Structured Review

Cell Signaling Technology Inc anti icam 1
Effect of 9t18:1 and 11t18:1 on gene expression of <t>ICAM-1,</t> VCAM-1 and IL-6 of leptin/9c11t-CLA treated HUVECs. ( A ) The effect of 11t18:1 on expression of ICAM-1, VCAM-1 and IL-6 in leptin treated HUVECs. HUVECs were treated or without leptin (75 nmol/L) for 24 h and then cultured with 11t18:1(25, 50, 100 μmol/L) for 24 h. ( B ) The effect of 9t18:1 on expression of ICAM-1, VCAM-1 and IL-6 in 9c11tCLA treated HUVECs. HUVECs were treated or non-treated with 9c11tCLA (5, 25, 50 μmol/L) and then cultured with 9t18:1 (100 μmol/L) for 24 h. ( C ) The effect of 11t18:1 + leptin on expression of ICAM-1, VCAM-1 and IL-6 in 9c11tCLA treated HUVECs. HUVECs were treated or non-treated with 9c11tCLA (5, 25, 50 μmol/L) and then cultured with the group of 11t18:1 (100 μmol/L) + leptin (75 nmol/L) for 24 h. ( D ) The effect of 9t18:1 on expression of ICAM-1, VCAM-1 and IL-6 of leptin treated HUVECs. HUVECs were treated or non-treated with leptin (75 nmol/L) and then cultured with 9t18:1 (100 μmol/L) and 11t18:1 (100 μmol/L) for 24 h. a–g Data were presented as mean ± SD, values not sharing a common superscript denote significant difference ( P
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1) Product Images from "9c11tCLA modulates 11t18:1 and 9t18:1 induced inflammations differently in human umbilical vein endothelial cells"

Article Title: 9c11tCLA modulates 11t18:1 and 9t18:1 induced inflammations differently in human umbilical vein endothelial cells

Journal: Scientific Reports

doi: 10.1038/s41598-018-19729-9

Effect of 9t18:1 and 11t18:1 on gene expression of ICAM-1, VCAM-1 and IL-6 of leptin/9c11t-CLA treated HUVECs. ( A ) The effect of 11t18:1 on expression of ICAM-1, VCAM-1 and IL-6 in leptin treated HUVECs. HUVECs were treated or without leptin (75 nmol/L) for 24 h and then cultured with 11t18:1(25, 50, 100 μmol/L) for 24 h. ( B ) The effect of 9t18:1 on expression of ICAM-1, VCAM-1 and IL-6 in 9c11tCLA treated HUVECs. HUVECs were treated or non-treated with 9c11tCLA (5, 25, 50 μmol/L) and then cultured with 9t18:1 (100 μmol/L) for 24 h. ( C ) The effect of 11t18:1 + leptin on expression of ICAM-1, VCAM-1 and IL-6 in 9c11tCLA treated HUVECs. HUVECs were treated or non-treated with 9c11tCLA (5, 25, 50 μmol/L) and then cultured with the group of 11t18:1 (100 μmol/L) + leptin (75 nmol/L) for 24 h. ( D ) The effect of 9t18:1 on expression of ICAM-1, VCAM-1 and IL-6 of leptin treated HUVECs. HUVECs were treated or non-treated with leptin (75 nmol/L) and then cultured with 9t18:1 (100 μmol/L) and 11t18:1 (100 μmol/L) for 24 h. a–g Data were presented as mean ± SD, values not sharing a common superscript denote significant difference ( P
Figure Legend Snippet: Effect of 9t18:1 and 11t18:1 on gene expression of ICAM-1, VCAM-1 and IL-6 of leptin/9c11t-CLA treated HUVECs. ( A ) The effect of 11t18:1 on expression of ICAM-1, VCAM-1 and IL-6 in leptin treated HUVECs. HUVECs were treated or without leptin (75 nmol/L) for 24 h and then cultured with 11t18:1(25, 50, 100 μmol/L) for 24 h. ( B ) The effect of 9t18:1 on expression of ICAM-1, VCAM-1 and IL-6 in 9c11tCLA treated HUVECs. HUVECs were treated or non-treated with 9c11tCLA (5, 25, 50 μmol/L) and then cultured with 9t18:1 (100 μmol/L) for 24 h. ( C ) The effect of 11t18:1 + leptin on expression of ICAM-1, VCAM-1 and IL-6 in 9c11tCLA treated HUVECs. HUVECs were treated or non-treated with 9c11tCLA (5, 25, 50 μmol/L) and then cultured with the group of 11t18:1 (100 μmol/L) + leptin (75 nmol/L) for 24 h. ( D ) The effect of 9t18:1 on expression of ICAM-1, VCAM-1 and IL-6 of leptin treated HUVECs. HUVECs were treated or non-treated with leptin (75 nmol/L) and then cultured with 9t18:1 (100 μmol/L) and 11t18:1 (100 μmol/L) for 24 h. a–g Data were presented as mean ± SD, values not sharing a common superscript denote significant difference ( P

Techniques Used: Expressing, Cell Culture

Effect of TAK242 on MAPKs phosphorylation in HUVECs treated with 9t18:1 and 11t18:1. ( A ) Effect of TAK242 on ICAM-1 expression. HUVECs were treated with TAK242 (0.5, 1, 1.5 μmol/L) for 30 min and then cultured with 9t18:1 for 24 h. ( B ) Effect of TAK242 on MAPKs phosphorylation in HUVECs treated with 9t18:1 and 11t18:1. HUVECs were treated with TAK242 (1 μmol/L) for 30 min and then cultured with TFA for 24 h. Values labeled with different letters in each set indicate significant differences ( p
Figure Legend Snippet: Effect of TAK242 on MAPKs phosphorylation in HUVECs treated with 9t18:1 and 11t18:1. ( A ) Effect of TAK242 on ICAM-1 expression. HUVECs were treated with TAK242 (0.5, 1, 1.5 μmol/L) for 30 min and then cultured with 9t18:1 for 24 h. ( B ) Effect of TAK242 on MAPKs phosphorylation in HUVECs treated with 9t18:1 and 11t18:1. HUVECs were treated with TAK242 (1 μmol/L) for 30 min and then cultured with TFA for 24 h. Values labeled with different letters in each set indicate significant differences ( p

Techniques Used: Expressing, Cell Culture, Labeling

2) Product Images from "Enhancement of Programmed Death Ligand 2 on Hepatitis C Virus Infected Hepatocytes by Calcineurin Inhibitors"

Article Title: Enhancement of Programmed Death Ligand 2 on Hepatitis C Virus Infected Hepatocytes by Calcineurin Inhibitors

Journal: Transplantation

doi: 10.1097/TP.0000000000000572

The effect of increased PD-L2 expression on immunological parameters. A, RT-PCR and Western blotting were performed to confirm the efficiency of PD-L2 containing plasmid transfection. The ability of the PD-L2 containing plasmid to induce PD-L2 mRNA expression was confirmed in two cell lines, with the more efficiently transfected Huh7 cells used for confirmation of protein expression. B, Representative flow cytometric analysis of Huh7 cells transfected with PD-L2 containing plasmid (green) and control plasmid (purple). Expressions of Fas, CD1d, HLA-class 1 and intercellular adhesion molecule-1 (ICAM-1) are shown. C, The mean fluorescence intensity (MFI) is presented as fold increase relative to control cells. The data represent mean ± SD of triplicate measurements. D, Immunohistochemical staining pattern of ICAM-1. Left panel is the representative staining pattern of ICAM-1 in liver tissue. Expression intensity was scored as follows: 0, negative; 1, weak expression; 2, moderate expression; 3, strong expression. Right: The concordance coefficients (κ statistics) were used to evaluate agreement between elevated PD-L2 expression and elevated ICAM-1 expression.
Figure Legend Snippet: The effect of increased PD-L2 expression on immunological parameters. A, RT-PCR and Western blotting were performed to confirm the efficiency of PD-L2 containing plasmid transfection. The ability of the PD-L2 containing plasmid to induce PD-L2 mRNA expression was confirmed in two cell lines, with the more efficiently transfected Huh7 cells used for confirmation of protein expression. B, Representative flow cytometric analysis of Huh7 cells transfected with PD-L2 containing plasmid (green) and control plasmid (purple). Expressions of Fas, CD1d, HLA-class 1 and intercellular adhesion molecule-1 (ICAM-1) are shown. C, The mean fluorescence intensity (MFI) is presented as fold increase relative to control cells. The data represent mean ± SD of triplicate measurements. D, Immunohistochemical staining pattern of ICAM-1. Left panel is the representative staining pattern of ICAM-1 in liver tissue. Expression intensity was scored as follows: 0, negative; 1, weak expression; 2, moderate expression; 3, strong expression. Right: The concordance coefficients (κ statistics) were used to evaluate agreement between elevated PD-L2 expression and elevated ICAM-1 expression.

Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Plasmid Preparation, Transfection, Flow Cytometry, Fluorescence, Immunohistochemistry, Staining

3) Product Images from "Anti-inflammatory effects of a Chinese herbal medicine in atherosclerosis via estrogen receptor β mediating nitric oxide production and NF-κB suppression in endothelial cells"

Article Title: Anti-inflammatory effects of a Chinese herbal medicine in atherosclerosis via estrogen receptor β mediating nitric oxide production and NF-κB suppression in endothelial cells

Journal: Cell Death & Disease

doi: 10.1038/cddis.2013.66

BSNXD suppresses adhesion molecules expression on HUVECs through ER β /NO/NF- κ B pathway. The primary HUVECs were exposed to control serum or 10% BSNXD-derived serum for 48 h; in the final 24 h culture the ox-LDL was added. The supernatants were collected, and MCP-1 concentrations were determined. The MCP-1 expression of HUVECs was determined by western blot analysis. The mRNA and protein expression levels of cell adhesion molecules (ICAM-1, VCAM-1 and E-selectin) were assessed by RT-PCR, western blot and FACS. The MCP-1( a ), ICAM-1 ( b ), VCAM-1 ( c ) and E-selectin ( d ) expression was greatly decreased after treatment with the 10% drug-derived serum. NOS inhibitor (L-NAME), ER β antagonist (R, RTHC) other than ER α antagonist (MPP) could block these effects induced by the drug-derived serum. Data are expressed as mean values±S.E.M. ( n =6). * P
Figure Legend Snippet: BSNXD suppresses adhesion molecules expression on HUVECs through ER β /NO/NF- κ B pathway. The primary HUVECs were exposed to control serum or 10% BSNXD-derived serum for 48 h; in the final 24 h culture the ox-LDL was added. The supernatants were collected, and MCP-1 concentrations were determined. The MCP-1 expression of HUVECs was determined by western blot analysis. The mRNA and protein expression levels of cell adhesion molecules (ICAM-1, VCAM-1 and E-selectin) were assessed by RT-PCR, western blot and FACS. The MCP-1( a ), ICAM-1 ( b ), VCAM-1 ( c ) and E-selectin ( d ) expression was greatly decreased after treatment with the 10% drug-derived serum. NOS inhibitor (L-NAME), ER β antagonist (R, RTHC) other than ER α antagonist (MPP) could block these effects induced by the drug-derived serum. Data are expressed as mean values±S.E.M. ( n =6). * P

Techniques Used: Expressing, Derivative Assay, Western Blot, Reverse Transcription Polymerase Chain Reaction, FACS, Blocking Assay

4) Product Images from "Deficiency in p53 is required for doxorubicin induced transcriptional activation of NF-?B target genes in human breast cancer"

Article Title: Deficiency in p53 is required for doxorubicin induced transcriptional activation of NF-?B target genes in human breast cancer

Journal: Oncotarget

doi:

NF-κB -driven transcription by doxorubicin differs among breast cancer cells Four genes ( IL8 , ICAM-1 , CXCL-1 and TNFAIP3 ) were chosen for validation of the microarray results by qRT-PCR. Cells were treated as for the microarray experiment: A, MDA.MB-231 B, SKBR3, BT-474 and MCF-7. The relative gene expression level is normalized to the RPLP0 gene. The graph shows the expression of each gene across different experimental conditions relative to their expression in control condition. C, ICAM-1 protein expression was determined in whole cell lysates of MDA-MB-231, SKBR3 and MCF-7 cells treated as for Fig 1e. α-tubulin served as the loading control. Cells treated with TNF-α were used as positive control.
Figure Legend Snippet: NF-κB -driven transcription by doxorubicin differs among breast cancer cells Four genes ( IL8 , ICAM-1 , CXCL-1 and TNFAIP3 ) were chosen for validation of the microarray results by qRT-PCR. Cells were treated as for the microarray experiment: A, MDA.MB-231 B, SKBR3, BT-474 and MCF-7. The relative gene expression level is normalized to the RPLP0 gene. The graph shows the expression of each gene across different experimental conditions relative to their expression in control condition. C, ICAM-1 protein expression was determined in whole cell lysates of MDA-MB-231, SKBR3 and MCF-7 cells treated as for Fig 1e. α-tubulin served as the loading control. Cells treated with TNF-α were used as positive control.

Techniques Used: Microarray, Quantitative RT-PCR, Multiple Displacement Amplification, Expressing, Positive Control

Doxorubicin induces p65/p50 nuclear translocation ex vivo in breast tumors and increases NF-κB-driven transcription A set of 20 human breast tumors were exposed ex vivo to vehicle, 20 µM MLN120B, 2 µg/ml doxorubicin or both during 24h and then formalin-fixed paraffin embedded (FFPE) tissues were prepared. A, Representative immunohistochemical images of FFPE sections control and doxorubicin treated stained with p65 and p50. B, Graph showing the IHC HScores for nuclear p65 and p50 stainining in all samples relative to control condition. C, Total RNA from ex vivo human breast tumors was extracted for analysis of the ICAM-1 and TNFAIP3 and CXCL-1 genes by qRT-PCR. The relative target gene expression level was also normalized to the RPLP0 expression in each sample. D, Representative immunohistochemical results of ICAM-1 staining in sections of the FFPE breast specimens; control (left panel), doxorubicin 5µM 24h (middle panel) and 20µM MLN120B plus doxorubicin 5µM 24 hours (right panel). E, Graph show the average of ICAM-1 expression determined by immunohistochemistry in all samples relative to control condition.
Figure Legend Snippet: Doxorubicin induces p65/p50 nuclear translocation ex vivo in breast tumors and increases NF-κB-driven transcription A set of 20 human breast tumors were exposed ex vivo to vehicle, 20 µM MLN120B, 2 µg/ml doxorubicin or both during 24h and then formalin-fixed paraffin embedded (FFPE) tissues were prepared. A, Representative immunohistochemical images of FFPE sections control and doxorubicin treated stained with p65 and p50. B, Graph showing the IHC HScores for nuclear p65 and p50 stainining in all samples relative to control condition. C, Total RNA from ex vivo human breast tumors was extracted for analysis of the ICAM-1 and TNFAIP3 and CXCL-1 genes by qRT-PCR. The relative target gene expression level was also normalized to the RPLP0 expression in each sample. D, Representative immunohistochemical results of ICAM-1 staining in sections of the FFPE breast specimens; control (left panel), doxorubicin 5µM 24h (middle panel) and 20µM MLN120B plus doxorubicin 5µM 24 hours (right panel). E, Graph show the average of ICAM-1 expression determined by immunohistochemistry in all samples relative to control condition.

