Journal: Journal of Molecular Medicine (Berlin, Germany)

Article Title: Nuclear factor IX promotes endometriosis progression through transcriptional activation of tetraspanin-2

doi: 10.1007/s00109-026-02665-x

Figure Lengend Snippet: Difference in TSPAN2 expression between EMs and ESCs. A Venny platform identified 33 intersecting targets of the NFIX target genes from KnockTF2.0 database ( GSE45492 , q < 0.1) and differentially expressed genes from our previous RNA sequencing database (q < 0.1). B Heatmap of the 33 intersecting targets. C The mRNA levels of TSPAN2 were measured using real-time PCR in paired EMs and ESCs ( n = 15, *** P < 0.001, t- test). D The protein levels of TSPAN2 in paired EMs and ESCs were analyzed with western blot ( n = 15, ** P < 0.01, t- test). E Immunohistochemical staining was performed with samples of paired eutopic endometrium and ectopic endometrium ( n = 6, original magnification was × 200). F ESCs were transfected with si-NFIX, and the expression levels of TSPAN2 were analyzed by real-time PCR and western blot ( n = 5; * P < 0.05, ** P < 0.01, t- test). G ESCs were transfected with pENTER-NFIX, and the expression levels of TSPAN2 were analyzed by real-time PCR and western blot ( n = 5; *** P < 0.001, t- test)

Article Snippet: Subsequently, the paraffin-coated sections were subjected to incubation using primary antibody against NFIX (1:500; Abclonal, A9390) and an antibody against TSPAN2 (1:200; Invitrogen, PA5-53355).

Techniques: Expressing, RNA Sequencing, Real-time Polymerase Chain Reaction, Western Blot, Immunohistochemical staining, Staining, Transfection

Journal: Journal of Molecular Medicine (Berlin, Germany)

Article Title: Nuclear factor IX promotes endometriosis progression through transcriptional activation of tetraspanin-2

doi: 10.1007/s00109-026-02665-x

Figure Lengend Snippet: TSPAN2 is involved in the modulation of ESCs proliferation and invasion by NFIX. A ESCs were harvested for western blot after transfection with si-TSPAN2. The knockdown efficiency was verified ( n = 3, ** P < 0.01; t- test). B The transfection efficiency of pENTER-TSPAN2 was verified by western blot ( n = 3, ** P < 0.01; t- test). C and D ESCs were transfected with the si-TSPAN2, then harvested for real-time RT-PCR and western blot ( n = 3, * P < 0.05; t- test). E and F ESCs were transfected with the pENTER-TSPAN2, then harvested for real-time RT-PCR and western blot ( n = 3, * P < 0.05, ** P < 0.01, *** P < 0.001, t- test). G The cell proliferation and invasion activities were detected by CCK8 and transwell invasion assay in ESCs with si-TSPAN2 transfection ( n = 3, * P < 0.05; t- test). H The cell proliferation and invasion activities were detected by CCK8 and transwell invasion assay in ESCs with pENTER-TSPAN2 transfection ( n = 3, * P < 0.05; t- test). I Real-time PCR and western blot were used to analyze the expression level of Cyclin B1, MMP9, NFIX and TSPAN2 in the co-transfected ESCs ( n = 3, * P < 0.05, ANOVA ). J The cell proliferation and invasion activities were measured in co-transfected ESCs ( n = 3, * P < 0.05, *** P < 0,001, **** P < 0.0001; ANOVA )

Article Snippet: Subsequently, the paraffin-coated sections were subjected to incubation using primary antibody against NFIX (1:500; Abclonal, A9390) and an antibody against TSPAN2 (1:200; Invitrogen, PA5-53355).

