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: Smart Medicine

Article Title: Mechanistic Insights Into NFIX‐Mediated DNA Recognition and Transcriptional Regulation in Skeletal Muscle

doi: 10.1002/smmd.70027

Figure Lengend Snippet: NFIX regulates skeletal muscle cell proliferation, apoptosis, and differentiation. (A) Schematic diagram illustrating the domain architecture of full‐length human NFIX (residues 1–502) and its DNA‐binding domain. (B) Single‐cell RNA‐seq analysis showing NFIX expression across various human cell types. (C) NFIX expression in skeletal muscle tissues from patients with Duchenne muscular dystrophy (DMD), inclusion body myositis (IBM), nemaline myopathy (NM), polymyositis (PM), and tibial muscular dystrophy (TMD) compared with healthy controls, based on multiple GEO datasets. (D–E) siRNA‐mediated knockdown of NFIX in immortalized human skeletal muscle cells, with depletion efficiency validated by qPCR (D) and Western blot analysis (E). (F, I) EdU incorporation assay showing reduced DNA synthesis in NFIX‐depleted cells compared with control cells (F), quantified as the percentage of EdU‐positive nuclei (I). Scale bar, 100 μm. (G, J) TUNEL assay indicating increased apoptosis in NFIX knockdown cells (G), with quantification of apoptotic nuclei (J). Scale bar, 100 μm. (H, K) Myogenic fusion assay showing reduced myotube formation in NFIX‐deficient cells (H), quantified as fusion index (K). Scale bar, 100 μm. Data in (D–K) are presented as mean ± SD, dots represent individual samples. Analysis by unpaired Student's t ‐test. For all panels, n = 3. * p < 0.05.

Article Snippet: Transfection was performed using RNATransMate reagent (Sangon Biotech, #E607402) with 5 nM small interfering RNA (siRNA) targeting NFIX (Sangon Biotech) or non‐targeting control siRNA (Sangon Biotech), following the manufacturer's instructions.

Techniques: Binding Assay, RNA Sequencing, Expressing, Knockdown, Western Blot, DNA Synthesis, Control, TUNEL Assay, Single Vesicle Fusion Assay

Journal: Smart Medicine

Article Title: Mechanistic Insights Into NFIX‐Mediated DNA Recognition and Transcriptional Regulation in Skeletal Muscle

doi: 10.1002/smmd.70027

Figure Lengend Snippet: NFIX‐mediated transcriptional regulation in human skeletal muscle cells. (A) Volcano plot showing differentially expressed genes (DEGs) in NFIX knockdown versus control human skeletal muscle cells, as determined by RNA‐seq. Significantly upregulated (red) and downregulated (blue) genes are indicated. (B) KEGG pathway enrichment analysis of DEGs, highlighting significant enrichment in immune and inflammatory responses, metabolic processes, and stress‐related pathways. (C) Heat map illustrating expression changes of representative NFIX‐regulated genes across enriched pathways. (D) qPCR validation of NMNAT2, PPARD, IL1RN, IL6, NDRG2, and EGR1. Data in (D) are presented as mean ± SD, dots represent individual samples. Analysis by unpaired Student's t ‐test. For all panels, n = 3. * p < 0.05.

Article Snippet: Transfection was performed using RNATransMate reagent (Sangon Biotech, #E607402) with 5 nM small interfering RNA (siRNA) targeting NFIX (Sangon Biotech) or non‐targeting control siRNA (Sangon Biotech), following the manufacturer's instructions.

