Immunofluorescence Analysis of Actin Cytoskeleton

HeLa cells were seeded in a glass-bottomed multi-well plate at 8.0 × 10⁴. After treatment with either ART or 8-p-ART, cells were fixed in p-formaldehyde at 4% v/v in PBS (Lonza; Basilea, Swiss), permeabilized with Triton X-100 at 0.5% v/v in PBS (Lonza; Basilea, Swiss), and then blocked with goat serum at 20% v/v in PBS (Lonza; Basilea, Swiss). Incubation with the respective antibodies against FLNA and FLNB was performed O/N at 4°C. F-actin detection was evaluated by phalloidin-FITC (5 μg/ml, Sigma-Aldrich; Saint Louis, MO, United States) for 30 min at RT in the dark. The staining with conjugated secondary antibodies (1:500, anti-mouse), the nuclei with DAPI (1:1000), and the subsequent confocal microscope analysis were performed as described in Belvedere et al. (2018a (link)) and Belvedere et al. (2018b (link)).