Ts2 ph

VM tube formation was assessed as described previously [23 (link),41 (link)]. Cells (3.6 × 10⁵) were seeded on a matrigel-polymerized 24-well plate and then treated with serum with or without MiA for 16 h at 37 °C. In siRNA-transfected cells, cells (3.6 × 10⁵) were seeded after 48 h transfection and then treated with serum. In CRISPR activation plasmid-transfected cells, cells (3.6 × 10⁵) were seeded after 48 h transfection without serum. Tubular shapes were counted after imaging using an inverted light microscope Ts2_PH (Nikon, Tokyo, Japan) at 40× magnification.

The cell invasion assay was performed using Transwell^® cell culture inserts with 8-μm pores (Corning Inc., NY, USA). The membrane filter of an insert was precoated with dilute matrigel (1:20, BD Biosciences) for 2 h at 37 °C. The lower chamber was filled with 700 μL of RPMI 1640 containing 10% FBS (Welgene), and 400 μL of a cell suspension (2 × 10⁵/well) with various concentrations (10, 20, and 40 μM) of EGCG in serum-free media was placed in the inserts. After incubation for 24 h at 37 °C, the inserts were fixed and stained with Diff-Quick (Sysmex, Kobe, Japan), and the cells on the upper surface of the membrane filter were removed with a cotton swab. Then, the invaded cells were captured using an inverted light microscope Ts2_PH at 200× magnification (Nikon, Tokyo, Japan) and quantified.

A 24-well plate (Cat: 30024, SPL, Pocheon, Korea) was used to perform the VM tube-formation assay. Before seeding the cells, the well plates were polymerized with equal amounts of Matrigel and PBS at 37 °C for 1 h. The cells (3 × 10⁵) were added to coated wells in 10% FBS media or serum-free medium, following which they were incubated at 37 °C under hypoxic or normoxic conditions for 11 h. Formed tubes (i.e., tubular shapes) were counted after imaging using a light microscope Ts2_PH (Nikon, Tokyo, Japan) at 40× magnification.