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

Journal: Developmental cell

Article Title: Cell-type-specific RNA polymerase II activity maps in intact tissues provide a gateway to mammalian gene regulatory mechanisms in vivo

doi: 10.1016/j.devcel.2025.09.017

Figure Lengend Snippet: (A) GC content of high-paused and low-paused gene groups in 5 bp bins around the TSS (±500 bp) and quantification. n = 2 experiments, n ≥ 429 genes per group. *** p ≤ 0.001. Student’s t test. Boxplot elements: center line, median; box limits, upper and lower quartiles; whiskers, 1.5× IQR (interquartile range). (B) ATAC-seq profile on promoters (TSS ± 1 kb) of low-, mid-, and high-paused, expressed and unexpressed genes identified by NB PReCIS-seq. (C) Overlap of enriched motifs for high- or low- paused genes across all in vivo stages. Tables depict significant motifs common to all in vivo conditions. See also extended – . (D) Localization metaplots for TF motifs enriched in high- (C) and low- (C ’ ) paused genes relative to the TSS across all in vivo stages. Shaded area represents mean ± SD of the normalized motif counts. (E and F) Example genome browser tracks of PReCIS-seq, ATAC-seq, and CUT&RUN for NRF1 (E) and KLF3 (F) and quantification of the corresponding CUT&RUN signal in the promoter regions (TSS ± 300 bp) of high-paused, low-paused, and unexpressed gene groups. n = 2 experiments, n ≥ 429 genes per group. *** p ≤ 0.001. Student’s t test. Boxplot elements: center line, median; box limits, upper, and lower quartiles; whiskers, 1.5 × IQR.

Article Snippet: Antibodies used for CUT&RUN include: H3K27ac (Active Motif, 39134), and Atlas Antibodies for NRF1 (HPA029329), KLF3 (HPA065054), BRD4 (HPA061646).

Techniques: In Vivo

Journal: Nature Communications

Article Title: NRF1-mediated innate immune response drives inflammaging

doi: 10.1038/s41467-025-66368-6

Figure Lengend Snippet: a The correlation between NRF1 and CDKN1A in human tissues, including the heart, liver, kidney, muscle, blood, and stomach. b The correlation between Nrf1 and Cdkn1a in mouse tissue, including the heart, liver, kidney, skin, white blood cells (WBC), and marrow. c Immunoblot analysis of NRF1 and senescent marker (P53, P21, P16, and γH2AX) in Primary MEF treated with Etoposide, Doxorubicin, or H 2 O 2 at the indicated time points. The band intensity of indicated proteins was quantitated using ImageJ. d Immunoblot analysis of NRF1 and senescent marker P21 in heart, liver, and kidney of C57BL/6 mice upon IR or NIR treatment ( n = 2 biologically independent mice in NIR, n = 3 biologically independent mice in IR). e SA-β-Gal staining in Primary MEF transfected with siNRF1 or siNC for 24 h, followed by treatment with DMSO or Etoposide for 24 h; mean and s.d. of fold change of SA-β-Gal-positive cells are indicated ( n = 6 biologically independent experiments). f Immunoblot analysis of NRF1 and senescent marker (e.g., P53, P21, P16, and γH2AX) in Primary MEF with siNRF1 or siNC for 24 h, followed by treatment with DMSO or Etoposide for 24 h. g , h EdU staining in Primary MEF transfected with siNC or siNRF1 for 24 h, followed by treatment with Etoposide for 24 h; mean and s.d. of fold change of EdU positive cells are indicated ( n = 6 biologically independent experiments). The white arrows indicate EdU-positive cells. i , j Ki67 staining in Primary MEF transfected with siNC or siNRF1 for 24 h, followed by treatment with Etoposide for 24 h; mean and s.d. of fold change of Ki67 positive cells are indicated ( n = 6 biologically independent experiments). The white arrows indicate Ki67-positive cells. k qPCR analyses SASP gene ( Il-6 , Cxcl1 , and Mmp13 ) mRNA in Primary MEF transfected with siNC or siNRF1 for 24 h, followed by treatment with Etoposide for 24 h ( n = 3 biologically independent experiments). All data represent means ± s.e.m; not significant (ns, p > 0.05), * p < 0.05, ** p < 0.01, and *** p < 0.001, determined by statistical analysis of the indicated comparison with two-way ANOVA with Tukey’s multiple-comparisons test ( e , h , j , k ).

Article Snippet: Nrf1 -/- and Nrf1 flox/flox mice generated by the CRISPR/Cas9-mediated genome editing were obtained from Cyagen Biosciences.

