Pparα

Liver tissues were fixed in 10% formaldehyde solution at 4 °C for paraffin sections or placed in 30% sucrose solution and embedded with liquid nitrogen-cooled isopentane for frozen sections. Paraffin sections of liver tissue were stained with hematoxylin and eosin (H&E) for light microscopy. For Oil Red O staining, frozen sections were incubated with the dye for 15 min and washed with 1× PBS. For immunofluorescence analysis, primary hepatocytes were stained overnight at 4 °C with antibodies against NCoR1 (Cell Signaling Technology) or PPARα (Santa Cruz Biotechnology). After washing with PBS, the sections were incubated with secondary antibodies (Alexa Fluor 488-conjugated goat anti-mouse IgG1 and Alexa Fluor 594-conjugated goat anti-rabbit IgM; Thermo Fisher Scientific) for 1 h at 37 °C and then counterstained with DAPI. NCoR1 and PPARα interactions were detected using a Duolink Proximity Ligation Assay kit (#DUO96010, Sigma-Aldrich).

Liver tissues were fixed in 10% formaldehyde solution at 4 °C for paraffin sections or placed in 30% sucrose solution and embedded with liquid nitrogen-cooled isopentane for frozen sections. Paraffin sections of liver tissue were stained with hematoxylin and eosin (H&E) for light microscopy. For Oil Red O staining, frozen sections were incubated with the dye for 15 min and washed with 1× PBS. For immunofluorescence analysis, primary hepatocytes were stained overnight at 4 °C with antibodies against NCoR1 (Cell Signaling Technology) or PPARα (Santa Cruz Biotechnology). After washing with PBS, the sections were incubated with secondary antibodies (Alexa Fluor 488-conjugated goat anti-mouse IgG1 and Alexa Fluor 594-conjugated goat anti-rabbit IgM; Thermo Fisher Scientific) for 1 h at 37 °C and then counterstained with DAPI. NCoR1 and PPARα interactions were detected using a Duolink Proximity Ligation Assay kit (#DUO96010, Sigma-Aldrich).

Mice livers or primary murine hepatocytes were subject to crosslinking in 1% of formaldehyde for 5 min followed by quenching with glycine for 5 min on ice. Cells were pelleted by centrifugation and resuspended in a lysis buffer (50 mM HEPES pH 8.0, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP40, 0.25% Triton X100). The pellets were then resuspended in a washing buffer (10 mM Tris pH 8.0, 1 mM EDTA, 0.5 mM EGTA, 200 mM NaCl), washed and resuspended in a shearing buffer (0.1% SDS, 1 mM EDTA, pH 8, 10 mM Tris HCl, pH 8) before sonication following the instruction of the manufacturer. Chromatin fragments were precipitated using specific antibodies and protein G beads, washed, and treated with proteinase K and RNase A. Purified chromatin immunoprecipitation (ChIP) DNA was then used for chromatin immunoprecipitation quantitative real-time PCR (ChIP-qPCR) analysis. The antibodies used for the ChIP-qPCR assay include PPARα (Millipore, MA, USA; MAB3890); RNAPII (Santa Cruz Biotechnology, TX, USA; sc-899); RNAPII-S2P (Active Motif, CA, USA; #61083); H3K27ac (Abcam, Cambridge, UK; ab4729); H3K4me1(Abcam, Cambridge, UK; ab8895); IgG (Santa Cruz Biotechnology, TX, USA; sc-2027), and REV-ERBα (Proteintech, IL, USA; 14506-1-AP). ChIPs were performed with each experimental point in triplicate, and each experiment was repeated three times. The primers are shown in Supplementary Table 1.

Total RNA was isolated from mice livers or hepatocytes. The complementary DNA (cDNA) was prepared, amplified, and measured in the presence of SYBR Green as previously described (30 (link)). The fluorescent values were collected and a melting curve analysis was performed. Gapdh was used as the internal reference to normalize the relative level of each transcript. The primers used are shown in Supplementary Table 1. Hepatocytes were analyzed by immunoblotting with PPARα (Millipore, MA, USA; MAB3890), REV-ERBα (cell signaling, #13418), and Bmal1 (cell signaling, #14020).

Nuclear and cytoplasmic extracts from cultured hepatocytes and mouse livers were prepared using the NE-PER nuclear and cytoplasmic extraction reagent kit (Pierce Biotechnology, Rockford, IL) according to the manufacturer's instructions. Protein content was determined using a BCA Protein Assay Kit (Shenergy Biocolor Bioscience & Technology Company, Shanghai, China). Protein from nuclear extracts (40–60 µg) or cytoplasmic extracts (60–80 µg) was electrotransferred onto a polyvinylidene fluoride membrane (Millipore, Billerica, MA, USA), and after incubation in 5% BSA for one hour, the blots were probed with the following antibodies at the dilution indicated: SREBP-1 (1∶200; Santa Cruz) and PPARα (1∶1000; Millipore) at 4°C for the entire night. Mouse anti-LMB1 antibody and anti-GAPDH antibody were obtained from Cwbiotech (Beijing, China) and were used to target endogenous control proteins in the nuclear and cytosolic fractions, respectively. After incubation with the appropriate secondary antibodies conjugated to horseradish peroxidase (HRP) (Amersham, Little Chalfont Bucks, UK) at 1∶5,000 for one hour at room temperature, the membranes were visualized using a HyGLO HRP detection kit (Denville, NJ, USA). Quantification of Western blots was performed using ImageJ software (developed at the National Institutes of Health, Bethesda, Maryland).