Techniques Used: Translocation Assay, Ex Vivo, Formalin-fixed Paraffin-Embedded, Immunohistochemistry, Staining, Quantitative RT-PCR, Expressing

p53 deficiency is required for NF-κB-driven transcription induced by doxorubicin A, B, graph shows the relative levels of ICAM-1 , TNFAIP-3 and CXCL-1 obtained by qRT-PCR (a) and ICAM-1 determined by IHC (b) taking into account all tumors in which p53 has been considered as WT or deficient status by immunohistochemistry. C, MEFs WT and p53-/- were preincubated 1h 30 min with MLN120B 205µM and exposed to 5µM doxorubicin for 4hours. qRT-PCR analysis of ICAM-1 was performed and normalized to the RPLP0 expression in each condition. p53 expression in MEFs was determined by WB, tubulin expression was used as loading control. D, MCF-7 were infected with 3 different lentiviral particles coding for; shCT and 2 shRNA against TP53 (shTP53.1 and shTP53.2); qRT-PCR of TP53 relative to RPLP0 and Western Blot determination of p53 in cell lysates from cells exposed to 5µM doxorubicin for 24h was were used as knocking down controls. E, MCF-7 shCT and shTP53 were exposed for 4hours to doxorubicin 5µM. Relative expression levels of ICAM-1 and TNFAIP3 were determined by qRT-PCR in each condition. RPLP0 expression was used for normalization. F, MDA-MB-231 were transfected with pCDNA3CT vector and pCDNA3fagp53. RNA was extracted from both transfected cell lines and RT-PCR was done to detect fag p53 expression. RPLP0 was used as loading control. G, qRT-PCR analysis of ICAM-1 , CXCL-1 and TNFAIP3 was performed in MDA-MB-231pcDNA3CT and pCDNA3 fagp53 after 4 hours of 5µM doxorubicin treatment. The relative target gene expression level was also normalized to the RPLP0 expression in each condition. The graph shows the results of the expression of the different genes in each condition relative to their expression in control condition. Mean and SE from triplicate experiments are indicated.
Figure Legend Snippet: p53 deficiency is required for NF-κB-driven transcription induced by doxorubicin A, B, graph shows the relative levels of ICAM-1 , TNFAIP-3 and CXCL-1 obtained by qRT-PCR (a) and ICAM-1 determined by IHC (b) taking into account all tumors in which p53 has been considered as WT or deficient status by immunohistochemistry. C, MEFs WT and p53-/- were preincubated 1h 30 min with MLN120B 205µM and exposed to 5µM doxorubicin for 4hours. qRT-PCR analysis of ICAM-1 was performed and normalized to the RPLP0 expression in each condition. p53 expression in MEFs was determined by WB, tubulin expression was used as loading control. D, MCF-7 were infected with 3 different lentiviral particles coding for; shCT and 2 shRNA against TP53 (shTP53.1 and shTP53.2); qRT-PCR of TP53 relative to RPLP0 and Western Blot determination of p53 in cell lysates from cells exposed to 5µM doxorubicin for 24h was were used as knocking down controls. E, MCF-7 shCT and shTP53 were exposed for 4hours to doxorubicin 5µM. Relative expression levels of ICAM-1 and TNFAIP3 were determined by qRT-PCR in each condition. RPLP0 expression was used for normalization. F, MDA-MB-231 were transfected with pCDNA3CT vector and pCDNA3fagp53. RNA was extracted from both transfected cell lines and RT-PCR was done to detect fag p53 expression. RPLP0 was used as loading control. G, qRT-PCR analysis of ICAM-1 , CXCL-1 and TNFAIP3 was performed in MDA-MB-231pcDNA3CT and pCDNA3 fagp53 after 4 hours of 5µM doxorubicin treatment. The relative target gene expression level was also normalized to the RPLP0 expression in each condition. The graph shows the results of the expression of the different genes in each condition relative to their expression in control condition. Mean and SE from triplicate experiments are indicated.

Techniques Used: Quantitative RT-PCR, Immunohistochemistry, Expressing, Western Blot, Infection, shRNA, Multiple Displacement Amplification, Transfection, Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction

5) Product Images from "Vaspin inhibits cytokine-induced nuclear factor-kappa B activation and adhesion molecule expression via AMP-activated protein kinase activation in vascular endothelial cells"

Article Title: Vaspin inhibits cytokine-induced nuclear factor-kappa B activation and adhesion molecule expression via AMP-activated protein kinase activation in vascular endothelial cells

Journal: Cardiovascular Diabetology

doi: 10.1186/1475-2840-13-41

AMPK activation mediates the inhibitory effects of vaspin on TNFα-induced NF-κB activation and expression of adhesion molecules. The effects of vaspin on TNFα-induced NF-κB promoter activity (A) and mRNA expression of the adhesion molecules ICAM-1 (B) , VCAM-1 (C) , E-selectin (D) , and MCP-1 (E) were measured at 24 hr after treatment of HAECs with 100 ng/mL vaspin in the presence of absence of 10 ng/mL TNFα. HAECs were transfected with control siRNA or AMPKα1 siRNA 24 hr before. Data shown are mean values ± SEM of five independent experiments. * p
Figure Legend Snippet: AMPK activation mediates the inhibitory effects of vaspin on TNFα-induced NF-κB activation and expression of adhesion molecules. The effects of vaspin on TNFα-induced NF-κB promoter activity (A) and mRNA expression of the adhesion molecules ICAM-1 (B) , VCAM-1 (C) , E-selectin (D) , and MCP-1 (E) were measured at 24 hr after treatment of HAECs with 100 ng/mL vaspin in the presence of absence of 10 ng/mL TNFα. HAECs were transfected with control siRNA or AMPKα1 siRNA 24 hr before. Data shown are mean values ± SEM of five independent experiments. * p

Techniques Used: Activation Assay, Expressing, Activity Assay, Transfection

Vaspin inhibits TNFα-induced expression of adhesion molecules. The effect of vaspin on TNFα-induced expression of ICAM-1 (A) , VCAM-1 (A) , E-selectin (B) , and MCP-1 (B) . The protein level and mRNA expression of each adhesion molecule was measured at 24 hr after treatment with 100 ng/mL vaspin in the presence or absence of 10 ng/mL TNFα. Data shown are representative Western blots (top panels) and mean values ± SEM of three independent experiments (bottom panels). The fold change in mRNA expression compared to levels in untreated cells is shown. * p
Figure Legend Snippet: Vaspin inhibits TNFα-induced expression of adhesion molecules. The effect of vaspin on TNFα-induced expression of ICAM-1 (A) , VCAM-1 (A) , E-selectin (B) , and MCP-1 (B) . The protein level and mRNA expression of each adhesion molecule was measured at 24 hr after treatment with 100 ng/mL vaspin in the presence or absence of 10 ng/mL TNFα. Data shown are representative Western blots (top panels) and mean values ± SEM of three independent experiments (bottom panels). The fold change in mRNA expression compared to levels in untreated cells is shown. * p

Techniques Used: Expressing, Western Blot

6) Product Images from "Reduction of Endothelial Nitric Oxide Increases the Adhesiveness of Constitutive Endothelial Membrane ICAM-1 through Src-Mediated Phosphorylation"

Article Title: Reduction of Endothelial Nitric Oxide Increases the Adhesiveness of Constitutive Endothelial Membrane ICAM-1 through Src-Mediated Phosphorylation

Journal: Frontiers in Physiology

doi: 10.3389/fphys.2017.01124

Immuno-precipitation assay conducted in non-denatured proteins showing that reduction of basal NO increases the adhesiveness of EC constitutive ICAM-1 via Src-dependent pathway. (A) Reduced basal NO increased EC ICAM-1 binding to its inhibitory antibody. mAb1A29. The application of AP-CAV (10 μM) or L-NMMA (100 μM) for 30 min (A1) or 6 h (A2) significantly increased ICAM-1 binding to its blocking antibody. Pre-treatment of ECs with Src kinase inhibitor, PP1 (10 μM), significantly attenuated the effect of AP-CAV, indicating the involvement of Src signaling (A3,A4) . (B) In contrast to the results shown in A, reduction of basal NO did not change ICAM-1 binding to antibody against total ICAM-1, indicating no changes in ICAM-1 expression. AP-CAV (B1) or L-NMMA (B2) treatment for 30 min with or without PP1 did not alter ICAM-1 binding to antibody against total ICAM-1. n = 3 for all IP and western blot assays. * P
Figure Legend Snippet: Immuno-precipitation assay conducted in non-denatured proteins showing that reduction of basal NO increases the adhesiveness of EC constitutive ICAM-1 via Src-dependent pathway. (A) Reduced basal NO increased EC ICAM-1 binding to its inhibitory antibody. mAb1A29. The application of AP-CAV (10 μM) or L-NMMA (100 μM) for 30 min (A1) or 6 h (A2) significantly increased ICAM-1 binding to its blocking antibody. Pre-treatment of ECs with Src kinase inhibitor, PP1 (10 μM), significantly attenuated the effect of AP-CAV, indicating the involvement of Src signaling (A3,A4) . (B) In contrast to the results shown in A, reduction of basal NO did not change ICAM-1 binding to antibody against total ICAM-1, indicating no changes in ICAM-1 expression. AP-CAV (B1) or L-NMMA (B2) treatment for 30 min with or without PP1 did not alter ICAM-1 binding to antibody against total ICAM-1. n = 3 for all IP and western blot assays. * P

Techniques Used: Immunoprecipitation, Binding Assay, Blocking Assay, Expressing, Western Blot

The role of Src signaling in NO-dependent phosphorylation of constitutive ICAM-1. (A) Western blot shows dose-dependent inhibition of phosphorylated ICAM-1 (p-ICAM-1) relative to total ICAM-1 by Src kinase inhibitor, PP1, after ECs were exposed to AP-CAV or L-NMMA. (B) Application of AP-CAV (10 μM) for 30 min significantly increased p-ICAM-1. PP1 at 5, 10, or 20 μM showed graded inhibition of increased p-ICAM-1. (C) L-NMMA (100 μM)-induced increases in p-ICAM-1 was inhibited by PP1 in a manner similar to that of AP-CAV. n = 3 for all experiments. * P
Figure Legend Snippet: The role of Src signaling in NO-dependent phosphorylation of constitutive ICAM-1. (A) Western blot shows dose-dependent inhibition of phosphorylated ICAM-1 (p-ICAM-1) relative to total ICAM-1 by Src kinase inhibitor, PP1, after ECs were exposed to AP-CAV or L-NMMA. (B) Application of AP-CAV (10 μM) for 30 min significantly increased p-ICAM-1. PP1 at 5, 10, or 20 μM showed graded inhibition of increased p-ICAM-1. (C) L-NMMA (100 μM)-induced increases in p-ICAM-1 was inhibited by PP1 in a manner similar to that of AP-CAV. n = 3 for all experiments. * P

Techniques Used: Western Blot, Inhibition

AP-CAV application shows no effect on ICAM-1 expression at whole cell and subcellular fractions of HUVECs. (A) Western blot showed no significant changes in ICAM-1 expression in whole cell (W), membrane (M), and cytosol (C) of HUVECs after AP-CAV (10 μM) treatment for 30 min or 6 h, respectively. VE-Cadherin, a membrane bound protein, that was only evident in the membrane and whole cell validated the cell fractionation procedure. (B) Summary of ICAM-1 expression in whole cell and subcellular fractions of HUVECs under control conditions. (C–E) Result summary showing no significant changes of ICAM-1 expression after AP-CAV (10 μM) treatment for 30 min or 6 h in whole cell and subcellular locations. n = 3 per group. ** P
Figure Legend Snippet: AP-CAV application shows no effect on ICAM-1 expression at whole cell and subcellular fractions of HUVECs. (A) Western blot showed no significant changes in ICAM-1 expression in whole cell (W), membrane (M), and cytosol (C) of HUVECs after AP-CAV (10 μM) treatment for 30 min or 6 h, respectively. VE-Cadherin, a membrane bound protein, that was only evident in the membrane and whole cell validated the cell fractionation procedure. (B) Summary of ICAM-1 expression in whole cell and subcellular fractions of HUVECs under control conditions. (C–E) Result summary showing no significant changes of ICAM-1 expression after AP-CAV (10 μM) treatment for 30 min or 6 h in whole cell and subcellular locations. n = 3 per group. ** P

Techniques Used: Expressing, Western Blot, Cell Fractionation

Reduced endothelial basal NO by AP-CAV increases the binding strength of live cell membrane ICAM-1 to its blocking antibody. (A) Force curves differ between groups (contacting surfaces with and without cultured ECs, probes with and without coated ICAM-1 blocking antibody, and cultured ECs with and without exposure to AP-CAV). The curve with a gradual decrease indicates the adhesive interaction between ICAM-1 blocking antibody (on probe) and ICAM-1 on EC membrane. (B) Result summary showing that 30 min application of AP-CAV (10 μM) significantly increased EC ICAM-1 adhesive strength. Values shown are median with 95% confidence interval. C) The distribution of each measured force value in control and AP-CAV treated cells. More than 60 measurements were acquired in three separate cell surface areas (60 μm × 60 μm each) in each cell group. Kolmogorov-Smirnov test was performed to compare the distribution differences between two groups. * P
Figure Legend Snippet: Reduced endothelial basal NO by AP-CAV increases the binding strength of live cell membrane ICAM-1 to its blocking antibody. (A) Force curves differ between groups (contacting surfaces with and without cultured ECs, probes with and without coated ICAM-1 blocking antibody, and cultured ECs with and without exposure to AP-CAV). The curve with a gradual decrease indicates the adhesive interaction between ICAM-1 blocking antibody (on probe) and ICAM-1 on EC membrane. (B) Result summary showing that 30 min application of AP-CAV (10 μM) significantly increased EC ICAM-1 adhesive strength. Values shown are median with 95% confidence interval. C) The distribution of each measured force value in control and AP-CAV treated cells. More than 60 measurements were acquired in three separate cell surface areas (60 μm × 60 μm each) in each cell group. Kolmogorov-Smirnov test was performed to compare the distribution differences between two groups. * P

Techniques Used: Binding Assay, Blocking Assay, Cell Culture

The increased adhesiveness of EC ICAM-1 induced by basal NO reduction is associated with Src signaling-mediated phosphorylation of constitutive ICAM-1. (A) Western blot showing phosphorylated ICAM-1 (p-ICAM-1) relative to total ICAM-1 significantly increased after HUVECs were exposed to AP-CAV (10 μM) or L-NMMA (100 μM) for 30 min (B) , or 6 h (C) . Pre-exposure cells to 10 μM PP1 significantly attenuated the effect (D,E) . n = 3 for western blot. * P
Figure Legend Snippet: The increased adhesiveness of EC ICAM-1 induced by basal NO reduction is associated with Src signaling-mediated phosphorylation of constitutive ICAM-1. (A) Western blot showing phosphorylated ICAM-1 (p-ICAM-1) relative to total ICAM-1 significantly increased after HUVECs were exposed to AP-CAV (10 μM) or L-NMMA (100 μM) for 30 min (B) , or 6 h (C) . Pre-exposure cells to 10 μM PP1 significantly attenuated the effect (D,E) . n = 3 for western blot. * P

Techniques Used: Western Blot

). (A) Video images of a perfused venule under control conditions and after AP-CAV (10 μM)-induced leukocyte adhesion, and the administration of a NO donor, sodium nitroprusside (SNP), in both perfusate (10 μM) and superfusate (20 μM) abolished AP-CAV-induced leukocyte adhesion. (B) AP-CAV induced dose-dependent increases in EC ICAM-1 binding to its blocking antibody mAb1A29. Confocal images of mAb1A29 (green) and vascular cell nuclei (red) immunofluorescence co-staining under control conditions, after AP-CAV perfusion, and after adding SNP to AP-CAV perfused vessels. (C) Perfusion of vessels with ICAM-1 inhibitory antibody, mAb1A29, significantly attenuated AP-CAV induced leukocyte adhesion ( n = 5 per group). * and used by original authors).
Figure Legend Snippet: ). (A) Video images of a perfused venule under control conditions and after AP-CAV (10 μM)-induced leukocyte adhesion, and the administration of a NO donor, sodium nitroprusside (SNP), in both perfusate (10 μM) and superfusate (20 μM) abolished AP-CAV-induced leukocyte adhesion. (B) AP-CAV induced dose-dependent increases in EC ICAM-1 binding to its blocking antibody mAb1A29. Confocal images of mAb1A29 (green) and vascular cell nuclei (red) immunofluorescence co-staining under control conditions, after AP-CAV perfusion, and after adding SNP to AP-CAV perfused vessels. (C) Perfusion of vessels with ICAM-1 inhibitory antibody, mAb1A29, significantly attenuated AP-CAV induced leukocyte adhesion ( n = 5 per group). * and used by original authors).

