Pgl3 basic

The human FOSL1 promoter region −1138 bp to −724 bp that covers P1 primers used for ChIP analysis and contains three GATA3 binding sites with consensus sequences, GATAA, at −944, −924, and −826 was inserted into the pGL3-basic (Promega), as pGL3-FOSL1. The human FOS promoter region −2286 bp to −679 bp that covers P1, P2, and P3 primers used for ChIP analysis and contains four GATA3 binding sites with consensus sequences, GATAA, at −930 and −1062, GATAG at −1646, and TGATTA at −2236 was inserted into the pGL3-basic (Promega), as pGL3-FOS. For promoter-luciferase reporter assay, we infected MDA-MB231 cells with pLvx-Flag (Empty) and pLvx-Flag-GATA3 (GATA3) and established empty- and GATA3-expressing stable cells, which were then transfected with Renilla vector (internal control), pGL3-basic, pGL3-FOSL1, or pGL3-FOS. 48 h after transfection, cell lysates were collected and subjected to luciferase assay using the Dual-Luciferase Reporter Assay System (Promega). Two independent transfection experiments were conducted, and each luciferase assay was performed in triplicates. Normalized data was calculated as the ratio of the firefly/Renilla luciferase activities.

The reported plasmid pLTR(Sp1)-luciferase was generated by PCR amplification of pNL4-3 using the sense primer 5′-CGGGGTACCCCGTGGAAGGGCTAATTTGGTCCC-3′ and the antisense primer 5′-CCGCTCGAGCGGCATCTCTCTCCTTCTAGCCTC-3′, digestion with KpnI and XhoI, and ligation into KpnI/XhoI-digested pGL3-Basic (Promega). Mutations to the NF-κB and IRF binding sites in pLTR(Sp1)-luciferase were generated using the QuikChange IIXL site-directed mutagenesis kit (Stratagene). Primers used for site-directed mutagenesis are listed in Table 1. The −158 LTR-luciferase construct was generated by deleting the LTR sequence upstream of position −158 (relative to the start site of transcription) of pNL4-3, which includes the AP-1 binding sites located in the U3 portion of the 5′ LTR, digestion of the resulting fragment with KpnI and XhoI, and ligation into KpnI/XhoI-digested pGL3-Basic (Promega).

Lipofectamine 3000 reagent (Thermo Fisher, Waltham, MA) was used for BMP reporter plasmid (Addgene, MA) transfection. Cells were disassociated by incubating with EDTA (0.02%) for 5 minutes. The disassociated cells were collected and centrifuged at 500g for 5 minutes. The cell pellet was resuspended into 2 ml of media and cell count was performed before replating cells at the density of 1.3 × 10⁵ cells into one well of a BD Matrigel coated 24 well plate one day before lipofection. For plasmid lipofection, 500 ng of pGL3‐Basic or pGL3 BRE Luciferase (Promega, Madison, WI) were used to transfect the cells in each well of 24‐well plate following manufacturer's recommendations. Cells that were transfected with empty vector (pGL3‐Basic) or BMP reporter (pGL3 BRE Luciferase) were cotransfected with empty Renilla vector (pRL‐Null) (Promega, Madison, WI).

The binding site of FOXA1 with CTGF was predicted on LASAGNA-search. Sequences of the wild-type CTGF promoter (-500 to -1) and mutant CTGF promoter (-399 to -390, CAGGGCAAAC to CACCGCTTAC) with recognition sites specific for the enzymes KpnI/XhoI were manufactured by Sangon Biotech (Shanghai). FOXA1 CDS flanked with BamHI/EcoRI was cloned as well. Luciferase reporter plasmids, pGL3-Basic-CTGF-w (wild type) and pGL3-Basic-CTGF-m (mutant), were constructed by having the primers flanked with KpnI/Xho cloned into the KpnI/Xho sites of pGL3-Basic vector (Promega). The FOXA1 overexpression plasmid was created by cloning the FOXA1 CDS sequence with BamHI/EcoRI into pcDNA3.1 (Invitrogen). pRL-TK vector (Promega), the Renilla luciferase plasmid, was adopted as an internal control reporter vector.

