Modulation of PIEZO1-GFP clusters determined by STORM superresolution microscopy. (A) TIRF microscopy of PIEZO1-GFP cells immunolabeled with an anti-GFP antibody conjugated to Alexa Fluor 647. Images of control cells and treated cells (cell boundary highlighted by a dashed line) show the PIEZO1-GFP signal (green) superimposed on the STORM signal (yellow arrowheads) from Alexa Fluor 647 (red; insets). Scale bar represents 5 µm or 1 μm in inset. The physical properties of the clusters shown in the insets were analyzed by cluster decomposition and are shown in the adjacent cluster maps (black arrowheads). (B) Left: Quantification of PIEZO1-GFP cluster area as violin plots in control and treatment conditions. Right: Relative frequency of cluster areas in square nanometers. (C) Left: Quantification of PIEZO1-GFP cluster perimeters as violin plots in control and treatment conditions. Right: Relative frequency of cluster perimeters in nanometers. For cluster area and perimeter: control PIEZO1-GFP (n = 34,741), MBCD (n = 19,159), cholesterol (n = 12,097), dynasore (n = 19,982); n = 1 represents a single PIEZO1-GFP cluster. (D) Left: Quantification of PIEZO1-GFP cluster density as violin plots in control and treatment conditions. Right: Relative frequency of cluster density in clusters per square micrometer. Control PIEZO1-GFP (n = 173), MBCD (n = 193), cholesterol (n = 126), dynasore (n = 82); n = 1 represents a single cell. All violin plots are marked with median (thick dashed line) flanked by 25th and 75th percentile (thin dashed lines). The median value of each plot is reported on the righthand side of each graph. One-way ANOVA; *, P < 0.05; ****, P < 0.00005; ns, not significant.