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$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 <t>NRF1</t> 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.$
Nrf1 Shrna, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "Proteomic profiling and genome-wide mapping of O-GlcNAc chromatin-associated proteins reveal an O-GlcNAc-regulated genotoxic stress response"

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

Journal: Nature Communications

doi: 10.1038/s41467-020-19579-y

$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.$
Figure Legend 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.

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

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.

Figure Legend 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.

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

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.

Figure Legend 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.

Techniques Used: 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

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.

Figure Legend 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.

Techniques Used: Binding Assay, Activity Assay

shRNA:

Article Title: Proteomic profiling and genome-wide mapping of O-GlcNAc chromatin-associated proteins reveal an O-GlcNAc-regulated genotoxic stress response
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. .. Transfection of the 293T, MCF-7 and MCF-7/ADR cells was performed with Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions.

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Genetic manipulation of L- Nrf1 expression in Nrf2 -deficient RAW 264.7 cells and osteoclast differentiation following cadmium exposure. The (A) mRNA (A) and (B) protein levels of L-NRF1 in RAW cells with lentiviral Nrf2 silencing treated with RANKL ( 50 ng / mL ) and M-CSF ( 30 ng / mL ) for indicated days in the presence of 20 nM Cd. n = 3 – 9 . Values are expressed as mean ± SD . Two-way ANOVA with Bonferroni multiple comparison posttest was used. * p < 0.05 vs. Scr with the same treatment; # p < 0.05 vs. the same genotype of D0. (C,D,I,J) Representative images (C) and (I) and quantifications (D) and (J) of TRAP staining of RAW cells with Nrf2-KD and L- Nrf1 knockdown or overexpression treated with RANKL ( 50 ng / mL ) and M-CSF ( 30 ng / mL ) for 5 d in the presence (i.e., Cd) or absence (i.e., Veh) of 20 nM Cd. For quantification, TRAP-positive multinucleated cells containing three or more nuclei on the images were counted. n = 3 – 4 . Values are expressed as mean ± SD . Two-way ANOVA with Bonferroni multiple comparison posttest was used. * p < 0.05 vs. the same genotype with Veh; # p < 0.05 vs. Scr with the same treatment; & p < 0.05 vs. KD with the same treatment. (E–H, K–N) The mRNA expression of Cathepsin K , H + - a t p a s e , and Atp6v0d2 (E–G) and (K-M), as well as the protein levels of NFATc1 (H) and (N) were measured by RT-qPCR and immunoblotting, respectively, in the cells treated as detailed in (C), (D), (I), and (J). n = 3 – 4 . Values are expressed as mean ± SD . Two-way ANOVA with Bonferroni multiple comparison posttest was used. * p < 0.05 vs. the same genotype with Veh; # p < 0.05 vs. Scr with the same treatment; & p < 0.05 vs. KD with the same treatment. Summary data are provided in Tables S50–S58. Quantifications of (B), (H), and (N) are found in Figure S10. Note: ANOVA, analysis of variance; Atp6v0d2, ATPase H positive Transporting V 0 Subunit D 2; D0–D5, the day following osteoclastogenic treatments; DKD, Nrf2 and L-Nrf1 double knockdown; KD, Nrf2 -knockdown (Nrf2-KD); KD+OE, Nrf2-KD and L-NRF1-741 overexpression; L-NRF1, long isoform nuclear factor-erythroid 2-related factor 1; M-CSF, macrophage colony-stimulating factor; MNC, multinucleated cells; NFATc1, nuclear factor of activated T cells, cytoplasmic, calcineurin dependent 1; Nrf2, nuclear factor erythroid 2-related factor 2; RANKL, receptor activator of nuclear factor kappa-B ligand; RAW 264.7, mouse leukemic monocyte/macrophage cells; RT-qPCR, reverse transcription quantitative polymerase chain reaction; Scr, Scramble; SD, standard deviation; Trap, tartrate-resistant acid phosphatase; Veh, vehicle.

