Figure 3.

E7 inhibition of STING‐triggered NF‐κB activation is related to its HPV pathogenicity, but not to Rb‐binding. (A) HEK293T cells were transfected with NF‐κB‐Luc, pRL‐TK Renilla, Myc‐cGAS, and STING‐Flag in the presence or absence of different pathogenic HPV E7 plasmids, as indicated. NF‐κB promoter transactivation and protein expression were analyzed. (B–C) HEK293T cells were transfected with Myc‐cGAS and STING‐Flag in the presence or absence of different pathogenic HPV E7 plasmids, as indicated. (B) Total mRNA was extracted and analyzed using RT‐qPCR to determine the transcription levels of the indicated genes. (C) Co‐immunoprecipitation analysis was performed. (D) Sequences of HPV18 E7 containing the LXCXE motif (wild type and mutant). (E) HEK293T cells were transfected with NF‐κB‐Luc, pRL‐TK Renilla, Myc‐cGAS, and STING‐Flag in the presence or absence of HPV18 E7 or its LXCXE motif mutant, as indicated. Promoter activities and protein expression were analyzed using a luciferase reporter assay and immunoblotting, respectively. (F–G) HEK293T cells were transfected with Myc‐cGAS and STING‐Flag in the presence or absence of HPV18 E7 or its LXCXE motif mutant, as indicated. (F) Total mRNA was extracted and analyzed using RT‐qPCR to determine the transcription levels of the indicated genes. (G) Coimmunoprecipitation analysis was performed. Statistical significance was determined using two‐sided unpaired t‐tests (*p < 0.05; **p < 0.01; NS, not significant). The results are shown for N = 3 independent experiments. mRNA, messenger RNA; NF‐κB, nuclear factor kappa‐light‐chain‐enhancer of activated B cells; RT‐qPCR, reverse‐transcription quantitative polymerase chain reaction; STING, stimulator of interferon gene.