FIG 5.

Depletion of DINO protects HPV16 E7-expressing cells from cell death induced by metabolic stress and DNA damage. Cell viability was assessed by resazurin assays of control, HPV16 E7-expressing, and HPV16 E6/E7-expressing HFKs that were either untreated (mock) or treated with 20 mM metformin for 96 h (A). TP53 and CDKN1A expression in HPV16 E7-expressing HFKs was determined by Western blotting (B). Expression of CDKN1A mRNA and validation of DINO depletion in these HFK populations were determined by qRT-PCR (C). Viabilities of HPV16 E7-expressing keratinocytes with expression of either of two DINO-specific shRNAs or a scrambled control shRNA were assessed in response to treatment with metformin for 3 days (D), to being fed regularly or being starved for 7 days (E), and to treatment with 0.125 μg/ml of the DNA-damaging chemotherapy agent doxorubicin for 3 days (F). Bar graphs present means and SEM (n = 3) calculated from a single representative experiment. ***, P < 0.001; **, P < 0.01; *, P < 0.05; #, P < 0.001 (Student's t test). Similar results were obtained in three independent experiments.