Techniques: Western Blot, Marker, Staining, Transfection, Comparison

Journal: Nature Communications

Article Title: NRF1-mediated innate immune response drives inflammaging

doi: 10.1038/s41467-025-66368-6

Figure Lengend Snippet: a Immunoblot analysis of NRF1 expression in Primary MEF infected by the retroviral vector GV138 encoding vector (NRF1-WT) or CRE (NRF1-KO) ( n = 3 biologically independent experiments). b , c RNA-sequencing (RNA-Seq) analysis of gene expression in proliferation (Prol) NRF1-WT and NRF1-KO Primary MEF ( n = 3 biologically independent experiments). b Volcano plot of the differentially expressed genes (DEGs, |log2FC| > 1, padj <0.05). Red, gray, and blue dots represent the significantly upregulated, no-different, and downregulated genes in NRF1-KO Primary MEF, respectively. c Left: heatmap analysis shows upregulated and downregulated genes. Right: Gene ontology (GO) enrichment analysis of upregulated or downregulated DEGs. Results show the top 10 significantly enriched GO terms in biological process (BP). d , e Heatmap of the altered “aging” and “inflammatory” response genes in NRF1-KO Primary MEF. f GSEA illustrates the enrichment of “RESPONSE TO VIRUS” and “ACTIVATION OF INNATE IMMUNE RESPONSE” genes in NRF1-KO Primary MEF. g Heatmap of the altered “response to interferon-beta” genes in NRF1-KO Primary MEF. h , i qPCR analysis of IFN-I, ISGs, and SASP mRNA in Primary MEF infected by the retroviral vector GV138 encoding vector (NRF1-WT) or CRE (NRF1-KO) ( n = 3 biologically independent experiments). j Immunoblot analysis of NRF1, phosphorylated and total TBK1 and STING in NRF1-WT and NRF1-KO Primary MEF, followed by treatment with cGAMP for 2 h ( n = 3 biologically independent experiments). (k) qPCR analysis of IFN-I and ISGs mRNA in NRF1-WT and NRF1-KO Primary MEF treated with cGAMP for 2 h ( n = 3 biologically independent experiments). All data represent means ± s.e.m; ** p < 0.01, and *** p < 0.001, determined by statistical analysis of the indicated comparison with two-way ANOVA with Tukey’s multiple-comparisons test ( h , i ) or two-way ANOVA with Tukey’s multiple-comparisons test ( k ).

Article Snippet: Nrf1 -/- and Nrf1 flox/flox mice generated by the CRISPR/Cas9-mediated genome editing were obtained from Cyagen Biosciences.

Techniques: Western Blot, Expressing, Infection, Retroviral, Plasmid Preparation, RNA Sequencing, Gene Expression, Virus, Activation Assay, Comparison

Journal: Nature Communications

Article Title: NRF1-mediated innate immune response drives inflammaging

doi: 10.1038/s41467-025-66368-6

Figure Lengend Snippet: a Overlapped genes between ChIP-Seq-based NRF1 target genes and down-regulated genes in Prol-KO or Sen-KO. b GO enrichment analysis of the overlapped genes shows significantly enriched GO terms in BP. c Prediction of NRF1 RE in the promoter region of Tbk1 and Irf3 . d qPCR analysis of Tbk1 and Irf3 mRNA in NRF1-WT and NRF1-KO Primary MEF ( n = 3 biologically independent experiments). e , f Luciferase assay of Tbk1 ( e ) and Irf3 ( f ) promoter-driven luciferase reporter in HEK 293T cells co-transfected with NRF1 plasmids for 24 h ( n = 4 biologically independent experiments). Schematic representation of Tbk1 ( e ) and Irf3 ( f ) promoter deletion constructs and putative NRF1 binding sites were marked in red. g , h ChIP-PCR analysis of the interaction between NRF1 proteins and Tbk1 or Irf3 promoters ( n = 3 biologically independent experiments). i EMSA assay using biotinylated or no-biotinylated oligonucleotide probes containing the predicted NRF1 binding site or mutant NRF1 binding site in the promoter of Tbk1 or Irf3 ( Tbk1 or Irf3 probe, cold probe, and mut-cold probe, respectively). The probes were incubated with HEK 293T cells nuclear extracts. The asterisk indicates the super-shifted band generated with antibody to NRF1. j , k Luciferase assay of Tbk1 ( j ) and Irf3 ( k ) promoter or mut-promotor driven luciferase reporter in HEK 293T cells co-transfected with NRF1 plasmids for 24 h ( n = 3 biologically independent experiments). All data represent means ± s.e.m; * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001, determined by statistical analysis of the indicated comparison with one-way ANOVA with a Tukey’s test for multiple-comparisons ( d , h , j , and k ).