Techniques: Knockdown, Control, RNA Sequencing, Expressing, Biomarker Discovery

Journal: Smart Medicine

Article Title: Mechanistic Insights Into NFIX‐Mediated DNA Recognition and Transcriptional Regulation in Skeletal Muscle

doi: 10.1002/smmd.70027

Figure Lengend Snippet: NFIX recognizes TGGCA motifs in a monomeric mode. (A) Motifs enriched in NFIX ChIP‐seq peaks from ENCODE (ENCFF726LLI) identified by HOMER: a TGGCA half‐site (top) and a palindromic dyad (bottom). (B) Size‐exclusion chromatography (Superdex 200 Increase) of full‐length NFIX and the DNA‐binding domain (NFIX DBD ); elution positions of standards are indicated. (C) Native PAGE analysis confirming the monomeric state of NFIX and NFIX DBD in solution. (D, G) Sequences of biotinylated DNA probes used in binding assays: DNA‐1 (TGGCA half‐site) and DNA‐2 (dyad). (E–F) BLI sensorgrams for NFIX (E) and NFIX DBD (F) binding to DNA‐1; global fits to a 1:1 Langmuir model. (H–I) BLI sensorgrams for NFIX (H) and NFIX DBD (I) binding to DNA‐2 under identical conditions.

Article Snippet: Transfection was performed using RNATransMate reagent (Sangon Biotech, #E607402) with 5 nM small interfering RNA (siRNA) targeting NFIX (Sangon Biotech) or non‐targeting control siRNA (Sangon Biotech), following the manufacturer's instructions.

Techniques: ChIP-sequencing, Size-exclusion Chromatography, Binding Assay, Clear Native PAGE

Journal: Smart Medicine

Article Title: Mechanistic Insights Into NFIX‐Mediated DNA Recognition and Transcriptional Regulation in Skeletal Muscle

doi: 10.1002/smmd.70027

Figure Lengend Snippet: Crystal structure of NFIX DBD bound to a palindromic duplex. (A) Overall structure of NFIX DBD (orange) in complex with an 18‐bp dsDNA (strand 1, cyan; strand 2, magenta). Orthogonal views depict major‐groove engagement and the binding orientation along one face of the helix. (B) Size‐exclusion chromatography of the purified complex shows a single symmetric peak consistent with a 1:1 NFIX DBD :DNA stoichiometry. (C) Superposition with the apo NFIX DBD structure (PDB 7QQE) reveals a ligand‐induced movement of the DNA‐binding loop. (D) Comparison with the previously deposited NFIX:dsDNA structure (PDB 7QQD) indicates that the present structure captures a specific, productive binding mode, whereas 7QQD places the DNA without base‐specific contacts.

Article Snippet: Transfection was performed using RNATransMate reagent (Sangon Biotech, #E607402) with 5 nM small interfering RNA (siRNA) targeting NFIX (Sangon Biotech) or non‐targeting control siRNA (Sangon Biotech), following the manufacturer's instructions.

Techniques: Binding Assay, Size-exclusion Chromatography, Purification, Comparison

Journal: Smart Medicine

Article Title: Mechanistic Insights Into NFIX‐Mediated DNA Recognition and Transcriptional Regulation in Skeletal Muscle

doi: 10.1002/smmd.70027

Figure Lengend Snippet: Structural basis of sequence‐specific DNA recognition by NFIX. (A) Detailed interactions of the NFIX DBD :dsDNA interface. NFIX DBD is shown as a ribbon with a semitransparent surface; the duplex is contoured with a composite‐omit 2m F o –D F c electron density map contoured at 2 σ . Insets highlight base‐specific contacts and phosphate‐backbone interactions formed by key residues. (B) Schematic diagram summarizing the protein–DNA contacts observed in the NFIX DBD :dsDNA structure. (C) Electrostatic potential surface of NFIX DBD at the DNA interface, illustrating the basic patch complementary to the DNA backbone. (D) Model of a hypothetical 2:1 NFIX DBD :dsDNA assembly generated by aligning two NFIX molecules to the symmetric half‐sites. Extensive steric clashes (indicated) argue against simultaneous dimeric occupancy of the palindromic motif on short B‐form dsDNA. (E) Model of a hypothetical NFIX dimer on nucleosomal chromatin at a dyad motif. Bending of nucleosomal DNA permits simultaneous binding of two NFIX molecules.

