*4.5. Immunofluorescence and Confocal Microscopy Analysis*

Cells cultured on coverslips were fixed with 4% paraformaldehyde in PBS for 10 min, permeabilized with 0.2% Triton X-100-PBS, blocked with 0.5% fetal bovine serum and 3% bovine serum albumin (BSA) in PBS and then incubated overnight at 4 ◦C with the corresponding primary antibodies. The following day, cells were washed with 0.05% Triton-X-100-PBS for 5 min and then with PBS alone three times, prior to be incubated for 1 h at room temperature with the appropriate fluorochrome-conjugated secondary antibody. For double immunolabeled samples, this was followed by overnight incubation at 4 ◦C with corresponding primary antibodies and the next day, cells were incubated with secondary fluorochrome-conjugated antibodies. Where indicated, F-actin was labelled using TRITC-conjugated Phalloidin (Sigma-Aldrich St. Louis, MO, USA) diluted 1:500 in PBS for 10 min at room temperature. Finally, coverslip preparations were incubated for 20 min at room temperature with 0.2 μg/mL diamidino-2-phenylindole (DAPI; Sigma Aldrich) for nuclei visualization, mounted on microscope slides with VectaShield (Vector Laboratories Inc., Burlingame, CA, USA) and further analyzed by confocal laser scanning microscopy (CLSM; Eclipse Ti Series, Nikon Corporation Healthcare Business Unit, Japan) using a 63× (NA = 1.2) oil-immersion objective. The analysis of digitized images was carried out using ImageJ, 1.49 software (Wayne Rasband National Institutes of Health, USA. http://imageJ.nih.gov.ij). For morphometric analysis of nuclei, raw images were calibrated and converted to 8-bit gray scale, to set up a threshold for nuclei selection. Then, nuclear area and circularity parameters were calculated, as described previously [58]. The nucleolar area (μm2) was calculated on maxima projection images, using a 3D objects counter, as described previously [59]. To quantify the fluorescence intensity of γ-H2AX (DNA-damage marker and H3K9me3 (heterochromatin marker) foci, the Find Maxima function from ImageJ was used, as previously described [60]. Data were plotted using Prism6 software.