Techniques Used: Binding Assay, Blocking Assay, Immunofluorescence, Staining

7) Product Images from "Reduction of Endothelial Nitric Oxide Increases the Adhesiveness of Constitutive Endothelial Membrane ICAM-1 through Src-Mediated Phosphorylation"

Article Title: Reduction of Endothelial Nitric Oxide Increases the Adhesiveness of Constitutive Endothelial Membrane ICAM-1 through Src-Mediated Phosphorylation

Journal: Frontiers in Physiology

doi: 10.3389/fphys.2017.01124

Immuno-precipitation assay conducted in non-denatured proteins showing that reduction of basal NO increases the adhesiveness of EC constitutive ICAM-1 via Src-dependent pathway. (A) Reduced basal NO increased EC ICAM-1 binding to its inhibitory antibody. mAb1A29. The application of AP-CAV (10 μM) or L-NMMA (100 μM) for 30 min (A1) or 6 h (A2) significantly increased ICAM-1 binding to its blocking antibody. Pre-treatment of ECs with Src kinase inhibitor, PP1 (10 μM), significantly attenuated the effect of AP-CAV, indicating the involvement of Src signaling (A3,A4) . (B) In contrast to the results shown in A, reduction of basal NO did not change ICAM-1 binding to antibody against total ICAM-1, indicating no changes in ICAM-1 expression. AP-CAV (B1) or L-NMMA (B2) treatment for 30 min with or without PP1 did not alter ICAM-1 binding to antibody against total ICAM-1. n = 3 for all IP and western blot assays. * P
Figure Legend Snippet: Immuno-precipitation assay conducted in non-denatured proteins showing that reduction of basal NO increases the adhesiveness of EC constitutive ICAM-1 via Src-dependent pathway. (A) Reduced basal NO increased EC ICAM-1 binding to its inhibitory antibody. mAb1A29. The application of AP-CAV (10 μM) or L-NMMA (100 μM) for 30 min (A1) or 6 h (A2) significantly increased ICAM-1 binding to its blocking antibody. Pre-treatment of ECs with Src kinase inhibitor, PP1 (10 μM), significantly attenuated the effect of AP-CAV, indicating the involvement of Src signaling (A3,A4) . (B) In contrast to the results shown in A, reduction of basal NO did not change ICAM-1 binding to antibody against total ICAM-1, indicating no changes in ICAM-1 expression. AP-CAV (B1) or L-NMMA (B2) treatment for 30 min with or without PP1 did not alter ICAM-1 binding to antibody against total ICAM-1. n = 3 for all IP and western blot assays. * P

Techniques Used: Immunoprecipitation, Binding Assay, Blocking Assay, Expressing, Western Blot

The role of Src signaling in NO-dependent phosphorylation of constitutive ICAM-1. (A) Western blot shows dose-dependent inhibition of phosphorylated ICAM-1 (p-ICAM-1) relative to total ICAM-1 by Src kinase inhibitor, PP1, after ECs were exposed to AP-CAV or L-NMMA. (B) Application of AP-CAV (10 μM) for 30 min significantly increased p-ICAM-1. PP1 at 5, 10, or 20 μM showed graded inhibition of increased p-ICAM-1. (C) L-NMMA (100 μM)-induced increases in p-ICAM-1 was inhibited by PP1 in a manner similar to that of AP-CAV. n = 3 for all experiments. * P
Figure Legend Snippet: The role of Src signaling in NO-dependent phosphorylation of constitutive ICAM-1. (A) Western blot shows dose-dependent inhibition of phosphorylated ICAM-1 (p-ICAM-1) relative to total ICAM-1 by Src kinase inhibitor, PP1, after ECs were exposed to AP-CAV or L-NMMA. (B) Application of AP-CAV (10 μM) for 30 min significantly increased p-ICAM-1. PP1 at 5, 10, or 20 μM showed graded inhibition of increased p-ICAM-1. (C) L-NMMA (100 μM)-induced increases in p-ICAM-1 was inhibited by PP1 in a manner similar to that of AP-CAV. n = 3 for all experiments. * P

Techniques Used: Western Blot, Inhibition

AP-CAV application shows no effect on ICAM-1 expression at whole cell and subcellular fractions of HUVECs. (A) Western blot showed no significant changes in ICAM-1 expression in whole cell (W), membrane (M), and cytosol (C) of HUVECs after AP-CAV (10 μM) treatment for 30 min or 6 h, respectively. VE-Cadherin, a membrane bound protein, that was only evident in the membrane and whole cell validated the cell fractionation procedure. (B) Summary of ICAM-1 expression in whole cell and subcellular fractions of HUVECs under control conditions. (C–E) Result summary showing no significant changes of ICAM-1 expression after AP-CAV (10 μM) treatment for 30 min or 6 h in whole cell and subcellular locations. n = 3 per group. ** P
Figure Legend Snippet: AP-CAV application shows no effect on ICAM-1 expression at whole cell and subcellular fractions of HUVECs. (A) Western blot showed no significant changes in ICAM-1 expression in whole cell (W), membrane (M), and cytosol (C) of HUVECs after AP-CAV (10 μM) treatment for 30 min or 6 h, respectively. VE-Cadherin, a membrane bound protein, that was only evident in the membrane and whole cell validated the cell fractionation procedure. (B) Summary of ICAM-1 expression in whole cell and subcellular fractions of HUVECs under control conditions. (C–E) Result summary showing no significant changes of ICAM-1 expression after AP-CAV (10 μM) treatment for 30 min or 6 h in whole cell and subcellular locations. n = 3 per group. ** P

Techniques Used: Expressing, Western Blot, Cell Fractionation

Reduced endothelial basal NO by AP-CAV increases the binding strength of live cell membrane ICAM-1 to its blocking antibody. (A) Force curves differ between groups (contacting surfaces with and without cultured ECs, probes with and without coated ICAM-1 blocking antibody, and cultured ECs with and without exposure to AP-CAV). The curve with a gradual decrease indicates the adhesive interaction between ICAM-1 blocking antibody (on probe) and ICAM-1 on EC membrane. (B) Result summary showing that 30 min application of AP-CAV (10 μM) significantly increased EC ICAM-1 adhesive strength. Values shown are median with 95% confidence interval. C) The distribution of each measured force value in control and AP-CAV treated cells. More than 60 measurements were acquired in three separate cell surface areas (60 μm × 60 μm each) in each cell group. Kolmogorov-Smirnov test was performed to compare the distribution differences between two groups. * P
Figure Legend Snippet: Reduced endothelial basal NO by AP-CAV increases the binding strength of live cell membrane ICAM-1 to its blocking antibody. (A) Force curves differ between groups (contacting surfaces with and without cultured ECs, probes with and without coated ICAM-1 blocking antibody, and cultured ECs with and without exposure to AP-CAV). The curve with a gradual decrease indicates the adhesive interaction between ICAM-1 blocking antibody (on probe) and ICAM-1 on EC membrane. (B) Result summary showing that 30 min application of AP-CAV (10 μM) significantly increased EC ICAM-1 adhesive strength. Values shown are median with 95% confidence interval. C) The distribution of each measured force value in control and AP-CAV treated cells. More than 60 measurements were acquired in three separate cell surface areas (60 μm × 60 μm each) in each cell group. Kolmogorov-Smirnov test was performed to compare the distribution differences between two groups. * P

Techniques Used: Binding Assay, Blocking Assay, Cell Culture

The increased adhesiveness of EC ICAM-1 induced by basal NO reduction is associated with Src signaling-mediated phosphorylation of constitutive ICAM-1. (A) Western blot showing phosphorylated ICAM-1 (p-ICAM-1) relative to total ICAM-1 significantly increased after HUVECs were exposed to AP-CAV (10 μM) or L-NMMA (100 μM) for 30 min (B) , or 6 h (C) . Pre-exposure cells to 10 μM PP1 significantly attenuated the effect (D,E) . n = 3 for western blot. * P
Figure Legend Snippet: The increased adhesiveness of EC ICAM-1 induced by basal NO reduction is associated with Src signaling-mediated phosphorylation of constitutive ICAM-1. (A) Western blot showing phosphorylated ICAM-1 (p-ICAM-1) relative to total ICAM-1 significantly increased after HUVECs were exposed to AP-CAV (10 μM) or L-NMMA (100 μM) for 30 min (B) , or 6 h (C) . Pre-exposure cells to 10 μM PP1 significantly attenuated the effect (D,E) . n = 3 for western blot. * P

Techniques Used: Western Blot

Perfusion of rat mesenteric venules with AP-CAV induced basal NO-dependent ICAM-1 mediated leukocyte adhesion. Intact venules were perfused by AP-CAV for 30 min followed by resuming blood flow in the same vessel for 10 min. Leukocyte adhesion was quantified when each vessel was recannulated with BSA-Ringer solution (Xu et al., 2013 ). (A) Video images of a perfused venule under control conditions and after AP-CAV (10 μM)-induced leukocyte adhesion, and the administration of a NO donor, sodium nitroprusside (SNP), in both perfusate (10 μM) and superfusate (20 μM) abolished AP-CAV-induced leukocyte adhesion. (B) AP-CAV induced dose-dependent increases in EC ICAM-1 binding to its blocking antibody mAb1A29. Confocal images of mAb1A29 (green) and vascular cell nuclei (red) immunofluorescence co-staining under control conditions, after AP-CAV perfusion, and after adding SNP to AP-CAV perfused vessels. (C) Perfusion of vessels with ICAM-1 inhibitory antibody, mAb1A29, significantly attenuated AP-CAV induced leukocyte adhesion ( n = 5 per group). * and † indicate a significant increase and decrease from the control, respectively (modified from Xu et al., 2013 and used by original authors).
Figure Legend Snippet: Perfusion of rat mesenteric venules with AP-CAV induced basal NO-dependent ICAM-1 mediated leukocyte adhesion. Intact venules were perfused by AP-CAV for 30 min followed by resuming blood flow in the same vessel for 10 min. Leukocyte adhesion was quantified when each vessel was recannulated with BSA-Ringer solution (Xu et al., 2013 ). (A) Video images of a perfused venule under control conditions and after AP-CAV (10 μM)-induced leukocyte adhesion, and the administration of a NO donor, sodium nitroprusside (SNP), in both perfusate (10 μM) and superfusate (20 μM) abolished AP-CAV-induced leukocyte adhesion. (B) AP-CAV induced dose-dependent increases in EC ICAM-1 binding to its blocking antibody mAb1A29. Confocal images of mAb1A29 (green) and vascular cell nuclei (red) immunofluorescence co-staining under control conditions, after AP-CAV perfusion, and after adding SNP to AP-CAV perfused vessels. (C) Perfusion of vessels with ICAM-1 inhibitory antibody, mAb1A29, significantly attenuated AP-CAV induced leukocyte adhesion ( n = 5 per group). * and † indicate a significant increase and decrease from the control, respectively (modified from Xu et al., 2013 and used by original authors).

