Lipidtox

Tissues were fixed and stained for EdU with the Click-iT EdU Alexa Fluor 555 Imaging Kit (Molecular Probes) and for neutral lipids using oil red O or LipidTOX (Molecular Probes). Live ex vivo imaging was used to detect oxidized DHE (Invitrogen) and peroxidated lipid (C11-BODIPY 581/591, Invitrogen). See Supplemental Experimental Procedures for details.

Cells were washed twice with PBS and fixed with 3.7% formaldehyde (Sigma) in PBS for 10 min, followed by a brief wash with PBS. LipidTOX (Molecular Probes H34476) at 1:500 dilution in PBS was incubated on cells for 30 min along with 5 μg/ml Hoechst 33342 (Molecular Probes), followed by a brief wash in PBS.
Oil Red O working solution was prepared by dissolving 0.35% w/v Oil Red O (Sigma) overnight in isopropanol followed by passage through a 0.45 μm filter. Two volumes of this solution were mixed with one volume of water by gentle rocking overnight at 4°C. The solution was passed through a 0.2 μm filter. Cells were fixed with 4% formaldehyde in PBS for 60 min, washed once with PBS, once with water and once with 66% isopropanol. The working Oil Red O solution was added to the fixed cells for 1 h, washed three times with water and imaged on a standard tissue culture microscope fitted with a Nikon CoolPix 5000 digital camera. The plates were air-dried and the dye was extracted with isopropanol. Absorbance of the extracts was measured at 500 nm wavelength in a CECIL CE2041 Spectrophotometer. Statistical analysis was addressed by performing ANOVA followed by post-hoc Tukey HSD correction using the free statistical software R [36 ].

Cells were obtained from ATCC and maintained according to ATCC guidelines. To simulate androgen deprivation, cells were kept in phenol-red free media supplemented with charcoal-stripped serum. Knockdown of ECI2 was performed with RNAiMAX reagent (Thermo Fisher Scientific) according to manufacturer's instructions. ECI2 targeting siRNAs were obtained from Qiagen (SI04202282 and SI04201848). Perhexiline was purchased from Sigma (catalogue number: SML0120-10MG). More detailed protocols for cell lysate preparation, Oil Red O staining (obtained from Sigma, catalogue number: O1391-250ML) and Lipid Tox staining (obtained from Life Technologies, catalogue number: H34475) are available in Supplementary Materials.

Mice were sacrificed and autopsied, and dissected tissue samples were fixed for 24 h in 4% paraformaldehyde, dehydrated, and embedded in paraffin. Paraffin blocks were sectioned at 4 mm and stained with H&E. Images were scanned by Pannoramic SCAN II (3DHISTECH, Hungary). BM adipocytes were quantified by intracellular staining of the FBM lipid with LipidTox (a fluorescent dye that stains neutral lipids; Life Technologies) and analyzed using ImageStream X Mark II, Luminex.