Journal: Aging

Article Title: Pharmacological recapitulation of the lean phenotype induced by the lifespan-extending sulfur amino acid-restricted diet.

doi: 10.18632/aging.206237

Figure Lengend Snippet: Figure 3. The SAAR diet and BSO exert tissue-specific effects on Nrf2 and Phgdh. The SAAR diet increased Nrf2 (A) and Phgdh (B) protein expressions in the liver, which ultimately resulted in higher serine concentrations (C). Unlike the SAAR diet, BSO did not increase Nrf2 and Phgdh in the liver but increased both in the kidneys (D, E). Regardless of the changes in Nrf2 and Phgdh, BSO increased serine concentrations in livers and kidneys (C–F). NAC reversed SAAR-induced changes in Nrf2, Phgdh, and serine (A–F). Note: Sample size = 5-6. Statistical methods are similar to those in Figure 2.

Article Snippet: Host species Antibody dilutions Primary Antibodies Nrf2 Cell Signaling Technology 20733 Rabbit 1:1000 in 5% BSAa Phgdh Cell Signaling Technology 13428 Rabbit 1:1000 in 5% milka β-Actin Sigma A5441 Mouse 1:15000 in 5% milka Vinculin Proteintech 66305-1-Ig Mouse 1:10000 in 5% milka Secondary Antibodies Anti-Rabbit-HRP Cell Signaling Technology 7074 Goat 1:4000 in 5% milk, 1hb Anti-Mouse-HRP Bio-Rad 170-6516 Goat 1:20000 in 5% milk, 30 minb aAll incubations were performed overnight at 4° C. bAll incubations were performed at room temperature. www.aging-us.com 22 AGING Supplementary Table 3.

Techniques:

Journal: bioRxiv

Article Title: Nrf2 is a central regulator of the metabolic landscape in macrophages and finetunes their inflammatory response

doi: 10.1101/2021.08.13.456204

Figure Lengend Snippet: (A) Experimental design and workflow (B) Validation of WT, Nrf2 and Keap1-KO genotypes (n = 3 biological replicates). (C) Volcano plot of unstimulated Nrf2-KO compared to WT (D) Volcano plot of unstimulated Keap1-KD compared to WT (E) Transcription factor (TF) enrichment of increased targets in unstimulated Keap1-KD compared to WT (F) Volcano plot of LPS-stimulated Nrf2-KO compared to WT (G) Volcano plot of LPS-stimulated Keap1-KD compared to WT (H) TF enrichment of increased targets in LPS-stimulated Keap1-KD compared to WT (C-D, F-G) (n = 3 biological replicates). Cut-offs - log 2 FC = 0.5; FDR < 0.05, determined using t statistics. (E, F) Determined by Enrichr using ENCODE and ChEA database (combined score – p value and z score).

Article Snippet: Immunoblotting was performed using primary antibodies generated against GAPDH (1:20000, Proteintech 60004-1-Ig), Keap1 (1:2000, Millipore MABS514), and Nrf2 and Nqo1 (1:1000, Cell Signaling #12721, #62262), all of which were diluted in Milk-PBST.

Techniques: Biomarker Discovery

Journal: bioRxiv

Article Title: Nrf2 is a central regulator of the metabolic landscape in macrophages and finetunes their inflammatory response

doi: 10.1101/2021.08.13.456204

Figure Lengend Snippet: (A) Flow cytometry analysis of cell size in unstimulated state (B) TF enrichment of decreased targets in unstimulated Keap1-KD compared to Nrf2-KO (C) TF enrichment of decreased targets in LPS stimulated Nrf2-KO compared to WT (D) Enrichment map of GO: biological processes of significantly increased proteins with LPS stimulation in WT (E) qPCR (n = 3 biological replicates) validation of 4-OI (125 μM) inhibition of IL-1β (A, E) (n = 3 biological replicates). Data are mean ± SEM. p value determined by one-way ANOVA, corrected for multiple comparisons by Tukey statistical test. p <0.05*; p <0.01**; p < 0.001***. (D) ORA by clusterProfiler, FDR correction by Bonferroni test (B-C) Determined by Enrichr using ENCODE and ChEA database (combined score – p value and z score).

