Migration, invasion and tube formation assay
The migration and invasion assay were performed as described
before(Mierke, 2019). To examine the effect of AZM on tube formation,
Matrigel (10 mg/mL, BD Biosciences, San Jose, CA, USA) was firstly
plated in 96-well culture plates and allowed to polymerise at 37 °C in
5% CO2 humidified for 30 min. Following the exposure of
conditioned medium of AZM-treated RA FLSs for 24 h, human umbilical vein
endothelial cells (HUVECs; 2×105 cells/mL) were
trypsinized, resuspended, and added to each chamber in sterile medium
(supplemented with 5% fetal bovine serum, 100 U/mL penicillin, and 80
U/mL streptomycin) with vascular endothelial growth factor (VEGF; 50
ng/mL, AF-100, Peprotech, Rocky Hill, NJ, USA), then incubated for 6 h
at 37 °C in 5% CO2. After that, the capillary-like tube
formation of each well in the culture plates was photographed. All
experiments were done in triplicate.