Journal: The Journal of Experimental Medicine

Article Title: IFNγ-induced memory in human macrophages is sustained by the durability of cytokine signaling itself

doi: 10.1084/jem.20250976

Figure Lengend Snippet: LPS and IFNγ both generate stimulus-specific de novo enhancers in human macrophages; however, only IFNγ-induced enhancers are durable. (A) Schematic of experimental design: Human macrophages were stimulated with either IFNγ (100 ng/ml), LPS (100 ng/ml), or LPS in the presence of 1 µM ruxolitinib for 8 h. Cells were subsequently washed and cultured for an additional 88 h. H3K4me1 CUT&Tag was performed at each time point. (B) Heatmap of Z-scored reads within H3K4me1 peaks induced by either LPS or IFNγ (L2FC > 2, FDR < 0.01). Clusters were generated by unsupervised k-means clustering. Each column represents a biological replicate from the same human donor. (C) Top enriched motifs in clusters from B. (D) Box/whisker plot quantifying log2 cpm of reads within IFNγ-induced peaks before and after cytokine washout. (E) Box/whisker quantifying log2 cpm of reads within LPS-induced peaks before and after cytokine washout. (F) Box/whisker quantifying log2 cpm of reads within peaks induced by both IFNγ and LPS (L2FC > 2, FDR < 0.01 for each) peaks before and after cytokine washout. (G) Z-scored heatmap of reads within CUT&Tag peaks of only IFNγ-induced peaks before and after cytokine washout. (H) Boxplot of log2 cpm of reads within peaks for each cluster identified in G. (I) Examples of genome browser tracks for each cluster in E. Box/whisker plots indicate interquartile range and 1.5× interquartile range. Statistical tests were determined by paired Wilcoxon test. *P < 0.05; ****P < 0.0001.

Article Snippet: In some conditions, ruxolitinib (1 μM), CHX (10 μg/ml), anti-IFNγ neutralizing antibody (hifng-mab7-02; InvivoGen at indicated concentrations for , or 506532; BioLegend, clone B27, RRID:AB_2801092 for all subsequent experiments at a concentration of 10 μM), or IgG1, κ Isotype control antibody (400166; BioLegend, clone MOPC-21, RRID:AB 11146992 at concentration of 10 μM), was spiked into complete RPMI media.

Techniques: Cell Culture, Generated, Whisker Assay

Journal: The Journal of Experimental Medicine

Article Title: IFNγ-induced memory in human macrophages is sustained by the durability of cytokine signaling itself

doi: 10.1084/jem.20250976

Figure Lengend Snippet: Ruxolitinib blocks LPS and IFNγ-induced Janus kinase signaling. (A) Human macrophages were pre-treated with increasing concentrations of ruxolitinib for 15 min and subsequently stimulated with IFNγ (100 ng/ml) or LPS (100 ng/ml) for 3 h. Whole-cell lysate western blots showing effect of ruxolitinib on STAT1 and STAT2 phosphorylation by each stimulus. Blot is representative of two replicates from separate human donors. (B and C) Quantification of pSTAT2 and (C) pSTAT1 band intensities in A. Source data are available for this figure: .

Article Snippet: In some conditions, ruxolitinib (1 μM), CHX (10 μg/ml), anti-IFNγ neutralizing antibody (hifng-mab7-02; InvivoGen at indicated concentrations for , or 506532; BioLegend, clone B27, RRID:AB_2801092 for all subsequent experiments at a concentration of 10 μM), or IgG1, κ Isotype control antibody (400166; BioLegend, clone MOPC-21, RRID:AB 11146992 at concentration of 10 μM), was spiked into complete RPMI media.

