Rtgf β2

Human vascular endothelial cells (HUVECs; Cambrex, Walkersville, MD, USA) were cultured for four to six passages in EGM-2 MV medium (Clonetics Co., San Diego, CA, USA) supplemented with 10% fetal bovine serum, 1 mg/mL hydrocortisone, 12 mg/mL bovine brain extract, 50 mg/mL gentamycin, 50 ng/mL amphotericin B, and 10 ng/mL epidermal growth factor. After 3 days of culture, cells were detached from dishes by the addition of 3 mM ethylenediaminetetraacetic acid solution and a minimal amount of trypsin. Cells (2 × 10⁵/well) were then placed in 8-well chamber slides with serum-free medium for 24 hours and washed twice with phosphate-buffered saline (PBS). Next, recombinant transforming growth factor (rTGF)-β2 (R&D Systems, Minneapolis, MN, USA) was added at a final concentration of 10 ng/mL, except where otherwise indicated. Cells were then incubated with or without AUDA (3 or 6 μM) for 48 hours. Primary human tubular epithelial cells (hTECs) were isolated from unaffected specimens of surgically resected kidneys from patients diagnosed with kidney cell carcinoma [14 (link)]. hTECs were cultured for four to six passages in EGM-2 MV medium under the same conditions used for HUVECs.