Journal: Environmental Health Perspectives

Article Title: Prolonged Cadmium Exposure and Osteoclastogenesis: A Mechanistic Mouse and in Vitro Study

doi: 10.1289/EHP13849

Figure Lengend Snippet: Genetic manipulation of L- Nrf1 expression in Nrf2 -deficient RAW 264.7 cells and osteoclast differentiation following cadmium exposure. The (A) mRNA (A) and (B) protein levels of L-NRF1 in RAW cells with lentiviral Nrf2 silencing treated with RANKL ( 50 ng / mL ) and M-CSF ( 30 ng / mL ) for indicated days in the presence of 20 nM Cd. n = 3 – 9 . Values are expressed as mean ± SD . Two-way ANOVA with Bonferroni multiple comparison posttest was used. * p < 0.05 vs. Scr with the same treatment; # p < 0.05 vs. the same genotype of D0. (C,D,I,J) Representative images (C) and (I) and quantifications (D) and (J) of TRAP staining of RAW cells with Nrf2-KD and L- Nrf1 knockdown or overexpression treated with RANKL ( 50 ng / mL ) and M-CSF ( 30 ng / mL ) for 5 d in the presence (i.e., Cd) or absence (i.e., Veh) of 20 nM Cd. For quantification, TRAP-positive multinucleated cells containing three or more nuclei on the images were counted. n = 3 – 4 . Values are expressed as mean ± SD . Two-way ANOVA with Bonferroni multiple comparison posttest was used. * p < 0.05 vs. the same genotype with Veh; # p < 0.05 vs. Scr with the same treatment; & p < 0.05 vs. KD with the same treatment. (E–H, K–N) The mRNA expression of Cathepsin K , H + - a t p a s e , and Atp6v0d2 (E–G) and (K-M), as well as the protein levels of NFATc1 (H) and (N) were measured by RT-qPCR and immunoblotting, respectively, in the cells treated as detailed in (C), (D), (I), and (J). n = 3 – 4 . Values are expressed as mean ± SD . Two-way ANOVA with Bonferroni multiple comparison posttest was used. * p < 0.05 vs. the same genotype with Veh; # p < 0.05 vs. Scr with the same treatment; & p < 0.05 vs. KD with the same treatment. Summary data are provided in Tables S50–S58. Quantifications of (B), (H), and (N) are found in Figure S10. Note: ANOVA, analysis of variance; Atp6v0d2, ATPase H positive Transporting V 0 Subunit D 2; D0–D5, the day following osteoclastogenic treatments; DKD, Nrf2 and L-Nrf1 double knockdown; KD, Nrf2 -knockdown (Nrf2-KD); KD+OE, Nrf2-KD and L-NRF1-741 overexpression; L-NRF1, long isoform nuclear factor-erythroid 2-related factor 1; M-CSF, macrophage colony-stimulating factor; MNC, multinucleated cells; NFATc1, nuclear factor of activated T cells, cytoplasmic, calcineurin dependent 1; Nrf2, nuclear factor erythroid 2-related factor 2; RANKL, receptor activator of nuclear factor kappa-B ligand; RAW 264.7, mouse leukemic monocyte/macrophage cells; RT-qPCR, reverse transcription quantitative polymerase chain reaction; Scr, Scramble; SD, standard deviation; Trap, tartrate-resistant acid phosphatase; Veh, vehicle.

Article Snippet: Lentivirus containing short-hairpin RNA (shRNA) targeting Nrf2 (SHVRS-NM_010902, Sigma-Aldrich), L- Nrf1 (SHCLND-NM_008686, Sigma-Aldrich) or Scramble (Scr) nontarget negative control (SHC002V, Sigma-Aldrich) (Table S2) were used to transfect RAW cells as described previously.

