Research Paper Volume 10, Issue 12 pp 4224—4240

ΔNp63 promotes IGF1 signalling through IRS1 in squamous cell carcinoma

Figure 2. p63 binds to the regulatory region of theIrs1gene. (A) p63 DNA-binding profiles in the Irs1 locus, obtained in NHEKs by ChIP-sequencing (ChIP-seq) using 4A4 and H129 anti-p63 antibodies in two normal human primary keratinocyte cell lines (K1 and K2) [87]. (B) ChIP analysis of p63 occupancy at the regulatory regions of the Irs1 gene. ChIP assays were performed in Fadu HNSCC cells using H129 anti-p63 antibody and control IgGs. PCR validation was performed using primers spanning the p63-binding sites located within the genomic regions identified by ChIP-seq assays. (C) Luciferase reporter assays of Irs1 regulatory regions (left panel). The pGL3 reporter vector (30 ng) and the pRL-CMV-Renilla luciferase plasmid (5 ng) were cotransfected with the empty pcDNA-HA vector or plasmids coding ΔNp63α, ΔNp63β, and ΔNp63γ (150 ng) into the p53 null human H1299 cell line. The luciferase activities of cellular extracts were measured 24 h after transfection. Cellular lysates were also analysed by western blot (right panel). Data are presented as mean ± SD and are representative of three independent experiments.