Expression of cortactin in human colon adenocarcinomas samples.

<p>(<b>A</b>) Total RNA was extracted from frozen samples of normal and tumor tissues and cortactin mRNA expression was determined using quantitative RT-PCR. The box plots depict relative quantities of cortactin normalized to GAPDH in normal and tumor tissues (n = 37 for normal samples, n = 44 for tumor samples). (<b>B</b>) DNA gel analysis of PCR products obtained from qPCR analysis (representative of 37 normal tissues, 5 benign tumors, and 39 malignant tumors). (<b>C</b>) The tissue lysates of matched pairs (N-normal, M-malignant) of colon specimens prepared from the same tumor samples as in (A) were analyzed the expression of pTyr⁴²¹-cortactin (pTyr⁴²¹-CTTN) and total cortactin by Western blotting. Representative results are shown from three matched pairs. β-actin was used as a loading control. (<b>D</b>) Representative pTyr⁴²¹-cortactin and total cortactin immunostaining of colon tumor specimens. Tissue array containing a series of colon carcinomas were stained for pTyr⁴²¹-CTTN and total cortactin. The staining was mostly cytoplasmic for total cortactin, whereas pTyr⁴²¹-CTTN showing increased staining intensity at the plasma membrane.</p

Curcumin impairs the physical interaction between cortactin and p120 catenin (CTNND1).

<p>HCT116 cells were treated with 50 µM curcumin for 15 min and pre-cleared lysates were immunoprecipitated using anti- pTyr⁴²¹-CTTN or anti-CTTN (total) rabbit polyclonal antibodies. Immunoprecipitated complexes were analyzed by Western blotting for the presence of CTNND1. Lower three panels demonstrate even input of CTTN, CTNND1 and GAPDH in the cell lysates used for co-immunoprecipitation. All images representative of three independent experiments.</p

Curcumin induces cortactin dephosphorylation in colon cancer cells.

<p>(<b>A</b>) T84, HCT116, SW480 and HT29 cells were treated for 15–60 min with DMSO (CTRL) or 50 µM curcumin and pTyr⁴²¹ –CTTN and total CTTN expression was analyzed by western blotting. GAPDH was used as a loading control. (<b>B</b>). Immunofluorescent analysis of pTyr⁴²¹ –CTTN (green) and total CTTN (cyan) in HCT116 cells treated with DMSO (top panels) or with 50 µM curcumin for 15 min (bottom panels). Nuclei (red) were counterstained with Sytox Red (Life Technologies). 40 X magnification. Further cropped and magnified images are provided as indicated by the dotted lines. (<b>C</b>). Western blot analysis of cortactin, actin and GAPDH proteins from DMSO and curcumin treated cell fractions of HCT116 cells. Total cell lysates were used to represent total protein input. Cytosolic and cytoskeletal proteins were extracted using Cell Fractionation kit (Cell Signaling, MA) and quantification of the blots are summarized in graphs. The images were scanned using C-Digit and quantified using Image Studio Digits (LI-COR Biosciences, NE). The data are expressed as a ratio to total protein (mean ± SD). * p<0.05 DMSO vs. curcumin; Student's T-test. All images are representative of three independent experiments. (<b>D</b>). The effects of curcumin in early (15 min) and late (30 min) response of HCT116 and SW480 cells to serum stimulation. The cells were serum-starved overnight and subsequently stimulated with serum-supplemented medium containing DMSO or 50 µM curcumin. pTyr⁴²¹ –CTTN and total CTTN were determined by Western blotting. Representative blots from three experiments are shown. GAPDH served as a loading control. In all cases, CTRL represents DMSO treatment for the longest incubation time in a respective study.</p

Curcumin dephosphorylates pTyr⁴²¹ –CTTN through activation of PTPN1 in colon cancer cells.

<p>(<b>A</b>) Expression of PTPN1 protein in HCT116, HT29 and SW480 cells. GAPDH served as a loading control. (<b>B</b>) PTPN1 activity in DMSO (CTRL) or curcumin (CUR)-treated HCT116 cells. Equal amounts of protein lysates were assayed for phosphatase activity against DADEpYLIPQQG peptide as substrate as described in Materials and Methods. PTPN1 activity calculating in U/mg protein is shown as means ± SEM from three separate experiments (* p<0.05, CTRL vs. CUR; Student's T-test). (<b>C</b>) Dephosphorylation of pTyr⁴²¹-CTTN by curcumin in the presence of PTPN1 inhibitor. Cells were subjected to treatment with 50 µM curcumin or DMSO without or with increasing concentrations of PTPN1 inhibitor for 15 min followed by western blot analysis of pTyr⁴²¹-CTTN. GAPDH served as a loading control. (<b>D</b>) Confocal immunofluorescence analysis image of pTyr⁴²¹-CTTN in HCT116 cells treated with DMSO (left panels) or curcumin (right panel) in the absence (top panels) or presence of 50 µM PTPN1 inhibitor. (<b>E</b>) Expression of CTTN in HTC116 cells treated as in (D) was evaluated by western blot analysis of cell surface biotinylated fraction and total cell lysate. GAPDH served as a loading control. All images are representative of three independent experiments.</p

Overexpression of cortactin promotes migration in colon cancer cells; inhibition by curcumin.

<p>Ectopic expression of cortactin was accomplished by adenoviral delivery (Ad-CTTN) and elevated expression confirmed by qRT-PCR (<b>A</b>) and western blotting (<b>B</b>). (<b>C</b>) Enhanced migration of HCT116, SW480, and HT29 cells transduced with Ad-CTTN. (<b>D</b>) HCT116, and SW480, but not HT29 cells treated with curcumin showed significantly reduced migration. * p<0.05 Control vs. Ad-CTTN or Control vs. Curcumin; Student's T-test. n.s. – not statistically significant. Bars represent mean ± SEM of three independent experiments.</p

Curcumin physically interacts with PTPN1.

