Journal: Bone & Joint Research

Article Title: Naringin targets TGF-β1-mediated angiogenesis to enhance the osteogenic effect of induced membrane

doi: 10.1302/2046-3758.155.BJR-2025-0412.R1

Figure Lengend Snippet: The effect of naringin on the expression of transforming growth factor β (TGF-β)/SMAD pathway-related factors in induced membrane. a) The protein level of TGF-β1, phosphorylated SMAD (p-SMAD)2 and p-SMAD3 was detected by western blot. b) Immunohistochemistry result of TGF-β1, p-SMAD2 and p-SMAD3. N = 6/group. Each value was presented as the mean (SD). *p < 0.05, **p < 0.01, ***p < 0.001 vs the control group; #p < 0.05, ##p < 0.01, ###p < 0.001 vs the L-Naringin group.

Article Snippet: Three small interfering RNA sequences targeting TGF-β1 (si-TGF-β1 #1, si-TGF-β1 #2, si-TGF-β1 #3) and a negative control sequence (si-TGF-β1 NC) were synthesized separately by Sangon Biotech (China).

Techniques: Expressing, Membrane, Western Blot, Immunohistochemistry, Control

Journal: Bone & Joint Research

Article Title: Naringin targets TGF-β1-mediated angiogenesis to enhance the osteogenic effect of induced membrane

doi: 10.1302/2046-3758.155.BJR-2025-0412.R1

Figure Lengend Snippet: Characterization of endothelial progenitor cells (EPCs) and transfection efficacy of small interfering RNA targeting transforming growth factor-β1 (si-TGF-β1). a) was the immunofluorescence result of cluster of differentiation (CD)34 and vascular endothelial growth factor receptor 2 (VEGFR2). b) EPCs could simultaneously absorb DiI-labelled acetylated low-density lipoprotein (Dil-Ac-LDL) and fluorescein isothiocyanate-labeled Ulex europaeus agglutinin I (FITC-UEA-I). Scale bar: 100 μm. c) Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was used to validate the silencing effect of si-TGF-β1 #1, #2 and #3. N = 5/group. d) Western blot was used to validate the silencing effect of si-TGF-β1 #1, #2 and #3. N = 5/group. Each value was presented as the mean (SD). ***p < 0.001 vs the si-TGF-β1 negative control (NC) group; ##p < 0.01, ###p < 0.001 vs the si-TGF-β1 #2 group.

Article Snippet: Three small interfering RNA sequences targeting TGF-β1 (si-TGF-β1 #1, si-TGF-β1 #2, si-TGF-β1 #3) and a negative control sequence (si-TGF-β1 NC) were synthesized separately by Sangon Biotech (China).

Techniques: Transfection, Small Interfering RNA, Immunofluorescence, Labeling, Reverse Transcription, Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot, Negative Control

Journal: Bone & Joint Research

Article Title: Naringin targets TGF-β1-mediated angiogenesis to enhance the osteogenic effect of induced membrane

doi: 10.1302/2046-3758.155.BJR-2025-0412.R1

Figure Lengend Snippet: The effect of naringin on the proliferation and viability of endothelial progenitor cells (EPCs). a) 5-ethynyl-2'-deoxyuridine (EdU) staining method was used to detect the effect of naringin on the viability of EPCs at different stages (24, 48, and 72 hours). Scale bar: 100 μm. N = 5/group. b) Cell Counting Kit-8 (CCK-8, Beyotime, China) method was used to detect the effect of naringin on the viability of EPCs at different stages (24, 48, and 72 hours). N = 5/group. Each value was presented as the mean (SD). *p < 0.05, ** p< 0.01, ***p < 0.001 vs the control group; #p < 0.05, ##p < 0.01 vs the small interfering RNA targeting transforming growth factor-β1 (si-TGF-β1) group.

Article Snippet: Three small interfering RNA sequences targeting TGF-β1 (si-TGF-β1 #1, si-TGF-β1 #2, si-TGF-β1 #3) and a negative control sequence (si-TGF-β1 NC) were synthesized separately by Sangon Biotech (China).

