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Hre luciferase

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The HRE-luciferase is a reporter gene construct that contains multiple copies of the hypoxia response element (HRE) sequence upstream of the luciferase gene. The HRE is a regulatory DNA sequence that responds to low oxygen levels (hypoxia) by activating transcription of the luciferase reporter gene. This construct can be used to monitor and quantify the cellular response to hypoxic conditions.

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Lab products found in correlation

Transit 2020 transfection reagent, by Mirus Bio (2 mentions) Odd luciferase, by Addgene (2 mentions) Dual luciferase reporter assay system, by Promega (2 mentions) Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions) Ab15148, by Abcam (1 mentions) Ab6302, by Abcam (1 mentions) Ab12327, by Abcam (1 mentions) On targetplus smartpool, by Thermo Fisher Scientific (1 mentions) Pgl2 basic, by Promega (1 mentions) Q5 site directed mutagenesis kit, by New England Biolabs (1 mentions) Pgl3 enhancer vector, by Promega (1 mentions) Ha ubiquitin, by Addgene (1 mentions) Mirus transit lt1, by Mirus Bio (1 mentions) D luciferin, by PerkinElmer (1 mentions) Victor3, by PerkinElmer (1 mentions) Penicillin, by Thermo Fisher Scientific (1 mentions) Streptomycin, by Thermo Fisher Scientific (1 mentions) Dmem medium, by Thermo Fisher Scientific (1 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions) Dual luciferase reporter assay, by Promega (1 mentions)

11 protocols using hre luciferase

1

Regulation of MRTFA-CTNNB1 Signaling

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The p3Xflag‐MKL1‐N200 expression vector was kindly provided by Professor R. Prywes (Colombia University, New York, NY, USA). It encodes a constitutively nuclear and active form of MRTFA (MRTFA‐CA). pcDNA3‐S33Y CTNNB1 (#19286), M50 Super 8x TOPFlash (TCF4‐Luc #12456) and HRE‐luciferase (#26731) were purchased from Addgene. Human MRTFA (ON‐TARGETplus® SMARTpool®) and esiRNA human HIF1A were purchased from Thermo Scientific and Sigma respectively. Antibodies against the following proteins were used: CTNNB1 (ab6302 Abcam), CDH1 (ab15148 Abcam), ERK (extracellular signal‐regulated kinase 1); K‐23 ESR1 (HC‐20, SC‐543, Santa Cruz Biotechnology), FHL2 (ab12327; Abcam), HIF1A (610958 BD Biosciences), MRTFA (ab113264 Abcam) and phalloidin‐iFluor 594 (lab176757; Abcam).
Jehanno C., Le Page Y., Flouriot G., Le Goff P, & Michel D. (2023). Synergistic activation of genes promoting invasiveness by dual deprivation in oxygen and nutrients. International Journal of Experimental Pathology, 104(2), 64-75.
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2

Engineered CD19-Targeted Vectors with HRE-MiniTK

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Constructs encoding truncated human CD19 (CD19t), a gift from Dr. Michael Jensen,16 (link) eGFP:ffluc-t2a-CD19t were inserted into epHIV7.2 by gibson assembly. The CD19t-t2a-huIL12 and eGFP:ffluc-t2a-huIL12 constructs were synthesized by GeneArt and inserted into the epHIV7.2 backbone. The HRE Luciferase (Addgene Plasmid #26731), containing three repeated HREs and HSV MiniTK, was modified by mutagenesis to remove the Nhe1 restriction site in the HRE by introducing point mutation at 5115G>A and 5120C>A. The HREs and MiniTK promoter were inserted into epHIV7.2 transgene vectors by Gibson Assembly, amplifying the HRE MiniTK promoter by PCR. Backbones were digested at NheI and RruI restriction sites to replace the minimal EF1α promoter with the HRE MiniTK promoter. The MiniTK promoter was inserted into epHIV7.2 transgene vectors by mutagenesis.
Chinn H.K., Gardell J.L., Matsumoto L.R., Labadie K.P., Mihailovic T.N., Lieberman N.A., Davis A., Pillarisetty V.G, & Crane C.A. (2022). Hypoxia-inducible lentiviral gene expression in engineered human macrophages. Journal for Immunotherapy of Cancer, 10(6), e003770.
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3

