FIG 6.

Nbs1 is necessary for productive viral replication. (A) DNA was harvested from CIN612 9E cells stably expressing a Scramble shRNA or Nbs1 shRNA at T₀ (undifferentiated) or after 48 and 96 h of differentiation in high-calcium medium. Southern blot analysis was performed to analyze viral genome amplification. The bar graph represents quantification of the episome copy number present at each time point, relative to T₀ shScramble, which was set to 1. Densitometry was performed using ImageJ. Ca, calcium. (B) Total protein was harvested from CIN612 9E shScramble and shNbs1 cells at T₀ or after 48 and 96 h of differentiation in high-calcium medium. Western blot analysis was performed using antibodies to Nbs1 and involucrin, and GAPDH as a loading control. (C) DNA was harvested from human foreskin keratinocytes stably maintaining HPV31 genomes (HFK-31) at T₀ or after 24 and 48 h of differentiation in methylcellulose (MC) and analyzed by Southern blotting for amplification of viral genomes. DNA samples were digested with BamHI (does not cut the viral genome; upper panel) or with HindIII to linearize viral genomes. The bar graph represents quantification of the episome copy number present at each time point, relative to T₀ shScramble (set to 1). Densitometry was performed using ImageJ. (D) Western blot analysis was performed on lysates harvested from HFK-31 cells at T₀ or after 24 and 48 h of differentiation in methylcellulose using antibodies to Nbs1 and involucrin. GAPDH was used as a loading control. All results are representative of observations of four or more independent experiments.