Journal: Nature Communications

Article Title: Gut-primed neutrophils activate Kupffer cells to promote hepatic injury in mouse sepsis

doi: 10.1038/s41467-025-65572-8

Figure Lengend Snippet: WT mice were subjected to CLP and the small intestines were harvested after 20 h. A The number (no.) of neutrophils in the gut epithelium. Experiments were performed 2 times, and all data were used for analysis. Data are expressed as mean ± SEM (n = 4 samples/group) and compared by paired two-tailed Student’s t test. P value: 1.11E-2. *p < 0.05 vs. Sham. B Representative microscopic images of the gut epithelium (original magnification, ×630; scale bar: 10 μm). Experiments were performed 3 times, and representative images are shown. Bone marrow - derived neutrophils (PMNs) were treated with or without LPS in the presence and absence of intraepithelial lymphocytes (IELs) at 1:1 ratio for 12 h to evaluate NETs. C Representative microscopic images from 3 independent experiments are shown (original magnification, ×200; scale bar: 100 μm). Ly6G (purple) serves as a neutrophil marker. D Representative dot plots following the gating strategy of NETs in Fig. and E frequency of NETs + (Cit-H3 + MPO + ) neutrophils assessed by flow cytometry are shown. Experiments were performed 3 times, and all data were used for analysis. Data are expressed as mean ± SEM (n = 6 samples/group) and compared by one-way ANOVA and SNK test. P values based on the order of appearance: 3.02E-7, 1.42E-6, 4.15E-5. *p < 0.05 vs. PBS, # p < 0.05 vs. LPS, † p < 0 . 05 vs. PBS+IELs. F The number of CD112 + neutrophils in the gut epithelium of sham and CLP mice. Experiments were performed 2 times, and all data were used for analysis. Data are expressed as mean ± SEM (n = 4 samples/group) and compared by paired two-tailed Student’s t test. P value: 1.11E-2. *p < 0.05 vs. Sham. Neutrophils were incubated with IELs and LPS in the presence of a vehicle (PBS, Veh.), isotype control (Iso.), or 10 μg/mL anti-CD112 Ab for 12 h to evaluate NETs by flow cytometry. G Representative dot plots following the gating strategy of NETs in Fig. and H frequency of NETs + neutrophils are shown. Experiments were performed 2 times, and all data were used for analysis. Data are expressed as mean ± SEM (n = 5 samples/group) and compared by one-way ANOVA and SNK test. P values based on the order of appearance: 6.21E-3, 2.52E-2. *p < 0.05 vs. Veh., # p < 0.05 vs. Iso. CLP cecal ligation and puncture.

Article Snippet: Neutrophils incubated with IELs and LPS were also treated with a vehicle (PBS), isotype control, or 10 μg/mL anti-CD112 Ab (Cat. No.: MAB3869; R&D Systems, Minneapolis, MN).

Techniques: Two Tailed Test, Derivative Assay, Marker, Flow Cytometry, Incubation, Control, Ligation

Journal: Nature Communications

Article Title: Gut-primed neutrophils activate Kupffer cells to promote hepatic injury in mouse sepsis

doi: 10.1038/s41467-025-65572-8

Figure Lengend Snippet: During sepsis, neutrophils interact with IELs via CD112 in the gut, resulting in increased NETosis via PAD4-mediated histone citrullination (Cit). NET-forming neutrophils migrate from the gut into the liver and activate PAR-1 on Kupffer cells by NET-contained proteases, such as NE. NET-activated Kupffer cells polarize to an iNOS-expressing M1 phenotype and produce proinflammatory mediators, such as IL-6 and TNF. This gut-liver crosstalk mediated by immune cells leads to sepsis-induced liver injury. PAD4, protein-arginine deiminase type-4; NET, neutrophil extracellular trap; PAR-1, protease-activated receptor-1; NE, neutrophil elastase; iNOS, inducible nitric oxide synthase. Created in BioRender. Murao, A. (2025) https://BioRender.com/fhkfis3 .

Article Snippet: Neutrophils incubated with IELs and LPS were also treated with a vehicle (PBS), isotype control, or 10 μg/mL anti-CD112 Ab (Cat. No.: MAB3869; R&D Systems, Minneapolis, MN).

