Journal: Autophagy

Article Title: A new transcription factor ATG10S activates IFNL2 transcription by binding at an IRF1 site in HepG2 cells

doi: 10.1080/15548627.2020.1719681

Figure Lengend Snippet: Potential ATG10S binding sites on the human IFNL2 promoter within a 1 kb sequence from the initiation codon. (A) Endogenous IFNL2 expression levels in HepG2 cells were detected using qRT-PCR (left) and immunoblot assay (right) through the regulation of ATG10S and IRF1 gene expression. ATG10 or ATG10S overexpression was achieved by transfection of plasmids with FLAG-tagged ATG10 or ATG10S; IRF1 overexpression and knockdown were through transfection of IRF1 5ʹ-capped mRNA and IRF1-siRNA respectively. Ctrl, non-transfection. Ctrl-siRNA, Control-siRNA. (B) Diagrams show the 2.1-kb genomic sequence of the IFNL2 promoter, 2 expression constructs directed by LP1 and LP2 segments (upper), and fluorescence microscopy images for GFP expression directed by the IFNL2 promoter fragments LP1 or LP2 induced by ATG10S in HepG2 cells (bottom). Scale bars: 220 µm. (C) Luciferase activity assay shows the different actions of ATG10 and ATG10S on IFNL2 LP1 transcription activity. RLU, relative light unit. (D) RT-PCR (left) and western blotting (right) examined ATG10- and ATG10S-mediated activation of the IFNL2 LP1 promoter. (E) Diagram of 3 GFP-expressing constructs directed by LP1 truncated segments (LP1-1, LP1-2, and LP1-3). (F) RT-PCR (upper panel) and western blot (bottom panel) were used to determine the effects of ATG10 and ATG10S on GFP expression directed by the LP1-1, LP1-2 or LP1-3 segments respectively. Ctrl, non-transfection; * indicates P < 0.05, *** indicates P < 0.001. The statistics data are expressed as the mean ± standard deviation (SD) (n = 3)

Article Snippet: To knockdown the expression of IRF1, IRF1 -siRNA was obtained from Santa Cruz Biotechnology, and siRNA transfection was performed as described in the user manual (sc-35706).

Techniques: Binding Assay, Sequencing, Expressing, Quantitative RT-PCR, Western Blot, Gene Expression, Over Expression, Transfection, Knockdown, Control, Construct, Fluorescence, Microscopy, Luciferase, Activity Assay, Reverse Transcription Polymerase Chain Reaction, Activation Assay, Standard Deviation

Journal: Autophagy

Article Title: A new transcription factor ATG10S activates IFNL2 transcription by binding at an IRF1 site in HepG2 cells

doi: 10.1080/15548627.2020.1719681

Figure Lengend Snippet: Transcription factor IRF1 Binding Domain (BD) is crucial for ATG10S promotion of IFNL2 transcription. (A) Diagrams of LP1-1-GFP constructs with IRF1-BD deletion (ΔBD) and four pairs of oligonucleotide deletions in the BD. All of the mutants were cloned into pEGFP-ΔCMV-N1 (GFP as a reporter gene) and pGL3-basic (luciferase gene as a reporter gene) vectors. (B) GFP expression directed by LP1-1 and LP1-1-ΔBD in HepG2 cells via co-transfected with ATG10 or ATG10S mRNA were analyzed by RT-PCR (upper) and western blotting (bottom), Ctrl, non-transfection control. (C) The luciferase activity driven by LP1-1 and LP1-1-ΔBD under co-transfected with ATG10 or ATG10S mRNA were examined by luciferase assay. (D–F) ATG10/ATG10S-mediated activations of the oligonucleotide-deleted LP1-1 mutants (LP1-1-BDΔ1, LP1-1-BDΔ1-2, LP1-1-BDΔ2-3, and LP1-1-BDΔ3) compared with LP1-1 were detected using RT-PCR (D), western blotting (E), and luciferase assay (F). RLU, relative light unit. * indicates P < 0.05, *** P < 0.001. All of the statistics data are expressed as the mean ± SD (n = 3)

Article Snippet: To knockdown the expression of IRF1, IRF1 -siRNA was obtained from Santa Cruz Biotechnology, and siRNA transfection was performed as described in the user manual (sc-35706).

