Loss of p53 inhibition by 16E6 restores IRF6 activity.

<p>(A) Table defining the 16E6 mutations that alter p53, E6AP or PDZ binding sites. (B) NIKs were co-transfected with IL-1β promoter luciferase construct along with the HPV16E6 WT and mutated constructs at the indicated concentration. n = 5. (C) Human primary keratinocytes were transfected with cas9/sgRNA for p53 or control and 36h later cells were examined for mRNA levels for p53, IL-1β or IRF6. n = 4. (D) siRNAE6AP (+) or siRNA scramble control (-) was transfected into PLXSN or 16E6 transduced human keratinocytes for 48 h, cells were harvested for protein and RNA. Western blot analysis for p53 and β-actin. Left top, RT-qPCR for IL-1β and, left below IRF6. n = 3, (E) NIKs were co-transfected with the IL-1β promoter and pLXSN, E6, p53 or E6 with p53. Luciferase activity was measured 48 h post infection. n = 4. (F) 16E6 transduced primary keratinocytes were transfected with vector control (-), p53 or IRF6 expression vectors. Twenty-four hours later cells were harvested and pro-IL-1β, p53 or IRF6 levels were examined by immunoblotting. n = 4. Data are representative of n independent experiments performed in triplicate. Shown are the mean ± SEM with ***, P < 0.0001, based on an one or two way (applicable to > 2 conditions) ANOVA test. For immunoblotting data, 1 out of 4 experiments is shown.