Cell Culture and HIV PI Treatment RAW264.7 cells were obtained from ATCC (Manassas, USA), cultured in RPMI 1640 medium (Sigma) supplemented with 10% fetal bovine serum (Gibco BRL), 100 U penicillin per milliliter, 100 mg of streptomycin per milliliter, and incubated in 10% CO2 in air at 37 8C. Cells were plated on six-well plates (100.000 for well). The medium was replaced after 24 h, and treated. Ritonavir and Atazanavir were dissolved in DMSO. In the first set of experiments HIV PI were directly added to culture medium (final concentration, 5 to 40 mM, DMSO 0.1%) alone for 16 hours at the end of incubation the cells were collected to evaluated cells dead and CD36 expression by flow cytometry. In a second set of experiments the cells were preincubated for 24 h with Genfibrozil (250mM) or CDCA (50mM) and then treated with ritonavir 10 mM for 16 h, at the end of incubation the cells were collected to evaluated CD36 expression by flow cytometry and for mRNA extraction. Total RNA (10 mg) was used for first-strand cDNA synthesis. The mRNA levels of, CD36, SREBP1-2, SHP and PPARc were determinate by quantitative real-time polymerase chain reaction (qRT-PCR). Measurement of Cholesterol up-take Mouse RAW264.7 macrophages were plated on six-well plates (100.000 for well). The medium was replaced after 24 h, and cells were preincubated for 24 h with Genfibrozil (250mM) or CDCA (50mM) and then treated with ritonavir 10 mM for 16 h, at the end of incubation 10mg/ml of low density lipoprotein acetylated Dil complex (Dil AcLDL) (Molecular Probe, Oregon, USA) was added for 3 hours at 37u. At the end of incubation, cells were collected and washed with PBS and the LDL up-take were measured by flow cytometer (Beckman Coulter, Fullerton, CA). Real-time PCR analysis Quantification of genes expression in tissues was performed by qRT-PCR as described previously [44]. Quantization of the expression of selected genes was performed by quantitative real- time PCR (qRT-PCR). Total RNA were obtained from livers and cells and isolated with TRIzol reagent (Invitrogen), incubated with DNase I and reverse-transcribed with Superscript II (Invitrogen) according to manufacturer specifications. For real-time PCR, 100 ng of template was used in a 25-ml reaction containing a 0.3 mM concentration of each primer and 12.5 ml of 26 SYBR Green PCR Master Mix (Bio-Rad Laboratories, Hercules, CA). All reactions were performed in triplicate using the following cycling conditions: 2 min at 95uC, followed by 50 cycles of 95uC for 10 s and 60uC for 30 s using an iCycler iQ instrument (Bio- Rad Laboratories). The mean value of the replicates for each sample was calculated and expressed as cycle threshold (CT). The amount of gene expression was then calculated as the difference (DCT) between the CT value of the sample for the target gene and the mean CT value of that sample for the endogenous control (GAPDH). Relative expression was calculated as the difference (DDCT) between the DCT values of the test and control samples for each target gene. The relative level of expression was measured as 22DDCT. All PCR primers were designed using the software PRIMER3-OUTPUT using published sequence data obtained from the NCBI database. Mouse primers were as follows: mSREBP1c: gatcaaagaggagccagtgc and tagatggtggctgctgagtg; mSREBP2: acagatgccaagatgcacaa and ttcagcaccatgttctcctg; mFAS: tgggttctagccagcagagt and accaccagagaccgttatgc; mHMGCoAS: ggtggatgggaagctgtcta and acatcatcgagggtgaaagg; mHMGCoAR: ccgaattgtatgtggcactg and ggtgcacgttccttgaagat; PPARc: gccagtttcgatccgtagaa and aatccttggccctctgagat; mFXR: tgtgagggctgcaaaggttt and acatccccatctctctgcac; FXR and Anti-HIV Therapy mSHP: tctcttcttccgccctatca and aagggcttgctggacagtta; mCD36: cggagacatgcttattgagaa and actctgtatgtgtaaggacct mABCA1: agccagaagggagtgtcaga and catgccatctcggtaaacct; mGAPDH: ctgagtatgtcgtggagtctac and gttggtggtgcaggatgcattg; m18S: accgcagctaggaataatgga and gcctcagttccgaaaacc. Nuclear and cytoplasmic extracts Nuclear and cytoplasmic extracts from Raw264.7 cells left untreated or administered with 10 mM Ritonavir alone or in combination with 50 mM CDCA or with 250 mM Gemfibrozil were made with the NE-PER kit (Pierce) according to the manufacturer’s protocol. Western Blotting. Total lysates from RAW264.7 cells were prepared by solubilization in NuPAGE sample buffer containing Sample reducing agent. Proteins were separated by polyacrylamide gel electrophoresis, transferred to nitrocellulose membranes (Bio-Rad, Hercules, CA) and than probed with primary anti-SREBP-1 antibody (Santa Cruz Biotech, Santa Cruz, CA). The anti-immunoglobulin G Rabbit (Bio-Rad) was used as a secondary antibody, and specific protein bands were visualized by chemoluminescence using Super Signal West FEMTO reagent (Pierce, Rockford, IL). Electrophoretic mobility shift assay (EMSA) Preparation of nuclear extract from RAW264.7 cells was done using NE-PER (Pierce). The probes used for EMSA (CD36-SRE wild type and mutated) were labelled with biotin using Biotin 39 end DNA labelling kit (Pierce) according to the manufacturer’s instructions. For EMSA, 5 mg of nuclear extract from RAW264.7 cells naive or treated with ritonavir alone or in combination with Gemfibrozil or CDCA were incubated with 15 fmol of the CD36-SRE probe, while 5 mg of nuclear extract from stimulated cells was incubated with CD36-SRE- mutated probe in a total volume of 20 mL of binding buffer (50 mmol/L NaCl, 10 mmol/L Tris-HCl, pH 7.9, 0.5 mmol/L EDTA, 10% glycerol, 1 mg of poly dI-dC) for 20 min at room temperature. For competition assays, an excess of CD36-SRE unlabeled oligonucleotides were pre-incubated with nuclear extract from treated cells for 15 min prior to the addition of the biotin-labelled CD36-SRE probe. For antibody-mediated super- shift assay, extracts from stimulated cells were pre-incubated with 1 mg anti-SREBP-1 antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) at room temperature for 20 min before the addition of the biotin-labelled CD36-SRE probe. The reactions were loaded on a 6% polyacrylamide non-denaturing gel in 0.56 Tris-borate-EDTA buffer and electrophoresed for 1 h at 100 V. The protein/DNA complexes were then transferred to positively charged nylon membrane (Pierce) and the supershift was detected using the Chemiluminescent Nucleic Acid Detection Module (Pierce). Additional methods are described in ‘‘Materials and Methods S1’’. Supporting Information Materials and Methods S1 Found at: doi:10.1371/journal.pone.0013238.s001 (0.03 DOC) MB Figure S1 In the absence of ritonavir, administering ApoE2/2 mice with an FXR and PPARa agonist reverts dyslipidemia. Gemfibrozil and CDCA reduced plasma levels of total cholesterol, LDL, and triacylglycerols in ApoE2/2 mice. In each panel, data are mean6 SE of 12 animal per group. *P,0.05 ApoE2/2 versus naive; # P,0.05 treatments versus ApoE2/2. Found at: doi:10.1371/journal.pone.0013238.s002 (0.31 MB TIF) PLoS ONE | www.plosone.org 12 October 2010 | Volume 5 | Issue 10 | e13238