Techniques: Western Blot, Transfection, Knockdown, Quantitative RT-PCR, Transwell Invasion Assay, Real-time Polymerase Chain Reaction, Expressing

Journal: Journal of Molecular Medicine (Berlin, Germany)

Article Title: Nuclear factor IX promotes endometriosis progression through transcriptional activation of tetraspanin-2

doi: 10.1007/s00109-026-02665-x

Figure Lengend Snippet: Identification of the critical binding site of NFIX on the TSPAN2 promoter in regulation. A Schematic presentation of 5 potential binding sites of NFIX on the TSPAN2 promoter, −1406bp to −1398bp (P1), −1289bp to −1281bp (P2), −1255bp to −1247bp (P3), −771bp to −763bp (P4) and −408bp to −400bp (P5). B Full length as well as 4 kinds of 5′-delete constructs of TSPAN2 promoter fragments were cloned upstream to luciferase reporter gene. The reporter plasmids were transiently transfected into ECC-1, and luciferase activities were measured ( n = 3, * P < 0.05, ** P < 0.01, t- test). C ESCs and EMs were subjected to ChIP using anti-NFIX antibody or IgG to validate the NFIX binding region of TSPAN2 promoter by real-time PCR ( n = 3, * P < 0.05, t- test)

Article Snippet: Subsequently, the paraffin-coated sections were subjected to incubation using primary antibody against NFIX (1:500; Abclonal, A9390) and an antibody against TSPAN2 (1:200; Invitrogen, PA5-53355).

Techniques: Binding Assay, Construct, Clone Assay, Luciferase, Transfection, Real-time Polymerase Chain Reaction

Journal: Scientific Reports

Article Title: MiR-744-5p inducing cell death by directly targeting HNRNPC and NFIX in ovarian cancer cells

doi: 10.1038/s41598-018-27438-6

Figure Lengend Snippet: miRNA correlation to potential targets on mRNA level of the TCGA ovarian serous cystadenocarcinoma dataset.

Article Snippet: For detection of target proteins the following antibodies were used: HNRNPC (1:1,000; #91327; Cell Signaling Technology), NFIX (1:250; HPA007533; Atlas Antibodies AB, Bromma, Sweden), Bcl2 (1:500; sc-509; Santa Cruz, Heidelberg, Germany), HNF-3α (FoxA1; 1:250; sc-514695; Santa Cruz Biotechnology), AR (1:250; sc-7305; Santa Cruz Biotechnology), Akt1/2/3 (1:500; sc-81434; Santa Cruz Biotechnology), p-Akt1/2/3 Ser473 (1:500; sc-514032; Santa Cruz Biotechnology) and GAPDH (1:5,000; MA5-15738; Thermo Fisher Scientific).

Techniques: Binding Assay

Journal: Scientific Reports

Article Title: MiR-744-5p inducing cell death by directly targeting HNRNPC and NFIX in ovarian cancer cells

doi: 10.1038/s41598-018-27438-6

Figure Lengend Snippet: HNRNPC and NFIX as direct targets for miR-744. To validate miR-744 mediated regulation of potential target genes, SKOV3 and OVCAR3 cells were transfected with miR-744 mimic or non-targeting siRNA (NT, negative control for cell death) as described in Fig. . After 48 h, potential target gene expression was analysed by qRT-PCR. The relative mRNA expression of potential target genes was normalized to PPIA and NT employing the Livak method ( a ). Significantly downregulated potential targets were further analysed by the Luciferase Reporter Assay ( b and c ) to proof direct binding of the miRNA to its predicted target sequence. Anti-miR-744 was used to confirm the binding sides of miR-744 to the target mRNAs ( d ). The binding sites in the 3′UTR (Table ), based on data of microRNA.org and TargetScanHuman , were cloned into a pMirGLO vector. Relative luciferase activity was measured 72 h after co-transfection of the pMirGLO vector with miR-744 mimic, miRNA inhibitor anti-miR-744 or non-targeting siRNA (NT) in HEK293T cells. The relative luciferase expression was normalized to the expression after co-transfection with NT. For Western Blot analysis, SKOV3 and OVCAR3 cells were harvested 60 h after miR-744 transfection ( e ). The representative blots were stained with antibodies against NFIX and HNRNPC. GAPDH was used as loading control. Statistical differences were tested using paired t-test [n = 3 biological and technical replicates; mean ± SD; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001].

