Fig. 3

Effect of NBDs on the autophagic flux of U-2OS cells. a LC3 turnover assays in basal conditions. Lysates from cells treated with vehicle (CTRL), 5 μM MC3181 or 5 μM NBDHEX for 24 h, incubated or not with 100 nmol/l BAF for 3 h before lysis, were subjected to western blot for LC3-II and β-actin. b LC3-II/β-actin ratios determined from the densitometric analysis of the autoradiograms shown in a expressed in arbitrary units (a.u.). c Autophagic flux indexes calculated from the data illustrated in b as the difference in LC3-II/actin ratios between samples plus and minus BAF, expressed in arbitrary units (a.u.). d Dose–response effect of NBDHEX on basal autophagic flux. Lysates from cells treated with vehicle (CTRL) or 0.5–5 μM NBDHEX for 24 h, incubated or not with 100 nmol/l BAF for 3 h before lysis, were subjected to western blot for LC3-II and β-actin. e LC3-II/β-actin ratios determined from the densitometric analysis of the autoradiograms shown in d. f Autophagic flux indexes calculated from the data illustrated in e as the difference in LC3-II/actin ratios between samples plus and minus BAF. g LC3 turnover assays in nutritional stress conditions. Lysates from cells treated with vehicle (CTRL) or 5 μM NBDs in the presence of 0.2 % FBS for 24 h, incubated or not with 100 nmol/l BAF for 3 h before lysis, were subjected to western blot for LC3-II and β-actin. Lysates from cells grown for 24 h in the presence of 10 % FBS, incubated or not with BAF as above, were included to assess basal flux levels. h LC3-II/β-actin ratios determined from the densitometric analysis of the autoradiograms shown in g. i Autophagic flux indexes calculated from the data illustrated in h as the difference in LC3-II/actin ratios between samples plus and minus BAF