Effect of cholesterol depletion via MBCD on transiently transfected WT human PIEZO1. (A) Boltzmann curve fits of normalized I/I_max values from all control and MBCD-treated cells stably or transiently expressing human PIEZO1 proteins. Data for the stable cell line is quantified in Fig. 1 F. (B) Quantification of midpoint activation pressure for transiently expressed WT human PIEZO1. PIEZO1 WT (n = 8 cells), PIEZO1 WT + MBCD (n = 7 cells). (C) Quantification of channel inactivation expressed as normalized steady-state current for transiently expressed WT human PIEZO1. PIEZO1 WT (n = 9 cells), PIEZO1 WT + 5 mM MBCD (n = 7 cells). Statistical analysis was performed using Student’s t test; ****, P < 0.0001. (D) TIRF imaging of transiently expressed hP1-GFP in PIEZO1-KO HEK293T cells (left) and stably expressed hP1-GFP from hP1-GFP-CellLine (right). Scale bar represents 10 µm. (E) Average GFP intensity detected in the basal membrane of transiently or stably expressed PIEZO1-GFP. Transient hP1-GFP (n = 662 cells), stable hP1-GFP (n = 756 cells), imaged over three separate sample preparations. Error bars indicate SEM; ****, P < 0.0001. (F) Normalized cell count histogram of the range of observed fluorescence intensities exhibited by cells over the course of the imaging experiments (bin size = 25 A.U.).