Inverted photomicroscope

Growth factor-reduced Matrigel (BD Biosciences, San Jose, CA, USA) was pipetted into prechilled 24-well culture plates (100 μl/well) and polymerized for 30 min at 37°C. HUVECs (4 × 10⁴) with various concentrations of MLN4924 were seeded onto the surface of the Matrigel. After 12 h of incubation, capillary tube formation was observed and digitized with an inverted photomicroscope (Olympus). The tubular networks were quantified using ImageJ.^{40 (link)}

H1299 and A549 cells with various transfections were cultured in the corresponding culture medium, and the supernatant was collected as the conditional medium. The Matrigel (BD Biosciences) was dissolved at 4°C overnight, and 24‐well plates were treated with 100 μl of Matrigel. Human umbilical vein endothelial cells (HUVECs; Gibco) cultured in the tumor‐conditioned medium were placed into 24‐well plated (4 × 10⁴ cells/well) coated with Matrigel for 12 h. Next, the images were observed using an inverted photomicroscope (Olympus). Tube formation was assessed according to the number of branches using Image J software (NIH). Five different fields in each group were used to count the number of branches, and the results are shown as the means of the values.