Fig 2: 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 3: 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 4: LXR isoform selectivity from CRT.Concentration-response for recruitment of SRC-1 to LXRα (dotted lines) and LXRβ (solid line), determined by CRT assay, showing mean and SD from triplicate experiments, normalized to T0 (100%) as a full LXR agonist (shown in turquoise for reference). GSK2033 (R) and SR9238 (S) titrations were run in the presence of 24HC (3 μM) and neither ligand stabilized the LXR:SRC1 complex alone (T,U).

Fig 5: ABCA1-luc reporter in CCF cells.Concentration-response for LXR ligands in astrocytoma cells measured after 24 hr incubation, normalized to T0 response (100%) showing mean and SD from triplicate experiments (see Fig. S2 for biological replicates).