Luciferase reporter assay

pGL4.42[luc2P/HRE/Hygro] vector was purchased from Promega (#E4001). While it was used as a hypoxia control, 4x HREs were excised using XhoI and BglII. Sequences of HRE8, HRE9, or mutated HRE8 or HRE9, as well as, MARE were ordered from IDT with sticky ends of overhangs of enzyme restriction. 100 μM of sense oligos and 100 μM of antisense oligos were mixed with annealing buffer and incubated for 5 min at 95 °C to anneal. Annealed oligos were diluted to 1:20 and ligated into a vector cut with two enzymes using T4 DNA ligase. The reaction was performed overnight at room temperature. Ligated DNA was transformed into DH5 alpha competent cells (ThermoFisher Scientific #18258012) and colonies were picked and grown to prep DNA using GeneJET plasmid mini prep kit (ThermoFisher Scientific #K0502). DNA with correct sequences were used for the luciferase assay.

10⁴ cells were seeded on a 96-well plate. Then 50 ng of pGL4 firefly luciferase vector with HRE or mutations, or IL11 promoter with MARE were transfected with 5 ng of renilla luciferase vector using X-tremeGENE9^TM DNA transfection reagent (MilliporeSigma #6365787001). 48 h after transfection cells were treated under normoxia or hypoxia.

1× PLB was prepared from Dual-Luciferase® Reporter assay System (ThermoFisher Scientific #E1910). After PBS washing, 20 μl of 1× PLB was added in each well and the culture plate was shaken at room temperature for 15 min. Cell lysates were mixed with 100 μl of LAR II and transferred into a white walled 96-well plate. Firefly luciferase activity was measured using the luminometer (Biotek Synergy H1). Then 100 μl of Stop & Glo reagents were mixed and renilla luciferase activity was measured using the same luminometer. Sequences are included in Supplementary Data ⁶.