Pgl2 basic vector

The 5′ flanking region of the SELENBP1 promoter (−1584 to −34) was amplified by PCR from human genomic DNA. The PCR product was cloned into pGL2-Basic vector (Promega, Madison, WI, U.S.A.) to construct pGL2-SELENBP1/1584. The 1584-Lck was constructed by inserting both 5′-flanking regions of SELENBP1 promoter from pGL2-SELENBP1/1584 and Lck (lymphocyte-specific protein tyrosine kinase) cDNA into pHYK [50 (link)]. Deletion constructs containing different lengths of the 5′-flanking sequences of the SELENBP1 gene were generated by PCR amplification from the 1584-Lck plasmid. To express HBx transiently, the HA-tagged HBx gene was cloned into pRcCMV (Invitrogen, Waltham, NY, U.S.A.) to give pRcCMV-HBx.

Primers used for the generation of 5′‐end truncated promoter fragments are described in Data S1. Fragments were digested with KpnI and XhoI and subcloned in the pGL3 basic vector (Promega). The deletion of the −560 to −275 fragment on the wild‐type 5′‐end truncated hsa‐miR‐4639 promoters were achieved with the primers described in Data S1 by overlapping PCR. Finally, the −560 to −275 fragment‐deleted promoter‐luciferase constructs were cloned in the pGL2‐basic vector (Promega). The promoter plasmid containing rs760632 A allele was amplified from the genomic DNA of PD patients with the primers used to amplify the −560 to −275 fragment. 980 μg of a reporter plasmid and 20 μg of pRL‐SV40, a Renilla luciferase vector as the internal control of firefly luciferase, were co‐transfected to SH‐SY5Y cells approximately 60% confluent in a 12‐well plate with lipofectamine 3000 (Invitrogen). For the rs760632 luciferase assay in Figure S4d, 490 μg of promoter plasmid containing rs760632 G allele and 490 μg of A allele was transfected to mimic the GA genotype. The luciferase assay was performed using a Microplate reader (Synergy Mx, Bio‐Tek) according to the manufacturer's instructions. The results presented are the relative luciferase activity generated from five independent experiments and normalized against the activity of Renilla luciferase from pRL‐SV40.

Genomic fragments of a 5′-transcriptional regulatory region of the 0.6 kb MK [12 (link)], the 0.5 kb Sur [13 (link)], or the 0.3 kb COX-2 [14 (link)] gene were cloned into pGL-2 basic vector (Promega, Madison, WI, USA) that contained the firefly luciferase gene. Plasmid DNA containing the respective genomic fragments, pGL-control vector (Promega) harboring the SV40 T antigen promoter-linked firefly luciferase gene, or pGL-basic vector without any transcriptional regulatory regions (Promega), and a control vector, the renilla luciferase gene fused with the herpes simplex virus-thymidine kinase gene promoter (pRL-TK, Promega), at a molar ratio of 10:1, was transfected into tumors with a lipofectin reagent (Life Technologies, Gaithersburg, MD, USA). Cell lysate on day 2 was assayed for the luciferase activity with the dual luciferase reporter assay (Promega). The firefly luciferase activity was standardized with the amounts of luminescence produced by renilla luciferase and the relative activity was expressed as a percentage of the SV40 T antigen promoter-mediated activity.

The tyrosinase promoter reporter construct contains a 2.3 kb fragment from mouse Tyr gene between positions -2236 and +63, in reference to the transcriptional start site, cloned in pGL2-Basic vector (Promega) upstream of the firefly luciferase gene (a generous gift of Dr. Ballotti) [28 (link)]. Expression vectors containing human MITF (NM_000248) in pcDNA3.1/myc-His(-) vector (Invitrogen) is a generous gift of Dr. Esumi [29 (link)]. The vector carrying the adeno-associated virus 2 (AAV2) genome and the transgene cassette encoding eGFP under control of a cytomegalovirus (CMV) promoter (pAAV2-CMV-eGFP) is a generous gift of Pr. Bennett [30 (link)]. Expression vectors containing rat Otx2 splice variants from the normalized libraries are identified by their internal identification numbers in retinaDB: LA0ACA144YK13CM1 (Otx2), LA0ACA6YL17.CONTIG (Otx2L) and LA0ACA63YE20CM1 (Otx2S). The plasmid pAAV2-CMV-Otx2S was constructed by replacing the eGFP in pAAV2-CMV-eGFP plasmid using NotI (5’) and BamHI (3’). The rat Otx2S fragment was amplified from plasmid LA0ACA63YE20CM1 via high fidelity PCR using the forward primer 5’-GTGTCCAGGCGGCCGCAAAAATGATGTCTTATCTAAA and reverse primer 5’-AATCGGATCCCGATATCTCACAAAACCTGGAATTTCCA.

For the construction of the luciferase plasmids, four copies of each kappaB site or mutant site (see below) were ligated (LigaFast rapid DNA ligation System, Promega, WI, USA) into a BglII and MluI-digested pGL2-Basic vector (Promega). The insert was generated by annealing a synthetic 5' phosphorylated oligonucleotides (Syntezza, Jerusalem, Israel). A control plasmid containing the consensus NF-kappaB sequence linked to the luciferase sequence was obtained from Clontech (pNF-kB-Luc, CA, USA). The CMVp65 and CMVdeltaNIkappaB plasmids have been previously described [35] .
The sequences of the kappaB sites inserted into the luciferase vector are as follows:
MGMT-kB1-Luc: 4XGTAAAGTCCCC
MGMT-Mut-kB1-Luc: 4XGTAAAGTCGGC
MGMT-kB2-Luc: 4XGGAACACCCC
MGMT-Mut-kB2-Luc: 4XGTAAAGTCGGC