Animals
Three 4-month-old large white pig were provided by Zhejiang Huateng Agricultural Technology Co., Ltd. Pigs were dissected for sampling after euthanatizing in CO2 euthanasia box. The longissimus dorsi muscle, subcutaneous adipose tissues, cardiac apex, the right lobe of liver, spleen, right kidney, the upper lobe of the right lung and intestine tenue (jejunum) were collected in liquid nitrogen and stored at − 80 °C until RNA extraction. Jiaxing University Animal Care Committee approved and verified all the experimental procedures and followed ARRIVE guidelines to perform the experiments [23].
Digital differential display (DDD) analysis
Digital Differential Display (DDD) is an algorithmic system for the identification of differentially expressed genes based on the relative abundance of expressed sequence tags (ESTs) from two or more contrasting cDNA libraries, which are deposited in the NCBI UniGene database (http://www.ncbi.nlm.nih.gov/UniGene/). DDD compares the number of assignments of ESTs from several different libraries, or pools of libraries, to a specific UniGene cluster. To account for the unequal number of ESTs in each library, DDD utilizes Fisher’s exact test to restrict the output to statistically significant differences (P ≤ 0.05) [9]. Gene expression levels of adipose tissue derived cDNA libraries (pool A) against the other organ-specific cDNA libraries (pool B including intestine, kidney, longissimus, lung, muscle, ovary, spleen, testis, thymus, uterus) were compared. Genes with statistically significant differential expression between adipose and non-adipose tissues were recorded. For these recorded genes, fold changes of expression levels were calculated by dividing the frequency of that gene in pool A (adipose tissue) to the frequency in pool B (non-adipose tissue). More than 10-fold changed genes were subjected to Gene Ontology (GO) and Kyoto Encyclopedia of Gene and Genome (KEGG) [24] pathway analysis using the online databases DAVID 6.8.
Q-PCR
Total RNA was isolated from pig tissues using Trizol reagent (Invitrogen, USA). The HiFiScript gDNA Removal cDNA Synthesis Kit (CWBiotech, Beijing, China) was sued for cDNA synthesis. Q- PCR was conducted using the 2 × plus SYBR real-time PCR mixture (BioTek, Beijing, China) on a QuantStudio 3 system (Thermo fisher, USA). The primer sequences are listed in Table S1. Relative gene expression levels were calculated using the 2-ΔΔCt method with GAPDH as the internal control.
Promoter analysis
The BDGP [25] online tool was used to predict core promoter sequence, and the type of organism selects eukaryote and the threshold score was set by default to 0.8. The JASPAR [26] website was used to identify the transcription factor binding sites, and the taxonomic group was set to vertebrata.
Vector construction
The pDsRed-Express-N1 vector was amplified by PCR using primers 5′- ATAGAGCTCCGCGG.
AACTCCATATATGGG-3′ and 5′-CTCGAGCTCAAGCTTCGAATTCTGC-3′, then digested by SacI and ligated to construct a new tool vector without CMV promoter. The LGALS12 promoter (− 4061 to + 121) was cloned into the new vector digested by SacI and SalI, and the primers were 5′-ATAGAGCTCAGCATACATGGAATACATGCTG-3′ and 5′-ATAGTCGACGCCCAACTGAGCC.
CTGAGAC-3′, namely pDsRed-L-4 kb vector. Next, the pDsRed-L-4 kb vector and the APOR coding sequence was amplified using primers 5′-CAGTGGTACCCAAAGAAGAAAAGTGTCA.
G-3′ and 5′-CTAACCGGTTACAGCTCCAGGGCCAATTTTATCTCTCC-3′, were digested by KpnI and AgeI, then ligated to construct pDsRed-L-4 kb-APOR vector.
Cell culture and transfection
Preadipocyte cell line 3 T3-L1 was purchased from ATCC, and cultured in DMEM containing 10% fetal bovine serum (FBS, Gibco, LOT2206993CP) to confluence (day 0), and then shifted to adipocyte differentiation medium, which was DMEM with 10% FBS, 0.5 mM dexamethasone, 20 nM insulin, and 0.5 mM isobutyl methylxanthine (IBMX) for 2 days (Day 3). From day 4 to day 8, cells were maintained in DMEM with 10% FBS and 20 nM insulin, and the medium was replaced every other day. Lipofectamine™ 2000 (11668027, Invitrogen, USA) was used for plasmid transfection at the indicated time points. Then, the medium was replaced with fresh medium in 24 hours.
Luciferase reporter assays
HEK293T were seeded in 24-well plates and co-transfected with LGALS12 promoter and transcription factor (PPARα、C/EBPα、CEBPβ、KLF4 and KLF15) vector by VigoFect (Vigorous Biotech, Beijing). The pGL3-Basic and pCR3.1 vector were used to insert promoter and transcription factor sequence, respectively. The pTK-Renila luciferase reporter (Promega) was included in all transfections for normalization. Luciferase activities were measured after transfection for 24 h using the dual-luciferase reporter assay system (Promega), and each combined set of vectors had three independent replicates. The measure was performed as described [27] with some modification. Tecan’s Spark multimode microplate reader was used to measure fluorescence intensity, and the comparison was conducted based on the ratio of firefly luciferase activity to Renilla luciferase activity.
Oil red O staining
The matured 3 T3-L1 cells were washed with PBS and fixed with 4% paraformaldehyde for 30 min at room temperature, then washed three times with PBS. The fixed cells were then covered with a mixture of Oil Red O solution (0.6% Oil Red O dye in isopropanol) and water at a 6:4 ratio for 30 min, followed by washing four times with PBS, and images were captured under an optical microscope (Leica Microsystems, Germany).
Statistical analysis
The data were obtained from at least three independent experiments, and presented as the means ± standard error (SE). GraphPad Prism 8.0 (GraphPad, San Diego, CA, USA) were used to conduct the statistical analysis, and the assessment of normality are Kolmogorov-Smirnov (K-S) test. Individual comparisons were assessed using Student’s t-test. P-values of less than 0.05 were considered to indicate significant differences, are displayed as * p < 0.05, ** p < 0.01, and *** p < 0.001.
