human pro il 1beta  (Sino Biological)

Journal: Communications Biology

Article Title: PCBP1 binding to single-stranded poly-cytosine motifs enhances cGAS sensing and impairs breast cancer development

doi: 10.1038/s42003-025-09456-z

Figure Lengend Snippet: A Kaplan–Meier analysis for TCGA breast cancer patients representing survival probability over time for low (blue), medium (white) and high (red) expression of PCBP1 mRNA. B Representation of the murine PyMT Pcbp1+/+ vs. PyMT Pcbp1–/– breast cancer models. Created in BioRender. Frereux, C. (2025) https://BioRender.com/gnssy36 C Immunohistochemical analysis of PCBP1 and hematoxylin/eosin staining of PyMT Pcbp1 +/+ and PyMT Pcbp1 –/– tumors (scale bars: 100 µm). D Representative images of all the tumors collected in one PyMT Pcbp1 +/+ mouse vs. one PyMT Pcbp1 –/– mouse. E Number of tumors, and F average total tumor burden per PyMT Pcbp1 +/+ ( n = 13), PyMT Pcbp1 +/− ( n = 12) and PyMT Pcbp1 –/– ( n = 21) mouse. (Mean ± SEM, two-tailed unpaired t test for two-by-two analysis and one-way ANOVA, **** P value < 0.0001 for all three groups analyzed.

Article Snippet: The final reaction was composed of 100 μM ATP, 100 μM GTP, 30 nM human His-tagged recombinant cGAS (cat. no. 22810, Cayman Chemical), 10 mM Tris-HCl pH 7.5, 2.5 mM MgCl 2 and 0.005% Tween20 and with or without 60 nM of His-tagged recombinant PCBP1 (cat. no. 11628-H07E, Sino Biological).

Techniques: Expressing, Immunohistochemical staining, Staining, Two Tailed Test

Journal: Communications Biology

Article Title: PCBP1 binding to single-stranded poly-cytosine motifs enhances cGAS sensing and impairs breast cancer development

doi: 10.1038/s42003-025-09456-z

Figure Lengend Snippet: A Volcano plot of the RNA-sequencing analysis representing the genes differentially expressed between PyMT Pcbp1 –/– ( n = 6) and PyMT Pcbp1 +/+ ( n = 5) mammary tumors. B Heat map showing the relative expression value (Log2 normalized counts CPM) of top 20 downregulated protein-coding genes ( p < 0.001) in PyMT Pcbp1 –/– versus PyMT Pcbp1 +/+ tumors ranked by fold change. C GSEA analysis with MSigDB M5 (ontology), based on RNA-sequencing results, representing the normalized enrichment score of significantly upregulated and downregulated gene sets in PyMT Pcbp1 –/– versus PyMT Pcbp1 +/+ tumors. D Enrichment plots of ‘Response to Type I Interferon’ (NES = −2.76, FDR q value < 0.0001) and ‘Type I Interferon Production’ (NES = −2.60, FDR q value < 0.0001) gene sets based on GSEA analysis (M5 ontology). E RT-qPCR analysis of Pcbp1 and several ISGs’ expression: Isg15, Irf7, Ifit11, Rsad2 and Bst2 , and of Ifnb1 in PyMT Pcbp1 –/– ( n = 23 except for Ifit1, Bst2, Rsad2 n = 16) and PyMT Pcbp1 +/+ ( n = 24, except for Ifit1, Bst2, Rsad2 n = 13) tumors (mean ± SEM, two-tailed unpaired t test). F Western blot analysis of HSP90, ISG15, IRF7 and a downstream activation marker of IFN signaling: phospho-STAT1 as well as total STAT1 in PyMT Pcbp1 +/+, +/–, and –/– tumors. G Immunohistochemical analysis of ISG15 in PyMT Pcbp1 +/+ and PyMT Pcbp1 –/– tumors (scale bars: 100 µm) and H its quantification (mean ± SEM, two-tailed unpaired t test). I Cross-correlation between the expression of PCBP1 and the interferon-stimulated genes ISG15, IRF3 and IRF7 in 1247 human breast tumor samples from the Cancer Genome Atlas BRCA RNA-sequencing database. J Correlation analysis between PCBP1 and ISG expression. ISG15 over IRF3 expression is provided as a positive correlation control (left panel). Gene expression is represented in Log2 (normalized count + 1), and Pearson’s rho ( r = ) and P value ( p = ) are provided for each correlation.

Article Snippet: The final reaction was composed of 100 μM ATP, 100 μM GTP, 30 nM human His-tagged recombinant cGAS (cat. no. 22810, Cayman Chemical), 10 mM Tris-HCl pH 7.5, 2.5 mM MgCl 2 and 0.005% Tween20 and with or without 60 nM of His-tagged recombinant PCBP1 (cat. no. 11628-H07E, Sino Biological).