Techniques: Staining, Cell Counting, CCK-8 Assay, Control, Small Interfering RNA

Journal: Bone & Joint Research

Article Title: Naringin targets TGF-β1-mediated angiogenesis to enhance the osteogenic effect of induced membrane

doi: 10.1302/2046-3758.155.BJR-2025-0412.R1

Figure Lengend Snippet: The effect of naringin on the migration, invasion and tube formation of endothelial progenitor cells (EPCs). a) Scratch wound was used to detect the invasion area of EPCs within 24 hours. Scale bar: 200 μm. b) Transwell assay was used to detect the number of migrated EPCs at 24 hours. Scale bar: 100 μm. c) The tube formation experiment detected the total tube length of EPCs. Scale bar: 100 μm. N = 5/group. Each value was presented as the mean (SD). **p < 0.01, ***p < 0.001 vs the control group; ###p < 0.001 vs the small interfering RNA targeting transforming growth factor-β1 (si-TGF-β1) group.

Article Snippet: Three small interfering RNA sequences targeting TGF-β1 (si-TGF-β1 #1, si-TGF-β1 #2, si-TGF-β1 #3) and a negative control sequence (si-TGF-β1 NC) were synthesized separately by Sangon Biotech (China).

Techniques: Migration, Transwell Assay, Control, Small Interfering RNA

Journal: Bone & Joint Research

Article Title: Naringin targets TGF-β1-mediated angiogenesis to enhance the osteogenic effect of induced membrane

doi: 10.1302/2046-3758.155.BJR-2025-0412.R1

Figure Lengend Snippet: The effect of naringin on angiogenic-osteogenic and transforming growth factor β (TGF-β)/SMAD pathway-related factors of endothelial progenitor cells (EPCs). a) The concentrations of platelet derived growth factor BB (PDGF-BB), vascular endothelial growth factor (VEGF), and slit guidance ligand 3 (SLIT3) in the supernatant of EPCs were detected by enzyme-linked immunosorbent assay. b) Alizarin red staining (ARS) was performed to detect mineralized nodules in osteoblasts. Scale bar: 50 μm. c) The protein level of p-SMAD2 and p-SMAD3 in EPCs was detected by western blot. d) was the immunofluorescence result of p-SMAD2. Scale bar: 100 μm. N = 5/group. Each value was presented as the mean (SD). ***p < 0.001 vs the control group; ##p < 0.01, ###p < 0.001 vs the small interfering RNA targeting transforming growth factor-β1 (si-TGF-β1) group.

Article Snippet: Three small interfering RNA sequences targeting TGF-β1 (si-TGF-β1 #1, si-TGF-β1 #2, si-TGF-β1 #3) and a negative control sequence (si-TGF-β1 NC) were synthesized separately by Sangon Biotech (China).

Techniques: Derivative Assay, Enzyme-linked Immunosorbent Assay, Staining, Western Blot, Immunofluorescence, Control, Small Interfering RNA

Journal: Bone & Joint Research

Article Title: Naringin targets TGF-β1-mediated angiogenesis to enhance the osteogenic effect of induced membrane

doi: 10.1302/2046-3758.155.BJR-2025-0412.R1

Figure Lengend Snippet: Interactions between naringin and transforming growth factor-β1 (TGF-β1). a) The basic chemical structure of naringin. b) 3D and 2D molecular docking patterns of naringin with TGF-β1. c) to g) Results of molecular dynamics simulation analysis illustrating root mean square deviation (RMSD), root mean square fluctuation (RMSF), radius of gyration (Rg), solvent-accessible surface area (SASA), and hydrogen-bond number for the TGF-β1-naringin complexes. h) Representative images of cellular thermal shift assay (CETSA) showing TGF-β1 thermal stability after naringin treatment. i) CETSA curve was performed using GraphPad Prism (GraphPad Software, USA). N = 5/group. Each value was presented as the mean (SD). *p < 0.05, **p < 0.01, ***p < 0.001 vs the dimethyl sulfoxide (DMSO) group.

