Primers used to make constructs to analyse promoter activity in the study.

<p>C2-A, C2-B, C2-C, C2-D, C2-E, C2-a, C2-b, C2-c and C2-d: Forward primers for construction of the luciferase reporter gene vectors pGL₃-A, pGL₃-B, pGL₃-C, pGL₃-D, pGL₃-E, pGL₃-a, pGL₃-b, pGL₃-c and pGL₃-d, respectively; C2-R: Reverse primer for construction of luciferase reporter gene vectors pGL₃-A, pGL₃-B, pGL₃-C, pGL₃-D, pGL₃-E, pGL₃-a, pGL₃-b, pGL₃-c and pGL₃-d.</p

The cell morphology of 293T under fluorescence microscope (Olympus, micropublisher 3.3RTV, 100×) transfected by CRABP2 overexpression vector and the expression of CRABP2 detected by Westernblot.

<p>(A) Visible image transfected with lentivirus; (B) Green fluorescence image transfected with lentivirus; (C) CRABP2 expression by westernblot.</p

The first deletion analysis of the promoter of the mouse <i>CRABP2</i> gene in C2C12 cells.

<p>C2C12 cells were co-transfected with various promoter regions fused to firefly luciferase and a Renilla luciferase expression control vector. The resulting firefly luciferase activity was then normalized to Renilla luciferase activity, and the relative values were presented as the fold-increase over the activity of the promoter-less pGL₃-basic vector. Values represent the mean ± SD of three independent experiments.</p

The expression of the <i>CRABP2</i> gene during differentiation was assessed by quantitative real-time PCR (qRT-PCR).

<p>The values were normalized to <i>GAPDH</i> mRNA expression level and the value of day 0 was set to 1. The error bars indicate the SD (n = 3).</p

The second deletion analysis of the promoter of the mouse <i>CRABP2</i> gene in C2C12 cells.

<p>C2C12 cells were co-transfected with various promoter regions fused to firefly luciferase and a Renilla luciferase expression control vector. The resulting firefly luciferase activity was then normalized to Renilla luciferase activity, and the relative values were presented as the fold-increase over the activity of the promoter-less pGL₃-basic vector. Values represent the mean ± SD of three independent experiments.</p

Binding activities of MyoD and Sp1 in nuclear extracts.

<p>(A) Assay of binding activity of MyoD in nuclear extracts; (B) Assay of binding activity of Sp1 in nuclear extracts. Lanes 1, 6: nuclear extracts from C2C12 myoblasts; lanes 2, 3, 4, 7, 8, 9: nuclear extracts from C2C12 myotubes after a 4 day induction with horse serum; lanes 5, 11: competition for binding by the unlabeled probe; lane 10: positive control.</p

The target gene expression in C2C12 cells transfected with the <i>CRABP2</i> lentivirus vector.

<p>(CON: Blank control; NC: Negative control; OE: Overexpression).</p

The cell morphology of C2C12 under fluorescence microscope (Olympus, micropublisher 3.3RTV, 200×) transfected by different lentivirus vector.

<p>(CON: Blank control; NC: Negative control; OE: Overexpression).</p

Transcriptional activation of the <i>CRABP</i>2 promoter was effected by <i>MyoD</i> and <i>Sp</i>1.

<p>(A) Transcriptional activation of the <i>CRABP2</i> promoter was potentiated by the MyoD expression plasmid. The empty vector or the core promoter plasmid pGL₃-E was cotransfected with the MyoD expression plasmid pcDNA3.1-MyoD into C2C12 cells. The over-expression of MyoD increased the <i>CRABP2</i> promoter activity (n = 3); (B) The transcriptional activation of the <i>CRABP2</i> promoter was repressed by the Sp1 site-directed mutation vector. The Sp1 binding site was required for <i>CRABP2</i> promoter function, and the transcription factor Sp1 facilitated the transcriptional activation of the <i>CRABP2</i> gene. Values represent the mean ± SD of three independent experiments.</p

Global Transcriptomic Profiling of Cardiac Hypertrophy and Fatty Heart Induced by Long-Term High-Energy Diet in Bama Miniature Pigs

<div><p>A long-term high-energy diet affects human health and leads to obesity and metabolic syndrome in addition to cardiac steatosis and hypertrophy. Ectopic fat accumulation in the heart has been demonstrated to be a risk factor for heart disorders, but the molecular mechanism of heart disease remains largely unknown. Bama miniature pigs were fed a high-fat, high-sucrose diet (HFHSD) for 23 months. These pigs developed symptoms of metabolic syndrome and showed cardiac steatosis and hypertrophy with a greatly increased body weight (2.73-fold, P<0.01), insulin level (4.60-fold, P<0.01), heart weight (1.82-fold, P<0.05) and heart volume (1.60-fold, P<0.05) compared with the control pigs. To understand the molecular mechanisms of cardiac steatosis and hypertrophy, nine pig heart cRNA samples were hybridized to porcine GeneChips. Microarray analyses revealed that 1,022 genes were significantly differentially expressed (P<0.05, ≥1.5-fold change), including 591 up-regulated and 431 down-regulated genes in the HFHSD group relative to the control group. KEGG analysis indicated that the observed heart disorder involved the signal transduction-related MAPK, cytokine, and PPAR signaling pathways, energy metabolism-related fatty acid and oxidative phosphorylation signaling pathways, heart function signaling-related focal adhesion, axon guidance, hypertrophic cardiomyopathy and actin cytoskeleton signaling pathways, inflammation and apoptosis pathways, and others. Quantitative RT-PCR assays identified several important differentially expressed heart-related genes, including STAT3, ACSL4, ATF4, FADD, PPP3CA, CD74, SLA-8, VCL, ACTN2 and FGFR1, which may be targets of further research. This study shows that a long-term, high-energy diet induces obesity, cardiac steatosis, and hypertrophy and provides insights into the molecular mechanisms of hypertrophy and fatty heart to facilitate further research.</p></div