Article Snippet: Nrf1 -/- and Nrf1 flox/flox mice generated by the CRISPR/Cas9-mediated genome editing were obtained from Cyagen Biosciences.

Techniques: ChIP-sequencing, Luciferase, Transfection, Construct, Binding Assay, Mutagenesis, Incubation, Generated, Comparison

Journal: Nature Communications

Article Title: NRF1-mediated innate immune response drives inflammaging

doi: 10.1038/s41467-025-66368-6

Figure Lengend Snippet: a Immunoblot analysis of TBK1, IRF3, and γH2AX in Primary MEF treated with ETO at the indicated time points ( n = 3 biologically independent experiments). b qPCR analysis of Tbk1, Irf3, and Cdkn1a mRNA in Primary MEF treated with ETO at the indicated time points ( n = 3 biologically independent experiments). c Immunoblot analysis of TBK1, IRF3, and γH2AX in Primary MEF treated with DOX at the indicated time points ( n = 3 biologically independent experiments). d qPCR analysis of Tbk1, Irf3, and Cdkn1a mRNA in Primary MEF treated with DOX at the indicated time points ( n = 3 biologically independent experiments). e Luciferase activity of Tbk1 and Irf3 promoter-reporter in HEK 293T cells overexpressing shNRF1 and then treated with ETO for 12 h ( n = 3 biologically independent experiments). f qPCR analysis of Tbk1 and Irf3 mRNA in NRF1-WT and NRF1-KO Primary MEF treated with ETO for 12 h ( n = 3 biologically independent experiments). g Immunoblot analysis of NRF1, pTBK1, TBK1, pP65, P65, IRF3, and senescent markers (e.g., P21, P16, and γH2AX) in NRF1-WT and NRF1-KO Primary MEF treated with ETO for 12 h. h The band intensity of indicated proteins was quantitated using ImageJ ( n = 3 biologically independent experiments). i , j qPCR analysis of IFN-I, ISGs, and SASP mRNA in NRF1-WT and NRF1-KO Primary MEF in NRF1-WT and NRF1-KO Primary MEF treated with ETO for 12 h ( n = 3 biologically independent experiments). All data represent means ± s.e.m; * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001, determined by statistical analysis of the indicated comparison with one-way ANOVA with a Tukey’s test ( b , d , e ) or two-way ANOVA with Tukey’s multiple-comparisons test for multiple comparisons ( f , h – j ).

Article Snippet: Nrf1 -/- and Nrf1 flox/flox mice generated by the CRISPR/Cas9-mediated genome editing were obtained from Cyagen Biosciences.

Techniques: Western Blot, Luciferase, Activity Assay, Comparison

Journal: Nature Communications

Article Title: NRF1-mediated innate immune response drives inflammaging

doi: 10.1038/s41467-025-66368-6

Figure Lengend Snippet: a Immunoblot analysis of (Flag-) NRF1, (Flag-) TBK1, (Flag-) IRF3, Flag-CRE, P21, and γH2AX in Primary MEF which were overexpressed with lentivirus packaging vector, Flag-NRF1, Flag-TBK1 or Flag-IRF3 and then treated with DMSO or ETO for 24 h ( n = 3 biologically independent experiments). b , c qPCR analysis of SASP, IFN-I, and ISGs mRNA in Primary MEF with various treatments ( n = 3 biologically independent experiments). d SA-β-Gal, EdU, and Ki67 staining in Primary MEF with indicated treatments. e – g Mean and s.d. of the fold change of SA-β-Gal, EdU, and Ki67 positive cells are indicated ( n = 8 biologically independent experiments for EdU and Ki67 staining statistics; n = 6 biologically independent experiments for SA-β-Gal staining statistics). h Immunoblot analysis of NRF1, TBK1, IRF3, P21, and γH2AX in NRF1-WT and NRF1-KO Primary MEF treated with IFN-β protein for 24 h ( n = 3 biologically independent experiments). i , j qPCR analysis of SASP, IFN-I, and ISGs mRNA in NRF1-WT and NRF1-KO Primary MEF treated with IFN-β protein for 24 h ( n = 4 biologically independent experiments). k , l SA-β-Gal staining in Primary MEF for the indicated treatment, followed by treatment with ETO for 24 h; mean and s.d. of the fold change of SA-β-Gal-positive cells are indicated ( n = 8 biologically independent experiments). All data represent means ± s.e.m; * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001, determined by statistical analysis of the indicated comparison with one-way ANOVA ( e – g , i , j , l ) or two-way ANOVA with Tukey’s multiple-comparisons test for multiple-comparisons ( b , c ).

