Regulation of EGFR Signal Transduction conditions for an additional 48 h. Select cultures were transfected using nucleofection with GFP-H-RasG12V prior to being seeded into the 96-well plate. Cell proliferation was measured using CyQUANT cell proliferation assay. Results are representative of 3 independent experiments. Data are expressed as mean6SEM (n = 9 samples per treatment). Statistical significance between treatments as indicated by different letters (P,0.05) was determined using ANOVA and Tukey’s test of contrast. No comparisons were made between proliferation of wild-type and EGFR2/2 cells. B) Putative model: Under control conditions, EGFR is enriched in liquid ordered lipid raft domains of the cell. Upon stimulation, ligand bound receptors dimerize and transphosphorylate. The phosphorylated tyrosine residues then serve as docking sites for adaptor proteins, which leads to the activation of downstream targets, including Ras. Signaling is efficient due to the lateral organization of EGFR and downstream signaling partners. Upon fatty acid treatment, the cell incorporates DHA into plasma membrane phospholipids. DHA is sterically incompatible with cholesterol and perturbs liquid ordered domains resulting in a change in the lateral organization of EGFR. Ligand-induced receptor dimerization and phosphorylation is increased, along with receptor ubiquitination, internalization, and degradation. The altered lateral and subcellular organization of EGFR results in inefficient cell signaling. Suppression of EGFR signaling causes altered cellular function, including reduced cell proliferation. doi:10.1371/journal.pone.0039682.g007 the EGF-stimulated recruitment of Grb2 to the plasma membrane (Figure 5A), indicating that DHA does not affect the ability of phosphorylated EGFR to recruit intracellular signaling mediators. We subsequently demonstrated that the breakpoint in the DHA- mediated suppression of EGFR signaling was at the level of Ras activation (Figure 5B). Our lab has previously shown that DHA suppresses the levels of activated GTP-bound Ras under basal conditions [75]. We have extended these observations by Figure 8. Fish oil feeding suppresses colonic epithelial EGFR signaling in mice. 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 mean6SEM 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 = 22225 mice per diet. Statistical significance between diets was determined using Student’s t-test. CO, corn oil; FO, fish oil. doi:10.1371/journal.pone.0039682.g008 PLoS ONE | www.plosone.org 12 June 2012 | Volume 7 | Issue 6 | e39682