(A) mPiezo1 mediates Ca2+ influx upon mechanical activation. Ratiometric Ca2+ imaging (Fura-2) of human embryonic kidney (HEK) 293T cells transiently transfected with Piezo1 or untransfected. Cells were subjected to a series of mechanical stimuli, by pressing a glass probe briefly onto the cell surface for 150 ms (arrows). For each consecutive stimulus, the travel distance of probe was increased by 1 μm (B) Yoda1 (25 μM) mediates Ca2+ responses (384-well FLIPR) in HEK cells transiently transfected with mPiezo1. When indicated, extracellular calcium was chelated by addition of EGTA, or cells were pretreated with thapsigargin to deplete intracellular calcium stores. Traces represent average ± SEM fluorescence of four wells. (C) Concentration-response profiles of mouse and human Piezo1 and Piezo2, transfected HEK293T cells assayed using FLIPR suggesting apparent EC50 of 17.1 and 26.6 μM for mouse and human Piezo1, respectively (95% confidence interval: 13.4 to 21.9, and 20.6 to 34.4), however, compound (in) solubility precludes meaningful conclusions with respect to EC50 (see text). (D) Chemical structure of Yoda1. The functional groups tested chlorines and thioether are highlighted.