Piezo1 activates noncanonical EGFR endocytosis and signaling

Cell culture 

Non-verified HeLa, A549, and MDCK-II cells were grown in DMEM with 10% FBS and 1% Pen/Strep  in a cell culture incubator at 37ºC with 5% CO₂. For all experiments, cells were seeded in growth medium. After 24h, growth medium was washed twice with PBS and replaced by starvation medium (DMEM+1%Pen/Strep, FBS omitted) for 24h before cell treatment. All cells tested negative for Mycoplasma contamination in periodic PCR tests (Sartorius, 20-700-20).

siRNA transfection

The day after seeding, 20%-40% confluent cells were transfected with Lipofectamine RNAiMax and OptiMEM following manufacturer’s instructions. siRNA pool (Horizon Discovery, siControl: D-001810-10-50, siPIEZO1: L-020870-03-0050, siCHC: L-004001-01-0020) final concentration was 10nM. siRNA transfection was repeated 24h later. The next day, cells were trypsinized and seeded on glass coverslips in growth medium before starvation overnight and treatment.

Chemicals and treatments

24h after serum starvation, cells were treated with DMSO , 10µM Yoda1 (Tocris, 5586, reconstituted in DMSO) or 10ng/mL EGF (Peprotech, 400-25, reconstituted in deionized water) for indicated durations. Experiments involving inhibitors included a 30-min pre-treatment with 1µM PD153035 (Sigma, SML0564), 200nM PP2 (Sigma, P0042), or 10 µM SB202190 (Abcam, ab120638), all reconstituted in DMSO. All treatments were prepared recycling starvation medium. 

Staining

After treatment, cells grown on glass coverslips were fixed in 4% PFA (28908) for 20min at 37°C, permeabilized with 0.5% Triton X-100 for 5min at RT and stained with primary (overnight, 4°C) and secondary (45min, RT) antibodies. Coverslips were mounted with Fluoromount-G (004958-02). All solutions prepared and washed with PBS. 

Primaries: pY1173-EGFR (1/250, R&D Systems, AF1095), pS1046/1047-EGFR (1/250, Abcam, ab76300), EEA1 (1/250, BD, 610457), phospho-cJUN (1/200, Abcam, ab32385), cFOS (1/200, SCBT, sc-166940). 

Secondaries: 1/250 Alexa Fluor (AF)-conjugated 488 Anti-Rabbit and 594 Anti-Mouse, supplemented with 1/500 AF647 Phalloidin and 30 μg/ml Hoechst.

All mixes prepared in 1% BSA in PBS.

Imaging

Samples were imaged at 1µm-thick Z displacements through 20X and 40X air and 60X oil objectives of a Nikon Eclipse Ti2-E microscope with a Yokogawa CSU-W1 spinning disk system coupled to an Andor DU-888 camera, and a Toptica multi-laser bed. Settings remained unchanged between conditions. 

Image analysis pY1173 internalization

Maximum Intensity Projections were built from raw ND2 files using the Extract Images and Make projections functions of the Process imagesmacro set developed by Christophe Leterrier in Fiji, available at https://github.com/cleterrier/Process_Images.