shrna mediated knockdown  (OriGene)

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: A custom FLAG primer synthesized at IDT: Forward: AGG ATG ACG ACG ATA AGG ACT ATA A Rev: CCC GCG CTG CCT TTA TC PRB: /5HEX/TCA TCT TTG /ZEN/TAG TCC TTG TCA TCA TCG TCC /3IABkFQ/ Antibodies: FLAG (Sigma Cat#F3165), NRF2 (Abcam Cat#ab62352), H2B (CST Cat#12364), SQSTM1 (CST Cat#88588), GCLC (CST Cat#48005) TXNRD1 (Abcam Cat#ab16840), NR0B1 (Cat#13538), MAFK (Abcam Cat#ab229766), CBP (CST Cat#7389), β-actin (CST Cat#3700).

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