Journal: Cell Reports Medicine

Article Title: Interferon-driven CAF reprogramming augments immunogenic response to neoadjuvant radiotherapy in colorectal cancer

doi: 10.1016/j.xcrm.2025.102251

Figure Lengend Snippet: Single-cell RNA sequencing analysis identifies IFN-γ-dependent CAFs enriched in rectal cancer responsive to radiotherapy (A) Schematic representation of the workflow for scRNA-seq and validation experiments conducted on rectal tumors pre- and post-RT ( n = 7 for each group). (B) Uniform manifold approximation and projection (UMAP) plot of all cells, representing eleven cell types. Cell clusters are colored by cell identity. (C) Reclustering of CAFs in the dataset visualized using UMAP, demonstrating five distinct CAF clusters: iCAFs (dark blue), myCAFs (orange), ilCAFs (light blue), SOX6 + CAFs (red), and CXCL1 + CAFs (purple). (D) Heatmap displaying differentially expressed genes across all CAF clusters. (E) GSEA depicting the top upregulated pathways and core enrichment genes in five distinct CAF clusters, with all pathways filtered by false discovery rate < 0.05. (F) Density plot of different CAF clusters pre- or post-RT. (G) Slingshot and tradeSeq trajectory analysis of RC CAF scRNA-seq data indicating predicted lineage trajectory. The trajectory path from iCAFs-ilCAFs is overlaid on the cluster-based UMAP and colored by pseudotime of this respective lineage. Trajectory analysis overlaid IRF1 expression vs. pseudotime scatterplot of iCAFs and ilCAFs along the lineage. (H) Quantitative PCR mRNA expression analysis of representative genes of ilCAFs ( IRF1 , CCL4 , STAT1 , and STING1 ), iCAFs ( C3 and CFD ), myCAFs ( RGS5 and MCAM ), SOX6 + CAFs ( CXCL14 and PDGFRA ), and CXCL1 + CAFs ( CXCL1 and CCL11 ) in primary CAFs treated with RT, compared to untreated controls ( n = 4 for each group). (I) Representative flow cytometric plots (top) and quantification (down) of IRF1 expression in CAFs pre- and post-RT ( n = 7 for each group). (J) Representative multiplex immunofluorescence image depicting the localization of ilCAFs (COL3A, PDPN, and IRF1) and tumor cells (pan-cytokeratin) in rectal tumors pre- and post-RT ( n = 5 for RT group, and n = 7 for untreated group). Scale bars, 50 μm. (K) Quantification of ilCAFs is shown in the adjacent bar graphs. (L) Kaplan-Meier survival curves of CRC patients with low (blue) and high (red) expression of ilCAFs in total CRC samples ( n = 165). Student’s t tests were performed for (F), (H), (I), (J), and (K). For (L) (survival curves), the log rank test was performed. Data are presented as mean ± SEM and are representative of at least three independent experiments. A p value less than 0.05 indicates statistical significance. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001.

Article Snippet: PE anti-mouse IRF1 Antibody , Santa Cruz , Cat#sc-514544; RRID: AB_2891129.

Techniques: RNA Sequencing, Biomarker Discovery, Expressing, Real-time Polymerase Chain Reaction, Multiplex Assay, Immunofluorescence

Journal: Cell Reports Medicine

Article Title: Interferon-driven CAF reprogramming augments immunogenic response to neoadjuvant radiotherapy in colorectal cancer

