Butyrate increases hPepT1 protein expression in Caco2-BBE monolayers.

<p>A) Caco2-BBE cells grown on filters were treated with 5 mM butyrate for 24 h and membrane and cytosol hPepT1 protein expression was analyzed by Western blot. Expressions of Na⁺/K⁺ ATPase and GAPDH were used as loading controls. B) Bar graphs represent the densitometric quantification of hPepT1 blots shown in (A). Values represent means±S.E. of four blots from independent experiments. **<i>P</i><0.005.</p

Ecto-phosphorylation of CD98 in CHO cell membranes enhances Jurkat cell-CHO cell interactions.

<p>CHO cells were transfected with the pcDNA3.1 TOPO/V5 vector (CHO/Vector) or V5-fused constructs of wild-type CD98 (CHO/CD98) or CD98 mutated at serines-305/307/309 (CHO/CD98[S^305/307/309A]) or sesrines-426/430 (CHO/CD98[S^426/430A]). (A) Western blot analysis of membrane extracts from transfected CHO cells using anti-CD98 or anti-V5 antibody (upper panel). (B) Cell binding assays of fluorescent-labeled Jurkat cells on transfected CHO cells were performed under the ecto-kinase assay condition as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003895#s2" target="_blank">Materials and Methods</a>. Data are means±S.E.M of three independent determinations. **P<0.005 vs CHO/CD98; ***P<0.001. (C) Ecto-phosphorylation of CD98 expressed in CHO cells by intact Jurkat cells. Ecto-kinase assays were performed by applying Jurkat cells in the ecto-kinase buffer containing 10 µCi [γ-³²P]ATP to CHO cells for 10 min at 37°C. After washes, CHO cells were lysed, immunoprecipitated for CD98 and analysed by SDS-PAGE. Radiolabeled bands were detected by autoradiography. Bar graphs show the relative intensity of radiolabeled bands in the upper panel. Data are means±S.E.M of three determinations. ***P<0.001 vs CHO/CD98.</p

Transcription factors Cdx2 and CREB are crucial for butyrate-induced hPepT1 promoter activity in Caco2-BBE cells.

<p>Caco2-BBE cells were transfected with different constructs of hPepT1 promoter that are point mutated at CREB, Sp1, Cdx2, or AP1 sites. Cells were then treated with 5 mM butyrate for 24 h and luciferase activity relative to hPepT1 promoter activity was measured. Data were normalized by Renilla activity and expressed as fold increase in response to butyrate. Values represent means±S.E. of three determinations. *<i>P</i><0.05; ***<i>P</i><0.001.</p

Lack of NPY inhibits DSS-induced inflammation.

<p>Mice were randomized into 4 groups: WT water or DSS (3%) and <i>NPY</i>^−/− water or 3% DSS and sacrificed on day 6. (A) Representative hematoxylin eosin (H/E) stained sections of colon from each group are shown. Arrows represent neutrophil infiltration and crypt damage. Magnification 10X and 40X. Disease severity was assessed as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003304#s2" target="_blank">Materials and Methods</a> and expressed in terms of (B) Histological score (C) Clinical score and (D) Myeloperoxidase activity. Each bar represents mean±S.E; n = 4, Significant difference from all other groups represented as * p<0.05, ** p<0.01.</p

Butyrate increases hPepT1-mediated inflammation in Caco2-BBE cells.

<p>Caco2-BBE cells were treated or not (vehicle) with 5 mM butyrate for 24 h. Cells were then washed, incubated in serum-free medium overnight and stimulated with 1 µM fMLP for the indicated times. Cell lysates were analyzed by Western blot using IκB-α antibody. Bar graphs represent the densitometric quantification of IκB-α blots. Values represent means±S.E. of four blots from independent experiments. **<i>P</i><0.005.</p

Butyrate increases hPepT1-mediated peptides uptake in Caco2-BBE monolayers.

<p>Caco2-BBE cells were grown on filters and apically treated with 5 mM butyrate for the indicated times. Uptake experiments were performed with 20 µM [¹⁴C]Glycine-Sarcosine±20 mM Glycine-Leucine at an apical pH of 6.2 and basolateral pH of 7.4 for 15 min at room temperature, and radioactivity was then measured. Values, expressed as fold increases compared with untreated cells, represent means±S.E. of three determinations. *<i>P</i><0.05; **<i>P</i><0.005 <i>vs</i> untreated cells. B) Caco2-BBE cells were grown on filters and apically treated with (○) or without (•) 5 mM butyrate for 24 h. Uptake experiments were performed using 20 nM, 120 nM, 10 µM, 100 µµ, 400 µµ and 1 mM of [³H]KPV. Results represent means±S.E. of three determinations.</p

Hematoxylin-stained colon sections of mice treated with DSS at 0, 1, 3, 5, 9 (withdraw after 5 days treatment, 4 days to recover) and 14 (withdraw after 5 days treatment, 9 days to recover) days, with magnification of 20 times.

