DHA reduces localization of EGFR to lipid rafts.

<p>A) YAMC cells were untreated (control) or treated with 50 µM BSA-complexed fatty acids (LA or DHA) for 72 h (12 flasks per treatment). For the final 16–18 h, cells were incubated with low serum media (0.5% FBS) with the same concentration of fatty acids. Cells were harvested from each flask, pooled (n = 12), and the plasma membrane (PM) was isolated. Following isolation, the plasma membrane was fractionated into 3 distinct fractions, high density membrane (HDM), intermediate density membrane (IDM), and lipid raft enriched membrane (LR) by gradient ultracentrifugation. Fractions were collected and an equal amount of protein from each fraction was analyzed by Western blotting using antibodies against EGFR, caveolin-1, and clathrin or using peroxidase conjugated cholera toxin B subunit (for GM-1). Quantification of band intensity was performed, and data are presented as the relative amount of EGFR in each fraction, with the sum of each fraction equaling 100. Western blots are representative of 2 independent experiments. C, control; LA, linoleic acid; DHA, docosahexaenoic acid; PM, plasma membrane; HDM, high density membrane; IDM, intermediate density membrane; LR, lipid raft enriched membrane. B) YAMC cells were treated with 50 µM BSA-complexed fatty acids for 72 h. Twenty-four h after initiating fatty acid treatment, cells were co-transfected with RFP-tH and EGFR-mGFP. Approximately 32 h after transfection, cells were incubated in low serum media (0.5% FBS) overnight prior to imaging. Images are representative of 4 independent experiments. Whole cell images of each individual channel and the merged images are shown on the left. High magnification images of the plasma membrane are shown on the right. Mander’s colocalization coefficient was calculated at the plasma membrane for the amount of EGFR-mGFP (green) colocalizing with RFP-tH (red) using Nikon Elements AR 3.2. The coefficient is the mean of n = 30–40 cells per treatment. Statistical significance between treatments (*<i>P</i><0.05) was determined using ANOVA and Tukey’s test of contrast. Bars,10 µm.</p

Fish oil feeding suppresses colonic epithelial EGFR signaling in mice.

<p>Carcinogen (AOM) and DSS-treated mice were fed a diet enriched in fish oil (high in DHA) or corn oil (contains no DHA) for a total of 15 weeks. A) Whole cell lysates were isolated from scraped colonic mucosa. Equal concentrations of protein were assessed by Western blotting for total and phosphorylated EGFR (Tyr1068). Additionally, EGFR was immunoprecipitated from mucosal lysates and Western blotted for phosphorylated tyrosine residues and EGFR. Also, whole cell lysates were probed by Western blotting for total and phosphorylated downstream mediators of EGFR signaling, including ERK1/2, STAT3, and Akt. Quantification of band volume was performed and data are presented as mean±SEM of the ratio of phosphorylated protein to total protein and normalized to CO, n = 12 mice per diet. B) Colon lesions were fixed in 4% paraformaldehyde, embedded in paraffin, stained with hematoxylin-eosin, and evaluated by a board certified pathologist. The mean colon tumor entity number, including adenomas and adenocarcinomas, per mouse in each diet treatment is presented, n = 22−25 mice per diet. Statistical significance between diets was determined using Student’s <i>t</i>-test. CO, corn oil; FO, fish oil.</p

DHA alterations to EGFR function require enrichment of DHA in the plasma membrane.

