Journal: iScience

Article Title: SART1 modulates poly-(ADP-ribose) chain accumulation and PARP1 chromatin localization

doi: 10.1016/j.isci.2024.111252

Figure Lengend Snippet:

Article Snippet: Mouse anti-PAR antibody , RD-Systems , 4335-MC-100.

Techniques: Virus, Recombinant, Lysis, Extraction, Protease Inhibitor, Transfection, Mutagenesis, Western Blot, Purification, Expressing, Plasmid Preparation, Software, Microscopy

Journal: Frontiers in Oncology

Article Title: Forkhead Box Q1 Is Critical to Angiogenesis and Macrophage Recruitment of Colorectal Cancer

doi: 10.3389/fonc.2020.564298

Figure Lengend Snippet: Inhibition of FOXQ1 induces downregulation of angiogenic factors and upregulation of angiogenic inhibitors. (A) Matrix distribution of 60 well-established angiogenic proteins on a Quantibody Human Angiogenesis Array (QAH-ANG-2 and QAH-ANG-3); each protein is distributed in quadruplicate horizontally. (B) Fluorescence detection of protein arrays for either cell lysate of HUVECs or conditioned medium (CM) of colorectal cancer (CRC) cells. CM was collected from either DLD1-shFOXQ1 or DLD1-shControl and added to HUVECs. After 48 h, HUVECs were harvested and lysed. Both the cell lysates and CM were collected, and the secretion of 60 angiogenic proteins were measured by hybridization with QAH-ANG-2 and QAH-ANG-3. The 17 green and 6 red rectangles on (A, B) show 17 angiogenesis factors and 6 angiogenic inhibitors within the detection range of the protein array. (C) Quantitative concentration of the 17 angiogenic factors and 6 angiogenic factors in cell lysates of HUVECs (pg/ml). (D) Quantitative concentration of the 17 angiogenic factors and 6 angiogenic factors in CM collected from either DLD1-shFOXQ1 or DLD1-shControl (pg/ml). The values above each bar on (C, D) represent quantitative concentration of corresponding protein, is the average of quadruplicates (n = 1 per group). (E) Western blot analyses for ANG, PDGF, PDGFRB, ANGPT1, PLAUR, tPA, VEGF, EGFR, HB-EGF, and Twist1 were performed with DLD1-shFOXQ1 and DLD1-shControl cell lysates. β-actin was used as the loading control. (F) Inhibition of FOXQ1 prevented the autocrine secretion of CCL2, whereas overexpression of FOXQ1 enhanced the secretion of CCL2, as determined by ELISA analysis. ** P <0.01, *** P <0.001 signifies a significant difference between the indicated groups (two-tailed, unpaired Student’s t -test). Bars represent mean ± S.E. of three independent experiments. HUVEC, human umbilical vein endothelial cell; CM, conditioned media.

Article Snippet: The antibody against EGFR (3265S) was purchased from Cell Signaling (Cold Spring Harbor, NY, USA), and antibodies against ANG (18302-1-AP), PDGFRB (13449-1-AP), ANGPT1 (23302-1-AP), PLAUR (10286-1-AP), tPA (10147-1-AP), VEGF (19003-1-AP), and β-actin (66009-1-Ig) were purchased from Proteintech (Wuhan, China). β-Actin was used as the loading control.

Techniques: Inhibition, Fluorescence, Hybridization, Protein Array, Concentration Assay, Western Blot, Control, Over Expression, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: Journal of thrombosis and haemostasis : JTH

Article Title: Transcriptional program induced by factor VIIa-tissue factor, PAR1 and PAR2 in MDA-MB-231 cells.

doi: 10.1111/j.1538-7836.2007.02603.x

Figure Lengend Snippet: Fig. 4. Differential gene regulation in U373 cells. (A) Flow cytometry of U373 cells probed with antiprotease-activated receptor 1 (PAR1), anti-PAR2 or antitissue factor (TF). (B–D) Total RNA from U373 cells treated with: media; 50 nM FVIIa; 50 nM FXa; 50 nM thrombin (FIIa); 10 lM PAR1 agon- ist peptide or 50 lM PAR2 agonist peptide for 1 or 6 h was isolated and subjected to real-time qPCR analysis. Regulation of the mRNA level was measured relative to media control (mean ± SEM; n = 2). Statistical significance (*P < 0.05; **P < 0.01; ***P < 0.001) was determined by t-test analysis.

Article Snippet: Cells were resuspended in FACS buffer at 4 C. Samples were incubated with 10 lg mL–1 monoclonal antibodies against TF (1F44A1, Novo Nordisk, Maalov, Denmark), PAR1 (ATAP-2) or PAR2 (SAM11) (Santa-Cruz Biotechnology, Santa Cruz, CA, USA) for 60 min at 4 C. Samples and controls were then washed and incubated with 50 lg mL–1 PE-conjugated goat antimouse IgG (Dako, Copenhagen, Denmark) in the dark for 30 min at 4 C, after which they were washed twice with FACS buffer and fixed for 2 h in the dark at 4 C in FACS buffer containing 0.5% paraformaldehyde and analyzed for fluorescence using a BDFACS CantoTM (Becton Dickinson, Brondby, Denmark).

