healthmedicinet

Animals

Five preweaning purebred male LW (body weight, 8.10?±?0.34 kg) and 6 EHL (body weight,
4.08?±?0.25 kg) piglets at the age of 25 days (d) were respectively obtained from
two neighboring pig breeding farms in Changzhou, Jiangsu Province, China. As large
animals, 5 to 6 pigs are enough for the lab experiments. The piglets were exsanguinated
after electric stunning by a licensed slaughterhouse staff on the respective farm.
Liver samples from the same regions were frozen in liquid nitrogen immediately after
collection and then stored at ?70 °C for further analysis. The Animal Ethics Committee
at Nanjing Agricultural University reviewed the protocol and approved this study specifically,
with the project number 2009ZX08009-138B. The slaughter and sampling procedures complied
with the “Guidelines on Ethical Treatment of Experimental Animals” (2006) No. 398
set by the Ministry of Science and Technology, China and “the Regulation regarding
the Management and Treatment of Experimental Animals” (2008) No.45 set by the Jiangsu
Provincial People’s Government.

RNA isolation, cDNA synthesis and real-time PCR

Total RNA was isolated from liver samples using Trizol Reagent (Invitrogen, USA) and
then purified with DNase I (RNase Free, D2215, Takara, Japan) according to the manufacturer’s
instructions. Concentration of the extracted RNA was measured using NanoDrop 1000
Spectrophotometer. Denaturing agarose electrophoresis was used to confirm RNA integrity.
Real-time PCR was carried out to examine DNA contamination. M-MLV reverse transcriptase
(Promega, Madison, WI, USA) were used to synthesize cDNA with 2 ?g of total RNA following
the manufacturer’s instructions. Two microlitres of diluted cDNA (1:50) were used
for real-time PCR using SYBR Green Real-time PCR Master Mix (TaKaRa, Japan) in Mx3000P
(Stratagene, USA). All primers (Table 1) were synthesized by Generay Biotech Co., Ltd. (Shanghai, China). Several reference
genes (18S, GAPDH, ACTB and PPIA) were tested for mRNA quantification. The expression stability was evaluated by geNorm
31] and peptidylprolyl isomerase A (PPIA) was shown to be the most stable and did not show difference in expression between
indigenous Chinese pig breeds and western breeds 32]. Therefore PPIA was chosen as the reference gene. The method of 2^-??Ct was used to analyze the real-time PCR data 33], and the mRNA levels were expressed as the fold change relative to the mean value
of LW pigs.

Table 1. Primers used in the present study

Protein extraction and Western blot analysis

Nuclearprotein extracts from 100 mg frozen liver tissue were prepared as previously
described with some modifications 34]. Briefly, approximately 100 mg frozen liver samples were homogenized in 1 mL of low-salt
buffer (20 mM HEPES pH 8.0, 10 mM KCl, 1 mM EDTA, 1 mM EGTA, 0.2 % NP-40, 10 % glycerol)
containing protease inhibitors cocktail (05892791001, Roche, Germany) and phosphatase
inhibitors cocktail (4906837001, Roche, Germany), and incubated for 10 min on ice.
After centrifugation for 1 min at 13,000 g at 4 °C, the remaining pellet was dissolved
in 100 ?L high-salt buffer (420 mM NaCl, 20 mM HEPES pH 8.0, 10 mM KCl, 1 mM EDTA,
1 mM EGTA, 20 % glycerol, protease inhibitor cocktail, phosphatase inhibitors cocktail),
kept on ice for 30 min, and then centrifuged for 10 min at 13,000 g at 4 °C. Supernatants
were taken as nuclear fractions. Protein concentration was measured with the BCA Protein
Assay Kit (Pierce, Rockford, IL, USA) according to the manufacturer’s instructions.
After denaturation, 40 ?g of protein, which was calibrated as the optimum loading
quantity, was electrophoresed in a 7.5 % or 10 % SDS-PAGE gel together with a protein
size ladder (SM0671, Fermentas), and the separated proteins were transferred onto
the nitrocellulose membranes (BioTrace, Pall Co., USA). Western blot analysis for
GR (sc-1004×, Santa Cruz, USA, diluted 1:2000), phospho-Ser²¹¹GR (ab55189, Abcam, USA, diluted 1:1000), AR (sc-815×, Santa Cruz, USA, diluted 1:2000)
and C/EBP? (sc-150×, Santa Cruz, USA, diluted 1:2000) was carried out according to
the recommended protocols provided by the manufacturers. Histone H1 (BS1655, bioworld,
USA, diluted 1:500) was used as a reference. Finally, the blots were detected by enhanced
chemiluminescence (ECL) using the LumiGlo substrate (Super Signal West Pico Trial
Kit, Pierce, USA). The ECL signals were recorded by the VERSADOC MP 4000 system (Bio-Rad,
USA) and analyzed with Quantity One software (Bio-Rad, USA).

The GR, phospho-Ser²¹¹GR, AR, C/EBP? and Histone H1 antibodies worked well in the detection of porcine proteins
in high specificity, and the main band appeared at the expected molecular weight.
So we cut the gel for these antibodies to include all 11 samples in one blot. The
Western blot results were repeated twice.

Transcription factors’ prediction and Chromatin immunoprecipitation (ChIP)

The glucocorticoid responsive element (GRE), androgen responsive element (ARE) and
C/EBP? binding sites were predicted on pig 3?-HSD gene promoter, by using TF Search
(http://www.cbrc.jp/research/db/TFSEARCH.html) and gene-regulation (http://www.gene-regulation.com/pub/programs.html) online resources.

ChIP analysis was performed according to our previous publication 21], 35]. In brief, 100 mg frozen liver samples were ground in liquid nitrogen and resuspended
in PBS. After cross-linking in 1 % formaldehyde, the reaction was stopped using 2.5 M
glycine. The pellets were washed using PBS and lysed with SDS lysis buffer containing
protease inhibitors cocktail. The chromatin was sonicated on ice to yield DNA fragments
of 200 to 500 bp in length. Then the protein-DNA complex was diluted in ChIP dilution
buffer containing protease inhibitors. After pre-clearance of the chromatin preparations
with salmon sperm DNA/ protein A/G plus beads (40 ?L, 50 % slurry, sc-2003, Santa
Cruz, CA, USA), the immunoprecipitation was performed with 4 ?g GR (sc-1004×, Santa
Cruz, USA), AR (sc-815×, Santa Cruz, USA), C/EBP? (sc-150×, Santa Cruz, USA) and normal
rabbit IgG (sc-2027, Santa Cruz, USA) (negative control) overnight at 4 °C, respectively.
Protein A/G plus beads (40 ?L, 50 % slurry) were added to capture the immunoprecipitated
chromatin complexes for 2 h at 4 °C. Finally, reverse cross-linking was performed
to release DNA fragments from the immunoprecipitated complex at 65 °C for overnight
and DNA was purified. Immunoprecipitated DNA was used as template for real-time PCR
and specific primers were designed to amplify genomic sequences at the promoter region
of 3?-HSD gene (Table 1). ChIP results were normalized to the rabbit normal IgG precipitation and shown as
fold enrichment.

Co-immunoprecipitation

Co-immunoprecipitation was performed as previously described with minor modifications
36]. Five hundred micrograms of total protein were precleared with 40 ?L of protein A/G
plus beads for 1 h at 4 °C, then centrifuged at 1,000?×?g for 5 min. The supernatants
were incubated with 4 ?g C/EBP? and GR antibodies and rotated overnight at 4 °C. Thereafter,
40 ?L of agarose beeds were incubated with the protein-antibody complexes for 2 h
at 4 °C. After centrifugation, the agarose beeds were washed and the immunoprecipitated
proteins were run on 7.5 % SDS-polyacrylamide gel for western blot analysis.

Statistical analysis

All data were presented as mean?±?SEM, and were analyzed using independent-samples
t-test with SPSS 21.0 for Windows. The mRNA or protein expression was expressed as the
fold change relative to LW. Difference was considered significant when P??0.05.