Journal: Communications Biology

Article Title: Erythroid lineage chromatin accessibility maps facilitate identification and validation of NFIX as a fetal hemoglobin repressor

doi: 10.1038/s42003-023-05025-4

Figure Lengend Snippet: a ATAC-seq profiles spanning Chr19:13,122,000–13,138,000 (hg19) of sorted BM (HbF low) and CB (HbF high) cell populations showing increased chromatin accessibility at the NFIX promoter in BM cells (red boxes). NFIX splice variants NM_002501 (line 1), NM_001271044 (line 2), and NM_001365985 (line 3) are shown at the top. b , c NFIX mRNA quantification in BM versus CB sorted cell populations and in HUDEP-2 (HbF low) versus HUDEP-1 (HbF high). Bars represent mean of two biological replicates. Asterisk denotes statistically significant data using Student’s t -test [ P -value < 0.05 for BM versus CB cells (paired t -test, b )]. d Western blot for NFIX protein. Histone H3 served as a normalizing control. Representative immunoblots from two biological replicates are shown.

Article Snippet: TaqMan® Probe IDs used in this study: Hs_00362215_g1 ( HBE ), Hs00361131_g1 ( HBG ), Hs_00426283_m1 ( HBD ), Hs_00747223_g1 ( HBB ), Hs_00958843_m1 ( NFIX ), Hs01093198_m1 ( BCL11A ), Hs_00252415_s1 ( ZBTB7A ), and Hs99999903_m1 ( ACTB ).

Techniques: Western Blot, Control

Journal: Communications Biology

Article Title: Erythroid lineage chromatin accessibility maps facilitate identification and validation of NFIX as a fetal hemoglobin repressor

doi: 10.1038/s42003-023-05025-4

Figure Lengend Snippet: a RT-qPCR validation of NFIX knockdown in BM cells shows a 90% reduction in NFIX transcripts. b ATAC-seq profiles spanning Chr11:5,245,000–5,277,000 (hg19) of BM control and NFIX knockdown cells showing reduced chromatin accessibility at the HBB promoter and increased chromatin accessibility at the HBG promoter relative to the control cells (BM day 7 of differentiation, red boxes). c Percent HBG promoter DNA methylation at CpG -162 and an average of all 6 CpGs tested shows decreased DNA methylation in NFIX knockdown BM cells relative to the control. d Knockdown of NFIX in BM cells results in induction of HBG mRNA. e NFIX knockdown increases the number of F-cells (BM day 10 of differentiation). f HPLC chromatograms show increased absolute HbF levels in NFIX knockdown cells (BM day 14 of differentiation). g Knockdown of NFIX in HUDEP-2 cells increases HBG mRNA, F-cells, and HbF relative to the control. Data shown are representative of N = 3 independent experiments ( a , e , f ) and N = 2 independent experiments ( b , c , d , g ) using distinct BM and CB donors and independent HUDEP-2 transductions.

Article Snippet: TaqMan® Probe IDs used in this study: Hs_00362215_g1 ( HBE ), Hs00361131_g1 ( HBG ), Hs_00426283_m1 ( HBD ), Hs_00747223_g1 ( HBB ), Hs_00958843_m1 ( NFIX ), Hs01093198_m1 ( BCL11A ), Hs_00252415_s1 ( ZBTB7A ), and Hs99999903_m1 ( ACTB ).

Techniques: Quantitative RT-PCR, Biomarker Discovery, Knockdown, Control, DNA Methylation Assay

Journal: Communications Biology

Article Title: Erythroid lineage chromatin accessibility maps facilitate identification and validation of NFIX as a fetal hemoglobin repressor

doi: 10.1038/s42003-023-05025-4

Figure Lengend Snippet: a RT-qPCR validation of NFIX overexpression in CB cells shows approximately 10-fold increase in NFIX transcripts relative to empty vector control cells. Bars represent mean of two biological replicates. b Western blot validation of NFIX overexpression in CB cells as determined by detection of the FLAG tag on day 10 of erythroid differentiation. c CB cells overexpressing NFIX have a reduction in HBG mRNA levels relative to other beta-like globins on day 7 of erythroid differentiation. Bars represent mean of two biological replicates. d CB cells overexpressing NFIX show reduced percentage of F-cells relative to empty vector control on day 14 of erythroid differentiation. e HPLC profiles demonstrate reduction in HbF protein in NFIX overexpressing CB cells relative to the empty vector control on day 14 of erythroid differentiation. Data are representative of N = 2 biological replicates using distinct CB donors and independent NFIX transductions.

Article Snippet: TaqMan® Probe IDs used in this study: Hs_00362215_g1 ( HBE ), Hs00361131_g1 ( HBG ), Hs_00426283_m1 ( HBD ), Hs_00747223_g1 ( HBB ), Hs_00958843_m1 ( NFIX ), Hs01093198_m1 ( BCL11A ), Hs_00252415_s1 ( ZBTB7A ), and Hs99999903_m1 ( ACTB ).

Techniques: Quantitative RT-PCR, Biomarker Discovery, Over Expression, Plasmid Preparation, Control, Western Blot, FLAG-tag

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: 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