Figure 2. HRAS(G12V) transforms Ink4a/Arf^−/− MECs to form triple-negative mammary tumors.

A, Left, Tumor-bearing mouse that was inoculated with HRAS(G12V)-introduced Ink4a/Arf^−/− MECs into the right inguinal mammary fat pad. Arrow indicates tumor. Right, Intraperitoneal image of the same mouse. Tumor is delineated with a dotted line. B, Tumor-free survival after mice underwent transplantation with 1.0×10⁵ mammosphere cells with each indicated profile. The P value was obtained by log-rank statistical analysis. C, Microscopic images of induced-tumor sections with hematoxylin and eosin staining (upper left and lower images) and periodic-acid Schiff (PAS) staining (upper right). A mitotic cell (arrow) and a PAS-positive mucus deposit (arrowhead) are visible in the upper images. Central necrosis (arrow) and pushing border (arrowheads) are visible in the lower images. D, Left, Immunohistochemical staining (IHC) of induced tumor with anti-HRAS antibody and with isotype rabbit IgG (Inset). Right, Western blot analysis of HRAS in Ink4a/Arf^−/− MECs and tumor cells. β-actin is shown as a loading control. E, F, IHC of induced tumor with antibodies (F) to keratin 14, keratin 18, EGFR, and phosphorylated ERK (p-ERK), and (E) to estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor-2 (HER2). The images in the right columns of E and F are positive controls except for p-ERK in (F). Controls for ER, PR, HER2, K14, and K18 are from mouse mammary ducts; for EGFR is from mouse endometrium. For p-ERK, the peritoneal wall in the same section is shown as a negative control. Scale bars, 50 μm.