Journal: Cell reports

Article Title: Cdc42 activity in Sertoli cells is essential for maintenance of spermatogenesis

doi: 10.1016/j.celrep.2021.109885

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit polyclonal anti-PARD3 (Par3) , Novus , Cat#NBP1-88861; RRID: AB_11056253.

Techniques: Recombinant, Microarray, Real-time Polymerase Chain Reaction, Software

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

Journal: PLoS ONE

Article Title: Calcium Mobilization And Protein Kinase C Activation Downstream Of Protease Activated Receptor 4 (PAR4) Is Negatively Regulated By PAR3 In Mouse Platelets

doi: 10.1371/journal.pone.0055740

Figure Lengend Snippet: Fura 2-loaded wild type (black circle), PAR3 −/− (gray circle), and PAR3 +/− (white square) platelets were activated with the indicated concentrations of: ( A ) thrombin, (0.001–100 nM, ( B ) AYPGKF (0–2 mM), ( C ) convulxin (0.01–100 nM), or 20 µM of ADP for 10 min at 37°C in the presence of 2 mM of CaCl 2 . The difference between the maximum increase and the basal intracellular Ca 2+ mobilization was measured. The results are the mean (± SD) of three independent experiments (* p <0.05).

Article Snippet: The cDNA for mouse PAR3 was purchased from Origene Technologies Inc. (Rockville, MD) and the cDNA for mouse PAR4 was isolated from a BaF3 cDNA library.

Techniques:

Journal: PLoS ONE

Article Title: Calcium Mobilization And Protein Kinase C Activation Downstream Of Protease Activated Receptor 4 (PAR4) Is Negatively Regulated By PAR3 In Mouse Platelets

doi: 10.1371/journal.pone.0055740

Figure Lengend Snippet: Flow cytometric analysis of PAR4 expression in wild type (WT) (black line), PAR3 −/− (gray line), and PAR4 −/− (shaded) mice platelets using anti-PAR4-FITC antibodies.

Article Snippet: The cDNA for mouse PAR3 was purchased from Origene Technologies Inc. (Rockville, MD) and the cDNA for mouse PAR4 was isolated from a BaF3 cDNA library.

Techniques: Expressing

Journal: PLoS ONE

Article Title: Calcium Mobilization And Protein Kinase C Activation Downstream Of Protease Activated Receptor 4 (PAR4) Is Negatively Regulated By PAR3 In Mouse Platelets

doi: 10.1371/journal.pone.0055740

Figure Lengend Snippet: Fura 2-loaded wild type (black) and PAR3 −/− (gray) platelets were incubated at 37°C for 5 min in the absence or the presence of 100 µM 2MeSAMP. After treatment, platelets were activated 100 nM thrombin ( A ,) or 2 mM AYPGKF ( B ) for 10 min at 37°C in the presence of 2 mM of CaCl 2 . The difference between the maximum increase and the basal intracellular Ca 2+ mobilization was measured. The results are the mean (± SD) of three independent experiments (* p <0.05).

Article Snippet: The cDNA for mouse PAR3 was purchased from Origene Technologies Inc. (Rockville, MD) and the cDNA for mouse PAR4 was isolated from a BaF3 cDNA library.

Techniques: Incubation

Journal: PLoS ONE

Article Title: Calcium Mobilization And Protein Kinase C Activation Downstream Of Protease Activated Receptor 4 (PAR4) Is Negatively Regulated By PAR3 In Mouse Platelets

doi: 10.1371/journal.pone.0055740

Figure Lengend Snippet: Fura 2-loaded wild type (black line) and PAR3 −/− (gray line) platelets were resuspended in Ca 2+ -free medium (0.1 mM EGTA was added at the time of experiment). Representative tracings are shown from platelets activated with the indicated concentrations of: ( A ) thrombin (1–100 nM), ( C ) AYPGKF (0.15–2 mM), or ( E ) 3 µM thapsigargin (TG). Quantitation of the change in peak Ca 2+ mobilization in platelets stimulated with: ( B ) thrombin, ( D ) AYPGKF, or ( F ) thapsigargin. The results are the mean (± SD) of three independent experiments (* p <0.05).

Article Snippet: The cDNA for mouse PAR3 was purchased from Origene Technologies Inc. (Rockville, MD) and the cDNA for mouse PAR4 was isolated from a BaF3 cDNA library.