Techniques: Expressing, Comparison, Staining, Knockdown, Over Expression, Quantitative RT-PCR, Western Blot, Reverse Transcription, Real-time Polymerase Chain Reaction, Standard Deviation

Effects of N -acetyl- l -cysteine (NAC) and mitoquinone mesylate (MitoQ) treatment on the protein levels of L-NRF1 and NFATc1 and osteoclast differentiation in Nrf2 -KD and Scr RAW 264.7 cells. The cells were treated with RANKL ( 50 ng / mL ) and M-CSF ( 30 ng / mL ) for 5 d in the presence of 20 nM cadmium (Cd) with NAC ( 2 mM ) or MitoQ ( 0.2 μ M ). (A,G) Representative images of immunoblotting of L-NRF1 (upper panels) and NFATc1 (lower panels). (B,C,H,I) Representative images of TRAP staining (B) and (H) and the quantification of TRAP-positive multinucleated cells containing three or more nuclei (C) and (I). n = 3 – 5 . Values are expressed as mean ± SD . Two-way ANOVA with Bonferroni multiple comparison posttest was used. * p < 0.05 vs. Scr with the same treatment; # p < 0.05 vs. the same genotype with Veh; & p < 0.05 vs. the same genotype with Cd alone. (D–F,J–L) mRNA expression of Cathepsin K , H + - a t p a s e , and Atp6v0d2 . n = 3 – 4 . Values are expressed as mean ± SD . Two-way ANOVA with Bonferroni multiple comparison posttest was used, * p < 0.05 vs. Scr with the same treatment; # p < 0.05 vs. the same genotype with Veh; & p < 0.05 vs. the same genotype with Cd alone. Summary data are provided in Tables S59–S66. Quantification of (A) and (G) is found in Figure S16. Note: ANOVA, analysis of variance; Atp6v0d2, ATPase H positive Transporting V 0 Subunit D 2; Cd, Cd alone; Cd+MitoQ, Cd and MitoQ co-treatment; Cd+NAC, Cd and NAC co-treatment; KD, Nrf2 -knockdown ( Nrf2 -KD); L-NRF1, long isoform nuclear factor-erythroid 2-related factor 1; M-CSF, macrophage colony-stimulating factor; MitoQ, MitoQ alone; NAC, NAC alone; NFATc1, nuclear factor of activated T cells, cytoplasmic, calcineurin dependent 1; RANKL, receptor activator of nuclear factor kappa-B ligand; Scr, Scramble; SD, standard deviation; Trap, tartrate-resistant acid phosphatase; Veh, vehicle.

Journal: Environmental Health Perspectives

Article Title: Prolonged Cadmium Exposure and Osteoclastogenesis: A Mechanistic Mouse and in Vitro Study

doi: 10.1289/EHP13849

Figure Lengend Snippet: Effects of N -acetyl- l -cysteine (NAC) and mitoquinone mesylate (MitoQ) treatment on the protein levels of L-NRF1 and NFATc1 and osteoclast differentiation in Nrf2 -KD and Scr RAW 264.7 cells. The cells were treated with RANKL ( 50 ng / mL ) and M-CSF ( 30 ng / mL ) for 5 d in the presence of 20 nM cadmium (Cd) with NAC ( 2 mM ) or MitoQ ( 0.2 μ M ). (A,G) Representative images of immunoblotting of L-NRF1 (upper panels) and NFATc1 (lower panels). (B,C,H,I) Representative images of TRAP staining (B) and (H) and the quantification of TRAP-positive multinucleated cells containing three or more nuclei (C) and (I). n = 3 – 5 . Values are expressed as mean ± SD . Two-way ANOVA with Bonferroni multiple comparison posttest was used. * p < 0.05 vs. Scr with the same treatment; # p < 0.05 vs. the same genotype with Veh; & p < 0.05 vs. the same genotype with Cd alone. (D–F,J–L) mRNA expression of Cathepsin K , H + - a t p a s e , and Atp6v0d2 . n = 3 – 4 . Values are expressed as mean ± SD . Two-way ANOVA with Bonferroni multiple comparison posttest was used, * p < 0.05 vs. Scr with the same treatment; # p < 0.05 vs. the same genotype with Veh; & p < 0.05 vs. the same genotype with Cd alone. Summary data are provided in Tables S59–S66. Quantification of (A) and (G) is found in Figure S16. Note: ANOVA, analysis of variance; Atp6v0d2, ATPase H positive Transporting V 0 Subunit D 2; Cd, Cd alone; Cd+MitoQ, Cd and MitoQ co-treatment; Cd+NAC, Cd and NAC co-treatment; KD, Nrf2 -knockdown ( Nrf2 -KD); L-NRF1, long isoform nuclear factor-erythroid 2-related factor 1; M-CSF, macrophage colony-stimulating factor; MitoQ, MitoQ alone; NAC, NAC alone; NFATc1, nuclear factor of activated T cells, cytoplasmic, calcineurin dependent 1; RANKL, receptor activator of nuclear factor kappa-B ligand; Scr, Scramble; SD, standard deviation; Trap, tartrate-resistant acid phosphatase; Veh, vehicle.

