Immunofluorescence Analysis of Cell Markers

IF assays were performed as previously described in [68 (link),69 (link)]. Briefly, treated and control HaCaT cells were seeded on glass coverslips at a density of 1.5 × 10⁵ cells/well in 24-well dishes, fixed with 3.7% PFA, and permeabilized with 0.5% Triton X-100. After blocking with 3% BSA, cells were incubated with primary antibody (E-cadherin and YB-1) for 1 h at RT followed by incubation with Alexa-Fluor conjugated secondary antibodies for 1 h in the dark. To visualize the actin cytoskeleton, cells were stained with TRITC-conjugated phalloidin. The cells were counterstained with DAPI for the visualization of the nucleus. Images were taken with a Zeiss confocal laser-scanning microscope Axio Observer. A x40 objective was used and image analysis was performed using ImageJ. All images were taken with the same setting. Image processing and analysis were performed with Fiji (ImageJ version 2.0) software. The stress granules formation experiment was performed as described in [70 (link)] and images were acquired using a Nikon TE Eclipse 2000. Antibodies of anti-YB-1 (12148 Abcam, Cambridge, UK), anti-E-Cadherin (610181 BD Transduction Laboratories™, MA, USA), Alexa Fluor 488 anti-rabbit and anti-mouse (Thermo-Fisher Scientific, Waltham, MA, USA), and DAPI (Sigma-Aldrich, Saint Louis, MO, USA) were used.