Article Snippet: Transfection was performed using RNATransMate reagent (Sangon Biotech, #E607402) with 5 nM small interfering RNA (siRNA) targeting NFIX (Sangon Biotech) or non‐targeting control siRNA (Sangon Biotech), following the manufacturer's instructions.

Techniques: Sequencing, Generated, Binding Assay

Journal: Smart Medicine

Article Title: Mechanistic Insights Into NFIX‐Mediated DNA Recognition and Transcriptional Regulation in Skeletal Muscle

doi: 10.1002/smmd.70027

Figure Lengend Snippet: NFIX activates skeletal muscle–related promoters via specific DNA recognition. (A) Mutational analysis of sequence‐recognition residues in NFIX DBD using BLI, comparing the dsDNA binding profiles of wild‐type NFIX DBD with R116A, K125A, and R116A/K125A mutants. (B) Quantification of relative DNA‐binding affinities, calculated as WT K D ÷ mutant K D × 100. (C–F) Dual‐luciferase assays with pGL3 reporters driven by the NDRG2 , EGR1 , IL1RN , and NMNAT2 promoters. WT NFIX robustly enhances reporter activity, whereas the DNA‐binding–defective double mutant (R116A/K125A) fails to activate transcription. Data are mean ± s.d. from ≥ 3 independent experiments; unpaired two‐tailed t ‐test: *** p < 0.001.

Article Snippet: Transfection was performed using RNATransMate reagent (Sangon Biotech, #E607402) with 5 nM small interfering RNA (siRNA) targeting NFIX (Sangon Biotech) or non‐targeting control siRNA (Sangon Biotech), following the manufacturer's instructions.

Techniques: Sequencing, Binding Assay, Mutagenesis, Luciferase, Activity Assay, Two Tailed Test

Journal: Smart Medicine

Article Title: Mechanistic Insights Into NFIX‐Mediated DNA Recognition and Transcriptional Regulation in Skeletal Muscle

doi: 10.1002/smmd.70027

Figure Lengend Snippet: NFIX regulates skeletal muscle cell proliferation, apoptosis, and differentiation. (A) Schematic diagram illustrating the domain architecture of full‐length human NFIX (residues 1–502) and its DNA‐binding domain. (B) Single‐cell RNA‐seq analysis showing NFIX expression across various human cell types. (C) NFIX expression in skeletal muscle tissues from patients with Duchenne muscular dystrophy (DMD), inclusion body myositis (IBM), nemaline myopathy (NM), polymyositis (PM), and tibial muscular dystrophy (TMD) compared with healthy controls, based on multiple GEO datasets. (D–E) siRNA‐mediated knockdown of NFIX in immortalized human skeletal muscle cells, with depletion efficiency validated by qPCR (D) and Western blot analysis (E). (F, I) EdU incorporation assay showing reduced DNA synthesis in NFIX‐depleted cells compared with control cells (F), quantified as the percentage of EdU‐positive nuclei (I). Scale bar, 100 μm. (G, J) TUNEL assay indicating increased apoptosis in NFIX knockdown cells (G), with quantification of apoptotic nuclei (J). Scale bar, 100 μm. (H, K) Myogenic fusion assay showing reduced myotube formation in NFIX‐deficient cells (H), quantified as fusion index (K). Scale bar, 100 μm. Data in (D–K) are presented as mean ± SD, dots represent individual samples. Analysis by unpaired Student's t ‐test. For all panels, n = 3. * p < 0.05.

Article Snippet: The single‐cell RNA sequencing data for the NFIX gene were obtained from the Human Protein Atlas (HPA) single‐cell database (gene ID: ENSG00000008441).

