Primary huvecs

Primary human RPTECs (Lonza, Walkersville, MD) were maintained in renal epithelial cell basal medium supplemented with REGM complex (hydrocortisone, human epidermal growth factor, epinephrine, triiodothyronine, transferrin, insulin, gentamicin sulfate, and 0.5% fetal bovine serum). Primary HUVECs (Clonetics, Walkersville, MD) were cultured in endothelial basal medium (HuMedia-EB2, Kurabo, Osaka, Japan) supplemented with HuMedia-EG (1.34 μg/ml hydrocortisone, 10 ng/ml human epidermal growth factor, 5 ng/ml human fibroblast growth factor-B, 10 μg/ml heparin, 50 μg/ml gentamicin, 50 ng/ml amphotericin B, and 2% fetal bovine serum). These cells were cultured in humidified conditions of 95% air/5% CO₂ at 37°C. The culture medium of RPTECs was changed every 3–4 days. HUVEC culture medium was changed every 1–2 days. To collect HUVEC-conditioned media (HUVEC-CM), which contains the soluble factor(s) produced by HUVEC, confluent HUVECs were cultured in HuMedia-EB2 supplemented with HuMedia-EG for 48 h. Culture supernatant were collected and then centrifuged at 1500 rpm for 5 min at 4°C.

Primary HUVECs, human EECs, human CAECs, human MCECs, and human AECs were obtained from Clonetics Inc. (Walkersville, MD, USA). Cells were grown in endothelial basal medium supplemented with 2% fetal bovine serum and penicillin (100 u/ml), and streptomycin (100 µg/ml). Cultured cells were used between passages 3 and 8. All cells were incubated in a humidified atmosphere of 5%CO₂ + 95% air at 37 °C. When 70–80% confluent, the cells were treated with different agents. For HEK293 cells, cells were cultured in M200 medium supplemented with 2% fetal bovine serum and penicillin (100 u/ml), and streptomycin (100 µg/ml). For lentivirus infection, cells were infected with lentivirus expressing AP-2α shRNA or circRNA-RBCK1 shRNA from Shanghai Genechem Co., Ltd. (Shanghai, China) overnight in antibiotics-free medium supplemented with 2% FBS. The target sequence of CircRNA-RBCK1 shRNA is TCTTGCAGCAGTGGGTGATTG. The target sequence of AP-2α shRNA is TCCCAGATCAAACTGTAATTA. These targets were designed by VectorBuilder Inc. The cells were then washed and incubated in fresh medium for an additional 12 h before experiments.

Human ovarian cancer cell lines (SKOV-3 and OVCAR-3) were purchased from the American Type Culture Collection (ATCC, Manassas, VA). They were grown in Dulbecco's modified Eagle's medium (DMEM; Life Technologies, Gaithersburg, MD) supplemented with 10% fetal bovine serum (FBS) and 100 U/ml penicillin/streptomycin at 37°C in a humidified 5% CO₂ incubator. Primary HUVECs (Clonetics, San Diego, CA) were grown on 0.3% gelatin-coated dishes (Sigma-Aldrich, St. Louis, MO) in EGM-2 BulletKit medium (Clonetics). Rapamycin was purchased from Cell Signaling Technology (Beverly, MA). Doxazosin and all other chemicals were purchased from Sigma-Aldrich. The following primary antibodies were used: anti-phospho-VEGFR-2 (Y1175), anti-VEGFR-2, anti-phospho-PI3K, anti-PI3K, anti-phospho-Akt, anti-Akt, anti-HIF-1α, anti-phospho-ERK1/2, anti-ERK1/2 (all from Santa Cruz Biotechnology, Santa Cruz, CA), anti-phospho-PDK1, anti-PDK1, anti-phospho-4E-BP1, anti-4E-BP1, anti-phospho-mTOR, anti-mTOR, anti-phospho-p70S6K, anti-p70S6K, PCNA, cyclin D1, survivin (all from Cell Signaling Technology), anti-CD31 (Abcam, Cambridge, UK), anti-VEGF₁₆₅ (Ab-1; Oncogene, Cambridge, MA), and anti-β-actin (Sigma-Aldrich).