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:

Journal: Animals : an Open Access Journal from MDPI

Article Title: An N-ethyl-N-Nitrosourea Mutagenesis Screen in Mice Reveals a Mutation in Nuclear Respiratory Factor 1 ( Nrf1) Altering the DNA Methylation State and Correct Embryonic Development

doi: 10.3390/ani11072103

Figure Lengend Snippet: MommeD46 is an enhancer of variegation. ( a ) Schematic representation of the ENU mutagenesis gene discovery pipeline. ( b ) Table indicating the average percentage of GFP positive erythrocytes ± standard deviation (SD) for Nrf1 +/+ and Nrf1 MommeD46/+ . Nrf1 MommeD46/MommeD46 were not viable at weaning. ( c ) Graph of the GFP fluorescence profile obtained by flow cytometry from a drop of blood of Nrf1 +/+ (wild-type) and Nrf1 MommeD46/+ (heterozygote). Statistical significance was determined by two-tailed Student’s t-test, **** indicates p < 0.00005.

Article Snippet: The anti-Nrf1 antibody from Novus Biologicals (Littleton, CO, USA) was incubated in the same solution overnight at 4 °C, 1:1000 dilution.

Techniques: Mutagenesis, Standard Deviation, Fluorescence, Flow Cytometry, Two Tailed Test

Journal: Animals : an Open Access Journal from MDPI

Article Title: An N-ethyl-N-Nitrosourea Mutagenesis Screen in Mice Reveals a Mutation in Nuclear Respiratory Factor 1 ( Nrf1) Altering the DNA Methylation State and Correct Embryonic Development

doi: 10.3390/ani11072103

Figure Lengend Snippet: MommeD46 harbors a point mutation in the third exon of Nrf1 ( a ) Schematic representation of the mutation in the NRF1 protein and conservation of the indicated amino acid sequence across different species. ( b ) Representative Sanger sequences traces of amplicons derived from Nrf1 +/− , Nrf1 MommeD46/+ and Nrf1 MommeD46/MommeD46 9.5 days post-coitum (dpc) embryos. ( c ) Graph of qRT-PCR relative expression values of Nrf1 ± SD in Nrf1 +/+ , Nrf1 MommeD46/+ and Nrf1 MommeD46/MommeD46 9.5 dpc embryos. Hprt is used as normalizer. ( d ) Western Blot of Nrf1 using whole protein extracts from Nrf1 +/+ , Nrf1 MommeD46/+ and Nrf1 MommeD46/MommeD46 9.5 dpc embryos. γ-Tubulin is used as loading control. D46 is an abbreviation of MommeD46 . Statistical significance was determined by one-way ANOVA and post hoc Tukey test, * indicates p < 0.05 and *** indicates p < 0.0005.

Article Snippet: The anti-Nrf1 antibody from Novus Biologicals (Littleton, CO, USA) was incubated in the same solution overnight at 4 °C, 1:1000 dilution.

Techniques: Mutagenesis, Sequencing, Derivative Assay, Quantitative RT-PCR, Expressing, Western Blot, Control

Journal: Animals : an Open Access Journal from MDPI

Article Title: An N-ethyl-N-Nitrosourea Mutagenesis Screen in Mice Reveals a Mutation in Nuclear Respiratory Factor 1 ( Nrf1) Altering the DNA Methylation State and Correct Embryonic Development

doi: 10.3390/ani11072103

Figure Lengend Snippet: Nrf1 is involved in DNA methylation. Methylation of the CpG sites in the HS-40 enhancer region of the GFP transgene in Nrf1 +/+ , Nrf1 MommeD46/+ and Nrf1 MommeD46/MommeD46 9.5 dpc embryos. Three embryos per group were analyzed. Dark circles indicate methylated sites and white circles represent unmethylated sites. Statistical differences between the groups were determined by one-way ANOVA and post hoc Tukey test, * indicates p < 0.05.

Article Snippet: The anti-Nrf1 antibody from Novus Biologicals (Littleton, CO, USA) was incubated in the same solution overnight at 4 °C, 1:1000 dilution.