doi: 10.1016/j.xcrm.2025.102251

Figure Lengend Snippet: RT induces enrichment of ilCAFs to enhance anti-tumor responses (A) Bar graph illustrating the upregulated pathways in ilCAFs post-RT compared to pre-RT based on GO enrichment analysis ( n = 7 per group), with all pathways filtered by adjusted p value (P adj ) < 0.05. (B) Correlation analysis between IRF1 expression in ilCAFs ( x axis) and the proportion of CD8 + T cell subsets (red, y axis) in scRNA-seq data. Spearman correlation analysis was performed to determine the correlation coefficient and two-sided p value. (C) Reclustering of CD8 T cells in the dataset visualized by UMAPs, demonstrating six distinct clusters: naive T (light green), effector T (blue), T EMRA (light purple), T RM (dark purple), T EX (red) and MAIT (orange). (D) UMAP nucleus densities of different CD8 T cell clusters pre- or post-RT. (E) Effector memory and exhaustion scores of effector T cells and T EMRA cells pre- and post-RT. (F) ELISA for CCL4 and CCL5 content in the supernatant of primary CAFs pre-RT and post-RT ( n = 3 per group). (G) Quantification of CD8 + T cells and GZMB + CD8 + T cells after CAFs were exposed to CCR5i combined with RT ( n = 5 per group). (H) Differential interaction strength between post-RT and pre-RT among ilCAFs and antigen-presenting cell subsets in RC scRNA-seq data. (I) GO enrichment analysis showing top upregulated pathways in cDC1s compared to other DCs pre-RT and post-RT in rectal cancers, with all pathways filtered by P adj < 0.05. (J and K) Representative multiplex immunohistochemistry staining images (left) and quantification (right) for COL3A1, IRF1 (J), or CCL4 (K) in tumors from day 12 RT-treated and untreated MC38-bearing mice ( n = 5 per group). Scale bars, 20 μm. (L–O) Evaluation of the impact of RT on the growth of established MC38 colorectal tumors co-inoculated with Irf1 −/− or WT fibroblasts in syngeneic mice. (L) Experimental design for the treatment of MC38 colorectal tumor-bearing C57BL/6 mice. (M) Average growth curve of colorectal cancer treated with vehicle and RT ( n = 5 per group). (N) Quantification of CD4 + T cells, CD8 + T cells, and cDCs among CD45 + cells in tumors at day 10 post-RT treatment ( n = 5 per group). (O) Modified Kaplan-Meier curves for each treatment cohort in the mouse model ( n = 5 mice per group). (P) Evaluation of the role of the IFN-γ pathway in RT-mediated tumor control in established MC38 tumor cells co-inoculated with fibroblasts in syngeneic mice. Average tumor growth curves of colorectal tumors treated with vehicle or anti-IFN-γ, with or without RT ( n = 5 per group). (Q) Evaluation of the impact of RT-induced CCL4-ilCAFs on tumor growth in established MC38 tumor cells co-inoculated with si- Ccl4 or WT fibroblasts in syngeneic mice. Average tumor growth curve of colorectal tumors treated with vehicle and RT ( n = 5 per group). (R) Evaluation of the impact of CCR5i combined with RT on tumor growth in established MC38 tumor cells co-inoculated with fibroblasts in syngeneic mice. Average tumor growth curve of colorectal cancer treated with vehicle and CCR5i ± RT ( n = 5 per group). One-way or two-way ANOVA was performed for (G), (M), (N), (P), (Q), and (R). For (O), the log rank test was performed. Data are presented as mean ± SEM and are representative of at least three independent experiments. A p value less than 0.05 indicates statistical significance. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001.

Article Snippet: PE anti-mouse IRF1 Antibody , Santa Cruz , Cat#sc-514544; RRID: AB_2891129.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Multiplex Assay, Immunohistochemistry, Staining, Modification, Control

Journal: Cell Reports Medicine

Article Title: Interferon-driven CAF reprogramming augments immunogenic response to neoadjuvant radiotherapy in colorectal cancer

doi: 10.1016/j.xcrm.2025.102251

Figure Lengend Snippet: ilCAFs mediate radiation-induced senescence via the IFN-γ/STAT1 pathway (A) Representative histogram plots (left) and quantification (right) showing mean fluorescence intensity (MFI) of IRF1 or CCL4 in CAFs isolated from treatment-naive patients with rectal cancer, analyzed by flow cytometry. Comparisons include vehicle control, RT, IFN-γ, IFN-γ + RT, STAT1 inhibitor (STAT1i), and RT + STAT1i, all evaluated at 48 h post-RT ( n = 3 per group). (B) Representative immunofluorescent staining images (left) and quantification (right) depicting the expression of α-SMA, IRF1, CCL4, or STAT1 in primary CAFs treated with vehicle, IFN-γ, IFN-γ ± RT, STAT1i, or STAT1i ± RT at specified time points ( n = 5 per group). Scale bars, 10 μm. (C) Western blot images showing the expression levels of cGAS, p-STAT1/STAT1, p-STING/STING, IRF1, and CCL4 in primary CAFs treated with IFN-γ (20 ng/mL) or STAT1i (5 μM) combined with RT for 48 h. (D) GSEA depicting the upregulated STING pathway signature in ilCAFs compared to other CAFs from rectal cancers. (E) Heatmap depicts a list of differentially expressed genes sourced from inflammatory modulation, type II IFN, and TNF-α between vehicle- and RT-treated groups. (F) Representative flow cytometric dot plots displaying the MFI of IRF1 or CCL4 in CAFs isolated from treatment-naive rectal cancer patients ( n = 3 per group), comparing vehicle control, RT, STING agonist SR717, and SR717 + RT at 48 h post-RT treatment. (G) Western blot images depicting the expression levels of cGAS, p-STAT1/STAT1, p-STING/STING, and IRF1 in primary CAFs treated with SR717 (3 μM). (H) Bubble plots illustrating upregulated pathways in fibroblasts co-cultured with tumor cell-derived media (TCMs) after RT + SR717 treatment compared to RT alone, based on GO biological processes. Pathways were filtered using adjusted p values (P adj < 0.05). (I) Quantification (right) of flow cytometry analysis of CD8 + T cells, GZMB + CD8 + T cells, CD11c + DCs, and CD103 + CD11c + cDC1s in T cells or DCs co-cultured with mouse fibroblasts from the indicated combination treatment groups ( n = 3 per group). For (A), (B), (F), and (I), one-way ANOVA was performed. Data are presented as mean ± SEM and are representative of at least three independent experiments. A p value less than 0.05 indicates statistical significance. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001.

