Journal: Scientific Reports

Article Title: FAK and Pyk2 activity promote TNF-α and IL-1β-mediated pro-inflammatory gene expression and vascular inflammation

doi: 10.1038/s41598-019-44098-2

Figure Lengend Snippet: FAK/Pyk2 inhibition blocks TNF-α-induced pro-inflammatory adhesion molecule expression in HAoECs. ( a ) HAoECs were treated with DMSO, a dual FAK/Pyk2 inhibitor (PF-271, 2.5 μM) or a Src inhibitor (Dasatinib, 1 μM) for 1 h prior to TNF-α (10 ng/ml, 6 h) stimulation. Cropped images of immunoblotting for VCAM-1, ICAM-1, E-selectin, active FAK (pY397 FAK), FAK, active Src (pY418 Src), Src, and β-actin as loading control are shown. Full length blots shown in Supplemental Fig. . ( b – d ) HAoECs were treated with DMSO, a dual FAK/Pyk2 inhibitor (PF-271, 2.5 μM), a Src inhibitor (Dasatinib, 1 μM), or a FAK inhibitor (PF-228, 10 μM) for 1 h prior to TNF-α (10 ng/ml, 6 h) stimulation. Expression levels of ( b ) VCAM-1, ( c ) ICAM-1, and ( d ) E-selectin were determined using ELISA (n = 3, ±SEM). ( e ) HAoECs were treated with DMSO, a dual FAK/Pyk2 inhibitor (PF-271, 2.5 μM) or a FAK inhibitor (PF-228, 10 μM) for 1 h prior to TNF-α (10 ng/ml, 6 h) stimulation. Cropped images of immunoblotting for VCAM-1, ICAM-1, E-selectin, pY397 FAK, FAK, pY402 Pyk2, Pyk2, and β-actin as loading control are shown. Full length blots shown in Supplemental Fig. . ( f ) HAoECs were transfected with either control, FAK siRNA (400 pmol, 36 h), or Pyk2 siRNA (400 pmol, 36 h) prior to TNF-α (10 ng/ml, 4 h) stimulation. Cropped images of immunoblotting for VCAM-1, ICAM-1, E-selectin, FAK, Pyk2, and β-actin as loading control are shown. Full length blots shown in Supplemental Fig. . **p < 0.01, ****p < 0.0001.

Article Snippet: HAoECs were pretreated for 1 h with either the FAK/Pyk2 inhibitor PF-271 (MedKoo), the FAK inhibitor PF-228 (MedKoo), the FAK/Pyk2 inhibitor PF-271 VS-6063 (MedChemExpress), the Src inhibitor Dasatinib (Selleckchem), the MEK inhibitor PD98059 (Tocris), or the JNK inhibitor SP600125 (Selleckchem) prior to stimulation with either TNF-α (R&D) or IL-1β (Miltenyi Biotec).

Techniques: Inhibition, Expressing, Western Blot, Control, Enzyme-linked Immunosorbent Assay, Transfection

Journal: Scientific Reports

Article Title: FAK and Pyk2 activity promote TNF-α and IL-1β-mediated pro-inflammatory gene expression and vascular inflammation