Transfected cells were assessed using a Dual-Luciferase Reporter Assay System (Promega Corp., Madison, WI, USA). The guanine-rich sequence, 5′-GGGCTGGGCTGGGCGGGG-3′, located in the c-Myb promoter region was synthesized to replace the sequence at the KpnⅠ site with NheⅠ of pGL3-basic (Promega Corp.), the vector of luciferase reporter gene 20 (link). According to the protocol supplied by the manufacturer, pGL3-basic has a number of consensus transcription factor binding sites that express luciferase in complex environments. We seeded 293 cells in 12-well plates and co-transfected them with 1.0 μg of modified pGL3 (pGL3-p) or pGL3-basic and 0.2 μg of pRL-SV40 (Promega Corp.) for 48 h as described by the manufacturer. The cells were then incubated with or without (0 mM) or with 1-, 0.5-, or 0.25-mM brucine for 24 h.

The subcloned chimeric constructs containing the human CYP11B2 genomic DNA and luciferase cDNA (pGL3-basic, Promega, Madison, WI) [14 (link),17 (link)] were used for the transient transfection studies: -1521/+2-luc (harboring the CYP11B2 5’-flanking region from -1521 to +2 relative to the transcription start site upstream of the luciferase cDNA in pGL3-basic); -747/+2-luc; -135/+2-luc; -106/+2-luc; -65/+2-luc. β-Galactosidase control plasmid in pCMV (pCMV-β-gal) was purchased from Clontech (Mountain View, CA). Murine nerve growth factor-induced clone B (NGFIB) and Nur-related factor 1 (NURR1) cDNA were cloned by PCR from murine pituitary AtT20 cell RNA and subcloned into the pcDNA3 expression vector (Invitrogen, Carlsbad, CA) (NGFIB-pcDNA3 and NURR1-pcDNA3). Murine RXRα and RXRβ cDNA previously subcloned into pcDNA1/Amp expression vector (Invitrogen) (RXRα-pcDNA1/Amp, RXRβ-pcDNA1/Amp) [18 (link)] were also used. Several vectors were mutated using a QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA); NBRE-1 element in -1521/+2-luc from 5’-AAAGGCTA-3’ (-766/-759) to 5’-gAAttCTA-3’ (-1521/+2-luc-NBRE-1-mut); Ad5 element in -1521/+2-luc from 5’-GACCTT-3’ (-129/-114) to 5’-GAtaTc-3’ (-1521/+2-luc-Ad5-mut); Ad1/CRE element in -1521/+2-luc from 5’TGACGTGA-3’ (-71/-64) to 5’-gGtaccGA-3’ (-1521/+2-luc-Ad1/CRE-mut) [14 (link),19 (link)].

Primers were designed to amplify a genomic region that encompasses potential GRHL2 regulatory regions, spanning all variants of interest. A 2,728-bp product (chr8:101,491,361–101,494,128) containing upstream sequence, exon 1, and partial intron 1 of the GRHL2 gene (Figure S1) was amplified from control genomic DNA, cloned into pGEM-TEasy (Promega) and sub-cloned into the promoter-less firefly luciferase reporter vector pGL3-Basic (Promega). Primers used for cloning incorporated KpnI and NheI restriction site to facilitate subcloning (forward 5′-GGTACCCAAGCTTTCCACGTCCTCC-3′ and reverse 5′-GCTAGCCAAAGTTACCGGGGAAAGCAA-3′). Variants identified in PPCD4-affected individuals were introduced by site-directed mutagenesis using a Q5 Site-Directed Mutagenesis Kit (New England Biolabs) and all constructs were verified by Sanger sequencing. Wild-type or mutant GRHL2 promoter pGL3-Basic plasmids (90 ng) were used to co-transfect HEK293 cells with 10 ng of pRL-CMV (CMV-promoter driven Renilla luciferase reporter, Promega) using TransIT-LT1 transfection reagent (Mirus). At 24 hr post-transfection, luciferase activity was measured using an Orion L Microplate Luminometer (Titertek Berthol) and a dual-luciferase reporter assay system (Dual-Glo Luciferase Assay System, Promega).