Conditioned medium, transwell assay and tube formation

The treated CAL27 cells (SUV39H1 siRNA or/and DAPT) were washed twice with phosphate buffer (PBS). Endothelial basal medium (EBM; Lonza, Walkersville, MD, USA) with serum removed was continued to grow for 24 h. The cleared supernatant was collected as conditioned medium (CM) and stored at −80 °C.

HUVECs cells were suspended in six-well culture plates, and grown to 90% confluence. The Transwell Boyden chamber (Corning Life Sciences, Corning, NY, USA) was used to measure migration of endothelial cells. Starved HUVECs cells (5 × 10⁴ cells/well) incubated in 100 μl of ECM were placed in the upper wells, whereas CM, as a chemo-attractant, was added to the lower wells. Then, cells were incubated for 12 h at 37 °C. HUVECs on the upper surface were carefully scrubbed off using a cotton swab, and cells adhering to the lower membrane were fixed with 4% formaldehyde, stained by crystal violet, and observed under microscope. The rat tail glue was configured first: rat tail glue was composed in the ratio of A:B:C = 9:1:1. Liquid A was 0.02N acetic acid plus 50× Collagen solution (0.02N acetic acid 7.8 μl + Collagen 130 μl + H₂O 6.3 ml), and liquid B was 10 × Eagle’s medium solution. C is a 2% sodium carbonate solution. Rat tail gum solution was added to 24-well plate, and the volume of 500 μl/well was added. The solution was set at 37 °C for 1 h, and after solidified, it was washed gently with PBS. HUVEC was inoculated with 5 × 10⁴ cells per well. After several hours of culture, the cells were adherent to the wall. Ordinary medium was replaced with conditioned medium collected after treatment. This should be stopped after 24 h. The supernatant was removed and discarded with gum then gently washed three times with PBS. Then, 4% paraformaldehyde should be used to fix for 10 min; wash with water and then gently wash with PBS. The culture plates were placed under an inverted microscope, and six fields of view were randomly selected to take photos to observe and analyze the number of tubes.