Techniques: RNA Sequencing, Expressing, Quantitative RT-PCR, Two Tailed Test, Western Blot, Activation Assay, Marker, Immunohistochemical staining, Control, Gene Expression

Journal: Communications Biology

Article Title: PCBP1 binding to single-stranded poly-cytosine motifs enhances cGAS sensing and impairs breast cancer development

doi: 10.1038/s42003-025-09456-z

Figure Lengend Snippet: A UMAP plot from snRNA-seq analysis showing the different annotated cell type clusters in PyMT Pcbp1 +/+ ( n = 2) and PyMT Pcbp1 –/– ( n = 2) mammary tumors. B UMAP plot representing the expression of Pcbp1, Isg15 and Oas2 in the annotated cell types in pooled PyMT Pcbp1 +/+ and PyMT Pcbp1–/– groups. C Volcano plot of the average log fold change versus –log10(FDR) for interferon-stimulated genes differentially expressed across all the annotated cell types from the snRNA-seq analysis. D GSEA analysis with M5 (ontology), based on single nuclei RNA-sequencing results, representing the normalized enrichment score of significantly upregulated and downregulated gene sets specifically in the mammary epithelial cells of PyMT Pcbp1 –/– versus PyMT Pcbp1 +/+ tumors. E RT-qPCR analysis of Isg15, Irf7, Ifit1 and Ifnb1 expression in Py8119 Pcbp1 +/+ ( n = 5), Pcbp1 –/– ( n = 5) and Pcbp1 –/– rescued with V5-tagged PCBP1 ( n = 3), as well as in EMT6 shScramble ( n = 7) versus sh PCBP1 cells ( n = 7). F Immunoblot analysis of PCBP1, ISG15 and HSP90 levels in Py8119 and EMT6 shScramble versus sh PCBP1 cell lines. Data are represented as mean ± SEM, two-tailed unpaired t test.

Article Snippet: The final reaction was composed of 100 μM ATP, 100 μM GTP, 30 nM human His-tagged recombinant cGAS (cat. no. 22810, Cayman Chemical), 10 mM Tris-HCl pH 7.5, 2.5 mM MgCl 2 and 0.005% Tween20 and with or without 60 nM of His-tagged recombinant PCBP1 (cat. no. 11628-H07E, Sino Biological).

Techniques: Expressing, RNA Sequencing, Quantitative RT-PCR, Western Blot, Two Tailed Test

Journal: Communications Biology

Article Title: PCBP1 binding to single-stranded poly-cytosine motifs enhances cGAS sensing and impairs breast cancer development

doi: 10.1038/s42003-025-09456-z

Figure Lengend Snippet: A Single-nuclei RNA-seq analysis representing the Log2FC expression of significantly regulated MHC class I molecules and beta-2 microglobulin ( B2m ) in PyMT Pcbp1 –/– mammary epithelial cells ( n = 2) compared to PyMT Pcbp1 +/+ ( n = 2). B Enrichment plots of ‘Interferon Gamma Response’ (NES = −3.06, FDR q value < 0.0001) and ‘TNFalpha Signaling via NFkappaB’ (NES = −1.82, FDR q value = 0.0011) gene sets from GSEA analysis with MSigDB Hallmark ( H ) based on bulk RNA-seq data from PyMT Pcbp1 –/– vs PyMT Pcbp1 +/+ mammary tumors. C Cytotoxic T-cell infiltration analysis in PyMT Pcbp1 +/+ ( n = 10) versus PyMT Pcbp1 –/– ( n = 8) tumors by flow cytometry using the CD3 and CD8 markers. D Dual immunohistofluorescence of CD3 and CD8 markers in Pcbp1 +/+ ( n = 2) vs. PyMT PCBP1 –/– ( n = 2) tumors with ( E ). Its quantification (three fields counted per sample, scale bars: 100 µm). F Cxcl9 and G Cxcl10 expression in normalized count per million from bulk RNA-sequencing analysis in PyMT Pcbp1 +/+ ( n = 5) vs PyMT Pcbp1 –/– ( n = 6) tumors. H RT-qPCR analysis of Cxcl10 expression (fold change) in Py8119 Pcbp1 −/− ( n = 6) and Pcbp1 −/− rescued with V5- PCBP1 WT ( n = 3) relative to Py8119 Pcbp1 +/+ ( n = 7). I RT-qPCR analysis of Cxcl10 in Py8119 Pcbp1 −/− ( n = 6) vs Py8119 Pcbp1 + /+ ( n = 7) following vehicle or 85 ng/mL IFN-β for 12 h. Data are represented as mean ± SEM, two-tailed unpaired t test.

