Prl sv40 renilla luciferase reporter vector

For M2H assays, 2.15 × 10⁵ HEK293 or 4 × 10⁵ A375 cells were transiently transfected with 1 µg firefly luciferase reporter plasmid (5xGAL4-TATA-luciferase, Addgene, 46756) (Sun et al., 1994 (link)) and 500 ng or 200 and 300 ng chimeric Gal4 and VP16 fusion protein vectors, respectively, in 12- or 6-well format using CaCl₂ or JetOptimus. To control for differences in transfection efficiency and extract preparation, 25 ng or 50 ng pRL-SV40 Renilla luciferase reporter vector (Promega, Madison, WI, USA) was co-transfected, respectively for HEK293 and A375 cells. Cultures were evenly split onto 12-well plates 24 hr after transfection for A375 cells. For hypoxia control experiments, 4 × 10⁵ A375 cells were transiently co-transfected with 500 ng firefly 5’/3’-hypoxia response element-dependent EPO promoter-driven luciferase reporter plasmid (Storti et al., 2014 (link)) and 50 ng pRL-SV40 Renilla luciferase reporter vector. Luciferase activities of duplicate wells were determined using the Dual Luciferase Reporter Assay System (Promega) as described before (Schörg et al., 2015 (link)). Reporter activities were expressed as relative firefly/Renilla luciferase activities. All reporter gene assays were performed at least three times independently.

A portion of the 3′UTR sequence containing two predicted binding sites for miR-218 was cloned in the pMiR reporter vector by using the following oligonucleotides: forward: GCGCACTAGT-CGCACAACCTCAAGGGAAACTC (containing the cloning site for Spe I); reverse: GCGCAAGCTT-GCAGCGTTCCTCAGATCTCAAG (containing the cloning site for Hind III). The assay was performed by using the Luciferase Reporter Assay System (Promega, Milan, Italy), following the manufacturer’s instructions. The 3’UTR-containing pmiR-Report was co-transfected with the Tet-O-FUW miRNA-overexpressing vector and the rtTA-expressing vector in HeLa cells. A pRL-SV40 Renilla luciferase reporter vector (Promega, Milan, Italy) was also used to quantify the transfection efficiency. Firefly luciferase luminescent signal was normalized on the Renilla luciferase signal (Promega, Milan, Italy). Control experiments using the empty pMiR-Report vector in presence or absence of the microRNA were performed.

CYGB promoter construct generation was described before^{25 (link)}. 3 × 10⁵ Hep3B or 3.5 × 10⁵ A375 cells were transiently transfected with 300 ng reporter plasmid and YFP-HIF-1α or YFP-HIF-2α as indicated, in a six-well format using JetOptimus (Polyplus). To control for differences in transfection efficiency and extract preparation, 25 ng pRL-SV40 Renilla luciferase reporter vector (Promega) was co-transfected. Cultures were evenly split onto 12-well plates 24 h after transfection. Luciferase activities of triplicate wells were determined using the Dual Luciferase Reporter Assay System (Promega) as described before^{56 (link),57 (link)}. Reporter activities were expressed as relative firefly/Renilla luciferase activities (R.L.U.). All reporter gene assays were performed four to eight times independently.