(A, B) Mammary gland tissue sections were prepared from parous Wap-Cre+/- Rosa26mTmG+/- female mice (n = 6) on day 21 post-weaning. These sections were stained with antibodies to cytokeratin 8 (CK8, red (A)) or cytokeratin 5 (CK5, red (B)), and GFP (green), and counterstained with DAPI (blue). Representative images are presented (upper panel, scale bar = 48 µm). Boxed area was magnified (lower panel, scale bar = 24 µm). (C) Summary of the study cohort showing the total number of Mapk8LoxP/LoxP Mapk9LoxP/LoxP (MEWT) and Wap-Cre+/- Mapk8LoxP/LoxP Mapk9LoxP/LoxP (MEKO) mice examined, the number of mice exhibiting mammary intraepithelial neoplasia (MIN) or tumors (*p=0.037, **p=0.0084; Fisher’s Exact Test) (left panel). The type of carcinoma is presented (right panel). (D) Extracts prepared from MEKO and Wap-Cre+/- Trp53LoxP/LoxP (JNKWT) tumor cells were subjected to immunoblot analysis using antibodies to JNK and α-Tubulin. The MEKO tumors examined were representative of adenocarcinoma (#1), tumors with characteristics of both adenocarcinoma and adenosquamous carcinoma (#2), and adenosquamous carcinoma (#3). (E, F) Representative hematoxylin and eosin (H and E) -stained sections of adenosquamous carcinomas (E) and adenocarcinomas (F) from MEKO female mice are presented. Scale bar = 100 µm. (G) Exome sequencing was performed on MEKO tumor cell lines (n = 3). Mammary tissue from a virgin female of the same genotype (Wap-Cre+/- Mapk8LoxP/LoxP Mapk9LoxP/LoxP) was used as the reference genome. Circos plots showing copy number variations (CNVs) in MEKO tumor cells are presented. The outermost ring shows chromosome ideograms. The next track indicates high (red) and moderate (yellow) impact single nucleotide variants and indels marked by rectangles and triangles, respectively. The innermost track shows chromosome amplifications and deletions, with red and blue lines indicating chromosomal fragments present at log2(ratio tumor/normal)>0.2 or log2(ratio tumor/normal)<−0.2, respectively.