doi: 10.1038/s41598-019-44098-2

Figure Lengend Snippet: FAK/Pyk2 activity promotes TNF-α-mediated pro-inflammatory molecule expression via transcriptional control. ( a ) HAoECs were treated with DMSO or PF-271 (2.5 μM) for 1 h prior to TNF-α (10 ng/ml) stimulation for 0 to 60 min. Cropped images of immunoblotting for pY397 FAK, FAK, active NF-κB (pS536 NF-κB), NF-κB, IκBα, active ERK (p-ERK), ERK, active JNK (p-JNK), JNK, active p38 (p-p38), p38, and β-actin as loading control are shown. Full length blots shown in Supplemental Fig. . ( b ) HAoECs were treated with DMSO or PF-271 (2.5 μM) for 1 h prior to TNF-α (10 ng/ml, 6 h) stimulation. RNA was collected, and RT-qPCR for cell adhesion molecules was performed (n = 3, ±SEM). ( c ) HAoECs were treated with DMSO or Dasatinib (1 μM) for 1 h prior to TNF-α (10 ng/ml, 6 h) stimulation. RNA was collected, and RT-qPCR for cell adhesion molecules was performed (n = 3, ±SEM). ( d ) HAoECs were treated with DMSO or PF-271 (2.5 μM) for 1 h prior to TNF-α (10 ng/ml, 24 h) stimulation. RNA was collected, and RT-qPCR was performed using an array for inflammatory cytokines, chemokines and their receptors. Shown is a selection of genes that is important in vascular inflammation and were reduced by PF-271 treatment upon TNF-α stimulation. ( e ) CXCL11, MCP-1, and IP-10 mRNA levels were verified by RT-qPCR (n = 3, ±SEM). ( f ) HAoECs were treated with DMSO or PF-271 (2.5 μM) for 1 h prior to TNF-α (10 ng/ml, 24 h) stimulation. Supernatant was collected, and MCP-1 protein expression was determined via ELISA (n = 3, ±SEM). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Article Snippet: HAoECs were pretreated for 1 h with either the FAK/Pyk2 inhibitor PF-271 (MedKoo), the FAK inhibitor PF-228 (MedKoo), the FAK/Pyk2 inhibitor PF-271 VS-6063 (MedChemExpress), the Src inhibitor Dasatinib (Selleckchem), the MEK inhibitor PD98059 (Tocris), or the JNK inhibitor SP600125 (Selleckchem) prior to stimulation with either TNF-α (R&D) or IL-1β (Miltenyi Biotec).

Techniques: Activity Assay, Expressing, Control, Western Blot, Quantitative RT-PCR, Selection, Enzyme-linked Immunosorbent Assay

Journal: Scientific Reports

Article Title: FAK and Pyk2 activity promote TNF-α and IL-1β-mediated pro-inflammatory gene expression and vascular inflammation

doi: 10.1038/s41598-019-44098-2

Figure Lengend Snippet: FAK/Pyk2 inhibition reduces TNF-α-induced in vitro inflammation. ( a ) HAoECs were treated with PF-271 (2.5 μM) for 1 h prior to TNF-α (10 ng/ml, 6 h) stimulation. Staining for VCAM-1 (red) and DAPI (blue) are shown. Scale bar (20 μm). ( b-c ) Monocyte adhesion assay. Primary mouse monocytes from bone marrow were labeled using Cell Tracker Green. HAoECs were treated with DMSO or PF-271 (2.5 μM) for 1 h prior to TNF-α (10 ng/ml, 6 h) stimulation. Monocytes were allowed to attach for 1 h prior to fixation. ( b ) Images of attached monocytes are shown. Scale bar (200 μm). ( c ) Attached monocytes were enumerated (n = 3, ±SEM). ( d ) HAoECs onto collagen I (10 μg/ml) coated Boyden chamber were treated with DMSO or PF-271 (2.5 μM) for 1 h prior to TNF-α (10 ng/ml, 6 h) stimulation. THP-1 cells were allowed to transmigrate the endothelial layer for 16 h. Transmigrated THP-1 cells were quantified (n = 3, ±SEM). ( e ) HAoECs were treated with PF-271 (2.5 μM) for 1 h prior to TNF-α (10 ng/ml, 6 h) stimulation. Staining of active FAK (pY397 FAK, red) or total FAK (green) are shown. Scale bar (20 μm). **p < 0.01, ****p < 0.0001.

Article Snippet: HAoECs were pretreated for 1 h with either the FAK/Pyk2 inhibitor PF-271 (MedKoo), the FAK inhibitor PF-228 (MedKoo), the FAK/Pyk2 inhibitor PF-271 VS-6063 (MedChemExpress), the Src inhibitor Dasatinib (Selleckchem), the MEK inhibitor PD98059 (Tocris), or the JNK inhibitor SP600125 (Selleckchem) prior to stimulation with either TNF-α (R&D) or IL-1β (Miltenyi Biotec).

