Pgl4.17 vector

The reporter plasmids were prepared by inserting the promoter fragments of the mouse Ren (−4094 ~ +33) and Cd38 (−4888 ~ +92) upstream of a firefly luciferase reporter gene in the pGL4.17 vector (Promega, Madison, WI, USA). The reporter plasmids were then transfected into mouse the As4.1 cells using Lipofectamine^® 3000 (Invitrogen, Waltham, MA, USA), as previously described [54 (link),55 (link),56 (link)]. The cells were then exposed to either 70 cycles/24 h of IH (mimicking the As4.1 JG cells of SAS patients) or normoxia for 24 h. After the cells were exposed to IH, they were harvested and the cell extracts were prepared in an extraction buffer (0.1 M potassium phosphate, pH 7.8/0.2% Triton X-100; Life Technologies). To monitor the transfection efficiency, the pCMV•SPORT-βgal plasmid (Life Technologies, Carlsbad, CA, USA) was co-transfected in all the experiments at a 1:10 dilution. The luciferase activity was measured using a Pica Gene luciferase assay system (Toyo-ink, Tokyo, Japan) and was normalized according to the β-galactosidase activity, as described in earlier works [37 (link),40 (link),41 (link),43 (link),50 (link),54 (link),55 (link),56 (link),57 (link),58 (link),64 (link)].

Genomic DNA was isolated from a male C57BL/6N mouse liver using the Genomic DNA isolation kit (QIAGEN, Germantown, MD, USA). The CD24 promoter region from −688 to −1 from the TSS was amplified from genomic DNA with the GC-Rich PCR system (Roche, Basil, Switzerland) using the primers indicated in Table 1. The promoter was cloned into the HindIII and BglII sites of the pGL4.17 vector (Promega, Madison, USA). The deletion constructs −469 to −1, −357 to −1, and −168 to −1 were generated using the Erase-a-base kit (Promega) according to the manufacturer's instructions. All sequences were verified by sequencing at The Centre for Applied Genomics (Toronto, ON, Canada).
Control or RasV12 cells (3 × 10⁴ cells/well in 24-well-plates) were transfected with 1 μg of the pGL4.17 vector with or without the CD24 promoter regions and 6.66 ng pRL-SV40 vector (Promega) using 2.5 μl Superfect transfection reagent (Qiagen), following the manufacturer's instructions. After 24 h, cells were lysed with 1X Passive Lysis Buffer and Firefly and Renilla Luciferase activity were measured using the Dual-Luciferase Reporter Assay kit (Promega).

The human EPHA2 proximal promoter region was amplified by the primers EPHA2-Promoter-F and EPHA2-Promoter-R (above). The PCR product was then cloned into the PCR 2.1-TOPO (Invitrogen, Grand Island, NY) vector. After sequencing for verification, the EPHA2 promoter region was then cloned into the PGL4.17 vector (Promega, Madison, WI) with the restriction enzymes of Hind III and XhoI (NEB, Ipswich, MA). HLE cells were grown to 80% confluence in 6-well plates. One ug of either PGL4-EPHA2 Promoter or PGL4 plasmid were transfected into HLE cells along with 30 ng pGL4.75 [hRluc/CMV] using a LipoJet Transfection Kit (SignaGen, Gaithersburg, MD). Forty-eight or Seventy-two hours after transfection luciferase activities were tested using dual-luciferase reporter assay system (Promega, Madison, WI) per the manufacturer’s suggested protocol.

Reporter plasmids were prepared by inserting the promoter fragments of human DBH (−1018–+10) and PNMT (−600–+67) up-stream of a firefly luciferase reporter gene in the pGL4.17 vector (Promega, Madison, WI, USA). The reporter plasmids were transfected into human NB-1 neuroblastoma cells using Lipofectamine^® 3000 (Invitrogen, Waltham, MA, USA), as previously described [5 (link),40 (link),43 (link),45 (link),46 (link),50 (link)], and the cells were exposed to either 70 cycles/24 h of IH or normoxia for 24 h. After the cells were exposed to IH, they were harvested, and cell extracts were prepared in extraction buffer (0.1 M potassium phosphate, pH 7.8/0.2% Triton X-100; Life Technologies, Carlsbad, CA, USA). To monitor the transfection efficiency, pCMV-SPORT-βgal plasmid (Life Technologies) was co-transfected in all experiments at a 1:10 dilution. Luciferase activity was measured using a PicaGene luciferase assay system (Toyo-ink, Tokyo, Japan), and was normalized by the β-galactosidase activity, as described previously [5 (link),40 (link),41 (link),43 (link),46 (link),49 (link),50 (link)].