Article Snippet: The final reaction was composed of 100 μM ATP, 100 μM GTP, 30 nM human His-tagged recombinant cGAS (cat. no. 22810, Cayman Chemical), 10 mM Tris-HCl pH 7.5, 2.5 mM MgCl 2 and 0.005% Tween20 and with or without 60 nM of His-tagged recombinant PCBP1 (cat. no. 11628-H07E, Sino Biological).

Techniques: RNA Sequencing, Expressing, Flow Cytometry, Immunohistofluorescence, Quantitative RT-PCR, Two Tailed Test

Journal: Communications Biology

Article Title: PCBP1 binding to single-stranded poly-cytosine motifs enhances cGAS sensing and impairs breast cancer development

doi: 10.1038/s42003-025-09456-z

Figure Lengend Snippet: A RT-qPCR analysis of Isg15, Irf7 and Ifit1 in Py8119 Pcbp1 +/+ and Pcbp1 –/– cells after vehicle or 85 ng/mL IFN-β treatment (mean ± SEM, two-tailed unpaired t test). B Immunoblot of cGAS-STING-type I IFN activation marker: p-STING, total STING, two ISGs: IRF3, ISG15, as well as HSP90 and V5, in Py8119 Pcbp1 +/+ cells vs Pcbp1 –/– or Pcbp1 –/– rescued with V5-tagged WT PCBP1 . C Immunoblot of the indicated markers in parental, shScramble and sh PCBP1 EMT6 mouse breast cancer cell line or ( D ) in HMLE human mammary epithelial cells. E Representation of four cell lines generated: Py8119 Pcbp1 +/+, Pcbp1 –/–, and Pcbp1 –/– cells rescued with either V5-tagged wild-type PCBP1 or a V5-tagged PCBP1 mutant in which all KH domain GxxG loops were mutated to GDDG to impair single-stranded nucleic acid binding. Expression of the V5-PCBP1 WT and GDDG-mutant constructs was confirmed by RT-PCR using primers targeting the wild-type or GDDG-mutant KH3 domain of Pcbp1 in forward and the V5 tag in reverse. Cells were then transfected with either a fluorescently labeled single-stranded (ssDNA) or double-stranded (dsDNA) HSV120 oligonucleotide containing poly-cytosine motifs. F cGAS activity assay measuring 2’3’-cGAMP production in cell lysates by ELISA (mean ± SEM, two-tailed unpaired t test, asterisks indicate statistical comparisons between each bar and the immediately preceding condition). G as well as its associated western blot on these same samples for the indicated markers after treatment with vehicle (lipofectamine 3000 only) or 1.5 µg of HSV120 ssDNA or 1.5 µg of HSV120 dsDNA +/− cGAS inhibitor (TDI-6570) for 20 hours.

Article Snippet: The final reaction was composed of 100 μM ATP, 100 μM GTP, 30 nM human His-tagged recombinant cGAS (cat. no. 22810, Cayman Chemical), 10 mM Tris-HCl pH 7.5, 2.5 mM MgCl 2 and 0.005% Tween20 and with or without 60 nM of His-tagged recombinant PCBP1 (cat. no. 11628-H07E, Sino Biological).

Techniques: Quantitative RT-PCR, Two Tailed Test, Western Blot, Activation Assay, Marker, Generated, Mutagenesis, Binding Assay, Expressing, Construct, Reverse Transcription Polymerase Chain Reaction, Transfection, Labeling, Activity Assay, Enzyme-linked Immunosorbent Assay

Journal: Communications Biology

Article Title: PCBP1 binding to single-stranded poly-cytosine motifs enhances cGAS sensing and impairs breast cancer development

doi: 10.1038/s42003-025-09456-z

Figure Lengend Snippet: A EMSA showing human PCBP1 (left) or human cGAS (center) or both proteins (right) binding to the single-stranded poly-cytosine HSV120 ssDNA tagged with Cyanine 5 (units are in pmoles). B Representation of the Cyanine 5-tagged Gmut-HSV120 ssDNA oligo used in the following EMSA, where the first cytosines of the polyC tracts have been mutated to guanines. C EMSA showing human PCBP1 or human cGAS binding to the Gmut-HSV120 ssDNA tagged with Cyanine 5. D EMSA showing human PCBP1 (left) or human cGAS binding (right) to the double-stranded poly-cytosine HSV120 dsDNA tagged with Cyanine 5 in forward and 5’6’FAM in reverse. E EMSA quantification F Streptavidin pulldown of biotinylated HSV120 dsDNA versus Gmut-HSV120 dsDNA oligos in Pcbp1 +/+ and –/– lysates followed by immunoblotting for cGAS, PCBP1 and HSP90 as a negative control in the pulldown and loading control in the input.