Techniques: Binding Assay, Construct, Clone Assay, Luciferase, Expressing, Transfection, Reverse Transcription Polymerase Chain Reaction, Western Blot, Control, Activity Assay

Journal: Autophagy

Article Title: A new transcription factor ATG10S activates IFNL2 transcription by binding at an IRF1 site in HepG2 cells

doi: 10.1080/15548627.2020.1719681

Figure Lengend Snippet: ATG10S can be transported into the nucleus, and bind on the IFNL2-core motif as a transcription factor. (A) Representative immunocytochemical images of ATG10 and ATG10S distribution in HepG2 cells and HCV replicon cells via using anti-FLAG antibody (FLAG labeled to ATG10 or ATG10S). Scale bars: 15 µm. (B) Prediction of NLS in ATG10 and ATG10S using online prediction software (http://nls-mapper.iab.keio.ac.jp/cgi-bin/NLS_Mapper_form.cgi). Based on the software, a score of a protein is greater than 3, indicating that the protein can distribute in both the nucleus and cytoplasm; a protein score less than 3 suggests the protein only localizes in the cytoplasm. (C) Interactions among ATG10S, KPNA1, KPNB1, and IRF1 in HepG2 and HCV subreplicon cells by co-IP. Cells transfected with the ATG10S or ATG10 constructs, the cell lysates were immunoprecipitated with anti-FLAG antibody and the proteins were detected by immunoblot assay. (D, E) Subcellular localization of ATG10S protein was determined by nuclear-cytoplasmic fractionation (D) and cell-IF (E) under KPNA1 or KPNB1 knockdown using KPNA1-siRNA or KPNB1-siRNA, respectively

Article Snippet: To knockdown the expression of IRF1, IRF1 -siRNA was obtained from Santa Cruz Biotechnology, and siRNA transfection was performed as described in the user manual (sc-35706).

Techniques: Labeling, Software, Co-Immunoprecipitation Assay, Transfection, Construct, Immunoprecipitation, Western Blot, Fractionation, Knockdown

Journal: Autophagy

Article Title: A new transcription factor ATG10S activates IFNL2 transcription by binding at an IRF1 site in HepG2 cells

doi: 10.1080/15548627.2020.1719681

Figure Lengend Snippet: ATG10S activates IFNL2 transcription via competition with IRF1 at the same core motif. (A, B) Activity of IFNL2 promoter LP1-1 was competitively induced by ATG10S and IRF1 in Luciferase activity assay (A) and qRT-PCR (B) using gene upregulation and downregulation of ATG10S and IRF1 genes. (C) DNA IP analysis of ATG10S binding to LP1-1 DNA with IRF1-siRNA compared with no IRF1-siRNA and Ctrl-siRNA groups in HepG2 cells. The cell lysates were immunoprecipitated with anti-FLAG antibody and LP1-1 DNA were detected by RT-PCR (left panel), IRF1 downregulation was confirmed by western blotting (right panel). (D) Luciferase activity analysis of LP1-1 sequential deletions (LP1-1-ΔBD, LP1-1-BDΔ1, LP1-1-BDΔ1-2, LP1-1-BDΔ2-3, and LP1-1-BDΔ3) groups compare to LP1-1 group (upper panel), and of the CM deletion and six-point mutants (LP1-1-ΔCM, LP1-1-CM1[C > T], LP1-1-CM2[A > G], LP1-1-CM3[A > C], LP1-1-CM4[G > T], LP1-1-CM5[A > G], and LP1-1-CM6[C > T]) groups compare to LP1-1 group (bottom panel) under IRF1 overexpression. (E) GFP transcription directed by LP1-1, LP1-1-ΔCM, LP1-1-CM1[C > T], LP1-1-CM2[A > G], LP1-1-CM3[A > C], LP1-1-CM4[G > T], LP1-1-CM5[A > G], and LP1-1-CM6[C > T] mutants under IRF1 overexpression were analyzed by RT-PCR. (F) A model of competitive binding loci for IRF1 and ATG10S on the CM and their corresponding inductive activity. + indicates endogenous IRF1 or ATG10S expression, ++ indicates IRF1 or ATG10S overexpression, – indicates endogenous IRF1 or ATG10S knockdown. * indicates P < 0.05, ** and ## indicates P < 0.01, *** and ### indicates P < 0.001. All data are expressed as the mean ± SD (n = 3)

Article Snippet: To knockdown the expression of IRF1, IRF1 -siRNA was obtained from Santa Cruz Biotechnology, and siRNA transfection was performed as described in the user manual (sc-35706).

