Figure 2. ShRNA silencing of ΔNp63-SIRT1-p53-Sp1 pathway.. Mouse epidermal keratinocytes (2x105 cells) from p63-/+ (samples 1-5) or overexpressing ΔNp63α(samples 6-10) were treated with control media (samples 1 and 6), SIRT1 inhibitor (Sirtinol, 100 μg/ml for 24 h; samples 2 and 7), or transfected with the SIRT1 shRNA (samples 3 and 8), p53 shRNA (samples 4 and 9), and sh-Sp1 RNA (samples 5 and 10). (A) Immunoblotting with indicated antibodies (dilutions: anti-ΔNp63, 1:500; anti-SIRT1, 1:300; anti-Sp1, 1:300; anti-p53, 1:500; anti-acetyl-p53, 1:400; anti-β-actin, 1:400). The vertical lines separate data obtained from independent protein gels. (B) mTERT promoter luciferase reporter assay. Mouse keratinocytes (1.0 x 105) were transfected with the pGL3-347-Luc plasmid (0.5 μg) or the pGL3 control plasmid (0.5 μg) by using FuGENE6 transfection reagent (Roche Diagnostics). 3 ng of the pRL-SV40 (Promega) was used as a normalization control. Measurements were performed by using the Dual Luciferase reporter assay system (Promega) and a BioOrbit 1251 luminometer. The activity of each TERT promoter fragment was expressed as a relative value. All of the data (mean +SD) were from at least three independent experiments.