Techniques: Binding Assay, RNA Sequencing, Expressing, Knockdown, Western Blot, DNA Synthesis, Control, TUNEL Assay, Single Vesicle Fusion Assay

Journal: Smart Medicine

Article Title: Mechanistic Insights Into NFIX‐Mediated DNA Recognition and Transcriptional Regulation in Skeletal Muscle

doi: 10.1002/smmd.70027

Figure Lengend Snippet: NFIX‐mediated transcriptional regulation in human skeletal muscle cells. (A) Volcano plot showing differentially expressed genes (DEGs) in NFIX knockdown versus control human skeletal muscle cells, as determined by RNA‐seq. Significantly upregulated (red) and downregulated (blue) genes are indicated. (B) KEGG pathway enrichment analysis of DEGs, highlighting significant enrichment in immune and inflammatory responses, metabolic processes, and stress‐related pathways. (C) Heat map illustrating expression changes of representative NFIX‐regulated genes across enriched pathways. (D) qPCR validation of NMNAT2, PPARD, IL1RN, IL6, NDRG2, and EGR1. Data in (D) are presented as mean ± SD, dots represent individual samples. Analysis by unpaired Student's t ‐test. For all panels, n = 3. * p < 0.05.

Article Snippet: The single‐cell RNA sequencing data for the NFIX gene were obtained from the Human Protein Atlas (HPA) single‐cell database (gene ID: ENSG00000008441).

Techniques: Knockdown, Control, RNA Sequencing, Expressing, Biomarker Discovery

Journal: Smart Medicine

Article Title: Mechanistic Insights Into NFIX‐Mediated DNA Recognition and Transcriptional Regulation in Skeletal Muscle

doi: 10.1002/smmd.70027

Figure Lengend Snippet: Structural basis of sequence‐specific DNA recognition by NFIX. (A) Detailed interactions of the NFIX DBD :dsDNA interface. NFIX DBD is shown as a ribbon with a semitransparent surface; the duplex is contoured with a composite‐omit 2m F o –D F c electron density map contoured at 2 σ . Insets highlight base‐specific contacts and phosphate‐backbone interactions formed by key residues. (B) Schematic diagram summarizing the protein–DNA contacts observed in the NFIX DBD :dsDNA structure. (C) Electrostatic potential surface of NFIX DBD at the DNA interface, illustrating the basic patch complementary to the DNA backbone. (D) Model of a hypothetical 2:1 NFIX DBD :dsDNA assembly generated by aligning two NFIX molecules to the symmetric half‐sites. Extensive steric clashes (indicated) argue against simultaneous dimeric occupancy of the palindromic motif on short B‐form dsDNA. (E) Model of a hypothetical NFIX dimer on nucleosomal chromatin at a dyad motif. Bending of nucleosomal DNA permits simultaneous binding of two NFIX molecules.

Article Snippet: The single‐cell RNA sequencing data for the NFIX gene were obtained from the Human Protein Atlas (HPA) single‐cell database (gene ID: ENSG00000008441).

Techniques: Sequencing, Generated, Binding Assay

Journal: Smart Medicine

Article Title: Mechanistic Insights Into NFIX‐Mediated DNA Recognition and Transcriptional Regulation in Skeletal Muscle

doi: 10.1002/smmd.70027

Figure Lengend Snippet: NFIX activates skeletal muscle–related promoters via specific DNA recognition. (A) Mutational analysis of sequence‐recognition residues in NFIX DBD using BLI, comparing the dsDNA binding profiles of wild‐type NFIX DBD with R116A, K125A, and R116A/K125A mutants. (B) Quantification of relative DNA‐binding affinities, calculated as WT K D ÷ mutant K D × 100. (C–F) Dual‐luciferase assays with pGL3 reporters driven by the NDRG2 , EGR1 , IL1RN , and NMNAT2 promoters. WT NFIX robustly enhances reporter activity, whereas the DNA‐binding–defective double mutant (R116A/K125A) fails to activate transcription. Data are mean ± s.d. from ≥ 3 independent experiments; unpaired two‐tailed t ‐test: *** p < 0.001.

Article Snippet: The single‐cell RNA sequencing data for the NFIX gene were obtained from the Human Protein Atlas (HPA) single‐cell database (gene ID: ENSG00000008441).

Techniques: Sequencing, Binding Assay, Mutagenesis, Luciferase, Activity Assay, Two Tailed Test