Techniques Used: Flow Cytometry, Binding Assay, Blocking Assay, Immunofluorescence, Staining, Modification

8) Product Images from "Exercise intervention attenuates hyperhomocysteinemia-induced aortic endothelial oxidative injury by regulating SIRT1 through mitigating NADPH oxidase/LOX-1 signaling"

Article Title: Exercise intervention attenuates hyperhomocysteinemia-induced aortic endothelial oxidative injury by regulating SIRT1 through mitigating NADPH oxidase/LOX-1 signaling

Journal: Redox Biology

doi: 10.1016/j.redox.2017.08.016

Exercise training repressed HHcy-activated endothelial inflammation and oxidative stress through the modulation of SIRT1. (A) The protein levels of SIRT1, p-AMPK, NOX-1, LOX-1, p-ERK and ICAM-1 in the aortic endothelium of the control group, the 1% methionine group, and the 1% methionine plus exercise group were investigated by Western blot analysis. (B, C, D, E, F, G) The bars represent the relative protein levels of SIRT1, p-AMPK, NOX-1, LOX-1, p-ERK and ICAM-1, which were normalized to β-actin, and indicate the mean ± SD (n = 8 in each group). *P
Figure Legend Snippet: Exercise training repressed HHcy-activated endothelial inflammation and oxidative stress through the modulation of SIRT1. (A) The protein levels of SIRT1, p-AMPK, NOX-1, LOX-1, p-ERK and ICAM-1 in the aortic endothelium of the control group, the 1% methionine group, and the 1% methionine plus exercise group were investigated by Western blot analysis. (B, C, D, E, F, G) The bars represent the relative protein levels of SIRT1, p-AMPK, NOX-1, LOX-1, p-ERK and ICAM-1, which were normalized to β-actin, and indicate the mean ± SD (n = 8 in each group). *P

Techniques Used: Western Blot

Exercise training repressed HHcy-activated endothelial inflammation. (A) The protein levels of p-ERK, ICAM-1, VCAM-1 in the aortic endothelium of the control group, the 1% methionine group, and the 1% methionine plus exercise group were investigated by Western blot analysis. Nuclear protein from endothelial tissues was prepared for NF-kBp65 and LaminB1 detection. (B, C, D, E) The bars represent the relative protein levels of p-ERK, ICAM-1 and VCAM-1, which were normalized to β-actin, and the relative protein level of NF-kBp65, which was normalized to Lamin B1. The data are presented as the mean ± SD (n = 8 in each group). *P
Figure Legend Snippet: Exercise training repressed HHcy-activated endothelial inflammation. (A) The protein levels of p-ERK, ICAM-1, VCAM-1 in the aortic endothelium of the control group, the 1% methionine group, and the 1% methionine plus exercise group were investigated by Western blot analysis. Nuclear protein from endothelial tissues was prepared for NF-kBp65 and LaminB1 detection. (B, C, D, E) The bars represent the relative protein levels of p-ERK, ICAM-1 and VCAM-1, which were normalized to β-actin, and the relative protein level of NF-kBp65, which was normalized to Lamin B1. The data are presented as the mean ± SD (n = 8 in each group). *P

Techniques Used: Western Blot

9) Product Images from "MiRNA-296-3p-ICAM-1 axis promotes metastasis of prostate cancer by possible enhancing survival of natural killer cell-resistant circulating tumour cells"

Article Title: MiRNA-296-3p-ICAM-1 axis promotes metastasis of prostate cancer by possible enhancing survival of natural killer cell-resistant circulating tumour cells

Journal: Cell Death & Disease

doi: 10.1038/cddis.2013.458

MiR-296-3p directly regulates ICAM-1 in PCa cells. ( a ) Screening of candidate miRNAs (in target cells) involved in the resistance to NK cells were performed using calcein release assay. Data are the mean±S.E.M. of three independent experiments. ( b ) The expression level of miR-296-3p and transcription level of ICAM-1 in P69 and M12 were analyzed by miRNA transcriptome sequencing and DGE sequencing, respectively. TPM means transcripts per million. ( c ) Real-time PCR in ΔΔCt method was performed to identify the expression levels of miR-296-3p in P69 and M12. One representative experiment out of three was shown. ( d ) The protein levels of ICAM-1 in P69 and M12 were determined by western blotting. One representative experiment out of three was shown. ( e ) The predicted miR-296-3p binding site (red) and their mutant (blue) in the 3′-UTR of ICAM-1 mRNA are indicated. Number shows position of nucleotides in the 3′-UTR of ICAM-1 mRNA. ( f ) HEK-293T cells were transfected with psiCHECK-2 containing WT or mutant ICAM-1 3′-UTR and miR-296-3p or mock control vector. The Renilla luciferase activity was normalized on the constitutive activity of firefly luciferase. Data are the mean±S.E.M. of three independent experiments. ( g ) The expression of membrane-bound or total ICAM-1 in P69 and M12 were analyzed by flow cytometry (upper panel) and western blotting (bottom panel), respectively. One representative experiment out of three was shown
Figure Legend Snippet: MiR-296-3p directly regulates ICAM-1 in PCa cells. ( a ) Screening of candidate miRNAs (in target cells) involved in the resistance to NK cells were performed using calcein release assay. Data are the mean±S.E.M. of three independent experiments. ( b ) The expression level of miR-296-3p and transcription level of ICAM-1 in P69 and M12 were analyzed by miRNA transcriptome sequencing and DGE sequencing, respectively. TPM means transcripts per million. ( c ) Real-time PCR in ΔΔCt method was performed to identify the expression levels of miR-296-3p in P69 and M12. One representative experiment out of three was shown. ( d ) The protein levels of ICAM-1 in P69 and M12 were determined by western blotting. One representative experiment out of three was shown. ( e ) The predicted miR-296-3p binding site (red) and their mutant (blue) in the 3′-UTR of ICAM-1 mRNA are indicated. Number shows position of nucleotides in the 3′-UTR of ICAM-1 mRNA. ( f ) HEK-293T cells were transfected with psiCHECK-2 containing WT or mutant ICAM-1 3′-UTR and miR-296-3p or mock control vector. The Renilla luciferase activity was normalized on the constitutive activity of firefly luciferase. Data are the mean±S.E.M. of three independent experiments. ( g ) The expression of membrane-bound or total ICAM-1 in P69 and M12 were analyzed by flow cytometry (upper panel) and western blotting (bottom panel), respectively. One representative experiment out of three was shown

Techniques Used: Release Assay, Expressing, Sequencing, Real-time Polymerase Chain Reaction, Western Blot, Binding Assay, Mutagenesis, Transfection, Plasmid Preparation, Luciferase, Activity Assay, Flow Cytometry, Cytometry

MiR-296-3p is upregulated and negatively correlated with ICAM-1 in human PCa tissues. ( a ) The expression levels of miR-296-3p in human prostate normal tissues (upper panel) and cancer tissues (bottom panel) were detected by ISH as described in Material and methods section. Representative staining tissue sections were presented and the numbers shown in images indicated the ratio of positive staining area. ( b ) Vertical coordinates indicated the percentage ratio of positive staining area to total tissue area and specimens were ranked into as high (upper 30%), middle (a range of 10%–30%) and low (below 10%) expression levels. Data were analyzed with the Mann–Whitney test to determine the statistic significance ( P
Figure Legend Snippet: MiR-296-3p is upregulated and negatively correlated with ICAM-1 in human PCa tissues. ( a ) The expression levels of miR-296-3p in human prostate normal tissues (upper panel) and cancer tissues (bottom panel) were detected by ISH as described in Material and methods section. Representative staining tissue sections were presented and the numbers shown in images indicated the ratio of positive staining area. ( b ) Vertical coordinates indicated the percentage ratio of positive staining area to total tissue area and specimens were ranked into as high (upper 30%), middle (a range of 10%–30%) and low (below 10%) expression levels. Data were analyzed with the Mann–Whitney test to determine the statistic significance ( P

Techniques Used: Expressing, In Situ Hybridization, Staining, MANN-WHITNEY

Schematic model of miR-296-3p-ICAM-1 axis promotes PCa metastasis by possible enhancing survival of NK cell-resistant CTC. During the early stage of EMT in the long-term malignant transition process from non-metastatic P69 to highly metastatic M12 in nude mice, 15 some of tumour cells acquired genetic changes leading to highly expressing miR-296-3p that directly targeted ICAM-1 gene. These heterogenous tumour cells might invade locally and afterward intravasate into blood vessels to form CTC. Compared with NKs-CTC or parental P69 cells, NKr-CTC with low expression of ICAM-1 on cell surfaces inhibited by miR-296-3p were hardly destroyed by NK cells in blood vessels. The survived NKr-CTC might successively complete the remaining events of the invasion-metastasis cascade including extravasation, micrometastasis, MET and metastatic colonization. CTC, circulating tumour cell; EMT, epithelial–mesenchymal transition; NKs-CTC, natural killer cell-sensitive circulating tumour cell; NKr-CTC, natural killer cell-resistant circulating tumour cells; MET, mesenchymal–epithelial transition
Figure Legend Snippet: Schematic model of miR-296-3p-ICAM-1 axis promotes PCa metastasis by possible enhancing survival of NK cell-resistant CTC. During the early stage of EMT in the long-term malignant transition process from non-metastatic P69 to highly metastatic M12 in nude mice, 15 some of tumour cells acquired genetic changes leading to highly expressing miR-296-3p that directly targeted ICAM-1 gene. These heterogenous tumour cells might invade locally and afterward intravasate into blood vessels to form CTC. Compared with NKs-CTC or parental P69 cells, NKr-CTC with low expression of ICAM-1 on cell surfaces inhibited by miR-296-3p were hardly destroyed by NK cells in blood vessels. The survived NKr-CTC might successively complete the remaining events of the invasion-metastasis cascade including extravasation, micrometastasis, MET and metastatic colonization. CTC, circulating tumour cell; EMT, epithelial–mesenchymal transition; NKs-CTC, natural killer cell-sensitive circulating tumour cell; NKr-CTC, natural killer cell-resistant circulating tumour cells; MET, mesenchymal–epithelial transition

Techniques Used: Mouse Assay, Expressing

MiR-296-3p-ICAM-1-NK signalling axis in control of pulmonary metastasis of M12 in vivo. ( a ) BALB/c athymic mice (nu/nu) at 8 weeks old were injected intravenously with 2.5 × 10 6 of M12 luc . The transferred M12 luc in lungs was detected by IVIS spectrum image system at 0, 24 and 48 h after injection and presented visually with various colour spots based on different intensity of luminescence. ( b ) The relative amounts of transferred M12 luc in lung were presented as percentage. The photon flux of M12 control cells at 0 h was set as a common denominator, by which all other photon flux values of M12 variants at three time points were divided. The data were representative of two independent experiments (five nude mice per group) and expressed as mean±S.E.M. ( c ) BALB/c athymic mice (nu/nu) at 8 weeks old were injected intraperitoneally with 50 μ l of NK cell-depleting anti-asialo GM1 antibody 1 day before intravenous injection of 2.5 × 10 6 of M12 luc . At 24 h after injection of M12 luc , 2.5 × 10 7 of expanded human NK cells were injected intravenously into nude mice. The transferred M12 luc in lungs were detected by IVIS spectrum image system at 0, 24 and 48 h, and ( d ) the data were processed and presented as previously described above
Figure Legend Snippet: MiR-296-3p-ICAM-1-NK signalling axis in control of pulmonary metastasis of M12 in vivo. ( a ) BALB/c athymic mice (nu/nu) at 8 weeks old were injected intravenously with 2.5 × 10 6 of M12 luc . The transferred M12 luc in lungs was detected by IVIS spectrum image system at 0, 24 and 48 h after injection and presented visually with various colour spots based on different intensity of luminescence. ( b ) The relative amounts of transferred M12 luc in lung were presented as percentage. The photon flux of M12 control cells at 0 h was set as a common denominator, by which all other photon flux values of M12 variants at three time points were divided. The data were representative of two independent experiments (five nude mice per group) and expressed as mean±S.E.M. ( c ) BALB/c athymic mice (nu/nu) at 8 weeks old were injected intraperitoneally with 50 μ l of NK cell-depleting anti-asialo GM1 antibody 1 day before intravenous injection of 2.5 × 10 6 of M12 luc . At 24 h after injection of M12 luc , 2.5 × 10 7 of expanded human NK cells were injected intravenously into nude mice. The transferred M12 luc in lungs were detected by IVIS spectrum image system at 0, 24 and 48 h, and ( d ) the data were processed and presented as previously described above

Techniques Used: In Vivo, Mouse Assay, Injection

MiR-296-3p-ICAM-1-NK signalling axis affects survival of M12 in peripheral blood. ( a ) Quantitative analysis of the residual GFP-positive M12 in peripheral blood of nude mice treated with normal rabbit serum. The cells gated on FSC-SSC plot were analyzed (left upper panel) and the number in oval represented percentage of GFP + /7-AAD − viable M12 variant cells (left lower panel). The percentage of GFP + M12 variants at three time points of 0, 24 and 36 h was normalized by being divided by percentage of GFP + cells in M12 control cells at 0 h. The data are representative of two independent experiments ( n =5) expressed as mean±S.E.M. (right graph). ( b and c ) Mice were intraperitoneally pre-treated anti-asialo GM1 antibody 1 day before intravenous inoculation of M12 variant cells. Quantitative analysis of residual GFP + M12 variants in peripheral blood of these mice transferred without ( b ) or with ( c ) human NK cells at 24 h after intravenous injection of tumour cells. The data were processed and presented as previously described
Figure Legend Snippet: MiR-296-3p-ICAM-1-NK signalling axis affects survival of M12 in peripheral blood. ( a ) Quantitative analysis of the residual GFP-positive M12 in peripheral blood of nude mice treated with normal rabbit serum. The cells gated on FSC-SSC plot were analyzed (left upper panel) and the number in oval represented percentage of GFP + /7-AAD − viable M12 variant cells (left lower panel). The percentage of GFP + M12 variants at three time points of 0, 24 and 36 h was normalized by being divided by percentage of GFP + cells in M12 control cells at 0 h. The data are representative of two independent experiments ( n =5) expressed as mean±S.E.M. (right graph). ( b and c ) Mice were intraperitoneally pre-treated anti-asialo GM1 antibody 1 day before intravenous inoculation of M12 variant cells. Quantitative analysis of residual GFP + M12 variants in peripheral blood of these mice transferred without ( b ) or with ( c ) human NK cells at 24 h after intravenous injection of tumour cells. The data were processed and presented as previously described

Techniques Used: Mouse Assay, Variant Assay, Injection

ICAM-1 contributes to the differential susceptibility of P69 and M12 to NK cells. ( a ) The expression levels of ligands on P69 (green lines) or M12 (blue lines) paired to NK cell receptors were analyzed by flow cytometer. Red and orange lines represent P69 and M12 with PE or APC-conjugated isotype matched IgG as negative controls, respectively. ( b ) The NK cell cytotoxicities to ICAM-1-knockdown P69 (P69 shICAM-1) and ICAM-1-overexpressing M12 (M12 ICAM-1) were assessed by calcein release assay. ( c ) The conjugation of NK cell to P69 shICAM-1 and M12 ICAM-1 were determined. Data are the mean±S.E.M. from three independent experiments. ( d ) The degranulation of NK cells against P69 shICAM-1 and M12 ICAM-1 was performed. Data are the mean±S.E.M. of three independent experiments. ( e ) NK cells were incubated with anti-CD11 α (a subunit of LFA-1) mAbs to block function of LFA-1 or isotype-matched IgG as a control for 30 min and then mixed with P69 or M12 at an E/T ratio of 5–1 for calcein release assay. Data are the mean±S.E.M. of three independent experiments
Figure Legend Snippet: ICAM-1 contributes to the differential susceptibility of P69 and M12 to NK cells. ( a ) The expression levels of ligands on P69 (green lines) or M12 (blue lines) paired to NK cell receptors were analyzed by flow cytometer. Red and orange lines represent P69 and M12 with PE or APC-conjugated isotype matched IgG as negative controls, respectively. ( b ) The NK cell cytotoxicities to ICAM-1-knockdown P69 (P69 shICAM-1) and ICAM-1-overexpressing M12 (M12 ICAM-1) were assessed by calcein release assay. ( c ) The conjugation of NK cell to P69 shICAM-1 and M12 ICAM-1 were determined. Data are the mean±S.E.M. from three independent experiments. ( d ) The degranulation of NK cells against P69 shICAM-1 and M12 ICAM-1 was performed. Data are the mean±S.E.M. of three independent experiments. ( e ) NK cells were incubated with anti-CD11 α (a subunit of LFA-1) mAbs to block function of LFA-1 or isotype-matched IgG as a control for 30 min and then mixed with P69 or M12 at an E/T ratio of 5–1 for calcein release assay. Data are the mean±S.E.M. of three independent experiments

Techniques Used: Expressing, Flow Cytometry, Cytometry, Release Assay, Conjugation Assay, Incubation, Blocking Assay

