TGF-β1 increases SERT levels on the luminal surface in 3D-culture of Caco-2 cells.

<p>Caco-2 cells in 3D culture system untreated or treated with TGF-β1 (10 ng/ml, 1h) were stained for SERT (green), Phalloidin (red) and DAPI (blue) and visualized by confocal microscopy. XY planar images and orthogonal XZ images Orthognal <i>xz</i> images were obtained with a Zeiss LSM 510 confocal microscope.</p

SERT function in response to TGF-β1 treatment of native mouse ileum.

<p>The distal small intestine was stripped off of the muscle layer, mounted in the Ussing chamber to expose tissue on both sides to Kreb’s solution supplemented with L-ascorbic acid (100 μM) and the monoamine oxidase inhibitor pargyline (100 μM). Tissues were treated with TGF-β1 for 1h from basolateral side and then incubated with ³[H]-5-HT for 30 min. Fluoxetine (10 μM) was added 30 min prior to TGF-β1 treatment from the apical side. Values represent mean ± SEM of 3 different experiments. *<i>P</i><0.05 compared with untreated control.</p

Expression of genes involved in lipid metabolism in ISR2 mice.

<p>Expression of HMG-CoA reductase, CYP51, PCSK9 and scd1 were assessed by real time PCR using gene specific primers and total RNA extracted from jejunum of ISR2 mice (TG) and wild type littermates (Control). The results are expressed as arbitrary unit (A.U.) and represent the Mean ± SE using 10–12 animals of each group. * P<0.05 as compared to WT mice.</p

Intestine-specific overexpression of active SREBP2.

<p>Total RNA was extracted from different tissues from ISR2 mice (TG) and their wild type littermates (WT) and the expression of SREBP2 was then assessed by real time PCR using gene specific primers. <b>A</b>: The expression of active SREBP2 transgene evaluated using a set of primers specific for the N-terminal of SREBP2 mRNA in the jejunum, ileum and colon. <b>B</b>: The expression of endogenous SREBP2 mRNA in jejunum, ileum and colon assessed using primers specific for the C-terminal of the SREBP2 gene. <b>C</b>: The expression of SREBP2 in the kidney and lung utilizing N- and C-terminal specific primers. <b>D</b>: The expression of SREBP1c in jejunum and ileum of ISR2 and WT mice. The presented data represent the expression of respective gene relative to the expression of GAPDH, used as an internal control. The results are expressed as arbitrary unit (A.U.) and represent Mean ± SE using 10–12 animals of each group. * P<0.05 as compared to WT mice.</p

Phenotypic characterization of ISR2 mice.

<p>P<0.05.</p

Serum levels of cholesterol and triglycerides in ISR2 mice.

<p><b>A</b>: Serum was collected from ISR2 (TG) and wild type (WT) mice. The levels of cholesterol and triglycerides were measured as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084221#s2" target="_blank">Materials and Methods</a>. The data are expressed as mg/dl and represent the Mean ± SE from 4 animals per group. * P<0.05 as compared to WT. <b>B</b>: Triglyceride levels were measured in different lipoprotein fractions prepared from ISR2 transgenic mice and their wild type littermates. The figure shows representative data showing a decrease in triglycerides of the VLDL from the ISR2 (open circles) mice as compared to their wild type littermates (closed triangles). Serum samples from three different ISR2 transgenic mice and three wild type littermates were pooled into one sample and lipoproteins were then obtained by the method of FPLC. <b>C</b>: Serum was collected from ISR2 (open circles) and wild type mice (closed triangles) for a total of 12 animals per group, and three animals were pooled into one aliquot for subsequent cholesterol measurement. Cholesterol levels were assessed in different lipoprotein fractions by the FPLC methods. The levels of cholesterol in different lipoprotein fractions are expressed as µg/fraction.</p

The levels of triglycerides and cholesterol in the jejunum of ISR2 mice.

<p>Total lipids were extracted from jejnunal mucosal scrapings from ISR2 (TG) and wild type (WT) mice and the levels of triglycerides, total cholesterol and cholesterol ester were measured as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084221#s2" target="_blank">Materials and Methods</a>. Data are presented as mg lipid/g of tissues and expressed as Mean ± SE from 8 mice per group. * P<0.05 as compared to WT.</p

Generation of intestine-specific active SREBP2 mice.

<p>The coding sequence for the N-terminal of SREBP2 (representing the active transcription factor) was cloned down-stream of villin promoter. <b>A</b>: A schematic representation of the transgene and the location of the G1 and G2 primers used for genotyping and the identification of positive transgenic mice (designated as ISR2) <b>B</b>: A representative of genotyping results showing the expected amplified PCR fragment from genomic DNA extracted from ISR2 mice (+) but not their wild type littermates (−).</p

Distribution of active SREBP2 in ISR2 mice.

<p>Total protein lysates were prepared form intestinal mucosal scraping as mentioned in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084221#s2" target="_blank">Materials and Methods</a>. <b>A</b>: A representative blot depicting the bands for active SREBP2 in ISR2 mice and their wild type littermates. Villin was used as a loading control. <b>B</b>: Villin staining (green) of the jejunum showing similar epithelial structure in ISR2 and wild type mice. <b>C</b>: immuno fluorescence staining of SREBP2 in jejunum of ISR2 and wild type mice. SREBP2 is stained with red and the nuclei with blue. The figure shows predominant cytoplasmic staining in wild type mice and increased colocalization of SREBP2 with the nuclei in ISR2 mice (white arrow).</p