Journal: Cells

Article Title: Mechanosensitive Channel PIEZO1 Senses Shear Force to Induce KLF2/4 Expression via CaMKII/MEKK3/ERK5 Axis in Endothelial Cells

doi: 10.3390/cells11142191

Figure Lengend Snippet: Inhibition of CaMKII suppresses PIEZO1/MEKK3-mediated KLF2/4 expression. ( A ) Control and Piezo1 -dificient MBMECs were pretreated with Fluo-4 AM (2 μM) for 1 h and then treated with Yoda1 (5 μM) for 10 min. Calcium imaging was analyzed under a fluorescence microscope. Scale bars, 50 μm. ( B ) MBMECs were pretreated with calcium chelator (BAPTA-AM, 10 μM) for 2 h and then treated with Yoda1 (5 μM) for the indicated time. KLF4 proteins were monitored by western blotting. ( C ) MBMECs were pretreated with L-type calcium current blocker, Ruthenium Red (RR, 10 μM), for 2 h and then treated with Yoda1 with the indicated concentration for 8 h. KLF4 proteins were monitored by western blotting. ( D ) MBMECs were treated with RR (10 μM) in presence of shear stress exposure for 5 days, and mRNA levels of Klf2 and Klf4 were measured by quantitative RT-PCR. ( E ) MBMECs were pretreated with CaMKII inhibitor (KN-93, 10 μM), PKC inhibitor (Bisindolylmaleimide I, 10 μM) and FAK inhibitor (TAE226, 10 μM) for 2 h and then treated with Yoda1 (5 μM) for 8 h. KLF4 proteins were monitored by western blotting. ( F ) MBMECs were exposed to differential shear stress for 5 days, and cell lysates were probed with indicated antibodies. ( G ) MBMECs were pretreated with KN-93 (10 μM) for 2 h and then treated with Yoda1 (5 μM) for 8 h. KLF4 proteins were monitored by western blotting. ( H ) Wild-type male ICR mice were injected with KN-93 (2 mg/kg) via tail vein every 12 h for 4 times, and thoracic aortas were collected for quantitative RT-PCR of Klf2 and Klf4 mRNA expression. n = 3 mice/group. Data are representative of three independent experiments and are presented as mean ± SEM of three technical replicates by an unpaired Student’s t -test ( D ). The immunoblot was measured using ImageJ to determine the relative intensities of the indicated bands, which were normalized using the internal loading control proteins. The relative intensities are shown as numbers. ST: static culture; UF: unidirectional laminar flow.

Article Snippet: After serum starvation overnight, MBMECs were treated with agonists, including Yoda1 (TargetMol), 2-APB (100 μM; TargetMol), GSK1016790A (10 μM; TargetMol) and ICILIN (10 μM; TargetMol) or pretreated with inhibitors, including KN-93 (10 μM, 2 h, TargetMol), TAE226 (10 μM, 2 h, TargetMol), Bisindolylmaleimide I (10 μM, 2 h, TargetMol), BIX02189 (10 μM, 12 h, TargetMol), BAPTA-AM (10 μM, 2 h, TargetMol) and Ruthenium Red (RR, 10 μM, 12 h, TargetMol).

Techniques: Inhibition, Expressing, Control, Imaging, Fluorescence, Microscopy, Western Blot, Concentration Assay, Shear, Quantitative RT-PCR, Injection