Article Snippet: For detection of target proteins the following antibodies were used: HNRNPC (1:1,000; #91327; Cell Signaling Technology), NFIX (1:250; HPA007533; Atlas Antibodies AB, Bromma, Sweden), Bcl2 (1:500; sc-509; Santa Cruz, Heidelberg, Germany), HNF-3α (FoxA1; 1:250; sc-514695; Santa Cruz Biotechnology), AR (1:250; sc-7305; Santa Cruz Biotechnology), Akt1/2/3 (1:500; sc-81434; Santa Cruz Biotechnology), p-Akt1/2/3 Ser473 (1:500; sc-514032; Santa Cruz Biotechnology) and GAPDH (1:5,000; MA5-15738; Thermo Fisher Scientific).

Techniques: Transfection, Negative Control, Targeted Gene Expression, Quantitative RT-PCR, Expressing, Luciferase, Reporter Assay, Binding Assay, Sequencing, Clone Assay, Plasmid Preparation, Activity Assay, Cotransfection, Western Blot, Staining, Control

Journal: Scientific Reports

Article Title: MiR-744-5p inducing cell death by directly targeting HNRNPC and NFIX in ovarian cancer cells

doi: 10.1038/s41598-018-27438-6

Figure Lengend Snippet: Predicted binding sides cloned into a pmirGlo Dual Luciferase miRNA target expression vector.

Article Snippet: For detection of target proteins the following antibodies were used: HNRNPC (1:1,000; #91327; Cell Signaling Technology), NFIX (1:250; HPA007533; Atlas Antibodies AB, Bromma, Sweden), Bcl2 (1:500; sc-509; Santa Cruz, Heidelberg, Germany), HNF-3α (FoxA1; 1:250; sc-514695; Santa Cruz Biotechnology), AR (1:250; sc-7305; Santa Cruz Biotechnology), Akt1/2/3 (1:500; sc-81434; Santa Cruz Biotechnology), p-Akt1/2/3 Ser473 (1:500; sc-514032; Santa Cruz Biotechnology) and GAPDH (1:5,000; MA5-15738; Thermo Fisher Scientific).

Techniques: Binding Assay, Clone Assay, Luciferase, Expressing, Plasmid Preparation, Sequencing

Journal: Scientific Reports

Article Title: MiR-744-5p inducing cell death by directly targeting HNRNPC and NFIX in ovarian cancer cells

doi: 10.1038/s41598-018-27438-6

Figure Lengend Snippet: Signalling pathways leading to apoptosis. Western Blot data based on literature research , after transfection of SKOV3 cells with miRNA-744 or with siRNA against NFIX ( a ) or HNRNPC ( b ) and qRT-PCR ( c ) confirmed the potential signalling pathway by reduced expression of NFIX and Bcl2 as well as diminished phosphorylation of AKT. By binding to NFIX and HNRNPC miRNA-744 is interfering with the intrinsic apoptotic pathway. Downregulation of HNRNPC resulted in enhanced expression and activation of PDCD4 and p70S6K ( e ). Reduced NFIX expression, induced by binding of miR-744, leads to reduced expression of Bcl2 leading to apoptosis ( d ). The expression analysis of miR-744 and miR-21 was normalized to the CT value of U6 snRNA . Statistical differences were tested using paired t-test. In order to analyse DNA fragmentation (cells in sub G0/G1, d ) SKOV3 cells were seeded and transfected as described in Fig. with miR-744 mimics, non-targeting siRNA (NT, negative control for cell death), or siRNA against NFIX or HNRNPC. Statistical analysis was performed by one-way ANOVA followed by Bonferroni post-test ( d ). [n = 3 technical replicates; mean ± SD; ** p < 0.01; *** p < 0.001; **** p < 0.0001]. The cells were harvested for Western Blot analysis 60 h after treatment (a and b). GAPDH was used as loading control.

Article Snippet: For detection of target proteins the following antibodies were used: HNRNPC (1:1,000; #91327; Cell Signaling Technology), NFIX (1:250; HPA007533; Atlas Antibodies AB, Bromma, Sweden), Bcl2 (1:500; sc-509; Santa Cruz, Heidelberg, Germany), HNF-3α (FoxA1; 1:250; sc-514695; Santa Cruz Biotechnology), AR (1:250; sc-7305; Santa Cruz Biotechnology), Akt1/2/3 (1:500; sc-81434; Santa Cruz Biotechnology), p-Akt1/2/3 Ser473 (1:500; sc-514032; Santa Cruz Biotechnology) and GAPDH (1:5,000; MA5-15738; Thermo Fisher Scientific).

