Characterization of RUNX1T1, an Adipogenesis Regulator in Ovine Preadipocyte Differentiation

Runt-related transcription factor 1 translocation partner 1 (RUNX1T1), a potential novel regulator of adipogenesis, exists in two splice variants: a long (RUNX1T1-L) and a short (RUNX1T1-S) isoform. However, there is no data showing the existence of RUNX1T1 in ovine subcutaneous fat at different stages of developmental and its role on ovine adipogenesis. Therefore, the objectives of this study were to evaluate the presence of RUNX1T1 in subcutaneous fat of five-day-old to 24-month-old sheep and to investigate the role of RUNX1T1 in ovine adipogenesis. In this study, we detected a 1829 bp cDNA fragment of RUNX1T1 which contains a 1815 bp coding sequence that encodes 602-amino acid and 14 bp of 5′ untranslated region, respectively. The amino acid sequence of RUNX1T1 has 31.18–94.21% homology with other species’ protein sequences. During fat development, the RUNX1T1 protein expression was higher in subcutaneous fat of 24-month-old Hu sheep. In addition, the expression of RUNX1T1-L mRNA decreased first, then subsequently increased during ovine preadipocyte differentiation. Knockdown of RUNX1T1-L in ovine preadipocytes promoted preadipocyte differentiation and lipid accumulation. Taken together, our data suggests that RUNX1T1 is an important functional molecule in adipogenesis. Moreover, it showed for the first time that RUNX1T1-L was negatively correlated with the ovine preadipocyte differentiation.


Introduction
Adipocyte differentiation is a complex process that involves two stages; (i) commitment of mesenchymal precursors to the fate of preadipocyte and (ii) terminal differentiation [1]. Under appropriate stimulation, preadipocytes undergo cellular morphology changes and eventually differentiate into round mature adipocytes [2]. Adipocyte differentiation involves an interaction of peroxisome proliferator-activated receptor gamma (PPARγ) with CCAAT/enhancer-binding protein (C/EBP) transcription factors [3,4]. Recent evidence indicated that Runt-related transcription factor 1 translocation partner 1 (RUNX1T1) acts as a potential novel regulator factor of adipogenesis and is actively involved in the mitotic clonal expansion phases of adipogenesis [5,6].
RUNX1T1 is a member of the runt-related transcription factor (RUNX) family of transcription factor, also known as eight twenty one (ETO) and myeloid translocation gene 8 (MTG8), which is involved in the regulation of proliferation and differentiation of hematopoietic stem cells [7,8]. Several studies have shown that the RUNX1T1 gene is widely expressed in many human tissues, with the highest expression levels in brain and heart [9,10]. Current research on RUNX1T1 mainly focuses on leukemia, cancer and neuronal differentiation [11][12][13], however, there are few reports about adipogenesis. In 3T3-L1 cells, RUNX1T1 exists in two splice variants; a long and a short isoform. In addition, the expression of RUNX1T1 isoforms is regulated by the gene fat mass and obesity-associated (FTO) [14]. Previous studies in mouse primary adipocytes have found that overexpression of the long isoform of RUNX1T1 (RUNX1T1-L) inhibits adipocyte differentiation [5], conversely, increased expression of the short isoform of RUNX1T1 (RUNX1T1-S) stimulates adipogenesis [6]. These reports further demonstrate the potential role of RUNX1T1 in regulating adipogenesis.
Notably, recent studies in 3T3-L1 cells and mice showed that FTO plays a role in early adipogenesis to increase the number of adipocytes by regulating alternative splicing of RUNX1T1 mRNA [6,14]. However, the mechanism by which RUNX1T1 affects adipogenesis has been elusive. In addition, little is known about the function of RUNX1T1 in fat development in large mammalian species. Therefore, we explored whether RUNX1T1 modulates preadipocyte differentiation derived from native cells of sheep. Although several evidences [9,10] have demonstrated the presence of RUNX1T1 in human and mouse tissues, there is no data available for ovine adipose tissue. Therefore, in the present study, we cloned the ovine RUNX1T1 coding sequence and further investigated whether RUNX1T1 exists in the adipose tissue of sheep.

