippet: VEGF-A promotes GATA3-mediated alteration of CX3CL1 expression and HPMo homing. a Heat map of the influence of inflammatory conditions on transcription factor binding to the promoter of CX3CL1 in HUVECs. HUVECs were stimulated with inflammatory regimens, then chromatin immunoprecipitation (ChIP) was performed with antibodies against indicated transcription factors and immunoprecipitated DNA fragments in parallel with total input DNA were analysed. Analysis was done by qPCR for the presence of the CX3CL1 promoter. The result is presented as percentage of input DNA. The data represent individual mean values of experimental replicates (3 experiments #1 to #3). b Analysis of the effect of inflammatory conditions on the binding of candidate transcription factors to the CX3CL1 promoter from a . Data are presented as mean ± s.d. ( n = 3 experiments). c Analysis of the binding of Nfκb and Gata3 to Cx3cl1 promoter in tumour associated endothelial cells. DLD1 and SKBR7 xenograft-bearing mice were treated either with the mixture of DC101 and bevacizumab (D/B mix) or control IgG for 24 h. Mice were killed, and tumours collected to isolate endothelial cells for ChIP analysis of Nfκb and Gata3. Data are presented as mean ± s.d. of experimental replicates ( n = 3). d Analysis of GATA3 downregulation in HUVEC with specific siRNA by western blotting and quantification. e Effect of GATA3 downregulation on CX3CL1 protein expression induced by TNF and TNF + IFNγ in HUVECs. f Quantification of GATA3 expression in e . g Role of GATA3 in HPMo transmigration induced by angiogenic factors-driven inflammation. The data are presented as mean ± s.d. of three independent experiments. * p

Techniques Used: Expressing, Binding Assay, Chromatin Immunoprecipitation, Immunoprecipitation, Real-time Polymerase Chain Reaction, Mouse Assay, Western Blot, Transmigration Assay

Blocking VEGF-A signalling inhibits homing of proangiogenic monocytes to tumours. a Analysis of Cx3cl1 protein expression in xenografts of SKBR7, DLD1 and HCT116 tumours. b Quantification of Cx3cl1 band intensity in a after normalisation with same sample Actin level. c Scheme of the study of VEGF-A implication in human monocyte recruitment in DLD1 xenograft. DLD1 xenograft-bearing mice were treated either with the mixture of DC101 and bevacizumab (D/B mix) or control IgG for 24 h before adoptive transfer of human pan monocytes. The mice were perfused with PBS-EDTA to wash out circulating and non-extravasated leucocytes before tumour collect and analysis. d Analysis of Cx3cl1 protein level in DLD1 tumour after 24 h of D/B mix treatment by western blotting. e Quantification of Cx3cl1 band intensity in d after normalisation with Pecam1 level in same samples. f Expression level of Cx3cl1 in tumour vasculature by immunofluorescence of DLD1 xenografts. Scale bar = 80 µm. g Quantification of Cx3cl1 in blood vessels. Data are presented as scatter dot plots with medians. Each dot represent the mean intensity of Cx3cl1 per µm 2 blood vessels from 10 sections of the same tumour. n = 5 tumours per group were used for this quantification. ** p
Figure Legend Snippet: Blocking VEGF-A signalling inhibits homing of proangiogenic monocytes to tumours. a Analysis of Cx3cl1 protein expression in xenografts of SKBR7, DLD1 and HCT116 tumours. b Quantification of Cx3cl1 band intensity in a after normalisation with same sample Actin level. c Scheme of the study of VEGF-A implication in human monocyte recruitment in DLD1 xenograft. DLD1 xenograft-bearing mice were treated either with the mixture of DC101 and bevacizumab (D/B mix) or control IgG for 24 h before adoptive transfer of human pan monocytes. The mice were perfused with PBS-EDTA to wash out circulating and non-extravasated leucocytes before tumour collect and analysis. d Analysis of Cx3cl1 protein level in DLD1 tumour after 24 h of D/B mix treatment by western blotting. e Quantification of Cx3cl1 band intensity in d after normalisation with Pecam1 level in same samples. f Expression level of Cx3cl1 in tumour vasculature by immunofluorescence of DLD1 xenografts. Scale bar = 80 µm. g Quantification of Cx3cl1 in blood vessels. Data are presented as scatter dot plots with medians. Each dot represent the mean intensity of Cx3cl1 per µm 2 blood vessels from 10 sections of the same tumour. n = 5 tumours per group were used for this quantification. ** p

Techniques Used: Blocking Assay, Expressing, Mouse Assay, Adoptive Transfer Assay, Western Blot, Immunofluorescence

Human tumours express a panel of cytokines and angiogenic factors. a Screening of human tumour cells-expressed cytokines (screened with human primers) in the xenografts ( n = 6 tumours per group pooled from two independent experiments). All molecule expression was normalised against the expression of housekeeping genes, namely GAPDH and ACTB . The expression levels of cytokines in DLD1 and SKBR7 xenografts are presented as relative (fold change) to their expression levels in HCT116 xenograft, which showed intermediate recruitment levels of proangiogenic monocytes. b Confirmation of cytokine expression level in tumour xenografts by ELISA. The protein levels of TNF, IFNγ and VEGF-A were quantified in protein extract of DLD1, HCT116 and SKBR7 xenografts ( n = 5 samples per tumour type) by ELISA. The data are presented as mean ± s.d., ** p -value
Figure Legend Snippet: Human tumours express a panel of cytokines and angiogenic factors. a Screening of human tumour cells-expressed cytokines (screened with human primers) in the xenografts ( n = 6 tumours per group pooled from two independent experiments). All molecule expression was normalised against the expression of housekeeping genes, namely GAPDH and ACTB . The expression levels of cytokines in DLD1 and SKBR7 xenografts are presented as relative (fold change) to their expression levels in HCT116 xenograft, which showed intermediate recruitment levels of proangiogenic monocytes. b Confirmation of cytokine expression level in tumour xenografts by ELISA. The protein levels of TNF, IFNγ and VEGF-A were quantified in protein extract of DLD1, HCT116 and SKBR7 xenografts ( n = 5 samples per tumour type) by ELISA. The data are presented as mean ± s.d., ** p -value