Techniques: Western Blot, Phospho-proteomics

Journal: The Journal of Experimental Medicine

Article Title: IFNγ-induced memory in human macrophages is sustained by the durability of cytokine signaling itself

doi: 10.1084/jem.20250976

Figure Lengend Snippet: IFNγ induces long-lasting transcription factor activity and chromatin accessibility after washout. Macrophages were treated with LPS, IFNγ, and LPS in the presence of ruxolitinib for 8 h, as in . Cells were washed and cultured for an additional 88 h. ATACseq was performed after 8 h of stimulation and 4 days after washout. (A) Heatmap of Z-scored reads within ATAC peaks induced by either LPS or IFNγ (L2FC > 2, FDR < 0.01). Clusters were generated by unsupervised k-means clustering. Each column represents a biological replicate from the same human donor. (B) Top enriched motifs in clusters from A. (C) Boxplot quantifying log2 cpm of reads within IFNγ-induced ATAC peaks before and after cytokine washout. (D) Boxplot quantifying log2 cpm of reads within LPS-induced ATAC peaks before and after cytokine washout. (E) Boxplot quantifying log2 cpm of reads within ATAC peaks induced by both IFNγ and LPS (L2FC > 2, FDR < 0.01 for each) peaks before and after cytokine washout. (F) Barplot quantifying percent of transcription factor-bound motifs within STAT1 and IRF1 (IFNγ) and IRF1 and NF-κB (LPS) within induced ATAC peaks in C and D for unstimulated, IFNγ/LPS-stimulated macrophages, and stimulated macrophages 4 days after washout. Motif binding predicted using TOBIAS ATACseq footprinting analysis. Results are average of two technical replicates from a single subject; error bars display standard deviation. (G) Human macrophages were stimulated with IFNγ (100 ng/ml), LPS (100 ng/ml), or IFNβ (10 ng/ml) for 8 h, washed, and then cultured for an additional 66 h. Cells were collected, and whole cell western blotting for phosphorylated STAT1 was performed at the indicated time points. Blot is representative of three replicates from two separate human donors. All box/whisker plots indicate interquartile range and 1.5× interquartile range. Statistical tests were determined by paired Wilcoxon test. ****P < 0.0001. Source data are available for this figure: .

Article Snippet: In some conditions, ruxolitinib (1 μM), CHX (10 μg/ml), anti-IFNγ neutralizing antibody (hifng-mab7-02; InvivoGen at indicated concentrations for , or 506532; BioLegend, clone B27, RRID:AB_2801092 for all subsequent experiments at a concentration of 10 μM), or IgG1, κ Isotype control antibody (400166; BioLegend, clone MOPC-21, RRID:AB 11146992 at concentration of 10 μM), was spiked into complete RPMI media.

Techniques: Activity Assay, Cell Culture, Generated, Binding Assay, Footprinting, Standard Deviation, Western Blot, Whisker Assay

Journal: The Journal of Experimental Medicine

Article Title: IFNγ-induced memory in human macrophages is sustained by the durability of cytokine signaling itself

doi: 10.1084/jem.20250976

Figure Lengend Snippet: Cell surface–bound IFNγ mediates persistent signaling regardless of cytokine manufacturer and can be degraded by trypsinization. (A) Quantification of pSTAT1 intensity normalized to 3H IFNγ in . (B) Human macrophages were treated with Escherichia coli sourced IFNγ purchased from PeproTech and mammalian-sourced IFNγ purchased from Sigma-Aldrich and ACRO. Cells were treated at 100 ng/ml for 8 h, washed, and cultured for an additional 48 h in regular media prior to collection. Cells were collected for immunoblot at the indicated times. Representative blot of duplicates from one human subject. (C) Quantification of pSTAT1 band intensity normalized to tubulin at each time point in B. (D) Human A549 airway epithelial cells were stimulated with 100 ng/ml IFNγ, washed, and cultured for an additional 72 h. Cells were collected for immunoblot at the indicated timepoints. Representative blot of two replicates. (E) Quantification of pSTAT1 band intensity normalized to tubulin at each time point in D. (F) Human macrophages were treated with 100 ng/ml IFNγ for 8 h, washed, and lifted by scraping after incubation with either PBS, 0.5 mM EDTA in PBS, or trypsin. Cells were replated and cultured for an additional 24 h in regular media. Cells were collected for immunoblot at the indicated times. (G) Quantification of pSTAT1 band intensity normalized to GAPDH for each condition in F. Statistical tests were determined by single-tailed t test. *P < 0.05; **P < 0.01. Source data are available for this figure: .

Article Snippet: In some conditions, ruxolitinib (1 μM), CHX (10 μg/ml), anti-IFNγ neutralizing antibody (hifng-mab7-02; InvivoGen at indicated concentrations for , or 506532; BioLegend, clone B27, RRID:AB_2801092 for all subsequent experiments at a concentration of 10 μM), or IgG1, κ Isotype control antibody (400166; BioLegend, clone MOPC-21, RRID:AB 11146992 at concentration of 10 μM), was spiked into complete RPMI media.

Techniques: Cell Culture, Western Blot, Incubation

Journal: The Journal of Experimental Medicine

Article Title: IFNγ-induced memory in human macrophages is sustained by the durability of cytokine signaling itself

doi: 10.1084/jem.20250976

Figure Lengend Snippet: Cell surface–bound IFNγ mediates persistent JAK/STAT signaling even after cytokine washout. (A) Human macrophages were stimulated with IFNγ (100 ng/ml) for 8 h, washed, and then cultured in regular media or media containing ruxolitinib (1 µM) or increasing concentrations of anti-IFNγ neutralizing antibody for an additional 28 h. Cells were collected, and whole cell western blotting for phosphorylated STAT1 and IRF1 was performed at indicated time points. Representative blot of duplicates from two separate subjects. (B) Human macrophages were stimulated with 100 ng/ml IFNγ for 3 h, washed, and then cultured in either ruxolitinib (1 µM), anti-IFNγ neutralizing antibody (10 µg/ml), or isotype control antibody (10 µg/ml) for 2 h. Samples were collected at the indicated times for immunoblot. Representative blot of duplicates from two separate subjects. (C) Quantification of pSTAT1 band intensities from B. (D) Human macrophages were stimulated with 100 ng/ml IFNγ for 8 h, washed, and cultured for an additional 88 h in regular media. Supernatants from stimulated macrophages were collected after the 8-h stimulation and 88 h after washout. This supernatant was used to stimulate fresh macrophages for 1 h in the presence/absence of ruxolitinib (1 µM) or anti-IFNγ neutralizing antibody (10 µg/ml). As a control, fresh macrophages were stimulated with media supplemented with 1 ng/ml IFNγ for 1 h. Representative blot of duplicates from two separate subjects. (E) Macrophages were left in regular media or pre-treated with 10 µg/ml CHX for 15 min and stimulated with 100 ng/ml IFNγ for 3 h. Treated macrophages were washed and subsequently cultured for 2 h in regular media, media supplemented with 10 µg/ml CHX, or anti-IFNγ neutralizing antibody (10 µg/ml) and collected for immunoblot. Duplicates from one subject are shown. (F) Quantification of pSTAT1 band intensities in E normalized to band intensity of macrophages treated with IFNγ for 3 h. (G) Human macrophages were stimulated with 100 ng/ml IFNγ for 8 h, washed, and cultured in regular media or media supplemented with 1 µM ruxolitinib for 16 h. After 16 h, cells were washed again and cultured in regular media for an additional 24 h. Cells were collected for immunoblot at indicated times. Representative blot of four replicates from two subjects. (H) Quantification of pSTAT1 band intensities in G normalized to band intensity of macrophages treated with IFNγ for 3 h. Statistical tests were determined by a single-tailed t test. *P < 0.05, **P < 0.01, and ***P < 0.001. Source data are available for this figure: .

Article Snippet: In some conditions, ruxolitinib (1 μM), CHX (10 μg/ml), anti-IFNγ neutralizing antibody (hifng-mab7-02; InvivoGen at indicated concentrations for , or 506532; BioLegend, clone B27, RRID:AB_2801092 for all subsequent experiments at a concentration of 10 μM), or IgG1, κ Isotype control antibody (400166; BioLegend, clone MOPC-21, RRID:AB 11146992 at concentration of 10 μM), was spiked into complete RPMI media.

Techniques: Cell Culture, Western Blot, Control

Journal: The Journal of Experimental Medicine

Article Title: IFNγ-induced memory in human macrophages is sustained by the durability of cytokine signaling itself

doi: 10.1084/jem.20250976

Figure Lengend Snippet: Extracellular IFNγ signaling sustains chromatin accessibility and ISG expression even after cytokine washout. (A) Schematic of experiments: Human macrophages were stimulated with 100 ng/ml IFNγ for 8 h, washed, and then cultured for an additional 88 h in regular media or media with 1 µM ruxolitinib for an additional 88 h. Cells were collected for ATACseq and RNAseq at the indicated time points. (B) Heatmap of Z-scored reads within ATAC peaks induced by IFNγ (L2FC > 2, FDR < 0.01) after 8 h of stimulation for 4 days after washout when cultured in regular media or media with 1 µM ruxolitinib. Clusters were generated by unsupervised k-means clustering. Each column represents a biological replicate from the same human donor. (C) Boxplot of log2CPM of reads within each peak for each cluster in B. (D) Heatmap of Log2 fold change in RNAseq reads of genes induced at least fivefold after 8 h of IFNγ stimulation. Log2 fold changes are shown after washout for cells cultured in regular media and media containing 1 µM ruxolitinib. Genes are clustered by persistent level of expression after washout (CPM after wash as percent of CPM at 8-h simulation). (E) Boxplot showing Log2 fold changes of individual genes by cluster in D. Box/whisker plots indicate interquartile range and 1.5× interquartile range. Statistical tests were determined by paired Wilcoxon test. (F) Macrophages were stimulated and washed as above in A; after washout, cells were cultured in media alone, media with 1 µM ruxolitinib, or 10 µg/ml anti-IFNγ neutralizing antibody for 88 h. Cells were collected 88 h after washout, and qPCR was performed for IDO1. Boxplots indicate 2 ΔΔCt normalized to HPRT. Error bars indicate standard deviation. Statistical tests determined by ordinary one way ANOVA. (G) qPCR for IRF1 as in F. ** P < 0.01; ***P < 0.001; ****P < 0.0001.

Article Snippet: In some conditions, ruxolitinib (1 μM), CHX (10 μg/ml), anti-IFNγ neutralizing antibody (hifng-mab7-02; InvivoGen at indicated concentrations for , or 506532; BioLegend, clone B27, RRID:AB_2801092 for all subsequent experiments at a concentration of 10 μM), or IgG1, κ Isotype control antibody (400166; BioLegend, clone MOPC-21, RRID:AB 11146992 at concentration of 10 μM), was spiked into complete RPMI media.

Techniques: Expressing, Cell Culture, RNA sequencing, Generated, Whisker Assay, Standard Deviation

Journal: The Journal of Experimental Medicine

Article Title: IFNγ-induced memory in human macrophages is sustained by the durability of cytokine signaling itself

