Foxm1 d12d5

Cells were seeded on poly-L-lysine coated culture cover glass (13 mm, Matsunami Glass Industry, Ltd., Osaka, Japan) placed in 24-well plate at 3.0 × 10⁴ cells/well. After an overnight culture, cells were treated with reagents for 24 hours. Cells were fixed with 4% paraformaldehyde for 20 minutes at room temperature. After permeabilized with 0.5% Triton-X for 20 minutes at room temperature, the cells were treated with 3% bovine serum albumin (Sigma-Aldrich, Saint Louis, MO, USA) for 30 minutes at room temperature. After this blocking procedure, cells were treated with a primary antibody for overnight at 4° C, and then a secondary antibody for 1 hour at room temperature. We used the following antibodies: FoxM1 (D12D5, Cell Signaling Technology, Inc., Danvers, MA, USA), Goat anti-rabbit IgG (H+L) Secondary Antibody, Alexa Flour 488 conjugate (Thermo Fisher Scientific, Inc., Waltham, MA, USA). Cover glass was mounted with DAPI Fluoromount-G (Southern Biotechnology Associates, Inc., Birmingham, AL, USA) on slide glass.

Cells were harvested by trypsinization and whole cell lysates prepared as described previously [35 (link)]. Primary antibodies, FOXM1 D12D5 (Cell Signaling, Danvers, MA, USA), β-Tubulin H-235 (Santa Cruz Biotechnology, USA), and VEFG-A ab46154 (Abcam, Cambridge, MA, USA), were detected using horseradish peroxidase-linked anti-rabbit or anti-mouse conjugates as appropriate (KPL, Gaithersburg, MD, USA), and proteins were visualized using enhanced chemiluminescence (ECL) Pierce ECL Western Blotting Substrate detection system (ThermoFisher, USA) with X-ray films. The obtained images were analyzed using ImageJ software v1.54h (National Institutes of Health, Bethesda, MD, USA). All samples were normalized for protein loading using β-tubulin.