Journal: Molecules : A Journal of Synthetic Chemistry and Natural Product Chemistry

Article Title: Docosahexaenoic Acid Induces Expression of Heme Oxygenase-1 and NAD(P)H:quinone Oxidoreductase through Activation of Nrf2 in Human Mammary Epithelial Cells

doi: 10.3390/molecules22060969

Figure Lengend Snippet: Possible involvement of Keap1 cysteine thiol modification in DHA-induced HO-1 and NQO1 expression. ( A ) MCF-10A cells were pretreated with or without a thiol reducing agent DTT (500 μM) for 1 h, followed by 18-h incubation with 25 μM DHA. ( B ) Cells were pretreated with another thiol reducing agent NEM (25 μM) for 1 h, followed by 18-h incubation with 25 μM DHA. ( C ) MCF-10A cells were transfected with HA-mock, HA-Keap1 WT, Keap1-C151S, Keap1-C273S, or Keap1-C288S expressing vector for 24 h, and incubated with DHA (25 μM) for another 9 h to determine the expression of HO-1 and NQO1. HA-Keap1 was used to ensure the equal expression of mutant vectors. Each blot is a representative of three different experiments. Columns, means (n = 3); bars, SD. *, p < 0.05 or **, p < 0.01. NS. non-significant. ( D ) MCF-10A cells were incubated with DHA (25 or 50 μM) for 6 h and then treated with RIPA lysis buffer. Cell lysates were incubated with BPM for 30 min, and then immunoprecipitated with avidin-agarose. The precipitated proteins were subjected to Western blot analysis with an anti-Keap1 antibody.

Article Snippet: Primary antibodies for Nrf2, Keap1, normal mouse IgG, PKCδ siRNA (sc-36253), and protein G plus-agarose were supplied by Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Modification, Expressing, Incubation, Transfection, Plasmid Preparation, Mutagenesis, Lysis, Immunoprecipitation, Avidin-Biotin Assay, Western Blot

Journal: Nature

Article Title: Itaconate is an anti-inflammatory metabolite that activates Nrf2 via alkylation of KEAP1

doi: 10.1038/nature25986

Figure Lengend Snippet: a–c, Metabolite levels and itaconate abundance in control (ctrl) versus LPS-induced (a, n = 12, 4 h; b, c, n = 5, 24 h) human (a) and mouse (b, c) macrophages. Red and blue dots represent metabolites significantly up- and downregulated by LPS, respectively. FDR, false discovery rate. d, Reactivity of itaconate with KEAP1 thiol group. e, g, LPS-induced Nrf2 (e, 24 h) and HMOX1 (g, 6 h) after treatment with OI as indicated. f, Nrf2 target gene expression in mouse macrophages with or without LPS (6 h) and OI (Nqo1, Gclm, Hmox1, n = 12; Gsr, Pgd, Taldo1, n = 6). h, NQO1 activity in mouse Hepa1c1c7 cells treated as indicated (48 h, n = 8). Data are mean ± s.e.m. P values calculated using one-way or two-way analysis of variance (ANOVA) for multiple comparisons or two-tailed Student’s t-test for paired comparisons. Blots are representative of three independent experiments. In the box plots, line shows mean. For gel source data, see Supplementary Fig. 1.

Article Snippet: Analysis of KEAP1 modification by OI Human embryonic kidney cells (HEK293T cells) were transfected with a pCMV6-KEAP1 vector (Myc-DDK-tagged mouse KEAP1) (OriGene).

Techniques: Expressing, Activity Assay, Two Tailed Test

Journal: Nature

Article Title: Itaconate is an anti-inflammatory metabolite that activates Nrf2 via alkylation of KEAP1

doi: 10.1038/nature25986

Figure Lengend Snippet: a, Itaconate transport by the indicated carriers(n = 4). HsAAC1, Homo sapiens ADP/ATP carrier; HsCTP, H. sapiens citrate carrier; HsODC, H. sapiens oxodicarboxylate carrier; HsOGC, H. sapiens 2-oxoglutarate carrier; ScAAC2, Saccharomyces cerevisiae ADP/ATP carrier; ScDIC, S. cerevisiae dicarboxylate carrier. b, Nrf2 and KEAP1 protein after co-transfection with Nrf2–V5, and the wild-type (WT) or Cys151Ser mutant KEAP1. c, Tandem mass spectrometry spectrum of Cys151-containing KEAP1 peptide after OI treatment. d, Metabolite (13C6-glucose (left), 13C5-glutamine (right)) tracing to itaconate-cysteine adduct with or without LPS (24 h, n = 5). AU, arbitrary units. e, LDHA Cys84 alkylation plus LPS (24 h) or OI (250 μM, 4 h) (n = 4). Data are mean ± s.e.m. (in d, e) or s.d. (in a). P values calculated using one-way ANOVA for multiple comparisons or two-tailed Student’s t-test for paired comparisons. Blots are representative of three independent experiments. For gel source data, see Supplementary Fig. 1.