<p>(<b>A</b>) Synthesis of biotinylated curcumin derivative. (<b>B</b>) Comparison of the effects of unmodified curcumin (CUR) and biotinylated curcumin (BIO-CUR) in equimolar concentrations (50 µM) on pTyr⁴²¹-CTTN in HCT116 cells treated for 15 min. GAPDH served as a loading control. (<b>C</b>) Western blot analysis (left panel) and quantitative densitometry of the PTPN1 protein from pull-down experiment with biotinylated crcumin. HCT116 cell lysates were prepared with RIPA buffer and combined with curcumin (CUR), biotin linker (compound 3), or biotinylated curcumin (BIO-CUR; compound 4; all at 50 µM) for 30 min at room temperature. Protein fraction recovered with streptavidin agarose beads was analyzed by western blotting for the presence of PTPN1. The data in the summary graph are expressed as fold change compared to linker treated samples from more extended exposures to visualize background PTPN1 signal (mean ± SD). * p<0.05 linker vs. biotinylated curcumin; Student's T-test. All images representative of three independent experiments.</p

Identification of cortactin as curcumin target in colon cancer cells.

<p>(<b>A</b>) Table with 13 plasma membrane-associated proteins identified by QTOF-MS/MS as significantly decreased in T84 cell monolayers treated with curcumin. Data shows average number unique peptides identified from three different experiments ± SD. (<b>B</b>) Quantitative analysis of cortactin expression in the T84 cells by M/S. The spectrum values were obtained from three different experiments. The quantification data for cortactin protein was derived from M/S using Scaffold proteome software (version Scaffold_3.1.2). Data are means ± SE, *<i>p</i><0.05 compared with untreated cells, Student's t test. (<b>C</b>) Confirmation of CTTN protein expression by western blotting with biotinylated cell surface protein fraction prepared from T84 cells treated with DMSO (CTRL) or 50 µM curcumin for 1–4 hours. CD71 (transferrin receptor) was used as a loading control. CTRL represents cells treated with DMSO for 4 hours.</p

Summary of beta diversity analysis.

<p>A non-parametric ANOSIM (Analysis of Similarity) method was applied to dataset presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0152044#pone.0152044.g003" target="_blank">Fig 3D</a> (PBS or adoptive T cell transfer in Rag2^-/- or DKO mice, from day 0 to day 13). Among single variables, only the genotype of the T cell recipient mice was significantly different based on a UniFrac beta diversity metric. In multivariate analysis, genotype was the single contributor to differences in beta diversity. Each row is a comparison of microbial diversity done with one or more factors, indicated by the labels. Beta diversity was calculated using Analysis of Similarity (ANOSIM) and significance was considered reached when both a and b were true as follows: a) p-value < 0.05 and b) R value > 0.3.</p

Changes in microbiota composition in Rag and DKO mice before and after adoptive T cell transfer.

<p>(<b>A</b>) Endpoint taxonomic analysis (day 13) of the relative abundance of bacterial families in fecal samples; (<b>B</b>) Endpoint qPCR analysis of selected clusters of <i>Clostridia</i>; (<b>C</b>) Endpoint analysis of alpha diversity (Margalef index and the number of observed OTUs); (<b>D</b>) Principal Coordinate Analysis (PCoA) of unweighted UniFrac distances between of microbiota samples (first two principal coordinate axes versus time shown) in Rag2^-/- and DKO mice with or without adoptive T cell transfer over the course of the experiment. Samples were collected every 2 days post injection. Prematurely ended lines (DKO+AT) represent mice sacrificed earlier due to a critical body weight loss (≥20%). Points represent individual mice. In <b>B</b>-<b>C</b>, statistical analyses were performed using one-way ANOVA (P value indicated in each panel) with Fisher’s LSD post-hoc test. Asterisks in all panels indicate statistical significance: * P < 0.05, ** P < 0.01, *** P< 0.001, **** P < 0.0001.</p

After adoptive T cell transfer, NHE3^-/-Rag2^-/- DKO mice develop severe colitis, which is alleviated by broad-spectrum antibiotic treatment.

<p>(<b>A</b>) Changes in body weight (% change of initial value 13 days post-transfer; boxes extend from 25th to 75th percentiles of values, whiskers represent min and max value, line within the box represents the mean value); (<b>B</b>) Histological colitis scoring (dots represent individual mice and lines mean value ± SD); (<b>C</b>) Representative H&E images of distal colon from respective experimental groups; (<b>D</b>) qPCR analysis of mucosal expression of proinflammatory cytokines in Rag/PBS (N = 10), Rag/AT (N = 8) and DKO/PBS (N = 9) and DKO/AT (N = 8) mice (bar graphs represent mean ± SD values); (<b>E</b>) Intestinal mucosal permeability measured by FITC-labeled dextran tracer flux (N = 3–6). Statistical analysis was performed using one-way ANOVA (ANOVA P value is indicated in each panel) with Fisher’s LSD post-hoc test. Asterisks in all panels indicate statistical significance: * P < 0.05, ** P < 0.01, *** P< 0.001, **** P < 0.0001. Brackets point to differences between indicated groups. Asterisks without brackets indicate a statistical difference from all other groups within the panel. Each experimental groups is color coded in a way consistent in the remaining figures.</p