dna-binding elisa transam nrf2 kit  (Active Motif)

Journal: Redox Biology

Article Title: Small molecule screen identifies pyrimethamine as an inhibitor of NRF2-driven esophageal hyperplasia

doi: 10.1016/j.redox.2023.102901

Figure Lengend Snippet: Effects of 11 hit compounds on cell viability and NRF2 activity in H1299-YFP cells, and cell proliferation and cell death in NRF2 Mut ESCC cells (KYSE70 and KYSE180).

Article Snippet: NRF2-ARE binding assays were performed with an ELISA-based TransAM NRF2 Assay Kit (Active Motif, Carlsbad, CA) according to the manufacturer's instructions.

Techniques: Activity Assay, Inhibition

Journal: Redox Biology

Article Title: Small molecule screen identifies pyrimethamine as an inhibitor of NRF2-driven esophageal hyperplasia

doi: 10.1016/j.redox.2023.102901

Figure Lengend Snippet: PYR inhibited NRF2 expression in the esophageal epithelium of Sox2CreER;LSL-Nrf2 E79Q/+ mice . (A) Experimental design to test the effect of PYR (30 mg/kg/day, p.o. ) on the NRF2 high phenotype in the mouse esophagus; (B) Expression of NRF2 and its target genes (GCLC, GCLM, PKM2) in the esophageal epithelium of Sox2CreER;LSL-Nrf2 E79Q/+ mice as detected with Western blotting; (C) Histology and expression of NRF2, NRF2 target genes, and BrdU in the esophageal epithelium of Sox2CreER;LSL-Nrf2 E79Q/+ mice as detected with IHC. Scale bar = 50 μm.

Article Snippet: NRF2-ARE binding assays were performed with an ELISA-based TransAM NRF2 Assay Kit (Active Motif, Carlsbad, CA) according to the manufacturer's instructions.

Techniques: Expressing, Western Blot

Journal: Redox Biology

Article Title: Small molecule screen identifies pyrimethamine as an inhibitor of NRF2-driven esophageal hyperplasia

doi: 10.1016/j.redox.2023.102901

Figure Lengend Snippet: High-throughput screening for NRF2 inhibitors from two compound libraries (1 μM) using NQO1-YFP H1299 cells . (A) Flow chart of the screening strategy; (B) Time-dependent changes of the YFP signal (NRF2 activity); (C) Time-dependent changes of the mCherry signal (cell viability); Negative control: exposure to a known NRF2 activator (CDDO, 200 nM); Positive control: exposure to the vehicle. (D) Prestwick library (1280 FDA-approved drug compounds); (E) Asinex library (34,560 compounds). Hit compounds were selected if the CDDO-induced NRF2 activity was inhibited by >50% and cell survival was >80%. Three compounds from the Asinex library were defined as borderline hits because their inhibition of NRF2 activity was >50%, yet their effects on cell survival were ∼80%.

Article Snippet: NRF2-ARE binding assays were performed with an ELISA-based TransAM NRF2 Assay Kit (Active Motif, Carlsbad, CA) according to the manufacturer's instructions.

Techniques: High Throughput Screening Assay, Activity Assay, Negative Control, Positive Control, Inhibition

Journal: Redox Biology

Article Title: Small molecule screen identifies pyrimethamine as an inhibitor of NRF2-driven esophageal hyperplasia

doi: 10.1016/j.redox.2023.102901

Figure Lengend Snippet: PYR and MIT downregulated NRF2 and NQO1 expression in NRF2 Mut -KYSE70 cells in a dose- and time-dependent manner. (A) Dose-dependent downregulation of the expression of nuclear NRF2 and cytoplasmic NQO1 by PYR; (B) Time-dependent downregulation of the expression of nuclear NRF2 and cytoplasmic NQO1 by PYR (10 μM); (C) Dose-dependent downregulation of NRF2 and NQO1 expression by MIT; (D) Time-dependent downregulation of NFR2 and NQO1 expression by MIT (10 nM). *P < 0.05, **P < 0.01.

Article Snippet: NRF2-ARE binding assays were performed with an ELISA-based TransAM NRF2 Assay Kit (Active Motif, Carlsbad, CA) according to the manufacturer's instructions.

Techniques: Expressing

Journal: Redox Biology

Article Title: Small molecule screen identifies pyrimethamine as an inhibitor of NRF2-driven esophageal hyperplasia

doi: 10.1016/j.redox.2023.102901

Figure Lengend Snippet: PYR shortened NRF2 half-life and promoted NRF2 ubiquitination in NRF2 Mut ESCC cells. (A) NRF2 half-life was shortened by PYR (10 μM) and MIT (20 nM), but not by MTX (50 nM), in NRF2 Mut -KYSE70 cells. (B) NRF2 half-life was reduced by PYR (10 μM) and MIT (20 nM) in NRF2 Mut -KYSE180 cells. (C) NRF2 half-life was not changed by PYR (10 μM) and MIT (20 nM) in NRF2 WT -KYSE450 cells. (D) PYR (10 μM), but not MIT (20 nM), promoted NRF2 ubiquitination in NRF2 Mut -KYSE70 cells in the presence or absence of MG132 (a proteasomal inhibitor). (E) PYR (10 μM), but not MIT (20 nM), promoted NRF2 ubiquitination in NRF2 Mut -KYSE180 cells in the presence or absence of MG132. (F) PYR (10 μM), but not MIT (20 nM), slightly promoted NRF2 ubiquitination in NRF2 WT -KYSE450 cells only in the presence of MG132. *P < 0.05.

Article Snippet: NRF2-ARE binding assays were performed with an ELISA-based TransAM NRF2 Assay Kit (Active Motif, Carlsbad, CA) according to the manufacturer's instructions.

Techniques:

Journal: Redox Biology

Article Title: Small molecule screen identifies pyrimethamine as an inhibitor of NRF2-driven esophageal hyperplasia

doi: 10.1016/j.redox.2023.102901

Figure Lengend Snippet: NRF2 high esophageal phenotype in Sox2CreER;LSL-Nrf2 E79Q/+ mice. (A) Schematic figure of the LSL-Nrf2 E79Q allele and experimental design. (B) Expression of NRF2, NRF2 target genes (GCLC and GCLM), and a keratinization marker (loricrin) in the esophageal epithelium of Sox2CreER;LSL-Nrf2 E79Q/+ mice as detected with Western blotting; (C) Histology of the esophageal epithelium at 4 and 6 weeks after tamoxifen activation (H&E) and expression of NRF2, loricrin, and a proliferation marker (BrdU) in the mouse esophageal epithelium as detected with IHC. (D) 18 F-FDG PET/CT of mouse esophagus before tamoxifen induction (Week 0) and after tamoxifen induction (Week 1). Transverse views of the esophagus (arrow, highlighted by the contrast agent) of one mouse (M313) are shown. ****P < 0.001.

Article Snippet: NRF2-ARE binding assays were performed with an ELISA-based TransAM NRF2 Assay Kit (Active Motif, Carlsbad, CA) according to the manufacturer's instructions.

Techniques: Expressing, Marker, Western Blot, Activation Assay, Positron Emission Tomography-Computed Tomography