Extended Data Fig. 8: UPP1 expression and uridine catabolism for energy production in the monocytic lineage.
From: Salvage of ribose from uridine or RNA supports glycolysis in nutrient-limited conditions
(a) Protein immunoblot of human THP1 monocytic cells treated with 100 nM PMA alone for 48 h followed by the addition of 100 ng/mL LPS or 1 mg/mL purified yeast RNA for another 48 h and immunoblotted with antibodies to UPP1 and TUBB. Western blot quantification is shown as fold of untreated cells (monocytes). (b) Protein immunoblot of human MCSF-matured PBMC treated with 5 µg/ml R848 for 24 h and immunoblotted with antibodies to UPP1 and Actin (n = 3 donors). Western blot quantification is shown as fold of untreated cells and relative to each donor. (c) Expression of UCK1 and UCK2 in THP1 cells as determined by qPCR after treatment as in (a). Data are shown as mean ± SEM with n = 4. (d) 13C5-uridine tracer analysis of UMP in THP1 cells treated as in (a) with the exception that glucose-free RPMI media containing 5 mM 13C5-uridine was used. Data are shown as mean ± SEM with n = 4. (e) Expression of Upp1 and Il1b in BMDM as determined by qPCR after co-treatment for 24 h with 5 µg/ml R848 and 5 µg/ml BMS-345541, an IKK inhibitor. Data are shown as mean ± SEM with two-sided t-test relative to untreated (n = 3 mice, P < 1.8 × 10−2, P < 4.5 × 10−2). (f) 13C5-uridine tracer analysis of citrate and lactate in THP1 cells treated as in (a) with the exception that glucose-free RPMI media containing 5 mM 13C5-uridine was used for the last 6 h. Data are shown as mean ± SEM with two-sided t-test relative to PMA-treated cells with n = 4, P < 4.7 × 10−4, P < 1.5 × 10−2, P < 1.2 × 10−2, P < 2.2 × 10−2. (g) 13C5-uridine tracer analysis of media lactate in human MCSF-matured PBMC cells treated as in (b) with the exception that glucose-free RPMI media containing 5 mM 13C5-uridine was used was used for the last 6 h. Data are shown as mean ± SEM, with n = 4 donors. TUBB and Actin loading controls were performed on the same gels. All metabolomics included 2 mM (RPMI) or 4 mM (DMEM) L-glutamine and 10% dialyzed FBS.
