Phalloidin flash 488

Immunofluorescence staining was performed on cells cultured in 24‐well plates fixated on 12‐mm round glasses using cytoskeleton F‐actin and nuclear DAPI staining. Briefly, cells were fixed with 2% formaldehyde for 10 minutes at RT, washed twice with PBS, and permeabilized with 0.1% Triton‐X in PBS for 15 minutes. Blocking of unspecific binding sites was done with 1% BSA solution for 20 minutes, and cells were stained for F‐actin with Phalloidin Flash‐488 (424201, Biolegend, San Diego, CA, USA) for 1h at room temperature (RT). Glasses were washed twice with PBS, and stained for the nucleus with DAPI (A4099,0005, PanReac AppliChem, Darmstadt, Germany) and submerged in PBS. Fluorescence images were obtained using the BioTek Cytation 3 imaging reader with corresponding Gen5 Image software version 3.04. Imaging was performed with bright field or with the DAPI, GFP, and Texas Red filter cubes using the 4x and 10x objectives. Images were obtained using the BioTek Cytation 3 imaging reader with corresponding Gen5 Image software version 3.04. Imaging was performed with bright field or with the DAPI, GFP and Texas Red filter cubes using the 4x and 10x objectives.

Immunofluorescence staining was performed on CRC-FX-R and CRC treatment-naïve cells cultured in 24-well plates using cytoskeleton (F-actin) and nuclear (DAPI) staining. First, the cells were fixed using a 2% formaldehyde solution for 10 min, then washed twice with PBS, followed by a permeabilization step using Triton-X at a concentration of 0.1% in PBS for 15min. Then, the cells were exposed to a 1% BSA solution to block unspecific binding sites for 20min. Cells were then stained for F-actin with Phalloidin Flash-488 diluted at a 1:200 ratio (424201, Biolegend, San Diego, CA, USA) for 20 min at room temperature, followed by a double washing step using PBS prior to DAPI staining (diluted at 1:5000) for 5 min. Cells were afterwards washed and kept submerged in PBS for further analysis. Fluorescence images were obtained using bright field or DAPI/GFP filters with 4× and 10× objectives on Gen5 Image software, using the Biotek Cytation 3.

Murine bladders were fixed in AntigenFix for 30 min and human bladder in 1% PFA overnight at 4 degrees. Samples were then rinsed in PBS for 5 min and transferred into 30% sucrose in PBS for 24 h. 30μm sections were permeabilized and blocked-in blocking buffer containing 0.1M TRIS, 0.1% Triton, 1% normal mouse serum, 1% normal rat serum, 1% BSA for 1h at room temperature. Staining was performed in blocking buffer for 2h at room temperature prior to washing in PBS and mounting in Fluoromount-G or Fluoromount-G with DAPI. When required, a secondary staining was performed in blocking buffer for 2h at room temperature prior to washing and mounting. Images were acquired using a TCS SP8 confocal microscope and raw images were processed using Imaris. Human antibody: anti-RORC2- PE (IC6006P, R&D); anti-CD3⁻ AF488 (UCHT1, Biolegend); anti-HLA-DR- AF647 (ab223907, Abcam) and Hoechst 33342 (29 hermofisher). Mouse antibody: anti-F4/80- AF647 (ab204467, Abcam); anti-GFP rabbit polyclonal (PABG1, Chromotek); anti-CD3⁻ AF488 (17A2, Biolegend); anti-CD3⁻ PE (145-2C11, Invitrogen) and anti-Ki67- PE (SolA15, Invitrogen). Dyes: Flash Phalloidin 488 (Bio- Legend), Hoechst 33258 (cat# 40044, Biotum), DAPI (in mounting medium, cat# 00-4959-52, Invitrogen).