Article Snippet: PE anti-mouse IRF1 Antibody , Santa Cruz , Cat#sc-514544; RRID: AB_2891129.

Techniques: Fluorescence, Isolation, Flow Cytometry, Control, Staining, Expressing, Western Blot, Cell Culture, Derivative Assay

Journal: Cell Reports Medicine

Article Title: Interferon-driven CAF reprogramming augments immunogenic response to neoadjuvant radiotherapy in colorectal cancer

doi: 10.1016/j.xcrm.2025.102251

Figure Lengend Snippet: STING knockout in CAFs modulates the stromal landscape and enhances T cell infiltration (A and B) Histogram plots (left) and quantification of MFI of IRF1 (A) or CCL4 (B) in primary fibroblasts isolated from treatment-naive wild-type (WT) and Tmem173 −/− (STING knockout) mice, analyzed by flow cytometry. Comparisons include vehicle, RT, STING agonist (SR717), and combination therapy (SR717 + RT) in Tmem173 −/− fibroblasts and vehicle and RT in WT fibroblasts post-RT (vehicle group in A: n = 4; all other groups: n = 3 per group). (C–G) Impact of RT on the growth of established MC38 colorectal tumors co-inoculated with Tmem173 −/− or WT fibroblasts in syngeneic C57BL/6 mice. (C) Schematic of the experimental design. (D) Average tumor growth curve. (E) Tumor weight at endpoint. (F) Tumor-draining lymph node sizes. (G) Modified Kaplan-Meier survival curves for each treatment group ( n = 5 per group). (H) Percentage of CD4 + T cells, CD8 + T cells, and cDC1s among CD45 + tumor-infiltrating cells at day 14 post-RT treatment ( n = 5 per group). (I) Representative multiplex immunofluorescence images (left) and quantification (right) of IRF1 + CAFs (green: COL1A, red: IRF1) and CCL4 + CAFs (green: COL1A, red: CCL4). Scale bars, 100 μm ( n = 5 per group). For (A), (B), (E), (F), (H), and (I), one-way ANOVA was performed. For (D), a two-way ANOVA was conducted. For (G), the log rank test was performed. Data are presented as mean ± SEM. Results are representative of at least three independent experiments. A p value less than 0.05 indicates statistical significance. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001.

Article Snippet: PE anti-mouse IRF1 Antibody , Santa Cruz , Cat#sc-514544; RRID: AB_2891129.

Techniques: Knock-Out, Isolation, Flow Cytometry, Modification, Multiplex Assay, Immunofluorescence

Journal: Journal of neuroimmune pharmacology : the official journal of the Society on NeuroImmune Pharmacology

Article Title: Sirtuin 1-chromatin-binding dynamics points to a common mechanism regulating inflammatory targets in SIV infection and in the aging brain

doi: 10.1007/s11481-017-9772-3

Figure Lengend Snippet: Genes with a role in inflammation that had in-promoter Sirt1-binding in Controls, which was decreased with SIV, and with SIVE, and fold change transcript values from gene array data in SIVE compared to SIV These genes may be kept under control by Sirt-1 and are expected to become unrepressed in association with infection-driven CNS pathology.

