Egm 2 bulletkit medium

Human umbilical vein endothelial cells (HUVECs) were purchased from Clonetics (San Diego, CA, USA) and cultured in EGM-2 Bullet Kit medium (Clonetics) supplemented with 2 % fetal bovine serum (FBS) and complete endothelial growth factors at 37 ° C in humidified 5 % CO₂. The cells were seeded into twenty-four well plates 5000 cells/cm2, and sub-confluent cell monolayers were used for experiments. A subset of sub-confluent HUVECs were used as controls and the remainder were treated with either 0.3 mM L-citrulline, 1 mM GSH, or a combination of each at 0.3 mM, and incubated for 24 h. To measure nitrite production by HUVECs, the culture medium was collected and centrifuged to remove any precipitated materials. Four wells for each condition were used and nitrite concentrations of supernatants from each well were determined by high performance liquid chromatography (HPLC) (ENO-20; Eicom, Kyoto, Japan) using our previous approach [12 (link)].

Human cervical carcinoma cell lines (CaSki and SiHa) and human embryonic kidney 293T (HEK293T) cells were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and were maintained in monolayer cultures according to ATCC recommendations. HUVECs were purchased from Clonetics (Walkersville, MD, USA), and seeded on 0.3% gelatin-coated dishes (Sigma, St. Louis, MO, USA) using the EGM-2 BulletKit medium (Clonetics). Wortmannin and LY294002, which are PI3K inhibitors, were obtained from Sigma (St. Louis, MO, USA). The following antibodies were used in this study: anti-HPV16 E6 (ab226447), anti-IgG (Abcam, Cambridge, UK), anti-IRF-1, anti-Flag, anti-cyclin D1, anti-CDK4, anti-NF-κB (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-Bax, anti-Bcl-xL, anti-Bcl-2, anti-p53, anti-VEGFR-1, anti-VEGFR-2, anti-phospho-VEGFR-2(Tyr-1175), anti-HIF-1α, anti-PI3K, anti-phospho-PI3K, anti-Akt, anti-phospho-Akt(Ser473), anti-PDK-1, anti-phospho-PDK-1(Ser241), anti-mTOR, anti-phospho-mTOR(Ser2448), anti-4E-BP1, anti-phospho-4E-BP1(Thr70) (Cell Signalling, Beverly, MA, USA), anti-p21, anti-VEGF (Ab-1; Oncogene, Cambridge, MA, USA), and anti-β-actin (Sigma, St. Louis, MO, USA).

The HER2+ BC MDA-MB-453 (MM-453) cell line and human umbilical vein endothelial cells (HUVECs) were obtained from the American Type Culture Collection (Manassas, VA, U.S.A.). The cells were cultured in DMEM supplemented with 10% FBS and EGM-2 BulletKit medium (Clonetics, MD, U.S.A.), respectively. Trastuzumab was purchased from Roche Pharma AG (Grenzach-Wyhlen, Germany). The animal experimental protocols was approved by the ethics committee of Shandong University of Traditional Chinese Medicine. Female pathogen-free nude mice (nu/nu, age: 5–6 weeks; weight: 20–25 g) were obtained from Laboratory Animal Center of Shandong University of Traditional Chinese Medicine (Jinan, Shandong, China). To establish HER2+ BC xenograft models, 5 × 10⁶ MM-453 cells in 100 μl medium were subcutaneously injected to the right flank of each mouse. All mice were monitored for body weight, activity, and tumor volume. While the tumor volume reached ∼50 mm³ (1/2L*W²), the xenograft models were considered successfully constructed. Xenograft models were randomly classified into four groups: Control, YHD, Trastuzumab, YHD+Trastuzumab (n=10/group). Trastuzumab was intraperitoneally injected at 2 mg/kg every 3 days; YHD was intragastrically administrated at 50 mg/kg everyday. Control group only received the vehicle. Experiments were performed for 4 weeks.