antibodies against irf1  (R&D Systems)

Journal: bioRxiv

Article Title: Pioneer factor IRF1 unlocks latent enhancers to rewire chromatin and immunometabolism in inflammatory macrophages

doi: 10.64898/2026.02.27.708404

Figure Lengend Snippet: (A) Experimental design for multiomic assessment of WT and IRF1 KO bone marrow–derived macrophages (BMDMs) response to IFNγ stimulation. Time-resolved profiling by ATAC-seq, ChIP-seq, Hi-ChIP, SLAM-seq and metabolomics via GC/LC-MS is performed. (B) Venn diagram summarizing ATAC-seq–identified accessible chromatin regions, filtered for high-confidence peaks and IFNγ-responsiveness (n=38,564); this set is used for downstream clustering and differential analyses. (C) Heatmap of normalized ATAC-seq signal (rows = individual accessible site; columns = time points), grouped into eight clusters by k-means clustering. Clusters C1-C3 show IRF1-depedent increase in accessibility in response to IFNγ; highlighted in red. PU.1 ChIP-seq binding signal is also shown, with Cluster C1 lacking detectable PU.1 occupancy. (D) Ribbon plots of relative ATAC-seq peak height (each peak scaled to its maximum) over matched time points; lines indicate mean accessibility and shaded ribbons show ± SD for WT (black) and IRF1 KO (red). (E) Boxplots of normalized ATAC-seq counts in WT BMDMs at heterochromatin regions, and at clusters C1–C8 and unresponsive ATAC-seq sites; median with interquartile range are shown.

Article Snippet: Membranes were blocked in 5% skim milk in PBS Tween-20 0.1% and incubated with primary antibodies against IRF1 (R&D Systems, Cat. #AF4715, 1:200) or GAPDH (Cell Signaling Technologies, Cat. #2118) as loading control, followed by appropriate HRP-conjugated secondary antibodies.

Techniques: Derivative Assay, ChIP-sequencing, HiChIP, Liquid Chromatography with Mass Spectroscopy, Binding Assay

Journal: bioRxiv

Article Title: Pioneer factor IRF1 unlocks latent enhancers to rewire chromatin and immunometabolism in inflammatory macrophages

doi: 10.64898/2026.02.27.708404

Figure Lengend Snippet: (A) Network diagrams of transcription factor motif frequency (node size) and co-occurrence (edge thickness) within ±100 bp of ATAC-seq peak centre for clusters C1-C3 and unresponsive sites. “IRF1–IRF1” denotes sites with ≥2 IRF motifs, and node/edge scales reflect motif frequency and co-occurrence. (B) Volcano plots of TOBIAS differential binding scores for 879 mammalian TFs in WT BMDMs comparing 0.5, 3 and 48 h post-IFNγ versus non-treated (0 h); significant TFs are highlighted [Bonferroni-corrected FDR < 0.05; log2 FC > |0.5|]. (C) Heatmap of centered TOBIAS TF footprinting intensity in WT BMDMs, grouped into four clusters by k-means clustering. (D) Density plots (top) and motif-centered TOBIAS footprint heatmaps (bottom) in WT and IRF1 KO BMDMs showing aggregated IRF1-centered footprinting signal at Cluster 1 sites (rows = individual sites; columns = base position around motif). (E) Representative Western blots in WT BMDMs showing IRF1 protein and GAPDH control across time points (0–48 h) post-IFNγ stimulation.

Article Snippet: Membranes were blocked in 5% skim milk in PBS Tween-20 0.1% and incubated with primary antibodies against IRF1 (R&D Systems, Cat. #AF4715, 1:200) or GAPDH (Cell Signaling Technologies, Cat. #2118) as loading control, followed by appropriate HRP-conjugated secondary antibodies.

Techniques: Binding Assay, Footprinting, Western Blot, Control

Journal: bioRxiv

Article Title: Pioneer factor IRF1 unlocks latent enhancers to rewire chromatin and immunometabolism in inflammatory macrophages

doi: 10.64898/2026.02.27.708404

Figure Lengend Snippet: (A) Heatmaps of IRF1 occupancy (ChIP-seq), ATAC-seq accessibility and H3K4me1, H3K4me3 and H3K27ac signals across in response to IFNγ for sites in Clusters 1–3 in WT and IRF1 KO BMDMs. (B) Hi-ChIP arc plots showing loop contacts (arc width represents number of contacts) between IRF1-bound enhancers and promoters at C8 and unresponsive sites in WT and IRF1 KO BMDMs. [FitHiChIP thresholds FDR < 0.1; loop FC > 6, CPM > 6] (C) Graph of the temporal changes for ChIP-seq and ATAC-seq signals at Cluster 1. Half-time (t½) to reach 50% of each signal’s maximum was calculated by normalizing each trajectory to its maximum and extracting the pseudo-time at half-max. (D) Heatmap of ChIP-seq for IRF1, BRG1, ARID1A, BRD9 and PHF10 across Clusters 1–3 at 0, 1, and 4 h post TLR4 activation. ( E ) BRG1 ChIP–qPCR enrichment (fold over input) at four enhancers ( Wdr7 (C1), Shtn1 (C2), Clic5 (C2) , Nos2 (C3)) in WT and IRF1 KO BMDMs, untreated and 4 h post-IFNγ. (F) Boxplots of normalized ATAC-seq counts in Clusters 1–3 in WT BMDMs TLR4 activated with LipidA, with or without and BRG1 inhibition (BRM014).

Article Snippet: Membranes were blocked in 5% skim milk in PBS Tween-20 0.1% and incubated with primary antibodies against IRF1 (R&D Systems, Cat. #AF4715, 1:200) or GAPDH (Cell Signaling Technologies, Cat. #2118) as loading control, followed by appropriate HRP-conjugated secondary antibodies.

Techniques: ChIP-sequencing, HiChIP, Activation Assay, ChIP-qPCR, Inhibition

Journal: bioRxiv

Article Title: Pioneer factor IRF1 unlocks latent enhancers to rewire chromatin and immunometabolism in inflammatory macrophages

doi: 10.64898/2026.02.27.708404

Figure Lengend Snippet: (A) Heatmaps of IRF1 ChIP–seq and normalized ATAC–seq at sites grouped by IRF1 signal strength (very strong to weak) in response to IFNγ. (B) Line plots of average IRF1 ChIP–seq signal in WT BMDMs for each binding-strength category. (C) Heatmap of relative enrichment of IRF1 binding classes across ATAC clusters (enrichment is relative to the maximum site overlap). (D) Stacked bar plots showing proportions of sites with 0, 1, 2 or ≥3 IRF1 motifs per ATAC cluster. (E) Aggregate plots of IRF1 motif frequency across ±100 bp around IRF1 peaks for each ATAC cluster. (F) Heatmap of IRF1 ChIP–seq signal at 3 h post–IFNγ for sites stratified by IRF1 motif count, as determined in D). (G) Scatter plot of fraction of sites forming IRF1 Hi-ChIP loops versus motif count, with a fitted trend line shown. [FitHiChIP thresholds FDR < 0.1; loop FC > 6, CPM > 6] (H) Genome browser tracks at the Jdp2 locus showing IRF1, PU.1 and H3K27ac ChIP–seq, Hi-ChIP interactions and ATAC–seq in WT and IRF1 KO BMDMs. The cluster to with each ATAC-seq peak belong is indicated [C1 = cluster 1; UR = Unresponsive]. Insets display the array of IRF1 motifs at the C1 site and a SLAM-seq Jdp2 expression plot across the IFNγ time course.

