jedi2  (Tocris)

Journal: bioRxiv

Article Title: Endothelial cell Piezo1 promotes vascular smooth muscle cell differentiation on large arteries

doi: 10.1101/2024.06.11.598539

Figure Lengend Snippet: A ) Schematic of the proposed role of Piezo1 in blood flow sensing, and the available antagonists and agonists to regulate its activity. B ) Schematic of our imaging set up of Tg(bact2:GCaMP6s) embryos for tracking relative Ca 2+ concentrations in the zebrafish embryo. C ) Representative images of the GCaMP6s signal pseudocolored for ease of viewing. Orange represents sites of highest signal intensity, while magenta represents sites of lowest signal intensity. The control panels indicate the GCaMP6s signal in response to DMSO addition, followed by images of the same region’s response to the addition of the indicated small molecule. White arrows indicate regions where the GCaMP6s signal is markedly changed. D ) To quantify the relative changes in Ca 2+ concentration, individual cells were measured for their GCaMP6s signal over a 5-minute period. Data from all cells was then Fourier transformed and plotted as a relationship between the control and indicated small molecule treatment (GsMTx4 on the left; Jedi2 on the right). N = 4 individual fish; 5 cells regions from each fish, for a total of 20 cells per condition.

Article Snippet: To modulate Piezo activity in vivo and in vitro, we treated the experimental units with the Piezo1 inhibitor GsMTx4 (Tocris, Cat. #: 4912) at a concentration of 50nM; and the Piezo1 activators Yoda1 (Tocris, Cat. #: 5586) at 10nM and Jedi2 (Tocris, Cat. #: 6614) at 400nM.

Techniques: Activity Assay, Imaging, Control, Concentration Assay, Transformation Assay

Journal: bioRxiv

Article Title: Endothelial cell Piezo1 promotes vascular smooth muscle cell differentiation on large arteries

doi: 10.1101/2024.06.11.598539

Figure Lengend Snippet: A ) Zebrafish embryos were treated with a dose response curve to Jedi2, starting at 24 hpf. mRNA was extracted at 48 hpf and converted to cDNA for RT-qPCR analysis of klf2a transcript levels. Data is presented as a fold change compared to the DMSO treated control siblings. B ) RT-qPCR analysis for klf2a transcript levels in zebrafish embryos treated with 10 nM Yoda1 or 50nM GsMTx4 from 24-48 hpf. Data is normalized to the DMSO control condition. Each dot represents an individual biological replicate: Yoda 1 (N=7), GsMTx4 (N=5). C ) Western blot analysis of KLF2 protein levels in HUVECs treated with Jedi2 or GsMTx4 for 16 hours, compared to tubulin loading controls. Representative of N=3 biological replicates.

Article Snippet: To modulate Piezo activity in vivo and in vitro, we treated the experimental units with the Piezo1 inhibitor GsMTx4 (Tocris, Cat. #: 4912) at a concentration of 50nM; and the Piezo1 activators Yoda1 (Tocris, Cat. #: 5586) at 10nM and Jedi2 (Tocris, Cat. #: 6614) at 400nM.

Techniques: Quantitative RT-PCR, Control, Western Blot

Journal: bioRxiv

Article Title: Endothelial cell Piezo1 promotes vascular smooth muscle cell differentiation on large arteries

doi: 10.1101/2024.06.11.598539

Figure Lengend Snippet: A ) Confocal images of the medial trunk of Tg(tagln:eGFP; kdrl:mCherry-CAAX) zebrafish treated with 10 nM of Yoda1, 400 nM of Jedi2 or DMSO as the vehicle control. vSMCs shown in green and the endothelium in magenta. Arrows highlight vSMCs associated with the dorsal aorta and cardinal vein. B,C ) Quantification of tagln positive vSMCs associated with (B) the dorsal aorta or (C) the cardinal vein following treatment with Yoda1, Jedi2, or DMSO (control). DMSO vs Yoda1 ( p=0.0004 ; N=21 and 25); DMSO vs Jedi2 ( p=0.0092 ; N=15 and 18). Statistical analysis was performed using a Kruskal-Wallis test with Dunn’s multiple comparison test. Scale bar = 50 um.

