Endothelial cell growth kit bbe

Vascular cell basal medium (ATCC PCS-100-030), endothelial cell growth kit-BBE (ATCC PCS-100-040), Penicillin-Streptomycin-Amphotericin B Solution (ATCC PCS-999-002), and Phenol red (ATCC PCS-999-001) were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). Dulbecco’s Modified Eagle Medium (DMEM), fetal bovine serum (FBS), and penicillin/streptomycin were purchased from Gibco (Gibco BRL, Life Technologies, Inc., NY, USA). D-glucose, Mannitol, polyvinylidenedifluoride (PVDF) membrane, and enhanced chemiluminescence (ECL) were purchased from Merck Millipore (Merck, Darmstadt, Germany). Primary antibodies against phosphorylated-PI3K, phosphorylated-Akt, total-Akt, phosphorylated-ERK1/2, total-ERK1/2, p53, caspase 3, caspase 9, Bax, Bcl 2, and β-Actin were purchased from Cell Signaling Technology (Cell Signaling Technology, Inc., Danvers, MA, USA). Other chemicals and reagents were purchased from Sigma Aldrich (Sigma, St. Louis, MO, USA).

Human umbilical vein endothelial cells (HUVECs) were obtained from the American Tissue Culture Collection (Rockville, MD, United States). They were maintained as monolayer cultures in 58, 1 cm² plastic culture plates in Vascular Cell Basal Medium (VCBM) (ATCC, Rockville, MD, United States) supplemented with endothelial cell growth kit-BBE (ATCC, Rockville, MD, United States) which consisted on 2% fetal bovine serum (FBS), 0.2% Bovine Brain Extract, 5 ng/ml rhEGF, 10 mM L-glutamine, 0.75 units/ml heparin sulfate, 1 μg/ml hydrocortisone hemisuccinate, 50 μg/ml ascorbic acid, penicillin (20 units/ml), and streptomycin (20 μg/ml) (Sigma-Aldrich, Madrid, Spain) at 37° in a humid atmosphere containing 5% CO₂. To avoid genetic mutation and low viability, no more than five-six passages of HUVECs were used (Alvarez-García et al., 2013b (link),c (link)).
MCF-7 human breast cancer cells were purchased from the American Tissue Culture Collection (Rockville, MD, United States). They were maintained as monolayer cultures in 75 cm² plastic culture flasks in Dulbecco’s Modified Eagle’s Medium (DMEM) (Sigma-Aldrich, Madrid, Spain) supplemented with 10% FBS (PAA Laboratories, Pasching, Austria), penicillin (20 units/ml) and streptomycin (20 μg/ml) (Sigma-Aldrich, Madrid, Spain) at 37° in a humid atmosphere containing 5% CO₂ (Alvarez-García et al., 2013a (link)).

HMEC-1 cells (ATCC^® CRL-3243TM, USA) were cultured in complete MCDB 131 medium (Thermofisher Scientific, Horsham, UK) containing 10% foetal bovine serum (FBS, Lonza, Basel; Switzerland), L-glutamine dissolved in sterile water supplemented with 15.7 mL/L sodium bicarbonate solution (7.5% w/v), 10 ng/mL epidermal growth factor (EGF); 1 μg/mL hydrocortisone, 100 μg/mL penicillin and 0.1 mg/mL streptomycin (all Sigma Aldrich, Dorset, UK). HUVECs were cultured in complete endothelium growth medium (ATCC, Virginia, USA) containing Endothelial Cell Growth Kit-BBE (ATCC, USA). Human colorectal adenocarcinoma cells (Caco-2, ATCC^® HTB-37TM, USA) were cultured in high-glucose Dulbecco’s Modified Eagle’s Medium (DMEM, Sigma Aldrich, USA) containing 10% FBS, L-glutamine dissolved in sterile water with 15.7 mL/L sodium bicarbonate solution (7.5% w/v), 100 μg/mL penicillin and 0.1 mg/mL streptomycin (all Sigma Aldrich, USA). Human primary neutrophils were isolated using Percoll density gradient and suspended in RPMI-1640 medium with 0.5% fatty-acid-free BSA, 2 mM L-glutamine, 100 U/mL penicillin and 0.1 mg/mL streptomycin (all Sigma Aldrich, USA).

The mouse lung endothelial cell line and the mouse dEC line were generated by immortalizing primary cells isolated from adult C57/BL6 mice using SV40 large T-antigen^{11 (link),30 (link)–33 (link)}. The endothelial cell lines were cultured in DMEM with 10% FBS, penicillin–Streptomycin (100 µ/ml and 100 μg/ml, respectively) in a humidified cell culture incubator set at 5% CO₂ and 37 °C. The primary HUVECs from ATCC (PCS-100-013) were cultured with the Endothelial Cell Growth Kit-BBE (ATCC® PCS-100-040) following the kit`s instruction.

Hoechst 33342 stains were purchased from Life Technologies (Cat no. H3570). Human Chordoma cell lines U-CH1 (Cat. no. CRL-3217) and U-CH2 (Cat. no. CRL-3218) were purchased from ATCC (Manassas, VA, USA). Cells were grown in Iscove’s Modified Dulbecco’s Medium (Cat. no. 30-2005, ATCC): RPMI-1640 Medium (Cat. no. 30-2001, ATCC) (4:1), 10% FBS (Cat. no. 30-2020, ATCC) supplemented with 2 mM L-glutamine (Cat. no. 30-2214, ATCC). HEK-293 (Cat. no. CRL-1573) and HUVEC (Cat. no. PCS-100-013) cells were purchased from ATCC and were grown in Eagle’s Minimum Essential Medium (Cat. no. 30-2003, ATCC) supplemented with 10% fetal bovine serum, and Vascular Cell Basal Medium (Cat. no. PCS-100-030, ATCC) supplemented with Endothelial Cell Growth Kit-BBE (Cat. no. PCS-100-040, ATCC), respectively. PSMB5 (Cat. no. 11903), PSMB8 (Cat. no. 13635), and β-Actin (Cat. no. 3700) antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Doxorubicin was purchased from Sigma (Cat. no. D1515). Compound libraries were provided by the Institute of Chemistry and Cell Biology (ICCB) Longwood, Harvard Medical School, Boston, MA, USA.