cDNA Cloning and Sequence Analysis
To obtain the RUNX1T1 cDNA fragment (1829 bp) of sheep, we designed a pair of PCR primers (Table S1) and used these to undertake reverse transcription of the mRNA from subcutaneous fat of Hu sheep (Figure 1), then submitted the obtained cDNA fragment to the GenBank (https://www.ncbi. nlm.nih.gov/genbank/;accessionnumberMH063277). The sequence analysis indicated that the coding sequence of RUNX1T1 was 1815 bp and encoded a 602-amino acid protein with a predicted molecular weight of 67 kDa ( Figure 2). Comparison of the predicted protein with the homologous proteins from other species-including bovine, mouse, and human-using DNAMAN (version 6.0, LynnonBiosoft, San Ramon, CA, USA) showed high homology with mouse (94.21%) and human (86.73%) RUNX1T1 and low sequence identity with bovine (31.18%) RUNX1T1 ( Figure 3). the highest expression levels in brain and heart [9,10]. Current research on RUNX1T1 mainly focuses on leukemia, cancer and neuronal differentiation [11][12][13], however, there are few reports about adipogenesis. In 3T3-L1 cells, RUNX1T1 exists in two splice variants; a long and a short isoform. In addition, the expression of RUNX1T1 isoforms is regulated by the gene fat mass and obesityassociated (FTO) [14]. Previous studies in mouse primary adipocytes have found that overexpression of the long isoform of RUNX1T1 (RUNX1T1-L) inhibits adipocyte differentiation [5], conversely, increased expression of the short isoform of RUNX1T1 (RUNX1T1-S) stimulates adipogenesis [6]. These reports further demonstrate the potential role of RUNX1T1 in regulating adipogenesis.
Notably, recent studies in 3T3-L1 cells and mice showed that FTO plays a role in early adipogenesis to increase the number of adipocytes by regulating alternative splicing of RUNX1T1 mRNA [6,14]. However, the mechanism by which RUNX1T1 affects adipogenesis has been elusive. In addition, little is known about the function of RUNX1T1 in fat development in large mammalian species. Therefore, we explored whether RUNX1T1 modulates preadipocyte differentiation derived from native cells of sheep. Although several evidences [9,10] have demonstrated the presence of RUNX1T1 in human and mouse tissues, there is no data available for ovine adipose tissue. Therefore, in the present study, we cloned the ovine RUNX1T1 coding sequence and further investigated whether RUNX1T1 exists in the adipose tissue of sheep.

cDNA Cloning and Sequence Analysis
To obtain the RUNX1T1 cDNA fragment (1829 bp) of sheep, we designed a pair of PCR primers (Table S1) and used these to undertake reverse transcription of the mRNA from subcutaneous fat of Hu sheep (Figure 1), then submitted the obtained cDNA fragment to the GenBank (https://www.ncbi.nlm.nih.gov/genbank/; accession number MH063277). The sequence analysis indicated that the coding sequence of RUNX1T1 was 1815 bp and encoded a 602-amino acid protein with a predicted molecular weight of 67 kDa ( Figure 2). Comparison of the predicted protein with the homologous proteins from other species-including bovine, mouse, and human-using DNAMAN (version 6.0, LynnonBiosoft, San Ramon, CA, USA) showed high homology with mouse (94.21%) and human (86.73%) RUNX1T1 and low sequence identity with bovine (31.18%) RUNX1T1 ( Figure 3).      The same amino acid residues in four, three and two species are highlighted in red, green, yellow respectively, while the amino acid residues present in only one species are highlighted in white. The apostrophes indicates that these amino acid sequences are absent.

Expression Pattern of RUNX1T1 Isoforms in Different Tissues of Hu Sheep
RUNX1T1 exists in two splice variants-a long and a short isoform-in 3T3-L1 cells [14]. These isoforms were also found in different tissues of Hu sheep (Figure 4b). The PCR product of the constitutive RUNX1T1-L is 456 bp, while RUNX1T1-S, skipping the sequence of 757 to 1006, is 211 bp (Figure 4a). The PCR products were verified by sequencing analysis. To evaluate the expression pattern of RUNX1T1 isoforms, we first analyzed mRNA levels of the RUNX1T1-L and RUNX1T1-S in various Hu sheep tissues. It was found that RUNX1T1-L was expressed at much higher levels than RUNX1T1-S in these tissues (Figure 4b; p < 0.5). In addition, the mRNA levels of RUNX1T1-L in testis and adipose tissue were higher than that in other tissues (Figure 4c; p < 0.5). However, the expression of RUNX1T1-S in adipose tissue was similar to that in liver, kidney, testis, and duodenum (Figure 4d,e; p > 0.5). It has been suggested that FTO can affect the expression pattern of short and long isoforms of RUNX1T1 by regulating the RUNX1T1 mRNA splicing [14]. We also found that the expression trend of FTO in different tissues is similar to that of RUNX1T1-L ( Figure 4f).

