FIG 2.

DINO causes elevated TP53 levels in HPV16 E7-expressing cells. TP53 depletion in both control (C) and HPV16 E7-expressing (E7) iHFKs was validated by Western blotting and assessment of the canonical TP53 transcriptional target CKDN1A protein (A) and mRNA (B) levels. DINO levels were determined in TP53-depleted iHFKs by qRT-PCR assays, and results are relative to levels in control siRNA-transfected iHFKs (B). For acute depletion of DINO, HPV16 E7-expressing iHFKs harboring doxycycline-inducible DINO shRNA expression vectors were treated with 1 μg/ml doxycycline for 48 h. HPV16 E7 iHFKs with acute expression of nontargeting shRNA sequences were used as negative controls. Levels of TP53 protein were determined by Western blotting (C). Expression of the TP53-responsive CDKN1A mRNA and MDM2 mRNA, as well as DINO depletion, was assessed by qRT-PCR (D). The bar graphs present means and SEM (n = 3) calculated from a single representative experiment. ***, P < 0.001; **, P < 0.01; *, P < 0.05 (Student's t test). Similar results were obtained in three independent experiments.