Article Snippet: Immunoblotting was performed using primary antibodies generated against GAPDH (1:20000, Proteintech 60004-1-Ig), Keap1 (1:2000, Millipore MABS514), and Nrf2 and Nqo1 (1:1000, Cell Signaling #12721, #62262), all of which were diluted in Milk-PBST.

Techniques: Flow Cytometry, Biomarker Discovery, Inhibition

Journal: bioRxiv

Article Title: Nrf2 is a central regulator of the metabolic landscape in macrophages and finetunes their inflammatory response

doi: 10.1101/2021.08.13.456204

Figure Lengend Snippet: (A) Enrichment map of GO: biological processes of Nrf2 positively regulated targets (Nrf2-KO vs WT) (B) Enrichment map of GO: biological processes of Nrf2 positively regulated targets (Keap1-KD vs WT) (C) Enrichment map of GO: biological processes of Nrf2 negatively regulated targets – Keap1-KD versus WT (A-C) ORA by clusterProfiler, FDR correction by Bonferroni test. (D) Heatmap of significantly altered metabolites (n = 3 biological replicates) and oxygen consumption rates (OCR) (representative of 3 biological replicates). p value determined by one-way ANOVA, corrected for multiple comparisons by Tukey statistical test. Cut off – FDR < 0.05.

Article Snippet: Immunoblotting was performed using primary antibodies generated against GAPDH (1:20000, Proteintech 60004-1-Ig), Keap1 (1:2000, Millipore MABS514), and Nrf2 and Nqo1 (1:1000, Cell Signaling #12721, #62262), all of which were diluted in Milk-PBST.

Techniques:

Journal: bioRxiv

Article Title: Nrf2 is a central regulator of the metabolic landscape in macrophages and finetunes their inflammatory response

doi: 10.1101/2021.08.13.456204

Figure Lengend Snippet: (A) Enrichment of Reactome pathways of Nrf2 positively regulated targets (Nrf2-KO vs WT) (B) Enrichment of Reactome pathways of Nrf2 positively regulated targets (Keap1-KD vs WT) (C) Enrichment of Reactome pathways of Nrf2 negatively regulated targets (Keap1-KD vs WT) (A-C) ORA by Enrichr and FDR correction by Bonferroni test. (D) Enrichment map of GO: biological processes of Nrf2 negatively regulated targets (Nrf2-KO vs WT). ORA by clusterProfiler, FDR correction by Bonferroni test. (E) PCA plot of metabolomics. Determined by Metaboanalyst 5.0. (F) Fold change of prototypical Nrf2 targets (Nrf2-KO vs WT or Keap1-KD vs WT). Data are mean ± SEM. p value determined by multiple t tests - one per row, corrected for multiple comparisons by Holm-Sidak test. p <0.05*; p <0.01**; p < 0.001***.

Article Snippet: Immunoblotting was performed using primary antibodies generated against GAPDH (1:20000, Proteintech 60004-1-Ig), Keap1 (1:2000, Millipore MABS514), and Nrf2 and Nqo1 (1:1000, Cell Signaling #12721, #62262), all of which were diluted in Milk-PBST.

Techniques:

Journal: bioRxiv

Article Title: Nrf2 is a central regulator of the metabolic landscape in macrophages and finetunes their inflammatory response

doi: 10.1101/2021.08.13.456204

Figure Lengend Snippet: (A) Enrichment map of GO: biological processes of Nrf2 positively regulated targets (Nrf2-KO vs WT) (B) Enrichment map of GO: biological processes of Nrf2 positively regulated targets (Keap1-KD vs WT) (A-B) ORA by clusterProfiler, FDR correction by Bonferroni test. (C) Heatmap of significantly altered metabolites (n = 3 biological replicates). p value determined by one-way ANOVA, corrected for multiple comparisons by Tukey statistical test. Cut off – FDR < 0.05. (D) Extracellular acidification rates (ECAR) (representative of 3 biological replicates) (E) Confocal microscopy of mitochondrial morphology using TOM20 (images are representative, bar plot n = 3 biological replicates). Data are mean ± SEM. p value determined by one-way ANOVA, corrected for multiple comparisons by Tukey statistical test. p <0.05*; p <0.01**; p < 0.001***.

