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. 2013 Sep 19;9(9):e1003789. doi: 10.1371/journal.pgen.1003789

Figure 6. Amphiregulin is a chemoattractant for fibroblasts and helps prevent necrosis and tumor cell death.

Figure 6

(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 AREG 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 AREG. 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 AREG 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 AREG. 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.