Article Snippet: Three small interfering RNA sequences targeting TGF-β1 (si-TGF-β1 #1, si-TGF-β1 #2, si-TGF-β1 #3) and a negative control sequence (si-TGF-β1 NC) were synthesized separately by Sangon Biotech (China).

Techniques: Solvent, Thermal Shift Assay, Software

Journal: Biomolecules

Article Title: Lysine Acetyltransferase 6A Drives M1 Macrophage Polarization Through Metabolic Reprogramming in Sepsis-Induced Acute Lung Injury

doi: 10.3390/biom16040609

Figure Lengend Snippet: Lysine acetyltransferase 6A (KAT6A) is upregulated in macrophages during sepsis. ( A ) Microarray-based transcriptomic analysis of peripheral blood samples from healthy individuals and sepsis patients in the GSE54514 dataset. Upregulated genes (red), downregulated genes (blue), and stable genes (grey) are indicated. ( B ) Expression levels of KAT6A in healthy individuals (n = 18) and sepsis patients (n = 26). ( C – H ) Single-cell RNA sequencing analysis of lung tissues from sham and CLP mice ( GSE207651 ). ( C ) Uniform Manifold Approximation and Projection (UMAP) visualization of cells from sham and CLP groups. ( D ) Bar chart depicting the relative abundances of different cell populations. ( E ) Bar chart of the relative abundances of cells in the mononuclear phagocyte (MPS) clusters. ( F ) Violin plot illustrates the distribution of KAT6A expression in macrophages from sham and CLP mice. ( G ) UMAP visualization of M1 and M2 macrophage subpopulations identified within the macrophage cluster. ( H ) Violin plots of KAT6A expression in M1 and M2 macrophage subsets. ( I ) Representative confocal microscopy images of KAT6A (red) and DAPI (blue) in peritoneal macrophages (PMs). scale bar: 20 µm (top); scale bar, 2 µm (bottom). ( J ) KAT6A expression in lung tissues from septic and sham-operated mice was analyzed by Western blot. The experiment was repeated three times independently with similar results. ( K ) Quantitative real-time PCR (qPCR) analysis of KAT6A mRNA levels in lung tissues from sepsis mice (n = 5) and sham-operated mice (n = 3). All data are mean ± SEM. * p < 0.05, *** p < 0.001, **** p < 0.0001 by two-tailed unpaired Student’s t test in panel ( K ). ns, not significant. Original Western blot images can be found in .

Article Snippet: Small interfering RNA targeting KAT6A (siKAT6A) was synthesized by Sangon Biotech (Shanghai, China).

Techniques: Microarray, Expressing, Single Cell, RNA Sequencing, Confocal Microscopy, Western Blot, Real-time Polymerase Chain Reaction, Two Tailed Test

Journal: Biomolecules

Article Title: Lysine Acetyltransferase 6A Drives M1 Macrophage Polarization Through Metabolic Reprogramming in Sepsis-Induced Acute Lung Injury