doi: 10.1084/jem.20250976

Figure Lengend Snippet: Macrophages from a second human donor show reversibility of IFNγ-induced chromatin accessibility and de novo enhancers. Human macrophages from a second human subject were stimulated with IFNγ (100 ng/ml) for 8 h. Cells were subsequently washed and cultured for an additional 88 h in standard media or media supplemented with 1 µM ruxolitinib. ATAC and H3K4me1 CUT&Tag was performed after 8 h of stimulation and 88 h after cytokine washout. (A) Boxplot quantifying log2 fold changes of reads within IFNγ-induced ATAC peaks after 8 h of IFNγ stimulation and after washout for each condition (L2FC > 2, FDR < 0.01). (B) Heatmap of Z-scored reads within ATAC peaks induced IFNγ after 8 h of stimulation and 4 days after washout for each condition. Clusters were generated by unsupervised k-means clustering. Each column represents a biological replicate from the same human donor. (C) Boxplot quantifying log2 fold changes of reads within IFNγ-induced H3K4me1 CUT&Tag peaks after 8 h of IFNγ stimulation and after washout for each condition (L2FC > 2, FDR < 0.01). (D) Barplot showing fraction of IFNγ-induced H3K4me1 peaks at 8 h that persist 4 days after washout in each condition. Persistence was defined as L2FC ≥ 0, FDR < 0.01. (E) Heatmap of Z-scored reads within H3K4me1 peaks induced IFNγ after 8 h of stimulation and 4 days after washout for each condition. Clusters were generated by unsupervised k-means clustering. Each column represents a biological replicate from the same human donor. (F) Boxplot of log2CPM of reads within each peak for each cluster in E. Box/whisker plots indicate interquartile range and 1.5× interquartile range. Statistical tests were determined by paired Wilcoxon test. ****P < 0.0001.

Article Snippet: In some conditions, ruxolitinib (1 μM), CHX (10 μg/ml), anti-IFNγ neutralizing antibody (hifng-mab7-02; InvivoGen at indicated concentrations for , or 506532; BioLegend, clone B27, RRID:AB_2801092 for all subsequent experiments at a concentration of 10 μM), or IgG1, κ Isotype control antibody (400166; BioLegend, clone MOPC-21, RRID:AB 11146992 at concentration of 10 μM), was spiked into complete RPMI media.

Techniques: Cell Culture, Generated, Whisker Assay

Journal: The Journal of Experimental Medicine

Article Title: IFNγ-induced memory in human macrophages is sustained by the durability of cytokine signaling itself

doi: 10.1084/jem.20250976

Figure Lengend Snippet: Durability of IFNγ-induced de novo enhancers is dependent on continued JAK/STAT signaling by IFNγ. (A) Schematic of experimental design: Human macrophages were stimulated with IFNγ (100 ng/ml) for 8 h. Cells were subsequently washed and cultured for an additional 88 h in standard media or media supplemented with 1 µM ruxolitinib or 8 µg/ml anti-IFNγ neutralizing antibody. H3K4me1 CUT&Tag was performed at each time point. (B) Boxplot quantifying log2 fold changes of reads within IFNγ-induced H3K4me1 CUT&Tag peaks after 8 h of IFNγ stimulation and after washout for each condition. (C) Barplot showing fraction of IFNγ-induced H3K4me1 peaks at 8 h that persist 4 days after washout in each condition. Persistence was defined as L2FC ≥ 0, FDR < 0.01. (D) . Heatmap of Z-scored reads within H3K4me1 peaks induced IFNγ (L2FC > 2, FDR < 0.01) after 8 h of stimulation and 4 days after washout for each condition. Clusters were generated by unsupervised k-means clustering. Each column represents a biological replicate from the same human donor. (E) Representative genome browser tracks of peaks from each cluster in D. All box/whisker plots indicate interquartile range and 1.5× interquartile range. Statistical tests were determined by paired Wilcoxon test. ***P < 0.001; ****P < 0.0001.

Article Snippet: In some conditions, ruxolitinib (1 μM), CHX (10 μg/ml), anti-IFNγ neutralizing antibody (hifng-mab7-02; InvivoGen at indicated concentrations for , or 506532; BioLegend, clone B27, RRID:AB_2801092 for all subsequent experiments at a concentration of 10 μM), or IgG1, κ Isotype control antibody (400166; BioLegend, clone MOPC-21, RRID:AB 11146992 at concentration of 10 μM), was spiked into complete RPMI media.

