Figure 5. Non-p-ΔNp63α induces the expression of CAD in SCC-11M cells upon cisplatin exposure. (A) Metabolite reaction of carbamoyl-phosphate synthetase 2 (CAD). Grey arrow indicates the decrease in SCC-11 cells, while black arrow indicates the increase in SCC-11M cells. (B, upper panel) qPCR expression analysis of CAD mRNA obtained from SCC-11 and SCC-11M cells treated with control medium (Con) and 10 μg/ml cisplatin (CIS). The values from SCC-11 cells treated with control medium designated as 1. The qPCR values presented as relative units (RU). (B, lower panel) Immunoblot analysis of CAD in SCC-11 and SCC-11M cells treated with control medium (Con) or cisplatin (CIS). Loading was normalized by β-actin expression. (C) ChIP assay of the binding of p-ΔNp63α and E2F1 to the CAD promoter (enrichment) in SCC-11M cells exposed to control medium (Con) and cisplatin (CIS). (D) Quantitative analysis of enrichment (ChIP/Input ratio) of the CAD promoter with the p-ΔNp63α and E2F1 transcription factors in SCC-11M cells treated with cisplatin. ChiP/Input ratio values presented as relative units (RU). SCC-11M cells were transfected with the scrambled and E2F1 siRNA and analyzed for ChIP/Input enrichment. (E) Luciferase reporter assay of the CAD promoter in SCC-11 and SCC-11M cells treated with control medium (Con) and cisplatin (CIS). SCC-11M cells were transfected with the scrambled and E2F1 siRNA and analyzed for the CAD promoter-driven luciferase activity presented as relative units (RU). The values from SCC-11 cells treated with control medium designated as 1. All experiments were performed in triplicates. p < 0.05.