Techniques Used: Expressing, Enzyme-linked Immunosorbent Assay

37) Product Images from "Reduced microRNA-503 expression augments lung fibroblast VEGF production in chronic obstructive pulmonary disease"

Article Title: Reduced microRNA-503 expression augments lung fibroblast VEGF production in chronic obstructive pulmonary disease

Journal: PLoS ONE

doi: 10.1371/journal.pone.0184039

MiR-503 inhibits VEGF protein release and mRNA expression of human lung fibroblasts by direct binding to the 3’ untranslated region (UTR) of VEGF mRNA. (A-D) Primary human fetal lung fibroblasts (HFL-1 cells) cultured in monolayer were transfected with miR-503 mimic (Black bar) or control miRNA (White bar) transfection reagent, as described in Materials and Methods. 24hr after transfection, the medium was changed to DMEM containing 0.2% FCS, with or without IL-1ß and TNF-α (1 ng/ml), TGF-ß1 (1 ng/ml), or PGE 2 (1 x 10 −7 M). (A, B) 3 days after transfection, the culture medium was harvested and assayed for (A) VEGF or (B) IL-8 (with or without IL-1ß and TNF-α) by ELISA. (A) Vertical axis: VEGF release (pg per 10 5 cells per 2 days). (B) Vertical axis: IL-8 release (pg per 10 5 cells per 2 days). Horizontal axis: culture condition. (C) 1 day after transfection, RNA was isolated and endogenous miR-503 expression was analyzed in the presence of control miRNA or miR-503 mimic by real-time qPCR. Vertical axis: level of miR503 expression, expressed as fold of 18s-rRNA values. (D) 2 days after transfection, RNA was isolated and assayed for VEGF mRNA by real-time qPCR. Vertical axis: level of VEGF mRNA expression, expressed as fold of 18s-rRNA values. Horizontal axis: culture conditions. * p
Figure Legend Snippet: MiR-503 inhibits VEGF protein release and mRNA expression of human lung fibroblasts by direct binding to the 3’ untranslated region (UTR) of VEGF mRNA. (A-D) Primary human fetal lung fibroblasts (HFL-1 cells) cultured in monolayer were transfected with miR-503 mimic (Black bar) or control miRNA (White bar) transfection reagent, as described in Materials and Methods. 24hr after transfection, the medium was changed to DMEM containing 0.2% FCS, with or without IL-1ß and TNF-α (1 ng/ml), TGF-ß1 (1 ng/ml), or PGE 2 (1 x 10 −7 M). (A, B) 3 days after transfection, the culture medium was harvested and assayed for (A) VEGF or (B) IL-8 (with or without IL-1ß and TNF-α) by ELISA. (A) Vertical axis: VEGF release (pg per 10 5 cells per 2 days). (B) Vertical axis: IL-8 release (pg per 10 5 cells per 2 days). Horizontal axis: culture condition. (C) 1 day after transfection, RNA was isolated and endogenous miR-503 expression was analyzed in the presence of control miRNA or miR-503 mimic by real-time qPCR. Vertical axis: level of miR503 expression, expressed as fold of 18s-rRNA values. (D) 2 days after transfection, RNA was isolated and assayed for VEGF mRNA by real-time qPCR. Vertical axis: level of VEGF mRNA expression, expressed as fold of 18s-rRNA values. Horizontal axis: culture conditions. * p

Techniques Used: Expressing, Binding Assay, Cell Culture, Transfection, Enzyme-linked Immunosorbent Assay, Isolation, Real-time Polymerase Chain Reaction

38) Product Images from "The carboxyl terminal trimer of procollagen I induces pro-metastatic changes and vascularization in breast cancer cells xenografts"

Article Title: The carboxyl terminal trimer of procollagen I induces pro-metastatic changes and vascularization in breast cancer cells xenografts

Journal: BMC Cancer

doi: 10.1186/1471-2407-9-59

VEGF-A expression in PICP-treated and untreated contro-lateral tumors . Micrographs are representative of the results obtained from the analysis of at least two series of contro-lateral tumors treated or untreated with PICP. On the top is indicated the day after implant when the tumors were explanted. The two topmost rows are analysis by ISH and the two lower rows are analysis by immunoperoxidase for VEGF-A. Bar is 25 μm.
Figure Legend Snippet: VEGF-A expression in PICP-treated and untreated contro-lateral tumors . Micrographs are representative of the results obtained from the analysis of at least two series of contro-lateral tumors treated or untreated with PICP. On the top is indicated the day after implant when the tumors were explanted. The two topmost rows are analysis by ISH and the two lower rows are analysis by immunoperoxidase for VEGF-A. Bar is 25 μm.

