pe anti mouse irf1 antibody  (Santa Cruz Biotechnology)

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 Virology

Article Title: Distinctive Roles for Type I and Type II Interferons and Interferon Regulatory Factors in the Host Cell Defense against Varicella-Zoster Virus

doi: 10.1128/JVI.01151-18

Figure Lengend Snippet: Comparative transcriptome analysis of IFN-α- versus IFN-γ-treated HELFs. (A) The scatter plot represents a summary of the differential expression of cell gene transcripts in IFN-α- versus IFN-γ-treated cells. The red dots indicate the genes that were significantly upregulated in the IFN-γ-treated cells, while the blue dots represent those that were upregulated in the IFN-α-treated cells. (B) A gene network map was constructed for genes that were exclusively upregulated in the IFN-γ-treated cells and not by IFN-α treatment compared to their expression in mock-treated HELFs. (C) Network map of the 17 of 93 genes that had significantly higher transcript levels in IFN-γ-treated cells than in IFN-α-treated cells. (D) Mean difference plots using IRF1 and IRF7 as examples of significant differences in expression levels between experimental groups, where the y axis is the log CPM expression level (***, P < 0.0005). UI, uninfected.

Article Snippet: PE mouse anti-human IRF1 , BD Biosciences , Flow cytometry.

Techniques: Expressing, Construct

Journal: Journal of Virology

Article Title: Distinctive Roles for Type I and Type II Interferons and Interferon Regulatory Factors in the Host Cell Defense against Varicella-Zoster Virus

doi: 10.1128/JVI.01151-18

Figure Lengend Snippet: IRF1 is critical for the IFN-γ-mediated host cell defense against VZV. (A) HELFs were stimulated with either IFN-α or IFN-γ for the indicated times and then analyzed for IRF1 expression compared to that in the untreated HELFs. Data were collected on an LSR II.UV analyzer using a 561-nm green laser for excitation of the phycoerythrin (PE)-IRF1 antibody. Data analysis was done using FlowJo (version 10.0.5) software, and data are represented as a histogram (left) showing IRF1 expression (x axis). The line graph (right) shows the proportion of cells expressing IRF1 at the indicated time points in IFN-α- and IFN-γ-treated cells. (B) HELFs were stimulated with IFN-γ for 24 h, following which cells were grown in the absence of any stimulation and subsequently analyzed for IRF1 expression. Data were collected and analyzed as described above. (C to E) IRF1-knockout (IRF1KO) stable Mel39 cells (gRNA1) were generated using the CRISPR-Cas9 system; gRNA4 Mel39 cells expressing the empty pLentiCRISPRv2 vector were used as a control. (C) The gRNA1, gRNA4, and wild-type (WT) cell lines were treated with IFN-γ for 24 h and evaluated for IRF1 expression knockdown by flow cytometry. (D) The gRNA1, gRNA4, and WT cell lines were inoculated with 50 PFU/ml of VZV after 24 h of IFN-γ pretreatment, and virus detection was done by immunohistochemistry at day 5 postinfection. (E) Growth curve analysis of VZV replication was carried out in the IRF1KO, control, and WT cells treated with either IFN-γ or IFN-α or untreated for 24 h prior to inoculation with VZV (500 PFU/ml). IFNs were withdrawn at the time of inoculation, and cells were harvested from replicate wells for each of the nine test conditions at days 1 to 6; virus titers were assessed using WT Mel39 cells. The data are representative of those from two similar independent experiments. (F) HELFs were infected with VZV-GFP for 48 hpi or mock infected and exposed to IFN-α or IFN-γ for 24 h or left untreated. Prior to fixation, cells were treated with 50 μM MG132 for 6 h. IRF1 expression was evaluated by flow cytometry using the phycoerythrin-IRF1 antibody followed by analysis with FlowJo software. The data are representative of those from five similar independent experiments. untxt and untx, untreated.

Article Snippet: PE mouse anti-human IRF1 , BD Biosciences , Flow cytometry.

Techniques: Expressing, Software, Knock-Out, Generated, CRISPR, Plasmid Preparation, Flow Cytometry, Immunohistochemistry, Infection

Journal: Journal of Virology

Article Title: Distinctive Roles for Type I and Type II Interferons and Interferon Regulatory Factors in the Host Cell Defense against Varicella-Zoster Virus

doi: 10.1128/JVI.01151-18

Figure Lengend Snippet: Antibodies used in the study

Article Snippet: PE mouse anti-human IRF1 , BD Biosciences , Flow cytometry.

Techniques: Western Blot, Immunofluorescence, Flow Cytometry, Immunohistochemistry

Journal: Journal of Virology

Article Title: Distinctive Roles for Type I and Type II Interferons and Interferon Regulatory Factors in the Host Cell Defense against Varicella-Zoster Virus

doi: 10.1128/JVI.01151-18

Figure Lengend Snippet: IRF1 guide RNA sequences

Article Snippet: PE mouse anti-human IRF1 , BD Biosciences , Flow cytometry.

Techniques: Sequencing