Figure 2

HBZ activates Dkk1 expression at the level of transcription. (A) Dkk1 mRNA stability in cells expressing HBZ wt or carrying the empty vector. Levels of Dkk1 mRNA were normalized to UBE2D2 mRNA following quantitative real-time PCR of reverse transcribed total cellular RNA. The graph shows relative Dkk1 mRNA levels from cells harvested at the indicated times following treatment with actinomycin D. Dkk1 mRNA levels were set to 100% at time 0 hours for both cell lines. The graph shows data averaged from two independent experiments. (B) Levels of histone H3 acetylation (acH3) at the DKK1 promoter in cells expressing HBZ wt or carrying the empty vector. ChIP assays were performed using an antibody directed against acH3, and relative levels of acH3 at indicated sites with respect to the mRNA start site (+ 1) were normalized to 1% of the input DNA following quantitative real-time PCR. The graph shows data averaged from three independent ChIP assays. (C) Levels of RNA polymerase II (Pol. II) enrichment at the DKK1 promoter in cells expression HBZ wt or carrying the empty vector, with data averaged from three independent ChIP assays. (D) Dkk1 mRNA stability in cells transiently transfected with HBZ wt or the empty vector, with data averaged from two independent transfection experiments. (E) Levels of Pol. II enrichment at the DKK1 promoter in cells transiently transfected with HBZ wt or the empty vector, with data averaged from two independent ChIP assays. (F) Western blot analysis of HBZ wt expression after transfection.