Techniques Used: Expressing, In Situ Hybridization

39) Product Images from "Fibulin-5 Is Up-regulated by Hypoxia in Endothelial Cells through a Hypoxia-inducible Factor-1 (HIF-1?)-dependent Mechanism *"

Article Title: Fibulin-5 Is Up-regulated by Hypoxia in Endothelial Cells through a Hypoxia-inducible Factor-1 (HIF-1?)-dependent Mechanism *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M110.162917

HIF-2α silencing does not alter hypoxia-induced FBLN5 up-regulation. A , HIF-1α and HIF-2α mRNA levels, determined by real-time PCR, were analyzed in HUVEC transfected with a HIF-2α-specific siRNA (siHIF-2α) or a
Figure Legend Snippet: HIF-2α silencing does not alter hypoxia-induced FBLN5 up-regulation. A , HIF-1α and HIF-2α mRNA levels, determined by real-time PCR, were analyzed in HUVEC transfected with a HIF-2α-specific siRNA (siHIF-2α) or a

Techniques Used: Real-time Polymerase Chain Reaction, Transfection

40) Product Images from "Coculture of Stem Cells from Apical Papilla and Human Umbilical Vein Endothelial Cell Under Hypoxia Increases the Formation of Three-Dimensional Vessel-Like Structures in Vitro"

Article Title: Coculture of Stem Cells from Apical Papilla and Human Umbilical Vein Endothelial Cell Under Hypoxia Increases the Formation of Three-Dimensional Vessel-Like Structures in Vitro

Journal: Tissue Engineering. Part A

doi: 10.1089/ten.tea.2014.0058

Expression of HIF-1α, VEGF, and ephrinB2 after inhibiton of HIF-1α expression by YC-1. (A) Western blotting showed that overexpression of HIF-1α protein was blocked by 80 μM YC-1 after 4 h under hypoxia. (B) Real-time PCR results showed that the upregulated mRNA expression of ephrinB2 in SCAPs was reduced by 80 μM YC-1 after 2 h under hypoxia. (C) VEGF mRNA and protein expression after inhibition of HIF-1α expression (I) Real-time PCR results showed that the increased mRNA expression of VEGF in SCAPs was inhibited by 80 μM YC-1 after 2 h under hypoxia. (II) ELISA showed that the VEGF protein concentration was also reduced by 80 μM YC-1 after 24 h under hypoxia.
Figure Legend Snippet: Expression of HIF-1α, VEGF, and ephrinB2 after inhibiton of HIF-1α expression by YC-1. (A) Western blotting showed that overexpression of HIF-1α protein was blocked by 80 μM YC-1 after 4 h under hypoxia. (B) Real-time PCR results showed that the upregulated mRNA expression of ephrinB2 in SCAPs was reduced by 80 μM YC-1 after 2 h under hypoxia. (C) VEGF mRNA and protein expression after inhibition of HIF-1α expression (I) Real-time PCR results showed that the increased mRNA expression of VEGF in SCAPs was inhibited by 80 μM YC-1 after 2 h under hypoxia. (II) ELISA showed that the VEGF protein concentration was also reduced by 80 μM YC-1 after 24 h under hypoxia.

Techniques Used: Expressing, Western Blot, Over Expression, Real-time Polymerase Chain Reaction, Inhibition, Enzyme-linked Immunosorbent Assay, Protein Concentration

Protein and mRNA expression of vascular endothelial growth factor (VEGF) in SCAP and HUVEC. (A) Enzyme-linked immunosorbent assay (ELISA) was performed to detect VEGF protein concentration in supernatant of SCAP, HUVEC, and coculture of them under normoxia and hypoxia. (B) Magnetic beads were used to separate HUVEC from SCAP after 3 h of coculture under normoxia and hypoxia. VEGF mRNA expression in each cell type was detected using Real-time polymerase chain reaction (PCR) assay. SCAP isolated culture was regarded as control group, which were standardized to 1 (* p
Figure Legend Snippet: Protein and mRNA expression of vascular endothelial growth factor (VEGF) in SCAP and HUVEC. (A) Enzyme-linked immunosorbent assay (ELISA) was performed to detect VEGF protein concentration in supernatant of SCAP, HUVEC, and coculture of them under normoxia and hypoxia. (B) Magnetic beads were used to separate HUVEC from SCAP after 3 h of coculture under normoxia and hypoxia. VEGF mRNA expression in each cell type was detected using Real-time polymerase chain reaction (PCR) assay. SCAP isolated culture was regarded as control group, which were standardized to 1 (* p

Techniques Used: Expressing, Enzyme-linked Immunosorbent Assay, Protein Concentration, Magnetic Beads, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, Isolation

Related Articles

Immunohistochemistry:

Article Title: Nephron-deficient Fvb mice develop rapidly progressive renal failure and heavy albuminuria involving excess glomerular GLUT1 and VEGF
Article Snippet: .. Immunohistochemical staining with various antibodies including GLUT1 (1:400, Alpha Diagnostic International, San Antonio, TX, USA), VEGF (1:200, Lab Vision, CA, USA), tenascin, phospho-PKCα (p-PKCα), and PKCβ1 (all 1:100, Santa Cruz Biotechnology, CA, USA), fibronectin (FN) and laminin (both 1:100, Sigma-Aldrich, St Louis, MO, USA), collagen IV (1:100, Santa Cruz Biotechnology, against α-1, α-3, and α-5 chains), CD2AP (1:100, Abcam, Cambridge, MA, USA), nephrin and podocin (both 1:100, kind gifts from Dr L Holzman, University of Michigan) was conducted using a Vectastain Elite ABC Kit (Vector Laboratories, Burlingame, CA, USA) with some modifications. .. Briefly, 5 µm sections were deparaffinized, and endogenous peroxidase activity was ablated by incubation in 3% hydrogen peroxide in TBS (pH 7.4).

