Journal: American journal of physiology. Cell physiology
Article Title: Activation of the Piezo1 channel stimulates protein kinase D and migration in human aortic endothelial cells
doi: 10.1152/ajpcell.00457.2025
Figure Lengend Snippet: A, Confluent ECs were incubated with PKD inhibitor CRT0066101 at 2.5 μM (CRT) for 1h prior to stimulation with 5μM Yoda1 for 10 min. The cultures were lysed with 2X SDS-PAGE sample buffer and analyzed by immunoblotting with antibodies that detect HDAC7 phoshorylated at Ser 486 , PKD1 phosphorylated at Ser 910 , HDAC7 and GAPDH (as a loading control). B , Bars display the quantification of HDAC7 phosphorylation. Mean ± SEM; n=3 independent experiments, **p<0.001 by Student’s t-test. C , Confluent ECs were treated without or with 2.5 μM CRT0066101 (CRT) for 1 h and then stimulated with Yoda1 for 60 min, as indicated. ECs were fixed and stained with an antibody that detects HDAC7 and images obtained by confocal microscopy. Scale bar = 15μm. D, Bars represent the percentage of ECs treated with Yoda1 (Y1), CRT0066101 (CRT) or their combination (Y1+CRT) with nuclear HDAC7 obtained from 5–6 fields with a total of ~30 individual cells in each field, mean ± SEM; n= 5 fields, **p<0.001 control vs Y1 and ## p<0.001 Y1 vs Y1 +CRT. E, Ratio of nuclear/cytoplasmic HDAC7 fluorescence in 5–6 fields with a total of ~30 individual cells in each field, mean ± SEM; n= 5 fields, **p<0.001 control vs Y1 and ## p<0.001 Y1 vs Y1 +CRT. The treatments were as described in panel D. F, Confluent ECs were incubated with 10 μg/ml mitomycin C for 2 h. A scratch wound was then created with a sterile 200-μl pipette tip and after washing, wounded cells were stimulated with Yoda1 or Yoda2 at 5μM for 18 h. The cultures were fixed with 4% paraformaldehyde and stained with Giemsa stain. Representative microscopy fields are shown. Images were captured at original magnification 10X. G, Bars represent relative migration (average of 5 fields per experiment) of ECs stimulated with Yoda1 or Yoda2 in the absence or presence of CRT0066101 (1 μM and 2.5 μM). Bars represent the means ± SEM n= 4 for Yoda1 and n=3 for Yoda2 independent experiments., *p<0.02, **p,0.01 by Student’s t-test.
Article Snippet: Piezo1 antibody (Proteintech Cat# 15939–1-AP, RRID:AB_2231460; final dilution 1:1000); HDAC7 antibody ( Proteintech Cat# 26207–1-AP, RRID:AB_288042626207; final dilution 1:1000) and Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 (Thermo Fisher Scientific Cat# A-11034, RRID:AB_2576217; final dilution 1:1000) were from Thermo Fisher Scientific (Waltham, MA).
Techniques: Activation Assay, Phospho-proteomics, Migration, Incubation, SDS Page, Western Blot, Control, Staining, Confocal Microscopy, Fluorescence, Sterility, Transferring, Giemsa Stain, Microscopy
Cell Signaling Technology Inc is a verified supplier 
Cell Signaling Technology Inc manufactures this product 
