Loss of p53 induces YY1 protein levels in normal HKc and YY1 levels increase during in vitro progression of HKc/HPV16. Exogenous YY1 induces the EGFR promoter. (a) Western blot analysis for YY1 and β-actin (as a loading control) of cell lysates of normal HKc stably transfected with the p53RNAi-1 expression plasmid, or an HPV16 E6 expression vector (pLXSN-16E6) or their respective controls (pSuper or pLXSN). (b) Immunofluorescence staining for YY1 (green) in normal HKc, HKc/HPV16 at passage 15 (HKc/HPV16 low passage), 127 (HKc/HPV16 high passage) and HKc/DR. Cell nuclei (blue) were stained with DAPI and confocal microscopy was performed. (c) Normal HKc were transiently co-transfected with the pGL3-EGFR promoter construct (1.5 μg per well) and increasing amounts of a YY1 expression vector or its control plasmid (pcDNA 3.1) and with pRL-Null (10 ng per well). Cells were harvested 48 h post-transfection for dual-luciferase assays. EGFR, epidermal growth factor receptor; HKc, human keratinocytes; YY1, Yin Yang 1.