2 fdg

iSLK. 219 cells were induced at 60% confluency with doxycycline (1 μg/mL, Sigma) and sodium butyrate (1 mM) in the absence (—) or presence of one of the sugar analogs: 2-DG (Sigma), 2-FDG (Carbosynth), and 2-DFM (Carbosynth) at 2 mM or 5 mM. After 72 hours of induction, cell-free virus-containing supernatants were collected and, after a serial dilution, were used to de novo infect HEK-AD293, which were pre-treated for 30 min with 8 μg/mL polybrene (Millipore). 72 hours after infection, infectious virion production was measured by counting the GFP+ HEK-AD293 by flow cytometry to determine the 50% tissue culture infective dose (TCID₅₀) (1 GFP(+) AdHEK293 cell = 1 infectious KSHV virion).

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).