Article Snippet: Immunoblotting was performed using primary antibodies generated against GAPDH (1:20000, Proteintech 60004-1-Ig), Keap1 (1:2000, Millipore MABS514), and Nrf2 and Nqo1 (1:1000, Cell Signaling #12721, #62262), all of which were diluted in Milk-PBST.

Techniques: Confocal Microscopy

Journal: bioRxiv

Article Title: Nrf2 is a central regulator of the metabolic landscape in macrophages and finetunes their inflammatory response

doi: 10.1101/2021.08.13.456204

Figure Lengend Snippet: (A) Enrichment map of GO: biological processes of Nrf2 negatively regulated targets (Nrf2-KO vs WT) (B) Enrichment map of GO: biological processes of Nrf2 negatively regulated targets (Keap1-KD vs WT) (A-B) ORA by clusterProfiler, FDR correction by Bonferroni test. (C) Enrichment of Reactome pathways of Nrf2 positively regulated targets (Nrf2-KO vs WT) (D) Enrichment of Reactome pathways of Nrf2 positively regulated targets (Keap1-KD vs WT) (C-D) ORA by Enrichr and FDR correction by Bonferroni test. (E) Confocal microscopy of mitochondrial morphology using Tom20 (images are representative, bar plot n = 3 biological replicates). Data are mean ± SEM. p value determined by one-way ANOVA, corrected for multiple comparisons by Tukey statistical test. (F) Log 2 FC from LPS stimulated proteomics (Nrf2 vs WT & Keap1-KD vs WT).

Article Snippet: Immunoblotting was performed using primary antibodies generated against GAPDH (1:20000, Proteintech 60004-1-Ig), Keap1 (1:2000, Millipore MABS514), and Nrf2 and Nqo1 (1:1000, Cell Signaling #12721, #62262), all of which were diluted in Milk-PBST.

Techniques: Confocal Microscopy

Journal: bioRxiv

Article Title: Nrf2 is a central regulator of the metabolic landscape in macrophages and finetunes their inflammatory response

doi: 10.1101/2021.08.13.456204

Figure Lengend Snippet: (A) Volcano plot of LPS with 4-OI compared to LPS only (WT) (B) Volcano plot of LPS with 4-OI compared to LPS only (Nrf2-KO) (C) TF enrichment of increased targets in LPS with 4-OI compared to LPS only (WT). (A-B) ORA by Enrichr, FDR correction by Bonferroni test. (C) Determined by Enrichr using ENCODE and ChEA database (combined score – p value and z score).

Article Snippet: Immunoblotting was performed using primary antibodies generated against GAPDH (1:20000, Proteintech 60004-1-Ig), Keap1 (1:2000, Millipore MABS514), and Nrf2 and Nqo1 (1:1000, Cell Signaling #12721, #62262), all of which were diluted in Milk-PBST.

Techniques:

Journal: bioRxiv

Article Title: Nrf2 is a central regulator of the metabolic landscape in macrophages and finetunes their inflammatory response

doi: 10.1101/2021.08.13.456204

Figure Lengend Snippet: (A) Enrichment map of GO: biological processes of 4-OI positively regulated targets (WT) (B) Enrichment map of GO: biological processes of 4-OI negatively regulated targets (WT) (C) Enrichment map of GO: biological processes of 4-OI positively regulated targets (Nrf2-KO) (A-C) ORA by clusterProfiler, FDR correction by Bonferroni test. (D) Enrichment of GO: biological processes of 4-OI negatively regulated targets (Nrf2-KO). (E) Enrichment of GO: biological processes of 4-OI negatively regulated targets (WT). (A-E) 4-OI used at 125 μM ( D-E ) ORA by Enrichr and FDR correction by Bonferroni test.

