primary antibody upp1  (Proteintech)

Journal: bioRxiv

Article Title: Cancer-driven neutrophil priming couples systemic epithelial regenerative programs with pre-metastatic niche formation

doi: 10.1101/2025.10.20.683483

Figure Lengend Snippet: a) Pathways enriched in mature bone marrow (BM) neutrophils from tumour bearing (TB) versus Naïve mice. Inflammatory and metabolic pathways are coloured in red and blue, respectively; enrichment P values are indicated. b) LC–MS–based metabolic profiling of control or neutrophil-conditioned media. Neutrophils were isolated from lung or spleen of naïve or TB (4T1 Balb/c) mice (n=4 per group) and incubated in Advanced DMEM/F12 medium. Data are shown as mean z-score per metabolite. c-d) Abundance of uridine (c) and uracil (d) in neutrophil-conditioned or control media generated from lung or splenic neutrophils isolated from naïve or TB (4T1 Balb/c) mice, incubated in uridine-containing Plasmax medium (n=4 mice per group), ordinary one-way ANOVA. e) Schematic representation of Uridine phosphorylase 1 (Upp1) enzymatic activity. f) Upp1 mRNA expression in lung and splenic neutrophils from naïve or TB (PyMT-FVB) mice lung (n=5 per group, spleen n=4 per group), two-tailed students t-test. g) (Left) representative image and (Right) quantification of UPP1 immunofluorescence intensity in lung neutrophils (LuNeu) isolated from naïve or tumour bearing (PyMT-FVB) mice. Data are represented a per cell mean intensity (n=4). Welch’s t-test. h) Plasma Uracil abundance from Naïve (Balb/c) IgG, TB (4T1) IgG or TB (4T1) αLy6G mice measured by LC-MS (Naïve IgG and TB IgG n=10, TB αLy6G n=5), ordinary one-way ANOVA. i) Upp1 expression in circulating neutrophils from healthy volunteers (HV) and patients with breast cancer (P). Data are normalized counts from bulk RNA-seq of purified neutrophils (n=14 per group), two-tailed Wilcoxon test. Hormone-receptor status of patients is indicated. j) Plasma uracil concentrations in matched samples from HV and P with breast cancer (n=17 per group), quantified by LC–MS, two-tailed Wilcoxon test.

Article Snippet: Primary antibody Upp1 (Proteintech 14186-1-AP, 1:200) in IF Wash Buffer 1% BSA was incubated overnight at 4 °C prior to washing with PBS.

Techniques: Liquid Chromatography with Mass Spectroscopy, Control, Isolation, Incubation, Generated, Activity Assay, Expressing, Two Tailed Test, Immunofluorescence, Clinical Proteomics, RNA Sequencing, Purification

Journal: bioRxiv

Article Title: Cancer-driven neutrophil priming couples systemic epithelial regenerative programs with pre-metastatic niche formation

doi: 10.1101/2025.10.20.683483

Figure Lengend Snippet: a) PHATE embedding of bone marrow (BM) scRNAseq data from Naïve WT (C57/Bl6), Tumour-bearing (TB) (PyMT-C57/Bl6) WT and TB UPP1KO (PyMT-C57/Bl6) mice. Cells are coloured by cluster. Neutrophil and neutrophil progenitor clusters are outlined with dashed line. b) Granulopoiesis neutrophil and progenitor clusters from (a) represented by PHATE embedding. Cells are coloured by neutrophil and progenitor subtype as indicated in the legend. c) UPP1 expression across the dataset, shown per cell on the PHATE embedding (left) and as normalized expression across neutrophil subclusters (right) . d) MELD transcriptional perturbation analysis showing per-cell relative likelihoods for TB WT versus TB UPP1KO samples. Red indicates higher likelihood associated with the TB WT condition. e) RNA velocity and transcriptional dynamics across pro-neutrophil (Pro-Neu) cells. (Top) RNA velocity vectors overlaid on PHATE embedding indicating differentiation trajectories within the granulopoiesis dataset; Pro-Neu population marked in red. (Bottom) Predicted transcription (Y-axis) and degradation (X-axis) rates for individual genes in Pro-Neu cells derived from scVelo dynamical modelling. Dashed lines and arrow encircle a group of ‘protected’ transcripts with low degradation rates specific to the TB WT condition. f) Velocity length as a measure of differentiation rate within Pro-Neu population from Naïve WT, TB WT and TB UPP1KO mice. Data represent average velocity length per cell within the Pro-Neu population. Cell numbers are indicated in red, Kruskall-wallace test. g) Predicted degradation rates of eukaryotic translation initiation factor (eIF) transcripts within the stable RNAs in Pro-Neu population from TB mice (highlighted in g). Mean degradation rate for all transcripts is indicated in blue and ‘protected’ transcripts with degradation rate < 0.1 are shown in purple. h-k) In vivo translation rate measured by OP-Puro incorporation in Naïve WT, TB WT and TB UPP1KO mice. OP-Puro fluorescence intensity was quantified by flow cytometry in GMPs (h), (CD115 - Siglech - CD11b + Ly6C + Ly6G + cKit + ) Neutrophil progenitors (i) immature Ly6G low (j) and mature Ly6G high (k) bone marrow neutrophils isolated (Naïve WT n=4, TB WT n=3 TB UPP1KO n=5). Ordinary one-way ANOVA. l) Pathway enrichment analysis in mature neutrophils (M-Neu; from panel b) comparing TB WT and TB UPP1KO mice. Enrichment P values are shown. Inflammatory pathways are indicated in red, metabolic pathways in blue and transcription in black. m) Schematic of GMP colony-formation assay. FACS-sorted GMPs from naïve mice (FVB) were plated one cell per well with or without uracil. After colonies established, single GMP-derived colonies were profiled by FACS for cell number (colony size) and neutrophil/monocyte content (Ly6G+ and Ly6C+ cell count). n) Colony size (left) and Ly6G⁺ cell number per colony (right) from GMP colony-formation assay with or without uracil (n=6 mice. 62 PBS and 66 uracil-treated colonies) Mann-Whitney test. o) Schematic of bone-marrow chimera experiment. CD45.2 recipient mice (C57/Bl6) were reconstituted with either WT or UPP1KO CD45.1 bone marrow (C57/Bl6), generating chimeras with 10% WT CD45.1 and 90% WT or UPP1KO CD45.2 cells. This setup allows profiling of WT CD45.1 cells within a WT or UPP1KO haematopoietic environment. Mire where transplanted with PyMT tumour cells (C57/Bl6). p) Ly6G and CD11b intensity in circulating WT or UPP1KO CD45.2+ (donor) neutrophils and in WT CD45.1+ (recipient) neutrophils from bone marrow chimeric mice. (WT n=24, UPP1KO n=23 mice) Welch’s t-test.

Article Snippet: Primary antibody Upp1 (Proteintech 14186-1-AP, 1:200) in IF Wash Buffer 1% BSA was incubated overnight at 4 °C prior to washing with PBS.

Techniques: Expressing, Derivative Assay, In Vivo, Fluorescence, Flow Cytometry, Isolation, Colony Assay, Cell Counting, MANN-WHITNEY