Techniques: DNA Methylation Assay, Methylation

Journal: Animals : an Open Access Journal from MDPI

Article Title: An N-ethyl-N-Nitrosourea Mutagenesis Screen in Mice Reveals a Mutation in Nuclear Respiratory Factor 1 ( Nrf1) Altering the DNA Methylation State and Correct Embryonic Development

doi: 10.3390/ani11072103

Figure Lengend Snippet: MommeD46 shows developmental defects, reduced weight and non-Mendelian inheritance. ( a ) Table showing the number of Nrf1 +/− , Nrf1 MommeD46/+ and Nrf1 MommeD46/MommeD46 offspring at 8.5 dpc, 9.5 dpc, 10.5 dpc, 14.5 dpc and 3 weeks after birth obtained from the heterozygous intercross. The respective percentages are between parentheses. ( b ) Table showing the number of normal and abnormal (empty decidua) offspring at 8.5 dpc, 9.5 dpc, 10.5 dpc and 14.5 dpc obtained from the heterozygous intercross. ( c ) Table including the average litter size ± SD obtained from the intercrosses between male Nrf1 MommeD46/+ and female Nrf1 +/+ , male Nrf1 +/+ and female Nrf1 MommeD46/+ , and between male Nrf1 MommeD46/+ and female Nrf1 MommeD46/+ . The number of litters analyzed for each intercross are indicated. ( d ) Table showing the number of pups obtained with genotypes Nrf1 +/+ and Nrf1 MommeD46/+ from the intercrosses between male Nrf1 MommeD46/+ and female Nrf1 +/+ and between male Nrf1 +/+ and female Nrf1 MommeD46/+ . The respective percentages appear in parenthesis. ( e ) Whisker plots indicating the relative weight ± SD of Nrf1 +/+ and Nrf1 MommeD46/+ mice at 3 weeks after birth, for each sex. ( f ) Representative images Nrf1 +/− , Nrf1 MommeD46/+ and Nrf1 MommeD46/MommeD46 embryos of 9.5 and 10.5 dpc. Scale bars indicate 1mm. ♂ and ♀ mean male and female, respectively. Statistical significance in ( a , d ) was determined by Chi–Square test and/or Fisher’s exact test, and in ( c , e ) by two-tailed Student’s t-test (for females) or Mann–Whitney U test (for males). Levene’s F-test determined significant differences on variances when p < 0.05. ns means not significant.

Article Snippet: The anti-Nrf1 antibody from Novus Biologicals (Littleton, CO, USA) was incubated in the same solution overnight at 4 °C, 1:1000 dilution.

Techniques: Whisker Assay, Two Tailed Test, MANN-WHITNEY

Journal: Molecular nutrition & food research

Article Title: Dietary wolfberry up-regulates carotenoid metabolic genes and enhances mitochondrial biogenesis in the retina of db/db diabetic mice

doi: 10.1002/mnfr.201200642

Figure Lengend Snippet: DNA copy numbers of cytochrome b (Cyt b) and β-actin were determined by real-time PCR using retinal genomic DNA and designed gene specific primers. Mitochondrial DNA copy number was expressed as Cyt b/β-actin (A). Mitochondrial mass in the retina was also determined by Western blot using antibody against Cox IV, a mitochondrial protein. The Cox IV protein level was normalized to β-actin in each sample (B). Retinal mitochondrial function integrity was tested by citrate synthase activity assay expressed as nmol/mg protein/min (C). Protein levels of TFAM in mitochondria were monitored by Western blot using anti-TFAM. Prohibitin was used as a loading control to normalize the TFAM values (D). Transcriptional and translational expression of transcription factors PGC-1α and NRF1 was analyzed by real time PCR (E, PGC-1α mRNA; G, NRF1 mRNA) and Western blot (F, PGC-1α protein; H, NRF1 protein). Data were normalized to β-actin. Representative Western blot images were shown. n=6. The statistical significance was at p<0.05 (*) and/or p<0.01 (**). mt DNA, mitochondrial DNA; Cyt b, cytochrome b; Cox IV, cytochrome c oxidase subunit IV; mt TFAM, mitochondrial transcription factor in mitochondria; NRF1, nuclear respiratory factor 1; PGC-1α, peroxisome proliferator-activated receptor γ coactivator-1α.

Article Snippet: Antibodies against GSTP1, BCO2, transcription factor A, mitochondrial (TFAM), and NRF1 were ordered from Proteintech (Chicago, IL).

