PIEZO1 Knockout HEK293 Cell Culture

To avoid the unspecific activation of endogenous PIEZO1, we used throughout this study HEK293 cells KO for PIEZO1 (ref. ^{25 (link)}), named HEK-P1KO. These cells were a gift from Dr. Ardem Patapoutian (The Scripps Research Institute, La Jolla, CA, USA) and Dr. Eric Honoré (Institut de Pharmacologie Moléculaire et Cellulaire, CNRS, Valbonne, France) and were not authenticated. HEK-P1KO cells were maintained in Dubecco’s Modified Eagle’s Medium–high glucose (DMEM) supplemented with GlutaMax (Gibco, Life Technologies), 10% heat inactivated-fetal bovine serum (Gibco, Life Technologies), 100 units mL⁻¹ penicillin and 100 μg mL⁻¹ streptomycin (Gibco, Life Technologies) incubated at 37 °C in a 5% CO₂ atmosphere. Cells were passaged twice a week using 0.05% trypsin-EDTA (Gibco, Life Technologies) and used between passages 15–25 for the experiments. Cells were seeded in poly-l-lysine-treated 9-mm coverslips for patch-clamp at 5% confluence and poly-l-lysine-treated 12 mm coverslips for calcium imaging at 10–20% confluence 1 day before transfection.
Transfection was carried out using the calcium phosphate method in 35-mm well plates containing the seeded cells in the corresponding coverslips. We transfected 1 or 4 μg (for mP1 or Y2464C respectively) of plasmid per 35-mm dish for cell-attached experiments, 0.27 μg for whole-cell, and 2 μg for calcium imaging, and 5 μg per 100-mm dish for cell-surface biotinylation assay (unless stated otherwise).