Regulation of Nix Transcription by Antioxidant Response Elements

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Chemicals were routinely purchased from chemical supply companies: TBHQ and H2O2 (Sigma-Aldrich); DMOG (Tocris). Plasmids were from the following companies: HRE-luciferase (Addgene #26731); pGL2-basic (Promega); and Nix-flag (OriGene). For Nix-1195/+114 promoter construct (P1), the sequence from genomic DNA of U87 cells was amplified with Nix-1195F-SmaI and Nix+114R-BglII (5’-AACCCGGGACAAGTCCATTTTTAAGTTC-3’ and 5’-TCGGAGATCTGGCAGGACTGC-3’). Nix-1195/−151 (P2) and Nix-290/+114 (P3) were amplified with Nix-1195F-SmaI and Nix-151R-BglII (5’-AACCCGGGACAAGTCCATTTTTAAGTT C-3’ and 5’-AGATCTATGGGATGAGTGATGCCAGT-3’); Nix-290F-NheI and Nix+114R-BglII (5’-GAGTCCTAAGAGCTAGCAAACAGATG-3’ and 5’-TCGGAGATCTGGCAGGACTGC-3’), respectively. These sequences (P1~P3) were subcloned into pGL2-basic vector. Nix-ΔARE mutants (P4~P6) were derived from pGL2-Nix-290/+114 (P3) by substituting the potential ARE sequences. The substitution was performed by Q5 site-directed mutagenesis kit (New England BioLabs) according to manufacturer’s instruction using with the following primers: for P4, ΔARE-B-F and ΔARE-B-R (5’-CCGGTCATAACAAGAATCACGCAAGAGTTC-3’ and 5’-ATGGGATGAGTGATGCCAGTGGCAG-3’); for P5, ΔARE-C-F and ΔARE-C-R (5’-GTCAGCCAATCTCGAAAGTCTCCACGTCCG-3’ and 5’-GCGCGCGCCGGGAACGAACTCTTG-3’); and, for P6, ΔARE-D-F and ΔARE-D-R (5’-GTGTTAATGCCAATGTGCAAGAGACGGTCCTG-3’ and 5’-AACAAGCCGAGTCCGCCGCCCCCTTTT C-3’).
Jung J., Zhang Y., Celiku O., Zhang W., Song H., Williams B.J., Giles A.J., Rich J.N., Abounader R., Gilbert M.R, & Park D.M. (2019). Mitochondrial NIX Promotes Tumor Survival in the Hypoxic Niche of Glioblastoma. Cancer research, 79(20), 5218-5232.
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4

Plasmid Construction and Validation

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HIF1α-pcDNA3, HA-Ubiquitin, HRE-luciferase, and pEGFP-FIH1 plasmid were purchased from Addgene. ANKDD1A-pcDNA3.1 plasmid was purchased from Vigene Biosciences (Shandong, China). ANKDD1A was cloned into pDsRed-N1 plasmid. HIF1α was cloned into p3 × flag-cmv-10 plasmid. Upstream promoter regions (−2000 bp from TSS) of the CA9 gene were amplified from U251 cells gDNA by PCR, PCR fragments were digested with Kpn1/HindIII and linked to the pGL3-Enhancer Vector (Promega, WI, USA) to create plasmid pGL3-CA9. The sequences and the orientation of the cloned fragments were confirmed by direct DNA sequencing. Different domains of ANKDD1A (ARK domain, DD domain) were cloned into p3 × flag-cmv-10 plasmid by PCR and different domains of FIH1 (F-N, F-JMJC, F-C) were cloned into pGEX-4T-2 plasmid by PCR described above.
Feng J., Zhang Y., She X., Sun Y., Fan L., Ren X., Fu H., Liu C., Li P., Zhao C., Liu Q., Liu Q., Li G, & Wu M. (2018). Hypermethylated gene ANKDD1A is a candidate tumor suppressor that interacts with FIH1 and decreases HIF1α stability to inhibit cell autophagy in the glioblastoma multiforme hypoxia microenvironment. Oncogene, 38(1), 103-119.
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5

Quantifying HIF-1 and PHD Activity

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HIF-1 and PHD activities were measured using a dual luciferase kit from Promega with HRE-luciferase (Plasmid #26731, Addgene) as reporter of HIF-1 activity and ODD-luciferase (Plasmid #18965, Addgene) as reporter of PHD activity. pRL Renilla luciferase (Promega) was used as internal control. Cells were co-transfected using the TransIT-2020 transfection reagent (Mirus Bio).
Cacace A., Sboarina M., Vazeille T, & Sonveaux P. (2016). Glutamine activates STAT3 to control cancer cell proliferation independently of glutamine metabolism. Oncogene, 36(15), 2074-2084.
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6

Hypoxia Response Element Luciferase Assay

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The hypoxia response element (HRE)‐Luciferase, a pGL2 vector containing three hypoxia response elements, was purchased from Addgene (26731). Renilla luciferase‐expressing vector pGL4.74‐Rluc was obtained from Promega (Madison, WI, USA). Cells were cotransfected with the vector or USP11 (HIF‐1α) and NC or siHIF‐1α (siUSP11). After being cultured for 48 h, cells were harvested for luciferase assays by the Dual‐Luciferase Reporter Assay System (Promega, USA).
Qiao L., Hu W., Li L., Chen X., Liu L, & Wang J. (2024). USP11 promotes glycolysis by regulating HIF‐1α stability in hepatocellular carcinoma. Journal of Cellular and Molecular Medicine, 28(2), e18017.
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7

