dexamethasone and 5 mg/ml insulin for two days, and then, with standard medium supplemented with 5 mg/ml insulin for 6 days. This medium was renewed every two days. After 8 days, the appearance of cytoplasmic 14709329 lipid accumulation was observed by OilRed-O staining. Briefly, cells were washed with phosphate-buffered saline, and then fixed with 3.7% formaldehyde for 2 minutes. After a wash with water, cells were stained with 60% filtered OilRed-O stock solution in 100 ml of isopropanol) for 1 hour at room temperature. Finally, cells were washed twice in water and photographed. Lipid accumulation was defined as percentage of cells that are Oil-Red-O positive. Biotechnology), C/EBPb, C/ EBPd, C/EBPa, FABP4 , adiponectin, eIF2a, eIF4E and actin. Reactive bands were detected with an ECL plus system. Luciferase assays The reporter containing the proximal part of the hPPARc2 promoter cloned in front of the luciferase gene was kindly provided by Dr. Johan Auwerx. The ratC/EBPawtpSG5 and ratC/EBPbwtpSG5 expression vectors were kindly provided by Dr. Achim Leutz. The reporter containing the ratC/EBPa promoter was kindly provided by Dr. Ana Perez-Castillo. The expression vectors pcDNA3-hFUS-DDIT3, BOS-hDDIT3 and pcDNA3NH2-hFUS were generated by cloning the corresponding cDNAs into the expression plasmids. For reporter assays, U2OS cells were transfected using Dual-Luciferase with normalization to Renilla luciferase, and mean6standard error was determined from at least three data points. U2OS cells were maintained in DMEM supplemented with 10% fetal bovine serum. RNA Extraction Total RNA from liposarcoma samples were isolated in two steps 
using TRIzol followed by Rneasy Mini-Kit purification following the manufacturer’s RNA Clean-up protocol with the optional Oncolumn Dnase treatment. Total RNA from liposarcoma cell lines was isolated using the Rneasy Mini-Kit. The MedChemExpress Solithromycin integrity and the quality of RNA were verified by electrophoresis and its concentration measured. Reverse Transcription-PCR To analyze expression of FUS-DDIT3 in human liposarcoma cell lines, CombitTA-FUS-DDIT3 MEFs, and mouse liposarcomas, RT-PCR was performed according to the manufacturer’s protocol in a 20-ml reaction containing 50 ng of random hexamers, 3 mg of total RNA, and 200 units of Superscript II RNase H- reverse transcriptase. The sequences of the specific primers, which amplifiy specifically the fusion region, were as follows: FUS-F1: 59-GGTTATGGCAATCAAGACCAG39 and DDIT3-B1: 59-CTTGCAGGTCCTCATACCAGG-39. The thermocycling parameters for the polymerase chain reaction were as follows: 30 cycles at 94uC for 1 min, 60uC for 1 min and 72uC for 1 min. The PCR products were confirmed by hybridization with specific probes. Amplification of b-actin served as a control to assess the quality of each RNA sample. CAT assays The CAT reporter containing the,2.5 kb proximal promoter region of the murine eIF4E promoter, pm4ECAT, was kindly provided by Dr. Emmett V. Schmidt. C3H10T1/2 cells were maintained in DMEM supplemented with 10% fetal bovine serum. The transfections were carried out using the Profection Mammalia Transfection System Kit. Cells 18347139 were harvested,60 hr later and extracts were assayed for CAT activity. Relative CAT activities were determined by comparing the ratios of acetylated/unacetylated chloramphenicol present in spots cut from the thin-layer chromatographs. Equivalent amounts of protein and a reaction time of 1 hr were used in all CAT assays, which kept all values within th
