Anti piezo1

Freshly isolated mouse aortic smooth muscle cells and HAoSMCs were seeded onto glass coverslips and kept at 37°C for 1–2 h while attached to the surface of the coverslips. After fixed in 4% PFA and repeated washing in glicine-PBS solution (100 mM, 15 min, 22°C), cells were permeabilized with Triton-X-100 (0.5 v/v%, 10 min, 22°C). After PBS washing (3x) non-specific biding sites were blocked with Carbo-Free Blocking Solution (SP-5040, VECTOR Laboratories, Burlingame, CA, United States). Immunostaining was performed using anti-Piezo1 (Thermo Fisher Scientific, Rockford, IL, United States, MA5-32876, mouse-IgG, monoclonal) primary antibody with Cy3 anti-mouse secondary antibody labelling (A10521, Life technologies, Eugene, OR, United States). Images were taken using Zeiss laser scanning confocal microscope (Zeiss LSM880 Airyscan; Zeiss, Oberkochen, Germany) using 405, 488 and 543 nm excitation wavelengths and 10x or 63x oil immersion objective. On HAoSMCs anti-Piezo1 and anti-alfa smooth muscle specific actin (αSMA) (Thermo Fisher Scientific, Rockford, IL, United States, MA5-32876, mouse-IgG, monoclonal; PA516697, rabbit-IgG, polyclonal, respectively) primary antibodies were applied.
Paraffin embedded human aorta samples were subjected to deparaffinization process, and the protocol described above was applied as staining procedure.

DIV5 cholangiocytes were harvested using TrypLE express and pelleted by centrifugation at 300 g for 4 min. Following protein extraction, immunoprecipitation was performed with the Dynabeads coimmunoprecipitation kit according to the manufacturer’s instructions. The primary antibodies, rabbit polyclonal anti-Piezo1 (Thermo Fisher Scientific) and rabbit polyclonal anti-Ki67 (7 µg/mg of beads), were covalently linked to magnetic beads. The extraction buffer was supplemented by adding 150 mM NaCl, 20 mM N-ethylmaleimide, 1.2% Triton X-100, and a cocktail of protease inhibitors without EDTA. Proteins were eluted from beads by adding Laemmli denaturation Sample Buffer and incubated 10 min at 70°C.

For Western blot experiments total cell lysates were isolated from control, calcified and gene silenced HAoSMC cell lines. The samples were subjugated to sonication. Protein concentration were measured. Samples were subjected to SDS-PAGE (10% gels loaded with 20 μg protein per lane) and transferred to nitrocellulose membranes (Bio-Rad). The protein-binding nitrocellulose membranes were then blocked with 5% dry milk-PBS solution. Proteins were probed with anti-Piezo1 (Thermo Fisher Scientific, Rockford, IL, United States, MA5-32876, mouse-IgG, monoclonal) or with anti-Runx2 (20700-1-AP; Proteintech, Rosemont, IL) primary antibodies followed by HRP-conjugated secondary antibody labeling. The primary-secondary antibody complexes were detected using an enhanced chemiluminescence Western blotting Pico or Femto kit (Thermo Scientific, Rockford, IL, United States) in a Fujifilm Labs-3000 dark box. According to the manufacturers’ descriptions, the band corresponding to the Piezo1 protein in cell lysates can be detected above 250 kDa, at ∼280–300 kDa, while that of the Runx2 protein is 57–60 kDa.