MiR-296-3p affects the sensitivity of PCa cells to NK cells. ( a ) NK cells were incubated with anti-CD11 α mAbs or isotype-matched IgG and then the cytotoxicity assay was performed against miR-296-3p-overexpressing P69 (P69 miR-296-3p) (left panel) and miR-296-3p-silencing M12 (M12 anti-miR-296-3p) (right panel). ( b ) The NK cell cytotoxicity (left upper panel), degranulation (right upper panel), perforin polarization (left bottom panel) and conjugate formation (right bottom panel) against miR-296-3p P69 and the ICAM-1-overexpressing miR-296-3p P69 (miR-296-3p+ICAM-1 P69) cells were performed as indicated. ( c ) The NK cell cytotoxicity (left upper panel), degranulation (right upper panel), perforin polarization (left bottom panel) and conjugate formation (right bottom panel) against the anti-miR-296-3p M12 and the ICAM-1-knockdown anti-miR-296-3p M12 (anti-miR-296-3p+shICAM-1 M12) cells were performed as indicated. ( d ) The conjugate formation between NK cells and P69 (left panel) or M12 (right panel) stable cell lines after blockade of CD11α on NK cells with anti-CD11 α mAbs (isotype-matched IgG as a control) were examined as previously described. All above data were the mean±S.E.M. from three independent experiments
Figure Legend Snippet: MiR-296-3p affects the sensitivity of PCa cells to NK cells. ( a ) NK cells were incubated with anti-CD11 α mAbs or isotype-matched IgG and then the cytotoxicity assay was performed against miR-296-3p-overexpressing P69 (P69 miR-296-3p) (left panel) and miR-296-3p-silencing M12 (M12 anti-miR-296-3p) (right panel). ( b ) The NK cell cytotoxicity (left upper panel), degranulation (right upper panel), perforin polarization (left bottom panel) and conjugate formation (right bottom panel) against miR-296-3p P69 and the ICAM-1-overexpressing miR-296-3p P69 (miR-296-3p+ICAM-1 P69) cells were performed as indicated. ( c ) The NK cell cytotoxicity (left upper panel), degranulation (right upper panel), perforin polarization (left bottom panel) and conjugate formation (right bottom panel) against the anti-miR-296-3p M12 and the ICAM-1-knockdown anti-miR-296-3p M12 (anti-miR-296-3p+shICAM-1 M12) cells were performed as indicated. ( d ) The conjugate formation between NK cells and P69 (left panel) or M12 (right panel) stable cell lines after blockade of CD11α on NK cells with anti-CD11 α mAbs (isotype-matched IgG as a control) were examined as previously described. All above data were the mean±S.E.M. from three independent experiments

Techniques Used: Incubation, Cytotoxicity Assay, Stable Transfection

10) Product Images from "Application of a Flow-Based Hollow-Fiber Co-Culture System to Study Cellular Influences under Hyperglycemic Conditions"

Article Title: Application of a Flow-Based Hollow-Fiber Co-Culture System to Study Cellular Influences under Hyperglycemic Conditions

Journal: Scientific Reports

doi: 10.1038/s41598-019-40555-0

Protein levels of human VEGF ( A ), ICAM-1 ( B ), and VCAM-1 ( C ) as detected by Western blot in cellular lysates. HRECs were co-cultured with PMNs for 28 days under normal glucose (NG) or high glucose (HG) conditions. One representative cropped image is provided for each Western blot. N = 5/group; results are expressed as mean ± SD. ** P
Figure Legend Snippet: Protein levels of human VEGF ( A ), ICAM-1 ( B ), and VCAM-1 ( C ) as detected by Western blot in cellular lysates. HRECs were co-cultured with PMNs for 28 days under normal glucose (NG) or high glucose (HG) conditions. One representative cropped image is provided for each Western blot. N = 5/group; results are expressed as mean ± SD. ** P

Techniques Used: Western Blot, Cell Culture

11) Product Images from "9c11tCLA modulates 11t18:1 and 9t18:1 induced inflammations differently in human umbilical vein endothelial cells"

Article Title: 9c11tCLA modulates 11t18:1 and 9t18:1 induced inflammations differently in human umbilical vein endothelial cells

Journal: Scientific Reports

doi: 10.1038/s41598-018-19729-9

Effect of 9t18:1 and 11t18:1 on gene expression of ICAM-1, VCAM-1 and IL-6 of leptin/9c11t-CLA treated HUVECs. ( A ) The effect of 11t18:1 on expression of ICAM-1, VCAM-1 and IL-6 in leptin treated HUVECs. HUVECs were treated or without leptin (75 nmol/L) for 24 h and then cultured with 11t18:1(25, 50, 100 μmol/L) for 24 h. ( B ) The effect of 9t18:1 on expression of ICAM-1, VCAM-1 and IL-6 in 9c11tCLA treated HUVECs. HUVECs were treated or non-treated with 9c11tCLA (5, 25, 50 μmol/L) and then cultured with 9t18:1 (100 μmol/L) for 24 h. ( C ) The effect of 11t18:1 + leptin on expression of ICAM-1, VCAM-1 and IL-6 in 9c11tCLA treated HUVECs. HUVECs were treated or non-treated with 9c11tCLA (5, 25, 50 μmol/L) and then cultured with the group of 11t18:1 (100 μmol/L) + leptin (75 nmol/L) for 24 h. ( D ) The effect of 9t18:1 on expression of ICAM-1, VCAM-1 and IL-6 of leptin treated HUVECs. HUVECs were treated or non-treated with leptin (75 nmol/L) and then cultured with 9t18:1 (100 μmol/L) and 11t18:1 (100 μmol/L) for 24 h. a–g Data were presented as mean ± SD, values not sharing a common superscript denote significant difference ( P
Figure Legend Snippet: Effect of 9t18:1 and 11t18:1 on gene expression of ICAM-1, VCAM-1 and IL-6 of leptin/9c11t-CLA treated HUVECs. ( A ) The effect of 11t18:1 on expression of ICAM-1, VCAM-1 and IL-6 in leptin treated HUVECs. HUVECs were treated or without leptin (75 nmol/L) for 24 h and then cultured with 11t18:1(25, 50, 100 μmol/L) for 24 h. ( B ) The effect of 9t18:1 on expression of ICAM-1, VCAM-1 and IL-6 in 9c11tCLA treated HUVECs. HUVECs were treated or non-treated with 9c11tCLA (5, 25, 50 μmol/L) and then cultured with 9t18:1 (100 μmol/L) for 24 h. ( C ) The effect of 11t18:1 + leptin on expression of ICAM-1, VCAM-1 and IL-6 in 9c11tCLA treated HUVECs. HUVECs were treated or non-treated with 9c11tCLA (5, 25, 50 μmol/L) and then cultured with the group of 11t18:1 (100 μmol/L) + leptin (75 nmol/L) for 24 h. ( D ) The effect of 9t18:1 on expression of ICAM-1, VCAM-1 and IL-6 of leptin treated HUVECs. HUVECs were treated or non-treated with leptin (75 nmol/L) and then cultured with 9t18:1 (100 μmol/L) and 11t18:1 (100 μmol/L) for 24 h. a–g Data were presented as mean ± SD, values not sharing a common superscript denote significant difference ( P

Techniques Used: Expressing, Cell Culture

Effect of TAK242 on MAPKs phosphorylation in HUVECs treated with 9t18:1 and 11t18:1. ( A ) Effect of TAK242 on ICAM-1 expression. HUVECs were treated with TAK242 (0.5, 1, 1.5 μmol/L) for 30 min and then cultured with 9t18:1 for 24 h. ( B ) Effect of TAK242 on MAPKs phosphorylation in HUVECs treated with 9t18:1 and 11t18:1. HUVECs were treated with TAK242 (1 μmol/L) for 30 min and then cultured with TFA for 24 h. Values labeled with different letters in each set indicate significant differences ( p
Figure Legend Snippet: Effect of TAK242 on MAPKs phosphorylation in HUVECs treated with 9t18:1 and 11t18:1. ( A ) Effect of TAK242 on ICAM-1 expression. HUVECs were treated with TAK242 (0.5, 1, 1.5 μmol/L) for 30 min and then cultured with 9t18:1 for 24 h. ( B ) Effect of TAK242 on MAPKs phosphorylation in HUVECs treated with 9t18:1 and 11t18:1. HUVECs were treated with TAK242 (1 μmol/L) for 30 min and then cultured with TFA for 24 h. Values labeled with different letters in each set indicate significant differences ( p

Techniques Used: Expressing, Cell Culture, Labeling

12) Product Images from "E2F1 renders prostate cancer cell resistant to ICAM-1 mediated antitumor immunity by NF-?B modulation"

Article Title: E2F1 renders prostate cancer cell resistant to ICAM-1 mediated antitumor immunity by NF-?B modulation

Journal: Molecular Cancer

doi: 10.1186/1476-4598-13-84

RNA Silencing of E2F1 Sensitizes Prostate Cancer Cells to ICAM-1 Mediated Anti-tumor Responses. A . E2F1 knockdown increases cytotoxicity through ICAM-1. DU145/sh-Con and DU145/sh-E2F1 cells were transiently transfected with duplex siRNA of ICAM-1. The DU145 derived cells were used as the target cells and the CIK cells as the effector cells. Both of them at various ratios of effector cells to target cells were mixed and cytotoxicity was measured by 51 Cr release. The results are shown as the mean ± SD of triplicate measurements. B . The expression of membrane ICAM-1 in the indicated cells was measured by FACS with ICAM-1 specific antibody. C . The expression of IL-1β, TNF-α, IL-6 and IL-8 in DU145/sh-Con and DU145/sh-E2F1 cells were measured by real-time PCR. The data presents the fold-induction of the levels of each tested cytokine in DU145/sh-E2F1 cells over DU145/sh-Con cell, and represent the mean ± SD of triplicate measurements. D . E2F1 knockdown inhibits tumor xenografts growth in vivo, DU145 cells were stably transfected with the control vectors or shRNA expressing plasmids targeting E2F1 or ICAM-1 respectively and injected subcutaneously into nude mice. Tumor volumes were measured and estimated by the formula: length (mm) X width (mm) X height (mm)/2. The results are shown as the mean value ± SD of at least five tumors in each group. E and F . E2F1 knockdown increases ICAM-1-mediated leucocytes infiltration. The infiltrating leucocytes to DU145-derived tumors in nude mice were stained by H E and indicated by arrows. The expression of E2F1 and ICAM-1 in these DU145-derived tumors was monitored by immunohistochemistry analysis, H E staining (D) and Western blot (E) .
Figure Legend Snippet: RNA Silencing of E2F1 Sensitizes Prostate Cancer Cells to ICAM-1 Mediated Anti-tumor Responses. A . E2F1 knockdown increases cytotoxicity through ICAM-1. DU145/sh-Con and DU145/sh-E2F1 cells were transiently transfected with duplex siRNA of ICAM-1. The DU145 derived cells were used as the target cells and the CIK cells as the effector cells. Both of them at various ratios of effector cells to target cells were mixed and cytotoxicity was measured by 51 Cr release. The results are shown as the mean ± SD of triplicate measurements. B . The expression of membrane ICAM-1 in the indicated cells was measured by FACS with ICAM-1 specific antibody. C . The expression of IL-1β, TNF-α, IL-6 and IL-8 in DU145/sh-Con and DU145/sh-E2F1 cells were measured by real-time PCR. The data presents the fold-induction of the levels of each tested cytokine in DU145/sh-E2F1 cells over DU145/sh-Con cell, and represent the mean ± SD of triplicate measurements. D . E2F1 knockdown inhibits tumor xenografts growth in vivo, DU145 cells were stably transfected with the control vectors or shRNA expressing plasmids targeting E2F1 or ICAM-1 respectively and injected subcutaneously into nude mice. Tumor volumes were measured and estimated by the formula: length (mm) X width (mm) X height (mm)/2. The results are shown as the mean value ± SD of at least five tumors in each group. E and F . E2F1 knockdown increases ICAM-1-mediated leucocytes infiltration. The infiltrating leucocytes to DU145-derived tumors in nude mice were stained by H E and indicated by arrows. The expression of E2F1 and ICAM-1 in these DU145-derived tumors was monitored by immunohistochemistry analysis, H E staining (D) and Western blot (E) .

Techniques Used: Transfection, Derivative Assay, Expressing, FACS, Real-time Polymerase Chain Reaction, In Vivo, Stable Transfection, shRNA, Injection, Mouse Assay, Staining, Immunohistochemistry, Western Blot

NF-κB binding sites are required for E2F1 regulation of ICAM-1 promoter. A . The histograph represent the human ICAM-1 promoter luciferase reporters used in this study. The location of each binding site on the promoter of ICAM-1 is indicated and labeled. The crossed boxes indicate the mutation sites on each construct. The mutated nucleotides corresponding to κB-1-mut and κB-2-mut are underlined. B and C . DU145/sh-Con and DU145/sh-E2F1 cells were co-transfected with duplex siRNA targeting p65 and either ICAM-1 wild type or mutant promoter reporters. The stars indicate the significant differences ( P
Figure Legend Snippet: NF-κB binding sites are required for E2F1 regulation of ICAM-1 promoter. A . The histograph represent the human ICAM-1 promoter luciferase reporters used in this study. The location of each binding site on the promoter of ICAM-1 is indicated and labeled. The crossed boxes indicate the mutation sites on each construct. The mutated nucleotides corresponding to κB-1-mut and κB-2-mut are underlined. B and C . DU145/sh-Con and DU145/sh-E2F1 cells were co-transfected with duplex siRNA targeting p65 and either ICAM-1 wild type or mutant promoter reporters. The stars indicate the significant differences ( P

Techniques Used: Binding Assay, Luciferase, Labeling, Mutagenesis, Construct, Transfection

RNA Silencing of E2F1 increases p65/p50 heterdimer binding to ICAM-1 promoter. A . ChIP assay was performed with cell lysates from DU145/sh-Con and DU145/sh-E2F1 cells. The chromatin was immunoprecipitated with anti-E2F1 and anti-p65 antibodies or normal IgG which served as a negative control. A pair of primers flanking the κB-1 binding site within the ICAM-1 promoter was used in PCR. PCR for the E2F1 binding site within the CDC2 promoter served as a positive control for detecting E2F1 binding activity. B . Real-time PCR was employed to the ChIP assay in (A). Relative occupancy values were calculated by determining the apparent immunoprecipitation efficiency (ratios of the amount of immunoprecipitated DNA to that of the input sample) and normalized to the level of p65 binding with ICAM-1 promoter in DU145/sh-Con cells, which was defined as 1.0. C . The relationship among E2F1 and p65/p50 heterodimer in DU145/sh-Con and DU145/sh-E2F1 cells was examined by coimmunoprecipitation analysis. Anti-p65 antibody was used for immunoprecipitation. The amounts of E2F1, p65 and p50 in the immunoprecipitates were detected by Western blot with the indicated specific antibodies.
Figure Legend Snippet: RNA Silencing of E2F1 increases p65/p50 heterdimer binding to ICAM-1 promoter. A . ChIP assay was performed with cell lysates from DU145/sh-Con and DU145/sh-E2F1 cells. The chromatin was immunoprecipitated with anti-E2F1 and anti-p65 antibodies or normal IgG which served as a negative control. A pair of primers flanking the κB-1 binding site within the ICAM-1 promoter was used in PCR. PCR for the E2F1 binding site within the CDC2 promoter served as a positive control for detecting E2F1 binding activity. B . Real-time PCR was employed to the ChIP assay in (A). Relative occupancy values were calculated by determining the apparent immunoprecipitation efficiency (ratios of the amount of immunoprecipitated DNA to that of the input sample) and normalized to the level of p65 binding with ICAM-1 promoter in DU145/sh-Con cells, which was defined as 1.0. C . The relationship among E2F1 and p65/p50 heterodimer in DU145/sh-Con and DU145/sh-E2F1 cells was examined by coimmunoprecipitation analysis. Anti-p65 antibody was used for immunoprecipitation. The amounts of E2F1, p65 and p50 in the immunoprecipitates were detected by Western blot with the indicated specific antibodies.