Techniques: Activity Assay, Luciferase, Quantitative RT-PCR, Binding Assay, Immunoprecipitation, Reverse Transcription Polymerase Chain Reaction, Western Blot, Over Expression, Expressing, Knockdown

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: bioRxiv

Article Title: IFNγ-induced memory in human macrophages is not sustained by epigenetic changes but the durability of the cytokine itself

doi: 10.1101/2025.06.12.659073

Figure Lengend Snippet: Macrophages were treated with LPS, IFNγ, and LPS in the presence of ruxolitinib for 8 hours as in . Cells were washed and cultured for an additional 88 hours. ATACseq performed after 8 hours of stimulation and 4 days post-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-kB (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 (100ng/mL), LPS (100ng/mL), or IFNβ (10ng/mL) for 8 hours, washed, and then cultured for an addition 66 hours. Cells were collected and whole cell western blotting for phosphorylated STAT1 was performed at indicated timepoints. Blot is representative of 3 replicates from 2 separate human donors. H. Human macrophages were stimulated with IFNγ (100ng/mL) for 8 hours, washed, and then cultured in regular media or media containing ruxolitinib (1µM) or increasing concentrations of anti-IFNγ neutralizing antibody for an additional 28 hours. Cells were collected and whole cell western blotting for phosphorylated STAT1 and IRF1 was performed at indicated timepoints. Blot is representative of 2 replicates from separate human donors. I. Heatmap of Z-scored reads within ATAC peaks induced by IFNγ (L2FC >2, FDR < 0.01) after 8 hours of stimulation for 4 days post-washout when cultured in regular media or media with 1µM ruxolitinib. Clusters were generated by unsupervised k -means clustering. Each column represents a biological replicate from the same human donor. J. Boxplot of log2CPM of reads within each peaks for each cluster in (I). All box/whisker plots indicate interquartile range and 1.5x interquartile range. Statistical tests determined by paired Wilcoxon test. *** p <0.001, **** p <0.0001

Article Snippet: The following primary antibodies were used: pSTAT1 pY701.4A (Santa Cruz Biotechnology, #136229) diluted at 1:10,000; IRF1 D5E4 (Cell Signaling Technologies, #8478) diluted at 1:1000; β-tubulin TUB2.1 (Sigma-Aldrich, T5201) diluted at 1:10,000.

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

Journal: bioRxiv

Article Title: IFNγ-induced memory in human macrophages is not sustained by epigenetic changes but the durability of the cytokine itself

doi: 10.1101/2025.06.12.659073

Figure Lengend Snippet: A. Human macrophages were treated with IFNγ (100ng/mL) for 8 hours, washed, and cultured for an additional 88 hours in the presence and absence of ruxolitinib (1µM). RNAseq was performed after 8 hours of IFNγ-stimulation and 4 days post-washout. Heatmap of Log2 fold change in RNAseq reads of genes induced at least 5-fold after 8 hours of IFNγ stimulation. Log2 fold changes are shown after washout for cells cultured in regular media and media containing 1µM ruxolitinib. Genes are clustered by persistent level of expression post washout (CPM post wash as percent of CPM at 8h simulation). B. Boxplot showing Log2 fold changes of individual genes by cluster in (B). Box/whisker plots indicate interquartile range and 1.5x interquartile range. Statistical tests determined by paired Wilcoxon test. *** p <0.001, **** p <0.0001 C. Macrophages were stimulated and washed as above in (A), after washout cells were cultured in media alone, media with 1µM ruxolitinib, or 8µg/mL anti-IFNγ neutralizing antibody for 88 hours. Cells were collected 88 hours after washout and qPCR performed for IDO1. Boxplots indicate 2 ΔΔCt normalized to HPRT. Errors bars indicate standard deviation. D. qPCR for IRF1 as in (C)

Article Snippet: The following primary antibodies were used: pSTAT1 pY701.4A (Santa Cruz Biotechnology, #136229) diluted at 1:10,000; IRF1 D5E4 (Cell Signaling Technologies, #8478) diluted at 1:1000; β-tubulin TUB2.1 (Sigma-Aldrich, T5201) diluted at 1:10,000.

Techniques: Cell Culture, Expressing, Whisker Assay, Standard Deviation

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: C57BL/6JCya- Irf1 em1/Cya , Cyagen Biosciences , N/A.

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: C57BL/6JCya- Irf1 em1/Cya , Cyagen Biosciences , N/A.

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: C57BL/6JCya- Irf1 em1/Cya , Cyagen Biosciences , N/A.

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: C57BL/6JCya- Irf1 em1/Cya , Cyagen Biosciences , N/A.

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