Article Snippet: Formalin-fixed, paraffin embedded brain tissue was sectioned and the detection of molecular markers was performed using antibodies against Iba-1 (AIF1 - WAKO, Richmond, VA), CD163 (Vector Labs, Burlingame, CA), Mac3 (BioLegend, San Diego, CA) and glial fibrillary acidic protein (GFAP, Dako, Carpinteria, CA), as well as IRF1 (Novus Biologicals, Littleton, CO), IRF7 (Novus), and IFIT1 (Novus), using standard procedures( Bortell et al. 2015 ).

Techniques: Control, Infection

Journal: Journal of neuroimmune pharmacology : the official journal of the Society on NeuroImmune Pharmacology

Article Title: Sirtuin 1-chromatin-binding dynamics points to a common mechanism regulating inflammatory targets in SIV infection and in the aging brain

doi: 10.1007/s11481-017-9772-3

Figure Lengend Snippet: Isolated microglia cells from controls and SIV-infected macaques were used to examine the transcriptional expression of AIF1, IRF7, IFIT1 and IRF1, using SyBr Green qRT-PCR. Values were normalized againt the expression of GAPDH. *p<0.05 compared to controls.

Article Snippet: Formalin-fixed, paraffin embedded brain tissue was sectioned and the detection of molecular markers was performed using antibodies against Iba-1 (AIF1 - WAKO, Richmond, VA), CD163 (Vector Labs, Burlingame, CA), Mac3 (BioLegend, San Diego, CA) and glial fibrillary acidic protein (GFAP, Dako, Carpinteria, CA), as well as IRF1 (Novus Biologicals, Littleton, CO), IRF7 (Novus), and IFIT1 (Novus), using standard procedures( Bortell et al. 2015 ).

Techniques: Isolation, Infection, Expressing, SYBR Green Assay, Quantitative RT-PCR

Journal: Journal of neuroimmune pharmacology : the official journal of the Society on NeuroImmune Pharmacology

Article Title: Sirtuin 1-chromatin-binding dynamics points to a common mechanism regulating inflammatory targets in SIV infection and in the aging brain

doi: 10.1007/s11481-017-9772-3

Figure Lengend Snippet: Cells of the THP1 macrophage cell line were stimulated with 6ug/ml of CpG ODN, and/or the 100uM of the Sirt-1 inhibitor Sirtinol for 24 hrs. RNA was extracted from the cell pellets for determination of AIF1, IRF7, IFIT1 and IRF1 using SyBr Green qRT-PCR. Values of 3 independent experiments were normalized against the expression of GAPDH. P values represent results from Bonferroni’s posthoc test, which followed ANOVA.

Article Snippet: Formalin-fixed, paraffin embedded brain tissue was sectioned and the detection of molecular markers was performed using antibodies against Iba-1 (AIF1 - WAKO, Richmond, VA), CD163 (Vector Labs, Burlingame, CA), Mac3 (BioLegend, San Diego, CA) and glial fibrillary acidic protein (GFAP, Dako, Carpinteria, CA), as well as IRF1 (Novus Biologicals, Littleton, CO), IRF7 (Novus), and IFIT1 (Novus), using standard procedures( Bortell et al. 2015 ).

Techniques: SYBR Green Assay, Quantitative RT-PCR, Expressing

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Anti-tumor activity of all-trans retinoic acid in gastric-cancer: gene-networks and molecular mechanisms

