Migration and invasion assay

Cell migration activity was examined with a modified Boyden chamber with 8 μm pores. EA.hy926 cells were supplemented with 200 μL serum-free medium and seeded on the upper compartment. The lower compartment was supplemented with 600 μL complete medium. H₂O₂ or Rb1 was added to both inserts and wells. Cells were fixed with methanol after incubation at 37°C for 8 h. Traversed cells on the lower side of the filter were stained with crystal violet and counted. The invasive behavior of EA.hy926 cells was determined with a BD BioCoat^TM Matrigel^TM invasion (BD Biosciences, San Jose, CA, USA). Serum-free medium was supplemented into the inserts and the wells. EA.hy926 cells were plated onto the inserts. H₂O₂ or Rb1 was added to both inserts and wells. Chambers were incubated for 24 h at 37°C. After fixation with methanol and staining with crystal violet, the invasive cells were counted.