<p>A) Control treated YAMC cells were incubated with low serum media overnight. Select cultures were then treated with 10 µM U0126 for 2 h. Untreated and U0126 treated cultures then underwent stimulation with 25 ng/mL EGF for 10 min. Lysates were assessed by Western blotting for phosphorylation of Erk1/2 and EGFR (Tyr 1068). B) Following stimulation with EGF, cells were subjected to chemical crosslinking prior to harvesting cell lysates. Cell lysate was assessed by Western blotting for EGFR. EGFR dimers were identified as bands with twice the molecular weight of EGFR monomers. C-D) For “wash out” experiments, YAMC cells were either untreated or treated with 50 µM DHA for 72 h. Select DHA treated cultures were then washed and incubated for an additional 40 h with either untreated media or 50 µM LA. For the final 16–18 h, cells were incubated with low serum media (0.5% FBS) followed by stimulation with 25 ng/mL EGF then harvested. C) Membrane lipids were extracted and enrichment of membrane phospholipids with DHA was quantified by capillary gas chromatography/mass spectrometry. D) Lysates were assessed by Western blotting for phosphorylated EGFR (Tyr1068). The blot is representative of 3 independent experiments. Data are presented as mean±SEM of phosphorylated EGFR, normalized to control. Statistical significance between treatments as indicated by different letters (<i>P</i><0.05) was determined using ANOVA and Tukey’s test of contrast. C, control; AA, arachidonic acid; EPA, eicosapentaenoic acid; DHA, docosahexaenoic acid; LA, linoleic acid.</p

DHA mediates increased EGFR internalization and degradation.

<p>YAMC cells were incubated with untreated media or media supplemented with 50 µM BSA-complexed DHA for a total of 72 h. For the final 16–18 h, cells were incubated with low serum media (0.5% FBS). A) Cell surface proteins were labeled with EZ-Link Sulfo-NHS-SS-Biotin followed by stimulation with 25 ng/mL EGF for 0–30 min. After stimulation, cells were washed and biotin remaining on the cell surface was cleaved. Cell lysates were harvested, and biotinylated EGFR was quantified by ELISA using streptavidin coated plates and anti-EGFR antibody. B) Cell surface EGFR was assessed by treating cells the same as in A, and harvesting without stimulating with EGF or cleaving cell surface biotin. C) Cells were stimulated with 25 ng/mL EGF for 0–30 min and harvested. EGFR was immunoprecipitated from the total cell lysate, assessed by Western blotting for ubiquitin, and quantification of band intensity was performed. All results are representative of at least 3 independent experiments. Data represent mean±SEM. In (A) and (C), data are normalized to time 0. In B), data are normalized to control (no fatty acid). Statistical significance between treatments (*<i>P</i><0.05. **<i>P</i><0.01) was determined using Student’s <i>t</i>-test. C, control; DHA, docosahexaenoic acid.</p

DHA impairs EGF-induced activation of Ras.

<p>A) YAMC cells were treated with 50 µM BSA-complexed fatty acids for 72 h. Twenty-four h after initiating fatty acid treatment, cells were transfected with Grb2-YFP. For the final 16–18 h, cells were incubated with low serum media (0.5% FBS) with the same concentration of fatty acids and imaged using TIRF microscopy. Cells were stimulated with 100 ng/mL EGF and imaged every 5 sec. Images are representative of 4 independent experiments (n = 22−25 cells/treatment). Changes in total surface intensity were quantified using Nikon Elements AR 3.2. Fluorescence images and the respective surface intensity plots are shown. Surface intensity plots were generated in Nikon Elements AR 3.2 (the scale is from blue (lowest intensity) to red/pink (highest intensity). Data are presented as mean±SEM normalized to control. Bars, 10 µM. B) YAMC cells were treated with 50 µM BSA-complexed fatty acids for 72 h. For the final 16–18 h, cells were incubated with low serum media (0.5% FBS) with the same concentration of fatty acids. Cells were stimulated with 25 ng/mL EGF for 2 min and harvested. GTP-bound Ras was isolated using a GST pull-down assay. Isolated GTP-bound Ras was then analyzed by Western blotting for pan Ras. Isolated GTP-bound Ras was additionally analyzed by Western blotting for H, K, and N-Ras. Blots are representative of 3 independent experiments. Quantification of band volume was performed. Data are expressed as mean±SEM (n = 3), normalized to control. Statistical significance between treatments (<i>P</i><0.05) as indicated by different letters was determined using ANOVA and Tukey’s test of contrast. C, control; LA, linoleic acid; DHA, docosahexaenoic acid.</p

DHA uniquely alters EGFR function.

