(A) ERα is expressed in murine cervical cancers. Archival PFA-fixed and paraffin-embedded female reproductive tracts of indicated mice treated with 17β-estradiol were stained for ERα (upper panel) or ERβ (lower panel). Stromal cells stained for ERα is shown in the inset. Shown are representatives of more than six cancers in each transgenic mouse. More than 700 cancer cells in at least three different areas of each cancer were examined for positive staining. Nuclei were counterstained with hematoxylin. Black lines indicate basement membrane separating epithelium from underlying stroma. (B) ERα is expressed in human cervical cancers. Formalin-fixed and paraffin-embedded human cervical tissue sections were stained as described in (A). Shown is representative of three HPV-positive and four HPV-negative cervical cancers.

(A) Estrogen-treated K14E7/ERα^−/− mice lack ASM. H&E-stained reproductive tracts of mice described in Table 1 were examined for the presence of ASM. Representatives of each genotype are shown and arrowheads indicate ASM. (B) E6 and E7 contribute to ASM independently. H&E-stained archival reproductive tracts of indicated mice that are treated with 17β-estradiol (+E₂) for 6 months (top panel) or untreated (-E₂) (middle panel) were analyzed for the presence of ASM (20). Reproductive tracts of ovariectomized and untreated K14E7 mice were also examined (bottom panel). Arrowheads indicate ASM and representatives of each group of mice are shown. Number of ASM-positive mice over total number of mice analyzed is shown at right lower corner of each image.

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(A) Endocervix and ectovervix are preserved in ERα^−/− mouse. Female reproductive tracts from mice of the indicated genotypes were stained with H&E. Asterisks in black, red, and blue denote the endocervical septum, endocervical canal, and ectocervix, respectively. (B) ERα^−/− mice retain cells positive for both p63 and K8. Shown are high power microscopic images of the transformation zone from paraffin-embedded sections of female reproductive tracts from mice of the indicated genotype that were doubly stained for p63 (red) and K8 (green). Dotted lines indicate basement membrane separating epithelium from underlying stroma. Note the presence of p63/K8 double positive reserve cells positioned underneath the K8-positive, p63-negative columnar epithelium in both the ER-sufficient and ER-deficient mouse tissues. (C) Shown are high power microscopic images of the transformation zone from a paraffin-embedded section of the female reproductive tract of an ERα^+/+ mice triply stained for p63 (blue), ERα (red), and K8 (green). Dotted lines indicate basement membrane separating epithelium from underlying stroma. Note in left panel that the reserve cells (indicated by arrowheads) underlying the K8 positive, p63-negative columnar epithelium again are double-positive for p63 and K8. Note in middle panel that reserve cells (see arrowheads) are positive for both K8 and ERα. Note in right panel (merge of K8, p63 and ERα staining) that the nuclei of the reserve cells (see arrowheads) are purple indicative of double positivity for both p63 and ERα (these cells again are also positive in the cytoplasm for K8).

(A) E7 is expressed in the cervix of ERα^−/− mice. Vaginal tissue lysates from mice of indicated genotype was immunoblotted with anti-HPV-16 E7 or anti-actin antibodies. (B) Mcm7 expression is enhanced in K14E7/ERα^−/− cervix compared to NTG/ERα^−/− cervix. Paraffin sections of female reproductive tracts from estrogen-treated mice of the indicated genotypes were stained for Mcm7 and nuclei were counterstained with hematoxylin. Shown are representatives of three mice in each genotype. Black lines indicate basement membrane separating epithelium from underlying stroma. (C) BrdU incorporation into DNA is increased in K14E7/ERα^−/− cervix compared to NTG/ERα^−/− cervix. Paraffin sections of female reproductive tracts from estrogen-treated mice of indicated genotypes were stained for BrdU. Nuclei were counterstained with hematoxylin. (D) Results shown in panel C were quantified (ERα^+/+ background, n = 3 for each genotype; ERα^−/− background, n = 10 for each genotype). More than 500 cells in five different areas of cervix per mouse were examined. P-values for two-sided Wilcoxon's rank sum test are shown.