PERK Inhibition in MCF10A Cells Favors Mammary Tumor Formation.

<p>(A) Western blot for ErbB1 (EGFR) and ErbB2 (Her2) levels in adhered β-Gal or PERKΔC cells show increased levels when PERK is inhibited. GAPDH was used as loading control. (B) β-Gal or PERKΔC cells were injected orthotopically into the contralateral abdominal mammary fat pads of 3 week-old female nude mice (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000615#s4" target="_blank">methods</a> for details). Post-implantation mice were monitored biweekly for tumor take and when detected tumor diameters were measured and the volume was calculated and plotted as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000615#s4" target="_blank">methods</a>. Note that none of the mice implanted with β-Gal-MCF10A cells developed tumor nodules. (C) Histology of mammary glands and tumors in mice implanted with β-Gal and PERKΔC expressing MCF10A cells. (C-a) H&E staining of a cleared mammary fat pad inoculated with β-Gal cells. Only adipose tissue and occasional stromal cells that were negative for β-galactosidase activity was observed. (C-b) H&E staining of a PERKΔC tumor lesion. Note the disorganization of the fibrotic-epithelial tissue. Arrows depict the presence of epithelial cells forming acinar or duct-like structures within the PERKΔC tumor lesion. (C-c) Higher magnification of intratumoral accumulations of cells without a defined architecture but comprised of a mixture of epithelial (larger nuclei and pink cytoplasm) and inflammatory cells (smaller darkly stained nuclei). (C-d and e) PERKΔC cells form acini-like structures with an empty lumen or show hyperplastic growth and a repopulated lumen (C-e, top left and right insets). (C-f) Anti-Myc (αMyc) staining in control β-Gal injected mammary fat pads. Note that only a light background signal is observed in adipocytes. (C-g) Histological section of a PERKΔC acinus-like structure stained with a non-specific IgG (arrow denotes the lack of staining in these epithelial cells) or with anti-Myc 9E10 mAb (C-h and C-i); arrows denote the brown staining generated by Myc-tag detection. (D) β-Gal (left panel) or PERKΔC (middle and right panels) cells grown on 2D coverslips and fixed and stained with a non-specific IgG (IgG) or with an anti-Myc 9E10 mAb (α-Myc). The Myc staining was characteristic of intracellular cistern distribution.