Journal: Communications Biology

Article Title: The purine metabolite inosine monophosphate accelerates myelopoiesis and acute pancreatitis progression

doi: 10.1038/s42003-022-04041-0

Figure Lengend Snippet: a RT–QPCR analysis of the mRNA expression of NRF1 and mtTFA in lineage −/low cells after treated with IMP for 24 h, n = 6. b – e Western blot analysis expression level of NRF1, mtTFA and PFAS in IMP-treated cells for 24 or 72 h compared with controls, n = 15–27. Data are presented as the means ± SEM. f – h ADSS, ATPase and IMPDH2 activity in lineage −/low cells which were stimulated with IMP for 0, 4, and 8 h, n = 6. i ATP and GTP production in lineage −/low cells after 24 and 72 h of IMP treatment, n = 6. j , k Oxygen consumption rate analysis of the Lineage −/low cells which were exposed to PBS or 15 mM IMP for 6 h, n = 6. One-way ANOVA with Dunnett was used to compare groups against control. IMP Inosine-5′-monophosphate, ADSS adenylosuccinate synthetase, IMPDH2 Inosine-5′-monophosphate dehydrogenase 2, ATP Adenosine triphosphate, GTP guanosine triphosphate, NRF1 nuclear respiratory factor 1, PFAS phosphoribosylformylglycinamidine synthase.

Article Snippet: Primary antibodies used in our experiments were as follows: Akt (cat. 9272, diluted 1:1000, CST, Danvers, MA, USA); phospho-Akt (Ser473) (D9E) (cat. 4060, diluted 1:1000, CST); S6 Ribosomal Protein (54D2) (cat. 2317, diluted 1:1000, CST); phospho-S6 Ribosomal Protein (Ser235/236) (cat. 2211, diluted 1:1000, CST); NRF1 (D9K6P) (cat. 46743, diluted 1:1000, CST); mtTFA (cat. ab272885, diluted 1:1000, Abcam, Cambridge, UK); PGC-1α (cat. 66369-1-Ig, diluted 1:1000, Proteintech, Chicago, IL, USA); β-actin (13E5) (cat. 4970, diluted 1:2000, CST).

Techniques: Quantitative RT-PCR, Expressing, Western Blot, Activity Assay, Control

Journal: Journal of Translational Medicine

Article Title: Didymin alleviates metabolic dysfunction-associated fatty liver disease (MAFLD) via the stimulation of Sirt1-mediated lipophagy and mitochondrial biogenesis

doi: 10.1186/s12967-023-04790-4

Figure Lengend Snippet: Antibodies

Article Snippet: NRF1 , Rabbit , Proteintech , 12482-1-AP , 1:1000.

Techniques:

Journal: Journal of Translational Medicine

Article Title: Didymin alleviates metabolic dysfunction-associated fatty liver disease (MAFLD) via the stimulation of Sirt1-mediated lipophagy and mitochondrial biogenesis

doi: 10.1186/s12967-023-04790-4

Figure Lengend Snippet: Didymin enhances mitochondrial biogenesis and function by activating Sirt1 in PA-treated AML12 cells. A MitoTracker Green staining for mitochondrial content in AML12 cells. (n = 3, Scale bar = 20 μm). B Mitochondrial oxygen consumption ratio (OCR) of AML12 cells (n = 4). C ROS concentration in AML12 cells (n = 4). D Immunoprecipitation of PGC-1α in AML12 cells showing acetylated PGC-1α level (n = 3). E Western blot analysis of NRF1, TFAM, NDUFB8, SDHB, and MTCO2 in AML12 cells (n = 3). Data are expressed as mean ± SD. *P < 0.05, **P < 0.01, ****P < 0.0001 PA vs. PA + Didymin. # P < 0.05, ## P < 0.01, ### P < 0.001, #### P < 0.0001 control vs. PA. PA palmitic acid, PGC-1α proliferative activated receptor γ coactivator 1α, NRF1 nuclear respiratory factor 1, TFAM mitochondrial transcription factor A

Article Snippet: NRF1 , Rabbit , Proteintech , 12482-1-AP , 1:1000.

