Journal: Cancers

Article Title: Pyrimethamine Restores KEAP1-Mediated Degradation of Select NRF2 Mutants in Esophageal Squamous Cell Carcinoma

doi: 10.3390/cancers18091354

Figure Lengend Snippet: NRF2 differentially regulates gene expression and cellular behavior in ESCC cells. Isogenic NRF2 null -KYSE70 cells were compared with NRF2 W24C -KYSE70 cells, and KEAP1 null -KYSE450 cells were compared with NRF2 WT -KYSE450 cells. ( A ) Expression of squamous differentiation and basal cell markers assessed by Western blotting. ( B ) Cell-cycle distribution determined by flow cytometry. ( C ) Cell proliferation measured by IncuCyte live-cell imaging. ( D ) Apoptosis quantified by IncuCyte analysis. Data are shown as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Original western blots are presented in .

Article Snippet: KYSE70 and NRF2 null -KYSE70 cell pellets ( n = 3 per cell type) were analyzed by Metabolon (Durham, NC, USA) with ultra-high-performance liquid chromatography–tandem mass spectroscopy.

Techniques: Gene Expression, Expressing, Western Blot, Flow Cytometry, Live Cell Imaging

Journal: Cancers

Article Title: Pyrimethamine Restores KEAP1-Mediated Degradation of Select NRF2 Mutants in Esophageal Squamous Cell Carcinoma

doi: 10.3390/cancers18091354

Figure Lengend Snippet: NRF2 activation reduces chemosensitivity and radiosensitivity, which is restored by PYR. ( A ) Cell viability of NRF2 W24C -KYSE70 and NRF2 null -KYSE70 cells following treatment with 5-FU or cisplatin. ( B ) Cell viability of NRF2 W24C -KYSE70 cells following co-treatment with PYR (10 μM) and 5-FU or cisplatin. ( C ) CI analysis of PYR with 5-FU or cisplatin in NRF2 W24C -KYSE70 cells. The dotted lines represent the expected additive effect (CI = 1) and the circles represent CI value for combination of drug dose. ( D ) Cell viability of NRF2 W24C -KYSE70 and NRF2 null -KYSE70 cells in 3D culture following radiation exposure. ( E ) Cell viability of NRF2 W24C -KYSE70 cells treated with PYR (10 μM) and radiation in 2D culture. Data are shown as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: KYSE70 and NRF2 null -KYSE70 cell pellets ( n = 3 per cell type) were analyzed by Metabolon (Durham, NC, USA) with ultra-high-performance liquid chromatography–tandem mass spectroscopy.

Techniques: Activation Assay

Journal: Cancers

Article Title: Pyrimethamine Restores KEAP1-Mediated Degradation of Select NRF2 Mutants in Esophageal Squamous Cell Carcinoma

doi: 10.3390/cancers18091354

Figure Lengend Snippet: PYR enhances KEAP1-dependent degradation of NRF2 W24C . ( A ) Dose-dependent reduction in NRF2 W24C protein levels in KYSE70 cells following 4 h PYR treatment. ( B ) Lack of NRF2 W24C reduction in KEAP1 null -KYSE70 cells following 4 h PYR treatment. ( C ) Co-IP analysis showing increased association between NRF2 W24C and KEAP1 following PYR treatment (10 μM, 2 h), but not between NRF2 WT and KEAP1. MTX (1 μM, 2 h) did not alter NRF2 W24C -KEAP1 association. ( D ) PLA showing increased NRF2 W24C -KEAP1 interactions following PYR treatment (10 μM, 4 h). Red dots represent PLA signal. ( E , F ) NRF2 protein levels following 72 h PYR treatment (10 μM) in KEAP1 null -KYSE70 and KEAP1 null -KYSE450 cells. Data are shown as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Original western blots are presented in .

Article Snippet: KYSE70 and NRF2 null -KYSE70 cell pellets ( n = 3 per cell type) were analyzed by Metabolon (Durham, NC, USA) with ultra-high-performance liquid chromatography–tandem mass spectroscopy.

Techniques: Co-Immunoprecipitation Assay, Western Blot

Journal: Cancers

Article Title: Pyrimethamine Restores KEAP1-Mediated Degradation of Select NRF2 Mutants in Esophageal Squamous Cell Carcinoma

doi: 10.3390/cancers18091354

Figure Lengend Snippet: Computational modeling suggests PYR may facilitate NRF2 W24C –KEAP1 interaction via the Kelch domain. ( A ) Predicted interaction interface between the DLG WT motif and the Kelch domain. ( B ) Predicted weakening of the DLG W24C –Kelch interaction. ( C ) Docking of PYR into a Kelch domain pocket increases predicted interaction surface area and shape complementarity for the DLG W24C –Kelch complex. ( D ) Close-up view of PYR positioned within the Kelch domain cavity. ( E ) Two-dimensional interaction map showing predicted contacts between PYR and Kelch domain residues.

Article Snippet: KYSE70 and NRF2 null -KYSE70 cell pellets ( n = 3 per cell type) were analyzed by Metabolon (Durham, NC, USA) with ultra-high-performance liquid chromatography–tandem mass spectroscopy.

Techniques: