NAMPT expression in gliomas and its role in cell proliferation. (A) Western blots show the NAMPT expression profiles of adherent (A172, U87, U251-HF, LN229, T98G, U373, U138, and HS683) and patient-derived glioma stem-like cell (GSC) (GSC262, GSC811, GSC5-22, GSC23, BT142, GSC272, GSC267, and GSC20) cell lines in comparison to the profile of normal human astrocyte (NHA). (B) Western blots illustrating the decrease in NAMPT expression in GSC811-Cas9-(NAMPT gRNA) and GSC5-22 Cas9-(NAMPT gRNA) cells caused by the addition of 1µg/µL doxycycline at 24, 48, 72, 96, and 120 h after doxycycline addition. (C) Comparison of cell proliferation between GSC811 and GSC811ΔNAMPT cells (left panel) and GSC5-22 and GSC5-22ΔNAMPT cells (right panel). (D) The results of an in vitro enzyme kinetics assay illustrate KPT-9274’s specific inhibition of NAMPT enzyme activity. (E) Graphs show the targeted effect of KPT-9274 in glioma cell lines LN229, U251-HF, GSC262, GSC811, and GSC5-22, but not in NHA, as indicated by decreased levels of the energy metabolites NAD, NADP, and ATP in comparison to respective vehicle (DMSO) control. (F) Rescue of U251-HF, LN229, GSC262, GSC811, and GSC5-22 viability by addition of the NAD salvage pathway intermediates NAD, NMN, and nicotinamide. Abbreviations: NMN, nicotinamide mononucleotide; NMNAT, nicotinamide mononucleotide adenylyl transferase; OD450, optical density measured at wavelength 450 nM; WT, wild type.