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. Author manuscript; available in PMC: 2019 Nov 21.
Published in final edited form as: Cell Rep. 2019 Nov 5;29(6):1660–1674.e7. doi: 10.1016/j.celrep.2019.10.005

Figure 4. Conditional Basal Epithelial Expression of E6 and E7 Induces Canonical Tp53/pRb-E2f1 Pathway Genes and Epithelial Tumorigenesis.

Figure 4.

(A) General strategy for generating KH mice by crossing a Cre recombinase strain driven by the human keratin 14 (KRT14) promoter (K) to our knockin HPV16 strain (H; top). Representative photographs of control littermates and the KH animals at 1–2 months (bottom).

(B) Quantification of oral volume calculated based on the formula for an ellipsoid volume less the oral aperture (n = 3, mean ± SEM; **p < 0.001).

(C) Quantitative real-time PCR analysis of E6 and E7 transcript copy numbers from dissected whole tongue of KH and control littermate mice calculated using respective standard curves (n = 3, mean ± SEM).

(D) Immunofluorescence (IF) analysis of neonatal and adult epithelial tissues. Representative sections from neonatal back skin (p0) and adult tongue (p45) were fixed in paraformaldehyde (PFA) and embedded, and sections were stained for epithelial differentiation markers or E7/ E2F1 targets. White arrowheads denote suprabasal events. Magnification, 400×; bar, 50 µm.

(E) Histologic analysis of epithelial tissues. Representative sagittal sections from adult tongue and cutaneous epithelia were formalin fixed and embedded, and sections were H&E stained. Magnification, 200×; bar, 200 µm.

(F) Quantification of cutaneous epithelial thickness calculated in adult KH and control littermate mice (n = 3, ***p < 0.0001).

(G) Quantitative real-time PCR analysis of select E6/Tp53 and E7/E2F1 host pathway genes in adult KH and control littermate mice. Relative fold expression is shown normalized to Ppib mRNA levels (n = 3, mean ± SEM).

(H) Epithelial E6 and E7 expression together with 4NQO treatment increases oral tumor development. Kaplan-Meier analysis of KH and control littermate mice provided with 4NQO (20 mg/ml) in the drinking water for 8 weeks followed by weekly oral cavity examination for gross pathologic changes. Tumor onset was significantly reduced (p = 0.0499) for KH mice (median tumor-free survival = 123 days) compared to control littermates.

Data presented in (B) and (F) are shown as mean ± SEM. Student’s t test (2 tailed) was used to determine significance. Related to Figure S5.