Zn²⁺ induces a large current in FLCs.

<p>(<b>A</b>) Membrane potentials were recorded in current clamp configuration. Zn²⁺ induced rapid membrane depolarization in FLCs cultured from wild-type mice, but not <i>Gpr39</i>^−/− cells. (<b>B</b>) Bar chart summarizing Zn²⁺ induced membrane depolarization in FLCs as in panel A. p<0.01. (<b>C</b> and <b>D</b>) Zn²⁺ induced a large current in wild-type. Current was sampled at +80 and −80 mV. (<b>C</b>) shows a time-series plot for a typical Zn²⁺-induced current in FLCs (n = 21). (<b>D</b>) shows the corresponding I-V relationship. (<b>E</b> and <b>F</b>) are similar experiments using FLCs isolated from GPR39 knockout mice and show that no Zn²⁺-induced current was observed in <i>Gpr39</i>^−/− FLCs (n = 11).</p

GPR39-expressing cells.

<p>(<b>A</b>) a bright field image of cobblestone-like cells. Scale bar = 100 µm. (<b>B</b>) <i>Gpr39</i> mRNA levels were evaluated by Taqman gene expression analysis in both muscle tissues and cultured cells. After 7 days in culture, <i>Gpr39</i> levels were up-regulated 163.9 fold in the cultures (n = 3). (<b>C</b>) Cells were loaded with Fura2-AM. Shown are ratiometric images of Ca²⁺ signals before and after 100 µM Zn²⁺ challenge. [Ca²⁺]<i>_i</i> is indicated on a rainbow scale with blue representing low [Ca²⁺]<i>_i</i> and orange/red high [Ca²⁺]<i>_i</i>. (<b>D</b>) Ca²⁺ signals were elicited in cells cultured from wild-type mice, but not <i>Gpr39</i>^−/− mice. (<b>E</b>) Zn²⁺ induces Ca²⁺ signals in Ca²⁺ free bath solution. (<b>F</b>) Cells were pre-incubated with or without 1 µM thapsigargin to deplete intracellular Ca²⁺ stores. Thapsigargin treatment not only abolished Zn²⁺ elicited Ca²⁺ signals, but also resulted in a decrease in [Ca²⁺]_i after Zn²⁺ application, the mechanism of which is still unknown.</p

GPR39 activation is functionally linked with TMEM16A channel opening.

<p>(<b>A</b>) Time courses for the current measured at +80 mV from individual cells. Both Ionomycin (1 µM) and Zn²⁺ evoked currents in FLCs from WT mice (n = 11, top trace), while Zn²⁺ was ineffective in <i>Gpr39</i>^−/− FLCs (n = 6, bottom trace). The data are summarized in panel (<b>B</b>). (<b>C</b>) Knockdown of <i>Tmem16a</i> by siRNA in FLCs were verified by Taqman PCR (94% reduction in <i>Tmem16a</i> expression), whereas <i>Gpr39</i> expression were unaffected (n = 3). (<b>D</b>) Zn²⁺-induced Ca²⁺ responses were measured in FLCs. No difference was observed between control and <i>Tmem16a</i> siRNA transfected cells (n = 54 and 67). In contrast, the size of Zn²⁺-induced current was reduced to about 32% in transfected cells (n = 7 and 12).</p

Community Structure Comparison: Libshuff and Parsimony Statistical Analysis of Colon-Associated Bacterial Populations.

<p>Community Structure Comparison: Libshuff and Parsimony Statistical Analysis of Colon-Associated Bacterial Populations.</p

Comparison of physical and histological parameters and bacterial load of WT and Nod2 KO littermates following DSS damage.

<p><b>A.</b> Timelines and histology assessment for individual mice. No significant difference was observed between the two genotypes for physical parameters (body weight loss, colon length: not shown) nor histological scores between the two groups (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030273#pone.0030273.s004" target="_blank">Figure S4</a> for data from 2 additional independent experiments). <b>B.</b> Colon, mesenteric lymph node and spleen tissue-associated bacterial loads assessed by FACS 42 days following DSS damage. ** = p≤0.01 by Anova with Bonferroni's multiple comparison test. <b>C.</b> Residence of commensal bacteria in the muscle layer in Nod2 KO mice. Examples of bacterial staining by EUB338 FISH probes are indicated by closed arrows in these representative images.</p

Richness, diversity and taxonomic analysis of the WT and Nod2 KO colon tissue-associated bacterial communities.

<p>Top panel: Incidence of phyla from treatment groups of WT and Nod2 KO littermates. Mean +/− SEM (n = 4–6) for each group is shown. * p≤0.05, ** p<0.01 by 2 way ANOVA with Bonferroni's multiple comparison test. Bottom panels: the Chao and Shannon estimates for richness and diversity were calculated from individual mice from each group as indicated (n = 4–6). The mean +/− SEM are shown. No differences were statistically significant by ANOVA.</p

Phylogenetic tree visualisation of full length 16S rRNA sequences from WT and Nod2 KO mouse colon tissue 42 days post DSS or control as indicated.

<p>See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030273#s4" target="_blank">Materials and Methods</a> for parameters.</p

Temporal physical, histological and biomarker analysis of DSS damage.

<p>Mice were administered DSS in their drinking water from day 0 to day 5 and individual parameters outlined in each panel assessed by criteria described previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030273#pone.0030273-Smith1" target="_blank">[41]</a>. * = p≤0.05, ** = p≤0.01, *** = p≤0.001 by 1-way Anova with Bonferroni's multiple comparison test. <b>A.</b> Body weight (physiological parameter for progression of inflammation) expressed as mean +/− SEM, n = 7. <b>B.</b> Fecal Score (combined weighted score of intestinal damage based on the presence of blood in the stool and subjective assessment of diarrhea) as a physiological indicator of colon tissue damage expressed as mean +/− SEM, n = 7. <b>C.</b> Colon length (physiological parameter for progression of inflammation) expressed as mean +/− SEM, n = 7. Statistical comparisons are versus Naive (no DSS-treatment) mice. <b>D.</b> Histology Score (quantitative assessment of microscopic tissue damage identical to that described previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030273#pone.0030273-Smith1" target="_blank">[41]</a>) expressed as mean +/− SEM, n = 7. Statistical comparisons are versus Naive (no DSS-treatment) mice. <b>E.</b> Temporal cytokine (as indicated) profiles in colon homogenates from treated mice expressed as mean +/− SEM, n = 7. Statistical comparisons by Anova/Bonferroni. @ = p≤0.05 vs day 5, # = p≤0.05 vs day 8.</p

Proximity and progression of host/commensal interactions following DSS damage.

<p><b>A.</b> Localisation of commensal bacteria by EUB338 FISH probe analysis of FFPE sections from DSS-treated mice. Open arrows highlight examples of luminal or mucosal bacteria, closed arrows indicate bacteria associated with the muscle layers. E, epithelium. M, muscle. Bar = 50 µm. <b>B.</b> Quantification of colon tissue bacteria load in individual mice by FACS following DSS damage. * = p≤0.05, ** = p≤0.01, *** = p≤0.001 vs day 0 (no DSS treatment) by 1-way Anova with Bonferroni's correction.</p

Infiltration of colon by immune cells assessed by immunohistochemistry following DSS damage.

<p>Top panel provides timeline for the experiment with arrows indicating times of histological and immunohistochemical assessment. Representative images for each of the cell markers and from each of the time points are shown. H&E (haematoxylin/eosin histochemical stain), CD3 (T cell marker), F4/80 (macrophage marker) and GR-1/Ly-6G (granulocyte marker).</p