Techniques: Staining, Concentration Assay, Immunoprecipitation, Western Blot, Control

Journal: Journal of Translational Medicine

Article Title: Didymin alleviates metabolic dysfunction-associated fatty liver disease (MAFLD) via the stimulation of Sirt1-mediated lipophagy and mitochondrial biogenesis

doi: 10.1186/s12967-023-04790-4

Figure Lengend Snippet: Didymin enhances mitochondrial biogenesis and function, promotes lipophagy, and inhibits apoptosis by activating Sirt1 in mice hepatocytes. A Western blot analysis of Sirt1 expression in hepatocytes (n = 3). B Western blot analysis of NRF1, TFAM, NDUFB8, SDHB, and MTCO2 in hepatocytes. C Transmission electron microscopy figures of hepatocytes, arrows show mitochondria (Scale bar = 0.6 μm). D Transmission electron microscopy figures of hepatocytes, arrows show autolysosomes (Scale bar = 0.6 μm). E Western blot analysis of LC3, Beclin1, P62, PLIN2, LAMP1, and ATG5 in hepatocytes (n = 3). F Western blot analysis of Cleaved PARP, Bax, Bcl2, Cleaved Caspase 3, and Caspase 3 proteins in hepatocytes (n = 3). G Immunofluorescence staining of γ-H2AX in hepatocytes (Scale bar = 50 μm). NRF1 nuclear respiratory factor 1, TFAM mitochondrial transcription factor A, PLIN2 perilipin 2, LAMP1 lysosomal associated membrane protein 1, Bax Bcl-2-associated X protein, Bcl2 B-cell lymphoma 2

Article Snippet: NRF1 , Rabbit , Proteintech , 12482-1-AP , 1:1000.

Techniques: Western Blot, Expressing, Transmission Assay, Electron Microscopy, Immunofluorescence, Staining, Membrane

Journal: Journal of Translational Medicine

Article Title: Didymin alleviates metabolic dysfunction-associated fatty liver disease (MAFLD) via the stimulation of Sirt1-mediated lipophagy and mitochondrial biogenesis

doi: 10.1186/s12967-023-04790-4

Figure Lengend Snippet: Schematic representation of Didymin alleviates MAFLD through the Sirt1 pathway. Didymin binds to SIRT1 protein and activates its deacetylase activity, which in turn deacetylates FoxO3a and PGC-1α and enhances their activity. FoxO3a further increases the expression level of SIRT1 and promotes the process of lipophagy. The increase in lipophagy activity can further inhibit cell apoptosis. PGC-1α enhances mitochondrial biosynthesis and improves mitochondrial function by promoting the transcription of NRF1 and TFAM. PGC-1α proliferative activated receptor γ coactivator 1α, NRF1 nuclear respiratory factor 1, TFAM mitochondrial transcription factor A, FoxO3a Foxkhead Box Class O 3a, PLIN2 perilipin 2, LAMP1 lysosomal associated membrane protein 1, Bax Bcl-2-associated X protein, Bcl2 B-cell lymphoma 2

Article Snippet: NRF1 , Rabbit , Proteintech , 12482-1-AP , 1:1000.

Techniques: Histone Deacetylase Assay, Activity Assay, Expressing, Membrane

Journal: Nature Communications

Article Title: Proteomic profiling and genome-wide mapping of O-GlcNAc chromatin-associated proteins reveal an O-GlcNAc-regulated genotoxic stress response

doi: 10.1038/s41467-020-19579-y

Figure Lengend Snippet: a The chromatin binding sites and enrichment of 33 responsive OCTFs generated through a motif enrichment analysis of MCF-7- and ADR-biased COGC-seq peaks are shown in a heat map. The color of the dots represents the TF motif enrichment. More TF-binding sites are indicated by a large dot size. b Average enrichment profiles of NRF1 ChIP-seq reads (MCF-7 and ADR cells) and published HCF-1 ChIP-seq reads (MCF-7, GSE91992 ) at differential quantitative COGC-seq peaks. c COGC-seq peaks in MCF-7 and ADR cells overlap with NRF1 and a published HCF-1 ( GSE91992 ) ChIP-seq dataset. d O-GlcNAc NRF1 was upregulated in MCF-7 cells after transient stimulation with 100 nM Adm. IP of NRF1 was performed, and the immunoprecipitated fractions were analyzed by immunoblotting for O-GlcNAc (CTD110.6). e The NRF1-HCF-1 interaction is increased in ADR cells compared with MCF-7 cells. NRF1 co-IP was performed, and the immunoprecipitated fractions were analyzed by immunoblotting for the indicated proteins. f NRF-1 is O-GlcNAcylated at Ser448/Ser451. After treatment with PugNAc (Pug, 100 μM) and glucose (Glu, 25 mM) for 24 h, MCF-7 cells stably expressing Flag-WT-NRF1 or Flag-AA-NRF1 were immunoprecipitated with anti-Flag magnetic beads. O-GlcNAcylation (CTD110.6) was analyzed by immunoblotting. WT, wild-type NRF1; AA, Ser447/Ser450 → Ala mutational NRF1. Mock, cells transfected with empty pCMVPuro64 vector. g O-GlcNAc promotes the interaction of HCF-1 and OGT with NRF1. Immunoblotting showing the PPIs of endogenous HCF-1 and OGT with NRF1 in ADR cells. ADR cells stably expressing Flag-WT-NRF1 or Flag-AA-NRF1 were immunoprecipitated with anti-Flag magnetic beads. h O-GlcNAc inhibition expedites the degradation of NRF1. ADR cells expressing Flag-WT-NRF1 or Flag-AA-NRF1 were incubated with 50 μM cycloheximide (CHX) for up to 12 h. The expression levels of Flag-NRF1 were monitored by immunoblotting. i O-GlcNAc enhances the chromatin binding of NRF1. The crosslinked chromatin proteins were extracted, and the levels of Flag-NRF1 were detected by immunoblotting. For ( d – i ), all blots are representative of at least two biologically independent experiments. a , d – i Source Data are provided as a Source Data file.