doi: 10.1186/s13046-023-02869-w

Figure Lengend Snippet: IRF1 and DHRS3 involvement in the anti-proliferative effects exerted by ATRA in HGC-27 cells. HGC-27 cells were transfected with two IRF1- targeting ( si-IRF1a / si-IRF1b ) and a control siRNA ( si-CTRL ). Twenty-four hours later, cells were treated with vehicle (DMSO) or ATRA (1µM) for 48 hours. A Western-blot analysis using anti- IRF1 , anti- DHRS3 and anti- βactin antibodies: the lanes marked as “no-siRNA” indicate p arental HGC-27 cells. B Cell-growth of transfected HGC-27 cells (MTS-assay): Mean+SD of 3 replicate cultures; values normalized for vehicle-treated cells (100%). The p-values (two-tailed Student's t-test) of the comparisons between ATRA-treated and vehicle-treated cells and the comparisons between the indicated groups are shown above each red column and above the diagram, respectively. C HGC-27 cells were infected with lentiviral particles containing 2 IRF1- targeting-shRNAs ( sh-IRF1a / sh-IRF1b ), one control-shRNA ( sh-CTRL1 ) or the pGreenPuro- vector ( pGR ). Following puromycin-selection, we isolated 4 green-fluorescent cell-populations characterized by pGR­ - , sh-CTRL -, sh-IRF1a - and sh-IRF1b ­-integration. The cell-populations were treated with vehicle or ATRA (1µM) for 48 hours and subjected to Western-blot analysis using anti- IRF1 , anti- DHRS3 and anti- βactin antibodies as in ( A ). D The pGR - , sh-CTRL -, sh-IRF1a­ - and sh-IRF1b -infected cell-populations were treated with vehicle or ATRA (0.1µM/1.0µM) for 3/6/9 days: “ no-sh ”=parental- HGC-27 cells. Cell-growth (MTS-assay): each value is the Mean+SD of 3 cultures; values are normalized as in ( B ). The p-values (two-tailed-Student's-t-test) of the comparisons between ATRA-treated and corresponding vehicle-treated cells are shown above each column. E HGC-27 cells were infected with lentiviral-particles containing two DHRS3 -targeting pGreenPuro -constructs ( sh-DHRS3a / sh-DHRS3b ), the pGreenPuro -vector ( pGR ) and a control shRNA ( sh-CTRL2 ). Following infection/puromycin-selection, we isolated 4 populations of green-fluorescent HGC-27 cells characterized by stable pGR/sh-CTRL / sh-DHRS3a / sh-DHRS3b -integration. The cell-populations were treated with vehicle or ATRA (1.0µM) for 48 hours and subjected to Western-blot analysis using anti- IRF1 /anti- DHRS3 /anti- βactin antibodies as in ( A ). F pGR/sh-CTRL / sh-DHRS3a / sh-DHRS3b- infected cell-populations were treated with vehicle or ATRA as in ( D ). Cell-growth (MTS-assay): Mean+SD of 3 cultures; values normalized as in ( D ). The p -values (two-tailed-Student’s-t-test) of the ATRA-treated/vehicle-treated cells comparison are shown above each column

Article Snippet: Western-blot experiments were conducted with anti- IRF1 (8478, Cell Signaling Technology; E-AB-12,522, Elabscience), anti- DHRS3 (FNab02372, FineTest, Wuhan, China) anti- β2-actin (SC-47,778, Santa Cruz) and anti-tubulin (T5168, Sigma-Aldrich) antibodies.

Techniques: Transfection, Control, Western Blot, MTS Assay, Two Tailed Test, Infection, shRNA, Plasmid Preparation, Selection, Isolation, Construct, Comparison

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: PLoS ONE

Article Title: Gene Regulatory Network Inference of Immunoresponsive Gene 1 ( IRG1 ) Identifies Interferon Regulatory Factor 1 ( IRF1 ) as Its Transcriptional Regulator in Mammalian Macrophages

doi: 10.1371/journal.pone.0149050

Figure Lengend Snippet: ( A ) Mouse and ( B ) human networks hierarchical layouts after calculating all possible paths between LPS and IRG1 from the contextualised networks. IRF1 , interferon regulatory factor 1; CEBPB, CCAAT/enhancer binding protein (C/EBP) beta; CEBPD, CCAAT/enhancer binding protein (C/EBP) delta; STAT1, signal transducer and activator of transcription 1; JUNB, Jun B proto-oncogene; PRDM1, PR domain containing 1 with ZNF domain; STAT4, signal transducer and activator of transcription 4; FOS, FBJ murine osteosarcoma viral oncogene homolog; ETS2, v-ets avian erythroblastosis virus E26 oncogene homolog 2; VDR, vitamin D (1,25-dihydroxyvitamin D3) receptor; RARA, retinoic acid receptor alpha; RUNX1, runt-related transcription factor 1. Solid line: activation; dashed line: inhibition.

Article Snippet: Affinity-purified goat anti-mouse IRF1 antibody (catalogue #: AF4715) and rabbit anti-goat IgG secondary antibody (catalogue #: HAF017) were obtained from R&D Systems Europe Ltd., United Kingdom, while rabbit anti-human IRG1 antibody (catalogue #: HPA040143) and normal rabbit IgG (catalogue #: sc-2027) were purchased from Sigma and Santa Cruz Biotechnology, respectively.

Techniques: Binding Assay, Virus, Activation Assay, Inhibition

Journal: PLoS ONE

Article Title: Gene Regulatory Network Inference of Immunoresponsive Gene 1 ( IRG1 ) Identifies Interferon Regulatory Factor 1 ( IRF1 ) as Its Transcriptional Regulator in Mammalian Macrophages

doi: 10.1371/journal.pone.0149050

Figure Lengend Snippet: Transcription factors gene essentiality metric scores.