Article Snippet: To modulate Piezo activity in vivo and in vitro, we treated the experimental units with the Piezo1 inhibitor GsMTx4 (Tocris, Cat. #: 4912) at a concentration of 50nM; and the Piezo1 activators Yoda1 (Tocris, Cat. #: 5586) at 10nM and Jedi2 (Tocris, Cat. #: 6614) at 400nM.

Techniques: Control, Comparison

Journal: bioRxiv

Article Title: Endothelial cell Piezo1 promotes vascular smooth muscle cell differentiation on large arteries

doi: 10.1101/2024.06.11.598539

Figure Lengend Snippet: A-C ) Confocal spinning disk images from Tg(kdrl:mCherry-CAAX) y171 zebrafish embryos treated with the indicated small molecule versus its respective control. Treatment started at 24 hpf, and imaging was done at 96 hpf. D,E ) Quantification of zebrafish heart rate (in beats per minute (BMP)) following treatment with Yoda1/Jedi2 (D) or GsMTx4 (E) versus their paired control.

Article Snippet: To modulate Piezo activity in vivo and in vitro, we treated the experimental units with the Piezo1 inhibitor GsMTx4 (Tocris, Cat. #: 4912) at a concentration of 50nM; and the Piezo1 activators Yoda1 (Tocris, Cat. #: 5586) at 10nM and Jedi2 (Tocris, Cat. #: 6614) at 400nM.

Techniques: Control, Imaging

Journal: bioRxiv

Article Title: Endothelial cell Piezo1 promotes vascular smooth muscle cell differentiation on large arteries

doi: 10.1101/2024.06.11.598539

Figure Lengend Snippet: A ) Schematic representation of our 3D cell culture model. HUVECs (EC) and GFP-HBVPs (pericyte) were seeded in a 5:1 ratio, respectively, in a 3D collagen type I gel and allowed to self-assemble for 72 hours. Piezo1 activity was modulated pharmacologically by treating the cell cultures with 200nM Jedi2 or 50nM of GsMTx4 versus DMSO (control) or by siRNA suppression of EC Piezo1 (siPiezo1) versus an siRNA control (siControl). After 72 hours of incubation, pericyte association with EC tubes was evaluated. B ) Quantification of pericyte colocalization with EC tubes under treatments with control (water) and GsMTx4. ( p=<0.0001 , N= 15 independent cultures). Statistical analysis was performed using a two-tail unpaired t-test. C ) Quantification of pericyte colocalization with EC tubes under treatments with control (DMSO) and Jedi2. ( p=<0.0001 , N=3-5 independent cultures). Statistical analysis was performed using a two-tail unpaired t-test. D) Quantification of pericyte colocalization with EC tubes following EC specific treatment with Piezo1 siRNA ( p=<0.0001 , N=30 independent cultures). Statistical analysis was performed using a two-tail unpaired t-test. E ) Representative images of EC/GFP-pericyte cocultures. EC tubes are outlined by the white dashed line, and GFP-pericytes are green in siControl and EC siPiezo1 conditions. F ) Representative cell tracking plots of GFP-pericytes in 3D cocultures over 48 hours. G,H ) Quantification of GFP-pericyte total motility in 3D cocultures, obtained from 48-hour time-lapse video tracking of cultures treated with (H) Jedi2 ( p=0.00275 ) or GsMTx4 (N=12-25 independent cells); or (G) EC siPiezo1 cultures ( p<0.0001 , N=24 independent cells). Statistical analysis was performed using one way ANOVA with a Tukey’s multiple comparison test. I,J ) Quantification of GFP-pericyte filipodia length in 3D cocultures (I) DMSO vs Jedi2 ( p<0.0001 , N=200 cells), (J) DMSO vs. GsMTx4 ( p<0.0001 , N=200 cells). Phenotypes were assessed after 72 hours in Jedi2 (I) GsMTx4 (J). Statistical analysis was performed using a two-tail Mann-Whtney test. Scale bar = 50 um.

Article Snippet: To modulate Piezo activity in vivo and in vitro, we treated the experimental units with the Piezo1 inhibitor GsMTx4 (Tocris, Cat. #: 4912) at a concentration of 50nM; and the Piezo1 activators Yoda1 (Tocris, Cat. #: 5586) at 10nM and Jedi2 (Tocris, Cat. #: 6614) at 400nM.

Techniques: Cell Culture, Activity Assay, Control, Incubation, Cell Tracking Assay, Comparison