Techniques: Western Blot, Staining, Comparison, Expressing, Knockdown, Standard Deviation

A schematic depiction of the potential molecular mechanisms underlying the cadmium (Cd) exposure-augmented osteoclastogenesis. NFATc1, which is a direct downstream transcriptional target of L-NRF1 and is also influenced by intracellular ROS levels, is a key driver controlling the osteoclastogenesis signaling cascade. NRF2, as a master transcription factor regulating many antioxidant genes, is involved in the regulation of osteoclast differentiation via the NRF2–antioxidants–ROS negative feedback loop. Cd exposure may stimulate intracellular ROS production via a complex mechanism and thus aggravate osteoclast differentiation, which can be mitigated by the NRF2-mediated antioxidant response. As a result, deficiency of Nrf2 augments Cd exposure-stimulated osteoclast differentiation in a ROS-dependent manner, in which ROS may up-regulate NFATc1 via L-NRF1-dependent and independent mechanisms. Note: Atp6v0d2, ATPase H positive Transporting V 0 Subunit D 2; KD, Nrf2 -knockdown ( Nrf2 -KD); KO, myeloid-specific Nrf2 knockout [ Nrf2 (M)-KO]; L-NRF1, long isoform nuclear factor-erythroid 2-related factor 1; NFATc1, nuclear factor of activated T cells, cytoplasmic, calcineurin dependent 1; Nrf2, nuclear factor erythroid 2-related factor 2; RANK, receptor activator of nuclear factor kappa-B; ROS, reactive oxygen species; TRAF6, tumor necrosis factor receptor–associated factor 6; WT, wild type.

Journal: Environmental Health Perspectives

Article Title: Prolonged Cadmium Exposure and Osteoclastogenesis: A Mechanistic Mouse and in Vitro Study

doi: 10.1289/EHP13849

Figure Lengend Snippet: A schematic depiction of the potential molecular mechanisms underlying the cadmium (Cd) exposure-augmented osteoclastogenesis. NFATc1, which is a direct downstream transcriptional target of L-NRF1 and is also influenced by intracellular ROS levels, is a key driver controlling the osteoclastogenesis signaling cascade. NRF2, as a master transcription factor regulating many antioxidant genes, is involved in the regulation of osteoclast differentiation via the NRF2–antioxidants–ROS negative feedback loop. Cd exposure may stimulate intracellular ROS production via a complex mechanism and thus aggravate osteoclast differentiation, which can be mitigated by the NRF2-mediated antioxidant response. As a result, deficiency of Nrf2 augments Cd exposure-stimulated osteoclast differentiation in a ROS-dependent manner, in which ROS may up-regulate NFATc1 via L-NRF1-dependent and independent mechanisms. Note: Atp6v0d2, ATPase H positive Transporting V 0 Subunit D 2; KD, Nrf2 -knockdown ( Nrf2 -KD); KO, myeloid-specific Nrf2 knockout [ Nrf2 (M)-KO]; L-NRF1, long isoform nuclear factor-erythroid 2-related factor 1; NFATc1, nuclear factor of activated T cells, cytoplasmic, calcineurin dependent 1; Nrf2, nuclear factor erythroid 2-related factor 2; RANK, receptor activator of nuclear factor kappa-B; ROS, reactive oxygen species; TRAF6, tumor necrosis factor receptor–associated factor 6; WT, wild type.

