2 fdg

iSLK.BAC16 cells were plated at 8,000 cells/well in a 96-well plate in triplicates. The next day all cells, except for the control cells, were induced with doxycycline (1 μg/mL, Sigma) and sodium butyrate (1 mM) alone or in addition to one of the sugar analogs: 2-DG (Sigma), 2-FDG (Carbosynth), or 2-DFM (Carbosynth) at 5 mM. Additionally, a Cytotox Red Reagent at 250 nM (Essen Bioscience) was added to all wells. Cells were incubated in an Incucyte Zoom (Essen Bioscience) to acquire green and red fluorescence images at 10×magnification every 4 hours. The death index was calculated for each data point with the formula: Red object count (dead cells)/Green object count (iSLK.BAC16 cells).

2-DG, 2-FDG, oxamate, Mannose, Tunicamycin and (2’Z, 3’E)-6-Bromoindirubin-3′-oxime (BIO) were purchased from Sigma-Aldrich (St. Louis MO). Brefeldin A was acquired from Calbiochem-EMD Millipore (Billerica, MA). Matrigel was obtained from BD Biosciences (Bedford, MA) and used at 7mg/ml. Human bFGF and VEGF were purchased from R&D Systems (Minneapolis, MN). Human microvascular endothelial cells from lung (HMVEC-L) was purchased from Lonza (Walkersville, MD) in 2007, used only till passage 5 and was authenticated by vendor. EBM-2 basal medium and the EGM2 and EGM2-MV supplements were purchased from Lonza (Walkersville, MD). Human umbilical vein endothelial cells (HUVECs, purchased 3-4 times yearly depending on usage and used till passage 5), MDA-MB-231(purchased in 2007) and HT-29 (purchased in 2012, 2014) were purchased from the ATCC (Manassas, VA) and were authenticated by vendor. For the endothelial cell experiments described below, “Unstimulated ECs” are defined as cells maintained with endothelial basal medium (EBM) with 1% FBS while “stimulated ECs” are cells that were starved overnight (EBM and 1% FBS), and then treated with either bFGF or VEGF (10 ng/ml) the day of the experiment. Endothelial cell media contains 1 gram of glucose per liter (5.5 mM).