Figure S5.
Cholesterol depletion via MBCD in N2A cells. N2A cells were cultured in 10% FBS-supplemented DMEM and treated with 5 mM MBCD in serum-free medium for 30 min at 37°C before experiments. Electrophysiological recordings were collected as described in Materials and methods. (A) Computer-controlled high-speed pressure clamp square pressure pulses were applied to untreated N2A cells in cell-attached mode at +65 mV pipette potential. Mechanically evoked currents from native PIEZO1 proteins are shown below the pressure trace. The first pressure pulse and its respective PIEZO1 current trace are highlighted in red. (B) MBCD-treated N2A cells display a loss-of-inactivation phenotype compared with control. (C) Combined normalized I/Imax values from all control and MBCD-treated cells in the experiment were fitted with a single Boltzmann curve per condition (see Materials and methods). (D) Average P50 values obtained from Boltzmann fits for each individual cell analyzed under control and MBCD conditions. (E) Comparison of normalized steady-state mechanosensitive currents from N2A cells in control and MBCD-treated conditions. Control (n = 13), MBCD (n = 13); **, P < 0.01, Student’s t test. (F) Comparison of the mean current density recorded from cell-attached patches of N2A cells, hP1-1591–GFP stable cells, and transiently expressed WT Piezo1 (N2A n = 13 cells; hP1-1591–GFP n = 12 cells; Transient WT Piezo1 = 12 cells; *, P < 0.05; one-way ANOVA). Error bars represent SEM.