Lsm 880 airyscan

5 μm thickness sections were fixed with 4% formaldehyde and quenched with 0.5% Triton X‐100 (Sigma‐Aldrich). For blocking, 1% BSA (Sigma‐Aldrich) and 0.3% Triton X‐100 (Sigma‐Aldrich) in PBS were used. Specification of antibodies is described in Table S2 and validation of the SIX2 antibodies is presented in Figure S1.
Microscopy was performed on a Nikon Eclipse Ci microscope (Tokyo, Japan) or the Zeiss® LSM 880—Airyscan (Carl Zeiss Microscopy GmbH, Germany) supported by Hercules AKUL/15/37_GOH1816N and FWO G.0929.15 to Pieter Vanden Berghe, KU Leuven. Images were processed and analyzed using Zeiss ZEN Black and Blue imaging software and Fiji/ImageJ.

PL45 cells (3-5 × 10⁵) were seeded in a confocal plate with 500 µL of culture medium and incubated for 24 hours at 37°C with 5% CO₂. The cell culture medium was then removed, and the cells were rinsed 3 times using phosphate buffered saline (PBS). The cells were fixed using 400 µL of 4% paraformaldehyde (pH 7.4) for 20 minutes at room temperature (RT, ~20°C). Then 500 µL of 3% Bovine Serum Albumin (BSA) (Sigma-Aldrich Corp., St. Louis, MO, USA) was added and incubated for 30 minutes at RT for blocking. The cells were washed and then 250 µL of diluted anti-hsp90α antibody (1:250) or biotine-Dimer-San A peptide was added and incubated for 24 hours at 4°C. The cells were washed, and the desired concentration of the fluorescent-dye-labeled secondary antibody (Abcam, Cambridge, UK) or Cy5 labeled avidin (Vector Laboratories, Burlingame, CA, USA) was added and protected from light at RT for 60 minutes. The supernatant was discarded, and the cells were washed, then a drop of antifade mounting medium with DAPI (Vector Laboratories, Burlingame, CA, USA) was added and incubated for 45 minutes. The target antigen was visualized by confocal fluorescence microscopy Zeiss LSM 880 Airyscan (Carl Zeiss AG, Oberkochen, Germany).

For detection of ASC specks and NF-κB p65 translocation, cells were seeded on coverslips the day before the following procedures. After the indicated treatment, the cells were fixed with 4% paraformaldehyde, permeabilized with PBS containing 0.1% Triton × 100, and blocked with 3% BSA in PBST. The cells were stained with ASC antibody and NF-κB p65 antibody overnight at 4°C. The next day, anti-rabbit antibodies (Alexa Fluor 488 and Alexa Fluor 594) were added for incubation at room temperature for 1 h. The nuclei were counterstained with DAPI. All of the images were acquired with a fluorescence microscope (Zeiss LSM880 Airyscan, Zeiss, Germany).

Freshly isolated urothelial cells from wild type, Trpv4 KO and Tlr4 KO mice were seeded in glass coverslips and exposed to LPS (20 μg/ml) for 30 min. After treatment, cells were fixed with cold paraformaldehyde and permeabilized with 0.2% Triton X-100. Primary antibody against p65 NF-κB (1:250; Cell Signaling #4764) or S534-phosphorylated p65 NF-κB (1:800; Cell Signaling #3033) was incubated overnight at 4°C, followed by anti-rabbit Alexa Fluor 633 (1:600; A21070, Invitrogen) for 1 h at room temperature. Coverslips were mounted in glass slides using DAPI-containing mounting solution (VectaShield, Vector Laboratories, Burlingame, CA, United States). Confocal images were obtained from ten randomly selected fields from three independent experiments using the optimal pinhole size for the 63X oil objective on a Zeiss LSM 880-Airyscan (Carl Zeiss AG, Oberkochen, Germany). Images were analyzed using Fiji software (17 (link)) as described before (18 (link)).

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.

Cells were seeded on glass coverslips in 24 well plates. Cells were fixed in 4% PFA for 10 min and subsequently permeabilized for 5 min with 0.5% X-100 Triton. Cells were blocked in 0.5% skin fish gelatine for 1 h. Antibodies were diluted in the blocking solution. Primary antibodies were goat anti-lamin B1 (C-20, Santa Cruz Biotechnology, Santa Cruz, CA, USA), and mouse anti-lamin A/C (4C11) Active Motif (Carlsbad, CA, USA). Secondary antibodies used were donkey anti-mouse and donkey anti-goat conjugated to Alexa Fluor 488 and Alexa Fluor 647 (Invitrogen, Waltham, MA, USA). Cytoplasmic loading was conducted by permeabilising live cells with a 750 μg/mL solution of saponin containing a secondary goat anti rabbit IgG Alexa Fluor 546 (80 μL stock solution per 1mL of detergent solution) at 4 °C for 5 min then fixed immediately with cold 4% PFA. Samples were imaged immediately. Fixed cells were imaged on the LSM5 Zeiss Inverted 510 META laser scanning microscope (Zeiss, Oberkochen, Germany) using a Plan Apo 63 × 1.4 NA oil immersion lens. Images were acquired using Zen2009 operating software (Zeiss). Samples of cytoplasmic loading and the hormone removal experiment were imaged on a Zeiss LSM880 Airyscan using a Plan Apo 40 × 1.3 NA oil immersion lens. Images were acquired and pre-processed using ZenBlack (Zeiss). Collected images were analysed in FIJI [30 (link)].