Time-lapse imaging of single cells exposed to treatments. Live hP1-1591–GFP cells were stained with CtxB–Alexa Fluor 555 and imaged periodically in 100-frame time lapses for up to ∼70 min. (A and B) We monitored the amount of colocalized PIEZO1-GFP/CtxB–Alexa Fluor 555 entities (A) and the diffusion dynamics of PIEZO1-GFP (B) at the single-cell level in response to the various treatments used in this study. The results are quantified above and expressed as relative fold-change (+ shift for increase, – shift for decrease) with respect to values recorded before drug exposure. Each data point represents a single cell. (C) Representative frames of image series collected before (left) and after (right) start of treatment (from top to bottom: 5 mM MBCD, 50 µg/ml cholesterol, 80 µM dynasore). Statistical assessment was performed using one-way ANOVA with multiple comparisons; *, P < 0.05; **, P < 0.005; ***, P < 0.0005; error bars indicate median ± 95% confidence interval.