doi: 10.3390/biom16040609

Figure Lengend Snippet: KAT6A inhibition suppresses glycolysis and reprograms macrophage metabolic activity. ( A – F ) PMs from C57BL/6J mice were pretreated with WM1119 or DMSO for 1 h, followed by stimulation with lipopolysaccharide (LPS, 100 ng/mL) for 6 h. Bulk RNA sequencing was performed on PMs to identify differentially expressed genes (DEGs) and enriched gene sets (n = 5). ( A ) Principal component analysis (PCA) of samples in different groups. ( B ) Volcano plot for DEGs. ( C ) Gene Set Enrichment Analysis (GSEA) plot showing enrichment score and normalized enrichment scores (NES) for glycolysis. ( D ) Heatmap shows expression of glycolytic genes. ( E ) Gene expression of glucose transporter 1 ( Glut1 ), hexokinase ( Hk ) 2 , Aldoc , Tpi1 , Aldh9a1 , Gapdh , Pgk1 , Pkm , Pgam1 , Eno1 and Ldha was measured by qPCR in PMs. Data are presented as fold change relative to the DMSO group (n = 3). ( F ) Lactate concentration in culture supernatants (n = 3). ( G ) Gene expression of Hif1α and c-Myc was measured by qPCR (n = 3). ( H – J ) Bone marrow-derived macrophages (BMDMs) from C57BL/6J mice were pretreated with WM1119 (100 μM) or DMSO for 1 h, followed by stimulation with LPS (100 ng/mL) for 24 h. ( H ) HIF1α and c-Myc expression were measured by Western blot, quantification of protein expression levels normalized to β-actin (n = 3). ( I , J ) GLUT1 and pyruvate kinase M2 (PKM2) expression was measured by Western blot, quantification of protein expression levels normalized to β-actin (n = 3). ( K – P ) BMDMs from C57BL/6J mice were pretreated with WM1119 (100 μM) or DMSO for 1 h, followed by stimulation with LPS (100 ng/mL) for 6 h. ( K ) BMDMs were incubated with 2-NBDG, a fluorescent glucose analogue, and glucose uptake was measured by flow cytometry. Mean fluorescence intensity (MFI) is shown (n = 6). ( L – P ) Extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) were assessed with a Seahorse XF96 analyzer. ( L , M ) ECAR was measured following sequential injections of glucose, oligomycin (Oligo), and 2-deoxyglucose (2-DG), and the derived parameters for glycolysis and glycolytic capacity were quantified (n = 10). ( N , O ) OCR was determined after consecutive addition of Oligo, carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP), and rotenone/antimycin A (R/A), with analyses summarizing basal respiration, ATP-linked respiration, maximal respiration, and spare respiratory capacity (n = 6). ( P ) Ratio of OCR to ECAR. Data are mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Statistics were done by one-way ANOVA followed by adjustments for multiple comparisons in panels ( E , G , K ), and two-tailed unpaired Student’s t test in panels ( F , M , O , P ). ns, not significant. Original Western blot images can be found in .

Article Snippet: Small interfering RNA targeting KAT6A (siKAT6A) was synthesized by Sangon Biotech (Shanghai, China).

Techniques: Inhibition, Activity Assay, RNA Sequencing, Expressing, Gene Expression, Concentration Assay, Derivative Assay, Western Blot, Incubation, Flow Cytometry, Fluorescence, Two Tailed Test

Journal: Biomolecules

Article Title: Lysine Acetyltransferase 6A Drives M1 Macrophage Polarization Through Metabolic Reprogramming in Sepsis-Induced Acute Lung Injury

doi: 10.3390/biom16040609

Figure Lengend Snippet: KAT6A inhibition attenuates inflammatory response in macrophages. ( A – D ) PMs and ( E , F ) BMDMs from C57BL/6J mice were pretreated with WM1119 at the indicated concentrations or DMSO for 1 h, followed by stimulation with LPS (100 ng/mL) for 6 h. ( A ) Experimental scheme in PMs. ( B ) qPCR analysis of Tnfα , Il-1β , Il-6 , Nos2 , Il-10 (n = 3). ( C , D ) Flow cytometric analysis of TNFα and IL-6 production in F4/80 + CD11b + cells (n = 3). ( E ) Experimental scheme in BMDMs. ( F ) qPCR analysis of Tnfα , Il-1β , Il-6 , Nos2 , Il-10 (n = 3). Data are mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Statistics were done by one-way ANOVA followed by adjustments for multiple comparisons in panels ( B – D , F ). ns, not significant.

Article Snippet: Small interfering RNA targeting KAT6A (siKAT6A) was synthesized by Sangon Biotech (Shanghai, China).

Techniques: Inhibition

Journal: Biomolecules

Article Title: Lysine Acetyltransferase 6A Drives M1 Macrophage Polarization Through Metabolic Reprogramming in Sepsis-Induced Acute Lung Injury

doi: 10.3390/biom16040609

Figure Lengend Snippet: KAT6A inhibition suppresses M1 macrophage polarization. BMDMs were pretreated with WM1119 at the indicated concentrations or DMSO for 1 h, followed by stimulation with LPS (100 ng/mL) and interferon-gamma (IFN-γ) (50 ng/mL) for 48 h. ( A ) Experimental scheme. ( B ) Representative flow cytometric analysis of surface markers CD86 expression on F4/80 + BMDMs (n = 5). ( C , D ) qPCR analysis of Cd86 , Tnfα , Il-1β , Il-6 , Nos2 , Il-10 expression (n = 3). RAW264.7 macrophages transfected with siKAT6A or scramble control were stimulated with LPS and IFN-γ for 48 h. ( E ) Knockdown of KAT6A was confirmed by Western blot (n = 3). ( F ) Representative flow cytometric analysis and quantification of 2-NBDG uptake (n = 3). ( G , H ) qPCR analysis of glycolytic genes Glut1 , Hk2 , Pkm , Eno1 and Ldha (n = 3). ( I ) Lactate concentration in culture supernatants (n = 3). ( J ) Immunoblot analysis of H3K9ac and H3K27ac acetylation. Representative bands of six biologically independent replicates. ( K ) Representative flow cytometric analysis and quantification of CD86 expression in RAW264.7 macrophages (n = 3). ( L ) Representative flow cytometric analysis and quantification of intracellular TNFα and NOS2 expression (n = 3). All data are mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Statistics were done by one-way ANOVA followed by adjustments for multiple comparisons in panels ( B – D ) and two-tailed unpaired Student’s t test in panels ( F – I , K , L ). ns, not significant. Original Western blot images can be found in .

Article Snippet: Small interfering RNA targeting KAT6A (siKAT6A) was synthesized by Sangon Biotech (Shanghai, China).

Techniques: Inhibition, Expressing, Transfection, Control, Knockdown, Western Blot, Concentration Assay, Two Tailed Test

Journal: Biomolecules

Article Title: Lysine Acetyltransferase 6A Drives M1 Macrophage Polarization Through Metabolic Reprogramming in Sepsis-Induced Acute Lung Injury

doi: 10.3390/biom16040609

Figure Lengend Snippet: KAT6A inhibition is associated with reduced PI3K-AKT-mTOR signaling and M1 macrophage polarization. ( A ) KEGG enrichment of environmental information processing in down-regulated DEGs derived from WM1119-treated PMs. DEGs were defined as described in . ( B ) GSEA plot showing enrichment score and NES for mTOR complex 1 (mTORC1) signaling. ( C ) KEGG enrichment of metabolism in down-regulated genes. ( D – I ) BMDMs from C57BL/6J mice were pretreated with WM1119 (100 μM) or DMSO for 1 h, followed by stimulation with LPS (100 ng/mL) for 24 h. Western blot images of the protein expression of ( D ) p-PI3K p85 , PI3K, p-AKT S473 and AKT and ( E ) p-mTOR S2448 , mTOR, and Raptor in the indicated groups. Quantification of protein expression levels normalized to β-actin (n = 3). BMDMs were stained for p-AKT S473 ( F , G ) and p-S6 S235/236 ( H , I ) and analyzed by flow cytometry. (n = 6). All data are mean ± SEM. **** p < 0.0001. Statistics were done by a two-tailed unpaired Student’s t test in panels ( G , I ). Original Western blot images can be found in .

Article Snippet: Small interfering RNA targeting KAT6A (siKAT6A) was synthesized by Sangon Biotech (Shanghai, China).

Techniques: Inhibition, Derivative Assay, Western Blot, Expressing, Staining, Flow Cytometry, Two Tailed Test

Journal: Biomolecules

Article Title: Lysine Acetyltransferase 6A Drives M1 Macrophage Polarization Through Metabolic Reprogramming in Sepsis-Induced Acute Lung Injury

doi: 10.3390/biom16040609

Figure Lengend Snippet: KAT6A inhibition protects against organ injury at 12 h after CLP-induced sepsis. ( A ) Scheme of the mouse experiment. C57BL/6J mice were randomly assigned to three groups. 4 h after receiving vehicle or WM1119 (50 mg/kg), mice were subjected to sham or CLP surgery. All mice were euthanized 12 h after surgery for organ injury assessment. ( B ) H&E staining and histological score of lung, liver and colon sections from the indicated groups. Scale bar: 100 µm (lung, liver), Scale bar: 50 µm (colon) (n = 3 for sham group, n = 5 for CLP group, n = 4 for CLP+WM1119 group). ( C ) qPCR of cytokine genes ( Tnfα , Il-1β , Il-6 , Nos2 , Il-10 ) in lung tissues from the indicated groups (n = 3 for sham group, n = 5 for CLP and CLP+WM1119 group). All data are mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Statistics were done by one-way ANOVA followed by adjustments for multiple comparisons in panels ( B , C ).

Article Snippet: Small interfering RNA targeting KAT6A (siKAT6A) was synthesized by Sangon Biotech (Shanghai, China).

Techniques: Inhibition, Staining

Journal: Biomolecules

Article Title: Lysine Acetyltransferase 6A Drives M1 Macrophage Polarization Through Metabolic Reprogramming in Sepsis-Induced Acute Lung Injury

doi: 10.3390/biom16040609

Figure Lengend Snippet: KAT6A inhibition protects against organ injury and promotes bacterial clearance at 24 h after CLP-induced sepsis. ( A ) Scheme of the mouse experiment. Mice were pretreated with WM1119 (50 mg/kg) or vehicle 4 h before CLP. Mice were euthanized 24 h after CLP, and tissues were harvested for analysis. ( B ) 24 h survival rate was recorded. ( C – E ) Representative H&E staining images of lung, liver and colon from septic mice treated with WM1119 or vehicle, with their respective histological scores quantified in ( F ) (n = 6). Scale bar: 100 μm ( C , D ), Scale bar: 50 μm ( E ). ( G , H ) Serum concentrations of aspartate aminotransferase (AST), alanine aminotransferase (ALT), creatine kinase (CK), urea, and creatinine (Crea) were measured in the indicated groups (n = 6). Bacterial burden was quantified as colony-forming units (CFU) per mL for peritoneal fluid ( I ) (n = 6) and CFU per gram for lung tissues ( J ) (n = 6). ( K ) Correlations between KAT6A mRNA expression levels and lung CFUs (n = 6 for each group). ( L ) The percentage of M1 macrophages (F4/80 + CD86 + ) in septic lung tissues treated with WM1119 or vehicle (n = 6). ( M ) qPCR of cytokine genes ( Tnfα , Il-6 , Nos2 , Il-1β ) in lung tissues treated with WM1119 or vehicle (n = 6). All data are mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Statistics were done by a two-tailed unpaired Student’s t test in panels ( B , F , G – J , L , M ). Correlation was assessed by the Pearson correlation coefficient in panel ( K ).

Article Snippet: Small interfering RNA targeting KAT6A (siKAT6A) was synthesized by Sangon Biotech (Shanghai, China).

Techniques: Inhibition, Staining, Expressing, 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: 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: Inflammation

Article Title: Inhibition of SPI1 by ADAP Regulates S100A8/A9 Signaling in Macrophages to Control the Development of Colitis

doi: 10.1007/s10753-025-02363-9

Figure Lengend Snippet: ADAP regulates inflammation via the transcription factor SPI1. A Prediction of SPI1 binding sites on the S100A8/A9 promoter. B Chromatin immunoprecipitation combined with CUT&RUN assay showing SPI1 binding to the S100A8/A9 promoter. C - D RT-qPCR and Western blot analysis of SPI1 expression in the colon from WT and Adap −/− mice with DSS treatment. E - F RT-qPCR and Western blot analysis of SPI1 expression in RAW264.7 cells with LPS stimulation (1 µg/mL, 24 h) in WT and Adap KD RAW264.7 cells. G Luciferase reporter assay assessing S100A8/A9 promoter activity in WT and Adap KD RAW264.7 cells after SPI1 knockdown and LPS stimulation (1 µg/mL, 24 h). H Western blot analysis of S100A8/A9 and NF-κB pathway proteins in RAW264.7 cells following SPI1 knockdown and stimulation with LPS (1 µg/mL, 24 h). Data are presented as mean ± SD from at least three independent experiments. Statistical analysis was performed using unpaired two-tailed Student’s t -test. ns, not significant, * p < 0.05, ** p < 0.01

Article Snippet: Small interfering RNA targeting SPI1 (si-SPI1) and the corresponding negative control (si-NC) were synthesized by Sangon Biotech (Shanghai, China).

Techniques: Binding Assay, Chromatin Immunoprecipitation, Quantitative RT-PCR, Western Blot, Expressing, Luciferase, Reporter Assay, Activity Assay, Knockdown, Two Tailed Test

Journal: Inflammation

Article Title: Inhibition of SPI1 by ADAP Regulates S100A8/A9 Signaling in Macrophages to Control the Development of Colitis

doi: 10.1007/s10753-025-02363-9

Figure Lengend Snippet: FBXW7 targets SPI1 for ubiquitin-proteasome-mediated degradation. A RAW264.7 cells were treated with LPS (1 µg/mL), chloroquine (CQ, 10 µM), or MG132 (10 µM) for 24 h. SPI1 protein levels were analyzed by Western blot. B WT or Adap KD RAW264.7 cells were left untreated or stimulated with LPS (1 µg/mL, 24 h), followed by Western blot analysis of FBXW7 expression. C HEK293T cells were transfected with Myc-FBXW7, Flag-SPI1, and His-Ub plasmids (1 µg each). At 48 h post-transfection, cell lysates were immunoprecipitated with anti-Flag antibodies and immunoblotted with anti-Ub, anti-Myc, and anti-Flag antibodies. D HEK293T cells were transfected with His-Ub (wild-type), His-Ub-K48R, His-Ub-K63R, Myc-FBXW7, or Flag-SPI1 plasmids. At 48 h post-transfection, lysates were immunoprecipitated with anti-Flag and immunoblotted with anti-Ub, anti-Myc, and anti-Flag antibodies

Article Snippet: Small interfering RNA targeting SPI1 (si-SPI1) and the corresponding negative control (si-NC) were synthesized by Sangon Biotech (Shanghai, China).

Techniques: Ubiquitin Proteomics, Western Blot, Expressing, Transfection, Immunoprecipitation

Journal: Inflammation

Article Title: Inhibition of SPI1 by ADAP Regulates S100A8/A9 Signaling in Macrophages to Control the Development of Colitis

doi: 10.1007/s10753-025-02363-9

Figure Lengend Snippet: Mechanism diagram: Inhibition of SPI1 by ADAP regulates S100A8/A9 signaling in macrophages to control the development of colitis. In inflammatory macrophages, ADAP upregulates FBXW7 expression and promotes ubiquitin-proteasome-mediated degradation of the transcription factor SPI1, thereby suppressing the expression of downstream effectors S100A8 and S100A9. ADAP deficiency disrupts this checkpoint, leading to SPI1 accumulation and aberrant overproduction of S100A8/A9, which triggers excessive inflammatory responses and exacerbates DSS-induced colitis

Article Snippet: Small interfering RNA targeting SPI1 (si-SPI1) and the corresponding negative control (si-NC) were synthesized by Sangon Biotech (Shanghai, China).

Techniques: Inhibition, Control, Expressing, Ubiquitin Proteomics