Article Snippet: Immunoblotting was performed using primary antibodies generated against GAPDH (1:20000, Proteintech 60004-1-Ig), Keap1 (1:2000, Millipore MABS514), and Nrf2 and Nqo1 (1:1000, Cell Signaling #12721, #62262), all of which were diluted in Milk-PBST.

Techniques:

Journal: bioRxiv

Article Title: Waves of transcription drive erythroid differentiation and launch the NRF2-activated antioxidant program

doi: 10.1101/2024.07.12.603281

Figure Lengend Snippet: A) Number of GATA-binding factor 1 (GATA1), GATA2, and nuclear factor erythroid 2-related factor 2 (NRF2) targets among induced and repressed genes, as detected with Enrichr. Significant p-values from Enrichr analysis are indicted. B) Time of NRF2 target gene induction upon erythroid differentiation. The colors indicate the three main pathways of antioxidant response: glutathione (GSH) production and metabolisms (light brown), thioredoxin (TXN)-based antioxidation (green) and NADPH production (blue). The bolded text indicates the earliest time point the gene’s nascent transcription is significantly induced. C) Schematic representation of the three main antioxidant pathways. Intermediates (bolded text) and hemin-induced NRF2 targets (rounded rectangles) are indicated. Dashed lines indicate intermediates not shown. D) Genome browser illustration of the induction of NRF2 target gene solute carrier family 7 member 11 ( SLC7A11 ) upon hemin-induced erythroid differentiation. RNA synthesis as PRO-seq reads per kilobase of gene body (gbRPK) are shown for each time point, indicating significant induction in red. E) Relative quantity of GSH and GSSG in K562 cells treated with 30 μM hemin for 60 min or 24 h. n=3. *p<0.05. F) Relative quantity of GSH and GSSG in K562 cells treated with the oxidative stress inducer menadione (MD) for 60 min. n=3. *p<0.05, **p<0.01. G) Schematic illustration comparing the ratio of protective GSH per oxidized GSSG (GSH/GSSG) in erythroid differentiation (left) versus oxidative stress (right). Related Supplementary Figures 5 and 6.

Article Snippet: The NRF2 antibody (D1Z9C, Cell Signaling Technology) was diluted in 5% milk-TBS-0.1% Tween20, applied to the membranes, and the membranes were incubated overnight at 4°C.

Techniques: Binding Assay

Journal: bioRxiv

Article Title: Waves of transcription drive erythroid differentiation and launch the NRF2-activated antioxidant program

doi: 10.1101/2024.07.12.603281

Figure Lengend Snippet: A-B) Hemin-induced RNA synthesis at NRF2 target genes A) glutamate-cysteine ligase modifier subunit ( GCLM ) and B) thioredoxin ( TXN ). C) Insets of e1 (left) and e4 (right) of the beta-globin LCR, shown in the main . The 5’-ends of nascent transcripts enrich at the precise transcription start nucleotide (+1 nt, TSN) and are indicated with asterisks. The 3’-ends of nascent transcripts (3’nt) show the active site of transcription. D) Inset of GCLM promoter region, showing the precise transcription start nucleotide (+1 nt, TSN), as identified from the 5’-ends of nascent RNAs (asterisk). NRF2 motif at the promoter of GCLM locates -26 nt from the +1 nt.

Article Snippet: The NRF2 antibody (D1Z9C, Cell Signaling Technology) was diluted in 5% milk-TBS-0.1% Tween20, applied to the membranes, and the membranes were incubated overnight at 4°C.

Techniques:

Journal: bioRxiv

Article Title: Waves of transcription drive erythroid differentiation and launch the NRF2-activated antioxidant program

