Figure 2.

p-ΔNp63α binds to the DICER1 promoter sequences and activates the DICER1 promoter activity upon CIS exposure. Wild-type ΔNp63α cells were exposed to Con or 10 μg/ml CIS for 24 h. (a) ChIP assay for the DICER1 promoter was performed using both anti-ΔNp63 and anti-p-ΔNp63α antibodies. A rabbit immunoglobulin (IgG) was used as a negative control for ChIP. CCAAT elements 1 and 2 were amplified using primers covering the specific regions (−1892 to −1191) and (−1421 to −721), respectively, and yielding the 700-bp PCR fragments. Positive controls (Inputs) and a negative control (ChIP using primers for the DICER1 promoter nonspecific region −2639 to −2301 yielding the 340-bp PCR product) were shown. (b) Luciferase reporter activity assay. Wild-type and mutated cells were transfected with 100 ng of the promoterless pGL3 plasmid or pGL3-DCR-Luc plasmid along with 1 ng of the Renilla luciferase plasmid. Wild-type ΔNp63α cells were also transfected with 100 ng of the ΔNp63α-S385G-FL expression cassette, whereas ΔNp63α-S385G cells were transfected with 100 ng of the ΔNp63α-FL expression cassette, as indicated. Cells were exposed to Con and 10 μg/ml CIS for 24 h. Luciferase reporter assays were conducted in triplicate (±S.D., P<0.01). Firefly luciferase activity values were normalized against Renilla luciferase values and resulting values obtained from wild-type ΔNp63α cells with the promoterless pGL3 plasmid and exposed to Con were designated as 1, while fold changes are shown as numerical values. Samples were also tested by immunoblotting with the indicated antibodies