Cdc42-overexpressing mammary glands display characteristics associated with stromal activation. (A) Representative images of H&E-stained longitudinally sectioned TEBs from 1 week dox-treated Cdc42-overexpressing and control mammary glands. Scale bar = 100 μm. Graph depicts average stromal thickness (± SEM) at neck region of TEBs (n = 7,5; *P = 0.008). (B) Representative images of F4/80-immunostained TEBs from 1 week dox-treated Cdc42-overexpressing and control mammary glands (n = 4 to 5 animals per genotype, with at least three TEBs analyzed per mouse). Scale bar = 50 μm. Graph shows percentage of F4/80-positive stromal cells (± SEM). (C) Representative images of Masson’s trichrome-stained TEBs (blue) (n = 4 animals per genotype, with an average of three TEBs analyzed per mouse). Scale bar = 50 μm. Graph depicts quantification of collagen deposition (*P= 0.009). (D) Table of qRT-PCR Super Array results showing genes that were upregulated at least 1.5 fold in stromal cells isolated from Cdc42-overexpressing compared to control mammary glands. Data are representative of three animals per genotype. (E) qRT-PCR and new primer sets were used to validate several genes identified in the Super Array. Graph shows fold change in mRNA expression levels (*P = 0.0003, **P = 0.04, ***P = 0.003, #P = 0.001, ##P = 0.005). Data are representative of three animals per group. Cdc42, cell division cycle 42; TEB, terminal end bud.