Fig. 6

DDA induces lethal autophagy in AML cells via LXRβ. a DDA-induced cell death in KG1 and HL60 cells was determined over time as in Fig. 1b. b Quantification of the Δ8-sterols that had accumulated in KG1 and HL60 cells treated without or with 2.5 µM DDA. c May–Grünwald–Giemsa staining of KG1 and HL60 cells treated or not with DDA. d Immunoblots of LC3 proteins in cells treated or not with 2.5 µM DDA, and E64 + pepstatin A (Pep). e Effect of the pharmacological inhibitor of autophagy Baf A1 on DDA cytotoxicity at 48 and 72 h. f DDA cytotoxicity in KG1 or HL60 cells permanently transfected with control shRNA (shC) or shRNA against VPS34 (shVPS34) after 72 h treatment. g Immunoblots of ATG3 and LC3 proteins in KG1 cells transfected with control scramble siRNA (siSC) or siRNA against ATG3 (siATG3). Seventy-two hours after transfection, cells were treated for 24 h with 5 µM DDA. h Analysis of DDA cytotoxicity in KG1 cells transfected with control scramble siRNA (siSC), siATG3, siATG7, or siBECN1. Seventy-two hours after transfection, cells were treated for 24 h with 5 µM DDA. i Analysis of DDA cytotoxicity in KG1 cells transfected with shCTRL, sh3LXRβ, or sh4LXRβ. Cells were treated for 24 h with 5 µM DDA or vehicle. j Immunoblots of LC3 protein expression in cells transfected with shCTRL, sh3LXRβ, or sh4LXRβ and treated with 5 µM DDA or vehicle for 24 h. Analysis of the cytotoxicity k and acridine orange-positive vesicles l in KG1 cells treated or not with 5 µM DDA, 2 µM TO, 2 µM GW, or 10 µM 22(R)HC. The presence of Nur77 and NOR1 is required in DDA cytotoxicity (m) and autophagy (n). Data from a, b, e, f, h, i, k, l, m, n are the means ± S.E.M. of three experiments performed in triplicate, *P < 0.05, **P < 0.01, ***P < 0.001 t-test. All images and densitometry values are representative of three independent experiments