Article Snippet: Nrf1 -/- and Nrf1 flox/flox mice generated by the CRISPR/Cas9-mediated genome editing were obtained from Cyagen Biosciences.

Techniques: Western Blot, Plasmid Preparation, Staining, Comparison

Journal: Nature Communications

Article Title: NRF1-mediated innate immune response drives inflammaging

doi: 10.1038/s41467-025-66368-6

Figure Lengend Snippet: a Immunoblot analysis and Phos-tag SDS–PAGE analysis of flag-NRF1 MEF treated with Etoposide for the indicated time points. b Immunoblot analysis of HEK 293T cells overexpressing Flag-NRF1 WT or Flag-NRF1 S393A and then treated with ETO for 3 h followed. c NRF1 protein sequence alignment analyzed by NetPhos-3.1 and the sequence proximal to S393 matched the known preferred consensus substrate motif of ATM. d Immunoblot analysis of Flag-NRF1 MEF was treated by Etoposide for 3 h, and then Co-IP was performed with an anti-flag antibody. e Immunoblot analysis of pATM S1981, ATM, pNRF1 S393, NRF1, and γH2AX in Flag-NRF1 MEF cells treated with Etoposide for the indicated time points. f Co-IP analysis of pNRF1 S393 signals in Primary MEF treated with vehicle or KU55933 (10 μM) for 3 h followed by ETO treatment for 3 h. g , h Native-PAGE analysis of dimerization and multimer of NRF1-WT, NRF1-S393A, and NRF1-S393D in HEK 293T cells; quantification by ImageJ of dimerized and multimer NRF1 is shown ( n = 3 biologically independent experiments). i EMSA assay using biotinylated oligonucleotide probes containing the predicted NRF1 binding site in the promoter of Tbk1 or Irf3 . The probes were incubated with HEK 293T cells nuclear extracts containing NRF1-WT, NRF1-S393A, or NRF1-S393D protein. j , k Immunoblot analysis of (Flag) NRF1, TBK1, IRF3, P53 and γH2AX in NRF1-KO Primary MEF overexpressed by lentivirus packaging vector, NRF1-WT, NRF1-393A or NRF1-S393D. The band intensity of indicated proteins was quantitated using ImageJ ( n = 3 biologically independent experiments). l qPCR analysis of Nrf1 , Tbk1 , Irf3 , SASP, IFN-I and ISGs mRNA in vector, NRF1-WT, NRF1-393A and NRF1-S393D Primary MEF ( n = 3 biologically independent experiments). m , n SA-β-Gal staining in vector, NRF1-WT, NRF1-393A, and NRF1-S393D Primary MEF; mean and s.d. of the fold change of SA-β-Gal-positive cells are indicated ( n = 8 biologically independent experiments). All data represent means ± s.e.m; * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001, determined by statistical analysis of the indicated comparison with one-way ANOVA with a Tukey’s test ( h , k , n ).

Article Snippet: Nrf1 -/- and Nrf1 flox/flox mice generated by the CRISPR/Cas9-mediated genome editing were obtained from Cyagen Biosciences.

Techniques: Western Blot, SDS Page, Sequencing, Co-Immunoprecipitation Assay, Clear Native PAGE, Binding Assay, Incubation, Plasmid Preparation, Staining, Comparison

Journal: Nature Communications

Article Title: NRF1-mediated innate immune response drives inflammaging

doi: 10.1038/s41467-025-66368-6

Figure Lengend Snippet: a Experimental design of NRF1-KD in 16 months mice: mice received both intraperitoneal injection (I.P) and intravenous injection (I.V) of AAV9-vector (8 × 10 11 V.G) or AAV9-shNRF1 (8 × 10 11 V.G) at week 0, 1, and 2 to generate control (CTR) or NRF1-KD mice, and then monitor weight change weekly and sacrificed at week 9 to detect the state of aging. b Immunoblot analysis of NRF1, P53, P21, P16, pTBK1 S172, TBK1, pSTING S365, STING, pP65 S536, P65, and TUBULIN in the heart tissue of CTR and NRF1-KD old mice ( n = 5 biologically independent mice). c immunohistochemistry analysis of P21 in the heart tissue of CTR and NRF1-KD mice. d The percentage of P21-positive area is calculated by Image-Pro Plus (right, n = 5 biologically independent mice). e immunohistochemistry analysis of IL-6 in the heart tissue of CTR and NRF1-KD mice. f The percentage of IL-6-positive area is calculated by Image-Pro Plus (right, n = 5 biologically independent mice). g , h ELISA quantification of IL-6 ( n = 5 biologically independent mice) and CXCL1 ( n = 6 biologically independent mice) in serum, heart, liver, and kidney tissues of CTR and NRF1-KD old mice. i qPCR analysis of Nrf1 , Tbk1 , Irf3 , Cdkn1a , Cdkn2a , SASP ( Il-6 , mmp13 , Cxcl1 , Ccl2 , and Ccl4 ), IFN-I ( Ifnb1 ), and ISGs ( Ifit3 , Cxcl10 ) mRNA in the heart, liver, and kidney tissue of CTR and NRF1-KD mice ( n = 6 biologically independent mice). All data represent means ± s.e.m; * p < 0.05, ** p < 0.01, and *** p < 0.001, determined by statistical analysis of the indicated comparison with two-tailed unpaired Student’s t test ( d , f , g – i ).

Article Snippet: Nrf1 -/- and Nrf1 flox/flox mice generated by the CRISPR/Cas9-mediated genome editing were obtained from Cyagen Biosciences.

Techniques: Injection, Plasmid Preparation, Control, Western Blot, Immunohistochemistry, Enzyme-linked Immunosorbent Assay, Comparison, Two Tailed Test

Journal: Nature Communications

Article Title: The SIAH2-NRF1 axis spatially regulates tumor microenvironment remodeling for tumor progression

doi: 10.1038/s41467-019-08618-y

Figure Lengend Snippet: Hypoxia transcriptionally regulates nuclear-encoded mitochondrial gene expression through NRF1. a , b Analysis of protein levels determined by immunoblotting using the indicated antibodies ( a ), and of mRNA levels, determined by real-time PCR (qRT-PCR) for the indicated genes ( b ), at the indicated time points in MDA-MB-231 cells cultured under hypoxia. qRT-PCR results were normalized to the housekeeping gene B2M . Detailed statistical data of ( a ) are shown in Supplementary Fig. . c MDA-MB-231 cells were pretreated with 25 µg ml −1 cycloheximide (CHX) for 2 h under normoxia, then cultured under hypoxic conditions for the indicated time points. Cells were harvested and analyzed by immunoblotting using the indicated antibodies. Detailed statistical data are shown in Supplementary Fig. . d Stable NRF1 -knockdown MDA-MB-231 cells cultured under hypoxia were analyzed by immunoblotting using the indicated antibodies at the indicated time points. Detailed statistical data are shown in Supplementary Fig. . e NRF1 siRNA was transfected into indicated cell lines and cultured under hypoxia for 36 h and then cell lysates were analyzed by immunoblotting using the indicated antibodies. For all panels, error bars indicate s.d., n = 3 biological replicates, average of n = 3 technical replicates for each biological replicate was used in ( b ). One-way ANOVA was used to compare data

Article Snippet: The following antibodies were used for immunohistochemistry: Prohibitin (1:200, Abcam, ab75766, clone EP2803Y), SIAH2 (1:40, Novus Biologicals, NB110-88113, clone 24E6H3) and NRF1 (1:100, Novus Biologicals, NBP1-89125).

Techniques: Gene Expression, Western Blot, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Cell Culture, Knockdown, Transfection

Journal: Nature Communications

Article Title: The SIAH2-NRF1 axis spatially regulates tumor microenvironment remodeling for tumor progression

doi: 10.1038/s41467-019-08618-y

Figure Lengend Snippet: SIAH2 promotes NRF1 polyubiquitination and degradation under hypoxia. a Hypoxia-mediated NRF1 degradation is inhibited by the proteasomal inhibitor MG132, but not by the autophagy inhibitor Bafilomycin A1 (BafA1). Right: densitometric quantification of NRF1 expressions. b MDA-MB-231 cells were incubated under normoxia or hypoxia for 24 h. Cells were treated with 10 µM MG132 for 6 h before harvesting. Cell lysates were immunoprecipitated with anti-NRF1 antibodies and then detected by western blotting with anti-NRF1 and anti-Ubiquitin antibodies. c Hypoxia-related ubiquitin E3 ligases were transiently co-transfected with Myc-NRF1 into HeLa cells for 24 h, and Myc-NRF1 protein levels were detected by western blotting with anti-Myc antibodies. d Direct interactions between bacterially expressed His-NRF1 and GST-SIAH2 in vitro. e Ectopic expression of SIAH2, but not SIAH2 RM increased NRF1 ubiquitination in vivo. f Ubiquitination of bacterially expressed His-NRF1 by purified SIAH2 but not by SIAH2 RM in vitro. g MDA-MB-231 cells were cultured under normoxia or hypoxia for 18 h, then treated with 10 µM MG132 and incubated under normoxia or hypoxia for another 6 h. Endogenous interactions between NRF1 and SIAH2 were analyzed by immunoprecipitation. h Wild-type or SIAH2 −/− MDA-MB-231 cells were transiently transfected with scramble or NRF1-targeted siRNA, and then cultured under normoxia or hypoxia for 36 h. Cells were harvested and analyzed by western blotting. Bottom: densitometric quantification of the indicated proteins. i Hypoxia-induced ubiquitination of NRF1 is abolished by depletion of SIAH2 in vivo. j Top: representative immunohistochemical staining of NRF1 and SIAH2 in normal breast tissue and breast cancer tissue from the tissue microarray. (Brown color indicates positive immune reaction; scale bars, 50 µm). Bottom: heat map showing quantitative analysis of the expression of NRF1 and SIAH2 proteins in normal breast tissues and breast cancer tissues. n = 158 breast tumors and n = 27 normal breast samples. Statistical analysis of the immunostaining results is shown in Supplementary Table , . For all panels, error bars indicate s.d., n = 3 biological replicates. Data were compared with two-tailed paired ratio t -tests

