Cell lines were plated at 5.104 cells/cm2 in Lab-Tek 4 chamber slides (Fisher Scientific). After 24 hours, cells were washed three times with 1X TBS (tris buffered saline), fixed with 4% paraformaldehyde for 10 minutes, permeabilized with fresh TST: 0.2 % Triton X-100 / 0.2% SVF for 5 min, washed in TBS, blocked in 10% BSA for 30 min, washed briefly in TBS and then stained with phalloidin-TRITC (1:50, Sigma, P1951), BAP1 (1:100, Santa Cruz, Sc-4), CDH2 (1:200), antibodies diluted in 1X TBS 1% BSA for 2 hour. Secondary FITC-conjugated goat anti-mouse (1:400, Dako, F0479) and anti-rabbit IgG (1:100, Dako, F0205) were diluted in 1X TBS 1% BSA and applied during 45 min after one TBS washes. Slides were mounted after once wash in VectaShield (H1200, Vector Laboratories) containing DAPI for the staining of nuclei. Images were captured by using upright widefield Apotome microscope (Zeiss) equipped with a Coolsnap HQ2 camera through x63 or x40 NA 1.4 oil-immersion objective lens and driven by Axiovision software (Zeiss). Images were then analyzed and merged using ImageJ software.
For CDH2 immunofluorescence, exposure time was not fixed for NCI-H226 cells: EV: 293.6ms, N2:363.2ms and N3: 354.4ms.
For CDH2 immunofluorescence, exposure time was not fixed for NCI-H226 cells: EV: 293.6ms, N2:363.2ms and N3: 354.4ms.