doi: 10.1101/2024.07.12.603281

Figure Lengend Snippet: A) The NRF2-recognized DNA motif as identified by MEME-ChIP from the unison of all hemin-induced enhancers (n= 3,757). B) CentriMo analysis of the probability for NRF2 and GATA1 motifs with respect to the enhancer center. C) Fraction of hemin-induced and unchanged enhancers that contain at least one NRF2 motif. The statistical analyses were performed with chi-square. **p<0.01, ***p<0.001. D) DNase I-seq, GATA1 ChIP-seq, transcribed enhancers, NRF2 motifs, and nascent transcription (PRO-seq) at the β-globin locus. The hypersensitive sites 1-4 (HS1-4), here denoted e1-4, indicate the erythroid-specific enhancers in the β-globin locus control region (LCR). The transcriptional profile of e1-4 and the nearest globin gene ( HBE1 ) is shown upon 0 min, 60 min and 24 h hemin treatment, and after 48 h recovery from the 60-min hemin treatment. Quantification of HBE1 (gbRPK; in black), and e1-4 transcription (eRPK; gold, brown, light green, dark green) are shown. E) Quantification of HBE1 (black), HBG1/2 (dark red), and e1-4 transcription (gold, brown, light green, and dark green, respectively) during erythroid differentiation. e1+e2+e3+e4 indicates the total transcription (sum eRPK) across enhancers e1-4. F) Insets of e1 (left) and e4 (right) from panel D , zooming into the GATA1 ChIP-seq summit points. Underlying architecture of transcription initiation (arrows), initiator motif, 5’-ends of eRNAs, GATA motifs and NRF2 motifs are indicated. The precise transcription start nucleotides (+1 nt) are identified from the 5’-ends of nascent transcripts shown in . Related .

Article Snippet: The NRF2 antibody (D1Z9C, Cell Signaling Technology) was diluted in 5% milk-TBS-0.1% Tween20, applied to the membranes, and the membranes were incubated overnight at 4°C.

Techniques: ChIP-sequencing, Control

Journal: bioRxiv

Article Title: Waves of transcription drive erythroid differentiation and launch the NRF2-activated antioxidant program

doi: 10.1101/2024.07.12.603281

Figure Lengend Snippet: A) Significantly (p<0.05) induced (red) or repressed (blue) mRNAs upon hemin-induced erythroid differentiation, as compared to untreated K562 cells. B) Nascent RNA synthesis (left) and mRNA expression (right) of phosphoglycerate dehydrogenase ( PHGDH ). C) NRF2 and γ-globin protein levels in K562 cells transfected with NRF2 siRNA or control siRNA (scr). β-tubulin (TUBB) serves as a loading control. D) Clustering of SHARE-seq (combinatorial scATAC-seq and scRNA-seq) data in human bone marrow. The distinct cell populations are indicated, and the erythroid lineage framed. E) Transcription factor scores of GATA1 and NRF2 during erythroid specification, showing GATA1 activity from erythroid progenitors to late erythroid cells, and NRF2 activation to peak in the late erythroid cells. The SHARE-seq data (D-E) is from reference and visualized in ACAMShiny ( https://buenrostrolab.shinyapps.io/ACAMShiny/ ). F) Summary of transcription regulation in erythroid differentiation, and comparison of transcription mechanisms, kinetics, and regulators upon differentiation versus stress. Erythroid differentiation launches slow but persistent changes in transcription that proceed in waves and are coordinated at the rate-limiting step of initiation. On the contrary, acute stress triggers instant but transient transcriptional reprogramming, coordinated primarily at the Pol II pause-release. Differentiating cells utilize lineage-specific (blue) and stress-inducible (orange) trans -activators to prime and execute transcriptional reprogramming. Ordered activity of GABPA, GATA1, TAL1, HEMGN and NRF2, drive globin expression. Additionally, NRF2 activates the antioxidant program including the synthesis of glutathione (GSH), thioredoxin (TXN) and NADPH biogenesis to prepare differentiating erythroid cells to oxidative stress, encountered as the mature, enucleated oxygen-transporting erythrocytes. Related Supplementary Figures 6-8.

Article Snippet: The NRF2 antibody (D1Z9C, Cell Signaling Technology) was diluted in 5% milk-TBS-0.1% Tween20, applied to the membranes, and the membranes were incubated overnight at 4°C.

