Time course and IF analysis of wild-type PIEZO1 expression in HEK293 cells. (A) Time-course experiments in an HEK293 cells doxycycline-inducible model of PIEZO1 demonstrated significant protein expression beginning at 16 hours and lasting for 96 hours postinduction. The time frame between 24 and 48 hours postinduction was used for all experiments unless otherwise indicated. (B) IF studies localized PIEZO1 expression to the cell membrane after doxycycline induction. 4′,6-Diamidino-2-phenylindole (DAPI) stain of nuclei (blue), phalloidin AF488 stain of F-actin (green), and ATT0647N FLAG-tagged PIEZO1 (red) images in uninduced and induced cells are shown. Scale bars, 10 µM. IAB, primary antibody, mouse anti-FLAG tag ATTO647N; IIAB, secondary antibody, goat anti-mouse; In, induced; Un, uninduced.