Nampt inhibition induces autophagic cell death of MM cells. (A) Electron microscopy of U266, RPMI-8226/S, and MM-1S cells treated with 10nM FK866 for 24 to 48 hours. Autophagic structures (autophagosomes) were observed on FK866 treatment as a double isolation membrane surrounding electron-dense cytoplasmatic material (arrows; direct magnification 2900× [untreated] and 9300 × [treated]). (B) Immunoblot analysis of LC3 and Beclin-1 protein levels in U266 and MM-1S cells treated with FK866 for indicated times or starved. Relative intensity of LC3 was calculated by normalizing the LC3-II intensity to β-actin using ImageJ Version 1.46 analysis software. (C) RPMI-8226/S and U266 cell lines were transfected with GFP-LC3 plasmid before treatment with FK866 or rapamycin (positive control). After 48 hours (FK866) or 24 hours (rapamycin), the autophagy flux was analyzed by immunofluorescence. Nuclei were counterstained with Hoechst. Fluorescence was recorded using a Nikon E800 epifluorescence microscope equipped with a Coolsnap CF color camera (Nikon; 40× magnification). Representative images from 2 independent experiments are shown for each point. (D) Quantification of percentage of GFP-LC3 puncta positive cells (mean ± SEM; n ≥ 50 cells per condition). (E) RPMI-8226/S-GFP-LC3⁺ and U266-GFP-LC3⁺cells were treated as described, washed briefly in 0.05% saponin in PBS or PBS alone, and then analyzed by flow cytometry for total GFP fluorescence. Data are representative of 2 independent experiments.