Article Snippet: SP1 siRNA (#sc-29487), KLF5 siRNA (#sc-37718), NRF1 shRNA (#sc-38105-SH) and NSMCE2 siRNA (#sc-77813) were purchased from Santa Cruz Biotechnology.

Techniques: Binding Assay, Generated, ChIP-sequencing, Immunoprecipitation, Western Blot, Co-Immunoprecipitation Assay, Stable Transfection, Expressing, Magnetic Beads, Transfection, Plasmid Preparation, Inhibition, Incubation

Journal: Nature Communications

Article Title: Proteomic profiling and genome-wide mapping of O-GlcNAc chromatin-associated proteins reveal an O-GlcNAc-regulated genotoxic stress response

doi: 10.1038/s41467-020-19579-y

Figure Lengend Snippet: a NRF-1 ChIP-seq signal in NRF-1 uniquely bound sites identified by overlapping MCF-7 and ADR NRF-1 peaks. Upper panel: Venn diagram showing the overlap of NRF-1 peaks in MCF-7 and ADR cells. The percentage of peaks annotated to promoter regions is indicated. Lower panel: Heat map representation of NRF-1 signal enrichment (red, low; blue, high) at NRF-1 uniquely bound sites. The enrichment levels were profiled ±3 kb from the peak center. b Heat map representation of COGC-seq signal enrichment (red, low; blue, high) at NRF-1 uniquely bound sites. The enrichment levels were profiled ±3 kb from the peak center. c Average enrichment profiles of published H3K27ac, H3K4me3 ( GSE97481 ), H3K27me3 ( GSE96363 ) and H3K4me1 ( GSE86714 ) ChIP-seq reads at NRF-1 uniquely bound sites. d The box plots showing the mRNA expression changes (RNA-seq FPKM) of NRF1-binding genes associated with MCF-7- and ADR-biased peaks. The box plots show the medians (black lines), 25th and 75th percentiles (boundaries), and minimum/maximum values (whiskers). The p value (0.0000006, two-sided unpaired Student’s t -test, calculated between multiple genes in each group) is indicated. n = 2 biologically independent RNA-seq replicates. Source Data are provided as a Source Data file. e Heat map representation of WT-NRF-1 and AA-NRF-1 signal enrichment (red, low; blue, high) at NRF-1 binding sites in MCF-7 and ADR cells. The enrichment levels were profiled ±3 kb from the peak center.

Article Snippet: SP1 siRNA (#sc-29487), KLF5 siRNA (#sc-37718), NRF1 shRNA (#sc-38105-SH) and NSMCE2 siRNA (#sc-77813) were purchased from Santa Cruz Biotechnology.

Techniques: ChIP-sequencing, Expressing, RNA Sequencing, Binding Assay

Journal: Nature Communications

Article Title: Proteomic profiling and genome-wide mapping of O-GlcNAc chromatin-associated proteins reveal an O-GlcNAc-regulated genotoxic stress response