Article Snippet: The following antibodies were used for immunohistochemistry: Prohibitin (1:200, Abcam, ab75766, clone EP2803Y), SIAH2 (1:40, Novus Biologicals, NB110-88113, clone 24E6H3) and NRF1 (1:100, Novus Biologicals, NBP1-89125).

Techniques: Incubation, Immunoprecipitation, Western Blot, Ubiquitin Proteomics, Transfection, In Vitro, Expressing, In Vivo, Purification, Cell Culture, Immunohistochemical staining, Staining, Microarray, Immunostaining, Two Tailed Test

Journal: Nature Communications

Article Title: The SIAH2-NRF1 axis spatially regulates tumor microenvironment remodeling for tumor progression

doi: 10.1038/s41467-019-08618-y

Figure Lengend Snippet: NRF1 Lys230 is responsible for SIAH2-mediated NRF1 ubiquitination and degradation under hypoxia. a Determination of the Lysine 230 of NRF1 is responsible for SIAH2-induced degradation. Quantification of NRF1 protein levels is shown below. b SIAH2 induces degradation of wild-type NRF1 in a dosage-dependent manner but has no effect on NRF1-K230R. c SIAH2 induces ubiquitination of NRF1 but not NRF1-K230R. d Purified SIAH2 promotes ubiquitination of bacterially expressed wild-type NRF1 but not NRF1-K230R in vitro. e Co-immunoprecipitation of exogenously expressed Myc-NRF1 or Myc-NRF1-K230R with Flag-SIAH2 RM . f Hypoxia-induced ubiquitination of NRF1 is abolished by expression of the NRF1-K230R mutant in vivo. g HeLa cells were transiently transfected with wild-type NRF1 or NRF1-K230R for 12 h, and then incubated under normoxia or hypoxia for an additional 36 h. Cells were harvested and analyzed by immunoblotting with the indicated antibodies. Quantification of NRF1 protein levels is shown below. For all panels, error bars indicate s.d., n = 3 biological replicates. Data were compared with two-tailed paired ratio t -tests

Article Snippet: The following antibodies were used for immunohistochemistry: Prohibitin (1:200, Abcam, ab75766, clone EP2803Y), SIAH2 (1:40, Novus Biologicals, NB110-88113, clone 24E6H3) and NRF1 (1:100, Novus Biologicals, NBP1-89125).

Techniques: Ubiquitin Proteomics, Purification, In Vitro, Immunoprecipitation, Expressing, Mutagenesis, In Vivo, Transfection, Incubation, Western Blot, Two Tailed Test

Journal: Nature Communications

Article Title: The SIAH2-NRF1 axis spatially regulates tumor microenvironment remodeling for tumor progression