Techniques: Knockdown, Knock-Out

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

(A) Graphic representation of the conserved miR-378 binding motifs within the 3’UTRs of Nrf1 between human and mouse. Complementary sequences to the seed region of miR-378 within the 3’UTRs are conserved among two species (highlighted in green). (B) mRNA levels of Nrf1 in livers of mice fed with SD (n=6) or HFD (n=6). (C) Luciferase activity of the reporter constructs containing either the WT or mutated 3’UTR of murine Nrf1 after treatment with miR-378 mimics. Hepa1–6 cells transfected with the empty luciferase reporter vector and scramble served as control. NS: no significance. (D) qRT-PCR and immunoblot analysis of Nrf1 after MC-TTR-miR-378 or MC-TTR-miR-378-MM (control) transfection into Hepa1–6 cells. (E) qRT-PCR and Western blot analysis of Nrf1 after miR-378-ASO transfection into Hepa1–6 cells (20 nM). Hepa1–6 cells that received scramble served as control. (F) Reduced protein and mRNA levels of Nrf1 after MC-TTR-miR-378 injection into dietary obese mice. Control mice were treated with the same dose of MC-TTR-miR-378-MM. Student’s t test was used for statistical analysis. The data shown are representative of experiments repeated three times and conducted in triplicate. Data represent mean ± SEM. P values are indicated.

Journal: Metabolism: clinical and experimental

Article Title: A Negative Feedback Loop Between microRNA-378 and Nrf1 Promotes the Development of Hepatosteatosis in Mice Treated with a High Fat Diet

doi: 10.1016/j.metabol.2018.03.023

Figure Lengend Snippet: (A) Graphic representation of the conserved miR-378 binding motifs within the 3’UTRs of Nrf1 between human and mouse. Complementary sequences to the seed region of miR-378 within the 3’UTRs are conserved among two species (highlighted in green). (B) mRNA levels of Nrf1 in livers of mice fed with SD (n=6) or HFD (n=6). (C) Luciferase activity of the reporter constructs containing either the WT or mutated 3’UTR of murine Nrf1 after treatment with miR-378 mimics. Hepa1–6 cells transfected with the empty luciferase reporter vector and scramble served as control. NS: no significance. (D) qRT-PCR and immunoblot analysis of Nrf1 after MC-TTR-miR-378 or MC-TTR-miR-378-MM (control) transfection into Hepa1–6 cells. (E) qRT-PCR and Western blot analysis of Nrf1 after miR-378-ASO transfection into Hepa1–6 cells (20 nM). Hepa1–6 cells that received scramble served as control. (F) Reduced protein and mRNA levels of Nrf1 after MC-TTR-miR-378 injection into dietary obese mice. Control mice were treated with the same dose of MC-TTR-miR-378-MM. Student’s t test was used for statistical analysis. The data shown are representative of experiments repeated three times and conducted in triplicate. Data represent mean ± SEM. P values are indicated.

Article Snippet: 2.2 Preparation of mini-circle expression vectors for miR-378 and shRNA of Nrf1 Mini-circle vectors were purchased from System Biosciences (Cat. MN511A-1).

Techniques: Binding Assay, Luciferase, Activity Assay, Construct, Transfection, Plasmid Preparation, Control, Quantitative RT-PCR, Western Blot, Injection

(A–B) FAO rate and Oil-Red staining of three groups of Hepa1–6 cells treated with scramble (control), MC-TTR-Nrf1-shRNA, miR-378-ASO or a combination of miR-378-ASO and MC-TTR-Nrf1-shRNA. Hepa1–6 cells were maintained on DMEM medium containing 0.5 mM oleate. OCR: oxygen consumption rate. (C–D) FAO rate and Oil-Red staining of three groups of Hepa1–6 cells treated with the empty vector (Control), MC-TTR-miR-378, MC-TTR-Nrf1 or a combination of MC-TTR-miR-378 and MC-TTR-Nrf1. Student’s t test was used for statistical analysis. The data shown are representative of experiments repeated three times and conducted in triplicate. Data represent mean ± SEM. **p < 0.01