Techniques: Flow Cytometry, Isolation, Control

Journal: International Journal of Molecular Sciences

Article Title: Thrombin Preconditioning Boosts Biogenesis of Extracellular Vesicles from Mesenchymal Stem Cells and Enriches Their Cargo Contents via Protease-Activated Receptor-Mediated Signaling Pathways

doi: 10.3390/ijms20122899

Figure Lengend Snippet: Expression of protease-activated receptors. After thrombin preconditioning (2 U) for 6 h, umbilical cord-derived mesenchymal stem cells (UCB-MSCs) were lysed and subjected to immunoblotting analysis with the indicated antibodies. ( A ) Immunoblot analysis of the expression of protease-activated receptors (PARs) in UCB-MSCs. After thrombin preconditioning, the levels of PARs in the UCB-MSCs were determined by immunoblotting analysis. ( B ) Bar graph showing quantification of the amounts of each PAR. ( C ) Lysates from UCB-MSCs treated with the indicated siRNAs for 24 h were subjected to immunoblot analysis. Cell lysates were analyzed by immunoblotting, and the protein levels were normalized to GAPDH. An asterisk (*) indicates a significant difference vs. naive EVs.

Article Snippet: Control and PAR3 siRNAs were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Expressing, Derivative Assay, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Thrombin Preconditioning Boosts Biogenesis of Extracellular Vesicles from Mesenchymal Stem Cells and Enriches Their Cargo Contents via Protease-Activated Receptor-Mediated Signaling Pathways

doi: 10.3390/ijms20122899

Figure Lengend Snippet: Protease-activated receptors, PAR1 and PAR3, are involved in extracellular vesicle production and phosphorylation of ERK and AKT in umbilical cord-derived mesenchymal stem cells after thrombin preconditioning. Thrombin-preconditioned umbilical cord-derived mesenchymal stem cells (UCB-MSCs) were lysed for immunoblotting analysis with the indicated antibodies. ( A ) After thrombin treatment and inhibition, changes in the protein levels of endosome markers in the UCB-MSCs were assessed by immunoblotting. ( B ) Bar graph showing quantification of Rab-5 and EEA1 levels. ( C ) After thrombin treatment and inhibition, changes in the protein levels of phosphorylated (p)ERK1/2, p AKT, ERK1/2, and AKT in the UCB-MSCs were assessed by immunoblotting. ( D ) Bar graph showing quantification of p ERK1/2 p AKT, ERK1/2, and AKT. ( E ) After thrombin treatment and inhibition, changes in the levels of endosome markers in the UCB-MSCs were determined by immunoblotting. ( F ) Bar graph showing quantification of Rab-5 and EEA1 levels. ( G ) After thrombin treatment, changes in the expression of p ERK1/2 and p AKT in the UCB-MSCs were determined by immunoblotting. ( H ) Bar graph showing quantification of p ERK1/2 and p AKT levels. ( I ) Bar graph showing quantification of the ratio of p ERK/ERK and p AKT/AKT. Data are presented as mean ± SD. An asterisk (*) indicates a significant difference vs. naive EVs, a number sign (#) indicates a significant difference vs. thrombin-treated UCB-MSC and, a dollar sign ($) indicates a significant difference vs. thrombin + PAR3 siRNA UCB-MSCs ( p < 0.05, two-sample t -test; n = 5 per analysis).

Article Snippet: Control and PAR3 siRNAs were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Phospho-proteomics, Derivative Assay, Western Blot, Inhibition, Expressing

Journal: International Journal of Molecular Sciences

Article Title: Thrombin Preconditioning Boosts Biogenesis of Extracellular Vesicles from Mesenchymal Stem Cells and Enriches Their Cargo Contents via Protease-Activated Receptor-Mediated Signaling Pathways

doi: 10.3390/ijms20122899

Figure Lengend Snippet: Protease-activated receptors, PAR1 and PAR3, are involved in endosome production by umbilical cord-derived mesenchymal stem cells and protein cargo contents in extracellular vesicles after thrombin preconditioning. ( A ) After thrombin preconditioning of umbilical cord-derived mesenchymal stem cells (UCB-MSCs), the number of endosomes was determined by labeling early endosomes with green fluorescent protein(GFP-green) and the nuclei with 4′,6-Diamidino-2-Phenylindole, Dihydrochloride (DAPI-blue). ( B ) Bar graph showing the quantified intensities of the endosome-GFP signal. ( C ) After treatment with thrombin and thrombin inhibitor, the number of extracellular vesicles (EVs) was determined using a NanoSightNS300. ( D ) The size and number distribution of EVs as measured and analyzed by Nanoparticle Tracking Analysis software. ( E ) After treatment with thrombin and thrombin inhibitor, the levels of VEGF, angiogenin, angiopoietin, and HGF in the EVs were measured by multiplex ELISA. Following treatment with thrombin and thrombin inhibition, EVs were isolated from the conditioned media of UCB-MSC cultures, and VEGF, angiogenin, angiopoietin, and HGF protein levels were assessed. An asterisk (*) indicates a significant difference vs. naive EVs, a number sign (#) indicates a significant difference vs. thrombin-treated UCB-MSC and a dollar sign ($) indicates a significant difference vs. thrombin + PAR3 siRNA UCB-MSC ( p < 0.05, two-sample t -test; n = 6 per analysis).

Article Snippet: Control and PAR3 siRNAs were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Derivative Assay, Labeling, Software, Multiplex Assay, Enzyme-linked Immunosorbent Assay, Inhibition, Isolation