Article Snippet: Affinity-purified goat anti-mouse IRF1 antibody (catalogue #: AF4715) and rabbit anti-goat IgG secondary antibody (catalogue #: HAF017) were obtained from R&D Systems Europe Ltd., United Kingdom, while rabbit anti-human IRG1 antibody (catalogue #: HPA040143) and normal rabbit IgG (catalogue #: sc-2027) were purchased from Sigma and Santa Cruz Biotechnology, respectively.

Techniques:

Journal: PLoS ONE

Article Title: Gene Regulatory Network Inference of Immunoresponsive Gene 1 ( IRG1 ) Identifies Interferon Regulatory Factor 1 ( IRF1 ) as Its Transcriptional Regulator in Mammalian Macrophages

doi: 10.1371/journal.pone.0149050

Figure Lengend Snippet: ( A, B ) RAW264.7 cells were transfected with siRNA negative (siRNA NEG) or siRNA specific to Irf1 (siRNA Irf1) 24 hours before treatment and RNA was extracted 2 hours after activation with LPS (10ng/ml). The bars show the mean of 3 biological replicates (± SEM) of ( A ) Irf1 and ( B ) Irg1 mRNA levels measured by real-time PCR normalised with L27 as the housekeeping gene. **p < 0.01, *p < 0.05. ( C ) Proteins were extracted from transfected cells after 4 hours of LPS stimulation. Western blot bands of IRF1, IRG1/CAD and β-ACTIN proteins are shown. ( D, E ) Metabolites were extracted from transfected cells after 4h of LPS stimulation and analysed by GC-MS. Itaconic acid levels in Irf1 silenced cells were calculated as the percentage relative to the non-specific transfected cells in ( D ) control and ( E ) LPS activated cells. Error bars and statistical significance were calculated from 3 biological replicates (± SEM). *p < 0.05.

Article Snippet: Affinity-purified goat anti-mouse IRF1 antibody (catalogue #: AF4715) and rabbit anti-goat IgG secondary antibody (catalogue #: HAF017) were obtained from R&D Systems Europe Ltd., United Kingdom, while rabbit anti-human IRG1 antibody (catalogue #: HPA040143) and normal rabbit IgG (catalogue #: sc-2027) were purchased from Sigma and Santa Cruz Biotechnology, respectively.

Techniques: Transfection, Activation Assay, Real-time Polymerase Chain Reaction, Western Blot, Gas Chromatography-Mass Spectrometry, Control

Journal: PLoS ONE

Article Title: Gene Regulatory Network Inference of Immunoresponsive Gene 1 ( IRG1 ) Identifies Interferon Regulatory Factor 1 ( IRF1 ) as Its Transcriptional Regulator in Mammalian Macrophages

doi: 10.1371/journal.pone.0149050

Figure Lengend Snippet: PBMCs-derived macrophages were transfected with siRNA negative (siRNA NEG) or siRNA specific to IRF1 (siRNA IRF1) 24 hours before treatment and RNA was extracted 6 hours after activation with LPS (10μg/ml) in independent donors (D1-D3). The bars show the mean of ( A ) IRF1 and ( B ) IRG1 mRNA levels of 3 technical replicates (± SEM) measured by real-time PCR normalised with L27 as the housekeeping gene. *p < 0.05.

Article Snippet: Affinity-purified goat anti-mouse IRF1 antibody (catalogue #: AF4715) and rabbit anti-goat IgG secondary antibody (catalogue #: HAF017) were obtained from R&D Systems Europe Ltd., United Kingdom, while rabbit anti-human IRG1 antibody (catalogue #: HPA040143) and normal rabbit IgG (catalogue #: sc-2027) were purchased from Sigma and Santa Cruz Biotechnology, respectively.

Techniques: Derivative Assay, Transfection, Activation Assay, Real-time Polymerase Chain Reaction

Journal: International Journal of Clinical and Experimental Pathology

Article Title: Malignant glomus tumor of the ileum mimicking GIST with distant metastasis without BRAF V600E mutation

doi:

Figure Lengend Snippet: Immunohistochemical characteristics of the tumor cells

Article Snippet: Calretinin , OrigeneTechnologies, Inc , TA356330 , 1:60 , -.

Techniques: Immunohistochemical staining, Staining