Techniques: Inhibition, In Vitro, Staining, Cell Adhesion Assay, Labeling

Journal: Scientific Reports

Article Title: FAK and Pyk2 activity promote TNF-α and IL-1β-mediated pro-inflammatory gene expression and vascular inflammation

doi: 10.1038/s41598-019-44098-2

Figure Lengend Snippet: FAK/Pyk2 inhibition blocks IL-1β-mediated pro-inflammatory adhesion molecule expression in HAoECs. ( a ) HAoECs were treated with DMSO, a dual FAK/Pyk2 inhibitor (PF-271, 2.5 μM) or a Src inhibitor (Dasatinib, 1 μM) for 1 h prior to IL-1β (20 ng/ml, 6 h) stimulation. Cropped images of immunoblotting for VCAM-1, ICAM-1, E-selectin, active FAK (pY397 FAK), FAK, active Src (pY418 Src), Src, and β-actin as loading control are shown. Full length blots shown in Supplemental Fig. . ( b ) HAoECs were treated with DMSO, a dual FAK/Pyk2 inhibitor (PF-271, 2.5 μM) or a FAK inhibitor (PF-228, 10 μM) for 1 h prior to IL-1β (20 ng/ml, 6 h) stimulation. Cropped images of immunoblotting for VCAM-1, ICAM-1, E-selectin, pY397 FAK, FAK, pY402 Pyk2, Pyk2, and β-actin as loading control are shown. Full length blots shown in Supplemental Fig. .

Article Snippet: HAoECs were pretreated for 1 h with either the FAK/Pyk2 inhibitor PF-271 (MedKoo), the FAK inhibitor PF-228 (MedKoo), the FAK/Pyk2 inhibitor PF-271 VS-6063 (MedChemExpress), the Src inhibitor Dasatinib (Selleckchem), the MEK inhibitor PD98059 (Tocris), or the JNK inhibitor SP600125 (Selleckchem) prior to stimulation with either TNF-α (R&D) or IL-1β (Miltenyi Biotec).

Techniques: Inhibition, Expressing, Western Blot, Control

Journal: Scientific Reports

Article Title: FAK and Pyk2 activity promote TNF-α and IL-1β-mediated pro-inflammatory gene expression and vascular inflammation

doi: 10.1038/s41598-019-44098-2

Figure Lengend Snippet: FAK/Pyk2 activity promotes IL-1β-mediated pro-inflammatory molecule expression via transcriptional control. ( a ) HAoECs were treated with DMSO or PF-271 (2.5 μM) for 1 h prior to IL-1β (20 ng/ml) stimulation for 0 to 60 min. Cropped images of immunoblotting for pY397 FAK, FAK, active NF-κB (pS536 NF-κB), NF-κB, IκBα, active ERK (p-ERK), ERK, active JNK (p-JNK1, 2, and 3), JNK, active p38 (p-p38), p38, and β-actin as loading control are shown. Full length blots shown in Supplemental Fig. . ( b ) HAoECs were treated with DMSO or PF-271 (2.5 μM) for 1 h prior to IL-1β (20 ng/ml, 6 h) stimulation. RNA was collected, and RT-qPCR for cell adhesion molecules was performed (n = 3, ±SEM). ( c ) HAoECs were treated with DMSO or PF-271 (2.5 μM) for 1 h prior to IL-1β (20 ng/ml, 24 h) stimulation. RNA was collected, and RT-qPCR using an array for inflammatory cytokines, chemokines and their receptors. Shown are a selection of genes that are important in vascular inflammation and were reduced by PF-271 treatment upon IL-1β stimulation. ( d ) CXCL11, MCP-1, and IP-10 mRNA levels were verified by RT-qPCR (n = 3, ±SEM). **p < 0.01, ***p < 0.001, ****p < 0.0001.

Article Snippet: HAoECs were pretreated for 1 h with either the FAK/Pyk2 inhibitor PF-271 (MedKoo), the FAK inhibitor PF-228 (MedKoo), the FAK/Pyk2 inhibitor PF-271 VS-6063 (MedChemExpress), the Src inhibitor Dasatinib (Selleckchem), the MEK inhibitor PD98059 (Tocris), or the JNK inhibitor SP600125 (Selleckchem) prior to stimulation with either TNF-α (R&D) or IL-1β (Miltenyi Biotec).