Hypoxia-Induced Luciferase Assay in 293T Cells

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293T cells (ATCC CRL-3216) were cultured in DMEM +10% FBS+10 mM HEPES +1X Glutamax. Cells were plated at 100,000 per well of a 6 well plate and rested for 24 hours. 293Ts were transfected with HRE-Luciferase (Addgene Plasmid #26731) using MirusTransIT-LT1 (Mirus) and incubated for 24 hours. Transfected 293Ts were then placed into the hypoxia chamber for 15 or 24 hours. Cells were lifted and plated in a 96 well plate at 10,000 cells/well in 200 uL of media for luciferase assay. 20 µL of D-Luciferin (Perkin Elmer) at 1.4 mg/mL in PBS was added to each well. Cells were incubated at 37°C for 10 min, and luminescence was determined with the Perkin Elmer Victor 3.
Chinn H.K., Gardell J.L., Matsumoto L.R., Labadie K.P., Mihailovic T.N., Lieberman N.A., Davis A., Pillarisetty V.G, & Crane C.A. (2022). Hypoxia-inducible lentiviral gene expression in engineered human macrophages. Journal for Immunotherapy of Cancer, 10(6), e003770.
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8

Measuring HIF-1 Transcriptional Activity

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T47D cells (American Type Culture Collection) were cultured in DMEM medium (Invitrogen), supplemented with 10% (v/v) fetal bovine serum (FBS) (Invitrogen), 100 U/mL penicillin, and 100 μg/mL streptomycin (Invitrogen) in a humidified atmosphere (5% CO2 and 95% air) at 37°C. Cells (5 × 106 cells) were co-transfected with the HRE-luciferase (Addgene) and Renilla plasmids using Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocol. The transfected cells were seeded in 96-well plates and cultured in DMEM with 10% FBS for 12 h. In the next day, the cells were treated with tested compounds (100 nM and 200 nM) for 1 h, and then were exposed to hypoxic (1% O2/5% CO2/94% N2) or normoxic (5% CO2/95% air) conditions at 37°C for 24 h. The cells were finally lysed, and luciferase activities of both HRE and Renilla were measured by Dual-Luciferase® reporter assay (Promega) kit according to manufacturer's instructions by using a multimode reader (Infinite 200 PRO, Tecan). HIF-1 transcriptional activity was shown by the ratio of firefly/Renilla luciferase activity. The data were repeated by three independent experiments.
Parhira S., Zhu G.Y., Jiang R.W., Liu L., Bai L.P, & Jiang Z.H. (2014). 2′-Epi-uscharin from the Latex of Calotropis gigantea with HIF-1 Inhibitory Activity. Scientific Reports, 4, 4748.
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9

Quantifying HIF-1 and PHD Activity

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HIF-1 and PHD activities were measured using a dual luciferase kit from Promega with HRE-luciferase (Plasmid #26731, Addgene) as reporter of HIF-1 activity and ODD-luciferase (Plasmid #18965, Addgene) as reporter of PHD activity. pRL Renilla luciferase (Promega) was used as internal control. Cells were co-transfected using the TransIT-2020 transfection reagent (Mirus Bio).
Cacace A., Sboarina M., Vazeille T, & Sonveaux P. (2016). Glutamine activates STAT3 to control cancer cell proliferation independently of glutamine metabolism. Oncogene, 36(15), 2074-2084.
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10

Hypoxia Signaling Pathway Analysis

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MDA-MB-231 TNBC cells were seeded into 24-well plates and transfected with 500 ng of ODD-Luciferase-pcDNA3 (Addgene Plasmid # 18965, a gift from William Kaelin) [32 (link)], or HRE-luciferase (Addgene Plasmid # 26731, a gift from Navdeep Chandel) [73 (link)] in conjunction with 500 ng Renilla pRL-SV40P construct (Addgene Plasmid #27163, a gift from Dr. Ron Prywes) [74 (link)] using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. After 18 h, cells were treated with either DMSO (vehicle), cisplatin (5 µM), metformin (25 µM), gefitinib (5 µM) or different combinations for 24 h, after which cells were lysed and both Firefly and Renilla luciferase activities were quantified using a Dual-Luciferase® Reporter Assay System (Promega, Madison, WI, USA) following the manufacturer’s instructions.
Sulaiman A., McGarry S., Chambers J., Al-Kadi E., Phan A., Li L., Mediratta K., Dimitroulakos J., Addison C., Li X, & Wang L. (2020). Targeting Hypoxia Sensitizes TNBC to Cisplatin and Promotes Inhibition of Both Bulk and Cancer Stem Cells. International Journal of Molecular Sciences, 21(16), 5788.
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