Techniques Used: Binding Assay, Chromatin Immunoprecipitation, Immunoprecipitation, Negative Control, Polymerase Chain Reaction, Positive Control, Activity Assay, Real-time Polymerase Chain Reaction, Western Blot

E2F1 knockdown enhances cell adhesion through ICAM-1. A . DU145/sh-Con and DU145/sh-E2F1 cells were pre-incubated with anti-ICAM-1 antibody or transient transfected with duplex siRNA of ICAM-1. PBMC labeled with Calcein-AM were added to the indicated cells. After washing three times, attached PBMC were captured using a fluorescence microscope at nine randomly selected fields of each well. B . The number of attached PBMC in the (A) was presented by the bar graph (white bar, DU145/sh-Con; gray bar, DU145/sh-E2F1). The data are shown as the mean value ± SD of triplicate measurement. The stars indicate the significant differences ( P
Figure Legend Snippet: E2F1 knockdown enhances cell adhesion through ICAM-1. A . DU145/sh-Con and DU145/sh-E2F1 cells were pre-incubated with anti-ICAM-1 antibody or transient transfected with duplex siRNA of ICAM-1. PBMC labeled with Calcein-AM were added to the indicated cells. After washing three times, attached PBMC were captured using a fluorescence microscope at nine randomly selected fields of each well. B . The number of attached PBMC in the (A) was presented by the bar graph (white bar, DU145/sh-Con; gray bar, DU145/sh-E2F1). The data are shown as the mean value ± SD of triplicate measurement. The stars indicate the significant differences ( P

Techniques Used: Incubation, Transfection, Labeling, Fluorescence, Microscopy

A schematic model whereby E2F1 regulates the ICAM-1 mediated anti-tumor immune circuit through NF-κB modulation. NF-κB binding site is required for E2F1 regulation of ICAM-1. E2F1 interacts with NF-κB forming an NF-κB/E2F1 complex. E2F1 acts as a suppressor to prevent the NF-κB p65/p50 complex from binding to ICAM-1 promoter. As a consequence, E2F1 interferes with the adhesion of monocytes onto prostate cancer cells, the sensitivity of tumor cells to the cytotoxic effect of cytokine-induced killer cells and the growth of prostate cancer cells. Targeted knockdown of E2F1 does not affect the expression of phosphorylation of NF-κB p65 and IκBα, but releases NF-κB p65 to facilitate p65/p50 heterodimerization and binding to the ICAM-1 promoter. Subsequently, ICAM-1 transcription and production are induced, resulting in the enhanced immune cells mediated cytotoxicity against prostate carcinoma cells.
Figure Legend Snippet: A schematic model whereby E2F1 regulates the ICAM-1 mediated anti-tumor immune circuit through NF-κB modulation. NF-κB binding site is required for E2F1 regulation of ICAM-1. E2F1 interacts with NF-κB forming an NF-κB/E2F1 complex. E2F1 acts as a suppressor to prevent the NF-κB p65/p50 complex from binding to ICAM-1 promoter. As a consequence, E2F1 interferes with the adhesion of monocytes onto prostate cancer cells, the sensitivity of tumor cells to the cytotoxic effect of cytokine-induced killer cells and the growth of prostate cancer cells. Targeted knockdown of E2F1 does not affect the expression of phosphorylation of NF-κB p65 and IκBα, but releases NF-κB p65 to facilitate p65/p50 heterodimerization and binding to the ICAM-1 promoter. Subsequently, ICAM-1 transcription and production are induced, resulting in the enhanced immune cells mediated cytotoxicity against prostate carcinoma cells.

Techniques Used: Binding Assay, Expressing

Expression of ICAM-1 is highly inversely correlated with E2F1 expression. A . ICAM-1 expression in DU145 cells. Whole cell lysates or total RNA were prepared from DU145 cells stably transfected with either the control vector or shRNA targeting E2F1. The expression of E2F1 and ICAM-1 was monitored by RT-PCR (upper) and Western blot (lower). B . ICAM-1 expression in DU145 cells was analyzed by real-time PCR. The stars indicate the significant differences ( P
Figure Legend Snippet: Expression of ICAM-1 is highly inversely correlated with E2F1 expression. A . ICAM-1 expression in DU145 cells. Whole cell lysates or total RNA were prepared from DU145 cells stably transfected with either the control vector or shRNA targeting E2F1. The expression of E2F1 and ICAM-1 was monitored by RT-PCR (upper) and Western blot (lower). B . ICAM-1 expression in DU145 cells was analyzed by real-time PCR. The stars indicate the significant differences ( P

Techniques Used: Expressing, Stable Transfection, Transfection, Plasmid Preparation, shRNA, Reverse Transcription Polymerase Chain Reaction, Western Blot, Real-time Polymerase Chain Reaction

13) Product Images from "DZ2002 ameliorates fibrosis, inflammation, and vasculopathy in experimental systemic sclerosis models"

Article Title: DZ2002 ameliorates fibrosis, inflammation, and vasculopathy in experimental systemic sclerosis models

Journal: Arthritis Research & Therapy

doi: 10.1186/s13075-019-2074-9

DZ2002 reduced ICAM-1 and VCAM-1 in vivo and in vitro. a Western blot of ICAM-1 and VCAM-1 in BLM-induced SSc mice skin (DZ2002 = 50 mg/kg). b Survival rate of HMEC-1 at different concentrations of DZ2002 treatment. c Evaluation of mRNA levels of molecules associated with the upregulation of endothelial adhesion molecules in HMEC-1 by quantitative real-time reverse transcription-PCR, such as ICAM-1, VCAM-1, VEGF, bFGF, and ET-1. d Western blot of ICAM-1 and VCAM-1 in the HMEC-1 cells (TNF-α = 40 ng/ml, DZ2002 = 200 μM). e Representative photomicrographs showing the stained THP-1 cells on HMEC-1 cell layer (× 40, scale bar = 100 μm). The HMEC-1 cells were stimulated with TNF-a (40 ng/ml) with or without DZ2002 (200 μM). After 6 h incubation, those stained THP-1 cells adhered on HMEC-1 cell layer were counted. Mean ± SEM. * P
Figure Legend Snippet: DZ2002 reduced ICAM-1 and VCAM-1 in vivo and in vitro. a Western blot of ICAM-1 and VCAM-1 in BLM-induced SSc mice skin (DZ2002 = 50 mg/kg). b Survival rate of HMEC-1 at different concentrations of DZ2002 treatment. c Evaluation of mRNA levels of molecules associated with the upregulation of endothelial adhesion molecules in HMEC-1 by quantitative real-time reverse transcription-PCR, such as ICAM-1, VCAM-1, VEGF, bFGF, and ET-1. d Western blot of ICAM-1 and VCAM-1 in the HMEC-1 cells (TNF-α = 40 ng/ml, DZ2002 = 200 μM). e Representative photomicrographs showing the stained THP-1 cells on HMEC-1 cell layer (× 40, scale bar = 100 μm). The HMEC-1 cells were stimulated with TNF-a (40 ng/ml) with or without DZ2002 (200 μM). After 6 h incubation, those stained THP-1 cells adhered on HMEC-1 cell layer were counted. Mean ± SEM. * P

Techniques Used: In Vivo, In Vitro, Western Blot, Mouse Assay, Polymerase Chain Reaction, Staining, Incubation

14) Product Images from "A Contrast in Pathogenic Responses between C57BL/6J and BALB/cJ Mice Using a Model of Retinal Injury"

Article Title: A Contrast in Pathogenic Responses between C57BL/6J and BALB/cJ Mice Using a Model of Retinal Injury

Journal: The American Journal of Pathology

doi: 10.1016/j.ajpath.2018.08.010

Increased inflammation detected in B6 versus BALB/c retinas after ischemia/reperfusion (I/R) injury. Retinal lysates were processed under normal conditions and at 2 and 10 days after injury for protein levels of select proinflammatory mediators: IL-1β ( A ), tumor necrosis factor (TNF)-α ( B ), phosphorylated NF-κB p65 at Ser-536 ( C ), vascular endothelial growth factor (VEGF; D ), intracellular adhesion molecule-1 (ICAM-1; E ), and cyclooxygenase-2 (COX-2; F ). One representative gel image is provided for each Western blot. Data are expressed as means ± SD ( A–F ). n = 5 independent experiments ( A–F ). ∗ P
Figure Legend Snippet: Increased inflammation detected in B6 versus BALB/c retinas after ischemia/reperfusion (I/R) injury. Retinal lysates were processed under normal conditions and at 2 and 10 days after injury for protein levels of select proinflammatory mediators: IL-1β ( A ), tumor necrosis factor (TNF)-α ( B ), phosphorylated NF-κB p65 at Ser-536 ( C ), vascular endothelial growth factor (VEGF; D ), intracellular adhesion molecule-1 (ICAM-1; E ), and cyclooxygenase-2 (COX-2; F ). One representative gel image is provided for each Western blot. Data are expressed as means ± SD ( A–F ). n = 5 independent experiments ( A–F ). ∗ P

Techniques Used: Western Blot

15) Product Images from "Fibroblast growth factor 21 delayed endothelial replicative senescence and protected cells from H2O2-induced premature senescence through SIRT1"

Article Title: Fibroblast growth factor 21 delayed endothelial replicative senescence and protected cells from H2O2-induced premature senescence through SIRT1

Journal: American Journal of Translational Research

doi:

FGF21 delayed H 2 O 2 -induced HUVEC senescence. A representative picture (× 100 magnification) (A) and histogram (B) of SA-β-gal staining of HUVECs at PDL8 (Control), cells treated with 50 μmol/L H 2 O 2 (H 2 O 2 ), and cells pre-cultivated with 5 ng/mL FGF21 for 12 h before H 2 O 2 treatment (H 2 O 2 + FGF21). (C) The relative ROS level of each group compared to the control group. (D) The γ-H2AX immunofluorescence detection of DNA damage (× 200 magnification) with red showing γ-H2AX foci and blue showed nuclei stained with DAPI. A representative picture (E) and histogram (F) of the levels of proteins VCAM-1, ICAM-1, SIRT1, P53, P21 detected by western blot analysis with the protein GAPDH as a reference. Values are the mean ± SD; n = 3 in each group. Different letters indicate differences between the treatment groups, P
Figure Legend Snippet: FGF21 delayed H 2 O 2 -induced HUVEC senescence. A representative picture (× 100 magnification) (A) and histogram (B) of SA-β-gal staining of HUVECs at PDL8 (Control), cells treated with 50 μmol/L H 2 O 2 (H 2 O 2 ), and cells pre-cultivated with 5 ng/mL FGF21 for 12 h before H 2 O 2 treatment (H 2 O 2 + FGF21). (C) The relative ROS level of each group compared to the control group. (D) The γ-H2AX immunofluorescence detection of DNA damage (× 200 magnification) with red showing γ-H2AX foci and blue showed nuclei stained with DAPI. A representative picture (E) and histogram (F) of the levels of proteins VCAM-1, ICAM-1, SIRT1, P53, P21 detected by western blot analysis with the protein GAPDH as a reference. Values are the mean ± SD; n = 3 in each group. Different letters indicate differences between the treatment groups, P

Techniques Used: Staining, Immunofluorescence, Western Blot

Related Articles

Enzyme-linked Immunosorbent Assay:

Article Title: The MK2/HuR signaling pathway regulates TNF-α-induced ICAM-1 expression by promoting the stabilization of ICAM-1 mRNA
Article Snippet: .. Antibodies raised against MK2 (#3042), phospho-MK2 (Thr334, #3041), ICAM-1 (#4915) was obtained from Cell Signaling (Danvers, USA); a monoclonal antibody against HuR (ab14371) was received from Abcam (Cambridge, USA); antibodies against β-actin (P30002) and histone (P30266) were from Abmart (Shanghai, China);IL-8 ELISA Kit (BMS204/3) was received from eBioscience (San Diego, USA). .. RevertAid First Strand cDNA Synthesis Kit (#K1622) was purchased from Fermentas UAB (Vilnius, Lithuania).

Incubation:

Article Title: Vaspin inhibits cytokine-induced nuclear factor-kappa B activation and adhesion molecule expression via AMP-activated protein kinase activation in vascular endothelial cells
Article Snippet: .. Membranes were incubated in blocking buffer and then with one or more of the following primary antibodies: anti-AMPK (#2532, Cell Signaling, 1:1000), anti-phosphorylated AMPK (#2531, Thr172, Cell Signaling, 1:1000), anti-acetyl-CoA carboxylase (#3662, ACC, Cell Signaling, 1:1000), anti-phosphorylated ACC (#3661, Ser79, Cell Signaling, 1:1000), anti-phosphorylated Akt (#9271, Cell Signaling, 1:1000), anti-ICAM-1 (#4915, Cell Signaling, 1:1000), anti-VCAM-1 (#12367, Cell Signaling, 1:1000), anti-E-selectin (NBP1-45545, Novus Biologicals, CO, USA), and anti-MCP-1 (ab25124, Abcam, Cambridge, UK, 1:1000) antibodies. .. For the inhibitor of NF-κB (IκB) experiments, membranes were incubated with anti-IκBα (#4812, Cell Signaling, 1:1000), anti-phosphorylated IκBα (#4812, Ser32, Cell Signaling, 1:1000), or anti-β-actin (A5316, Sigma, St. Louis, MO, USA, 1:10,000) antibodies.

Article Title: Reduction of Endothelial Nitric Oxide Increases the Adhesiveness of Constitutive Endothelial Membrane ICAM-1 through Src-Mediated Phosphorylation
Article Snippet: .. The membranes were then incubated at room temperature in Odyssey blocking buffer (LiCor BioSciences) for 1 h, followed by incubations with primary antibodies [1:1,000 for anti-VE-Cadherin (Cat # 2500, Cell Signaling), anti ICAM-1 (1:1,000, Cat # 4915S, Cell Signaling or Cat # 554966, BD Pharmingen), and anti-β-actin (Cat # 8457, Cell Signaling), 1:500 for anti-p-ICAM-1 (phospho Y512, ab51033, Abcam)] at 4°C overnight, and secondary antibodies [1:10,000 for IRDye800-conjugated anti-mouse and anti-rabbit antibodies (Cat # 925-68071 and 925-32211, LiCor BioSciences)] at room temperature for 1 h. Images were visualized using a LiCor Odyssey Scanner and the bands were quantified using Image Studio software (LiCor BioSciences). ..