Techniques: Cell Culture, Generated, Whisker Assay

Journal: The Journal of Experimental Medicine

Article Title: IFNγ-induced memory in human macrophages is sustained by the durability of cytokine signaling itself

doi: 10.1084/jem.20250976

Figure Lengend Snippet: IFNγ exposed macrophages exhibit potentiated inflammatory gene expression upon LPS restimulation. (A) Human macrophages were stimulated with IFNγ (100 ng/ml) for 8 h. Cells were subsequently washed and cultured for an additional 88 h in standard media, or media supplemented with 1 µM ruxolitinib, at which time they were stimulated with 10 ng/ml LPS and cultured for an additional 12 h. RNAseq was performed at each time point. (B) Heatmap of log2 fold change in reads of LPS genes potentiated by IFNγ before treatment. Log2 fold changes are normalized to PBS-treated controls 88 h after washout prior to LPS stimulation (Naïve 0H). Potentiated genes defined as those reaching fivefold increase in reads after LPS stimulation and at least a twofold greater expression in IFNγ pre-treated cells compared with PBS. Genes are clustered by expression level 88 h after IFNγ washout. The top cluster of genes showed L2FC < 0.5 in IFNγ-treated cells compared with PBS treated; the bottom cluster showed L2FC >0.5 compared with PBS trained. (C) Heatmap quantifying extent of IFNγ-induced potentiation. The difference in L2FC for a given genes between PBS and IFNγ treated is quantified for each gene in F. (D) Example of CPM for a potentiated gene that showed basal expression equivalent to that of PBS-treated cells: CSF3 . (E) Example of CPM for a potentiated gene that showed basal expression higher than that of PBS-treated cells: IDO1 . (F) IFNγ-induced H3K4me1 CUT&Tag peaks (as defined in ) were linked to protein-coding genes within ±20 kb of a gene’s TSS. Promoter-proximal (±1 kb of TSS) peaks were excluded from analysis. Analysis was limited to LPS-induced genes. The mean “IFNγ potentiation” of each gene was calculated (defined as the average of the delta log2 fold change for each gene in C between IFNγ-trained and untrained conditions across all time points). The mean IFNγ potentiation value was plotted against the fold change of the enhancer induced 4 days after IFNγ washout. (G) Mean IFNγ potentiation and enhancer fold change in the presence of ruxolitinib as calculated in F.

Article Snippet: In some conditions, ruxolitinib (1 μM), CHX (10 μg/ml), anti-IFNγ neutralizing antibody (hifng-mab7-02; InvivoGen at indicated concentrations for , or 506532; BioLegend, clone B27, RRID:AB_2801092 for all subsequent experiments at a concentration of 10 μM), or IgG1, κ Isotype control antibody (400166; BioLegend, clone MOPC-21, RRID:AB 11146992 at concentration of 10 μM), was spiked into complete RPMI media.

Techniques: Gene Expression, Cell Culture, RNA sequencing, Expressing

Journal: The Journal of Experimental Medicine

Article Title: IFNγ-induced memory in human macrophages is sustained by the durability of cytokine signaling itself