doi: 10.1038/s41467-019-08618-y

Figure Lengend Snippet: SIAH2 regulates nuclear-encoded mitochondrial gene expression through NRF1. a Wild-type or SIAH2 −/− MDA-MB-231 cells were transiently transfected with scramble or NRF1 -targeted siRNA then cultured under normoxia or hypoxia for 36 h. Cells were harvested and analyzed by western blotting. Bottom: densitometric quantification of the indicated proteins. b Statistical analysis of qRT-PCR data from cells treated as in ( a ). qRT-PCR results were normalized to the housekeeping gene B2M . c Stable NRF1 -knockdown MDA-MB-231 cells reconstituted with wild-type NRF1 or the NRF1-K230R mutant, together with mock and NRF1 -knockdown MDA-MB-231 cells, were cultured under normoxia or hypoxia for 36 h and analyzed by immunoblotting. Bottom: densitometric quantification of the indicated proteins. d Cells treated as in ( c ) were analyzed by qRT-PCR and the data were statistically compared. qRT-PCR results were normalized to the housekeeping gene B2M . e MDA-MB-231 cells stably expressing K230R-NRF1 were cultured under hypoxia and then analyzed by immunoblotting using the indicated antibodies at the indicated time points. Detailed statistical data are shown in Supplementary Fig. . For all panels, error bars indicate s.d., n = 3 biological replicates, average of n = 3 technical replicates for each biological replicate was used in ( b ) and ( d ). The two-tailed paired ratio t -test was used to compare data in (a-d) and one-way ANOVA was used to compare data in ( e )

Article Snippet: The following antibodies were used for immunohistochemistry: Prohibitin (1:200, Abcam, ab75766, clone EP2803Y), SIAH2 (1:40, Novus Biologicals, NB110-88113, clone 24E6H3) and NRF1 (1:100, Novus Biologicals, NBP1-89125).

Techniques: Gene Expression, Transfection, Cell Culture, Western Blot, Quantitative RT-PCR, Knockdown, Mutagenesis, Stable Transfection, Expressing, Two Tailed Test

Journal: Nature Communications

Article Title: The SIAH2-NRF1 axis spatially regulates tumor microenvironment remodeling for tumor progression

doi: 10.1038/s41467-019-08618-y

Figure Lengend Snippet: The SIAH2-NRF1 axis facilitates hypoxia-induced metabolic reprogramming. a – d Wild-type, SIAH2 −/− and SIAH2 −/− / NRF1 siRNA MDA-MB-231 cells were cultured under normoxia or hypoxia for 36 h, and concentrations of prostaglandin E2 (PGE2) within cells ( a ), glucose ( b ), lactate ( c ) in the culture medium and mRNA levels of PDHB ( d ) were analyzed. qRT-PCR results were normalized to the housekeeping gene B2M . e Diagram showing the NRF1 binding site in PDHB gene and oligonucleotides used in the ChIP assay. f ChIP assay was performed with IgG and antibody against NRF1 and indicated genes were analyzed by qRT-PCR. qRT-PCR results were normalized to the input. g – i MDA-MB-231 cells, either mock-treated or stably expressing wild-type NRF1 or the NRF1-K230R mutant, were cultured under normoxia or hypoxia for 36 h, and concentrations of prostaglandin E2 (PGE2) ( g ), glucose ( h ) and lactate ( i ) were analyzed. j Indicated cells were cultured under normoxia or hypoxia for 36 h, cells were stained with 100 nM Nonyl Acrdine Orange (NAO) and analyzed. k Wild-type or K230R stably expressed MDA-MB-231 cells were cultured under normoxia or hypoxia for 36 h. Cells were harvested and analyzed by western blotting with indicated antibodies. Right: densitometric quantification of the indicated proteins. l Cells from ( k ) were collected and PDH activity was measured. m A proposed model of the SIAH2-NRF1 axis in regulating hypoxia-induced metabolic reprogramming. For all panels, error bars indicate s.d., n = 3 biological replicates, average of n = 3 technical replicates for each biological replicate was used in ( a – d ), ( f – i ) and ( l ). The two-tailed unpaired student t -test was used in ( a – c ) and ( f – i ). Two-tailed paired ratio t -test was used in ( d ) and ( k – l )

Article Snippet: The following antibodies were used for immunohistochemistry: Prohibitin (1:200, Abcam, ab75766, clone EP2803Y), SIAH2 (1:40, Novus Biologicals, NB110-88113, clone 24E6H3) and NRF1 (1:100, Novus Biologicals, NBP1-89125).

Techniques: Cell Culture, Quantitative RT-PCR, Binding Assay, Stable Transfection, Expressing, Mutagenesis, Staining, Western Blot, Activity Assay, Two Tailed Test

