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

Journal: Theranostics

Article Title: Nrf2 and HIF1α converge to arsenic-induced metabolic reprogramming and the formation of the cancer stem-like cells

doi: 10.7150/thno.42903

Figure Lengend Snippet: Activation of Nrf2 and Nrf2-dependent HIF1α expression in the cells treated by iAs. A. Time-dependent activation of Nrf2 and HIF1α. B. Involvement of JNK in iAs-induced Nrf2 as well as HIF1α. C. Luciferase reporter gene activities of Nrf2 and HIF1α in the cells treated with 1 μM iAs for the indicated times. D. Lentiviral transfection of Keap1 inhibited iAs-induced Nrf2 activation and HIF1α expression. E. Knockout of Nrf2 by CRISPR-Cas9 gene editing prevented HIF1α induction by iAs. Red asterisk denotes the non-specific (N.S.) band.

Article Snippet: Human Keap1 overexpression clone lentiviral particle (RC202189L4V) and Lentiviral control particle (PS100093V) were purchased from Origene Technologies Inc. BEAS-2B cells were seeded in 24-wells plate (5 × 10 4 cells per well) and incubated for 20 hours, and then infected with Keap1 lentiviral overexpression particle and Lentiviral control particle for 20 hours, respectively.

Techniques: Activation Assay, Expressing, Luciferase, Transfection, Knock-Out, CRISPR

Journal: Theranostics

Article Title: Nrf2 and HIF1α converge to arsenic-induced metabolic reprogramming and the formation of the cancer stem-like cells

doi: 10.7150/thno.42903

Figure Lengend Snippet: iAs induces expression of a number of stemness genes through Nrf2 and/or HIF1α activation. A. Enrichment status of Nrf2 and HIF1α (pointed by red arrows) induced by iAs on these indicated stemness genes as determined by ChIP-seq. B. De novo and/or known Nrf2 motif(s) induced by iAs on the promoter or gene body of MYC, CD44, KLF4, and EGFR that had been linked to the stemness of CSCs. C. Nrf2 inhibition by lentiviral Keap1 transfection prevented induction of MYC and SOX2 by iAs. This Nrf2 inhibition also reduced KLF4 expression. D. Nrf2 knockout blocked iAs-induced expression of MYC, SOX2, BACH1, TBC1D7, and decreased expression of KLF4.

Article Snippet: Human Keap1 overexpression clone lentiviral particle (RC202189L4V) and Lentiviral control particle (PS100093V) were purchased from Origene Technologies Inc. BEAS-2B cells were seeded in 24-wells plate (5 × 10 4 cells per well) and incubated for 20 hours, and then infected with Keap1 lentiviral overexpression particle and Lentiviral control particle for 20 hours, respectively.

Techniques: Expressing, Activation Assay, ChIP-sequencing, Inhibition, Transfection, Knock-Out

Journal: Theranostics

Article Title: Nrf2 and HIF1α converge to arsenic-induced metabolic reprogramming and the formation of the cancer stem-like cells

doi: 10.7150/thno.42903

Figure Lengend Snippet: Activation of Nrf2 and Nrf2-dependent HIF1α expression in the cells treated by iAs. A. Time-dependent activation of Nrf2 and HIF1α. B. Involvement of JNK in iAs-induced Nrf2 as well as HIF1α. C. Luciferase reporter gene activities of Nrf2 and HIF1α in the cells treated with 1 μM iAs for the indicated times. D. Lentiviral transfection of Keap1 inhibited iAs-induced Nrf2 activation and HIF1α expression. E. Knockout of Nrf2 by CRISPR-Cas9 gene editing prevented HIF1α induction by iAs. Red asterisk denotes the non-specific (N.S.) band.

Article Snippet: Human Keap1 overexpression clone lentiviral particle (RC202189L4V) and Lentiviral control particle (PS100093V) were purchased from Origene Technologies Inc. BEAS-2B cells were seeded in 24-wells plate (5 × 10 4 cells per well) and incubated for 20 hours, and then infected with Keap1 lentiviral overexpression particle and Lentiviral control particle for 20 hours, respectively.

Techniques: Activation Assay, Expressing, Luciferase, Transfection, Knock-Out, CRISPR

Journal: Theranostics

Article Title: Nrf2 and HIF1α converge to arsenic-induced metabolic reprogramming and the formation of the cancer stem-like cells

doi: 10.7150/thno.42903

Figure Lengend Snippet: iAs induces expression of a number of stemness genes through Nrf2 and/or HIF1α activation. A. Enrichment status of Nrf2 and HIF1α (pointed by red arrows) induced by iAs on these indicated stemness genes as determined by ChIP-seq. B. De novo and/or known Nrf2 motif(s) induced by iAs on the promoter or gene body of MYC, CD44, KLF4, and EGFR that had been linked to the stemness of CSCs. C. Nrf2 inhibition by lentiviral Keap1 transfection prevented induction of MYC and SOX2 by iAs. This Nrf2 inhibition also reduced KLF4 expression. D. Nrf2 knockout blocked iAs-induced expression of MYC, SOX2, BACH1, TBC1D7, and decreased expression of KLF4.

Article Snippet: Human Keap1 overexpression clone lentiviral particle (RC202189L4V) and Lentiviral control particle (PS100093V) were purchased from Origene Technologies Inc. BEAS-2B cells were seeded in 24-wells plate (5 × 10 4 cells per well) and incubated for 20 hours, and then infected with Keap1 lentiviral overexpression particle and Lentiviral control particle for 20 hours, respectively.

Techniques: Expressing, Activation Assay, ChIP-sequencing, Inhibition, Transfection, Knock-Out