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. 2020 Jul 15;5(4):e00398-20. doi: 10.1128/mSphere.00398-20

FIG 3.

FIG 3

Beta-3 E6/E7 alter p53 function by a mechanism similar to that seen with HR HPV16. (A) HFKs transduced with E6/E7 from beta-3 types with HPV16 E6/E7 or nontransduced HFKs were treated for 8 h with the DNA-damaging agent doxorubicin (DOXO) used at the final concentration of 2 μg/ml or with DMSO as a control and were collected. Levels of p53 and β-actin were determined by IB using specific antibodies. Band intensities were quantified, normalized to β-actin levels, and then calibrated against the corresponding protein band levels obtained in the scramble samples (right). Results shown are the means of data from 3 independent experiments performed in primary HFKs from 2 donors (*, P < 0.05; **, P < 0.01). (B) Total RNA extracted from cells treated as described for panel A was retrotranscribed and used as a template for RT real-time PCR analysis of PUMA and p21 gene expression, normalized to GAPDH levels. Results shown in the histogram are the means of data from 3 independent experiments performed in one donor. (C) Nontransduced HFKs or HPV16, HPV49, HPV75, and HPV76 E6/E7-transduced HFKs were treated for 4 h with a proteasome inhibitor (MG132). Levels of p53 and β-actin were determined by IB using specific antibodies. Band intensities were quantified, normalized to β-actin levels, and then calibrated against the corresponding protein band levels obtained in the scramble samples (right). Results shown are the means of data from 2 (HFKs) independent experiments performed in primary HFKs from 2 donors (*, P < 0.05; **, P < 0.01). (D) HPV16, HPV49, HPV75, and HPV76 E6/E7-transduced HFKs were transiently transfected with siRNA directed against E6AP (siRNA) or with scramble (Scr). Levels of p53, E6AP, and β-actin were determined by IB using specific antibodies (left). Band intensities were quantified, normalized to β-actin levels, and then calibrated against the corresponding protein band levels obtained in the scramble samples (right). Results shown are the means of data from 2 (HPV16) or 3 independent experiments performed in primary HFKs from 2 donors (*, P < 0.05; **, P < 0.01; ***, P < 0.005). (E) The indicated GST or GST/E6 fusion proteins were incubated with 1.5 mg of HNC136 total protein extract. Levels of MAML1, E6AP, and GST were determined by IB using specific antibodies.