Expression Pattern of RUNX1T1 Isoforms in Different Tissues of Hu Sheep
RUNX1T1 exists in two splice variants-a long and a short isoform-in 3T3-L1 cells [14]. These isoforms were also found in different tissues of Hu sheep (Figure 4b). The PCR product of the constitutive RUNX1T1-L is 456 bp, while RUNX1T1-S, skipping the sequence of 757 to 1006, is 211 bp (Figure 4a). The PCR products were verified by sequencing analysis. To evaluate the expression pattern of RUNX1T1 isoforms, we first analyzed mRNA levels of the RUNX1T1-L and RUNX1T1-S in various Hu sheep tissues. It was found that RUNX1T1-L was expressed at much higher levels than RUNX1T1-S in these tissues (Figure 4b; p < 0.5). In addition, the mRNA levels of RUNX1T1-L in testis and adipose tissue were higher than that in other tissues (Figure 4c; p < 0.5). However, the expression of RUNX1T1-S in adipose tissue was similar to that in liver, kidney, testis, and duodenum (Figure 4d,e; p > 0.5). It has been suggested that FTO can affect the expression pattern of short and long isoforms of RUNX1T1 by regulating the RUNX1T1 mRNA splicing [14]. We also found that the expression trend of FTO in different tissues is similar to that of RUNX1T1-L ( Figure 4f). (a) Primer I was designed for identifying RUNX1T1 isoforms, primer II was used for qPCR of RUNX1T1-L mRNA. The red line represents the region which is present in the long isoform and absent in the short isoform. Depending on isoforms, bands of either 456 bp or 211 bp can be detected (b). Real-time PCR of mRNA for RUNX1T1-L (178 bp; (c)) and FTO (f) in different tissues. Expression of the RUNX1T1-S (211 bp) in different tissues were determined by semi-qRT-PCR (d,e). Expression of gene was normalized to that of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and relative to the expression in brain. Quantitative data are represented as the mean ± SEM (n = 5). (a) Primer I was designed for identifying RUNX1T1 isoforms, primer II was used for qPCR of RUNX1T1-L mRNA. The red line represents the region which is present in the long isoform and absent in the short isoform. Depending on isoforms, bands of either 456 bp or 211 bp can be detected (b). Real-time PCR of mRNA for RUNX1T1-L (178 bp; (c)) and FTO (f) in different tissues. Expression of the RUNX1T1-S (211 bp) in different tissues were determined by semi-qRT-PCR (d,e). Expression of gene was normalized to that of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and relative to the expression in brain. Quantitative data are represented as the mean ± SEM (n = 5).

Expression Pattern of RUNX1T1 Isoforms and Adipogenic Marker Genes in Subcutaneous Fat Tissue at Different Developmental Stages
Results of Real-time PCR showed that the mRNA levels of adipogenic marker genes, PPARγ and adiponectin (ADIPOQ) were increased during development stages (Figure 5g), which indicates cells were undergoing differentiation. It has been suggested that RUNX1T1 plays an important role in regulating adipogenesis [6]. The temporal expression of RUNX1T1 mRNA and protein in subcutaneous fat tissue at different developmental stages was detected by Real-time PCR (Figure 5a-c) and Western blot analysis (Figure 5e,f), respectively. The long and short isoform of RUNX1T1 in subcutaneous fat showed similar expression pattern amongst different age groups (p > 0.5). Likewise, the expression of FTO in different age groups were similar (Figure 5d). However, the RUNX1T1 protein in subcutaneous fat of 24-month-old Hu sheep was higher than other developmental stages (p < 0.05). The original western blots image of RUNX1T1 and GAPDH were shown in Figure S1. In addition, we found that RUNX1T1 protein was located in the cytoplasm of subcutaneous fat by immunofluorescence (Figure 5h-j).