Techniques: Western Blot, Transfection, Quantitative RT-PCR, Expressing, Phospho-proteomics, Binding Assay, Activation Assay, Negative Control, Control

Journal: PLoS ONE

Article Title: Microbial Environment Affects Innate Immunity in Two Closely Related Earthworm Species Eisenia andrei and Eisenia fetida

doi: 10.1371/journal.pone.0079257

Figure Lengend Snippet: Bacterial composition in forest soil and in compost.

Article Snippet: C8 , Staphylococcus epidermidis ATCC 14990(T) , L37605 , 100.00.

Techniques:

Journal: Cells

Article Title: The Transcription Factor Nfix Requires RhoA-ROCK1 Dependent Phagocytosis to Mediate Macrophage Skewing during Skeletal Muscle Regeneration

doi: 10.3390/cells9030708

Figure Lengend Snippet: Nfix is mainly expressed by anti-inflammatory MPs. ( a ) Percentage of F4/80 + MPs positive for Nfix in Tibialis Anterior muscles (TA) of WT mice injected by CTX at D2, D4 and D7, post-injury. Immunostaining for F4/80 (green), Nfix (red) and DAPI (blue) at D4 and D7 after CTX injection; ( b ) Percentage of Ly6C + and Ly6C - sorted MPs positive for Nfix in TA muscles of WT mice injected by CTX at D2, D4 and D7 post-injury; ( c ) Percentage of Nfix + MPs after M1 and M2c polarization (with IFNγ and IL10, respectively). * p < 0.05; *** p < 0.001; for (b) * p < 0.05 Ly6C+ vs. Ly6C + at D4 and D7; # p < 0.05 Ly6C − D7 vs. D2. Results are means ± SEM of at least three independent experiments. Scale bar = 50 μm.

Article Snippet: For Nfix-F4/80 double immunolabeling, cryosections were labelled with antibodies against F4/80 (1:400, Novus Biologicals NB300-605) overnight at 4 °C and Nfix labelling using (1:200, Novus Biologicals NBP2-15039) the antibody was performed for 2 h at 37 °C.

Techniques: Muscles, Injection, Immunostaining

Journal: Cells

Article Title: The Transcription Factor Nfix Requires RhoA-ROCK1 Dependent Phagocytosis to Mediate Macrophage Skewing during Skeletal Muscle Regeneration

doi: 10.3390/cells9030708

Figure Lengend Snippet: MPs lacking Nfix are unable to adopt an anti-inflammatory phenotype in vivo and in vitro. ( a ) Representative FACS (Fluorescence-Activated Cell Sorting) gate of pro- and anti-inflammatory CD64 + MP populations in TA of Nfix fl/fl and LysM CRE :Nfix fl/fl mice at D2 after CTX injection. (CD64 + /Ly6C + and CD64 + /Ly6C − respectively); ( b ) Ratio of Ly6C +/ Ly6C − MPs sorted from TA of Nfix fl/fl and LysM CRE :Nfix fl/fl mice at D2, D4 and D7, after CTX injection; ( c ) WT BMDM (Bone Marrow Derived Macrophages) were transduced by shScramble and shNfix lentiviral vectors and then polarized into M1 and M2c MPs with IFNγ and IL10 treatment, respectively. MPs were immunolabeled for pro-inflammatory markers (TNFα, iNOS and CCl3) and anti-inflammatory markers (ArgI, TGFβ, CD163 and CD206). The number of positive cells is expressed as percentage out of total cells. * p < 0.05; ** p < 0.01; *** p < 0.001 vs. shScramble M1 MPs. # p < 0.05, ## p < 0.01, ### p < 0.001 vs. shScramble M2c MPs. Results are means ± SEM of at least three independent experiments.

Article Snippet: For Nfix-F4/80 double immunolabeling, cryosections were labelled with antibodies against F4/80 (1:400, Novus Biologicals NB300-605) overnight at 4 °C and Nfix labelling using (1:200, Novus Biologicals NBP2-15039) the antibody was performed for 2 h at 37 °C.