Techniques: Quantitation Assay

Journal: PLoS ONE

Article Title: Calcium Mobilization And Protein Kinase C Activation Downstream Of Protease Activated Receptor 4 (PAR4) Is Negatively Regulated By PAR3 In Mouse Platelets

doi: 10.1371/journal.pone.0055740

Figure Lengend Snippet: The HEK293 cells were transfected with: ( A ) PAR4-Luc (1 µg) and PAR3-GFP (0–2.5 µg), ( B ) PAR3-Luc (1 µg) and PAR3-GFP (0–2.5 µg), or ( C ) PAR4-Luc (1 µg) and PAR4-GFP (0–2.5 µg). As a control experiment, the HEK293 cells were transfected with: ( D ) PAR3-Luc (1 µg) and rho-GFP (0–0.12 µg), or ( E ) PAR4-Luc (1 µg) and rho-GFP (0–0.12 µg). Forty-eight hours post-transfection, the cells were analyzed for GFP expression, Luc expression, and BRET. The curves were plotted as the ratio of GFP to Luc and all points from 3–6 independent experiments were analyzed by global fit to a hyperbolic or linear curve. The surface expression of PAR3 and PAR4 in the HEK293 cells was determined by flow cytometry. ( G ) V5-PAR4-GFP and V5-PAR3-GFP were detected with a V5 tag antibody conjugated to Alexa Fluor 647. ( H ) HA-PAR4-LUC and HA-PAR3-LUC were detected with a HA tag antibody conjugated to Alexa Fluor 647. The results are the mean (± SD) of two independent experiments. The number of PAR4 and PAR3 molecules on the HEK293 cells surface is calculated from the V5 or HA antibody standard curve using quantitative flow cytometry ( F ).

Article Snippet: The cDNA for mouse PAR3 was purchased from Origene Technologies Inc. (Rockville, MD) and the cDNA for mouse PAR4 was isolated from a BaF3 cDNA library.

Techniques: Transfection, Control, Expressing, Flow Cytometry

Journal: bioRxiv

Article Title: Distributed neural computation and the evolution of the first brains

doi: 10.1101/2025.10.03.680388

Figure Lengend Snippet: a) Simplified phylogeny of animals shows that acoel brains are likely intermediate between cnidarian diffuse nets and the centralized brains of typical bilaterians. b) Photograph of juvenile Hofstenia miamia . c) Staining with voltage dye reveals a superficial network of dense neuropil (blue arrow) that extends into a sparser posterior nerve net (green arrow). d) Close-up view of neuropil stained sparsely with tubulin dye (orange) reveals that the neuropil (orange) contains many neurites running in parallel, with cellular clusters (cyan) interspersed between neurite bundles. Sensory neurons (likely clusters of H1 cells; bright orange) are set within many of these patches. e) Cross-section of brain stained with a Par3 antibody reveals that the brain has two layers: superficial neuropil, and deeper cell bodies that project outward. f) Staining with an ERK antibody (z-projected segmentation overlaid) shows that brain interneurons can be multipolar, with a central cell body generating multiple neurites. g) Cross-section of brain stained with an antibody against β-catenin reveals another sensory neuron class (possibly H2 ) with two projections that innervate brain neuropil. h) Electron microscopy cross-section shows the fine organization of the brain, confirming the relative configuration of tissue types within the head. The superficial neuropil (previously ‘layer 1’) is visible immediately beneath the skin, while neural cell bodies (previously ‘layer 2’) lie deeper in the tissue, internal to body wall muscle (green). Together, these layers compose the brain. i) Electron microscopy close-up of the brain shows dense neuropil; the box is a 6.7×6.7µm square. j) Segmenting neural projections within the highlighted box in (i) reveals over 400 neurites in a single section of neuropil. k) Segmentation of cellular clusters within neuropil allows quantification of brain structure and its variability. l) Quantifying the numbers of cellular clusters across brains reveals that, although cluster numbers increase with age (i.e. days after hatching) and size (i.e. head width, a good proxy for overall body size ), worms vary widely in how many clusters they possess. Linear regression p<0.0001, n=49. Scale bars: 200µm (c), 50µm (d,e), 20µm (f,g), 10µm (h).

Article Snippet: Primary antibodies used: Par3 (St. John’s Laboratory #STJ94951, 1:200), pERK (Cell Signaling Technologies #4370T, 1:200) , FMRFamide (EMDMillipore #AB15348, 1:1000) .

Techniques: Staining, Electron Microscopy