Cells were fixed with 4% paraformaldehyde (Carlo Erba, Milan, Italy), permeabilized by 0.5% Triton X-100 (Sigma-Aldrich, St Louis, MO, USA) and incubated for 1 h at 4 °C with the following antibodies, at a final concentration of 0.1 mg/mL: anti-AMOT1 (Santa Cruz Biotechnology, Inc. Dallas, TX, USA; sc-166924), anti-YAP (Santa Cruz Biotechnology, Inc., Dallas, TX, USA) or anti-p-YAP (Cell Signaling Technology). For F-actin detection, cells were stained with Biotin-XX Phalloidin (Thermo Fisher Scientific, Waltham, MA, USA; #B7474). After washings, cells were incubated for 30 min at 37 °C with a secondary antibody conjugated with Cy5 (Abcam) or Streptavidin-Cy5 (Thermo Fisher Scientific, Waltham, MA, USA). Cell samples were washed twice in PBS and immediately acquired by a cytometer.
For flow cytometry studies, samples were acquired with a FACScalibur cytometer (BD Biosciences Inc., San Diego, CA, USA) equipped with a 488 nm argon laser and with a 635 nm red diode laser. At least 20,000 events were acquired, recorded and analyzed using CellQuest software (BD Biosciences, San Diego, CA, USA). The expression level of the analyzed proteins by flow cytometry was reported as median fluorescence.
For flow cytometry studies, samples were acquired with a FACScalibur cytometer (BD Biosciences Inc., San Diego, CA, USA) equipped with a 488 nm argon laser and with a 635 nm red diode laser. At least 20,000 events were acquired, recorded and analyzed using CellQuest software (BD Biosciences, San Diego, CA, USA). The expression level of the analyzed proteins by flow cytometry was reported as median fluorescence.