Block ace

Cells were fixed by 4% paraformaldehyde (Wako) for 30 min at room temperature. They were blocked and permeabilized in 4% (w/v) Block Ace (AbD Serotec)/cell staining buffer (20 mM Tris-HCl [pH 7.5], 150 mM NaCl, and 0.2% Triton X-100) for 30 min at room temperature. Then, cells were incubated with primary antibodies in 0.4% (w/v) Block Ace/cell staining buffer overnight at 4°C, and subsequently incubated with appropriate secondary antibodies with Hoechst 33342 (10 μg/mL; Sigma-Aldrich) in 0.4% (w/v) Block Ace/cell staining buffer for 1.5 hr at room temperature. Detailed conditions of antibodies are given in Table S6. Fluorescence images were acquired on an IX73 (Olympus) or IN-Cell Analyzer (GE Healthcare).

Proximities between actin and vimentin or nestin filaments were examined by Duolink in situ Proximity ligation assay (PLA) (Sigma-Aldrich). Cells were cultured and fixed with 4% paraformaldehyde for 15 min. After permeabilization with cold acetone for 1 min, the cells were washed with a blocking solution containing 0.4% Block Ace (DS Pharma Biomedical) in PBS, incubated with mouse monoclonal anti-actin antibodies (1:400) (AC-40, Sigma-Aldrich, St. Louis, MO, USA) and rabbit monoclonal anti-vimentin (1:500) (EPR3776, Abcam, Cambridge, UK) or rabbit monoclonal anti-nestin antibodies (1:1000) (SAB4200347, Sigma-Aldrich, St. Louis, MO, USA) diluted in 0.4% Block Ace for 1 h at room temperature. All other steps were performed according to manufacturer's instructions. To evaluate the number of signals from each PLA reaction, fluorescence spots on the basal membranes of 30 cells were counted.