Expression Pattern of RUNX1T1 Isoforms and Adipogenic Marker Genes in Subcutaneous Fat Tissue at Different Developmental Stages
Results of Real-time PCR showed that the mRNA levels of adipogenic marker genes, PPARγ and adiponectin (ADIPOQ) were increased during development stages (Figure 5g), which indicates cells were undergoing differentiation. It has been suggested that RUNX1T1 plays an important role in regulating adipogenesis [6]. The temporal expression of RUNX1T1 mRNA and protein in subcutaneous fat tissue at different developmental stages was detected by Real-time PCR (Figure 5ac) and Western blot analysis (Figure 5e,f), respectively. The long and short isoform of RUNX1T1 in subcutaneous fat showed similar expression pattern amongst different age groups (p > 0.5). Likewise, the expression of FTO in different age groups were similar (Figure 5d). However, the RUNX1T1 protein in subcutaneous fat of 24-month-old Hu sheep was higher than other developmental stages (p < 0.05). The original western blots image of RUNX1T1 and GAPDH were shown in Figure S1. In addition, we found that RUNX1T1 protein was located in the cytoplasm of subcutaneous fat by immunofluorescence (Figure 5h-j). of RUNX1T1 isoforms (a-c) and FTO mRNA (d) and RUNX1T1 protein (e) in the subcutaneous fat of Hu sheep at different ages were measured using Real-time PCR and Western blot (5 d, 3 M, 6 M and 24 M). d means day, M means month. Semi-quantitative analysis of the relative RUNX1T1 protein expression levels in the subcutaneous fat at different developmental stages was undertaken (f). Real-time PCR was performed on different developmental stages of subcutaneous fat to determine the expression level of marker genes (g), and immunofluorescence localization of RUNX1T1 in the subcutaneous fat of 24-month-old Hu sheep (h-j). Blue color indicates 4 ,6-diamidino-2-phenylindole (DAPI) staining of the nuclei (h). Red color indicates the expression of RUNX1T1 (i). Pictures in (g) and (h) were merged (j). Scale bars = 100 µm. One-way ANOVA, and bars with different letters are significantly different (p < 0.05). Quantitative data are shown as mean ± SEM (n = 5).

Expression Pattern of RUNX1T1 Isoforms during Ovine Preadipocyte Differentiation
To obtain an ovine adipogenesis model, we successfully isolated ovine primary adipocytes (Figure 6c). Based on an in vitro ovine adipogenesis model, we observed that the expression of RUNX1T1-S and FTO were increasing during day 0 to 8 of adipogenic differentiation (Figure 6a,b; p < 0.05). However, the RUNX1T1-L expression was decreased first and then increased during preadipocyte differentiation (p < 0.05).

Expression Pattern of RUNX1T1 Isoforms during Ovine Preadipocyte Differentiation
To obtain an ovine adipogenesis model, we successfully isolated ovine primary adipocytes (Figure 6c). Based on an in vitro ovine adipogenesis model, we observed that the expression of RUNX1T1-S and FTO were increasing during day 0 to 8 of adipogenic differentiation (Figure 6a,b; p < 0.05). However, the RUNX1T1-L expression was decreased first and then increased during preadipocyte differentiation (p < 0.05). Figure 6. Expression patterns of RUNX1T1 isoforms during ovine preadipocyte differentiation. The temporal expression pattern of RUNX1T1 isoforms and FTO during ovine preadipocyte differentiation (a,b); image of newly isolated and adipogenic differentiated ovine preadipocytes (c). Scale bars = 100 µ m. One-way ANOVA followed by comparison of all time points to day 0 with Tuckey post hoc analysis, * p < 0.05. Quantitative data are represented as the mean ± SEM (n = 4).

