Cell culture media

Cell culture media, serum, and cell culture supplements were purchased from ATCC; Manassas, VA, USA, Millipore; USA, Invitrogen; NY, USA, and PAA; North America. Antibodies against VE‐cadherin (ab33168), Flk‐1 (ab9530), VEGFA (ab51745), STAT3 (ab119351), JAG‐1 (ab7771), NOTCH‐1 (ab27526), SMA; Smooth Muscle Actin (ab5831), and SM22 (ab14106) were purchased from Abcam; Cambridge, UK. The Antibody against GAPDH (sc‐25778) was purchased from Santa Cruz. Antibodies against CD31 were purchased from ABBIOTEC; San Diego, USA (250590) and Santa Cruz; Heidelberg, Germany (sc‐1506). The secondary antibodies for immunostaining anti‐mouse Alexa 488 and anti‐rabbit Alexa 488 were purchased from Invitrogen. The secondary antibodies for Western blotting were purchased from DakoCytomation; Glostrup, Denmark and Abcam. Recombinant mouse VEGF was purchased from R and D Systems; Abingdon, UK.

MCF-7 cells (ATCC, Manassas, VA) were grown in EMEM with 10% FBS and 10 μg/mL bovine insulin, in an incubator at 37°C with 5% CO₂[7 (link), 8 (link)]. The cells were arrested in the G1-phase by serum-deprivation for 48 h, in a medium consisting of DMEM and 4 mM L-glutamine, harvested, separated into nuclear and cytoplasmic fractions (Cell Lytic™ NuCLEAR™ extraction kit, Sigma, St. Louis, MO), digested with trypsin (Promega Corporation (Madison, WI) at 37°C for 24 h (50:1 substrate:enzyme ratio), and analyzed by nano-liquid chromatography (LC)-MS/MS with a linear trap quadrupole (LTQ/Thermo Electron Corporation, San Jose, CA) mass spectrometer. FACS analysis was performed with a Beckman Coulter EPICS XL-MCL analyzer (Brea, CA, USA). The protein content was measured by the Bradford assay on a SmartSpec Plus spectrophotometer (Bio-Rad, Hercules, CA). The sample analyzed by MS contained 2 μg/μL MCF-7 proteins. LC separations were performed with an Agilent 1100 LC system (Palo Alto, CA) and in-house prepared nano-separation columns (100 μm i.d. x 12 cm) packed with 5 μm Zorbax SB-C18 particles. Common reagents were purchased from Sigma, cell culture media from ATCC and Invitrogen (Carlsbad, CA), and HPLC-solvents from Fisher Scientific (Fair Lawn, NJ). Sample preparation and LC-MS/MS analysis protocols were described in detail in previous manuscripts [7 (link), 8 (link)].

The MDCK (NBL-2) cell line was purchased from American Type Culture Collection (ATCC, Manassas, VA, USA) in 2002, grown for a few passages and then cryopreserved in liquid nitrogen. MDCK cell aliquots used in this study were sub-cultured for a maximum of 15 passages. Cell culture media and fetal bovine serum were also purchased from ATCC. MDCK cells were grown in Eagle Minimum Essential Medium with Earle’s Balanced Salt Solution, 10% fetal bovine serum and 1% penicillin-streptomycin (Life Technologies, Burlington, ON, Canada) in a humidified incubator at 37°C and 5% CO₂. MDCK cells seeded at 70,000 cells/cm² were allowed to grow for two days before nucleic acid isolation.

All reagents and standards were purchased by Sigma-Aldrich Chemicals Co. (St. Louis, MO) and Merck (Darmstadt, Germany). Cell culture media and all other supplements were purchased by ATCC^® (American Type Culture Collection) Manassas, VA 20108 USA.