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

Fig 3: LXR are targets of DDA and LXRβ is required for its cytotoxicity in melanoma cells. a LXR transcriptional activity was analyzed using transient transfection reporter assays. Tranfected cells were treated with 10 µM 22(R)HC with or without DDA. b Competition binding assays on LBD-LXRα or LBD-LXRβ. c SPR sensorgrams showing the binding of DDA to the LBD-LXRβ. d Molecular docking of DDA with the LBD-LXRβ. Amino acid side chains that interact with DDA are represented (in black). The names of the amino acids known to interact with known LXR ligands are colored in blue. Gray: carbon atoms, white: hydrogen atoms, red: oxygen atoms, blue: nitrogen atoms, yellow: sulfur atoms. e Structures of DDA analogs assayed in the LXR reporter assay. f Analysis of LXRβ-dependent agonistic or antagonistic activities by DDA and analogs. g the stimulation of LC3, Nur77, and NOR1 protein expression by DDA is LXRβ-dependent. h ChIP-qPCR of LXRβ on the SCD1, LC3A, LC3B, SREBP1, NR4A1, NR4A3, ABCA1, and LDLR enhancers on SKMEL-28 cells treated or not with 10 µM 22(R)HC or 2.5 µM DDA. i ChIP-qPCR of LXRβ on the TFEB enhancer on SKMEL-28 cells treated or not with 10 µM 22(R)HC or 2.5 µM DDA. j Real-time PCR of TFEB expression in SKMEL-28 cells treated or not with 5 or 10 µM 22(R)HC, and 2 or 5 µM DDA. k Luciferase reporter gene assays with the TFEB promoter-luciferase construct in HEK293T. Cells were treated with increasing DDA concentrations. l Analysis of DDA cytotoxicity in cells transfected with control siRNA (shSC) or siLXRβ. Cells were treated with 2.5 µM DDA. m Analysis of the cytotoxicity of cells treated with or without 2 µM DDA, 5 µM 22(R)HC, 0.5 µM TO, 1 µM GW or 2 µM DDA + 10 µM 22(R)HC, 0.5 µM TO, or 1 µM GW. n TO reversed DDA induction of autophagic vesicles. Cells were treated for 24 h with or without 2 µM DDA, 0.5 µM TO, or 0.5 µM TO + 2 µM DDA. Cells were stained with MDC and observed by fluorescence microscopy. Data from a, b, f, j, k, l, m are the means ± S.E.M. of three independent experiments performed in triplicate (*P < 0.05, **P < 0.01, ***P < 0.001, t-test)

Fig 4: NLAI binding to LXR and coregulator recruitment. Concentration–response for recruitment of SRC-1 to LXRα (dotted lines) and LXRβ (solid line), determined by CRT assay, showing mean and SD, normalized to T0 as a full LXR agonist.

Fig 5: Corepressor binding to LXR isoforms: ligand dependence.Concentration-response from CRT measurements of: NCOR2 binding to LXRα (dashed lines) (A); NCOR2 binding to LXRβ (solid lines) (B); SMRT2 binding to LXRα (dashed lines) (C); SMRT2 binding to LXRβ (solid lines) (D). Data shown mean and SD from at least triplicate measurements. (See Fig. S7 for alternative layout).