RUNX1T1-L Knockdown Promoted Lipid Accumulation and Ovine Preadipocyte Differentiation
It has been suggested that overexpression of RUNX1T1-L attenuates lipid accumulation [14]. Based on previous findings, and the data of the RUNX1T1-L expression pattern in ovine preadipocyte and adipose tissue, we proposed that RUNX1T1-L may play a negative regulatory role in ovine preadipocyte differentiation. To verify this hypothesis, we performed RUNX1T1-L knockdown in ovine preadipocyte using siRUNX1T1-L. At 48 h after transfection, we found that siRUNX1T1-L transfection significantly decreased the expression of RUNX1T1-L in cells (Figure 7a; p < 0.05), Figure 6. Expression patterns of RUNX1T1 isoforms during ovine preadipocyte differentiation. The temporal expression pattern of RUNX1T1 isoforms and FTO during ovine preadipocyte differentiation (a,b); image of newly isolated and adipogenic differentiated ovine preadipocytes (c). Scale bars = 100 µm. One-way ANOVA followed by comparison of all time points to day 0 with Tuckey post hoc analysis, * p < 0.05. Quantitative data are represented as the mean ± SEM (n = 4).

RUNX1T1-L Knockdown Promoted Lipid Accumulation and Ovine Preadipocyte Differentiation
It has been suggested that overexpression of RUNX1T1-L attenuates lipid accumulation [14]. Based on previous findings, and the data of the RUNX1T1-L expression pattern in ovine preadipocyte and adipose tissue, we proposed that RUNX1T1-L may play a negative regulatory role in ovine preadipocyte differentiation. To verify this hypothesis, we performed RUNX1T1-L knockdown in ovine preadipocyte using siRUNX1T1-L. At 48 h after transfection, we found that siRUNX1T1-L transfection significantly decreased the expression of RUNX1T1-L in cells (Figure 7a; p < 0.05), however increased the expression of RUNX1T1-S (Figure 7b; p < 0.05). Oil Red-O staining and extraction assays showed that RUNX1T1-L knockdown promoted lipid accumulation in ovine adipocytes (Figure 7c,d). Furthermore, we performed Real-time PCR measurement and observed that on day 8 of differentiation, the mRNA levels of adipogenic marker genes, PPARγ, C/EBPα, ADIPOQ and lipoprotein lipase (LPL) were upregulated to different extents by RUNX1T1-L interference (Figure 7e; p < 0.05).

Discussion
Sheep is an apropos animal model of adult metabolic disease research as well as the primary meat source of human diet in many countries [15,16]. However, studies on the function of RUNX1T1 in ovine adipogenesis are rare. Although the two isoforms of RUNX1T1 have distinctive expression patterns during preadipocyte differentiation, the potential function of this gene in fat development, Figure 7. Adipogenic differentiation of ovine preadipocytes with RUNX1T1-L knockdown. siRUNX1T1-L and siNC were introduced into ovine preadipocytes when the density reached about 70%, and 48 h later, cells were subject to adipogenic differentiation (a). The expression of RUNX1T1-L was determined at 48 h post-transfection by Real-time PCR. Moreover, the expression of RUNX1T1-S was analyzed by semi-qRT-PCR (b). Ovine preadpocytes were transfected with siRUNX1T1-L or siNC and differentiated into mature adipocytes (c). Scale bars = 100 µm. After 8 days of differentiation, intracellular lipid droplets were stained using Oil Red-O. Lipid content was indirectly determined by measuring the optical density (OD) value at 510 nm on a spectrophotometer (d). Real-time PCR was performed on day 6 of adipogenic differentiation to determine the mRNA level of maker genes (e). Independent Students' t-test. * p < 0.05 against siNC. Quantitative data are represented as the mean ± SEM (n = 4).

Discussion
Sheep is an apropos animal model of adult metabolic disease research as well as the primary meat source of human diet in many countries [15,16]. However, studies on the function of RUNX1T1 in ovine adipogenesis are rare. Although the two isoforms of RUNX1T1 have distinctive expression patterns during preadipocyte differentiation, the potential function of this gene in fat development, such as ovine adipogenesis, has not received sufficient attention. In this study, the ovine RUNX1T1 gene was cloned for the first time, and the coding sequence of this gene was obtained. In addition, we found that RUNX1T1 can affect ovine preadipocyte differentiation.
In the present study, we cloned the RUNX1T1 gene from the subcutaneous fat of Hu sheep. The results showed that the coding sequence of RUNX1T1 was 1815 bp and encoded 602-amino acid. However, the RUNX1T1 coding sequence encodes 271, 579, and 662 amino acids in cattle, mice, and humans, respectively [17,18]. The amino acid sequence of sheep RUNX1T1 is highly homologous to RUNX1T1 in mice (94.21%) and humans (86.73%), however has poor homology to RUNX1T1 in cattle (31.18%). As a member of the conserved RUNX transcription factor family, RUNX1T1 exists in two splice variants; a long and a short isoform. Indeed, we found that the coding sequence of RUNX1T1-L is 245 bp longer than that of RUNX1T1-S [19]. In general, RUNX1T1 is abundantly expressed in the heart and brain of human [20]. However, we found that RUNX1T1-L is abundant in sheep testis and adipose tissue in the present study. These discrepancies may be caused by species differences.
As a white fat tissue, subcutaneous fat is characterized by mature adipocytes containing large and unilocular lipid droplets. It is also an active endocrine organ involved in various activities, such as insulin sensitivity, glucose tolerance, lipid metabolism and deposition [21,22]. Moreover, previous studies have found that subcutaneous fat deposition is related to meat quality [23]. The growth and development of adipose tissue includes two important stages: (i) adipocyte proliferation and (ii) adipocyte hypertrophy [24]. Proliferation of adipocytes proceeds in late gestation, while adipocyte hypertrophy mainly occurs postnatally [25]. Previous studies have confirmed that PPARγ plays an essential role in maintaining mature adipocyte function [26,27]. In the present study, PPARγ and ADIPOQ were used as biomarkers of mature adipocytes, and their expression increased with increasing developmental stages, indicating more lipid droplet deposition in mature adipocytes. In addition, the expression level of RUNX1T1 protein in subcutaneous fat increased with age. These results indicate that RUNX1T1 may play an important role in fat development in sheep.
Although there are few reports on the role of RUNX1T1 in adipogenesis, RUNX1T1 has been studied in-depth for its sophisticated functions since its initial identification [28,29]. In the formation of blood vessels, RUNX1T1 was confirmed to serve as an important angiogenic driver for vasculogenesis [30]. However, previous studies reported that RUNX1T1 has pleiotropic effects on fat development such as inhibiting CCAAT enhancer binding protein beta (C/EBPβ) activity during early adipogenesis and modulating preadipocyte differentiation through alternative splicing of RUNX1T1 [5,31]. FTO is a nucleic acid demethylase that can demethylate N 6 -methyladenosine [32], thereby controlling alternative splicing of RUNX1T1 mRNA [14]. Indeed, the expression of RUNX1T1-S increased during the differentiation of ovine preadipocytes, which was consistent with the expression of the FTO gene. These results indicated RUNX1T1 may be a regulator of ovine preadipocyte differentiation.
The two isoforms of RUNX1T1 are generally considered to have different functions in regulating adipogenesis [5,6]. Though previous studies did not distinguish differences between the long and short isoforms, overexpression of RUNX1T1 was found to suppress adipogenesis by inhibiting the transcriptional cascade that results in adipocyte formation [5]. Considering that the expression level of the long isoform of RUNX1T1 is higher than that of the short isoform in ovine adipose tissue and preadipocytes, the predominance of RUNXT1-L in regulating the process of adipogenesis is indicated.
To the best of our knowledge, this is the first study to assess the effect of the long isoform of RUNX1T1 on ovine preadipocyte differentiation. In the present study, we observed that knockdown of RUNX1T1-L resulted in the loss of the long isoform, whereas the short isoform of RUNX1T1 increased. The increase of short isoform may be due to the compensating effects of RUNX1T1-L interference [33]. In addition, knockdown of RUNX1T1-L promoted ovine preadipocyte differentiation and lipid accumulation. The predominant effect of RUNX1T1-L knockdown on preadipocyte differentiation may be related to the activation of C/EBPβ [5] and the increased expression of the short isoform [14]. RUNX1T1-L may bind to C/EBPβ, thereby blocking its DNA-binding activity and transcription of its downstream genes (such as PPARγ) [34], inhibiting preadipocyte differentiation. However, the mechanism by which the long isoform of RUNX1T1 effects preadipocyte differentiation is unclear. Thus, further studies are still required to determine the role of the long isoform of RUNX1T1 in ovine preadipocyte differentiation.

Material and Methods
All experimental design and procedures were performed in accordance with the approved Guidelines for Animal Experiments of Nanjing Agricultural University, China and were approved by the Animal Care and Use Committee of Nanjing Agricultural University, China (Approval ID: SYXK2011-0036; date: 6 December 2011).

Sample Collections
The experiment was carried out at the Jiangsu Taizhou Helen Sheep Industry Co., Ltd. A total of twenty Hu sheep were selected and divided into four groups according to the age. At the end of experiment, animals were slaughtered by exsanguination. At 30 min post-slaughter, brain, heart, liver, kidney, longissimus, gluteus, duodenum, testis, subcutaneous fat and perirenal fat were sampled. Samples were immediately frozen in liquid nitrogen for further study. One portion of sample was made into frozen sections for immunofluorescence; the other was stored at −80 • C for further analysis of gene and protein expression.

Primary Adipocyte Culture
Ovine primary adipocytes were isolated from the cervical subcutaneous fat tissue of 7-day-old Hu lambs under sterile conditions. The tissues were then minced and digested with collagenase (Sigma-Aldrich, St Louis, MO, USA) in Krebs-Ringer 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid buffer (HEPES) for 1 h at 37 • C. The digested tissue was filtered with a 200 µm nylon mesh and then centrifuged at 1200 rpm for 5 min to obtain preadipocytes. The cell pellet was re-suspended and washed with phosphate buffered solution (PBS, Gibco, Grand Island, NY, USA) and finally cultured in Dulbecco modified eagle (DMEM, Gibco) containing 10% fetal bovine serum (Gibco) and 1% penicillin/streptomycin (Gibco). Preadipocyte differentiation was induced by supplementing the growth culture medium with 0.5 mM 3-isobutyl-1-methylxanthine (IBMX), 1µM dexamethasone, 20 µM rosiglitazone and 10 µg/mL insulin for 4 days. After this period, the culture medium was supplemented with 10 µg/mL insulin only. The whole adipogenic process of ovine preadipocytes took 8-10 days.

Oil Red-O Staining and Extraction Assay
Ovine adipocytes were washed with PBS and fixed in 10% formaldehyde formalin for 30 min at room temperature. After washing three times in PBS, cells were stained with a filtered 1% Oil Red-O (Sigma-Aldrich, St Louis, MO, USA) solution at room temperature for 30 min, washed again in PBS and visualized under an inverted microscope (Olympus, Tokyo, Japan). To quantify intracellular lipid content, the lipid droplets were extracted from Oil Red-O-stained adipocytes with pure isopropanol. The optical density of solution (OD value) was measured at 510 nm on a spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA).

RNA Isolation and cDNA Synthesis
Total RNA was extracted from different tissues and cells using Trizol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's protocol. The RNA quality (purity and integrity) was determined using ND-2000 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA) and 1% agarose gel electrophoresis. Thereafter, cDNA was synthesized using reverse transcription reagent kits (Takara, Dalian, China).

Cloning of RUNX1T1
To obtain the coding sequence of RUNX1T1 in Hu Sheep, a pair of specific primers RUNX1T1-CDS were designed using Primer 5.0 software (PREMIER Biosoft, Palo Alto, CA, USA) ( Table S1). The ovine subcutaneous fat cDNA was amplified by PCR. The PCR conditions were as follows; 94 • C for 5 min, 35 cycles of 98 • C for 10 s, 60 • C for 45 s, 72 • C for 45 s and 72 • C for 7 min. All PCR products were separated using 1.5% agarose gel. After purification, the target PCR products were cloned into a pClone007 Blunt Vector (TSINGKE Biological Technology, Beijing, China) and then transformed into Escherichia coli DH5a cells. Positive clones were randomly selected and sequenced at TSINGKE Biological Technology.

Real-time PCR Analysis for mRNA Expression
Real-time PCR was performed according to the protocol described in our previous study [35] for gene expression quantification. Gen Bank accession numbers and sequences of the corresponding Real-time PCR primer are listed in Table S1. At the same time, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used to normalize the mRNA expression level. The relative gene expression of RUNX1T1-L, FTO, PPARγ, C/EBPα, ADIPOQ, and LPL were calculated with the comparative, efficiency-corrected 2 −∆∆Ct method.

Semi-Quantitative Reverse Transcription Polymerase Chain Reaction (Semi-qRT-PCR)
The expression level of RUNX1T1-S was determined by semi qRT-PCR method. The PCR was carried out in 20 µL reaction volume containing 1 µL cDNA, 10 µL LA Taq PCR Master Mix (Takara, Dalian, China), 7.8 µL nuclease-free water and 0.6 µL each of forward and reverse primer pairs (10 pmol). To amplify ovine GAPDH (internal control) and RUNX1T1-S, a denaturing cycle of 10 min at 95 • C, followed by 30 cycles of (95 • C for 10 s, an annealing step at 60 • C for 30 s and extension step at 72 • C for 45 s), was performed for each PCR. The PCR products were separated by 1% agarose gel electrophoresis. Following, the images of the semi-qRT-PCR stained with ethidium bromide were analyzed using ImageJ 1.50i software (National Institutes of Health, Bethesda, MD, USA). The band intensity of the genes of interest was normalized to GAPDH.

Western Blot Analysis for Protein Expression
Protein samples were prepared using protein lysis buffer (Radio Immunoprecipitation Assay; Beyotime, Shanghai, China) with phenylmethanesulfonyl fluoride (PMSF; Beyotime). After incubation on ice for at least 20 min, the lysates were centrifuged to remove insoluble material, and protein concentrations were estimated by a BCA protein assay kit (Beyotime).
Approximately 20 µg of the protein lysates from each treatment group were run on 12% sodium dodecyl sulfate (SDS) polyacrylamide gel and transferred to polyvinylidene fluoride membranes (Millipore; Billerica, MA, USA). Membranes were blocked for 1 h at room temperature in 5% (w/v) fat-free milk, and then incubated overnight at 4 • C with corresponding primary antibodies to RUNX1T1 (1:1000, Proteintech, Chicago, IL, USA) and GAPDH (1:8000, Proteintech) as an internal control. After washing with Tris buffered saline with Tween (TBST), membranes were incubated with the appropriate secondary antibody (1:1000, horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG for RUNX1T1; 1:1000, HRP-labeled goat anti-mouse IgG for GAPDH, Beyotime) for 1 h at room temperature. The protein bands were visualized with an ImageQuant LAS 4000 (Fujifilm, Tokyo, Japan) and quantified using ImageJ 1.50i software (National Institutes of Health).

Immunofluorescence
The frozen sections of subcutaneous fat stored at −20 • C until staining. The frozen sections prepared from the refrigerator were melted on a ventilated stage for 20 min. The frozen sections were fixed with 4% (v/v) paraformaldehyde for 1 h at 4 • C, washed three time with PBS, and then permeabilized with 0.2% (v/v) Triton X-100/PBS for 15 min. After treating with 3% (w/v) bovine serum albumin (BSA)/PBS for 30 min at room temperature, frozen sections were incubated overnight at 4 • C with primary rabbit anti-RUNX1T1 antibody (Proteintech) diluted 1:100 in 1% (w/v) BSA/PBS. After washing three times with PBS, the frozen sections were incubated with 594-conjugated donkey anti-rabbit antibody (1:200 dilution; Abcam, Cambridge, UK) in the dark for 2 h at room temperature. Subsequently, samples were stained with DAPI (Beyotime, Shanghai, China) for 10 min at room temperature. All samples were examined under a confocal laser scanning microscope (Zeiss LSM 710 META, Jena, Germany).

Statistical Analysis
All experiments were carried out in triplicate. All values were expressed as the mean ± SEM. Statistical analysis was performed using the SPSS software (version 24.0, SPSS Inc., Chicago, IL, USA) by either an independent Student's t-test or one-way analysis of variance (ANOVA) with Tuckey post hoc analysis. For all analyses, p < 0.05 is indicated as a statistically significant.

Conclusions
In conclusion, this study showed that the expression of the long isoform of RUNX1T1 was higher in the subcutaneous fat of Hu sheep. In addition, an increase in the long isoform of RUNX1T1 was observed during ovine preadipocyte differentiation, strongly indicating that the long isoform of RUNX1T1 may be involved in ovine adipogenesis. This is the first report that RUNX1T1-L knockdown promoted preadipocyte differentiation and lipid accumulation, indicating a negative relationship between RUNX1T1-L expression and ovine preadipocyte differentiation. However, little is known about the mechanism by which the long isoform of RUNX1T1 regulates preadipocyte differentiation. Therefore, further studies are needed to elucidate the role of the long isoform of RUNX1T1 in preadipocyte differentiation.