A metabolite screen of pancreatic cells shows that pancreatic cancer cells metabolize uridine-derived ribose via UPP1, supporting redox balance, survival and proliferation.
Cell culture The PDA cell lines A549, HT1080, HCT116 and U2OS and human pancreatic nestin-expressing cells were purchased from the American Type Culture Collection (ATCC) or the German Collection of Microorganisms (DSMZ). The human pancreatic stellate cells (hPSC) and mouse PDA cell lines KPC 7940b and MT3-2D were provided under a material transfer agreement by R. Hwang, G. Beatty and D. Tuveson, respectively. iKras cell lines A9993 and iKRAS 9805 were derived as described33. The identity of cell lines was confirmed by STR profiling, and lines were routinely tested for mycoplasma using MycoAlert (Lonza, LT07-318). For routine propagation, unless otherwise indicated, all cell lines were cultured in high-glucose DMEM (Gibco, 11965092) supplemented with 10% FBS (Corning, 35-010-CV) at 37 °C and 5% CO 2 . PBS (Gibco, 10010023) was used for cell washing steps unless otherwise indicated. For treatments, the following inhibitors were used: MYC, fedratinib (MedChemExpress, HY-10409) and 10058-F4 (Cayman Chemical, 15929); MEK1, trametinib (Selleckchem, S2673). Biolog metabolic assay In the initial phenotypic screen, the 22 cell lines were grown in 96-well PM-M1 and PM-M2 plates (Biolog, 13101 and 13102). The assay was set up such that one well was used per test metabolite substrate, accompanied by three replicates of positive (glucose) and negative (blank) control wells. The RMA from substrate catabolism in the cells was measured using Biolog Redox Dye Mix MB. In brief, the cell lines were counted, and their viability assessed using Trypan Blue Dye (Invitrogen, T10282). The cells were then washed two times with Biolog Inoculating fluid IF-M1 (Biolog, 72301) to remove residual culture medium. Then, a cell suspension containing 20,000 cells per 50 µl was prepared in Biolog IF-M1 containing 0.3 mM glutamine and 5% dialysed FBS (dFBS) (Hyclone GE Life Sciences, SH30079.01) and plated into PM-M1 and PM-M2 96-well plates at 50 µl per well. Plates were incubated for 24 h at 37…
Nwosu, Zeribe C., Department Of Molecular, Integrative Physiology, University Of Michigan, Ann Arbor, Ward, Matthew H., Department Of Chemistry, Washington University In St Louis