2.6. F-Actin Staining

F-actin protein was labeled by fluorescent phallotoxin, a bicyclic peptide showing a high binding affinity toward actin small filament. The staining procedure was performed according to the manufacturer’s instructions (Molecular Probes, Invitrogen, Eugene, OR, USA). Briefly, for each sample, 2 × 10⁵ cells were seeded on cover glasses in MEM supplemented with 10% FBS at 37 °C for 24 h. Then, samples were fixed in 4% paraformaldehyde in PBS for 20 min at 4 °C and subsequently permeabilized by 0.1% Triton-X in PBS for 5 min at RT. Coverslips were stained with fluorescent phallotoxin diluted to 1:40 in 1% bovine serum albumin (BSA) for 20 min at RT. After three washes in PBS and distilled water, coverslips were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) and then mounted with the permanent mountant ProLong gold (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA). Images were acquired by the fluorescence microscope Eclipse E800 (Nikon, Tokyo, Japan).

The quantitative analysis of phallotoxin-stained areas was assessed by area, counting five fields for each of the three slides per sample at 60× magnification by the Leica Qwin 3.0 software (Leica Microsystems Srl, Cambridge, UK), which allowed the phallotoxin-stained area to be selected and measured.