<p>(A): Distal colon; (B) Proximal colon.</p

Butyrate transcriptionally up-regulates hPepT1 expression in Caco2-BBE cells.

<p>Caco2-BBE cells were treated with 5 mM butyrate for 24 h and hPepT1 mRNA levels were assessed by A) semi-quantitative RT-PCR and B) real-time RT-PCR. Values represent means±S.E. of three determinations. **<i>P</i><0.005 <i>vs</i> control. To examine butyrate effect on the stability of hPepT1 mRNA, cells were pre-incubated with 5 µg/ml Actinomycin D (AcD) for 30 min and then treated with butyrate for 24 h. C) Total RNA was analyzed by Northern blot using a probe specific to the hPepT1 transcript. RNA loading controls were shown as bottom panel. D) hPepT1 mRNA levels were quantified using real-time RT-PCR. Values expressed as normalized cycling threshold values relative to untreated (0 h) cells represent means±S.E. of three determinations.</p

Phosphorylation of recombinant CD98 increases its interaction with Caco2-BBE cells and promotes epithelial cell adhesion.

<p>(A) Recombinant wild-type CD98 or its mutated forms CD98[S^305/307/309A] and CD98[S^426/430A] (2 µg each) were applied to <i>in vitro</i> kinase assays using 50 µM ATP and ecto-protein kinases (ePKs) purified from Jurkat cells in the presence or absence of 10 µM of the ePK inhibitor K252b. Caco2-BBE cells were seeded on the electric cell-substrate impedance-sensing electrodes coated with these samples (2×10⁵ cells/electrode). Capacitance was measured at 40 kHz, 1 V. Each microscopic image taken 8-h post-seeding is a representative of quadruplicate electrodes. Bars, 50 µm. Half time (<i>t</i>_1/2) and spreading rate (<i>s</i>) of the cells were determined for each electrode. Data are means±S.E.M of three determinations. (*<i>P</i><0.05; **<i>P</i><0.005) <i>vs</i> CD98+ePKs (black bar). (B) Cell attachment assays were performed in 96-well high-binding plates. Wells were coated with 10 µg/ml of BSA (control), recombinant wild-type CD98 or its mutated form CD98[S^305/307/309A] and CD98[S^426/430A]. Some wells were coated with the proteins pre-applied to <i>in vitro</i> kinase assays with ePKs from Jurkat cells in the presence or absence of K252b. After incubation for 3 h at 37°C, the plate was washed and blocked for 1 h at 37°C. Caco2-BBE cells were added (2×10⁵ cells/well) for 3 h at 37°C. After washes, attached cells were quantified by a hexosaminidase-based calorimetric reaction. Data were subtracted from background, and the numbers of attached cells were determined from a standard curve generated using known numbers of Caco2-BBE cells. Data are means±S.E.M of three determinations. (*<i>P</i><0.05; **<i>P</i><0.005) <i>vs</i> CD98+ePKs; ***<i>P</i><0.001; NS, not statically significant.</p

The extracellular C-terminal tail of CD98 expressed in CHO cell membranes is important for Jurkat cell-CHO cell interactions.

<p>(A) Schematic representation of variant CD98 constructs used in this study: chimeras of CD98 with CD69 (C98-T98-E69 contains the cytoplasmic and transmembrane domains of CD98 and the extracellular domain of CD69; C69-T69-E98 contains the cytoplasmic and transmembrane domains of CD69 and the extracellular domain of CD98). (B) and (C) Western blot analysis using anti-CD98 (B), anti-CD69 (C) or anti-V5 (C) antibody of membrane extracts from CHO cells stably transfected with the variant constructs. (D) Cell binding assays of fluorescent-labeled Jurkat cells on transfected CHO cells under the ecto-kinase assay condition as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003895#s2" target="_blank">Materials and Methods</a>. Data are means±S.E.M of three determinations. *<i>P</i><0.05; (***<i>P</i><0.001; NS, not statically significant) <i>vs</i> CHO/CD98.</p