Journal: Metabolism: clinical and experimental

Article Title: A Negative Feedback Loop Between microRNA-378 and Nrf1 Promotes the Development of Hepatosteatosis in Mice Treated with a High Fat Diet

doi: 10.1016/j.metabol.2018.03.023

Figure Lengend Snippet: (A–B) FAO rate and Oil-Red staining of three groups of Hepa1–6 cells treated with scramble (control), MC-TTR-Nrf1-shRNA, miR-378-ASO or a combination of miR-378-ASO and MC-TTR-Nrf1-shRNA. Hepa1–6 cells were maintained on DMEM medium containing 0.5 mM oleate. OCR: oxygen consumption rate. (C–D) FAO rate and Oil-Red staining of three groups of Hepa1–6 cells treated with the empty vector (Control), MC-TTR-miR-378, MC-TTR-Nrf1 or a combination of MC-TTR-miR-378 and MC-TTR-Nrf1. Student’s t test was used for statistical analysis. The data shown are representative of experiments repeated three times and conducted in triplicate. Data represent mean ± SEM. **p < 0.01

Article Snippet: 2.2 Preparation of mini-circle expression vectors for miR-378 and shRNA of Nrf1 Mini-circle vectors were purchased from System Biosciences (Cat. MN511A-1).

Techniques: Staining, Control, shRNA, Plasmid Preparation

(A) Diagram of hepatic-specific miR-378 expression vector construction (MC-TTR-miR-378). (B) Organ distribution of miR-378 after a single intravenous injection of MC-TTR-miR-378 (n=6). Control mice received MC-TTR-miR-378-MM (n=6). (C–D) Oil-Red staining of livers and hepatic lipid content from two groups of mice treated with MC-TTR-miR-378 (n=10) or MC-TTR-miR-378-MM (n=10). Eight-week-old wild-type male C57Bl/6 mice were maintained on a HFD (Open Source D12492: 60% Kcal fat) for 8 weeks. After 8 weeks of HFD administration, mice were injected with either MC-TTR-miR-378 or MC-TTR-miR-378-MM (control) for another 8 weeks. (E) Reduced FAO in hepatocytes of mice treated with MC-TTR-miR-378. (F) MC-TTR-miR-378 treatment reduced mRNA levels of Nrf1, Cpt1a, Ppara, Acad1, and Vlcad1 in livers of HFD-fed mice. Mann-Whitney test was used to evaluate the statistical significance. Data represent mean ± SEM. *p < 0.05; **p < 0.01; and ***p < 0.001.

Journal: Metabolism: clinical and experimental

Article Title: A Negative Feedback Loop Between microRNA-378 and Nrf1 Promotes the Development of Hepatosteatosis in Mice Treated with a High Fat Diet

doi: 10.1016/j.metabol.2018.03.023

Figure Lengend Snippet: (A) Diagram of hepatic-specific miR-378 expression vector construction (MC-TTR-miR-378). (B) Organ distribution of miR-378 after a single intravenous injection of MC-TTR-miR-378 (n=6). Control mice received MC-TTR-miR-378-MM (n=6). (C–D) Oil-Red staining of livers and hepatic lipid content from two groups of mice treated with MC-TTR-miR-378 (n=10) or MC-TTR-miR-378-MM (n=10). Eight-week-old wild-type male C57Bl/6 mice were maintained on a HFD (Open Source D12492: 60% Kcal fat) for 8 weeks. After 8 weeks of HFD administration, mice were injected with either MC-TTR-miR-378 or MC-TTR-miR-378-MM (control) for another 8 weeks. (E) Reduced FAO in hepatocytes of mice treated with MC-TTR-miR-378. (F) MC-TTR-miR-378 treatment reduced mRNA levels of Nrf1, Cpt1a, Ppara, Acad1, and Vlcad1 in livers of HFD-fed mice. Mann-Whitney test was used to evaluate the statistical significance. Data represent mean ± SEM. *p < 0.05; **p < 0.01; and ***p < 0.001.

