Fig. 1

Nur77 and NOR1 are required for the induction of cell death by DDA in melanoma. a Chemical structure of DDA and C17. b B16F10 and SKMEL-28 cells were treated or not for 24, 48, or 72 h with DDA or C17. DDA- or C17-induced cell death was determined by a trypan blue assay and expressed as the percentage cell death relative to control (vehicle). c Cells were treated with 5 µM DDA for 48 h or 72 h with or without 500 µM vitamin E (Vit E), 50 µM z-VAD-fmk, 50 µM z-DEVD-fmk, 1 µg/ml actinomycin D (Act D) or 2.5 µg/ml cycloheximide (CHX), and cell death was measured as in b. d DDA induced the accumulation of free sterols in cells. Cells were treated with solvent vehicle or 2.5 µM DDA for 48 h, then fixed and stained with filipin and analyzed by fluorescence microscopy. e Representative EM images of B16F10 (panels 1–3) and SKMEL-28 (panels 2–4) cells treated for 24 h with 2.5 µM DDA. Ly: multilamellar body-derived lysosomes, AP: autophagosomes, AL: autolysosomes, N: nucleus, C: cytoplasm. Bars: 250 nm for panel 1 and 100 nm for inserts; 500 nm for panel 2 and 100 nm for inserts; 500 nm for panel 3; and 250 nm for panel 4. f Heat map depicting transcription of genes encoding nuclear receptors (NR) and their co-regulators, using PCR arrays in B16F10 and SKMEL-28 cells treated or not with 2.5 µM DDA for 5 h, using PCR arrays. g Immunoblot for Nur77 and NOR1 protein expression in melanoma cells treated or not with DDA for 24 h. h Analysis of DDA cytotoxicity in B16F10 and SKMEL-28 cells transfected with control scramble siRNA (siSC), siNur77 and/or siNOR1. 72 h after transfection, cells were treated or not for 24 h with 2.5 µM DDA. Cell death is expressed as a percentage relative to the level of cell death induced by DDA in cells transfected with a scramble control siRNA (siSC). Data from b–d and h are the means ± S.E.M. of three independent experiments performed in triplicate, **P < 0.01, ***P < 0.001, t-test. All images and densitometry values are representative of three independent experiments