ef1α promoter  (Addgene inc)

Journal: EMBO Reports

Article Title: NRF2 supports non-small cell lung cancer growth independently of CBP/p300-enhanced glutathione synthesis

doi: 10.1038/s44319-025-00463-z

Figure Lengend Snippet: ( A ) Alignment of metazoan NRF2 Neh4 and Neh5 segments with Neh4/5mut substitutions indicated. Mutation of QD in Neh5 was derived from (Zhang et al, ). ( B ) Western blot of input (left) and FLAG-immunoprecipitations (right) of FLAG-tagged WT, Neh4/5mut, ΔNeh1, or luciferase control expressing HEK293 cells co-transfected with or without GFP-tagged CBP. Data are representative of three independent experiments. ( C ) Western blotting of H460-shNRF2-BIND-luc, -BIND-WT NRF2, -BIND-Neh4/5mut, or -BIND-ΔNeh1 cells treated with water (control) or 2.5 ng/mL dox for 24 h probing with FLAG, NRF2, or β-actin antibodies. Representative data from three independent experiments is shown. ( D ) Crystal violet staining of H460-shNRF2-BIND-luc, -BIND-WT NRF2, -BIND-Neh4/5mut, or -BIND-ΔNeh1 treated with water (control) or 2.5 ng/mL dox for 8 days. Representative data from three independent experiments is shown. ( E ) CellTiter-Glo measurements of H460-shNRF2-BIND-luc, -BIND-WT NRF2, -BIND-Neh4/5mut, or -BIND-ΔNeh1 treated with water (control) or 2.5 ng/mL dox for 5 days. Data are expressed as a fold of control values, with SD indicated and are derived from three individual experiments. ( F ) Quantification of scratch assay wound healing in A549shNRF2-BIND cells +/−2.5 ng/mL dox imaged over 7 days on the Incucyte system. Wound healing rate is shown as the ratio of the scratch area at day 7 vs day 0, with a ratio of 1 representing no migration of cells into the scratch area. Data are derived from three individual replicates.

Article Snippet: EF1ɑ-/TRE3G-promoter-driven Renilla luciferase and NRF2 (WT NRF2, Neh4/5mut, SV40-NLS-ΔNeh1) constructs , GenScript/this work , N/A.

Techniques: Mutagenesis, Derivative Assay, Western Blot, Luciferase, Control, Expressing, Transfection, Staining, Wound Healing Assay, Migration

Journal: EMBO Reports

Article Title: NRF2 supports non-small cell lung cancer growth independently of CBP/p300-enhanced glutathione synthesis

doi: 10.1038/s44319-025-00463-z

Figure Lengend Snippet: ( A ) Domain structure of NRF2 indicating point mutations in the Neh4/5mut protein. ( B , C ) FLAG IP/MS on nuclear fractions from A549 cells expressing Renilla luciferase, WT NRF2, or Neh4/5mut proteins. Differential enrichment between WT NRF2 vs Renilla luciferase ( B ) and Neh4/5mut vs WT NRF2 ( C ) are shown. Data are derived from four individual replicates. ( D ) qRT-PCR from RNA isolated from A549-shNRF2-BIND-luc, -BIND-WT NRF2, -BIND-Neh4/5mut, or -BIND-ΔNeh1 cells treated with water (control) or 2.5 ng/mL dox for 24 h using indicated primers normalized to RPL13A as a housekeeping gene. Exogenous NRF2 constructs are codon optimized and thus not detected by NFE2L2 primer. Data are generated in triplicate with mean +/− SD shown. Representative data from three independent experiments is shown. ( E ) Western blot of cells from ( D ) probing with FLAG, NRF2, or β-actin antibodies. Representative data from three independent experiments is shown. ( F ) Correlation of transcriptomic changes induced by rescue with WT NRF2 vs. Neh4/5mut in A549-shNRF2-BIND-WT NRF2 and -BIND-Neh4/5mut cells after 24 h of dox (2.5 ng/mL) treatment. Values show limma-voom log 2 fold changes between 24 and 0 h of treatment for each NRF2 construct, normalized by subtracting the log 2 fold change between 24 and 0 h of Luciferase. Highlighted points are NRF2 signature genes colored by cosine similarity between WT and Neh4/5mut. Cosine values of 1 represent the same angle and hence no differential expression between Neh4/5mut and WT NRF2, with more divergence as values decrease. ( G ) Western blot of cells from A549-shNRF2-BIND-luc, -BIND-WT NRF2 or -BIND-Neh4/5mut cells treated with water (control) or 2.5 ng/mL dox for 48 h, probing for FLAG, NRF2, H2B, SQSTM1, GCLC, NR0B1, and TXNRD1 antibodies. Representative data from three independent experiments is shown. .

Article Snippet: EF1ɑ-/TRE3G-promoter-driven Renilla luciferase and NRF2 (WT NRF2, Neh4/5mut, SV40-NLS-ΔNeh1) constructs , GenScript/this work , N/A.

Techniques: Protein-Protein interactions, Expressing, Luciferase, Derivative Assay, Quantitative RT-PCR, Isolation, Control, Construct, Generated, Western Blot, Quantitative Proteomics

Journal: EMBO Reports

Article Title: NRF2 supports non-small cell lung cancer growth independently of CBP/p300-enhanced glutathione synthesis

doi: 10.1038/s44319-025-00463-z

Figure Lengend Snippet: ( A ) Incucyte growth curves of A549-shNRF2-BIND-luc, -BIND-WT NRF2, -BIND-Neh4/5mut, or -BIND-ΔNeh1 cells treated with water (control) or 2.5 ng/mL dox. Representative data from two independent experiments is shown, with mean and SEM derived from 16 images/well indicated. ( B ) Crystal violet staining of A549-shNRF2-BIND-luc, -BIND-WT NRF2, -BIND-Neh4/5mut, or -BIND-ΔNeh1 cells treated with water (control) or 2.5 ng/mL dox for 8 days. Representative data from three independent experiments is shown. ( C ) Correlation between A-485 mean viability and NRF2 dependency as defined by NFE2L2 Chronos score. NRF2 activation score, defined as the average RPKM of NRF2 target genes, is overlaid. Line represents linear regression between mean viability and NRF2 Chronos. ( D ) NRF2 activation score vs GEMINI Score (Zamanighomi et al, ) for co-knockout of CREBBP and EP300 . GEMINI scores represent the added effect of a gene pair interaction, with a score of zero indicating no additional effect beyond each gene’s individual contribution. .

Article Snippet: EF1ɑ-/TRE3G-promoter-driven Renilla luciferase and NRF2 (WT NRF2, Neh4/5mut, SV40-NLS-ΔNeh1) constructs , GenScript/this work , N/A.

Techniques: Control, Derivative Assay, Staining, Activation Assay, Knock-Out