Techniques: Activity Assay, Expressing, Control, Western Blot, Quantitative RT-PCR, Selection

Journal: Scientific Reports

Article Title: FAK and Pyk2 activity promote TNF-α and IL-1β-mediated pro-inflammatory gene expression and vascular inflammation

doi: 10.1038/s41598-019-44098-2

Figure Lengend Snippet: FAK/Pyk2 activity controls vascular inflammation in vivo mouse carotid ligation model. Ligated carotid arteries from 8-week-old ApoE −/− mice were harvested after fed a high fat diet (HFD) for 1 week with or without PF-271. Normal arteries from ApoE −/− mice fed with normal chow were used as control. ( a ) Sections were stained with VCAM-1 (red), vWF (green), and DAPI (blue). Scale bar (20 μm). ( b ) Macrophage infiltration was monitored with anti-CD68 (red) staining. Anti-α-SMA (green) and DAPI (blue) were used as counterstain. Scale bar (20 μm). ( c ) FAK activity was evaluated by staining anti-pY576/577 FAK (red). Anti-α-SMA (green) and DAPI (blue) were used as counterstain. Scale bar (20 μm). ( d ) Cropped images of immunoblotting of lung lysates of active FAK (pY397 FAK), total FAK, and β-actin as loading control are shown. Full length blots shown in Supplemental Fig. .

Article Snippet: HAoECs were pretreated for 1 h with either the FAK/Pyk2 inhibitor PF-271 (MedKoo), the FAK inhibitor PF-228 (MedKoo), the FAK/Pyk2 inhibitor PF-271 VS-6063 (MedChemExpress), the Src inhibitor Dasatinib (Selleckchem), the MEK inhibitor PD98059 (Tocris), or the JNK inhibitor SP600125 (Selleckchem) prior to stimulation with either TNF-α (R&D) or IL-1β (Miltenyi Biotec).

Techniques: Activity Assay, In Vivo, Ligation, Control, Staining, Western Blot

Journal:

Article Title: Development of an Immunoglobulin M (IgM) Capture Enzyme-Linked Immunosorbent Assay for Detection of Equine and Swine IgM Antibodies to Vesicular Stomatitis Virus

doi: 10.1128/CDLI.8.3.475-481.2001

Figure Lengend Snippet: Titration of mouse anti-VSV antibodies. IgG antibodies were separated from ascitic fluid collected from mice infected with VSV-NJ (F38-51-NJ) or VSV-IN (F39-52-IN). Their levels of binding to VSV-NJ and VSV-IN antigens were detected by an I-ELISA for VSV-NJ (A) and VSV-IN (B). A cutoff value of an OD410 of 1.0 (horizontal lines) was used to determine the end point titer of the antibodies.

Article Snippet: The microtiter serum neutralization (MTSN) test was performed according to a method previously described ( 14 ) for testing neutralizing serum antibodies to VSV-NJ strain VS 20-022-522 and VSV-IN strain VS 20-001-022 obtained from the American Type Culture Collection (Manassas, Va.).

Techniques: Titration, Infection, Binding Assay, Enzyme-linked Immunosorbent Assay

Journal:

Article Title: Development of an Immunoglobulin M (IgM) Capture Enzyme-Linked Immunosorbent Assay for Detection of Equine and Swine IgM Antibodies to Vesicular Stomatitis Virus

doi: 10.1128/CDLI.8.3.475-481.2001

Figure Lengend Snippet: Sensitivities of sera and IgM antibodies to VSV-NJ (A) and VSV-IN (B) measured in the VSV-NJ MC-ELISA (A) and VSV-IN MC-ELISA (B). Serum samples and purified IgM antibodies were collected from horses experimentally infected with VSV-NJ (H-1) and VSV-IN (H-2) at 0 DPI (0d) and 6 DPI (6d). An OD410 of 0.3 (horizontal lines) was selected as the cutoff.

Article Snippet: The microtiter serum neutralization (MTSN) test was performed according to a method previously described ( 14 ) for testing neutralizing serum antibodies to VSV-NJ strain VS 20-022-522 and VSV-IN strain VS 20-001-022 obtained from the American Type Culture Collection (Manassas, Va.).

Techniques: Enzyme-linked Immunosorbent Assay, Purification, Infection

Journal: Scientific Reports

Article Title: Activation of PAR2 by tissue factor induces the release of the PTEN from MAGI proteins and regulates PTEN and Akt activities

doi: 10.1038/s41598-020-77963-6

Figure Lengend Snippet: The influence of incubation of cell lines with rec-TF and PAR2-activation on PTEN activity and Akt inhibition. Cells (2 × 10 5 ) were treated with ( A ) rec-TF (65 pg/ml) for 1 h, or ( B ) PAR2-AP for 30 min, lysed in PhosphoSafe buffer (150 µl) and the lipid-phosphatase activity of PTEN measured using the echelon PTEN Activity ELISA kit (n = 3; *p < 0.05 vs. the respective untreated samples). Akt activity was also determined in the samples treated with ( C ) rec-TF or ( D ) PAR2-AP as above, and measured using the Akt Kinase activity assay kit according to manufacturer's instructions (n = 3; *p < 0.05 vs. the respective untreated samples). Samples of MDA-MB-231, LoVo and CaCo-2 cells were pre-incubated with a PAR2 blocking antibody (SAM-11, 20 µg/ml) or a control isotype antibody. The cells were treated with recombinant TF and ( E ) PTEN activity and ( F ) Akt activity measured as above (n = 3; *p < 0.05 vs. the respective control isotype antibody).

Article Snippet: Cells (2 × 10 5 ) were treated with ( A ) rec-TF (65 pg/ml) for 1 h, or ( B ) PAR2-AP for 30 min, lysed in PhosphoSafe buffer (150 µl) and the lipid-phosphatase activity of PTEN measured using the echelon PTEN Activity ELISA kit (n = 3; *p < 0.05 vs. the respective untreated samples).

Techniques: Incubation, Activation Assay, Activity Assay, Inhibition, Enzyme-linked Immunosorbent Assay, Kinase Assay, Blocking Assay, Recombinant

Journal: Scientific Reports

Article Title: Activation of PAR2 by tissue factor induces the release of the PTEN from MAGI proteins and regulates PTEN and Akt activities

doi: 10.1038/s41598-020-77963-6

Figure Lengend Snippet: The influence of long-term treatment of cell lines with rec-TF on cellular PTEN antigen levels and Akt activity. Equal number of cells were lysed in PhosphoSafe buffer (150 µl) and ( A ) the level of PTEN antigen measured using a PTEN ELISA kit (n = 5; *p < 0.05 vs. the samples on first day 0). ( B ) The Akt activity was also measured using the Akt Kinase activity assay kit (n = 3; *p < 0.05 vs. the samples on first day 0). Cells (2 × 10 5 ) were cultured in the recommended media and supplemented every 2 days with rec-TF (65 pg/ml) up to 14 days. ( C ) The number of cells in the treated and untreated samples were determined using crystal violet staining (n = 3; *p < 0.05 vs. the samples on first day 0). In addition, cells were lysed in PhosphoSafe buffer and ( D ) the level of PTEN antigen and ( E ) the Akt kinase activity measured. (n = 3; *p < 0.05 vs. the samples on first day 0).

Article Snippet: Cells (2 × 10 5 ) were treated with ( A ) rec-TF (65 pg/ml) for 1 h, or ( B ) PAR2-AP for 30 min, lysed in PhosphoSafe buffer (150 µl) and the lipid-phosphatase activity of PTEN measured using the echelon PTEN Activity ELISA kit (n = 3; *p < 0.05 vs. the respective untreated samples).

Techniques: Activity Assay, Enzyme-linked Immunosorbent Assay, Kinase Assay, Cell Culture, Staining