Journal: bioRxiv

Article Title: Selective ablation of Nfix in Macrophages preserves Muscular Dystrophy by inhibiting FAPs-dependent fibrosis

doi: 10.1101/2021.05.12.443809

Figure Lengend Snippet: a) Percentage of F4/80 + MPs positive for Nfix in Tibialis Anterior muscles (TA) and diaphragm (Dia) of Sgca null mice at 2 and 6 months of life. b) Percentage of F4/80 + MPs positive for Nfix in Tibialis Anterior muscles (TA) and diaphragm (Dia) of mdx mice at 2 and 6 months of life. c) Percentage of Ly6C + and Ly6C - sorted MPs positive for Nfix from TA of Sgca null mice at 2, 3 and 4 months of life. Statistical significance was determined using a two-tailed Student’s t -test, one-way Anova test or two-way Anova test. * p<0,05; ** p<0,01 ; *** p<0,001 ; for c) * p<0,05 Ly6C - 2m vs 4m. 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 NBP2-15038) antibody was performed for 2 hr at 37°C.

Techniques: Muscles, Two Tailed Test

Journal: bioRxiv

Article Title: Selective ablation of Nfix in Macrophages preserves Muscular Dystrophy by inhibiting FAPs-dependent fibrosis

doi: 10.1101/2021.05.12.443809

Figure Lengend Snippet: a) TGFβ1 expression by RT-qPCR of Nfix fl/fl :α (-/-) and ΦNfix (-/-) :α (-/-) TA muscles at 2, 4 and 6 months of life. b) Nfix fl/fl :α (-/-) and ΦNfix (-/-) :α (-/-) TA muscles were digested using Collagenase B and CD45 - cells were selected with magnetic beads. CD45 - cells were incubated with CD31-FITC, CD45-FITC, ITAG7(a7)-APC and Sca1-PeCy7 antibodies. FAPs are CD31 - /CD45 - /a7 - /Sca1 + . c) Nfix fl/fl :α (-/-) and ΦNfix (-/-) :α (-/-) TA muscles were digested using Collagenase B and CD45 + cells were selected with magnetic beads. CD45 + cells were incubated with CD64-APC and Ly6C-PE. MPs are CD64 + cells. Representative FACS gate of Ly6C + and Ly6C - MPs from Nfix fl/fl :α (-/-) and ΦNfix (-/-) :α (-/-) TA muscles at 2 months of life. d) Number of FAPs and MPs in Nfix fl/fl :α (-/-) and ΦNfix (-/-) :α (-/-) TA muscles at 2 and 4 months of life. e) Percentage of Ly6C + and Ly6C - MPs in Nfix fl/fl :α (-/-) and ΦNfix (-/-) :α (-/-) TA muscles at 2 and 4 months of life. Statistical significance was determined using a two-tailed Student’s t -test, one-way Anova test or two-way Anova test.* 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 NBP2-15038) antibody was performed for 2 hr at 37°C.

Techniques: Expressing, Quantitative RT-PCR, Muscles, Magnetic Beads, Incubation, Two Tailed Test

Journal: bioRxiv

Article Title: Selective ablation of Nfix in Macrophages preserves Muscular Dystrophy by inhibiting FAPs-dependent fibrosis