Article Snippet: 2.2 Preparation of mini-circle expression vectors for miR-378 and shRNA of Nrf1 Mini-circle vectors were purchased from System Biosciences (Cat. MN511A-1).

Techniques: Expressing, Plasmid Preparation, Injection, Control, Staining, MANN-WHITNEY

(A) H&E and Oil-Red staining of livers from three groups of mice treated with scramble control (n=10), miR-378-ASO (n=10) or a combination of miR-378-ASO and MC-TTR-Nrf1-shRNA (n=10). (B) Hepatic lipid content in livers of the above three groups of mice. Mann-Whitney test was used to evaluate the statistical significance. (C) FAO in hepatocytes from livers of the above three groups of mice. (D) mRNA levels of Acox1, Ppara, Cpt1a, Acad1, and Vlcat1 in livers from the three groups of mice. Student’s t test was used for statistical analysis. Data represent mean ± SEM. Data represent mean ± SEM. **p < 0.01.

Journal: Metabolism: clinical and experimental

Article Title: A Negative Feedback Loop Between microRNA-378 and Nrf1 Promotes the Development of Hepatosteatosis in Mice Treated with a High Fat Diet

doi: 10.1016/j.metabol.2018.03.023

Figure Lengend Snippet: (A) H&E and Oil-Red staining of livers from three groups of mice treated with scramble control (n=10), miR-378-ASO (n=10) or a combination of miR-378-ASO and MC-TTR-Nrf1-shRNA (n=10). (B) Hepatic lipid content in livers of the above three groups of mice. Mann-Whitney test was used to evaluate the statistical significance. (C) FAO in hepatocytes from livers of the above three groups of mice. (D) mRNA levels of Acox1, Ppara, Cpt1a, Acad1, and Vlcat1 in livers from the three groups of mice. Student’s t test was used for statistical analysis. Data represent mean ± SEM. Data represent mean ± SEM. **p < 0.01.

Article Snippet: 2.2 Preparation of mini-circle expression vectors for miR-378 and shRNA of Nrf1 Mini-circle vectors were purchased from System Biosciences (Cat. MN511A-1).

Techniques: Staining, Control, shRNA, MANN-WHITNEY

(A) Schematic representation of Nrf1 binding site within the promoter of miR-378. (B–C) Luciferase activity of the reporter construct containing either the WT or mutated miR-378 promoter after treatment with miR-378 mimics. In addition to the luciferase reporter vector, Hepa1–6 cells were also transfected with miR-378 mimics or scramble (control). NS: no significance. (D) Levels of mature miR-378 and Pri-miR-378 in Hepa1–6 cells after Nrf1 overexpression. Hepa1–6 cells were transfected with MC-TTR-Nrf1 expression vector or empty vector (Control). (E) Levels of mature miR-378 and Pri-miR-378 in Hepa1–6 cells after Nrf1 knockdown. Hepa1–6 cells were transfected with MC-TTR-Nrf1-shRNA expression vector or empty vector (Control). (F) In vivo ChIP assays were performed using genomic DNA isolated from mouse livers treated with HFD; and the binding of Nrf1 to the endogenous promoter of miR-378 was detected using specific Nrf1 antibodies; N.S.: non-specific control, which is located 10 kb downstream of predicted Nrf1 binding site. Student’s t test was used for statistical analysis. The data shown are representative of an experiment repeated three times and conducted in triplicate. Data represent mean ± SEM. *p < 0.05; **p < 0.01.

