Yoda1

All treatments were performed in suspension on washed isolated RBCs diluted 12 times in DMEM (~42,000 RBCs/µL). After each treatment, RBCs were centrifuged at 200× g for 2 min and resuspended in chemical agent-free DMEM, except when otherwise stated. For Piezo1 activation, RBCs were pre-conditioned at the appropriate temperature for 20 min and then incubated with the allosteric modulator Yoda1 (Biotechne, Minneapolis, MD, USA) for 30 s. Except when otherwise stated, the Yoda1 concentration was 50 nM for immunofluorescence experiments and 100 nM for all other experiments. To induce a PKC-mediated Ca²⁺ permeability [56 (link)], RBCs were incubated for 20 min at 37 °C with 3 µM phorbol 12-myristate 13-acetate (PMA; Sigma-Aldrich, Saint-Louis, MO, USA) and 30 nM of the protein phosphatase inhibitor calyculin A from Discodermia calyx (CalA; Sigma-Aldrich). These agents were maintained throughout the whole experiment. To deplete membrane chol, RBCs were incubated with the chol-removing agent methyl-β-cyclodextrin (mβCD; Sigma-Aldrich) for 15 min at 0.6 mM at 37 °C, except when otherwise stated. To replete membrane chol after depletion, RBCs were incubated with 7.5 µg/mL of mβCD:chol (Sigma-Aldrich) for 60 min and then in a mβCD:chol-free medium for an additional 90 min to allow time for newly inserted chol to distribute in the appropriate membrane regions.

Yoda1 (Tocris), staurosporine (Sigma), PD98059 (Merck), SP600125 (Cambridge Bioscience), and SB203580 (Merck) were all solubilized in DMSO. ATP, gadolinium, and ruthenium red were all obtained from Sigma-Aldrich and dissolved in H₂O. Dooku1 and compound 2e (16 (link)) were synthesized at the University of Leeds and solubilized in DMSO.

Yoda1, ruthenium red, and Dooku1 were purchased from Tocris (Avonmouth, Bristol, UK). Capsaicin was purchased from Sigma (St. Louis, MO, USA) and the stock solutions were made with 99.5% ethanol. All stock solutions were stored at −20 °C.

HUVECs were plated on a thin-layered Matrigel-coated plate for 1 h to allow cells to attach. The cells were then loaded with Calcium 6-QF (Molecular Devices) in a Minimum Essential Medium–Hanks’ balanced salt solution (HBSS; 1:1) for 30 min at 37 °C in a CO₂ incubator (32 (link)). The cells were washed gently with HBSS buffer with 2 mM Ca²⁺. The cells were imaged by using a Zeiss Axio observer Z1 with a 20× objective, and images were captured at 400-ms intervals. HBSS buffer with 2 mM Ca²⁺ was used during imaging. The intracellular calcium elevation of individual cells was analyzed with MetaMorph software (Molecular Devices). Cells overloaded with dye or only faintly fluorescent were excluded from analysis. The chemicals used in calcium imaging experiments included Yoda1 (Tocris; 5586), GsMTx4 (Abcam; ab141871), 5,6- eicosatrienoic acid (Santa Cruz Biotechnology; sc-221066), arachidonic acid (Sigma; A3611), HC067047 (Tocris; 4100), GSK1016790A (Sigma; G0798), AACOCF3 (Tocris; 1462), and YM26734 (Tocris; 2522).

After overnight serum starvation, cells were treated with dimethyl sulfoxide (DMSO; 276855), 10 μM Yoda1 (Tocris, 5586, reconstituted in DMSO) or EGF (10 ng/ml; PeproTech, 400-25, reconstituted in deionized water) for indicated durations. Experiments involving inhibitors included a 30-min pretreatment with EGFR kinase inhibitor 1 μM PD153035 (Sigma-Aldrich, SML0564), SFK inhibitor 200 nM PP2 (Sigma-Aldrich, P0042), or p38 inhibitor 10 μM SB202190 (Abcam, ab120638), all reconstituted in DMSO. All treatments were prepared reusing starvation medium.
Shear stress was delivered to cells seeded on microfluidic chambers (Ibid 80166, I Luer 0.2 mm height, polymer coating) using a peristaltic pump (Thermo Fisher Scientific, 16609762) with a starvation medium flow rate of 4 ml/min. According to the manufacturer’s instructions, shear stress (τ) is the product of flow rate (ϕ, 4 ml/min), medium’s dynamical viscosity [η ≈ 0.0073 dynes·s/cm² for serum-free DMEM (87 (link))] and a chamber-dependent correction factor (512.9), yielding ≈ 15 dynes/cm², a shear stress value shown to activate Piezo1 (9 (link), 10 (link)).

HMVEC were loaded with Fluo-4 AM (1 mM; Invitrogen) per the manufacturer’s protocols. Images were acquired and analyzed with CellSens Dimensions (Olympus Corp.). The solution contained 140 mM NaCl, 2 mM CaCl₂, 1 mM MgCl₂, 10 mM HEPES, 10 mM glucose, and 20 mM mannitol (320 mOsm) (pH 7.3). Increases in fluorescence were achieved by perfusing 2 µM of Yoda1 (Tocris Bioscience) and washed with control solution. Cells with a large baseline fluorescence signal (>20 arbitrary units) were excluded from the analysis.