Fig. 1

Expressions of PFKFB family members and effect of carfilzomib under normoxic and hypoxic conditions. A Gene expression profiles of PFKFB family members (PFKFB3 and PFKFB4) were analyzed by comparing GEO data (GSE80140) for the normoxic (n = 4) and hypoxic groups (n = 4). *p < 0.05, **p < 0.01 vs. normoxia. n.s.: not significant. B Myeloma cells (U266) were cultured in RPMI 1640 medium under normoxia or hypoxia for 24 h. PFKFB3 and PFKFB4 expressions were examined by immunoblot analysis. β-actin was the loading control. Results represent the mean of three independent experiments. C U266 cells were cultured under hypoxia for the indicated amounts of time. Total extracts were examined by immunoblot analysis using antibodies against phospho-p38 MAPK (Thr180/Tyr182), p38 MAPK, HIF1α, PFKFB3, PFKFB4, and β-actin. D U266 cells were cultured under normoxia or hypoxia and incubated with the indicated concentrations of carfilzomib for 72 h. Cell growth was evaluated using Cell Counting Kit-8. *p < 0.05 vs. normoxia group. E U266 and RPMI8226 cells were cultured under normoxia or hypoxia and incubated with the indicated concentrations of carfilzomib for 48 h. Caspase 3/7 activity was determined using the Caspase-Glo® 3/7 Assay System. Luminescence signals were measured using the Empire Multimode Plate Reader. F U266 cells were cultured under normoxia or hypoxia for 2 h and incubated with the indicated concentration of FM19G11 or SB203580. Phospho- and NF-κB were analyzed using the NF-kB p65 (Phospho) [pS536] Human InstantOne™ ELISA Kit. G, H U266 cells were cultured under normoxia or hypoxia and incubated with the indicated concentrations of PFK158 or 5MPN for 72 h. Cell growth was evaluated using Cell Counting Kit-8. *p < 0.05 vs. untreated cells