Colonic miRNA expression levels are modulated in villin-hPepT1 and FVB WT mice with and without DSS.

<p>miRNAs expressed with the a <i>p</i>-value of <0.01 and signal >500 in microRNA microarray were analyzed by qRT-PCR (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087614#pone-0087614-g002" target="_blank">Fig. 2A</a>). miRNA 3077, miRNA 1934, miRNA 2145 were not associated with any target mRNAs so these three miRNAs were excluded from further analysis (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087614#pone-0087614-g002" target="_blank">Fig. 2B</a>) and the mature mmu-miR-1937a, mmu-miR-1937b, and mmu-miR-1937c are fragments of tRNA (<a href="http://www.mirbase.org" target="_blank">www.mirbase.org</a>) so we eliminated these from further analyses (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087614#pone-0087614-g002" target="_blank">Fig. 2C</a>). Difference in miRNA expression noted as * <i>p</i><0.05, ** <i>p</i><0.001 and *** <i>p</i><0.0001. Values represent means ± SEM of n = 6/group.</p

DAVID functional annotation clustering of villin-hPepT1 and FVB WT at the basal level.

<p>The common proteins were clustered into 30 groups, but based on enrichment score >1.0. 11 groups were considered to be the potential targets with a total of 1100 genes.</p

DAVID functional annotation clustering of villin-hPepT1 and FVB WT treated with DSS.

<p>The common proteins obtained from the matchminer software were clustered in to 15 groups but based on enrichment score >1.0, a total of 4 groups were considered to be the potential targets with a total of 270 genes.</p

MiR-23b directly targets the Marcksl-1 3′UTR in macrophages.

<p>Marcksl-1 3′UTR was cloned into a luciferase reporter gene (Luc-Marcksl-1 3′UTR), and transfected into RAW 264.7 cells in the presence or absence of miRNA 23b precursor. The targeting of miRNAs to Marcksl-1 3′UTR was then assessed by measuring luciferase activity in these cells. The miRNA 23b co-transfected with Luc-Marcksl-1 3′UTR significantly reduced luciferase activity in macrophage cells after 24 h of transfection. In contrast, Luc-Marcksl-1 3′UTR had an increase of luciferase activity. Values represent means ± SEM of n = 3 and ***<i>p</i><0.0001.</p

hPepT1 regulates miRNA 132, miRNA 200b and miRNA 23b expression in colon- specific manner.

<p>Upon exposure to DSS or normal drinking water, intestinal epithelial PepT1 overexpression did not affect the expression level of miRNA 132 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087614#pone-0087614-g003" target="_blank">Fig. 3A</a>), miRNA 200b (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087614#pone-0087614-g003" target="_blank">Fig. 3B</a>) and miRNA 23b (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087614#pone-0087614-g003" target="_blank">Fig. 3C</a>) in liver or spleen. Values represent means ± SEM of n = 6/group.</p

Clustering graph of miRNAs.

<p>microRNA microarray results demonstrated that villin-hPepT1 and FVB WT mice treated with and without DSS expressed different miRNA profiles. miRNAs expressed with a <i>p</i> value of <0.01 and signal >500.</p

Secretory miRNA in colon culture medium.

<p>miRNA 23b in conditioned medium was measured by miRNA plate assay chemiluminescence method (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087614#pone-0087614-g005" target="_blank">Fig. 5A</a>) and qRT-PCR (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087614#pone-0087614-g005" target="_blank">Fig. 5B</a>). Cy3 labelled miRNA 23b uptake by macrophage (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087614#pone-0087614-g005" target="_blank">Fig. 5C</a>). miRNA 23b uptake at 4°C (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087614#pone-0087614-g005" target="_blank">Fig. 5D</a>), miRNA 23b uptake at pH 7.0 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087614#pone-0087614-g005" target="_blank">Fig. 5E</a>) and pH 6.5 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087614#pone-0087614-g005" target="_blank">Fig. 5F</a>). F-actin (FITC - green), nuclear staining (DAPI - blue) and Cy3 labelled miRNA 23b (TRITC -filter setting used) are stained and separate pictures were taken at 60× for each filter and merged respectively. Values represent means ± SEM of n = 6/group. *<i>p</i><0.05, **<i>p</i><0.001 and ***<i>p</i><0.0001. Scale bar = 10 µm.</p

DSS induces colitis.

<p>(A) Mouse body weight changes during DSS treatment compared to that of control animals (drinking water). C57BL/6 mice were exposed to 3% (w/v) DSS in the drinking water for the indicated numbers of days. Body weight changes are shown as means±SEMs. * P<0.05, ** P<0.01. (B) Hematoxylin-stained colonic sections of mice treated with DSS; the mice were sacrificed on day 8. (C) Associated histological scores. *** P<0.001. (D) Macroscopic inflammation was assessed using a mouse colonoscope. Photographs were obtained on the day of sacrifice and (E) an endoscopic score was calculated. *** P<0.001. (F) KC mRNA expression was measured in test and control animals. *** P<0.001. (G) Determination of MPO enzymatic activity as an index of neutrophil infiltration into injured tissue. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032084#s3" target="_blank">Results</a> are expressed as MPO –fold increases compared to those of control mice and represent means±SEMs of three independent determinations. *** P<0.01.</p

Dextran-loaded nanoparticles but not free dextran induce colitis.

<p>Mouse body weight changes during treatment with DSS supplemented with dextran-loaded NPs (“dextran-loaded NPs”) or empty NPs (“DSS 3%”) compared to those of control mice gavaged with empty NPs suspended in water. C57BL/6 mice drank a 3% (w/v) DSS solution, or water, for the indicated numbers of days. (A) Body weight changes are shown as means±SEMs. * P<0.05. (B) Colon length (mm) of DSS-treated mice given dextran-loaded or empty NPs, compared to that of control animals (drinking water only). * P<0.05. (C) Determination of MPO enzymatic activity as an index of neutrophil infiltration into injured tissue. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032084#s3" target="_blank">Results</a> are expressed as units of MPO per µg of protein, are compared to the values in control mice, and are means±SEMs of three independent determinations. ** P<0.01, *** P<0.001. (D) Dextran-loaded NPs can significantly increase production of IL-1β and IL-6, compared to use of empty NPs, in animals drinking DSS (3% w/v). mRNA expression was measured in test and control animals. * P<0.05. (E) Hematoxylin-stained colon sections of mice receiving daily gavage with dextran-loaded or empty NPs (“DSS”) in the interval during which the drinking water contained DSS (3% w/v); thus on day 7. (F) Histological scores. (G) Macroscopic inflammation was assessed using a mouse colonoscope. Photographs were obtained on the day of sacrifice and, (H), endoscopic scores were calculated.</p

DSS and fatty acids form nano-lipocomplexes that disrupt Caco2-BBE monolayer resistance.

<p>(A) Confluent cultures were obtained after 48 h of growth and epithelial resistance was assessed by continuous resistance measurement at pH 7.4 (cellular pH) or pH 6.2 (colonic pH). The epithelial resistances of cultures treated with dodecanoate alone (10 mM) or with 3% (w/v) DSS (1 or 10 mM dodecanoate), at pH 6.2 or pH 7.4, were assessed by ECIS. (B) Light scattering measurement of the diameters (nm) of particles formed by DSS (3% w/v) alone; DSS (3% w/v) with Ca⁺⁺ (1 mM); and DSS (3% w/v) with Ca⁺⁺ (1 mM) and butyrate (1 or 10 mM) or dodecanoate (1 or 10 mM).</p