doi: 10.1084/jem.20250976

Figure Lengend Snippet: Sustained JAK/STAT signaling is required for long-term IFNγ-induced potentiated and tolerized gene expression responses. (A) Human macrophages were stimulated with IFNγ (100 ng/ml) for 8 h. Cells were subsequently washed and cultured for an additional 88 h in standard media, or media supplemented with 1 µM ruxolitinib, at which time they were stimulated with 10 ng/ml LPS and cultured for an additional 6 h. RNAseq was performed at each time point. (B) Heatmap of log2 fold change in reads of LPS-induced genes potentiated by IFNγ treatment. Log2 fold changes are normalized to PBS-treated controls 88 h after washout prior to LPS stimulation (Naïve 0H). Potentiated genes are defined as LPS-induced genes reaching at least fourfold increase for macrophages cultured in ruxolitinib and at least a twofold greater expression in IFNγ pre-treated cells compared with PBS in two contiguous time points. Genes are clustered by expression level 88 h after IFNγ washout: the top cluster of genes showed L2FC < 0.5 in IFNγ-treated cells compared with PBS treated; the bottom cluster showed L2FC >0.5 compared with PBS trained. (C) Heatmap quantifying extent of IFNγ-induced potentiation. The difference in L2FC for a given gene between PBS and IFNγ treated is quantified for each gene in F. (D) Boxplot quantifying difference in L2FC for IFNγ pre-treated and PBS-pre-treated cells at each time point. Box/whisker plots indicate interquartile range and 1.5× interquartile range. Statistical tests were determined by paired Wilcoxon test. ****P < 0.0001. (E) Example of fold change for potentiated gene: CCR7 . (F) Heatmap of log2 fold change in reads of LPS-induced genes tolerized by IFNγ. Tolerance is defined as twofold reduction in transcription in two contiguous time points with IFNγ before treatment and at least fourfold induction by LPS in the presence of ruxolitinib. (G) Heatmap quantifying extent of IFNγ-induced tolerance as in C. (H) Example of fold change for potentiated gene: PTX3 .

Article Snippet: In some conditions, ruxolitinib (1 μM), CHX (10 μg/ml), anti-IFNγ neutralizing antibody (hifng-mab7-02; InvivoGen at indicated concentrations for , or 506532; BioLegend, clone B27, RRID:AB_2801092 for all subsequent experiments at a concentration of 10 μM), or IgG1, κ Isotype control antibody (400166; BioLegend, clone MOPC-21, RRID:AB 11146992 at concentration of 10 μM), was spiked into complete RPMI media.

Techniques: Gene Expression, Cell Culture, RNA sequencing, Expressing, Whisker Assay

Journal: Neoplasia (New York, N.Y.)

Article Title: Microbe-associated molecular patterns differentially mediate carcinogenic alterations of the breast tissue in the context of obesity

doi: 10.1016/j.neo.2026.101284

Figure Lengend Snippet: LPS and flagellin induce DNA double-strand breaks in breast acini in a TLR-dependent manner. A. Representative comet images from neutral comet assays performed on 3D cultures of breast acini treated with vehicle (control), 1 μg/ml LPS, 1 μg/ml LTA, and 10 ng/ml flagellin for 72 h. Bleomycin (BLM; 20 mU/ml; 2h) was used as positive control. B. Comet assay quantification. n=300 acini from 3 biological replicates. C. Representative images for immunofluorescence staining of 53BP1 (red) in acini treated with MAMPs ± TLR inhibitors. Nuclei are counterstained with DAPI (blue). D-F. Quantification of 53BP1 foci numbers in acini treated with LPS ± TLR4 inhibitor (D), with LTA ± TLR2 inhibitor (E) or with flagellin ± TLR5 inhibitor (F). ns is not significant. *P<0.05, **P<0.01, ***P<0.001.

Article Snippet: For TLR blockade, cells were pre-treated for 1 h with 5 μg/ml of TLR4 neutralizing antibody (InvivoGen; mabg-htlr4), TLR2 neutralizing antibody (InvivoGen; maba2-htlr2-2), or TLR5 neutralizing antibody (InvivoGen; maba2-htlr5).

Techniques: Control, Positive Control, Single Cell Gel Electrophoresis, Immunofluorescence, Staining

Journal: Neoplasia (New York, N.Y.)