Techniques: In Vivo, In Vitro, Fluorescence, FACS, Injection, Derivative Assay, Immunolabeling

Journal: Cells

Article Title: The Transcription Factor Nfix Requires RhoA-ROCK1 Dependent Phagocytosis to Mediate Macrophage Skewing during Skeletal Muscle Regeneration

doi: 10.3390/cells9030708

Figure Lengend Snippet: Nfix is expressed after phagocytosis and drive MP phenotypical switch. ( a ) Phagocytosis assay of M1 and M2c Nfix fl/fl and LysM CRE :Nfix fl/fl MPs cocultured 8h with apoptotic mpc. Representative FACS gate of phagocytotic M2c Nfix fl/fl and LysM CRE :Nfix fl/fl MPs (CD64 + CellVue + ) and percentage of phagocytotic M1 and M2c MPs coming from Nfix fl/fl and LysM CRE :Nfix fl/fl BMDM; ( b ) WT MPs were cocultured 16h with apoptotic mpcs. Representative FACS gate of non-phagocytotic (CD64 + CellVue − ) and phagocytotic (CD64 + CellVue + ) WT MPs. Quantification of Nfix expression realized by RT-qPCR on sorted non-phagocytotic and phagocytotic WT MPs and quantification of MPs positive for Nfix (Nfix + /F4/80 + ) realized by IF on non-phagocytotic and phagocytotic WT MPs; ( c ) WT MPs were cocultured for 16 h with apoptotic mpcs, with or without addition of Cytochalasin D. Quantification of F4/80 + MPs were positive for Nfix on a total of F4/80 + MPs; ( d ) Western blot of Nfix expression in WT MPs treated with DMSO (Dimethyl sulfoxide) or Y27632 for 16 h and quantification. Vinculin was used to normalize; ( e ) WT MPs were treated with DMSO or Y27632 for 16 h and were immunolabeled for pro-inflammatory markers (iNOS and TNFα) and anti-inflammatory markers (TGFβ and CD163). The number of positive cells is expressed as percentage out of total cells; ( f ) LysM CRE :Nfix fl/fl MPs were treated with DMSO or Y27632 for 16 h and were immunolabeled for pro-inflammatory markers (iNOS and TNFα) and anti-inflammatory markers (TGFβ and CD163). The number of positive cells is expressed as percentage out of total cells. * p < 0.05, *** p < 0.001. Results are means ± SEM of at least three independent experiments.

Article Snippet: For Nfix-F4/80 double immunolabeling, cryosections were labelled with antibodies against F4/80 (1:400, Novus Biologicals NB300-605) overnight at 4 °C and Nfix labelling using (1:200, Novus Biologicals NBP2-15039) the antibody was performed for 2 h at 37 °C.

Techniques: Phagocytosis Assay, Expressing, Quantitative RT-PCR, Western Blot, Immunolabeling

Journal: bioRxiv

Article Title: Discovering cancer stem-like cells using Spatial transcriptomic analysis: Nuclear factor I X as a novel therapeutic target for gastric cancer

doi: 10.1101/2024.03.31.587468

Figure Lengend Snippet: Identification of NFIX as a key gene associated with cancer stemness in gastric cancer. (A) Gene Ontology enrichment analysis of the top 50 genes of cancer stem cell-like cells. (B) The overlap of the Venn diagram showed that there are 16 candidate targeted genes predicted by Cluster 0, Monocle3, and CytoTRACE. (C) Feature plots of each candidate gene. (D) Spatial plot of NFIX.

Article Snippet: The sections were incubated with rabbit polyclonal anti- NFIX antibody (NBP2-15039, 1:200; Novus Biologicals, LLC, USA) for 1 h at room temperature, followed by incubation with EnVision+ peroxidase-conjugated anti-rabbit secondary antibody for 1 h. For the color reaction, the sections were incubated with the Dako Liquid DAB+ Substrate Chromogen System (#K3468, Santa Clara, CA, USA) for 3 min.

