Figure 2.

Cell death by NAMPT inhibition or KD is linked to activation of the autophagy machinery. (a) Lysates from FK866-treated or NAMPT KD cells were subjected to western blot analysis using antibodies against Atg12, Atg7, Atg5, Beclin 1, LC3A, LC3B, mTOR and p-mTOR. (b) U2OS cells stably transfected with GFP-LC3 were exposed to FK866 (5 nM) for 48 h and examined by microscopy for the presence of GFP-LC3 puncta. (c and d) Effect of 3-MA (10 mM) and Z-VAD-FMK (10 μM) pre-treatment (2 h) on FK866-induced decrease in viability (based on Trypan blue staining) of U2OS cells. (e and f) U2OS cells (p53 wild-type (wt)) with Atg5 KD or Atg7 KD were exposed to FK866 (5 nM) for 48 h and analysed for (e) viability or (f) LC3A-II levels. Statistical analysis for the cell viability study was performed with one-way analysis of variance (ANOVA) followed by paired ‘t'-test; *^{, #}P-values ⩽0.05 obtained by comparing the respective data with the corresponding controls. Results represent three independent experiments. sh, short hairpin