Article Title: Microbe-associated molecular patterns differentially mediate carcinogenic alterations of the breast tissue in the context of obesity

doi: 10.1016/j.neo.2026.101284

Figure Lengend Snippet: Blockade of TLR4 and TLR5 mitigates DNA damage in the mammary glands. A. Schematic of the experiment. B-D-F. Representative images for TLR2 (B), TLR4 (D), and TLR5 (F) IHC signals in mammary glands of mice treated with saline, mismatch morpholino (MM) and the corresponding TLR morpholino (TLR M). Hematoxylin was used as a counterstain. Image analysis and quantification of TLR2/4/5-DAB positive areas is shown in the graphs (C,E,G). H. Representative comet images of the neutral comet assay performed on mammary cells treated with vehicle, MM, and TLR morpholinos. I. Quantification of comet assay results for the aforementioned mice groups. ns is not significant. *P<0.05, **P<0.01.

Article Snippet: For TLR blockade, cells were pre-treated for 1 h with 5 μg/ml of TLR4 neutralizing antibody (InvivoGen; mabg-htlr4), TLR2 neutralizing antibody (InvivoGen; maba2-htlr2-2), or TLR5 neutralizing antibody (InvivoGen; maba2-htlr5).

Techniques: Saline, Neutral Comet Assay, Single Cell Gel Electrophoresis

Journal: bioRxiv

Article Title: In differentiated HL-60 neutrophil-like cells, MRP1- (ABCC1-) mediated glutathione efflux stimulated by BzATP and P2X 7 receptor signalling regulates exosome release through nSMase activity

doi: 10.64898/2026.02.26.708162

Figure Lengend Snippet: Immuno-transmission electron microscopy was used to reveal isolated CD63-positive exosomes, using anti-CD9 and 5 nm immunogold-conjugated secondary antibody (A) and their size range and peak modal size measured by NTA (B). Optiprep density gradient centrifugation showed the exosomes to have a density of 1.10-1.12 g/ml (C). Western analysis showed 5-day differentiated HL-60 cells (NLCs) to have increased expression of CD63 (D); image J 1.52p was used to calculate fold-changes of deglycosylated CD63 expression, band intensities normalised to β-actin and compared relative to control. The exosomal expression of EV markers was also compared with their parental cells (E). NLCs exposed to increasing BzATP concentrations up to 300 μM, were assayed for iGSH and concentration of released exosomes (F) and nSMAse activity (G) and in the presence of nSMAse inhibitor, GW4869 (F, inset). For (D) and (E) bars represent the mean ± SEM of n=3 independent biological replicates. Relationship between BzATP and iGSH, exosomes and nSMAse activity shown by linear regression fit, statistical significance of correlation indicated by R 2 or r value and associated p-value.

Article Snippet: Neutral sphingomyelinase (nSMAse) activity in NLCs was measured using the Neutral Sphingomyelinase (nSMase) activity kit from Echelon Biosciences Inc. (cat no. K-1800); the coupled enzymatic reactions starting with nSMase hydrolysing sphingomyelin to phosphorylcholine and ceramide, eventually producing a blue product measured at 595 nm.

Techniques: Transmission Assay, Electron Microscopy, Isolation, Gradient Centrifugation, Western Blot, Expressing, Control, Concentration Assay, Activity Assay

Journal: bioRxiv

Article Title: In differentiated HL-60 neutrophil-like cells, MRP1- (ABCC1-) mediated glutathione efflux stimulated by BzATP and P2X 7 receptor signalling regulates exosome release through nSMase activity

doi: 10.64898/2026.02.26.708162

Figure Lengend Snippet: Model for MRP1- (ABCC1-) mediated glutathione efflux in neutrophil-like cells in which P2X 7 Rs stimulated by BzATP regulates nSMAse activity and exosome release.

Article Snippet: Neutral sphingomyelinase (nSMAse) activity in NLCs was measured using the Neutral Sphingomyelinase (nSMase) activity kit from Echelon Biosciences Inc. (cat no. K-1800); the coupled enzymatic reactions starting with nSMase hydrolysing sphingomyelin to phosphorylcholine and ceramide, eventually producing a blue product measured at 595 nm.

Techniques: Activity Assay