Techniques:

Journal: bioRxiv

Article Title: Discovering cancer stem-like cells using Spatial transcriptomic analysis: Nuclear factor I X as a novel therapeutic target for gastric cancer

doi: 10.1101/2024.03.31.587468

Figure Lengend Snippet: Correlation of NFIX expression with poor prognosis in gastric cancer (GC). (A-D) Representative immunohistochemical images of NFIX. (A and B) Immunohistochemical staining of NFIX in non-neoplastic gastric mucosa. Original magnification: (A) 100×; scale bars, 200 µm and (B) 400×; scale bars, 50 µm. (C and D) Immunohistochemical staining of NFIX in GC. Original magnification: (C) 100×; scale bars, 200 µm and (D) 400×; scale bars, 50 µm. (E) Overall survival probability in the 127 GC cases. (F) Overall survival probability in the public RNA-seq dataset.

Article Snippet: The sections were incubated with rabbit polyclonal anti- NFIX antibody (NBP2-15039, 1:200; Novus Biologicals, LLC, USA) for 1 h at room temperature, followed by incubation with EnVision+ peroxidase-conjugated anti-rabbit secondary antibody for 1 h. For the color reaction, the sections were incubated with the Dako Liquid DAB+ Substrate Chromogen System (#K3468, Santa Clara, CA, USA) for 3 min.

Techniques: Expressing, Immunohistochemical staining, Staining, RNA Sequencing

Journal: bioRxiv

Article Title: Discovering cancer stem-like cells using Spatial transcriptomic analysis: Nuclear factor I X as a novel therapeutic target for gastric cancer

doi: 10.1101/2024.03.31.587468

Figure Lengend Snippet: Role of NFIX in gastric cancer (GC) cells: proliferation, stemness, and kinase activity regulation. (A) Western blot analysis of NFIX in five non-neoplastic or GC cell lines. (B) Western blot analysis of NFIX in MKN-45 cells transfected with the negative control or NFIX siRNA. (C) Effect of NFIX knockdown on cell growth in MKN-45 cells transfected with the negative control or NFIX siRNA. (D and E) Wound-healing assay in MKN-45 cells transfected with the negative control or NFIX siRNA. (D) The mean percentage of wound closure. (E) Representative image. (F) Number and size of spheroids formed by MKN-45 cell lines transfected with negative control or NFIX siRNA. (G) The heatmap shows the gene expression profile of 3 independent sets of MKN-45 cells transfected with the negative control or NFIX siRNA by RNA sequencing analysis. Data are color-coded to reflect the gene expression level. (H) Volcano plot comparing MKN-45 cells transfected with the negative control and that with NFIX siRNA. (I) Gene Ontology enrichment analysis of the top 50 genes.

Article Snippet: The sections were incubated with rabbit polyclonal anti- NFIX antibody (NBP2-15039, 1:200; Novus Biologicals, LLC, USA) for 1 h at room temperature, followed by incubation with EnVision+ peroxidase-conjugated anti-rabbit secondary antibody for 1 h. For the color reaction, the sections were incubated with the Dako Liquid DAB+ Substrate Chromogen System (#K3468, Santa Clara, CA, USA) for 3 min.

Techniques: Activity Assay, Western Blot, Transfection, Negative Control, Knockdown, Wound Healing Assay, Gene Expression, RNA Sequencing

Journal: bioRxiv

Article Title: Discovering cancer stem-like cells using Spatial transcriptomic analysis: Nuclear factor I X as a novel therapeutic target for gastric cancer

doi: 10.1101/2024.03.31.587468

Figure Lengend Snippet: Proliferative capacity and stemness in non-gastric cancer cell lines. (A) Western blot analysis of NFIX in HFE-145 cells transfected with the negative control or NFIX siRNA. (C) Effect of NFIX knockdown on cell growth in HFE-145 cells transfected with the negative control or NFIX siRNA. (C and D) Wound-healing assay in HFE-145 cells transfected with the negative control or NFIX siRNA. (C) The mean percentage of wound closure. (D) Representative image. (E) Number and size of spheroids formed by MKN-45 cell lines transfected with negative control or NFIX siRNA.

Article Snippet: The sections were incubated with rabbit polyclonal anti- NFIX antibody (NBP2-15039, 1:200; Novus Biologicals, LLC, USA) for 1 h at room temperature, followed by incubation with EnVision+ peroxidase-conjugated anti-rabbit secondary antibody for 1 h. For the color reaction, the sections were incubated with the Dako Liquid DAB+ Substrate Chromogen System (#K3468, Santa Clara, CA, USA) for 3 min.