Article Snippet: Analysis of KEAP1 modification by OI Human embryonic kidney cells (HEK293T cells) were transfected with a pCMV6-KEAP1 vector (Myc-DDK-tagged mouse KEAP1) (OriGene).

Techniques: Cotransfection, Mutagenesis, Mass Spectrometry, Two Tailed Test

Journal: Nature

Article Title: Itaconate is an anti-inflammatory metabolite that activates Nrf2 via alkylation of KEAP1

doi: 10.1038/nature25986

Figure Lengend Snippet: Mass spectrometry analysis of itaconate-induced cysteine alkylation a , Cysteine/lysine residue(s) in KEAP1 modified by OI as determined by tandem mass spectrometry. b , Cysteine residues modified by itaconate in BMDMs treated with LPS identified using tandem mass spectrometry. c , Cysteine residues modified by itaconate in BMDMs treated with OI identified using tandem mass spectrometry.

Article Snippet: Analysis of KEAP1 modification by OI Human embryonic kidney cells (HEK293T cells) were transfected with a pCMV6-KEAP1 vector (Myc-DDK-tagged mouse KEAP1) (OriGene).

Techniques: Mass Spectrometry, Modification, Sequencing

Journal: bioRxiv

Article Title: Metabolic Reprogramming by Histone Deacetylase Inhibition Selectively Targets NRF2-activated tumors

doi: 10.1101/2023.04.24.538118

Figure Lengend Snippet: A. Schematic indicating domains of the full-length and the ΔNeh2 over-active mutant isoforms of NRF2 protein¹. B. NRF2 protein levels upon of overexpression of NRF2ΔNeh2 in KP cells (NRF2). C. Expression of NRF2 target genes upon overexpression of NRF2ΔNeh2 in KP cells. D. Viability assay of KP cells treated with the indicated concentrations of Romidepsin for 72h. E. NRF2 protein levels of KP cells carrying dox-inducible NRF2ΔNeh2 upon treatment with DMSO, KI696 or doxycycline (DOX) and of KPK cells overexpressing luciferase (LUC) or KEAP1. F. Viability assay of KP and KPK cells treated with the indicated concentrations for 5 days. G. Growth of subcutaneous KP tumors in C57/BL6 mice treated with Romidepsin or vehicle, and tumor weight at the end of treatment ( H ).

Article Snippet: NRF2ΔNeh2, Keap1 (mouse and human), and KEAP1 R470C overexpression constructs were generated by the Papagiannakopoulos lab. For CRISPR/Cas9 gene knock-out, we used the lentiCas9-blast plasmid (Addgene) and the pUSEPR vector for sgRNA (U6-sgRNA-EFS-Puro-P2A-TurboRFP in pLL3-based lentiviral backbone).

Techniques: Mutagenesis, Over Expression, Expressing, Viability Assay, Luciferase

Journal: bioRxiv

Article Title: Metabolic Reprogramming by Histone Deacetylase Inhibition Selectively Targets NRF2-activated tumors

doi: 10.1101/2023.04.24.538118

Figure Lengend Snippet: A. Bar graph indicating IC50s to various HDAC inhibitors, derived from 3-day viability experiments (n=3) in KP cells carrying dox-inducible NRF2ΔNeh2 pre-treated with DMSO, KI696 or DOX for 1 week (Statistical significance determined by paired t-test). B . HDAC inhibitor AUC (area under curve) comparison of KEAP1 WT and mutant NSCLC cell lines from the DepMap Prism repurposing secondary screen 19Q4. C and D . Protein levels of phosphorylated H2A.X and cleaved caspase 3 upon treatment with Romidepsin at a concentration were the difference in cell viability between control and NRF2 activated cells is more pronounced.

Article Snippet: NRF2ΔNeh2, Keap1 (mouse and human), and KEAP1 R470C overexpression constructs were generated by the Papagiannakopoulos lab. For CRISPR/Cas9 gene knock-out, we used the lentiCas9-blast plasmid (Addgene) and the pUSEPR vector for sgRNA (U6-sgRNA-EFS-Puro-P2A-TurboRFP in pLL3-based lentiviral backbone).