Article Title: Exosome-Transmitted miR-25 Induced by H. pylori Promotes Vascular Endothelial Cell Injury by Targeting KLF2
Article Snippet: .. The PVDF membranes were incubated with VCAM-1 (Cell Signaling Technology, CST, Boston, USA, #32653); ICAM-1 (CST, #4915); CD63 (Abcam, Shanghai, China, ab134045); KLF2 (Abcam, ab203591); or GAPDH (CST, #5174) antibodies overnight at 4°C, and then incubated with secondary antibodies (goat anti-rabbit or mouse) (ZSGB-BIO, Beijing, China) for 1 h. The PVDF membranes were then exposed in a chemiluminescence instrument (Bio-Rad ChemiDoc XRS+, USA) using the SuperSignal West Dura Extended Duration Substrate Kit (Thermo, Scientific, Beijing, China). .. Cell and Bacterial Culture The GES-1 and HEK293 cell lines were purchased from the American Type Culture Collection (ATCC).

Blocking Assay:

Article Title: Vaspin inhibits cytokine-induced nuclear factor-kappa B activation and adhesion molecule expression via AMP-activated protein kinase activation in vascular endothelial cells
Article Snippet: .. Membranes were incubated in blocking buffer and then with one or more of the following primary antibodies: anti-AMPK (#2532, Cell Signaling, 1:1000), anti-phosphorylated AMPK (#2531, Thr172, Cell Signaling, 1:1000), anti-acetyl-CoA carboxylase (#3662, ACC, Cell Signaling, 1:1000), anti-phosphorylated ACC (#3661, Ser79, Cell Signaling, 1:1000), anti-phosphorylated Akt (#9271, Cell Signaling, 1:1000), anti-ICAM-1 (#4915, Cell Signaling, 1:1000), anti-VCAM-1 (#12367, Cell Signaling, 1:1000), anti-E-selectin (NBP1-45545, Novus Biologicals, CO, USA), and anti-MCP-1 (ab25124, Abcam, Cambridge, UK, 1:1000) antibodies. .. For the inhibitor of NF-κB (IκB) experiments, membranes were incubated with anti-IκBα (#4812, Cell Signaling, 1:1000), anti-phosphorylated IκBα (#4812, Ser32, Cell Signaling, 1:1000), or anti-β-actin (A5316, Sigma, St. Louis, MO, USA, 1:10,000) antibodies.

Article Title: Reduction of Endothelial Nitric Oxide Increases the Adhesiveness of Constitutive Endothelial Membrane ICAM-1 through Src-Mediated Phosphorylation
Article Snippet: .. The membranes were then incubated at room temperature in Odyssey blocking buffer (LiCor BioSciences) for 1 h, followed by incubations with primary antibodies [1:1,000 for anti-VE-Cadherin (Cat # 2500, Cell Signaling), anti ICAM-1 (1:1,000, Cat # 4915S, Cell Signaling or Cat # 554966, BD Pharmingen), and anti-β-actin (Cat # 8457, Cell Signaling), 1:500 for anti-p-ICAM-1 (phospho Y512, ab51033, Abcam)] at 4°C overnight, and secondary antibodies [1:10,000 for IRDye800-conjugated anti-mouse and anti-rabbit antibodies (Cat # 925-68071 and 925-32211, LiCor BioSciences)] at room temperature for 1 h. Images were visualized using a LiCor Odyssey Scanner and the bands were quantified using Image Studio software (LiCor BioSciences). ..

Software:

Article Title: Reduction of Endothelial Nitric Oxide Increases the Adhesiveness of Constitutive Endothelial Membrane ICAM-1 through Src-Mediated Phosphorylation
Article Snippet: .. The membranes were then incubated at room temperature in Odyssey blocking buffer (LiCor BioSciences) for 1 h, followed by incubations with primary antibodies [1:1,000 for anti-VE-Cadherin (Cat # 2500, Cell Signaling), anti ICAM-1 (1:1,000, Cat # 4915S, Cell Signaling or Cat # 554966, BD Pharmingen), and anti-β-actin (Cat # 8457, Cell Signaling), 1:500 for anti-p-ICAM-1 (phospho Y512, ab51033, Abcam)] at 4°C overnight, and secondary antibodies [1:10,000 for IRDye800-conjugated anti-mouse and anti-rabbit antibodies (Cat # 925-68071 and 925-32211, LiCor BioSciences)] at room temperature for 1 h. Images were visualized using a LiCor Odyssey Scanner and the bands were quantified using Image Studio software (LiCor BioSciences). ..

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    Cell Signaling Technology Inc antibody against icam 1
    p-STAT3 upregulates <t>ICAM-1</t> expression in GSC11 cells under hypoxic conditions ( A ) Western blotting revealed that ICAM-1 is expressed in multiple human glioma stem cell lines. ( B ) Western blotting revealed that the ICAM-1 expression level in GSC11 cells increased in a time-dependent manner under hypoxic conditions. ( C ) Western blotting revealed that ICAM-1 was expressed in GSC11 cells treated with or without LY, U0126, SU, or AZD1480 under hypoxic conditions. ( D ) Western blotting revealed the p-STAT3 expression level in the nuclear fraction of GSC11 cells treated with or without AZD1480 under hypoxic conditions.
    Antibody Against Icam 1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 91/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc anti icam 1
    Effect of 9t18:1 and 11t18:1 on gene expression of <t>ICAM-1,</t> VCAM-1 and IL-6 of leptin/9c11t-CLA treated HUVECs. ( A ) The effect of 11t18:1 on expression of ICAM-1, VCAM-1 and IL-6 in leptin treated HUVECs. HUVECs were treated or without leptin (75 nmol/L) for 24 h and then cultured with 11t18:1(25, 50, 100 μmol/L) for 24 h. ( B ) The effect of 9t18:1 on expression of ICAM-1, VCAM-1 and IL-6 in 9c11tCLA treated HUVECs. HUVECs were treated or non-treated with 9c11tCLA (5, 25, 50 μmol/L) and then cultured with 9t18:1 (100 μmol/L) for 24 h. ( C ) The effect of 11t18:1 + leptin on expression of ICAM-1, VCAM-1 and IL-6 in 9c11tCLA treated HUVECs. HUVECs were treated or non-treated with 9c11tCLA (5, 25, 50 μmol/L) and then cultured with the group of 11t18:1 (100 μmol/L) + leptin (75 nmol/L) for 24 h. ( D ) The effect of 9t18:1 on expression of ICAM-1, VCAM-1 and IL-6 of leptin treated HUVECs. HUVECs were treated or non-treated with leptin (75 nmol/L) and then cultured with 9t18:1 (100 μmol/L) and 11t18:1 (100 μmol/L) for 24 h. a–g Data were presented as mean ± SD, values not sharing a common superscript denote significant difference ( P
    Anti Icam 1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 92/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 92 stars, based on 13 article reviews
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    Image Search Results


    p-STAT3 upregulates ICAM-1 expression in GSC11 cells under hypoxic conditions ( A ) Western blotting revealed that ICAM-1 is expressed in multiple human glioma stem cell lines. ( B ) Western blotting revealed that the ICAM-1 expression level in GSC11 cells increased in a time-dependent manner under hypoxic conditions. ( C ) Western blotting revealed that ICAM-1 was expressed in GSC11 cells treated with or without LY, U0126, SU, or AZD1480 under hypoxic conditions. ( D ) Western blotting revealed the p-STAT3 expression level in the nuclear fraction of GSC11 cells treated with or without AZD1480 under hypoxic conditions.

    Journal: Oncotarget

    Article Title: Targeting intercellular adhesion molecule-1 prolongs survival in mice bearing bevacizumab-resistant glioblastoma

    doi: 10.18632/oncotarget.18859

    Figure Lengend Snippet: p-STAT3 upregulates ICAM-1 expression in GSC11 cells under hypoxic conditions ( A ) Western blotting revealed that ICAM-1 is expressed in multiple human glioma stem cell lines. ( B ) Western blotting revealed that the ICAM-1 expression level in GSC11 cells increased in a time-dependent manner under hypoxic conditions. ( C ) Western blotting revealed that ICAM-1 was expressed in GSC11 cells treated with or without LY, U0126, SU, or AZD1480 under hypoxic conditions. ( D ) Western blotting revealed the p-STAT3 expression level in the nuclear fraction of GSC11 cells treated with or without AZD1480 under hypoxic conditions.

    Article Snippet: Blots were incubated with the primary antibody against ICAM-1 (1:1000; Cell signaling Technology, Danvers, MA), GFP (1:1000, CST), p-STAT3 (1:1000, Cell signaling), Tubulin (1:3000; Sigma), p-AKT (1: 1000, Cell signaling Technology, Danvers, MA), AKT (1: 1000, Cell signaling).

    Techniques: Expressing, Western Blot

    ICAM-1 knockdown inhibits macrophage infiltration into tumor in bevacizumab-treated mice ( A ) and ( B ) Immunofluorescence staining with F4/80 (red) (A) and the bar graph of F4/80 positive cells in each high powered microscopic field (B) revealed that shRNA ICAM-1generated tumor had less F4/80 positive macrophage infiltration compared with GSC11GFP generated tumor. Representative photomicrographic images are shown (magnification ×200). ( C ) Immunofluorescence staining for Nestin (green) and F4/80 revealed that blocking ICAM-1 by treatment with ICAM-1 antibody 10 µg/ml or 20 µg/ml in GSC11 cells reduced GSC11 binding to macrophages. ( D ) The bar graph represents the ratio of Nestin positive to F4/80 positive. Data showed that blocking ICAM-1 decreased GSC capacity of binding to macrophages.

    Journal: Oncotarget

    Article Title: Targeting intercellular adhesion molecule-1 prolongs survival in mice bearing bevacizumab-resistant glioblastoma

    doi: 10.18632/oncotarget.18859

    Figure Lengend Snippet: ICAM-1 knockdown inhibits macrophage infiltration into tumor in bevacizumab-treated mice ( A ) and ( B ) Immunofluorescence staining with F4/80 (red) (A) and the bar graph of F4/80 positive cells in each high powered microscopic field (B) revealed that shRNA ICAM-1generated tumor had less F4/80 positive macrophage infiltration compared with GSC11GFP generated tumor. Representative photomicrographic images are shown (magnification ×200). ( C ) Immunofluorescence staining for Nestin (green) and F4/80 revealed that blocking ICAM-1 by treatment with ICAM-1 antibody 10 µg/ml or 20 µg/ml in GSC11 cells reduced GSC11 binding to macrophages. ( D ) The bar graph represents the ratio of Nestin positive to F4/80 positive. Data showed that blocking ICAM-1 decreased GSC capacity of binding to macrophages.

    Article Snippet: Blots were incubated with the primary antibody against ICAM-1 (1:1000; Cell signaling Technology, Danvers, MA), GFP (1:1000, CST), p-STAT3 (1:1000, Cell signaling), Tubulin (1:3000; Sigma), p-AKT (1: 1000, Cell signaling Technology, Danvers, MA), AKT (1: 1000, Cell signaling).

    Techniques: Mouse Assay, Immunofluorescence, Staining, shRNA, Generated, Blocking Assay, Binding Assay

    ICAM-1 is overexpressed in bevacizumab-resistant GSC11 xenografts ( A ) Quantitative real-time PCR, in which GAPDH mRNA expression was used as internal control, revealed that the fold-change in the RNA expression level of ICAM-1 in xenografts from bevacizumab-treated mice was significantly higher than that in vehicle-treated control mice. ( B ) Immunohistochemical analysis revealed that GSC11 xenografts from mice treated with vehicle only (Control) had lower ICAM-1 expression than those from mice treated with bevacizumab (Bev) did. Representative images are shown; cell membranes expressing ICAM-1 are brown (indicated by red arrows; magnification × 400). ( C ) Western blotting revealed that ICAM-1 was expressed in the GSC11 xenografts regardless of whether mice were treated with vehicle (Control) or bevacizumab (Bev).

    Journal: Oncotarget

    Article Title: Targeting intercellular adhesion molecule-1 prolongs survival in mice bearing bevacizumab-resistant glioblastoma

    doi: 10.18632/oncotarget.18859

    Figure Lengend Snippet: ICAM-1 is overexpressed in bevacizumab-resistant GSC11 xenografts ( A ) Quantitative real-time PCR, in which GAPDH mRNA expression was used as internal control, revealed that the fold-change in the RNA expression level of ICAM-1 in xenografts from bevacizumab-treated mice was significantly higher than that in vehicle-treated control mice. ( B ) Immunohistochemical analysis revealed that GSC11 xenografts from mice treated with vehicle only (Control) had lower ICAM-1 expression than those from mice treated with bevacizumab (Bev) did. Representative images are shown; cell membranes expressing ICAM-1 are brown (indicated by red arrows; magnification × 400). ( C ) Western blotting revealed that ICAM-1 was expressed in the GSC11 xenografts regardless of whether mice were treated with vehicle (Control) or bevacizumab (Bev).

    Article Snippet: Blots were incubated with the primary antibody against ICAM-1 (1:1000; Cell signaling Technology, Danvers, MA), GFP (1:1000, CST), p-STAT3 (1:1000, Cell signaling), Tubulin (1:3000; Sigma), p-AKT (1: 1000, Cell signaling Technology, Danvers, MA), AKT (1: 1000, Cell signaling).