Incubation:

Article Title: Survivin Modulates Squamous Cell Carcinoma-Derived Stem-Like Cell Proliferation, Viability and Tumor Formation in Vivo
Article Snippet: .. Then, slides were incubated for 15 min with 0.5% bovine serum albumin and 5% goat serum, and for 60 min at 37 °C with the rabbit polyclonal anti-human Survivin antibody (1:50, Novus, Bloomington, MN, USA) or HIF1α (1:500, Novus, Bloomington, MN, USA), VEGF (1:50, Thermo Fisher Scientific, Waltham, MA, USA), CD51 (1:50, Abcam, Cambridge, UK). .. After four washes in PBS, samples were incubated for 60 min with the anti-rabbit secondary antibody, Alexa Fluor 546 (1:100, Thermo Fisher Scientific, Waltham, MA, USA).

Article Title: C-Kit Promotes Growth and Migration of Human Cardiac Progenitor Cells via the PI3K-AKT and MEK-ERK Pathways
Article Snippet: .. For bFGF and VEGF treatments, CPCs were incubated with 50 ng/ml bFGF (Peprotech) or 20 ng/ml VEGF (Invitrogen) either alone or in combination with SCF as indicated. ..

Proliferation Assay:

Article Title: Fibroblast Growth Factor Receptor-Dependent and -Independent Paracrine Signaling by Sunitinib-Resistant Renal Cell Carcinoma
Article Snippet: .. ECM was replaced by EC proliferation assay medium (M199 [Life Technologies] with 10% FBS and 1% P/S)-containing vehicle, sunitinib (100 nM), or dovitinib (LC Laboratories) (500 nM) without or with VEGF (Life Technologies) (100 ng/ml) and/or FGF2 (PeproTech) (50 ng/ml) as indicated. .. To determine the 50% inhibitory concentration (IC50 ) of dovitinib for RCC cells, dovitinib was serially diluted in DMSO and cells were incubated for 4 days and assayed as described above.

Expressing:

Article Title: Granulocyte-macrophage colony-stimulating factor-transfected bone marrow stromal cells for the treatment of ischemic stroke ☆
Article Snippet: .. Reverse transcription-PCR detection of vascular endothelial growth factor mRNA To measure vascular endothelial growth factor mRNA expression, total RNA samples were extracted from the ischemic region at baseline (before MCAO; normal tissue), 24 hours after MCAO (before implantation), and 48 hours after MCAO in all groups using Trizol reagent (Invitrogen, Carlsbad, CA, USA). .. Vascular endothelial growth factor primers are as follows: forward: 5’-GTC CAA TTG AGA CCC TGG TG-3’; reverse: 5’-CTA TGT GCT GGC TTT GGT GA-3’.

Article Title: Prognostic significance of Cytokeratin 20-positive lymph node vascular endothelial growth factor A mRNA and chromodomain helicase DNA binding protein 4 in pN0 colorectal cancer patients
Article Snippet: .. Validation of vascular endothelial growth factor A (VEGF-A) mRNA and transcription factor 20 (TCF20) mRNA in the second cohort of PELS TaqMan gene expression assays of specific primers and MGB probes were purchased from Applied Biosystems for the detection of VEGF-A mRNA (Hs00900054_m1) and TCF20 mRNA (Hs00390028_m1). .. QRT-PCR was performed in a reaction volume of 50 μL using Taqman Universal PCR Master Mix (Cat. no. 4304437; Applied Biosystems), and 10 μL of cDNA was used for each reaction.

Article Title: Inhibition of androgen-responsive LNCaP prostate cancer cell tumor xenograft growth by dietary phenethyl isothiocyanate correlates with decreased angiogenesis and inhibition of cell attachment
Article Snippet: .. TaqMan gene expression assay primers and probes for human prostate specific antigen (PSA) and glyceraldehydes-3-phosphate dehydrogenase (G3PDH), proliferating cell nuclear antigen (PCNA), Ki-67, integrin β1 (ITGB1), integrin α2 (ITGA2), integrin α5 (ITGA5), integrin α6 (ITGA6), mouse and human vascular endothelial growth factor (VEGF) A, mouse PECAM-1 and mouse and human TATA binding protein were purchased from Applied Biosystems (Foster City, CA). .. G3PDH was used as a housekeeping gene for calculation of relative expression levels in vitro and mouse or human TATA binding protein was used for tumor samples.

Staining:

Article Title: Nephron-deficient Fvb mice develop rapidly progressive renal failure and heavy albuminuria involving excess glomerular GLUT1 and VEGF
Article Snippet: .. Immunohistochemical staining with various antibodies including GLUT1 (1:400, Alpha Diagnostic International, San Antonio, TX, USA), VEGF (1:200, Lab Vision, CA, USA), tenascin, phospho-PKCα (p-PKCα), and PKCβ1 (all 1:100, Santa Cruz Biotechnology, CA, USA), fibronectin (FN) and laminin (both 1:100, Sigma-Aldrich, St Louis, MO, USA), collagen IV (1:100, Santa Cruz Biotechnology, against α-1, α-3, and α-5 chains), CD2AP (1:100, Abcam, Cambridge, MA, USA), nephrin and podocin (both 1:100, kind gifts from Dr L Holzman, University of Michigan) was conducted using a Vectastain Elite ABC Kit (Vector Laboratories, Burlingame, CA, USA) with some modifications. .. Briefly, 5 µm sections were deparaffinized, and endogenous peroxidase activity was ablated by incubation in 3% hydrogen peroxide in TBS (pH 7.4).

