Plasmids for NRF2 and HIF1α Regulation

For transient transfections the following plasmids were used: pGL3-8xARE^{42 (link)}, or pGLHIF1.3^{43 (link)}, pGL4.74 renilla luciferase (#E6921, Promega), pGFP-NRF2 (pcDNA3-EGFP-C4-Nrf2, #21549, Addgene), Flag-NRF2 (pCDNA3.1 FLAG NRF2, #36971, Addgene), Flag-Keap1 (#28023, Addgene), p(HA)HIF1alpha (P402A,P564A) (#52636, Addgene). To generate plasmid GFP-NRF2-S40A, a modified QuickChange protocol was used^{44 (link)} to perform a PCR using pGFP-NRF2 as the template and primers NRF2-S40A-for (5ʹ-GTCGAGAAGTATTTGACTTCGCCCAGCGACGGAAA-GAGTATGAGCTGGAAAA-3ʹ) and NRF2-S40A-rev (5ʹ-GGCGAAGTCAAATAC-TTCTCGACTTACTCCAAGA TCTATATCTTGCCTCCAAAGTATGTCA-3ʹ). pLKO.1-shRNA-NRF2 tet-on (tetracycline inducible) contained the sequence 5ʹ-GCTCCTACTGTGATGTGAAAT-3ʹ of NRF2 mRNA (GenBank acc. no. NM_001145412), pLKO.1-shRNA-PERK tet-on (tetracycline inducible) contained the sequence 5ʹ-GGAACGACCTGAAGCTATAAA-3ʹ of PERK mRNA (GenBank acc. no. NM_004836).

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Küper A., Baumann J., Göpelt K., Baumann M., Sänger C., Metzen E., Kranz P, & Brockmeier U. (2021). Overcoming hypoxia-induced resistance of pancreatic and lung tumor cells by disrupting the PERK-NRF2-HIF-axis. Cell Death & Disease, 12(1), 82.

Publication 2021

pGL4.74 renilla luciferase pcDNA3-EGFP-C4-Nrf2 Flag-NRF2 ( pCDNA3.1 FLAG NRF2 Flag-Keap1

Hif1alpha Keap1 Mrna Nrf2 Pgl3 Pgl4 Plasmid Primers Promega Renilla luciferase Shrna Tetracycline Transfections Transient

Corresponding Organization :

Other organizations : University of Duisburg-Essen

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Variable analysis

independent variables

PGL3-8xARE
PGLHIF1.3
PGFP-NRF2
Flag-NRF2
Flag-Keap1
P(HA)HIF1alpha (P402A,P564A)
GFP-NRF2-S40A
PLKO.1-shRNA-NRF2 tet-on
PLKO.1-shRNA-PERK tet-on

dependent variables

Luciferase activity

control variables

PGL4.74 renilla luciferase

controls

Positive control: pGL4.74 renilla luciferase
Negative controls not explicitly mentioned

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