Pmir report firefly luciferase reporter vector

The ΔNp63 and MIR944 promoter regions were amplified from human genomic DNA by PCR using the primer sets listed in Supplementary Table S3. PCR products were cloned into the pGL3-Basic vector (Promega, Madison, WI, USA). The mutant constructs were generated using the QuikChange Site-Directed Mutagenesis kit (Stratagene, La Jolla, CA, USA), according to the manufacturer's instructions, using the primer sets listed in Supplementary Table S3. Human primary keratinocytes or WM266-4 melanoma cells were transfected with each of the constructed luciferase constructs. All cells were cotransfected with the β-galactosidase expression vector (pSV-β-galactosidase). ΔNp63 expression vectors were purchased from Addgene (Cambridge, MA, USA). TFAP2A and TFAP2C expression vectors were constructed with the primers listed in Supplementary Table S3.
Partial segments of the 3′ UTR of MAPK1, FGF2, NRAS, RRAS2 mRNA containing the predicted miR-944-binding sequences were PCR-amplified using the primers listed in Supplementary Table S3. The PCR products were cloned into the pMIR-REPORT Firefly Luciferase reporter vector (Ambion, Austin, TX, USA). Site-directed mutagenesis was performed by overlapping PCR the primers listed in Supplementary Table S3.