Fig. 6: HEK293T PIEZO1 activity is influenced by F-actin mobility.

a, b Schematic illustration of fMPA on adhered (a) and suspended (b) HEK293T WT. To ensure HEK293T WT cells firmly adhered to the surface during aspiration, the cover glass was coated with fibronectin (FN). c, d 3D confocal images of adhered (c) and suspended (d) HEK293T WT cells. Both lateral and top view of the cells with F-actin (cyan) and DNA (orange) labeled are shown to illustrate the F-actin distribution in the cell. Three ROI lines were drawn to quantify F-actin signal along the line. e, f Normalized F-actin intensity along the ROIs in adhered (e) and suspended (f) cells. F-actin signal along the line showed that F-actin concentrated at the cell cortex to form organized structure when the cell is adhered to the FN-coated surface, indicated by the signal peaks on either end of the line (arrow pointed). g Snapshots of aspirated HEK293T cell that was adhered to FN-coated coverglass. The Ca²⁺ and F-actin intensities were concurrently imaged using a FV3000 confocal microscope. The contour of the attached HEK293T cell was outlined (white dash line) in the calcium and transmitted channels. White arrows showed that the pointed F-actin signal was immobilized during the whole aspiration process. h Ca²⁺ ∆F_max comparison between adhered (red) and suspended (black) HEK293T WT cells aspirated by micropipette with different tip angles. The number of data points (n number) is marked, and data are presented as violin plot, and analyzed by Welch’s ANOVA test. i Activity of PIEZO1 in suspended and adhered HEK293T cells. A left shift in the Boltzmann distribution was noticed when the cell was suspended comparing to when cells were adhered. Quantified P_1/2 demonstrated a lower gating pressure was required when cells were suspended. The number of measured cells is marked, and the data is presented as mean ± s.e.m. a, b are created with BioRender.com released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license https://creativecommons.org/licenses/by-nc-nd/4.0/deed.en.