    Techniques: Real-time Polymerase Chain Reaction, Expressing, RNA Expression, Mouse Assay, Immunohistochemistry, Western Blot

    ICAM-1 knockdown prolongs survival in mice with bevacizumab-resistant glioblastoma ( A ) Kaplan-Meier survival analysis revealed that when mice bearing shRNA ICAM-1 #4 xenografts prolonger survival compared with the mice bearing GSC11 GFP after treatment with bevacizumab. H E staining analysis ( B ) and the bar graph of tumor volume ( C ) revealed that the size of the tumor which from mice bearing shRNA ICAM-1 #4 xenografts significantly decreased compared with the tumor from the mice bearing GSC11 GFP after with or without treatment of bevacizumab for 5 weeks. Representative images are shown ( D ) for the immunofluorescence staining of ICAM-1 (green) revealing that ICAM-1 expression was significantly lower in tumor from mice bearing shRNA ICAM-1 xenografts compared with tumor from mice bearing GSC11GFP after treatment with or without bevacizumab. Representative photomicrograph images are shown (magnification × 200). ( E ) The bar graph from the percentage of ICAM-1 staining in tumor area (D) revealed that ICAM-1 expression was blocked in tumor from mice bearing shRNA ICAM-1#4 compared with tumor from the mice bearing GSC11 GFP ( * P

    Journal: Oncotarget

    Article Title: Targeting intercellular adhesion molecule-1 prolongs survival in mice bearing bevacizumab-resistant glioblastoma

    doi: 10.18632/oncotarget.18859

    Figure Lengend Snippet: ICAM-1 knockdown prolongs survival in mice with bevacizumab-resistant glioblastoma ( A ) Kaplan-Meier survival analysis revealed that when mice bearing shRNA ICAM-1 #4 xenografts prolonger survival compared with the mice bearing GSC11 GFP after treatment with bevacizumab. H E staining analysis ( B ) and the bar graph of tumor volume ( C ) revealed that the size of the tumor which from mice bearing shRNA ICAM-1 #4 xenografts significantly decreased compared with the tumor from the mice bearing GSC11 GFP after with or without treatment of bevacizumab for 5 weeks. Representative images are shown ( D ) for the immunofluorescence staining of ICAM-1 (green) revealing that ICAM-1 expression was significantly lower in tumor from mice bearing shRNA ICAM-1 xenografts compared with tumor from mice bearing GSC11GFP after treatment with or without bevacizumab. Representative photomicrograph images are shown (magnification × 200). ( E ) The bar graph from the percentage of ICAM-1 staining in tumor area (D) revealed that ICAM-1 expression was blocked in tumor from mice bearing shRNA ICAM-1#4 compared with tumor from the mice bearing GSC11 GFP ( * P

    Article Snippet: Blots were incubated with the primary antibody against ICAM-1 (1:1000; Cell signaling Technology, Danvers, MA), GFP (1:1000, CST), p-STAT3 (1:1000, Cell signaling), Tubulin (1:3000; Sigma), p-AKT (1: 1000, Cell signaling Technology, Danvers, MA), AKT (1: 1000, Cell signaling).

    Techniques: Mouse Assay, shRNA, Staining, Immunofluorescence, Expressing

    ICAM-1 knockdown suppresses cell invasion in vitro and in vivo ( A ) and ( B ) GSC11GFP control and shRNA ICAM-1#4 cells treated with or without bevacizumab were subjected to a transwell migration assay. Photomicrographs of representative samples from the assay (A) and the bar graph of quantitative absorbance (590 nm) (B) revealed that shRNA ICAM-1#2 and shRNA ICAM-1 #4 cells significantly were less invaded compared with GSC GFP after cells treated with or without bevacizumab. Representative photomicrographs from three independent experiments are shown (magnification ×200) ( * P

    Journal: Oncotarget

    Article Title: Targeting intercellular adhesion molecule-1 prolongs survival in mice bearing bevacizumab-resistant glioblastoma

    doi: 10.18632/oncotarget.18859

    Figure Lengend Snippet: ICAM-1 knockdown suppresses cell invasion in vitro and in vivo ( A ) and ( B ) GSC11GFP control and shRNA ICAM-1#4 cells treated with or without bevacizumab were subjected to a transwell migration assay. Photomicrographs of representative samples from the assay (A) and the bar graph of quantitative absorbance (590 nm) (B) revealed that shRNA ICAM-1#2 and shRNA ICAM-1 #4 cells significantly were less invaded compared with GSC GFP after cells treated with or without bevacizumab. Representative photomicrographs from three independent experiments are shown (magnification ×200) ( * P

    Article Snippet: Blots were incubated with the primary antibody against ICAM-1 (1:1000; Cell signaling Technology, Danvers, MA), GFP (1:1000, CST), p-STAT3 (1:1000, Cell signaling), Tubulin (1:3000; Sigma), p-AKT (1: 1000, Cell signaling Technology, Danvers, MA), AKT (1: 1000, Cell signaling).

    Techniques: In Vitro, In Vivo, shRNA, Transwell Migration Assay

    Effect of 9t18:1 and 11t18:1 on gene expression of ICAM-1, VCAM-1 and IL-6 of leptin/9c11t-CLA treated HUVECs. ( A ) The effect of 11t18:1 on expression of ICAM-1, VCAM-1 and IL-6 in leptin treated HUVECs. HUVECs were treated or without leptin (75 nmol/L) for 24 h and then cultured with 11t18:1(25, 50, 100 μmol/L) for 24 h. ( B ) The effect of 9t18:1 on expression of ICAM-1, VCAM-1 and IL-6 in 9c11tCLA treated HUVECs. HUVECs were treated or non-treated with 9c11tCLA (5, 25, 50 μmol/L) and then cultured with 9t18:1 (100 μmol/L) for 24 h. ( C ) The effect of 11t18:1 + leptin on expression of ICAM-1, VCAM-1 and IL-6 in 9c11tCLA treated HUVECs. HUVECs were treated or non-treated with 9c11tCLA (5, 25, 50 μmol/L) and then cultured with the group of 11t18:1 (100 μmol/L) + leptin (75 nmol/L) for 24 h. ( D ) The effect of 9t18:1 on expression of ICAM-1, VCAM-1 and IL-6 of leptin treated HUVECs. HUVECs were treated or non-treated with leptin (75 nmol/L) and then cultured with 9t18:1 (100 μmol/L) and 11t18:1 (100 μmol/L) for 24 h. a–g Data were presented as mean ± SD, values not sharing a common superscript denote significant difference ( P

    Journal: Scientific Reports

    Article Title: 9c11tCLA modulates 11t18:1 and 9t18:1 induced inflammations differently in human umbilical vein endothelial cells

    doi: 10.1038/s41598-018-19729-9

    Figure Lengend Snippet: Effect of 9t18:1 and 11t18:1 on gene expression of ICAM-1, VCAM-1 and IL-6 of leptin/9c11t-CLA treated HUVECs. ( A ) The effect of 11t18:1 on expression of ICAM-1, VCAM-1 and IL-6 in leptin treated HUVECs. HUVECs were treated or without leptin (75 nmol/L) for 24 h and then cultured with 11t18:1(25, 50, 100 μmol/L) for 24 h. ( B ) The effect of 9t18:1 on expression of ICAM-1, VCAM-1 and IL-6 in 9c11tCLA treated HUVECs. HUVECs were treated or non-treated with 9c11tCLA (5, 25, 50 μmol/L) and then cultured with 9t18:1 (100 μmol/L) for 24 h. ( C ) The effect of 11t18:1 + leptin on expression of ICAM-1, VCAM-1 and IL-6 in 9c11tCLA treated HUVECs. HUVECs were treated or non-treated with 9c11tCLA (5, 25, 50 μmol/L) and then cultured with the group of 11t18:1 (100 μmol/L) + leptin (75 nmol/L) for 24 h. ( D ) The effect of 9t18:1 on expression of ICAM-1, VCAM-1 and IL-6 of leptin treated HUVECs. HUVECs were treated or non-treated with leptin (75 nmol/L) and then cultured with 9t18:1 (100 μmol/L) and 11t18:1 (100 μmol/L) for 24 h. a–g Data were presented as mean ± SD, values not sharing a common superscript denote significant difference ( P

    Article Snippet: Anti-ICAM-1, anti-p38, anti-p-p38, anti-SAPK/JNK, anti-p-SAPK/JNK, anti-ERK 1/2, anti-p-ERK1/2, anti-TLR4 and anti-SCD-1 were purchased from Cell Signaling Technology (USA).

    Techniques: Expressing, Cell Culture

    Effect of TAK242 on MAPKs phosphorylation in HUVECs treated with 9t18:1 and 11t18:1. ( A ) Effect of TAK242 on ICAM-1 expression. HUVECs were treated with TAK242 (0.5, 1, 1.5 μmol/L) for 30 min and then cultured with 9t18:1 for 24 h. ( B ) Effect of TAK242 on MAPKs phosphorylation in HUVECs treated with 9t18:1 and 11t18:1. HUVECs were treated with TAK242 (1 μmol/L) for 30 min and then cultured with TFA for 24 h. Values labeled with different letters in each set indicate significant differences ( p

    Journal: Scientific Reports

    Article Title: 9c11tCLA modulates 11t18:1 and 9t18:1 induced inflammations differently in human umbilical vein endothelial cells

    doi: 10.1038/s41598-018-19729-9

    Figure Lengend Snippet: Effect of TAK242 on MAPKs phosphorylation in HUVECs treated with 9t18:1 and 11t18:1. ( A ) Effect of TAK242 on ICAM-1 expression. HUVECs were treated with TAK242 (0.5, 1, 1.5 μmol/L) for 30 min and then cultured with 9t18:1 for 24 h. ( B ) Effect of TAK242 on MAPKs phosphorylation in HUVECs treated with 9t18:1 and 11t18:1. HUVECs were treated with TAK242 (1 μmol/L) for 30 min and then cultured with TFA for 24 h. Values labeled with different letters in each set indicate significant differences ( p

    Article Snippet: Anti-ICAM-1, anti-p38, anti-p-p38, anti-SAPK/JNK, anti-p-SAPK/JNK, anti-ERK 1/2, anti-p-ERK1/2, anti-TLR4 and anti-SCD-1 were purchased from Cell Signaling Technology (USA).

    Techniques: Expressing, Cell Culture, Labeling

    The effect of increased PD-L2 expression on immunological parameters. A, RT-PCR and Western blotting were performed to confirm the efficiency of PD-L2 containing plasmid transfection. The ability of the PD-L2 containing plasmid to induce PD-L2 mRNA expression was confirmed in two cell lines, with the more efficiently transfected Huh7 cells used for confirmation of protein expression. B, Representative flow cytometric analysis of Huh7 cells transfected with PD-L2 containing plasmid (green) and control plasmid (purple). Expressions of Fas, CD1d, HLA-class 1 and intercellular adhesion molecule-1 (ICAM-1) are shown. C, The mean fluorescence intensity (MFI) is presented as fold increase relative to control cells. The data represent mean ± SD of triplicate measurements. D, Immunohistochemical staining pattern of ICAM-1. Left panel is the representative staining pattern of ICAM-1 in liver tissue. Expression intensity was scored as follows: 0, negative; 1, weak expression; 2, moderate expression; 3, strong expression. Right: The concordance coefficients (κ statistics) were used to evaluate agreement between elevated PD-L2 expression and elevated ICAM-1 expression.

    Journal: Transplantation

    Article Title: Enhancement of Programmed Death Ligand 2 on Hepatitis C Virus Infected Hepatocytes by Calcineurin Inhibitors

    doi: 10.1097/TP.0000000000000572

    Figure Lengend Snippet: The effect of increased PD-L2 expression on immunological parameters. A, RT-PCR and Western blotting were performed to confirm the efficiency of PD-L2 containing plasmid transfection. The ability of the PD-L2 containing plasmid to induce PD-L2 mRNA expression was confirmed in two cell lines, with the more efficiently transfected Huh7 cells used for confirmation of protein expression. B, Representative flow cytometric analysis of Huh7 cells transfected with PD-L2 containing plasmid (green) and control plasmid (purple). Expressions of Fas, CD1d, HLA-class 1 and intercellular adhesion molecule-1 (ICAM-1) are shown. C, The mean fluorescence intensity (MFI) is presented as fold increase relative to control cells. The data represent mean ± SD of triplicate measurements. D, Immunohistochemical staining pattern of ICAM-1. Left panel is the representative staining pattern of ICAM-1 in liver tissue. Expression intensity was scored as follows: 0, negative; 1, weak expression; 2, moderate expression; 3, strong expression. Right: The concordance coefficients (κ statistics) were used to evaluate agreement between elevated PD-L2 expression and elevated ICAM-1 expression.

    Article Snippet: Anti-PD-L2 (diluted 1:100; R & D Systems, Minneapolis, MN) and anti-ICAM-1 (diluted 1:30; Cell Signaling Technology, Danvers, MA) were used.

    Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Plasmid Preparation, Transfection, Flow Cytometry, Fluorescence, Immunohistochemistry, Staining

    BSNXD suppresses adhesion molecules expression on HUVECs through ER β /NO/NF- κ B pathway. The primary HUVECs were exposed to control serum or 10% BSNXD-derived serum for 48 h; in the final 24 h culture the ox-LDL was added. The supernatants were collected, and MCP-1 concentrations were determined. The MCP-1 expression of HUVECs was determined by western blot analysis. The mRNA and protein expression levels of cell adhesion molecules (ICAM-1, VCAM-1 and E-selectin) were assessed by RT-PCR, western blot and FACS. The MCP-1( a ), ICAM-1 ( b ), VCAM-1 ( c ) and E-selectin ( d ) expression was greatly decreased after treatment with the 10% drug-derived serum. NOS inhibitor (L-NAME), ER β antagonist (R, RTHC) other than ER α antagonist (MPP) could block these effects induced by the drug-derived serum. Data are expressed as mean values±S.E.M. ( n =6). * P

    Journal: Cell Death & Disease

    Article Title: Anti-inflammatory effects of a Chinese herbal medicine in atherosclerosis via estrogen receptor β mediating nitric oxide production and NF-κB suppression in endothelial cells

    doi: 10.1038/cddis.2013.66

    Figure Lengend Snippet: BSNXD suppresses adhesion molecules expression on HUVECs through ER β /NO/NF- κ B pathway. The primary HUVECs were exposed to control serum or 10% BSNXD-derived serum for 48 h; in the final 24 h culture the ox-LDL was added. The supernatants were collected, and MCP-1 concentrations were determined. The MCP-1 expression of HUVECs was determined by western blot analysis. The mRNA and protein expression levels of cell adhesion molecules (ICAM-1, VCAM-1 and E-selectin) were assessed by RT-PCR, western blot and FACS. The MCP-1( a ), ICAM-1 ( b ), VCAM-1 ( c ) and E-selectin ( d ) expression was greatly decreased after treatment with the 10% drug-derived serum. NOS inhibitor (L-NAME), ER β antagonist (R, RTHC) other than ER α antagonist (MPP) could block these effects induced by the drug-derived serum. Data are expressed as mean values±S.E.M. ( n =6). * P

    Article Snippet: Equal amounts of proteins were then separated by 10% of SDS-polyacrylamide gel electrophoresis, and the proteins were transferred to a PVDF membrane by electrotransfer for 1.5 h. The membranes were blocked with 5% non-fat-milk in TBS-T, and then incubated with anti-ERα (sc-8002; Santa Cruz Biotechnology), anti- ERβ (sc-8974; Santa Cruz Biotechnology), anti-eNOS (Cell Signaling Technology Inc., Beverly, MA, USA), anti-MCP-1 (Santa Cruz Biotechnology), anti-ICAM-1 (Cell Signaling Technology), anti-VCAM-1 (Santa Cruz Biotechnology), anti-E-selectin (Santa Cruz Biotechnology), mouse monoclonal antibodies for human CCR2 (Abcam), anti-CD11a antibody (LFA-1) (Abcam), Anti-Integrin α 4 antibody (VLA-4) (Abcam) or anti-β -actin (Cell Signaling Technology) overnight at 4°C followed by secondary horseradish peroxidase-conjugated antibody (Rockland Co., Gilbertsville, PA, USA).

    Techniques: Expressing, Derivative Assay, Western Blot, Reverse Transcription Polymerase Chain Reaction, FACS, Blocking Assay