FIG 3.

E7-dependent IL-18BP transcription is mediated by a proximal GAS element in the IL-18BP promoter. (A) Primary human foreskin keratinocytes stably expressing empty plasmid (pBabe) or 16E7 were treated with IFN-γ (10 ng/ml). RNA was extracted over a 24-h time course and analyzed by qRT-PCR for IL-18BP and U6snRNA expression. Expression levels are shown as the fold IL-18BP mRNA increase compared to untreated controls. (B) As for panel A except that supernatants were taken for measurement of secreted IL-18BP protein by ELISA. (C) Primary foreskin keratinocytes were transfected with the indicated IL-18BP promoter reporter plasmids. After 24 h, cells were untreated or stimulated with IFN-γ (10 ng/ml) for 12 h and activity measured using a dual-luciferase system. Results are expressed as the means ± SD from four independent experiments. (D) Primary normal human foreskin keratinocytes (NHK) and keratinocytes stably harboring the HPV18 genome were treated with IFN-γ (10 ng/ml) in the presence of DMSO (mock) or lestaurtinib (Lest) (100 nM), a small-molecule inhibitor of the Jak-STAT signaling pathway, for 24 h and supernatants taken for measurement of secreted IL-18BP protein by ELISA. Results are expressed as the means ± SD from three independent experiments. **, P < 0.01. Western blot analysis from representative samples of lysates taken from these cells demonstrated that treatment with lestaurtinib did not reduce E7 expression. GAPDH was used as a protein loading control.