Techniques: Western Blot, Transfection, Negative Control, Knockdown, Wound Healing Assay

Journal: bioRxiv

Article Title: Discovering cancer stem-like cells using Spatial transcriptomic analysis: Nuclear factor I X as a novel therapeutic target for gastric cancer

doi: 10.1101/2024.03.31.587468

Figure Lengend Snippet: Confirmation of genetic variation using gastric cancer cell lines. (A) Heatmap of genes found to be related in RNA sequence. Quantitative reverse transcription-polymerase chain reaction analysis of NFIX, RASSF2, MIR21, STRADB and ADARA2A genes in MKN-45 cells transfected with the negative control or NFIX siRNA.

Article Snippet: The sections were incubated with rabbit polyclonal anti- NFIX antibody (NBP2-15039, 1:200; Novus Biologicals, LLC, USA) for 1 h at room temperature, followed by incubation with EnVision+ peroxidase-conjugated anti-rabbit secondary antibody for 1 h. For the color reaction, the sections were incubated with the Dako Liquid DAB+ Substrate Chromogen System (#K3468, Santa Clara, CA, USA) for 3 min.

Techniques: Sequencing, Reverse Transcription, Polymerase Chain Reaction, Transfection, Negative Control

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: Repressors NFI and NFY Participate in Organ-Specific Regulation of von Willebrand Factor Promoter Activity in Transgenic Mice

doi: 10.1161/atvbaha.110.206680

Figure Lengend Snippet: Figure 4. Immunofluorescent analyses of NFI iso- form expression in organs of mice. Sections, 5 m, from OCT frozen heart, kidney, lung, and brain of a control C57Bl/6 mouse that were arranged on tissue arrays were treated with anti- bodies specific to NFIA, NFIB, NFIC, and NFIX iso- forms of NFI (red) and simultaneously with anti- PECAM (green) antibodies to detect endothelial cells, as described in the “Methods” section. The results are representative of 2 independent experi- ments for each NFI antibody plus PECAM antibod- ies (magnification 600). The insets show magni- fied endothelial cell nuclei (4,6-diamidino-2- phenylindole).

Article Snippet: Primary antibodies used were polyclonal rabbit antibodies specifically detecting NFIA, NFIB, NFIC, NFIX (Aviva System Biology, San Diego, CA), anti-β galactosidase antibody (Fitzgerald, Concord, MA) and goat polyclonal anti- mouse PECAM antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA).

Techniques: Expressing, Control

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: Repressors NFI and NFY Participate in Organ-Specific Regulation of von Willebrand Factor Promoter Activity in Transgenic Mice

doi: 10.1161/atvbaha.110.206680

Figure Lengend Snippet: Figure 5. ChIP detects the association of NFI and YY1 with their cognate binding sites and chromatin looping. ChIP and quantitative PCR analyses were performed on LMEC and HEK 293 cells, as described in the “Methods” section. A, Schematic of VWF chromatin looping that could bring NFI in proximity to the intron 51 YY1-binding site. B and C, Cross-linked chromatin from LMEC (B) and HEK 293 (C) cells were immunoprecipitated with antibodies specific to NFIA, NFIB, NFIC, NFIX, and YY1. Rabbit IgG antibody was used as a negative control. Immuno- precipitates were subjected to quantitative RT-PCR using prim- ers that specifically amplified an 89-bp upstream region of the VWF promoter containing the NFI binding site (NFI primers) or a 182-bp fragment of the VWF intron 51 sequence containing the YY1-binding site (I51 primers). Results for each antibody are presented as fold increase vs control IgG antibody, and all ChIP represents analyses of 2 to 4 independent ChIP experiments for each antibody.

Article Snippet: Primary antibodies used were polyclonal rabbit antibodies specifically detecting NFIA, NFIB, NFIC, NFIX (Aviva System Biology, San Diego, CA), anti-β galactosidase antibody (Fitzgerald, Concord, MA) and goat polyclonal anti- mouse PECAM antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA).

Techniques: Binding Assay, Real-time Polymerase Chain Reaction, Immunoprecipitation, Negative Control, Quantitative RT-PCR, Sequencing, Control