Article Snippet: The final reaction was composed of 100 μM ATP, 100 μM GTP, 30 nM human His-tagged recombinant cGAS (cat. no. 22810, Cayman Chemical), 10 mM Tris-HCl pH 7.5, 2.5 mM MgCl 2 and 0.005% Tween20 and with or without 60 nM of His-tagged recombinant PCBP1 (cat. no. 11628-H07E, Sino Biological).

Techniques: Binding Assay, Western Blot, Negative Control, Control

Journal: Communications Biology

Article Title: PCBP1 binding to single-stranded poly-cytosine motifs enhances cGAS sensing and impairs breast cancer development

doi: 10.1038/s42003-025-09456-z

Figure Lengend Snippet: A Schematic representation of C4-YSD (poly-C-rich) and Gmut-YSD (cytosines mutated to guanines) DNA duplexes. B Electrophoretic mobility shift assays showing in vitro binding of increasing concentrations of recombinant human PCBP1 (left) or human cGAS (right) to Cy5/5′6-FAM-labeled C4-YSD DNA (units are in pmoles). C EMSA showing in vitro binding of increasing concentrations of recombinant human PCBP1 or human cGAS to Cy5/5′6-FAM-labeled Gmut-YSD DNA. D EMSA quantification. E Streptavidin pulldown assays from Py8119 Pcbp1 WT or Pcbp1 KO cell lysates incubated with biotin-labeled C4-YSD or Gmut-YSD followed by immunoblotting for cGAS, PCBP1 and HSP90 as a negative control in the pulldown and loading control in the input. F In vitro co-immunoprecipitation of recombinant human His-cGAS in the presence of recombinant human GST-PCBP1 and with or without C4-YSD followed by His-tag and PCBP1 immunoblotting. G – I Human cGAS (30 nM) enzymatic activity (initial velocity, v ₀) measured in 30 seconds or 5 minutes reactions using sub-saturating (15 nM) or saturating (500 nM) C4-YSD concentrations in the absence or presence of human PCBP1 (60 nM). J Human cGAS (30 nM) enzymatic activity (initial velocity, v ₀) measured in 30 seconds using sub-saturating Gmut-YSD concentrations (15 nM, K ) or using sub-saturating HSV120 dsDNA concentrations (15 nM) in the absence or presence of human PCBP1 (60 nM). L Michaelis–Menten kinetics of human cGAS activity (30 nM) using increasing concentrations of C4-YSD DNA in the absence or presence of human PCBP1 (60 nM). Data are represented as mean ± SEM, two-tailed unpaired t test.

Article Snippet: The final reaction was composed of 100 μM ATP, 100 μM GTP, 30 nM human His-tagged recombinant cGAS (cat. no. 22810, Cayman Chemical), 10 mM Tris-HCl pH 7.5, 2.5 mM MgCl 2 and 0.005% Tween20 and with or without 60 nM of His-tagged recombinant PCBP1 (cat. no. 11628-H07E, Sino Biological).

Techniques: Electrophoretic Mobility Shift Assay, In Vitro, Binding Assay, Recombinant, Labeling, Incubation, Western Blot, Negative Control, Control, Immunoprecipitation, Activity Assay, Two Tailed Test

Journal: Communications Biology

Article Title: PCBP1 binding to single-stranded poly-cytosine motifs enhances cGAS sensing and impairs breast cancer development

doi: 10.1038/s42003-025-09456-z

Figure Lengend Snippet: PCBP1 binds cytosolic DNA containing accessible single-stranded poly-cytosine motifs and increases cGAS affinity for these nucleic acids, resulting in reduced K 1/2 and enhanced production of 2’3’-cGAMP at sub-saturating DNA concentrations. Elevated 2’3’-cGAMP drives downstream STING activation, leading to increased type I interferon and chemokine (CXCL9/10) secretion. This promotes CD8⁺ T-cell infiltration, enhances MHC-I expression on tumor cells, and facilitates tumor cell killing, thereby impairing tumorigenesis. Created in BioRender. Frereux, C. (2025) https://BioRender.com/hvrezkp .

Article Snippet: The final reaction was composed of 100 μM ATP, 100 μM GTP, 30 nM human His-tagged recombinant cGAS (cat. no. 22810, Cayman Chemical), 10 mM Tris-HCl pH 7.5, 2.5 mM MgCl 2 and 0.005% Tween20 and with or without 60 nM of His-tagged recombinant PCBP1 (cat. no. 11628-H07E, Sino Biological).

Techniques: Activation Assay, Expressing