Binding Assay:

Article Title: Inhibition of androgen-responsive LNCaP prostate cancer cell tumor xenograft growth by dietary phenethyl isothiocyanate correlates with decreased angiogenesis and inhibition of cell attachment
Article Snippet: .. TaqMan gene expression assay primers and probes for human prostate specific antigen (PSA) and glyceraldehydes-3-phosphate dehydrogenase (G3PDH), proliferating cell nuclear antigen (PCNA), Ki-67, integrin β1 (ITGB1), integrin α2 (ITGA2), integrin α5 (ITGA5), integrin α6 (ITGA6), mouse and human vascular endothelial growth factor (VEGF) A, mouse PECAM-1 and mouse and human TATA binding protein were purchased from Applied Biosystems (Foster City, CA). .. G3PDH was used as a housekeeping gene for calculation of relative expression levels in vitro and mouse or human TATA binding protein was used for tumor samples.

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    Thermo Fisher gene exp epo mm00433126 m1
    Roxadustat stimulates circulating <t>EPO</t> production without affecting plasma VEGF or the growth or onset of NeuYD tumors. Notes: Changes in body weight ( A ; n=20), tumor onset ( B ; n=20), tumor doubling times ( C ; n=11–14), and plasma cytokines ( D ; 6 h post-final dose, n=20) are shown for NeuYD mice treated with vehicle (●, solid black lines, open bars), 40 mg/kg roxadustat (▼, solid gray lines, light gray bars), and 80 mg/kg roxadustat (▲, dashed lines, dark gray bars). Final hematology was not available for this study. Data indicate mean ± SE ( A, D ) or median plus data for individual biologic replicates ( C ). * p
    Gene Exp Epo Mm00433126 M1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 85/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher vegf a mrna level
    <t>VEGF-A</t> expression following modification of PPARgamma activity in A549 lung adenocarcinoma cell line tranfected with PPRE control or reporter plasmid. a, Mimicking beta-catenin dependent canonical Wnt pathway activation using 10 mM LiCl led to significant decrease in PPRE reporter activity. b, 10 mM LiCl treatment induced VEGF-A <t>mRNA</t> expression compared to PPRE control cells and also c, 10 mM LiCl increased VEGF-A protein levels. Error bars, SEM. One-way ANOVA, post hoc Bonferroni; n = 4. Scale bars, 20 μm. d, VEGF-A mRNA expression decreased after 10 μM PPARgamma agonist treatment (RSG), while 10 μm PPARgamma specific antagonist (GW9662) increased VEGF-A transcript levels. Independent samples t -test, n = 3. e, VEGF-A protein level shows similar pattern after 10 μm RSG and 10 μm GW9662 treatment. Fluorescence intensity are representations of three different experiments as mean ± SEM. One-way ANOVA, post hoc Bonferroni; n = 3. Scale bars, 20 μm. P
    Vegf A Mrna Level, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher vegf mrna
    The effect long-term DMI, FLX or TIA treatment on the expression of <t>VEGF</t> <t>mRNA</t> copies in the (A) hippocampus, (B) amygdala and (C) hypothalamus of female rats subjected to chronic social instability stress. Data is presented as the mean ± standard error of the mean. Differences between groups were statistically analyzed using one-way analysis of the variance followed by a Tukey's range test. *P
    Vegf Mrna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 89/100, based on 17 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Roxadustat stimulates circulating EPO production without affecting plasma VEGF or the growth or onset of NeuYD tumors. Notes: Changes in body weight ( A ; n=20), tumor onset ( B ; n=20), tumor doubling times ( C ; n=11–14), and plasma cytokines ( D ; 6 h post-final dose, n=20) are shown for NeuYD mice treated with vehicle (●, solid black lines, open bars), 40 mg/kg roxadustat (▼, solid gray lines, light gray bars), and 80 mg/kg roxadustat (▲, dashed lines, dark gray bars). Final hematology was not available for this study. Data indicate mean ± SE ( A, D ) or median plus data for individual biologic replicates ( C ). * p

    Journal: Hypoxia

    Article Title: Induction of erythropoiesis by hypoxia-inducible factor prolyl hydroxylase inhibitors without promotion of tumor initiation, progression, or metastasis in a VEGF-sensitive model of spontaneous breast cancer

    doi: 10.2147/HP.S130526

    Figure Lengend Snippet: Roxadustat stimulates circulating EPO production without affecting plasma VEGF or the growth or onset of NeuYD tumors. Notes: Changes in body weight ( A ; n=20), tumor onset ( B ; n=20), tumor doubling times ( C ; n=11–14), and plasma cytokines ( D ; 6 h post-final dose, n=20) are shown for NeuYD mice treated with vehicle (●, solid black lines, open bars), 40 mg/kg roxadustat (▼, solid gray lines, light gray bars), and 80 mg/kg roxadustat (▲, dashed lines, dark gray bars). Final hematology was not available for this study. Data indicate mean ± SE ( A, D ) or median plus data for individual biologic replicates ( C ). * p

    Article Snippet: VEGF and EPO mRNA levels were determined from total kidney mRNA by reverse transcription-PCR and normalized to β-actin mRNA (ABI; Mm00433126_m1, EPO; Mm00437304_m1, VEGF; and 4352341E, β-actin).

