Fig. 2

DDA induces Nur77- and NOR1-dependent lethal autophagy in melanoma cells. a DDA triggers the accumulation of autophagic vesicles. Cells were treated for 24 h with or without 2.5 µM DDA then stained with monodansylcadaverine (MDC) and observed by fluorescence microscopy. MDC-specific activity was measured by fluorescence photometry. b Cells were treated for 24 h with solvent vehicle or increasing concentrations of DDA then analyzed for autophagic protein expression by immunoblotting. Blots are representative of three independent experiments. c Long-life protein degradation was determined in cells treated with solvent vehicle (control) or 1 µM DDA for 18 h in the presence or absence of the autolysosomal inhibitors bafilomycin A1 (Baf A1) and hydroxychloroquine (HCQ). Autophagic activity was measured as the level of degradation of long-lived proteins. Starvation for 18 h in Hank’s balanced salt solution (HBSS) was used as a positive control. d Immunoblots of LC3 proteins from cells treated for 24 h with or without 2.5 µM DDA and with or without E64 + pepstatin A (10 µg/ml). Images are representative of three independent experiments. e DDA induced the formation of punctate LC3 cells. Cells were transfected with a plasmid-expressing GFP-LC3 and then treated for 24 h with the solvent vehicle or 2.5 µM DDA, with or without E64 + pepstatin A (10 µg/ml) and observed by fluorescent microscopy. The percentage of GFP-LC3-positive cells with GFP-LC3 puncta was calculated. f Analysis of DDA cytotoxicity in cells transfected with scramble siRNA (siSC), siATG7, siVPS34, or siBECN1. Seventy-two hours after transfection, cells were treated or not for 24 h with 2.5 µM DDA. g Analysis of DDA cytotoxicity in SKMEL-28 cells permanently transfected with control shRNA (shCTRL) or shRNA against VPS34 (shVPS34). Cells were treated for 72 h with solvent vehicle (CTRL) or 2.5 µM DDA. h Cells were treated with 2.5 µM DDA for 24, 48 and 72 h in the presence or absence of the autolysosome inhibitors Baf A1 or HCQ. Cell death is expressed as in Fig. 1a. Data from a, c, e, f, g are the means ± S.E.M. of three experiments performed in triplicate, *P < 0.05, **P < 0.01, ***P < 0.001, t-test