Journal: Nature Communications

Article Title: The SIAH2-NRF1 axis spatially regulates tumor microenvironment remodeling for tumor progression

doi: 10.1038/s41467-019-08618-y

Figure Lengend Snippet: NRF1 degradation is required for tumor maintenance and TAM polarization. a – c Images ( a ), growth curves ( b ) and weights ( c ) of xenograft tumors derived from MDA-MB-231 cells with the indicated modifications. Tumors were established in mice by subcutaneous injection of cells. d , e The indicated xenograft tumor tissues were analyzed by hematoxylin-eosin staining ( d ) and tissue cross-sections were quantified necrotic area ( e ). Scale bars, 500 µm. f The indicated xenograft tumor tissues were stained with anti-ARG1 (red) and anti-TOMM20 (green) antibodies together with DAPI (blue). Scale bars, 50 µm. g <3 KDa fractions from DMEM or indicated cell-conditioned medium were used to stimulate bone-marrow derived macrophages. ARG1 mRNA expression was analyzed by qRT-PCR and normalized to ATCB as housekeeping gene. h Tissues derived from mouse spontaneous breast cancer were stained with DAPI (blue) together with anti-ARG1 (red) and anti-TIMM23 (green) or anti-GLUT1 (red) antibodies. Scale bars, 25 µm. For all panels, error bars indicate s.d. For panel ( a – f ) and ( h ), n = 5 mice per group. For panel ( g ), n = 3 biological replicates, average of n = 3 technical replicates for each biological replicate was used. The two-tailed unpaired student t -test was used to compare data

Article Snippet: The following antibodies were used for immunohistochemistry: Prohibitin (1:200, Abcam, ab75766, clone EP2803Y), SIAH2 (1:40, Novus Biologicals, NB110-88113, clone 24E6H3) and NRF1 (1:100, Novus Biologicals, NBP1-89125).

Techniques: Derivative Assay, Injection, Staining, Expressing, Quantitative RT-PCR, Two Tailed Test

Journal: Nature Communications

Article Title: The SIAH2-NRF1 axis spatially regulates tumor microenvironment remodeling for tumor progression

doi: 10.1038/s41467-019-08618-y

Figure Lengend Snippet: NRF1 accumulation enhances FADD-dependent apoptosis and impairs efferocytosis in vivo. a – c The indicated xenograft tumor tissues were stained with anti-IBA1 (red) and TUNEL (green) ( a ) and quantified TUNEL + apoptotic cells (ACs) ( b ), and ratio of free ACs: macrophage-associated ACs ( c ). Scale bars, 25 µm. d Diagram showing the NRF1 binding site in FADD gene and oligonucleotides used in the ChIP assay. e ChIP assay was performed with IgG and antibody against NRF1 and indicated genes were analyzed by qRT-PCR. qRT-PCR results were normalized to the input. f The indicated cells were cultured under normoxia or hypoxia for 36 h and FADD mRNA levels were statistical analyzed. qRT-PCR results were normalized to the housekeeping gene B2M . g Wild-type or K230R stably expressed MDA-MB-231 cells were cultured under normoxia or hypoxia for 36 h. Cells were harvested and analyzed by western blotting with anti-FADD and anti-ACTIN antibodies. Right: densitometric quantification of FADD protein levels. h Three groups of fresh frozen tissues from indicated xenograft tumors were analyzed by western blotting with the anti-FADD and anti-ACTIN antibodies. i Wild-type or K230R stably expressed MDA-MB-231 cells were cultured under normoxia or hypoxia for 24 h and then were treated with 50 ng mL −1 TRAIL for additional 12 h. Cells were harvested and analyzed by western blotting with indicated antibodies. Bottom: densitometric quantification of cleaved Caspase-3 and cleaved PARP1 protein levels. For all panels, error bars indicate s.d. For panel ( a – c ), n = 5 mice, average of n = 5–10 pictures per mouse were statistically analyzed. For other panels, n = 3 biological replicates, average of n = 3 technical replicates for each biological replicate was used in ( e – f ). The two-tailed unpaired student t -test was used in ( b – c ) and ( e ). The two-tailed paired ratio t -test was used in ( f – g ) and ( i )

Article Snippet: The following antibodies were used for immunohistochemistry: Prohibitin (1:200, Abcam, ab75766, clone EP2803Y), SIAH2 (1:40, Novus Biologicals, NB110-88113, clone 24E6H3) and NRF1 (1:100, Novus Biologicals, NBP1-89125).

Techniques: In Vivo, Staining, TUNEL Assay, Binding Assay, Quantitative RT-PCR, Cell Culture, Stable Transfection, Western Blot, Two Tailed Test

Journal: Nature Communications

Article Title: The SIAH2-NRF1 axis spatially regulates tumor microenvironment remodeling for tumor progression

doi: 10.1038/s41467-019-08618-y

Figure Lengend Snippet: A proposed model of the SIAH2-NRF1 axis in regulating the formation of pro-tumor microenvironments

Article Snippet: The following antibodies were used for immunohistochemistry: Prohibitin (1:200, Abcam, ab75766, clone EP2803Y), SIAH2 (1:40, Novus Biologicals, NB110-88113, clone 24E6H3) and NRF1 (1:100, Novus Biologicals, NBP1-89125).

Techniques: