Journal: Redox Report : Communications in Free Radical Research

Article Title: Neutrophil extracellular traps drive osteoporosis via NCF2-dependent signaling: integrated transcriptomics with mechanistic validation

doi: 10.1080/13510002.2025.2534745

Figure Lengend Snippet: Integrative transcriptomic and experimental validation of NET formation in osteoporosis. Schematic overview of the study design. A rat model of postmenopausal osteoporosis (OVX) was established and subjected to transcriptomic analysis (RNA-seq) to identify differentially expressed genes (DEGs) enriched in neutrophil extracellular traps (NETs) formation pathways. Mechanistic insights into NETosis were explored, including activation of neutrophils, NOX complex assembly, and release of NETs containing NE, MPO, and CitH3. For in vitro validation, primary osteoblasts isolated from 7-day-old rats were treated with NET-conditioned medium derived from PMA-induced bone marrow neutrophils or HL-60–derived neutrophil-like cells. siRNA-mediated knockdown of NCF2 was performed to assess its role in NETs formation and subsequent osteogenic inhibition.

Article Snippet: IF method: For immunofluorescence staining, CitH3 (Cell Signaling, Rabbit mAb #97272), NE (Thermo Fisher, PA5-88916) and MPO (Thermo Fisher, MPO-101AP) were used as primary antibodies.

Techniques: Biomarker Discovery, RNA Sequencing, Activation Assay, In Vitro, Isolation, Derivative Assay, Knockdown, Inhibition

Journal: Redox Report : Communications in Free Radical Research

Article Title: Neutrophil extracellular traps drive osteoporosis via NCF2-dependent signaling: integrated transcriptomics with mechanistic validation

doi: 10.1080/13510002.2025.2534745

Figure Lengend Snippet: Validation of NETs formation in bone tissue and neutrophils. (A) Triple immunofluorescence of bone-marrow neutrophils showing DAPI (blue), NE (green), CitH3 (red) and MPO (yellow) in unstimulated controls and PMA-induced NETs. Scale bar = 50 µm. (B) Representative Western blots of NE (29 kDa), MPO (60 kDa) and CitH3 (17 kDa) in rat bone tissue (Sham vs Model) and in primary neutrophils (Control vs NETs); GAPDH served as loading control. (C–E) Densitometric quantification of bone samples; (F–H) densitometry of neutrophil samples. (I–K) Percentage of CitH3-, MPO- and NE-positive areas in immunofluorescence images. (L) Extracellular DNA in culture supernatants quantified on a UV–visible micro-spectrophotometer. (M) Serum NETS level determined by MPO–DNA ELISA. (N) High-magnification confocal images illustrating co-localisation of NE, CitH3 and MPO in extracellular NETS fibres. Scale bar = 20 µm. Data are mean ± SD (n = 3); ** P < 0.01, **** P < 0.0001 versus respective controls (unpaired two-tailed t-test).

Article Snippet: IF method: For immunofluorescence staining, CitH3 (Cell Signaling, Rabbit mAb #97272), NE (Thermo Fisher, PA5-88916) and MPO (Thermo Fisher, MPO-101AP) were used as primary antibodies.

Techniques: Biomarker Discovery, Immunofluorescence, Western Blot, Control, Spectrophotometry, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: Redox Report : Communications in Free Radical Research

Article Title: Neutrophil extracellular traps drive osteoporosis via NCF2-dependent signaling: integrated transcriptomics with mechanistic validation

doi: 10.1080/13510002.2025.2534745

Figure Lengend Snippet: Knockdown of NCF2 decreases NETS formation and reverses NETs-mediated suppression of osteoblast differentiation. (A) Representative Western blot confirming NCF2 knockdown efficiency in HL-60 neutrophil-like cells. (B, C) Quantification of NCF2 protein and mRNA expression levels (**** P < 0.0001). (D) Triple immunofluorescence staining (DAPI, NE, MPO, CitH3) illustrating reduced NETS formation upon NCF2 knockdown; scale bar = 50 µm. (E–G) Quantitative analysis of NE-, MPO-, and CitH3-positive areas (**** P < 0.0001). (H) Extracellular DNA concentrations in supernatants measured by UV spectrophotometry (**** P < 0.0001). (I, J) ALP staining at day 7 (scale bar = 200 µm) and Alizarin red S staining of mineralisation at day 21 (scale bar = 200 µm) after exposure to indicated conditioned media. (K–M) Western blot and quantitative densitometric analysis of ALP and OCN protein expression levels normalised to GAPDH (*** P < 0.001, **** P < 0.0001). Data represent mean ± SD of three independent experiments; statistical analysis was performed by one-way ANOVA with Tukey’s multiple comparisons test.

Article Snippet: IF method: For immunofluorescence staining, CitH3 (Cell Signaling, Rabbit mAb #97272), NE (Thermo Fisher, PA5-88916) and MPO (Thermo Fisher, MPO-101AP) were used as primary antibodies.

Techniques: Knockdown, Western Blot, Expressing, Immunofluorescence, Staining, Spectrophotometry

Journal: Nature cell biology

Article Title: A chromosome folding intermediate at the condensin-to-cohesin transition during telophase

doi: 10.1038/s41556-019-0406-2

Figure Lengend Snippet: a, Classification of cell cycle stages based on DAPI staining and tubulin organization. Scale bar = 5μm. b, Localization of Lamin A/C, NCAPH, and CTCF during different cell cycle stages shown in panel a. Scale bar = 5μm. c, Quantification of CTCF and NCAPH colocalization with chromatin in single cells at different cell cycle stages. Left plot represents data from all cells with color indicating cell cycle stage. Right plots represent the data separated into each individual cell cycle stage. d, Top: Western blot analysis of chromatin-associated proteins purified from HeLaS3-NCAPH-dTomato cells at different time points after release from prometaphase. Bottom: Quantification of the western blot shown above. NCAPH and Rad21 were analyzed on the same gel. The samples for Histone H3 analysis were run on another gel. Four independent experiments were performed with similar results. Source Data for microscopy are provided in Source data . Unprocessed blots are provided in Source data .

Article Snippet: Membranes were then incubated with specified primary antibody diluted 1:1000 in 4% milk/PBS-T overnight at 4°C [Histone H3 (ab1791), Rad21 (ab154769), RFP (cross-reacts with dTomato for NCAPH-dTomato, Rockland 600-401-379), SMC2 (ab10412), SMC4 (ab17958), NCAPD3 (ab70349), NCAPG2 (ab70350), Lamin A (ab26300), CTCF (Cell Signaling 2899), RNA polymerase II CTD repeat phospho S2 (ab5095)].

Techniques: Staining, Western Blot, Purification, Microscopy

Journal: Nature cell biology

Article Title: A chromosome folding intermediate at the condensin-to-cohesin transition during telophase

doi: 10.1038/s41556-019-0406-2

Figure Lengend Snippet: a, Western blot analysis of chromatin-associated proteins purified from HeLa S3 cells at different time points after release from prometaphase. b, Quantification of the western blot shown in panel a. Protein levels were normalized to Histone H3 levels from the same samples. c, Summary of cellular and chromosomal events as cells exit mitosis and enter G1. Top: Schematic diagrams indicate the cellular events from prometaphase into late G1. Compartment type is indicated by color: blue = A, orange = B. Red lines represent tubulin and dashed gray lines represent lamina. Bottom: Models of chromosome conformation during early mitosis, telophase, cytokinesis, and interphase. Green bar indicates abundance of condensins I and II on the chromatin at the corresponding cell cycle stages. Yellow bar indicates cohesin abundance on the chromatin at the corresponding cell cycle stages. Unprocessed blots are provided in Source Data as Unprocessed Blots . Four independent experiments were performed with similar results. Source Data are provided in Source data .

Article Snippet: Membranes were then incubated with specified primary antibody diluted 1:1000 in 4% milk/PBS-T overnight at 4°C [Histone H3 (ab1791), Rad21 (ab154769), RFP (cross-reacts with dTomato for NCAPH-dTomato, Rockland 600-401-379), SMC2 (ab10412), SMC4 (ab17958), NCAPD3 (ab70349), NCAPG2 (ab70350), Lamin A (ab26300), CTCF (Cell Signaling 2899), RNA polymerase II CTD repeat phospho S2 (ab5095)].

Techniques: Western Blot, Purification

Journal: Frontiers in immunology

Article Title: Non-canonical Glucocorticoid Receptor Transactivation of gilz by Alcohol Suppresses Cell Inflammatory Response.

doi: 10.3389/fimmu.2017.00661

Figure Lengend Snippet: Figure 3 | Alcohol dehydrogenase (ADH) inhibitor fomepizole enhances GR nuclear translocation. (A) Western blots of nuclear GR and Histone H3. MM6 cells were exposed to alcohol (0 or 50 mM) or dexamethasone (Dex, 1 µM) for 24 h in the presence or absence of the ADH inhibitor fomepizole (4 µM). Cell nuclei from each treatment were isolated and subjected to Western blot analysis. Histone H3 was also assessed for loading normalization. (B) Statistical data from three independent experiments. The intensities of the GR and Histone H3 bands were quantified by densitometry. Ratio of nuclear GR over the corresponding Histone H3 for each treatment was obtained and graphed. Significant difference was judged by Student’s t-test (p < 0.01; n = 3).

Article Snippet: Rabbit polyclonal antibodies against human GR (Santa Crus Technology, Santa Cruz, CA, USA) and Histone H3 (Cell Signaling Technology, Danvers, MA, USA) were used.

Techniques: Translocation Assay, Western Blot, Isolation