    Techniques: Mouse Assay

    Oral administration of HIF-PHI FG-4497 to normal FVB mice induces transient increases in kidney EPO mRNA and circulating EPO protein. Notes: Mean time-course plots and individual data-points for each time-point (n=3) are shown as well as the 95% CI (gray area) for 24 h vehicle controls. Abbreviations: CI, confidence interval; EPO, erythropoietin; HIF-PHI, hypoxia-inducible factor prolyl hydroxylase inhibitor; mRNA, messenger RNA; VEGF, vascular endothelial growth factor.

    Journal: Hypoxia

    Article Title: Induction of erythropoiesis by hypoxia-inducible factor prolyl hydroxylase inhibitors without promotion of tumor initiation, progression, or metastasis in a VEGF-sensitive model of spontaneous breast cancer

    doi: 10.2147/HP.S130526

    Figure Lengend Snippet: Oral administration of HIF-PHI FG-4497 to normal FVB mice induces transient increases in kidney EPO mRNA and circulating EPO protein. Notes: Mean time-course plots and individual data-points for each time-point (n=3) are shown as well as the 95% CI (gray area) for 24 h vehicle controls. Abbreviations: CI, confidence interval; EPO, erythropoietin; HIF-PHI, hypoxia-inducible factor prolyl hydroxylase inhibitor; mRNA, messenger RNA; VEGF, vascular endothelial growth factor.

    Article Snippet: VEGF and EPO mRNA levels were determined from total kidney mRNA by reverse transcription-PCR and normalized to β-actin mRNA (ABI; Mm00433126_m1, EPO; Mm00437304_m1, VEGF; and 4352341E, β-actin).

    Techniques: Mouse Assay

    VEGF-A expression following modification of PPARgamma activity in A549 lung adenocarcinoma cell line tranfected with PPRE control or reporter plasmid. a, Mimicking beta-catenin dependent canonical Wnt pathway activation using 10 mM LiCl led to significant decrease in PPRE reporter activity. b, 10 mM LiCl treatment induced VEGF-A mRNA expression compared to PPRE control cells and also c, 10 mM LiCl increased VEGF-A protein levels. Error bars, SEM. One-way ANOVA, post hoc Bonferroni; n = 4. Scale bars, 20 μm. d, VEGF-A mRNA expression decreased after 10 μM PPARgamma agonist treatment (RSG), while 10 μm PPARgamma specific antagonist (GW9662) increased VEGF-A transcript levels. Independent samples t -test, n = 3. e, VEGF-A protein level shows similar pattern after 10 μm RSG and 10 μm GW9662 treatment. Fluorescence intensity are representations of three different experiments as mean ± SEM. One-way ANOVA, post hoc Bonferroni; n = 3. Scale bars, 20 μm. P

    Journal: BMC Cancer

    Article Title: Increased Wnt5a in squamous cell lung carcinoma inhibits endothelial cell motility

    doi: 10.1186/s12885-016-2943-4

    Figure Lengend Snippet: VEGF-A expression following modification of PPARgamma activity in A549 lung adenocarcinoma cell line tranfected with PPRE control or reporter plasmid. a, Mimicking beta-catenin dependent canonical Wnt pathway activation using 10 mM LiCl led to significant decrease in PPRE reporter activity. b, 10 mM LiCl treatment induced VEGF-A mRNA expression compared to PPRE control cells and also c, 10 mM LiCl increased VEGF-A protein levels. Error bars, SEM. One-way ANOVA, post hoc Bonferroni; n = 4. Scale bars, 20 μm. d, VEGF-A mRNA expression decreased after 10 μM PPARgamma agonist treatment (RSG), while 10 μm PPARgamma specific antagonist (GW9662) increased VEGF-A transcript levels. Independent samples t -test, n = 3. e, VEGF-A protein level shows similar pattern after 10 μm RSG and 10 μm GW9662 treatment. Fluorescence intensity are representations of three different experiments as mean ± SEM. One-way ANOVA, post hoc Bonferroni; n = 3. Scale bars, 20 μm. P

    Article Snippet: VEGF-A mRNA level was determined using real-time quantitative PCR, while miR-27b expression was measured by Taqman MicroRNA Assay (Thermo Fisher Scientific, Waltham, USA).

    Techniques: Expressing, Modification, Activity Assay, Plasmid Preparation, Activation Assay, Fluorescence

    The effect of Wnt5a on VEGF-A induced endothelial cell activation and motility. a, CD105 mRNA expression is significantly higher in primary AC compared to SCC samples. Error bars, SEM. One-way ANOVA, post hoc Bonferroni; n = 11 and n = 12 per groups. b, Flow cytometric analysis of CD105 protein expression in CD31 positive endothelial cells in primary AC and SCC samples. n = 6 per groups. c, Flow cytometric analysis of CD105 levels in normal and high VEGF-A microenvironment in 3D lung aggregate tissues has also shown an increase of activation marker CD105 in VEGF-A high tissues. The double positive (CD105/CD31) cell population was considered as activated endothelial cells. Independent samples t -test, n = 6. 1 μg/ml rhWnt5a treatment had no effect on the VEGF-A induced endothelial cell activation measured by the double positive (CD105/CD31) cell population identified by flow cytometric analysis. Independent samples t -test, n = 6. d, Localization of endothelial cells was identified by immunoflurescent staining of CD31 and analyzed by confocal microscopy in 3D lung tissue aggregates. In VEGF-A normal microenvironment endothelial cells remained diffuse in the tissue. Under VEGF-A excess endothelial cell migrated towards the source (VEGF-A high fibroblasts) of the signal in the center of the aggregate tissue. 1 μg/ml rhWnt5a treatment of VEGF-A high tissue aggregates inhibited endothelial cell accumulation in the center of the aggregate. Bar chart represents the quantification of endothelial cell distribution. Relative area of CD31+ endothelial cells are compared to total field. Percentages were calculated as relative area of endothelial cells/area of total field *100. Error bars; SEM. Independent samples t -test n = 3. Representative images of three independent experiments are shown. Scale bars, 50 μm. e , HMVEC-L transwell migration assay. Endothelial cells migrate significantly faster towards VEGF-A high fibroblast, while 1 μg/ml rhWnt5a can reverse the effect of elevated VEGF-A level. Scale bar 100 μm. One-way ANOVA, n = 3. P