Techniques: Expressing, Transfection, Control, Activity Assay, Activation Assay, Comparison

Journal: bioRxiv

Article Title: Waves of transcription drive erythroid differentiation and launch the NRF2-activated antioxidant program

doi: 10.1101/2024.07.12.603281

Figure Lengend Snippet: A) Spearman’s rank correlation (rho) of each biological mRNA-seq replicate pair, measured as counts across exons of each gene. B) Genome browser showing mRNA levels of HSPA1A and HSPA1B genes in replicates 1, 2 and 3. C) mRNA expression of the NRF2 target gene solute carrier family 7 member 11 ( SLC7A11 ). Level of mRNA expression (FPM) is indicated in each condition, red denoting significant induction. D) Induced mRNA expression of NRF2 target genes in the glutathione (GSH) metabolism and synthesis (light brown), thioredoxin (TXN) based antioxidation (green) and NADPH regeneration (blue) pathways. The first time point an mRNA is detected induced is shown in bold. E) Number of genes associated with changed RNA synthesis within 24 h (PRO-seq), and mRNA expression at 48 h (RNA-seq) of erythroid differentiation. F) mRNA expression of the fetal hemoglobin genes HBG1 and HBG2 , and the embryonic hemoglobin gene HBE1 as measured with mRNA-seq upon hemin treatment, and after 4 h or 48 h recovery from a transient hemin exposure.

Article Snippet: The NRF2 antibody (D1Z9C, Cell Signaling Technology) was diluted in 5% milk-TBS-0.1% Tween20, applied to the membranes, and the membranes were incubated overnight at 4°C.

Techniques: Expressing, RNA Sequencing

Journal: bioRxiv

Article Title: Waves of transcription drive erythroid differentiation and launch the NRF2-activated antioxidant program

doi: 10.1101/2024.07.12.603281

Figure Lengend Snippet: A) mRNA expression of platelet-derived growth factor subunit B (PDGFB) upon hemin-induced erythroid differentiation. B) Immunoblot of γ-globin (γ-glob) and NRF2 protein levels in K562 cells treated with hemin for the indicated time point. For recovery, the hemin was removed, and the cells cultured in hemin-free media. β-tubulin (TUBB) was used as a loading control. C-D) Expression on C) GATA1 and D) NRF2 across human tissues, as identified by the Human Protein Atlas. E) GATA1 interactions reported by the Human Protein Atlas. GATA1 and NFE2L2/NRF2 are highlighted. Image credit for C-E: Modified from Human Protein Atlas .

Article Snippet: The NRF2 antibody (D1Z9C, Cell Signaling Technology) was diluted in 5% milk-TBS-0.1% Tween20, applied to the membranes, and the membranes were incubated overnight at 4°C.

Techniques: Expressing, Derivative Assay, Western Blot, Cell Culture, Control, Modification

Journal: bioRxiv

Article Title: Waves of transcription drive erythroid differentiation and launch the NRF2-activated antioxidant program

doi: 10.1101/2024.07.12.603281

Figure Lengend Snippet: A) Single cell mRNA expression of GABPA, GATA1 and HEMGN, and NRF2 target genes HBB, GCLC and GCLM in indicated human bone marrow cells. The mRNA expression is shown in distinct cell clusters that corresponds to the bone marrow populations shown in the main . B) DORC score for GATA1, TAL1, HEMGN and NRF2 in human bone marrow cells, showing their ordered activation during the erythroid lineage specification. The mRNA expression (scRNA-seq) in A , and the DORC score (scATAC-seq and scRNA-seq) in B originate from SHARE-seq data from the reference . The DORC score in erythroid lineage was visualized in ACAMShiny ( https://buenrostrolab.shinyapps.io/ACAMShiny/ ) and shows the same regions as the main . C) Protein expression in human erythrocytes as reported by quantitative mass spectrometry. The peptide counts are shown for all expressed proteins, NRF2 targets genes in the antioxidant pathways, and globins. The quantitative mass spectrometry in panel C originates from reference .

Article Snippet: The NRF2 antibody (D1Z9C, Cell Signaling Technology) was diluted in 5% milk-TBS-0.1% Tween20, applied to the membranes, and the membranes were incubated overnight at 4°C.

Techniques: Expressing, Activation Assay, Mass Spectrometry