Article Snippet: Membranes were blocked in 5% skim milk in PBS Tween-20 0.1% and incubated with primary antibodies against IRF1 (R&D Systems, Cat. #AF4715, 1:200) or GAPDH (Cell Signaling Technologies, Cat. #2118) as loading control, followed by appropriate HRP-conjugated secondary antibodies.

Techniques: ChIP-sequencing, Binding Assay, HiChIP, Expressing

Journal: bioRxiv

Article Title: Pioneer factor IRF1 unlocks latent enhancers to rewire chromatin and immunometabolism in inflammatory macrophages

doi: 10.64898/2026.02.27.708404

Figure Lengend Snippet: (A) Line plots of nascent RNA-seq log2 fold-change (FC) for genes within ±10 kb of ATAC cluster regions in WT and IRF1 KO BMDMs in response to IFNγ stimulation. (B) Heatmap of GO biological process enrichment for genes within ±50 kb of ATAC peaks. Categories with clusterProfiler FDR < 0.05 for at least one cluster are shown. (C) Line plots of nascent RNA counts per million (CPM; mean ± SD) for selected genes across IFNγ time points; WT vs IRF1 KO comparison by two-way ANOVA and pairwise post-hoc testing at each time point. (D) Genome browser tracks at the Kmt2c locus showing IRF1 and PU.1 ChIP-seq, Hi-ChIP arcs and ATAC-seq signal for WT and IRF1 KO BMDMs. [UR = Unresponsive] (E) Line plots of RNA-seq CPM (mean ± SD) for selected genes at 0, 1 and 4 h post-LipidA treatment in WT BMDMs, with BRM014 treatment at the 4 h time point. [Student T-test; n = 3] (F) Bar plot of log2 odds ratio of downregulated genes (FC < 0.5 and FDR < 0.05) after BRM014 treatment (4 h Lipid A) across clusters. * < 0.05, ** < 0.01, *** < 0.001

Article Snippet: Membranes were blocked in 5% skim milk in PBS Tween-20 0.1% and incubated with primary antibodies against IRF1 (R&D Systems, Cat. #AF4715, 1:200) or GAPDH (Cell Signaling Technologies, Cat. #2118) as loading control, followed by appropriate HRP-conjugated secondary antibodies.

Techniques: RNA Sequencing, Comparison, ChIP-sequencing, HiChIP

Journal: bioRxiv

Article Title: Pioneer factor IRF1 unlocks latent enhancers to rewire chromatin and immunometabolism in inflammatory macrophages

doi: 10.64898/2026.02.27.708404

Figure Lengend Snippet: (A) Bar plot of the proportion of genes in selected metabolic pathways that harbor IRF1 ChIP-seq peaks; red intensity denotes average number of peaks per gene in each pathway. (B) Genome browser tracks of the Hk1 locus showing normalized IRF1 and PU.1 ChIP-seq, Hi-ChIP interactions and ATAC-seq; an inset shows the annotated intragenic enhancer and promoter contact. [UR = Unresponsive] (C) Line plots of nascent RNA CPM (mean ± SD) for selected genes in glycolysis, PPP and TCA pathways WT and IRF1 KO BMDMs; two-way ANOVA and post-hoc testing; * < 0.05, ** < 0.01, *** < 0.001. (D) Oxygen consumption rates (OCR; fmol mm⁻² s⁻¹) for untreated and IFNγ–stimulated WT and IRF1 KO BMDMs [n = 4/group]; adjacent heatmap shows Student t-test p-values for each time point measured. ( E ) Ribbon plots of relative glycolysis metabolite intensity (mean ± SD) detected by GC-MS in response to IFNγ in WT and IRF1 KO BMDMs [n = 3/group]. (F) Diagram of glycolysis, pentose phosphate pathway (PPP) and Krebs cycle highlighting genes and pathway components significantly dysregulated in IRF1 KO BMDMs for at least 1 timepoint.

Article Snippet: Membranes were blocked in 5% skim milk in PBS Tween-20 0.1% and incubated with primary antibodies against IRF1 (R&D Systems, Cat. #AF4715, 1:200) or GAPDH (Cell Signaling Technologies, Cat. #2118) as loading control, followed by appropriate HRP-conjugated secondary antibodies.

Techniques: ChIP-sequencing, HiChIP, Gas Chromatography-Mass Spectrometry

Journal: bioRxiv

Article Title: Pioneer factor IRF1 unlocks latent enhancers to rewire chromatin and immunometabolism in inflammatory macrophages

doi: 10.64898/2026.02.27.708404

Figure Lengend Snippet: (A) Volcano plots from differential metabolite abundance analysis for GC-MS data (n=3/group), comparing 48 h versus 0 h in WT cells (left) and WT versus IRF1 KO at 48 h (right). (B) Top: bar plots of normalized GC-MS intensity for sedoheptulose 7-P at 3 h post IFNγ, xylulose at 12 h, and erythrose 4-P at 48 h. Bottom: ribbon plots of normalized MS signal over time with mean ± SD. (C) Top: normalized LC-MS GSH intensity at 24 h post-IFNγ stimulation. Bottom: ribbon plots of GSH/GSSG ratios over time (mean ± SD) calculated from normalized LC-MS intensities [n=3/group]. (D) Genome browser tracks at the Acod1 locus showing normalized IRF1 and PU.1 ChIP-seq, Hi-ChIP interactions and ATAC-seq [UR = Unresponsive]. Adjacent panels show Acod1 nascent RNA expression and itaconic acid levels. (E) Ribbon plots of normalized GC-MS signal for TCA metabolites in response to IFNγ in WT and IRF1 KO BMDMs. (F) Diagram of glycolysis, PPP and TCA cycle metabolic pathways with dysregulated intermediates denoted in red.

Article Snippet: Membranes were blocked in 5% skim milk in PBS Tween-20 0.1% and incubated with primary antibodies against IRF1 (R&D Systems, Cat. #AF4715, 1:200) or GAPDH (Cell Signaling Technologies, Cat. #2118) as loading control, followed by appropriate HRP-conjugated secondary antibodies.

Techniques: Gas Chromatography-Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, ChIP-sequencing, HiChIP, RNA Expression

Journal: bioRxiv

Article Title: Pioneer factor IRF1 unlocks latent enhancers to rewire chromatin and immunometabolism in inflammatory macrophages

doi: 10.64898/2026.02.27.708404

Figure Lengend Snippet: (A) Schematic of experimental timeline for the long-term wash-and-rest assay. Cells are plated for seven days, pulsed with 24 h IFNγ (400 U/mL) at specified times (24 h, 48 h, 6 d) with defined washout intervals and a final 1 h re-stimulation. On day 7, cells are harvested for ChIP-seq (IRF1, H3K4me1, H3K27ac and H3K9me2). (B) Heatmaps of normalized ChIP-seq signal for IRF1, H3K4me1, and H3K27ac at Clusters 1–3. (C) Aggregate coverage plots of H3K4me1 ±1 kb from ATAC peak centers for UT, 24 h IFNγ, 6 d washout and 6 d + 1 h restimulation; insets show putative nucleosomal configurations. (D) Bar plots of fold-change in H3K27ac (mean ± SEM) comparing naïve and IFNγ-trained cells after 1 h restimulation; statistical comparison using Wilcoxon test. (E) Volcano plot of H3K4me1 differential enrichment for Cluster 1–3 (control versus IFNγ washout); points = enhancers, color key: red = increased, blue = decreased, yellow = pioneered genes; labeled enhancers meet log₂FC > 1 and CPM > 5. (F) Hif1a locus showing normalized IRF1, H3K27ac, and H3K4me1 ChIP-seq, and ATAC-seq in WT and IRF1 KO BMDMs [UR = Unresponsive]. Normalized SLAM-seq nascent RNA expression for Hif1a is shown; * p < 0.05.

Article Snippet: Membranes were blocked in 5% skim milk in PBS Tween-20 0.1% and incubated with primary antibodies against IRF1 (R&D Systems, Cat. #AF4715, 1:200) or GAPDH (Cell Signaling Technologies, Cat. #2118) as loading control, followed by appropriate HRP-conjugated secondary antibodies.

Techniques: ChIP-sequencing, Comparison, Control, Labeling, RNA Expression

Journal: bioRxiv

Article Title: Pioneer factor IRF1 unlocks latent enhancers to rewire chromatin and immunometabolism in inflammatory macrophages

doi: 10.64898/2026.02.27.708404

Figure Lengend Snippet: (A) Experimental design for multiomic assessment of WT and IRF1 KO bone marrow–derived macrophages (BMDMs) response to IFNγ stimulation. Time-resolved profiling by ATAC-seq, ChIP-seq, Hi-ChIP, SLAM-seq and metabolomics via GC/LC-MS is performed. (B) Venn diagram summarizing ATAC-seq–identified accessible chromatin regions, filtered for high-confidence peaks and IFNγ-responsiveness (n=38,564); this set is used for downstream clustering and differential analyses. (C) Heatmap of normalized ATAC-seq signal (rows = individual accessible site; columns = time points), grouped into eight clusters by k-means clustering. Clusters C1-C3 show IRF1-depedent increase in accessibility in response to IFNγ; highlighted in red. PU.1 ChIP-seq binding signal is also shown, with Cluster C1 lacking detectable PU.1 occupancy. (D) Ribbon plots of relative ATAC-seq peak height (each peak scaled to its maximum) over matched time points; lines indicate mean accessibility and shaded ribbons show ± SD for WT (black) and IRF1 KO (red). (E) Boxplots of normalized ATAC-seq counts in WT BMDMs at heterochromatin regions, and at clusters C1–C8 and unresponsive ATAC-seq sites; median with interquartile range are shown.

Article Snippet: 20 million BMDMs from WT and Irf1−/− mice were plated in 15 cm tissue culture-treated dishes as described above and stimulated with IFNγ (400 U/mL, R&D Systems) for 0 – 48 h. Crosslinking and chromatin immunoprecipitation were performed following the Dovetail Genomics Hi-ChIP protocol using the IRF1 antibody (R&D Systems, AF4715).

Techniques: Derivative Assay, ChIP-sequencing, HiChIP, Liquid Chromatography with Mass Spectroscopy, Binding Assay

Journal: bioRxiv

Article Title: Pioneer factor IRF1 unlocks latent enhancers to rewire chromatin and immunometabolism in inflammatory macrophages

doi: 10.64898/2026.02.27.708404

Figure Lengend Snippet: (A) Network diagrams of transcription factor motif frequency (node size) and co-occurrence (edge thickness) within ±100 bp of ATAC-seq peak centre for clusters C1-C3 and unresponsive sites. “IRF1–IRF1” denotes sites with ≥2 IRF motifs, and node/edge scales reflect motif frequency and co-occurrence. (B) Volcano plots of TOBIAS differential binding scores for 879 mammalian TFs in WT BMDMs comparing 0.5, 3 and 48 h post-IFNγ versus non-treated (0 h); significant TFs are highlighted [Bonferroni-corrected FDR < 0.05; log2 FC > |0.5|]. (C) Heatmap of centered TOBIAS TF footprinting intensity in WT BMDMs, grouped into four clusters by k-means clustering. (D) Density plots (top) and motif-centered TOBIAS footprint heatmaps (bottom) in WT and IRF1 KO BMDMs showing aggregated IRF1-centered footprinting signal at Cluster 1 sites (rows = individual sites; columns = base position around motif). (E) Representative Western blots in WT BMDMs showing IRF1 protein and GAPDH control across time points (0–48 h) post-IFNγ stimulation.

Article Snippet: 20 million BMDMs from WT and Irf1−/− mice were plated in 15 cm tissue culture-treated dishes as described above and stimulated with IFNγ (400 U/mL, R&D Systems) for 0 – 48 h. Crosslinking and chromatin immunoprecipitation were performed following the Dovetail Genomics Hi-ChIP protocol using the IRF1 antibody (R&D Systems, AF4715).

Techniques: Binding Assay, Footprinting, Western Blot, Control

Journal: bioRxiv

Article Title: Pioneer factor IRF1 unlocks latent enhancers to rewire chromatin and immunometabolism in inflammatory macrophages

doi: 10.64898/2026.02.27.708404

Figure Lengend Snippet: (A) Heatmaps of IRF1 occupancy (ChIP-seq), ATAC-seq accessibility and H3K4me1, H3K4me3 and H3K27ac signals across in response to IFNγ for sites in Clusters 1–3 in WT and IRF1 KO BMDMs. (B) Hi-ChIP arc plots showing loop contacts (arc width represents number of contacts) between IRF1-bound enhancers and promoters at C8 and unresponsive sites in WT and IRF1 KO BMDMs. [FitHiChIP thresholds FDR < 0.1; loop FC > 6, CPM > 6] (C) Graph of the temporal changes for ChIP-seq and ATAC-seq signals at Cluster 1. Half-time (t½) to reach 50% of each signal’s maximum was calculated by normalizing each trajectory to its maximum and extracting the pseudo-time at half-max. (D) Heatmap of ChIP-seq for IRF1, BRG1, ARID1A, BRD9 and PHF10 across Clusters 1–3 at 0, 1, and 4 h post TLR4 activation. ( E ) BRG1 ChIP–qPCR enrichment (fold over input) at four enhancers ( Wdr7 (C1), Shtn1 (C2), Clic5 (C2) , Nos2 (C3)) in WT and IRF1 KO BMDMs, untreated and 4 h post-IFNγ. (F) Boxplots of normalized ATAC-seq counts in Clusters 1–3 in WT BMDMs TLR4 activated with LipidA, with or without and BRG1 inhibition (BRM014).

Article Snippet: 20 million BMDMs from WT and Irf1−/− mice were plated in 15 cm tissue culture-treated dishes as described above and stimulated with IFNγ (400 U/mL, R&D Systems) for 0 – 48 h. Crosslinking and chromatin immunoprecipitation were performed following the Dovetail Genomics Hi-ChIP protocol using the IRF1 antibody (R&D Systems, AF4715).

Techniques: ChIP-sequencing, HiChIP, Activation Assay, ChIP-qPCR, Inhibition

Journal: bioRxiv

Article Title: Pioneer factor IRF1 unlocks latent enhancers to rewire chromatin and immunometabolism in inflammatory macrophages

doi: 10.64898/2026.02.27.708404

Figure Lengend Snippet: (A) Heatmaps of IRF1 ChIP–seq and normalized ATAC–seq at sites grouped by IRF1 signal strength (very strong to weak) in response to IFNγ. (B) Line plots of average IRF1 ChIP–seq signal in WT BMDMs for each binding-strength category. (C) Heatmap of relative enrichment of IRF1 binding classes across ATAC clusters (enrichment is relative to the maximum site overlap). (D) Stacked bar plots showing proportions of sites with 0, 1, 2 or ≥3 IRF1 motifs per ATAC cluster. (E) Aggregate plots of IRF1 motif frequency across ±100 bp around IRF1 peaks for each ATAC cluster. (F) Heatmap of IRF1 ChIP–seq signal at 3 h post–IFNγ for sites stratified by IRF1 motif count, as determined in D). (G) Scatter plot of fraction of sites forming IRF1 Hi-ChIP loops versus motif count, with a fitted trend line shown. [FitHiChIP thresholds FDR < 0.1; loop FC > 6, CPM > 6] (H) Genome browser tracks at the Jdp2 locus showing IRF1, PU.1 and H3K27ac ChIP–seq, Hi-ChIP interactions and ATAC–seq in WT and IRF1 KO BMDMs. The cluster to with each ATAC-seq peak belong is indicated [C1 = cluster 1; UR = Unresponsive]. Insets display the array of IRF1 motifs at the C1 site and a SLAM-seq Jdp2 expression plot across the IFNγ time course.

Article Snippet: 20 million BMDMs from WT and Irf1−/− mice were plated in 15 cm tissue culture-treated dishes as described above and stimulated with IFNγ (400 U/mL, R&D Systems) for 0 – 48 h. Crosslinking and chromatin immunoprecipitation were performed following the Dovetail Genomics Hi-ChIP protocol using the IRF1 antibody (R&D Systems, AF4715).

Techniques: ChIP-sequencing, Binding Assay, HiChIP, Expressing

Journal: bioRxiv

Article Title: Pioneer factor IRF1 unlocks latent enhancers to rewire chromatin and immunometabolism in inflammatory macrophages

doi: 10.64898/2026.02.27.708404

Figure Lengend Snippet: (A) Line plots of nascent RNA-seq log2 fold-change (FC) for genes within ±10 kb of ATAC cluster regions in WT and IRF1 KO BMDMs in response to IFNγ stimulation. (B) Heatmap of GO biological process enrichment for genes within ±50 kb of ATAC peaks. Categories with clusterProfiler FDR < 0.05 for at least one cluster are shown. (C) Line plots of nascent RNA counts per million (CPM; mean ± SD) for selected genes across IFNγ time points; WT vs IRF1 KO comparison by two-way ANOVA and pairwise post-hoc testing at each time point. (D) Genome browser tracks at the Kmt2c locus showing IRF1 and PU.1 ChIP-seq, Hi-ChIP arcs and ATAC-seq signal for WT and IRF1 KO BMDMs. [UR = Unresponsive] (E) Line plots of RNA-seq CPM (mean ± SD) for selected genes at 0, 1 and 4 h post-LipidA treatment in WT BMDMs, with BRM014 treatment at the 4 h time point. [Student T-test; n = 3] (F) Bar plot of log2 odds ratio of downregulated genes (FC < 0.5 and FDR < 0.05) after BRM014 treatment (4 h Lipid A) across clusters. * < 0.05, ** < 0.01, *** < 0.001

Article Snippet: 20 million BMDMs from WT and Irf1−/− mice were plated in 15 cm tissue culture-treated dishes as described above and stimulated with IFNγ (400 U/mL, R&D Systems) for 0 – 48 h. Crosslinking and chromatin immunoprecipitation were performed following the Dovetail Genomics Hi-ChIP protocol using the IRF1 antibody (R&D Systems, AF4715).

Techniques: RNA Sequencing, Comparison, ChIP-sequencing, HiChIP

Journal: bioRxiv

Article Title: Pioneer factor IRF1 unlocks latent enhancers to rewire chromatin and immunometabolism in inflammatory macrophages

doi: 10.64898/2026.02.27.708404

Figure Lengend Snippet: (A) Bar plot of the proportion of genes in selected metabolic pathways that harbor IRF1 ChIP-seq peaks; red intensity denotes average number of peaks per gene in each pathway. (B) Genome browser tracks of the Hk1 locus showing normalized IRF1 and PU.1 ChIP-seq, Hi-ChIP interactions and ATAC-seq; an inset shows the annotated intragenic enhancer and promoter contact. [UR = Unresponsive] (C) Line plots of nascent RNA CPM (mean ± SD) for selected genes in glycolysis, PPP and TCA pathways WT and IRF1 KO BMDMs; two-way ANOVA and post-hoc testing; * < 0.05, ** < 0.01, *** < 0.001. (D) Oxygen consumption rates (OCR; fmol mm⁻² s⁻¹) for untreated and IFNγ–stimulated WT and IRF1 KO BMDMs [n = 4/group]; adjacent heatmap shows Student t-test p-values for each time point measured. ( E ) Ribbon plots of relative glycolysis metabolite intensity (mean ± SD) detected by GC-MS in response to IFNγ in WT and IRF1 KO BMDMs [n = 3/group]. (F) Diagram of glycolysis, pentose phosphate pathway (PPP) and Krebs cycle highlighting genes and pathway components significantly dysregulated in IRF1 KO BMDMs for at least 1 timepoint.

Article Snippet: 20 million BMDMs from WT and Irf1−/− mice were plated in 15 cm tissue culture-treated dishes as described above and stimulated with IFNγ (400 U/mL, R&D Systems) for 0 – 48 h. Crosslinking and chromatin immunoprecipitation were performed following the Dovetail Genomics Hi-ChIP protocol using the IRF1 antibody (R&D Systems, AF4715).

Techniques: ChIP-sequencing, HiChIP, Gas Chromatography-Mass Spectrometry

Journal: bioRxiv

Article Title: Pioneer factor IRF1 unlocks latent enhancers to rewire chromatin and immunometabolism in inflammatory macrophages

doi: 10.64898/2026.02.27.708404

Figure Lengend Snippet: (A) Volcano plots from differential metabolite abundance analysis for GC-MS data (n=3/group), comparing 48 h versus 0 h in WT cells (left) and WT versus IRF1 KO at 48 h (right). (B) Top: bar plots of normalized GC-MS intensity for sedoheptulose 7-P at 3 h post IFNγ, xylulose at 12 h, and erythrose 4-P at 48 h. Bottom: ribbon plots of normalized MS signal over time with mean ± SD. (C) Top: normalized LC-MS GSH intensity at 24 h post-IFNγ stimulation. Bottom: ribbon plots of GSH/GSSG ratios over time (mean ± SD) calculated from normalized LC-MS intensities [n=3/group]. (D) Genome browser tracks at the Acod1 locus showing normalized IRF1 and PU.1 ChIP-seq, Hi-ChIP interactions and ATAC-seq [UR = Unresponsive]. Adjacent panels show Acod1 nascent RNA expression and itaconic acid levels. (E) Ribbon plots of normalized GC-MS signal for TCA metabolites in response to IFNγ in WT and IRF1 KO BMDMs. (F) Diagram of glycolysis, PPP and TCA cycle metabolic pathways with dysregulated intermediates denoted in red.

Article Snippet: 20 million BMDMs from WT and Irf1−/− mice were plated in 15 cm tissue culture-treated dishes as described above and stimulated with IFNγ (400 U/mL, R&D Systems) for 0 – 48 h. Crosslinking and chromatin immunoprecipitation were performed following the Dovetail Genomics Hi-ChIP protocol using the IRF1 antibody (R&D Systems, AF4715).

Techniques: Gas Chromatography-Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, ChIP-sequencing, HiChIP, RNA Expression

Journal: bioRxiv

Article Title: Pioneer factor IRF1 unlocks latent enhancers to rewire chromatin and immunometabolism in inflammatory macrophages

doi: 10.64898/2026.02.27.708404

Figure Lengend Snippet: (A) Schematic of experimental timeline for the long-term wash-and-rest assay. Cells are plated for seven days, pulsed with 24 h IFNγ (400 U/mL) at specified times (24 h, 48 h, 6 d) with defined washout intervals and a final 1 h re-stimulation. On day 7, cells are harvested for ChIP-seq (IRF1, H3K4me1, H3K27ac and H3K9me2). (B) Heatmaps of normalized ChIP-seq signal for IRF1, H3K4me1, and H3K27ac at Clusters 1–3. (C) Aggregate coverage plots of H3K4me1 ±1 kb from ATAC peak centers for UT, 24 h IFNγ, 6 d washout and 6 d + 1 h restimulation; insets show putative nucleosomal configurations. (D) Bar plots of fold-change in H3K27ac (mean ± SEM) comparing naïve and IFNγ-trained cells after 1 h restimulation; statistical comparison using Wilcoxon test. (E) Volcano plot of H3K4me1 differential enrichment for Cluster 1–3 (control versus IFNγ washout); points = enhancers, color key: red = increased, blue = decreased, yellow = pioneered genes; labeled enhancers meet log₂FC > 1 and CPM > 5. (F) Hif1a locus showing normalized IRF1, H3K27ac, and H3K4me1 ChIP-seq, and ATAC-seq in WT and IRF1 KO BMDMs [UR = Unresponsive]. Normalized SLAM-seq nascent RNA expression for Hif1a is shown; * p < 0.05.

Article Snippet: 20 million BMDMs from WT and Irf1−/− mice were plated in 15 cm tissue culture-treated dishes as described above and stimulated with IFNγ (400 U/mL, R&D Systems) for 0 – 48 h. Crosslinking and chromatin immunoprecipitation were performed following the Dovetail Genomics Hi-ChIP protocol using the IRF1 antibody (R&D Systems, AF4715).

Techniques: ChIP-sequencing, Comparison, Control, Labeling, RNA Expression

Journal: Journal of Translational Medicine

Article Title: Luteolin ameliorates steroid-induced osteonecrosis of the femoral head via a gut microbiota–L-Carnitine–IFIH1 axis

doi: 10.1186/s12967-026-07793-z

Figure Lengend Snippet: L-Carnitine selectively downregulates IFIH1 and its expression tracks SONFH severity. ( A ) Spearman correlation heatmap between four hub genes (OAS1A, HERC6, IFIH1, IFI44) and histologic/micro-CT indices; IFIH1 shows the strongest positive association with disease severity. ( B - G ) qRT-PCR of JAK1 ( B ), STAT1 ( C ), IRF1 ( D ), OAS1A ( E ), IFIH1 ( F ) and IFI44 ( G ) in ROS17/2.8 osteoblasts under Control, Dex, and Dex + L-Carnitine conditions ( n = 3). ( H - M ) The same analyses were performed in HMEC-1 endothelial cells ( n = 3). ( N ) Western blot for JAK1, STAT1, IRF1, OAS1A/OAS1, IFIH1, and IFI44 in ROS17/2.8 and HMEC-1 confirms that Dex robustly induces IFIH1 and that L-Carnitine attenuates this induction, with little consistent effect on other IFN-I markers ( n = 3). Data are mean ± SEM; ns, P ≥ 0.05; * P < 0.05; ** P < 0.01; *** P < 0.001. SONFH, steroid-induced osteonecrosis of the femoral head; Dex, dexamethasone

Article Snippet: Equal protein amounts (20 μg) were separated on 10% SDS-PAGE and transferred to PVDF membranes (Millipore, USA; cat. no. IPVH00010) using wet transfer at 100 V for 70 min. Membranes were blocked with 5% non-fat milk/TBST for 1 h at 25 °C, then incubated overnight at 4 °C with primary antibodies against: JAK1 (Proteintech, China; cat. no. 66466-1-Ig; 1:6000), STAT1 (Proteintech, China; cat. no. 10144-2-AP; 1:6000), IRF1 (Proteintech, China; cat. no. 11335-1-AP; 1:1000), OAS1 (FineTest, China; cat. no. FNab10795; 1:1000), IFI44 (Bioswamp, China; cat. no. PAB35743 ; 1:1000), IFIH1 (Bioswamp, China; cat. no. PAB31693 ; 1:1000) and GAPDH (Bioss, China; cat. no. bs-2188R; 1:6000).

Techniques: Expressing, Micro-CT, Quantitative RT-PCR, Control, Western Blot

Journal: Cell Research

Article Title: Targeting PTPN13 with 11-amino-acid peptides of C-terminal APC prevents immune evasion of colorectal cancer

doi: 10.1038/s41422-025-01206-4

Figure Lengend Snippet: a GSEA using GO pathways was performed between shAPC or scramble control transfected CT26 subcutaneous tumors, using gene sets associated with type II interferon response and antigen processing and presentation. b Pathway responsive genes for activity inference from gene expression (progeny) analysis performed between tumors formed in Apc-silenced and control groups. c Total cell lysates from a series of IFNγ concentrations were subjected to immunoblot analysis with antibodies to the indicated proteins. Data represent three independent experiments. d Irf1, Lmp2, Tap1, Tap2, MHC-I , and B2m mRNA expression (RT-qPCR) in CT26-shApc or CT26-scramble cells. n = 6 per group, one-way ANOVA. e Flow cytometry histogram and levels of the MHC-I complex on the surfaces of the indicated cells pretreated for 24 h with IFNγ (100 ng/mL) or BSA and stained with anti-H-2Kd/2Dd antibody. Data were calculated from three independent experiments. One-way ANOVA. f MC38-OVA-shAPC cells were stimulated with IFNγ (100 ng/mL) or BSA for 24 h, and the numbers of H-2Kb-OVA 257-264 positive cells and MFI were detected by flow cytometry. Data were calculated from three independent experiments. One-way ANOVA. g Numbers of OVA-tetramer positive CD8 + T cells in TILs of MC38-OVA-shAPC subcutaneous tumors as detected by flow cytometry. n = 3 for each group, one-way ANOVA. h Irf1, Lmp2, Tap1, Tap2, H2-D1, H2K1 , and B2m mRNA expression (RT-qPCR) in the indicated cells exposed to IFNγ (50 ng/mL) for 12 h before collection from three independent experiments. One-way ANOVA. i APC-silenced CT26 cells were transfected with Stat1 R274Q and Irf1 overexpression lentivirus, then subcutaneously xenotransplanted to Balb/c mice; tumor growth was monitored at the indicated times. n = 8 for each group, two-way ANOVA. j Scatterplot showing numbers of CD8 + cells in the indicated groups. n = 8, 7, 8 for each group, one-way ANOVA. k AKP organoids were transfected with Stat1 R274Q and Irf1 overexpression lentivirus, then orthotopically inoculated into C57BL/6 mice; tumor growth was monitored and scored by colonoscopy. n = 6 for each group, one-way ANOVA. l Representative immunofluorescence staining of CK (red) and CD8 (yellow) in tumor tissues, with scatterplot showing numbers of CD8 + cells in three groups. n = 6 for each group, one-way ANOVA. Data were calculated from three independent experiments. All data are mean ± SEM, * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: They were then permeabilized in 0.5% Triton for 20 min and blocked in IF buffer (PBS, 0.2% Triton, 0.05% Tween, and 1% BSA) for 1 h. Cells were incubated in anti-IRF1 primary antibody (1:200, #13063, Cell Signaling Technologies) overnight in IF buffer, then washed three times with TBS 0.1% Tween.

Techniques: Control, Transfection, Activity Assay, Gene Expression, Western Blot, Expressing, Quantitative RT-PCR, Flow Cytometry, Staining, Over Expression, Immunofluorescence

Journal: Cell Research

Article Title: Targeting PTPN13 with 11-amino-acid peptides of C-terminal APC prevents immune evasion of colorectal cancer

doi: 10.1038/s41422-025-01206-4

Figure Lengend Snippet: a Apc-silenced CT26 cells transfected with negative control (N.C) or two single guide RNAs (sgRNAs) targeting Ptpn13 were stimulated with different concentrations of IFNγ. Total cell lysates were subjected to immunoblot analysis with antibodies to the indicated proteins. Data are representative of three independent experiments. b Irf1, Lmp2, Tap1, Tap2, MHC-I , and B2m mRNA expression was determined by RT-qPCR in Ptpn13-knockout or negative control sgRNA-transfected CT26-shAPC cells with 12 h exposure to IFNγ (50 ng/mL). n = 6 per group, one-way ANOVA. c Flow cytometry histogram and levels of the MHC-I complex on the surfaces of the indicated cells pretreated for 24 h with IFNγ (100 ng/mL) or BSA and stained with anti-H-2Kd/2Dd antibody. Data were calculated from three independent experiments. One-way ANOVA. d Ptpn13-knockout or negative control sgRNA-transfected MC38-OVA-shAPC cells were stimulated with IFNγ (100 ng/mL) or BSA for 24 h, and the numbers of H-2Kb-OVA 257-264 positive cells and MFI were detected by flow cytometry. Data were calculated from three independent experiments. One-way ANOVA. e Numbers of OVA-tetramer positive CD8 + T cells in TILs of Ptpn13-knockout or negative control sgRNA-transfected MC38-OVA-shAPC subcutaneous tumors, as detected by flow cytometry. n = 3 for each group, one-way ANOVA. f Irf1, Lmp2, Tap1, Tap2, MHC-I , and B2m mRNA expression was determined by RT-qPCR in intestinal tumors of TAM- or oil-treated APV mice. n = 6 per group, one-way ANOVA. g MFI of H-2Kb/2Db positive cells in intestinal tumors of TAM- or oil-treated APV mice as detected by flow cytometry. n = 6 per group, unpaired t -test. h Scatterplot showing correlation between MFI of HLA-ABC and Ptpn13 IF staining in CRC primary tissues. n = 80, Pearson’s r . i, j CRC-patient-derived organoids (PDO) were cultivated and transfected with Ptpn13-knockout or negative control sgRNA. i Representative immunofluorescence staining of Epcam (green), HLA-ABC (red) and CD8 (cyan). j MFI of HLA-ABC IF staining in Ptpn13-knockout or negative control sgRNA-transfected PDOs. One-way ANOVA. Data were calculated from three independent experiments. All data are mean ± SEM, * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: They were then permeabilized in 0.5% Triton for 20 min and blocked in IF buffer (PBS, 0.2% Triton, 0.05% Tween, and 1% BSA) for 1 h. Cells were incubated in anti-IRF1 primary antibody (1:200, #13063, Cell Signaling Technologies) overnight in IF buffer, then washed three times with TBS 0.1% Tween.

Techniques: Transfection, Negative Control, Western Blot, Expressing, Quantitative RT-PCR, Knock-Out, Flow Cytometry, Staining, Derivative Assay, Immunofluorescence

Journal: Cell Research

Article Title: Targeting PTPN13 with 11-amino-acid peptides of C-terminal APC prevents immune evasion of colorectal cancer

doi: 10.1038/s41422-025-01206-4

Figure Lengend Snippet: a Flag-tagged Stat1 expression vector (0.5 μg) was transfected into CT26 cells. Total cell lysates were immunoprecipitated with anti-Flag and immunoblotted with anti-PTPN13. b Schematic diagram showing the GST-fused PTPN13 motifs and His-tagged STAT1 used in the GST pull-down assays. c GST pull-down assays examining the interactions between GST-fused PTPN13 fragments and His-tagged STAT1 protein. Data are representative of three independent experiments. d Phosphorylated STAT1 was immunoprecipitated with anti-Flag from IFNγ-stimulated 293T transfectants and incubated with 0.2 mg/mL recombinant GST-PTPase domain of PTPN13. Immunoprecipitates were immunoblotted with anti-phospho-STAT1. Equal loading was verified by reprobing with anti-STAT1. e Total cell lysates of CT26 cells were immunoprecipitated with anti-APC and immunoblotted with anti-PTPN13. Data are representative of three independent experiments. f CT26 cells were treated with the indicated concentrations of IFNγ, and cell lysates were immunoprecipitated with anti-APC and immunoblotted with anti-PTPN13. g A Flag-tagged Stat1 vector (0.5 μg) was transfected into CT26-shApc cells or their control cells. Total cell lysates from the indicated cells were immunoprecipitated with anti-Flag and immunoblotted with anti-PTPN13. Data are representative of three independent experiments. h Alphafold3-predicted binding pattern of human APC (brown) and the PDZ2a domain of PTPN13 (cyan). The APC V2843 residue is labeled, and APC Q2829–V2843 residues are shown as sticks and colored in yellow. Hydrogen bonds are shown as yellow dotted lines. i CT26 cells were transfected with HA-tagged Apc-WT or Apc V2860A mutant plasmids, and total cell lysates were immunoprecipitated with anti-HA and immunoblotted with anti-PTPN13 and anti-CTNNB1. Data are representative of three independent experiments. j IFNγ (50 ng/mL) was administered to CT26 cells transfected with Apc-WT or Apc V2860A mutant plasmids for 12 h, and Apc, Lgr5, Axin2, Irf1, Lmp2, Tap1, Tap2, H2-D1, H2K1 , and B2m mRNA expression was detected by RT-qPCR. Data are representative of three independent experiments. One-way ANOVA. k CRISPR/Cas9-based establishment of APC V2860A point mutation. l CT26 cells transfected with APC-WT or APC V2860A mutant plasmids (CT26-APC V2860A -1/2) were incubated with or without IFNγ at the indicated concentrations for 2 h, and total cell lysates were subjected to immunoblot analysis. m IFNγ (50 ng/mL) was administered to CT26 cells transfected with Apc-WT or Apc V2860A mutant plasmids (CT26-APC V2860A -1/2) for 12 h, and Apc, Lgr5, Axin2, Irf1, Lmp2, Tap1, Tap2, H2-D1, H2K1 , and B2m mRNA expression was detected by RT-qPCR. Data are representative of three independent experiments. One-way ANOVA. n CT26 cells harboring gRNA-induced mutant APC V2860A (CT26-APC V2860A -1/2) and the WT control were subcutaneously injected (2 × 10 6 cells) into Balb/c mice, and tumor growth was monitored. n = 8 for each group, two-way ANOVA. o Immunofluorescence of CD8 + cell infiltration in subcutaneous tumors of CT26-APC V2860A and the control group. n = 8 for each group, one-way ANOVA. Data are representative of three independent experiments. All data are mean ± SEM, * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: They were then permeabilized in 0.5% Triton for 20 min and blocked in IF buffer (PBS, 0.2% Triton, 0.05% Tween, and 1% BSA) for 1 h. Cells were incubated in anti-IRF1 primary antibody (1:200, #13063, Cell Signaling Technologies) overnight in IF buffer, then washed three times with TBS 0.1% Tween.

Techniques: Expressing, Plasmid Preparation, Transfection, Immunoprecipitation, Incubation, Recombinant, Control, Binding Assay, Residue, Labeling, Mutagenesis, Quantitative RT-PCR, CRISPR, Western Blot, Injection, Immunofluorescence

Journal: Cell Research

Article Title: Targeting PTPN13 with 11-amino-acid peptides of C-terminal APC prevents immune evasion of colorectal cancer

doi: 10.1038/s41422-025-01206-4

Figure Lengend Snippet: a Schematic diagram showing the indicated residues at the APC C terminus. b Kinetics of the interaction between PDZ-2a and the indicated residues of APC were explored by surface plasmon resonance-based binding assays. c Binding affinities of PDZ2a to APC C-terminal peptides of different lengths, as measured by an FP assay. d The 2.1-Å complex structure of the PDZ2a domain (1364–1446 aa) and the APC11 peptide. PDZ2a is shown in cyan and presented as a surface diagram, whereas the peptide is shown in yellow and presented as a stick diagram. e Detailed interactions between the APC11 peptide and PDZ2a within the complex. The PDZ2a residues involved are labeled and shown as magenta sticks, and the peptide-interacting water molecules are shown as green balls. Hydrogen bonds are shown as yellow dotted lines. f Binding affinity of PDZ2a to the WT APC11 peptide and the APC11M mutant (V2843A) as measured by an FP assay. g GST-fused STAT1 was incubated with HA-tagged PDZ2a and with TAT-APC11 or TAT-APC11M peptides, immunoprecipitated with GST beads, and immunoblotted with anti-GST and anti-HA antibodies. Data are representative of three independent experiments. h CT26 cells transfected with a Flag-tagged Stat1 vector were incubated with 25 μM TAT-HA2, with or without 50 μM TAT-APC11 or TAT-APC11M, for 4 h. Total cell lysates were immunoprecipitated with anti-Flag and immunoblotted with anti-PTPN13. Data are representative of three independent experiments. i Apc-silenced CT26 cells were incubated with 25 μM TAT-HA2, with or without 50 μM TAT-APC11 or TAT-APC11M, for 2 h and then treated with or without IFNγ at the indicated concentrations for 2 h. Total cell lysates were subjected to immunoblot analysis. Data are representative of three independent experiments. j Irf1, Lmp2, Tap1, Tap2, H2-D1, H2K1 , and B2m mRNA expression (RT-qPCR) in Apc-silenced CT26 cells exposed to IFNγ (50 ng/mL) for 12 h before collection from three independent experiments. One-way ANOVA. k Apc-silenced CT26 cells were incubated with 25 μM TAT-HA2, with or without 50 μM TAT-APC11 or TAT-APC11M, for 2 h and then treated with or without IFNγ (100 ng/mL) for 24 h. FACS histogram and quantification of the MHC-I complex on the surfaces of the indicated cells stained with anti-H-2Kd/2Dd antibody or isotype control antibodies. Data were calculated from three independent experiments. One-way ANOVA. l Apc-silenced MC38-OVA 257-264 cells were incubated with 25 μM TAT-HA2, with or without 50 μM TAT-APC11 or TAT-APC11M, for 2 h and then treated with or without IFNγ (100 ng/mL) for 24 h. FACS histogram and quantification of OVA 257-264 -specific MHC-I complex on the surfaces of the indicated cells stained with anti-H-2Kb/SIINFEKL antibody or isotype control antibodies. Data represent three independent experiments. One-way ANOVA. m DLD1 cells were incubated with 25 μM TAT-HA2, with or without 50 μM TAT-APC11 or TAT-APC11M, for 2 h and then treated with or without IFNγ at the indicated concentrations for 2 h. Total cell lysates were subjected to immunoblot analysis. Data are representative of three independent experiments. n IRF1, LMP2, TAP1, TAP2, HLA-A, HLA-B, HLA-C , and B2M mRNA expression (RT-qPCR) in the indicated cells after exposure to IFNγ (50 ng/mL) for 12 h before collection from three independent experiments. One-way ANOVA. All data are mean ± SEM, * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: They were then permeabilized in 0.5% Triton for 20 min and blocked in IF buffer (PBS, 0.2% Triton, 0.05% Tween, and 1% BSA) for 1 h. Cells were incubated in anti-IRF1 primary antibody (1:200, #13063, Cell Signaling Technologies) overnight in IF buffer, then washed three times with TBS 0.1% Tween.

Techniques: SPR Assay, Binding Assay, FP Assay, Labeling, Mutagenesis, Incubation, Immunoprecipitation, Transfection, Plasmid Preparation, Western Blot, Expressing, Quantitative RT-PCR, Staining, Control

Journal: Cell Discovery

Article Title: Luminal hormone-responsive cells tune the regenerative remodeling of mammary glands in large mammals

doi: 10.1038/s41421-025-00848-3

Figure Lengend Snippet: a Violin plot displaying the expression level of top transcription factors (TFs) in luminal subtypes at −4W and +1W. b Heatmap showing the regulon activities of the top TFs in luminal subtypes at −4W. c Immunohistochemical staining for IRF1 in the goat mammary gland at −4W and +1W. Nuclei were counterstained with hematoxylin. Scale bars, 50 μm. d Quantification of IRF1-positive cells in c . n = 8 sections from 4 goats. e Representative images of immunohistochemical staining for PR in the goat mammary organoids treated with or without IFNγ. Nuclei were counterstained with hematoxylin. Scale bars, 50 μm. f Quantification of PR-positive cells in e . n = 5 domes per group. g Representative images of carmine-stained mammary gland whole mounts in WT and IRF1-KO mice at 9 weeks. Scale bars, 0.4 mm. h − k Automatic quantification of the number junctions ( h ), tips ( i ), branches ( j ) and lumen diameters ( k ) of mammary tissues in f . n = 6 mice in wild type and n = 3 in IRF1-KO mice. n = 30 and n = 15 ductal lumens in WT and IRF1-KO mice, respectively. l , m Immunohistochemical staining ( l ) and quantification ( m ) of PR and ER in mammary tissues from WT or IRF1-KO mice at 9 weeks. Nuclei were counterstained with hematoxylin ( l ). n = 4 mice per group. Scale bars, 10 μm. n , o Immunohistochemical staining ( n ) and quantification ( o ) of PR and ER in mammary tissues from WT or IRF1-KO mice during RR. Nuclei were counterstained with hematoxylin. n = 5 mice per group. Scale bars, 10 μm. p Pre-ranked GSEA graphical output for the enrichment in IRF1-KO mice mammary glands of the gene set estrogen response early from the Molecular Signatures Database Hallmarks collection. n = 3 mice per group. q Heatmap representing the log 2 fold change expression of hormone-driven genes in IRF-KO compared to WT at 9 weeks. The data are presented as the mean ± SEM. The P values of two-sided Student’s t -tests are shown in d , f , h – k , m , o .

Article Snippet: Bead-bound nuclei were then incubated overnight at 4 °C with IRF1 rabbit antibody (1:100 dilution, 11335-1-AP, Proteintech).

Techniques: Expressing, Immunohistochemical staining, Staining

Journal: Cell Discovery

Article Title: Luminal hormone-responsive cells tune the regenerative remodeling of mammary glands in large mammals

doi: 10.1038/s41421-025-00848-3

Figure Lengend Snippet: a The genomic loci with IRF1 motifs are selected and the ATAC-seq signal intensity is shown in heatmaps. The average signal intensity is shown on top. b Heatmap displaying the transcriptional level of genes presumably bound by IRF1. n = 3 goats per group. c Heatmaps showing the signal intensity of IRF1 CUT&Tag in goat mammary tissues at −4W and +1 W. The average signal intensity is shown on top. d ATAC-seq and IRF1 CUT&Tag profiles at the ESRRB locus in −4W and +1 W are shown. The differential regions between −4W and +1 W with IRF1 motifs are highlighted in yellow. e UMAP plot showing the specific expression of ESRRB in LumHR cells by scRNA-seq data. f , g Immunohistochemical staining and quantification of ESRRB in goat mammary tissues at −4W and +1 W. Representative images of Immunohistochemical staining ( f ). Nuclei are counterstained with hematoxylin. n = 10 sections from 5 goats per group. Scale bars, 20 μm. Two-sided Student’s t -test. h Luciferase reporter assays in goat mammary epithelial cells. Cells are transfected with WT IRF1 motif (IRF1-MWT) or IRF1-motif site mutation (IRF1-MM) vector and treated with IFNγ or not. n = 4 biological replicates. Two-way ANOVA test. i , j Immunohistochemical staining and quantification of ESRRB in mouse WT or IRF1-KO mammary tissues under RR. Representative images of Immunohistochemical staining ( i ). Nuclei are counterstained with hematoxylin. n = 4 mice per group. Scale bars, 50 μm. Two-sided Student’s t -test. k The proposed model in the current study is that a reduction of LumHR cells triggered by IRF1-ESRRB signaling upregulation promotes the accumulation of LumSecP during RR in ruminants. LumHR cells control the differentiation of LumSecP to LumSec cells through the PRLR pathway and regulate the cell composition of luminal lineages during RR. Created with BioRender.com.

Article Snippet: Bead-bound nuclei were then incubated overnight at 4 °C with IRF1 rabbit antibody (1:100 dilution, 11335-1-AP, Proteintech).

Techniques: Expressing, Immunohistochemical staining, Staining, Luciferase, Transfection, Mutagenesis, Plasmid Preparation, Control