Nampt

Western blot assays were performed as previously described (24 (link)). Briefly, proteins (20 μg/sample) were separated using SDS–polyacrylamide gel electrophoresis and electroblotted to nitrocellulose membranes (Bio-Rad). Membranes were probed overnight with the following antibodies: phosphor-AKT (Ser 473), phosphor-AKT (Thr 308), AKT, N-MYC, phosphor-ACC (Ser 79), ACC, phosphor-GSK3b (Ser 9) GAPDH (obtained from Cell Signaling Technology), NAMPT, NAPRT (obtained from GeneTex) or α-tubulin (obtained from Abcam). Membranes were then washed, reacted with horseradish peroxidase–conjugated secondary antibodies and visualized using enhanced chemiluminescence (Thermo Scientific). Relative levels of NAMPT and NAPRT as well as quantitation of cleaved caspase 3 (active) and parp cleavage (indicative of caspase activation) were determined using Image J (imagej.nih.gov).

Cells were cultured on 4-well plates (Nunc), fixed in 4% paraformaldehyde, washed, blocked and permeabilized with a 5% bovine serum albumin (BSA) with 0.6% Triton-× 100 and then treated with the primary antibodies NAMPT and NAPRT (GeneTex). For analysis of stem cell markers in NB1691-sp cells, cells were plated on laminin/poly-L-Lysine (Sigma-Aldrich) coated plates and treated with Bmi1 or Musashi primary antibodies (Cell Signaling Technology). Cells were then treated with Alexa Fluor 594 -conjugated secondary followed by Prolong Gold Antifade Reagent with DAPI (Thermo Fisher Scientific). Samples were examined under an EVOS FLoid Cell Imaging Station fluorescent microscope (Thermo Fisher Scientific).