Journal: Metabolism: clinical and experimental

Article Title: A Negative Feedback Loop Between microRNA-378 and Nrf1 Promotes the Development of Hepatosteatosis in Mice Treated with a High Fat Diet

doi: 10.1016/j.metabol.2018.03.023

Figure Lengend Snippet: (A) Schematic representation of Nrf1 binding site within the promoter of miR-378. (B–C) Luciferase activity of the reporter construct containing either the WT or mutated miR-378 promoter after treatment with miR-378 mimics. In addition to the luciferase reporter vector, Hepa1–6 cells were also transfected with miR-378 mimics or scramble (control). NS: no significance. (D) Levels of mature miR-378 and Pri-miR-378 in Hepa1–6 cells after Nrf1 overexpression. Hepa1–6 cells were transfected with MC-TTR-Nrf1 expression vector or empty vector (Control). (E) Levels of mature miR-378 and Pri-miR-378 in Hepa1–6 cells after Nrf1 knockdown. Hepa1–6 cells were transfected with MC-TTR-Nrf1-shRNA expression vector or empty vector (Control). (F) In vivo ChIP assays were performed using genomic DNA isolated from mouse livers treated with HFD; and the binding of Nrf1 to the endogenous promoter of miR-378 was detected using specific Nrf1 antibodies; N.S.: non-specific control, which is located 10 kb downstream of predicted Nrf1 binding site. Student’s t test was used for statistical analysis. The data shown are representative of an experiment repeated three times and conducted in triplicate. Data represent mean ± SEM. *p < 0.05; **p < 0.01.

Article Snippet: 2.2 Preparation of mini-circle expression vectors for miR-378 and shRNA of Nrf1 Mini-circle vectors were purchased from System Biosciences (Cat. MN511A-1).

Techniques: Binding Assay, Luciferase, Activity Assay, Construct, Plasmid Preparation, Transfection, Control, Over Expression, Expressing, Knockdown, shRNA, In Vivo, Isolation

(A) Levels of mature miR-378 and Pri-miR-378 in livers of HFD-fed mice received MC-TTR-Nrf1-shRNA (n=9) or MC-TTR-miR-378-MM (Control, n=9). (B) Oil-Red staining of livers excised from the above two groups of mice. (C) Hepatic lipid content in the above two groups of mice. (D) Reduced FAO rate in hepatocytes of mice treated with MC-TTR-Nrf1-shRNA compared to control mice. (E) A negative feedback loop circuit between miR-378 and Nrf1 maintains the pathogenesis of hepatosteatosis in dietary obese mice. Once this circuit is triggered, it maintains activation of miR-378 biogenesis and suppression of Nrf1, which subsequently impairs fatty acid oxidation and promotes hepatosteatosis. Mann-Whitney test was used to evaluate the statistical significance. Data represent mean ± SEM. *p < 0.05; **p < 0.01.

Journal: Metabolism: clinical and experimental

Article Title: A Negative Feedback Loop Between microRNA-378 and Nrf1 Promotes the Development of Hepatosteatosis in Mice Treated with a High Fat Diet

doi: 10.1016/j.metabol.2018.03.023

Figure Lengend Snippet: (A) Levels of mature miR-378 and Pri-miR-378 in livers of HFD-fed mice received MC-TTR-Nrf1-shRNA (n=9) or MC-TTR-miR-378-MM (Control, n=9). (B) Oil-Red staining of livers excised from the above two groups of mice. (C) Hepatic lipid content in the above two groups of mice. (D) Reduced FAO rate in hepatocytes of mice treated with MC-TTR-Nrf1-shRNA compared to control mice. (E) A negative feedback loop circuit between miR-378 and Nrf1 maintains the pathogenesis of hepatosteatosis in dietary obese mice. Once this circuit is triggered, it maintains activation of miR-378 biogenesis and suppression of Nrf1, which subsequently impairs fatty acid oxidation and promotes hepatosteatosis. Mann-Whitney test was used to evaluate the statistical significance. Data represent mean ± SEM. *p < 0.05; **p < 0.01.

Article Snippet: 2.2 Preparation of mini-circle expression vectors for miR-378 and shRNA of Nrf1 Mini-circle vectors were purchased from System Biosciences (Cat. MN511A-1).

Techniques: shRNA, Control, Staining, Activation Assay, MANN-WHITNEY

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