Techniques: Derivative Assay, Comparison, Mutagenesis, Concentration Assay, Control

Journal: bioRxiv

Article Title: Metabolic Reprogramming by Histone Deacetylase Inhibition Selectively Targets NRF2-activated tumors

doi: 10.1101/2023.04.24.538118

Figure Lengend Snippet: A. Normalized RNA-seq read counts (by DESeq2) of amino-acid transporters involved in glutamine uptake. B . IHC staining of ATF4 and quantitation ( C ) in KP tumors after 17 days of treatment with DMSO or Romidepsin (related to ). D . Volcano plot showing gene effect difference between KEAP1 wild-type and mutant NSCLC cell line from the CERES DepMap dataset. E . Western blots indicating levels of indicated proteins upon CRISPR/Cas9 KO in KP cells - associated with competition assay in 4F. F . Quantitation of IHC staining of c-MYC in KP EV tumors (related to ). G . Bedgraphs of H4ac and BRD4 binding at indicated genes.

Article Snippet: NRF2ΔNeh2, Keap1 (mouse and human), and KEAP1 R470C overexpression constructs were generated by the Papagiannakopoulos lab. For CRISPR/Cas9 gene knock-out, we used the lentiCas9-blast plasmid (Addgene) and the pUSEPR vector for sgRNA (U6-sgRNA-EFS-Puro-P2A-TurboRFP in pLL3-based lentiviral backbone).

Techniques: RNA Sequencing, Immunohistochemistry, Quantitation Assay, Mutagenesis, Western Blot, CRISPR, Competitive Binding Assay, Binding Assay

Journal: bioRxiv

Article Title: Metabolic Reprogramming by Histone Deacetylase Inhibition Selectively Targets NRF2-activated tumors

doi: 10.1101/2023.04.24.538118

Figure Lengend Snippet: A . Schematic of CRISPR-mediated KO of NRF2, and NRF2 protein levels upon of NRF2 KO in a population of A549 cells (sgNRF2). B . Expression of NRF2 target genes upon NRF2 KO in A549 cells. C . Fold-Change expression of indicated genes upon Romidepsin treatment of A549 cells determined by RT-qPCR. D . Western blot showing levels of MYC and H4ac upon Romidepsin treatment of A549 cells 0.5nM for 24h. E . Viability assay of A549 cells treated with the indicated concentrations of Romidepsin for 72h. F . Growth of subcutaneous A549 tumors in nude mice treated with Romidepsin or vehicle, and tumor weight at the end of treatment ( G ). H-I . Relative growth curves of PDX tumors that are wild-type or mutant for KEAP1 treated with vehicle or Romidepsin.

Article Snippet: NRF2ΔNeh2, Keap1 (mouse and human), and KEAP1 R470C overexpression constructs were generated by the Papagiannakopoulos lab. For CRISPR/Cas9 gene knock-out, we used the lentiCas9-blast plasmid (Addgene) and the pUSEPR vector for sgRNA (U6-sgRNA-EFS-Puro-P2A-TurboRFP in pLL3-based lentiviral backbone).

Techniques: CRISPR, Expressing, Quantitative RT-PCR, Western Blot, Viability Assay, Mutagenesis

Journal: bioRxiv

Article Title: Metabolic Reprogramming by Histone Deacetylase Inhibition Selectively Targets NRF2-activated tumors

doi: 10.1101/2023.04.24.538118

Figure Lengend Snippet: A. NRF2 protein levels upon overexpression of wild-type or R470C mutant KEAP1 in NCI-H2009 cells. B . Viability assay of H2009 cells treated with the indicated concentrations of Romidepsin for 72h. C . Tumor volumes of PDX tumors at the indicated days of treatment and tumor weights at the experiment endpoint (related to 6H-I). D . IHC staining of c-MYC and quantitation in PDX tumors after 17 days of treatment with Vehicle (VEH) or Romidepsin (related to ).

Article Snippet: NRF2ΔNeh2, Keap1 (mouse and human), and KEAP1 R470C overexpression constructs were generated by the Papagiannakopoulos lab. For CRISPR/Cas9 gene knock-out, we used the lentiCas9-blast plasmid (Addgene) and the pUSEPR vector for sgRNA (U6-sgRNA-EFS-Puro-P2A-TurboRFP in pLL3-based lentiviral backbone).

Techniques: Over Expression, Mutagenesis, Viability Assay, Immunohistochemistry, Quantitation Assay