    Journal: BMC Cancer

    Article Title: Increased Wnt5a in squamous cell lung carcinoma inhibits endothelial cell motility

    doi: 10.1186/s12885-016-2943-4

    Figure Lengend Snippet: The effect of Wnt5a on VEGF-A induced endothelial cell activation and motility. a, CD105 mRNA expression is significantly higher in primary AC compared to SCC samples. Error bars, SEM. One-way ANOVA, post hoc Bonferroni; n = 11 and n = 12 per groups. b, Flow cytometric analysis of CD105 protein expression in CD31 positive endothelial cells in primary AC and SCC samples. n = 6 per groups. c, Flow cytometric analysis of CD105 levels in normal and high VEGF-A microenvironment in 3D lung aggregate tissues has also shown an increase of activation marker CD105 in VEGF-A high tissues. The double positive (CD105/CD31) cell population was considered as activated endothelial cells. Independent samples t -test, n = 6. 1 μg/ml rhWnt5a treatment had no effect on the VEGF-A induced endothelial cell activation measured by the double positive (CD105/CD31) cell population identified by flow cytometric analysis. Independent samples t -test, n = 6. d, Localization of endothelial cells was identified by immunoflurescent staining of CD31 and analyzed by confocal microscopy in 3D lung tissue aggregates. In VEGF-A normal microenvironment endothelial cells remained diffuse in the tissue. Under VEGF-A excess endothelial cell migrated towards the source (VEGF-A high fibroblasts) of the signal in the center of the aggregate tissue. 1 μg/ml rhWnt5a treatment of VEGF-A high tissue aggregates inhibited endothelial cell accumulation in the center of the aggregate. Bar chart represents the quantification of endothelial cell distribution. Relative area of CD31+ endothelial cells are compared to total field. Percentages were calculated as relative area of endothelial cells/area of total field *100. Error bars; SEM. Independent samples t -test n = 3. Representative images of three independent experiments are shown. Scale bars, 50 μm. e , HMVEC-L transwell migration assay. Endothelial cells migrate significantly faster towards VEGF-A high fibroblast, while 1 μg/ml rhWnt5a can reverse the effect of elevated VEGF-A level. Scale bar 100 μm. One-way ANOVA, n = 3. P

    Article Snippet: VEGF-A mRNA level was determined using real-time quantitative PCR, while miR-27b expression was measured by Taqman MicroRNA Assay (Thermo Fisher Scientific, Waltham, USA).

    Techniques: Activation Assay, Expressing, Flow Cytometry, Marker, Staining, Confocal Microscopy, Transwell Migration Assay

    Transcript analysis of Wnt5a, miRNA and VEGF-A in primary human lung cancer samples of AC and SCC and 3D in vitro lung aggregate cultures. a, Wnt5a mRNA is significantly upregulated in SCC compared to both normal lung and AC specimens. Error bars, SEM. One-way ANOVA, post hoc Bonferroni; n = 11 and n = 12 per groups. b, Immunohistochemical staining of Wnt5a in primary resected AC and SCC samples, n = 3 per groups. c, miR-27b and miR-200b expression levels are significantly lower in AC compared to SCC. Error bars; SEM. Independent samples t -test, n = 5 per groups. d, miR-27b is up-regulated by rhWnt5a in 3D lung aggregate cultures, while neither miR-27b nor miR-200b was affected by rhWnt11. Error bars; SEM. One-way ANOVA, post hoc Bonferroni, n = 6. e, VEGF-A and miR-27b expression levels after 10 μM RSG (PPARgamma agonist) and 10 μM GW9662 (PPARgamma antagonist) and combination treatment with rhWnt5a. Error bars; SEM. Independent samples t -test n = 3. P

    Journal: BMC Cancer

    Article Title: Increased Wnt5a in squamous cell lung carcinoma inhibits endothelial cell motility

    doi: 10.1186/s12885-016-2943-4

    Figure Lengend Snippet: Transcript analysis of Wnt5a, miRNA and VEGF-A in primary human lung cancer samples of AC and SCC and 3D in vitro lung aggregate cultures. a, Wnt5a mRNA is significantly upregulated in SCC compared to both normal lung and AC specimens. Error bars, SEM. One-way ANOVA, post hoc Bonferroni; n = 11 and n = 12 per groups. b, Immunohistochemical staining of Wnt5a in primary resected AC and SCC samples, n = 3 per groups. c, miR-27b and miR-200b expression levels are significantly lower in AC compared to SCC. Error bars; SEM. Independent samples t -test, n = 5 per groups. d, miR-27b is up-regulated by rhWnt5a in 3D lung aggregate cultures, while neither miR-27b nor miR-200b was affected by rhWnt11. Error bars; SEM. One-way ANOVA, post hoc Bonferroni, n = 6. e, VEGF-A and miR-27b expression levels after 10 μM RSG (PPARgamma agonist) and 10 μM GW9662 (PPARgamma antagonist) and combination treatment with rhWnt5a. Error bars; SEM. Independent samples t -test n = 3. P

    Article Snippet: VEGF-A mRNA level was determined using real-time quantitative PCR, while miR-27b expression was measured by Taqman MicroRNA Assay (Thermo Fisher Scientific, Waltham, USA).

    Techniques: In Vitro, Immunohistochemistry, Staining, Expressing

    The effect long-term DMI, FLX or TIA treatment on the expression of VEGF mRNA copies in the (A) hippocampus, (B) amygdala and (C) hypothalamus of female rats subjected to chronic social instability stress. Data is presented as the mean ± standard error of the mean. Differences between groups were statistically analyzed using one-way analysis of the variance followed by a Tukey's range test. *P

    Journal: Experimental and Therapeutic Medicine

    Article Title: Alterations in VEGF expression induced by antidepressant drugs in female rats under chronic social stress

    doi: 10.3892/etm.2017.4022

    Figure Lengend Snippet: The effect long-term DMI, FLX or TIA treatment on the expression of VEGF mRNA copies in the (A) hippocampus, (B) amygdala and (C) hypothalamus of female rats subjected to chronic social instability stress. Data is presented as the mean ± standard error of the mean. Differences between groups were statistically analyzed using one-way analysis of the variance followed by a Tukey's range test. *P

    Article Snippet: TaqMan Gene Expression Assay primers and probes for amplification of VEGF mRNA were obtained from Thermo Fisher Scientific, Inc. RT-qPCR was performed using TaqMan One-Step RT-PCR Master Mix Reagents and the ABI Prism 7700 Sequence Detection system (both Thermo Fisher Scientific, Inc.).

    Techniques: Expressing

    Effect of CoCl 2 -induced hypoxia on HIF-1α, IGF-1R and VEGF protein expression. (A) The HepG2 cells were treated with 0, 50, 100, 200, 400 or 800 μmol/l CoCl 2 for 12 h. (B) The HepG2 cells were treated for 0,4,8,12, 24 or 48 h with 200 μmol/l CoCl 2 . A significant difference in HIF-1α, IGF-1R and VEGF mRNA expression was observed when cells were treated with 200 and 400 μmol/l CoCl 2 , and for 8, 12, 24 and 48 h, compared with the control (P

    Journal: Oncology Letters

    Article Title: Effect of hypoxia on hypoxia inducible factor-1α, insulin-like growth factor I and vascular endothelial growth factor expression in hepatocellular carcinoma HepG2 cells

    doi: 10.3892/ol.2015.2879

    Figure Lengend Snippet: Effect of CoCl 2 -induced hypoxia on HIF-1α, IGF-1R and VEGF protein expression. (A) The HepG2 cells were treated with 0, 50, 100, 200, 400 or 800 μmol/l CoCl 2 for 12 h. (B) The HepG2 cells were treated for 0,4,8,12, 24 or 48 h with 200 μmol/l CoCl 2 . A significant difference in HIF-1α, IGF-1R and VEGF mRNA expression was observed when cells were treated with 200 and 400 μmol/l CoCl 2 , and for 8, 12, 24 and 48 h, compared with the control (P

    Article Snippet: Quantitative PCR analysis of HIF-1α, IGF-1R and VEGF mRNA levels was performed using the One-step RT-PCR kit from Thermo Fisher Scientific, according to the manufacturer’s instructions.

    Techniques: Expressing

    Effect of CoCl 2 - induced hypoxia on the expression of HIF-1α, IGF-1R and VEGF mRNA. (A) The HepG2 cells were treated with 0, 50, 100, 200, 400 or 800 μmol/l CoCl 2 for 12 h. (B) The HepG2 cells were treated for 0, 4, 8, 12, 24 or 48 h with 200 μmol/l CoCl 2 . A significant difference in HIF-1α, IGF-1R and VEGF protein expression was observed when cells were treated with 200 and 400 μmol/l CoCl 2 , and for 12, 24 and 48 h, compared with the control (P

    Journal: Oncology Letters

    Article Title: Effect of hypoxia on hypoxia inducible factor-1α, insulin-like growth factor I and vascular endothelial growth factor expression in hepatocellular carcinoma HepG2 cells

    doi: 10.3892/ol.2015.2879

    Figure Lengend Snippet: Effect of CoCl 2 - induced hypoxia on the expression of HIF-1α, IGF-1R and VEGF mRNA. (A) The HepG2 cells were treated with 0, 50, 100, 200, 400 or 800 μmol/l CoCl 2 for 12 h. (B) The HepG2 cells were treated for 0, 4, 8, 12, 24 or 48 h with 200 μmol/l CoCl 2 . A significant difference in HIF-1α, IGF-1R and VEGF protein expression was observed when cells were treated with 200 and 400 μmol/l CoCl 2 , and for 12, 24 and 48 h, compared with the control (P

    Article Snippet: Quantitative PCR analysis of HIF-1α, IGF-1R and VEGF mRNA levels was performed using the One-step RT-PCR kit from Thermo Fisher Scientific, according to the manufacturer’s instructions.

    Techniques: Expressing