doi: 10.1038/s41467-020-19579-y

Figure Lengend Snippet: a Effect of NRF1 O-GlcNAc modification on the indicated gene transcription levels in ADR cells. The gene mRNA levels in ADR cells expressing Flag-WT-NRF1 or Flag-AA-NRF1 were analyzed by quantitative PCR (qPCR). b Left panel: IGV tracks showing the signals at the promoter regions of the representative genes. Right panel: Validation of O-GlcNAc NRF1 binding peaks by ChIP-qPCR. qPCR amplification was performed. Each bar represents the fold enrichment of binding relative to the input. IgG and random primers that could not specifically bind the indicated gene promoter regions (off target) were used as negative controls. Mock, cells transfected with empty pCMVPuro64 vector. c O-GlcNAc inhibition reduces the transcriptional activity of NRF1. 293T cells were transfected with a reporter vector consisting of luciferase cDNA fused to the NSMCE2 promoter. The pGL3-basic vector (Mock) was used as a control. d ADR cells were transfected with NSMCE2 siRNA (siNSMCE2) or scrambled siRNA (siScr) and treated with increasing doses of Adm for 48 h. The cell viability was then assessed. Representative images of cell viability determined by crystal violet staining are shown. Results were reproduced in two biologically independent experiments. The protein levels of NSMCE2 were monitored by immunoblotting. All blots are representative of at least two biologically independent experiments. For ( a - c ), the data are presented as the means ± SEM., ( d ) replicates are represented. ( a – d ) n = 3 biologically independent experiments, * p < 0.05, ** p < 0.01, *** p < 0.001 (two-sided unpaired Student’s t -test). p values: 0.010099, 0.001375, 0.046814, 0.020148 ( a ); 0.010191, 0.000117, 0.000159, 0.001032 ( b ); 0.0065 (WT-NRF1 vs. WT-NRF1 L01), 0.0002 (WT-NRF1 vs. AA-NRF1), 0.0000858 (WT-NRF1 vs. AA-NRF1 L01) ( c ); 0.001575 ( d ). a – d Experiments were repeated independently two times with similar results. Source Data are provided as a Source Data file.

Article Snippet: SP1 siRNA (#sc-29487), KLF5 siRNA (#sc-37718), NRF1 shRNA (#sc-38105-SH) and NSMCE2 siRNA (#sc-77813) were purchased from Santa Cruz Biotechnology.

Techniques: Modification, Expressing, Real-time Polymerase Chain Reaction, Biomarker Discovery, Binding Assay, ChIP-qPCR, Amplification, Transfection, Plasmid Preparation, Inhibition, Activity Assay, Luciferase, Control, Staining, Western Blot

Journal: Nature Communications

Article Title: Proteomic profiling and genome-wide mapping of O-GlcNAc chromatin-associated proteins reveal an O-GlcNAc-regulated genotoxic stress response

doi: 10.1038/s41467-020-19579-y

Figure Lengend Snippet: Genotoxicity provokes O-GlcNAc (G) elevation and dynamic changes in multiple OCTF genomic binding sites. The activity of multiple OCTFs, including NRF1, modulates a network of transcriptome upregulation to induce a holistic effect on cell fates in response to genotoxic stress.

Article Snippet: SP1 siRNA (#sc-29487), KLF5 siRNA (#sc-37718), NRF1 shRNA (#sc-38105-SH) and NSMCE2 siRNA (#sc-77813) were purchased from Santa Cruz Biotechnology.

Techniques: Binding Assay, Activity Assay

Journal: International Journal of Molecular Sciences

Article Title: Maternal Low-Protein Diet Leads to Mitochondrial Dysfunction and Impaired Energy Metabolism in the Skeletal Muscle of Male Rats

doi: 10.3390/ijms252312860

Figure Lengend Snippet: Figure 1. Effects of LP programming on the expression of genes involved in mitochondrial dynamics and biogenesis in the skeletal muscle of control and LP rats. The mRNA levels of mitochondrial dynamic genes: (a) Mfn1; (b) Mfn2; (c) Opa1; (d) Fis1; (e) Drp1 (f) Tfam, (g) Pgc1A (h) Pgc1B (i) Essra (j) Nrf1 were analyzed by qPCR. The mRNA expressions of each gene were normalized to the average of internal controls. Data represent mean ± SEM (* p < 0.05, ** p < 0.01); n = 5.

Article Snippet: Details of primary antibodies and their dilutions are as follows: and Gapdh (Cat #97166, 1:1000), Vdac1 (Cat #4661, 1:1000), Opa1 (Cat #80471, 1:1000), Nrf1 (Cat #46743, 1:1000), Sirt1 (Cat #9475, 1:1000), Erra (Cat #13826, 1:1000), Cox-IV (Cat #4850, 1:5000) were obtained from cell signaling, Danvers, MA, USA; Fis1 (Cat # sc-376447, 1:1000), Mfn2 (Cat # sc-515647, 1:1000) were obtained from Santa Cruz Biotechnology, Dallas, TX, USA.

Techniques: Expressing, Control