Journal: Methods and Protocols

Article Title: Efficient Ex Vivo Screening of Agents Targeting Thrombospondin1-Induced Vascular Dysfunction Using a Digital Multiwire Myograph System

doi: 10.3390/mps4040074

Figure Lengend Snippet: Native thrombospondin 1 (TSP1) from different sources inhibited endotheliumdependent vasorelaxation with different efficacies. ( A ) Representative force vs. time traces recordings of isolated murine thoracic aorta responses to phenylephrine followed acetylcholine stimulations in the presence of Pierce TM —TSP1 or vehicle control, i.e., Krebs buffer (PE: phenylephrine; Ach: acetylcholine; the numbers marked in the recording are the logarithm value of concentrations of the stimulants). Concentration response curves of isolated mouse thoracic aorta exposed to Pierce TM —TSP1 ( B ) or Athens—TSP1 ( C ) at 2.2, 4.4, or 6.6 nM. Pierce TM —TSP1 induced impairment of vasodilation in a slightly different pattern than Athens—TSP1 (*, p < 0.05, TSP1 vs. control; ***, p < 0.0001, TSP1 vs. control). ( D ) The same concentration TSP1, e.g., 4.4 nM, from three sources inhibited vasorelaxation, two of which produced greater suppression of vasorelaxation induced by acetylcholine 1 × 10 −7 and 3 × 10 −7 M (*, p < 0.05, Athens—TSP1 vs. control; ***, p < 0.0001, Pierce TM —TSP1 or Novus—TSP1 vs. control). ( E ) An equivalent inhibition of vasorelaxation was reached when Novus—TSP1 or Athens—TSP1 at 2.2 nM and Pierce TM —TSP1 at 4.4 nM was applied (***, p < 0.0001, TSP1 vs. control).

Article Snippet: Therefore, it was essential to examine the bioactivity of native human TSP1 available from different commercial vendors (Thermo Scientific, Novus Biologicals, and Athens Research & Technology), specifically in terms of modulation of vasoactivity, in order to determine which TSP1 protein products can be used for the candidate drug screening assays afterwards.

Techniques: Isolation, Control, Concentration Assay, Produced, Inhibition

Journal: Methods and Protocols

Article Title: Efficient Ex Vivo Screening of Agents Targeting Thrombospondin1-Induced Vascular Dysfunction Using a Digital Multiwire Myograph System

doi: 10.3390/mps4040074

Figure Lengend Snippet: Thrombospondin 1 (TSP1)−impaired endothelium-dependent vasorelaxation was prevented by treatment with the proposed drug candidate, but not by blocking signal-regulatory protein α (SIRP α). Representative force vs. time traces recording from endothelium intactness test to acetylcholine-induced vasorelaxation by isolated murine thoracic aorta pre-treated with the proposed drug candidate 6.6 nM ( A ), CD47 antibody (CD47 ab) 1 µg/mL ( B ), or SIRPα antibody (SIRPα ab) 2 µg/mL ( C ) in the presence of Pierce TM —TSP1 4.4 nM (PE: phenylephrine; Ach: acetylcholine; the numbers marked in the recording are the logarithmic values of stimulant concentrations). Prior to receiving any treatment, the vessel segments were pre-contracted with 1 × 10 −6 M PE followed by 1 × 10 −6 M Ach-stimulated endothelium-dependent vasorelaxation to confirm endothelial intactness. ( D ) Concentration response curves of isolated mouse thoracic aorta exposed to TSP1, in the presence of vehicle, CD47-based drug candidate, or CD47 antibody. ( E ) Concentration response curves of isolated mouse thoracic aorta exposed to TSP1, in presence of vehicle or SIRPα antibody (***, p < 0.0001, TSP1, TSP1 + drug candidate, or TSP1 + SIRPα ab vs. control; ###, p < 0.0001, TSP1 + drug candidate or TSP1 + CD47 antibody vs. TSP1).

Article Snippet: Therefore, it was essential to examine the bioactivity of native human TSP1 available from different commercial vendors (Thermo Scientific, Novus Biologicals, and Athens Research & Technology), specifically in terms of modulation of vasoactivity, in order to determine which TSP1 protein products can be used for the candidate drug screening assays afterwards.

Techniques: Blocking Assay, Isolation, Concentration Assay, Control

Journal: Methods and Protocols

Article Title: Efficient Ex Vivo Screening of Agents Targeting Thrombospondin1-Induced Vascular Dysfunction Using a Digital Multiwire Myograph System

doi: 10.3390/mps4040074

Figure Lengend Snippet: Acute TSP1 challenge-induced reduction in endothelium-dependent vasorelaxation was significantly restored by therapeutic treatment with the proposed CD47-based drug candidate. Representative force vs. time traces recording from endothelium intactness test to acetylcholine-induced vasorelaxation by isolated murine thoracic aorta exposed to acute TSP1 (Pierce TM —TSP1 4.4 nM) challenge for 15 min, followed by treatment with the proposed CD47-based drug candidate 13.2 nM ( A ) or CD47 antibody (CD47 ab) 1 µg/mL ( B ) for 45 min (PE: phenylephrine; Ach: acetylcholine; the numbers marked in the recording are the logarithmic values of stimulant concentrations). Prior to receiving any treatment, the vessel segments were pre-contracted with 1 × 10 −6 M PE followed by 1 × 10 −6 M Ach-stimulated endothelium-dependent vasorelaxation to confirm endothelial intactness. ( C ) Concentration response curves of isolated mouse thoracic aorta exposed to TSP1, in the presence of vehicle, CD47-based drug candidate, or CD47 antibody (***, p < 0.0001, TSP1 or TSP1 + drug candidate vs. control; #, p < 0.05, TSP1 + drug candidate vs. TSP1; ###, p < 0.0001, TSP1 + CD47 antibody vs. TSP1).

Article Snippet: Therefore, it was essential to examine the bioactivity of native human TSP1 available from different commercial vendors (Thermo Scientific, Novus Biologicals, and Athens Research & Technology), specifically in terms of modulation of vasoactivity, in order to determine which TSP1 protein products can be used for the candidate drug screening assays afterwards.

Techniques: Isolation, Concentration Assay, Control

Journal: bioRxiv

Article Title: Early-life stress triggers long-lasting organismal resilience and longevity via tetraspanin

doi: 10.1101/2023.07.25.550452

Figure Lengend Snippet: (A) Representative confocal images showing co-localization of the marker Akt-PH::GFP, which binds to intestinal apical plasma membrane inner leaflet PIP2/3, with endogenous TSP-1 tagged with wrmScarlet by CRISPR. (B) Fluorescent intensity correlation analysis showing close proximity of Akt-PH::GFP and TSP-1::wrmScarlet. (C) Representative confocal images showing non-co-localization of the marker ERm::GFP, which labels intestinal ER membrane, with endogenous TSP-1 tagged with wrmScarlet by CRISPR. Scale bars: 10 µm. (D) Fluorescent intensity correlation analysis for ERm::GFP and TSP-1::wrmScarlet. (E) Representative confocal time-series images showing stability of tetraspanin webs formed by endogenous TSP-1::wrmScarlet, with enlarged views in (F). Scale bars: 10 µm.

Article Snippet: The NC membrane was initially blocked with 5% nonfat milk and 2% BSA (A4503, Sigma (v/v)) in Tris buffered saline with 0.1% Tween 20 (93773, Sigma) (TBS-T) at room temperature for 1 h. native TSP-1::GFP were detected using antibodies against GFP (1:1000, 66002-1-Ig, Proteintech) at cold room overnight.

Techniques: Marker, Clinical Proteomics, Membrane, CRISPR