Amphiregulin is a chemoattractant for fibroblasts and helps prevent necrosis and tumor cell death.

<p>(A) Photomicrographs of mouse fibroblasts that have transversed a Boyden chamber in response to different concentrations of amphiregulin in the opposing chamber. Migration was measured 5 hours post plating. Scale bars represent 100 µm. (B) Amphiregulin promotes migration of mouse embryonic fibroblasts (MEFs) in a scratch wound-healing assay (photomicrographs at indicated time after initiation of assay). (C) Quantification of the Boyden migration assay. Data are expressed as the mean ± SEM. Asterisks indicate a significant difference (p<0.05) between experimental and control groups. (D) Quantification of migration in scratch wound healing assay (cells that had moved into the scratched area as percent of control). Asterisk indicates a significant difference between the control and experimental group (p<0.05). (E) Quantitative RT-PCR analysis of α-SMA expression (relative to GAPDH) in HFFF2 fibroblasts (gray bars) and Wi38 fibroblasts (red bars) upon treatment with amphiregulin. Asterisk indicates a significant difference between untreated HFFF2 fibroblasts and those treated at 50 and 100 ng/ml (p value 0.01 and 0.001, respectively). All data are expressed as the mean ± SEM. (F) Necrosis in Cal51+HFFF2 tumors with control shRNA or with shRNA targeting <i>AREG</i> as visualized by H&E staining. Dashed lines indicate necrotic areas. Scale bars represent 1 mm. (G) Quantification of necrosis in Cal51+HFFF2 tumors with control shRNA or with shRNA targeting <i>AREG</i>. Necrotic area was calculated from five different tumors per group. Data are expressed as the mean ± SEM. Asterisks indicate a significant difference (p = 3e-4) between experimental and control groups. (H) EGFR activation (phosphorylation) in Cal51+HFFF2 tumors with control shRNA or with shRNA targeting <i>AREG</i> detected by immunostaining using an antibody to phospho-EGFR (Tyr1068). Scale bars represent 50 µm. (I) Quantification of phospho-EGFR positive tumor cells in Cal51+HFFF2 tumors with control shRNA or with shRNA targeting <i>AREG</i>. Areas positive for pEGFR were calculated from five different fields of five different tumors per group. Asterisk indicates that the experimental group is significantly different than the control (p = 0.007). Data are expressed as the mean ± SEM. (J) Effects of amphiregulin on the viability of Cal51 cells plated on non-adhesive plates and cultured for 24 hours (anoikis assay). Viability was determined by measuring calcein AM uptake. Asterisk indicates that the experimental group is significantly different than the control (p<0.05). Data are expressed as the percentage of viable cells normalized to control with the error bars representing SEM.