24 well chamber

Capillary tube formation assays were carried out as described by Qiu et al [52] (link). Briefly, HUVEC cells (3×10⁴ cells/well) were cultured on Matrigel in a 96-well plate for 24 hrs with 100 µg/mL of P0-P5, P1C-P5C or blank control. The enclosed capillary networks of tubes were photographed by a microscope (Olympus, CKX41, Japan) and the numbers of capillary tubes formed were counted at different time intervals.
The transwell cell migration assay was performed by using a 24-well chamber (Costar, Cambridge, MA, U.S.A.) as the outer chamber and polycarbonate filters (8 µm pores) as the inner chamber. HUVECs were starved overnight in serum-free F12 medium, harvested by trypsinization and 5×10⁴ cells were seeded into the inner chambers in F12 (1%FBS) media with 100 µg/mL P5 or blank control. The outer chambers contained the F12 media with 10% FBS. After incubation for 18 hrs at 37°C, the cells on the lower surface of the filter were fixed and stained with 0.1% crystal violet. Images of the migrated cells were taken using a microscope (Olympus, CKX41, Japan). The migrated cells were quantified at 595 nm after extraction with 10% acetic acid. Migrated cells in the outer chambers were also quantified independently by incubating the out chambers in F12 media with 10% FBS for 8 days followed by resazurin quantification.

Human EECs were cultured in 24-well plates and transfected with miR-125b mimics or NC according to the manufacturer’s instructions (RiboBio). After 48 h, the cells were digested with trypsase and resuspended in serum-free medium. Migration and invasion assays were performed in a 24-well chamber (Corning). For invasion assays, before 5 × 10⁵/ml cells were seeded on the upper chamber, matrigel (BD) diluted 1:8 with serum-free culture medium was added to a membrane with 8 μm pores and incubated at 37 °C for 1 h. For the migration assays, 1 × 10⁵/ml cells were seeded on the upper chamber without matrigel. For both assays, 600 μl complete medium was added to the lower chamber. After incubation for 24 h, cells that passed through the membrane were fixed and stained with crystal violet (Sigma-Aldrich). The number of cells that crossed the membrane was counted in five random fields at 100x. Each experiment was performed in triplicate.