Techniques: Real-time Polymerase Chain Reaction, Western Blot, Activity Assay, Control, Expressing

Journal: Molecular nutrition & food research

Article Title: Dietary wolfberry up-regulates carotenoid metabolic genes and enhances mitochondrial biogenesis in the retina of db/db diabetic mice

doi: 10.1002/mnfr.201200642

Figure Lengend Snippet: Hyperglycemia and/or subsequent hypoxia in db/db diabetic mice causes inhibition of lutein and zeaxnathin metabolic gene expression and decreases in protein levels of BCO2, AMPKα2, and TFAM in mitochondria, which leads to mitochondrial dysfunction and subsequent retinal degeneration in diabetes (marked with double lines). Dietary wolfberry and/or the bioactive components (wolfberry bioactives) primarily activates AMPKα2 in mitochondria and nuclei, which then triggers increased expression of genes related to lutein and zeaxanthin metabolism (SR-BI, GSTP1, and BCO2) and mitochondrial biogenesis (PGC-1α, NRF1, and TFAM), and decreases in cell stress responses (HIF-1α, VEGF, and HSP60), resulting in attenuation of hypoxia, enhancement of mitochondrial function, and subsequent retinal neuroprotection in db/db diabetic mice (marked with single lines).

Article Snippet: Antibodies against GSTP1, BCO2, transcription factor A, mitochondrial (TFAM), and NRF1 were ordered from Proteintech (Chicago, IL).

Techniques: Inhibition, Gene Expression, Expressing

Journal: Frontiers in Nutrition

Article Title: Lactobacillus improves meat quality in Sunit sheep by affecting mitochondrial biogenesis through the AMPK pathway

doi: 10.3389/fnut.2022.1030485

Figure Lengend Snippet: Primers used for RT-PCR.

Article Snippet: Primary antibodies against β-actin, and HRP-labeled sheep anti-rabbit (1: 50000, Wuhan Bode, cat. No. BA1054) were n (1: 1000, Wuhan Bode, cat. No. BM0627), AMPK (1: 1000, Cell Signaling, cat. No. 2532), p-AMPK (1: 1000, Cell Signaling, cat. No. 2535), PGC-1α (1: 2000, Abcam Affinity, cat. No. ab106814), NRF1 (recombinant nuclear respiratory factor 1) (1:2000, Affinity, cat. No. AF5298), TFAM (mitochondrial transcription factor A) (1: 1000, Affinity, cat. No. AF0531), and COX IV (Cytochrome C oxidase IV) (1: 1000, Abcam, cat. No. ab33985) and the secondary antibodies HRP-labeled sheep anti-mouse (1: 50000, Wuhan Bode, cat. No. BA1051), HRP-labeled rabbit anti-sheep (1: 50000, Wuhan Bode, cat. No. BA1060) used to detect protein expression.

Techniques:

Journal: Frontiers in Nutrition

Article Title: Lactobacillus improves meat quality in Sunit sheep by affecting mitochondrial biogenesis through the AMPK pathway

doi: 10.3389/fnut.2022.1030485

Figure Lengend Snippet: Effects of Lactobacillus on the expression of mitochondrial biogenesis-related proteins and genes in Sunit sheep. (A–E) Effects of Lactobacillus on the gene and protein expression of AMPK, PGC-1α, NRF1, TFAM, COX IV. Different letters indicate significant differences between the groups ( P < 0.05), and the same or no letters indicate insignificant differences between the groups ( P > 0.05).

Article Snippet: Primary antibodies against β-actin, and HRP-labeled sheep anti-rabbit (1: 50000, Wuhan Bode, cat. No. BA1054) were n (1: 1000, Wuhan Bode, cat. No. BM0627), AMPK (1: 1000, Cell Signaling, cat. No. 2532), p-AMPK (1: 1000, Cell Signaling, cat. No. 2535), PGC-1α (1: 2000, Abcam Affinity, cat. No. ab106814), NRF1 (recombinant nuclear respiratory factor 1) (1:2000, Affinity, cat. No. AF5298), TFAM (mitochondrial transcription factor A) (1: 1000, Affinity, cat. No. AF0531), and COX IV (Cytochrome C oxidase IV) (1: 1000, Abcam, cat. No. ab33985) and the secondary antibodies HRP-labeled sheep anti-mouse (1: 50000, Wuhan Bode, cat. No. BA1051), HRP-labeled rabbit anti-sheep (1: 50000, Wuhan Bode, cat. No. BA1060) used to detect protein expression.

Techniques: Expressing

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