Influence of oleic acid on PYY secretion in the apical and basolateral media.

<p>Oleic acid at the concentrations of 0.7 mM and 1.4 mM was added either to the apical or basolateral medium. Following the 2-h incubation period, both apical and basolateral media were assessed for their PYY content. Values are expressed as means ± SD for n = 3 separate experiments in each group. CTR, Control; Baso, basolateral; OA, Oleic Acid.</p

Detection of Y1 receptor (NPY1R) in Caco-2/15 cells in culture.

<p>Following differentiation, Caco-2/15 cells were homogenized and apical and basolateral membranes were isolated. Aliquots were fractionated by SDS-PAGE and electrotransferred onto nitrocellulose membranes. The blots were then incubated with the polyclonal antibody overnight at 4°C. Immunocomplexes were revealed by means of horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin and an enhanced chemiluminescence kit. Y1 receptor mass was quantitated by use of an HP Scanjet scanner equipped with a transparency adapter and software. Values are expressed as means ± SD for n = 3 separate experiments in each group.</p

Effects of the administration of PYY (1–36) to the apical or basolateral medium on the protein expression of LDL-receptor (LDL-R).

<p>Caco-2/15 cells were cultured for 24 h in MEM as described in the legend of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040992#pone-0040992-g002" target="_blank">Figure 2</a>. Western blot was used to analyse the protein expression of LDL-R. Data represented means ± SD for n = 3 separate experiments in each group and are reported as % of control values representing 100%.</p

Effects of the administration of PYY (1–36) to the apical or basolateral medium on cholesterol synthesis.

<p>After 24 h incubation with [¹⁴C]-acetate, cells were homogenized. Lipids were extracted in chloroform/methanol and separated by TLC. The free cholesterol (FC) and cholesteryl ester (CE) bands were scraped off the plate and counted. Data represent means ± SD for n = 3 separate experiments in each group. *P<0.05 vs. controls, **P<0.01 vs. controls.</p

Effects of the administration of PYY (1–36) to the apical or basolateral medium on lipoproteins output.

<p>Differentiated Caco-2/15 cells were cultured for 24 h in MEM containing 50 nM or 200 nM of PYY in their apical or basolateral medium, in the presence of [¹⁴C]-oleic acid for 24 h. Thereafter, the media were ultracentrifuged to isolate lipoproteins at their specific densities. Radioactivity incorporated into each fractions was further determined. Data were analyzed as dpm/mg of total protein but were reported as percent difference relative to control. Data represent means ± SD for n = 3 separate experiments in each group. * P<0.05 vs. controls. CM<b>;</b> Chylomicrons (A), VLDL; Very-low density lipoprotein (B), LDL; low density lipoprotein (C) and HDL; high density lipoprotein (D).</p

Effects of the administration of PYY (1–36) to the apical or basolateral medium on RXR and LXR gene expression in Caco-2/15 cells.

<p>PCR analysis was performed on Caco-2/15 cell line at 14 days post-confluence to analyze mRNA of RXRα (A), RXRβ (B), LXRα (C), LXRβ (D). Values represent means ± SD for n = 3 separate experiments in each group and are reported as % of control values representing 100%. *P<0.05 vs. controls, **P<0.01 vs. controls.</p

Effects of the administration of PYY (1–36) to the apical or basolateral medium on the protein expression of ACAT-2.

<p>Caco-2/15 cell line was cultured for 24 h in MEM described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040992#pone-0040992-g002" target="_blank">Figure 2</a>. Western blot was used to analyze the protein expression of ACAT-2. Values are means ± SD for n = 3 separate experiments in each group and are reported as percent. *P<0.01 vs. controls.</p

Effects of the administration of PYY (1–36) to the apical or basolateral medium on the protein expression of transporters mediating cholesterol absorption.

<p>Caco-2/15 cells were cultured for 24 h in MEM as described in the legend of Figure2. Western blot was used to analyze the protein expression of NPC1L1 (A), SR-BI (B) and CD36 (C). Values are means ± SD for n = 3 separate experiments in each group and are reported as % of control values representing 100%. *P<0.01 vs. controls.</p

Effects of the administration of PYY (1–36) to the apical or basolateral medium on the synthesis of apolipoproteins (apo).

<p>Epithelial cells at 14 days post-confluence were incubated with [³⁵S]-methionine in the presence of PYY (1–36) (50 or 200 nM) and unlabeled oleic acid for 24 h to stimulate apo biogenesis. At the end of the labelling period, cells were washed, homogenized, and centrifuged. Supernatants from the cell homogenates were then reacted with excess antibodies for 18 h at 4°C to precipitate specific apos. Immune complexes were washed and analyzed by linear 4–15% SDS-PAGE. After electrophoresis, gels were sliced and counted for radioactivity. Data represent means ± SD for n = 3 separate experiments in each group. Values are reported as % of control values representing 100%. *P<0.05 vs. controls.</p

Effects of the administration of PYY (1–36) to the apical or basolateral medium on the protein expression of ABCG5 and ABCG8.

<p>Caco-2/15 cells were cultured for 24 h in MEM as described in the legend of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040992#pone-0040992-g002" target="_blank">Figure 2</a>. Western blot was used to analyse the protein expression of ABCG5 (A) and ABCG8 (B). Data represent means ± SD for n = 3 separate experiments in each group and are reported as % of control values representing 100%.</p