<p>YAMC cells were treated with control or 50 µM BSA-complexed fatty acids (LA or DHA) for 72 h. For the final 16–18 h, cells were incubated in low serum media (0.5% FBS) with the same concentration of fatty acids. Cells were stimulated for 0–30 min with 25 ng/mL EGF and subsequently harvested. Equal concentrations of protein from the whole cell lysates were analyzed by Western blotting for A) phosphorylated EGFR, B) phosphorylated ERK1/2, and C) phosphorylated STAT3. Each immunoblot is representative of 3 independent experiments. Quantification of band volume was performed and data are presented as mean±SEM and normalized to time 0 (n = 3). Statistical significance between treatments (*<i>P</i><0.05; **<i>P</i><0.01) was determined using Student’s <i>t</i>-test. D) Additionally, YAMC cells were treated with 50 µM BSA-complexed fatty acids (AA, EPA, or DHA) for a total of 72 h. For the final 16–18 h, cells were incubated in low serum (0.5% FBS) then stimulated for 10 min with 25 ng/mL EGF. Whole cell lysates were separated assessed by Western blotting for phosphorylated (Tyr1068) EGFR. A representative blot from 3 independent experiments is presented. Data are expressed as mean±SEM of phosphorylated EGFR, normalized to control (n = 3). Statistical significance between treatments (<i>P</i><0.05) is indicated by different letters and was determined using ANOVA and Tukey’s test of contrast. C, control; LA, linoleic acid; DHA, docosahexaenoic acid; AA, arachidonic acid; EPA, eicosapentaenoic acid.</p

DHA increases EGFR phosphorylation but suppresses activation of downstream signaling.

<p>YAMC cells were untreated (control) or treated with 50 µM BSA-complexed fatty acids (LA or DHA) for 72 h. For the final 16–18 h, cells were incubated in low serum media (0.5% FBS) with the same concentration of fatty acids. Cells were either unstimulated or stimulated for 10 min with 25 ng/mL EGF and subsequently harvested. A) Equal amounts of protein from whole cell lysates were Western blotted for total and phosphorylated (Tyr1068) EGFR. Additionally, EGFR was immunoprecipitated from total cellular lysates prior to Western blotting for EGFR and phosphorylated tyrosine resides. B) Whole cell lysates were analyzed by Western blotting for total and phosphorylated downstream mediators of EGFR signaling, including ERK1/2, STAT3, S6kinase, and Akt. Each blot is representative of 3–4 independent experiments with 3 replicates of each treatment per experiment. Quantification of band volume was performed and data are presented as mean±SEM. Data are expressed as the ratio of the phosphorylated protein to total protein and normalized to control. Statistical significance between treatments (*<i>P</i><0.05; **<i>P</i><0.01) was determined using ANOVA and Tukey’s test of contrast. C, control; LA, linoleic acid; DHA, docosahexaenoic acid.</p

Effect of diet on splenic CD4⁺ T cell polarization.

<p>Splenic CD4⁺ cells were purified by positive selection and cultured for 3 d under A) Th17 or B) Treg polarizing conditions (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049739#s2" target="_blank">Materials and Methods</a>, n = 3−4 TNBS treated mice/dietary group). Bars represent mean values ± SEM. Bars not sharing a common letter are significantly different (P≤0.05).</p

Visceral adipose tissue mRNA expression¹.

1<p>Values are means ± SEM (n = 5−10/dietary group). Data were analyzed by two-way ANOVA (main effects: diet and treatment). For all genes, there was no effect of treatment (i.e., TNBS versus vehicle, <i>P</i>>0.05), therefore, only the main effect of diet is shown. Within individual genes, values not sharing a lower case letter denote significant differences (<i>P</i>≤0.05). Data were normalized to ribosomal 18S.</p

Colon histological disease scores for TNBS-treated mice.

<p>Colonic mucosal injury (0–3) and inflammation (0–3) scores were assessed in a blinded manner by a board-certified pathologist (B. Weeks) and combined for a total score (0–6). Representative images (100 × magnification) are shown for the HF, HF-FO and LF TNBS-treated groups, respectively (panels A-C) and a representative image of a HF vehicle control (panel D) is shown. E) Combined injury/inflammation histological score within the distal colon (n = 10−14 TNBS treated mice/diet). Data were analyzed using the Kruskal-Wallis test followed by Wilcoxon two-sample testing, and bars represent median values. Bars not sharing a common letter are significantly different (P≤0.05).</p