Fitc or tritc phalloidin

Cells were seeded on to glass coverslips in 24-well plates at 10,000/cm² and exposed to different concentrations of ribavirin analogs or ribavirin (20 or 50 μg/mL) for 1, 3 and 7 days of culture. At each time-point, cells were fixed with 3% paraformaldehyde (PFA) in PBS and washed with PBS containing 0.5% BSA. They were permeabilized with 0.1% Triton X100 in PBS and incubated for 2 h at room temperature with anti-αv integrins (sc-9969, Santa-Cruz) or anti-eIF4E (610270, BD PharMingen) antibodies. After washing, the coverslips were incubated with the appropriate fluorescent secondary antibodies: Alexa Fluor 555-conjugated anti-mouse antibody (A21424, Invitrogen) or Alexa Fluor 488-conjugated anti-mouse antibody (BD Transduction Laboratories). The actin cytoskeleton was stained with FITC- or TRITC-phalloidin (P5282 or P1981, Sigma Aldrich). For each assay, cell nuclei were stained with DAPI (4,6-diamidino-2-phenylindole dihydrochloride, D9542, Sigma Aldrich). Coverslips were mounted in Prolong-Gold Antifade Reagent (P36930, Invitrogen) and examined by laser scanning confocal microscopy (LSM710, Zeiss). Controls, in which primary antibodies were replaced with PBS, were negative. Fluorescence microscopy figures were processed using Fiji software [17 (link)].