doi: 10.1101/2021.05.12.443809

Figure Lengend Snippet: a) Immunostaining for PDGFRα (green) and Hoechst (blue) of Nfix fl/fl :α (-/-) and ΦNfix (-/-) :α (-/-) TA muscles at 2 months of life, and number of PDGFRα + cells by mm 2 . Immunostaining for F4/80 (green) and Hoechst (blue) of Nfix fl/fl :α (-/-) and ΦNfix (-/-) :α (-/-) TA muscles at 2 months of life, and number of F4/80 + cells by mm 2 . b) Western Blot of P-Smad3 and tot-Smad3 in Nfix fl/fl :α (-/-) and ΦNfix (-/-) :α (-/-) TA muscles at 2 months of life and quantification. Western blot were realized in double or membrane were stripped and vinculin was used to normalize. c) Strategy to sort MPs and FAPs by using CD45 magnetic beads on Nfix fl/fl :α (-/-) and ΦNfix (-/-) :α (-/-) hindlimb muscles at 2 and 4 months of life. d) Immunostaining for TNFα (red) or TGFβ (green) and Hoechst (blue) on cytospined sorted MPs and for aCaspase3 (red) or Ki67 (green) and Hoechst (blue) on cytospined sorted FAPs. e) Percentage of TNFα or TGFβ positive MPs and aCaspase3 or Ki67 positive FAPs sorted from Nfix fl/fl :α (-/-) and ΦNfix (-/-) :α (-/-) hindlimb muscles at 2 and 4 months of life. Statistical significance was determined using a two-tailed Student’s t -test or two-way Anova test. * p<0,05 ; ** p<0,01. Results are means ± SEM of at least three independent experiments. Scale bar = 50 μm for FAPs in a), and 100 μm for MPs in a), and b) and d).

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 NBP2-15038) antibody was performed for 2 hr at 37°C.

Techniques: Immunostaining, Muscles, Western Blot, Membrane, Magnetic Beads, Two Tailed Test

Journal: bioRxiv

Article Title: Selective ablation of Nfix in Macrophages preserves Muscular Dystrophy by inhibiting FAPs-dependent fibrosis

doi: 10.1101/2021.05.12.443809

Figure Lengend Snippet: a) 10 weeks old Sgca null and ΦNfix (-/-) :α (-/-) mice were i.p. injected 3 times per week for 2 weeks with physiological water or Y27632 ROCK inhibitor and analyzed. b) Number of F4/80 + cells per mm 2 of Sgca null and ΦNfix (-/-) :α (-/-) TA muscles of mice i.p. injected with physiological water or Y. c) Percentage of double Nfix + /Pax7 + cells and number of Pax7 + cells/mm 2 of Sgca null and ΦNfix (-/-) :α (-/-) TA muscles of mice i.p. injected with physiological water or Y. d) Percentage of Nfix + myofibers, CSA and percentage of perinucleated and centronucleated (from 1 to 3 + nuclei) myofibers of Sgca null and ΦNfix (-/-) :α (-/-) TA muscles of mice i.p. injected with physiological water or Y. Statistical significance was determined using one-way Anova test or two-way Anova test.* vs Sgca null: * p<0,05 ; ** p<0,01. $ vs Sgca null + Y: $ p<0,05. Results are means ± SEM of at least three independent experiments. Scale bar = 50μm.

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 NBP2-15038) antibody was performed for 2 hr at 37°C.

Techniques: Injection, Muscles

Journal: bioRxiv

Article Title: Selective ablation of Nfix in Macrophages preserves Muscular Dystrophy by inhibiting FAPs-dependent fibrosis

doi: 10.1101/2021.05.12.443809

Figure Lengend Snippet: a) Hematoxylin-eosin and Milligan Trichrome staining of Sgca null and ΦNfix (-/-) :α (-/-) TA muscles i.p. injected with physiological water or Y27632 (called Y). b) Immunostaining for Nfix (red), F4/80 (green) and Hoechst (blue) of Sgca null and ΦNfix (-/-) :α (-/-) TA muscles i.p. injected with physiological water or Y, and percentage of double Nfix + /F4/80 + cells. c) Immunostraining for Collagen I (green) of Sgca null and ΦNfix (-/-) :α (-/-) TA muscles i.p. injected with physiological water or Y, and percentage of Collagen I + area. d) Immunostaining for Lam (red), PDGFRα (green) and Hoechst (blue) of Sgca null and ΦNfix (-/-) :α (-/-) TA muscles i.p. injected with physiological water or Y, and number of PDGFRα + cells by mm 2 . Statistical significance was determined using a two-tailed Student’s t -test or one-way Anova test. * vs Sgca null: * p<0,05 ; ** p<0,01. $ vs Sgca null +Y: $$ p<0,01 ; $$$ p<0,001. 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 NBP2-15038) antibody was performed for 2 hr at 37°C.

Techniques: Staining, Muscles, Injection, Immunostaining, Two Tailed Test

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