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: Py8119 (cat. no. CRL-3278, ATCC), and EMT6 (gifted) cells were cultured in Dulbecco’s modified Eagle’s medium high glucose (cat. no. SH30081.01, GE Healthcare Life Sciences) supplemented with 10% fetal bovine serum (cat. no. SH30071.03, Cytiva).

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 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: Py8119 (cat. no. CRL-3278, ATCC), and EMT6 (gifted) cells were cultured in Dulbecco’s modified Eagle’s medium high glucose (cat. no. SH30081.01, GE Healthcare Life Sciences) supplemented with 10% fetal bovine serum (cat. no. SH30071.03, Cytiva).

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