Pgl3 promoter luciferase reporter vector

HEK-293T cells (ATCC, Manassas, VA, United States)⁴ were cultured in Dulbecco’s Modified Eagle Medium containing 10% fetal bovine serum in a humidified incubator at 37°C with 5% CO₂ in air under saturated humidity.
The pGL3-KLF4 3′-untranslated region (3′-UTR) reporter vector was constructed by cloning sequences containing KLF4 promoter sites into the pGL3-Promoter luciferase reporter vector (Promega). Next, the pGL3-KLF4 3′-UTR reporter vector were co-transfected with oe-NC or oe-EZH2 into human HEK-293T cells using Lipofectamine 3000 (Invitrogen) for 48 h. The cell lysate was subsequently collected to measure the firefly and Renilla luciferase activities using a dual-luciferase reporter gene assay kit (Promega) with Renilla luciferase activity employed for standardization.

The pCI-neo-V5DLX3^WT plasmid, expressing mouse DLX3 under a constitutive CMV promoter, was kindly provided by Dr. Maria Morasso (Developmental Skin Biology Section, NIAMS-NIH, DC, USA). The pCI-neo-V5DLX3^TDO plasmid (with the TDO mutation, c.571_574delGGGG) was constructed by GeneChem Technology (Shanghai, China). Constructs of pGLEnam-E1, pGLEnam-E2, pGLAmelx-E2, pGLKlk4-E1, and pGLOdam-E2 were made by cloning the amplified PCR products of the corresponding enhancer regions (with potential DLX3 binding sites) into the pGL3-Promoter Luciferase Reporter Vector (Promega, Madison, WI, USA). Mutations of the potential DLX3 binding sites (TAATT changed to TGGTT) on pGLEnam-E1, pGLAmelx-E2, and pGLOdam-E2 were performed by TransGen Tech (Beijing, China), generating the constructs Mut pGLEnam-E1, Mut pGLAmelx-E2, and Mut pGLOdam-E2. The mutations were verified by DNA sequencing.

DNA fragments containing the EPAS1-derived EREs were generated by PCR using the primers listed in Supplementary Table 1B, and cloned into the pGL3-promoter luciferase reporter vector (Promega Corporation, Madison, WI, USA). A cDNA encoding human ERα cloned into the pCMV5 mammalian expression vector was kindly provided by A. Odermatt (Basel, Switzerland). Human GATA-2, GATA-3, and GATA-4 cloned into pcDNA3.1 were kindly provided by C. Dame (Berlin, Germany). Dual luciferase reporter gene assays were performed as described previously [64 (link)].

For the highly conserved SVs of EPAS1, MB, DISC1, VWC2 and SNX3, we extracted sequences containing the entire SV and the 200 bp flanking sequences from the genomes of yak and cattle respectively, and inserted them into the pGL3-promoter luciferase reporter vector (Promega, USA) before checking by sequencing (TsingKe Biotech or BIORN Life Science Co, Ltd, Nanjing, China). The HEK293T and PC12 cell lines were purchased American Tissue Culture Collection (ATCC, Rockville, MD, USA). The cell line was authenticated by short tandem repeat analysis. The cells were maintained in DMEM (GIBCO, USA) supplemented with 10% FBS (GIBCO) and 1% Antibiotic-Antimycotic (GIBCO) at 37 °C, 5% CO₂. These constructs as well as the pRL-TK plasmid as the internal control were transfected into HEK293T (EPAS1, MB) or PC12 (other genes) cells. After 24 h, transcriptional activity was investigated by the Dual-Luciferase Reporter Assay System (Promega) using a Multimode microplate reader (Spark Tecan, Switzerland). Each experiment was independently performed three times. All data are expressed as means ± SD, and statistical differences between groups were determined by two-sided Student’s t-test, and a p-value < 0.05 was considered as a significant difference.