Tetramethylrhodamine phalloidin

A cell spreading assay was performed as previously described.24 (link) MDA-MB-231 controls and AIF1L-overexpressing cells were resuspended in serum-free medium. A total of 5×10⁴ cells were added to Matrigel-coated glass coverslips. Sixty minutes after plating, cells were fixed with 4% paraformaldehyde for 20 minutes at room temperature. After washing with PBS, 0.1% Triton X-100 (in PBS) was added for 10 minutes at room temperature. Cells were blocked with 10% normal goat serum for 10 minutes at room temperature. Cells were incubated with 0.5 µM tetramethylrhodamine-phalloidin (Sigma-Aldrich) for 1 hour at room temperature and counterstained with 4′,6-diamidino-2-phenylindole to visualize nuclei. Coverslips were imaged using epifluorescence microscopy (Nikon Corporation, Tokyo Japan). Images were processed with ImageJ software to measure the areas of the cell and nucleus. Circularity and aspect ratio were used to measure cell shape as previously described.25 (link),26 (link) Both cell circularity and aspect ratio were used to measure the roundness of a cell. Over 50 cells were quantified per sample. The experiment was performed in three technical replicates.

For staining of the actin filaments, a 5 mg/mL stock solution of tetramethylrhodamine–phalloidin (Sigma P1951) was prepared in methanol and used according to the manufacturer's instructions.
Immunofluorescence staining for phospho‐Met, c‐Met, and HGF were carried out as described previously.35 Staining was carried out using primary antibodies against phospho‐Met (Y‐1234/1235) (cs‐3129), c‐Met (sc‐161), and HGF (sc‐1387) prepared in blocking solution. Cells were visualized using an Olympus BX50 fluorescence microscope (Olympus, Tokyo, Japan).