FIG 1.

High-risk HPV-positive keratinocytes exhibit higher levels of Rad51 and BRCA1 proteins in an E7-dependent manner. (A) Whole-cell lysates were isolated from uninfected primary human foreskin keratinocytes (HFKs) and CIN612 9E cells, as well as HFKs stably maintaining HPV31 (HFK-31) or HPV16 (HFK-16) genomes. Immunoblotting was performed using antibodies specific to Rad51 and BRCA1, with GAPDH serving as a loading control. (B) Densitometry was performed across at least three independent experiments examining Rad51 and BRCA1 protein levels by immunoblotting in HFKs and CIN612 and HFK-31 cells using ImageJ software. Protein levels were normalized to the GAPDH loading control. Error bars represent means ± standard errors. *, P < 0.05; **, P < 0.01. (C) Western blot analysis of Rad51 and BRCA1 protein levels in whole-cell lysates from HFKs stably transduced with HPV31 E6-, E7-, or E6/E7-expressing retroviral vectors. Tubulin was used as a loading control. Densitometry was performed across at least four independent experiments using ImageJ software. Protein levels were normalized to the respective loading control. Error bars represent means ± standard errors. *, P < 0.05; **, P < 0.01; N.S., not significant. (D) Whole-cell lysates were harvested from control HFKs as well as HFKs stably expressing HPV31 E7 or E6/E7 at T₀ (undifferentiated) or after differentiation in methylcellulose (24 and 48 h). Western blot analysis was performed using antibodies to Rad51 and BRCA1 with GAPDH as a loading control. Densitometry was performed using ImageJ software. Protein levels were normalized to the GAPDH loading control. Densitometry was performed across at least four independent experiments. Error bars represent means ± standard errors. *, P < 0.05; **, P < 0.01.