Techniques: Expressing

Journal: Nature Communications

Article Title: Cancer cell plasticity defines response to immunotherapy in cutaneous squamous cell carcinoma

doi: 10.1038/s41467-024-49718-8

Figure Lengend Snippet: a Percentage of PD-L1 + cells within full epithelial cancer cells from epithelial cSCCs ( n = 27), EpCAM high , EpCAM low , and EpCAM − cancer cells from mixed cSCCs ( n = 23), and full mesenchymal cancer cells from mesenchymal cSCCs ( n = 27). b Percentage of CD112 + cells within the indicated cancer cells ( n = 12 tumors per group). c Percentage of Gal9 + cells within full epithelial cancer cells from epithelial cSCCs ( n = 27), EpCAM high , EpCAM low , and EpCAM − cancer cells from mixed cSCCs ( n = 17), and full mesenchymal cancer cells from mesenchymal cSCCs ( n = 21). d Percentage of CD80 + cells within full epithelial cancer cells from epithelial cSCCs ( n = 24), EpCAM high , EpCAM low , and EpCAM − cancer cells from mixed cSCCs ( n = 18), and full mesenchymal cancer cells from mesenchymal cSCCs ( n = 21). e Percentage of CD155 + cells within full epithelial cancer cells from epithelial cSCCs ( n = 10), EpCAM high , EpCAM low , and EpCAM − cancer cells from mixed cSCCs ( n = 9), and full mesenchymal cancer cells from mesenchymal cSCCs ( n = 12). f , h Representative immunofluorescence images of Ecad + (green), f CD80 + or h CD155 + (red), and DAPI nuclear (blue) staining in the indicated patient cSCCs. Scale bar, 100 µm. g Percentage of CD80 + cancer cells relative to total cancer cells in the indicated patient cSCCs ( n = 4 per group). i Percentage of CD155 + cancer cells relative to total cancer cells in epithelial ( n = 4), mixed ( n = 5), and mesenchymal ( n = 4) patient cSCCs. Each dot indicates the average quantification of at least five fields from different tumor regions. j Percentage of CD80 − Ecad + , CD80 + Ecad + , and CD80 + Ecad − cancer cells relative to total cancer cells in the indicated patient cSCCs ( n = 4 per group). k Percentage of CD155 − Ecad + , CD155 + Ecad + , and CD155 + Ecad − cancer cells relative to total cancer cells in epithelial ( n = 4), mixed ( n = 5), and mesenchymal ( n = 4) patient cSCCs. Data are represented as the mean ± SD ( a – e ) or ± SEM ( g , i , j , k ), and n values indicate independent tumors ( a – e , g , i ). P values are determined by one-way ANOVA with Dunnett’s ( a – e ) or Tukey’s ( g , i ) multiple comparison tests. See Supplementary Fig. for the gating strategy ( a – e ). Source data are provided as a Source Data file.

Article Snippet: For cell-surface staining, cells were blocked with 1 mg/ml IgG (Sigma, I5381) and stained with a cocktail of cell-surface antibodies in staining buffer (5% FBS in PBS) for 30 min at 4 °C: from Biolegend, CD11b-APC 1:250 (M1/70, 101211), CD11b-PE/Cy7 1:250 (M1/70, 101215), CD152 (CTLA-4)-PE/Cy7 1:250 (UC10-4B9, 106313), CD155-PE/Cy7 1:200 (TX56, 131511), CD223 (LAG-3)-PE/Cy7 1:250 (C9B7W, 125225), CD226 (DNAM-1)-PE/Cy7 1:250 (10E5, 128811), CD25-PE/Cy7 1:200 (PC61, 102015), CD274 (PD-L1)-PE/Cy7 1:200 (10F.9G2, 124313), CD279 (PD-1)-APC/Cy7 1:250 (29F.1A12, 135223), CD28-PE/Cy7 1:250 (37.51, 102125), CD3ε-APC 1:200 (145-2C11, 100311), CD366 (TIM-3)-PE/Cy7 1:250 (B8.2C12, 134009), CD4-PE/Cy7 1:200 (RM4-5, 100528), CD49f (α6-integrin)-FITC 1:10 (GoH3, 313605), CD69-PE/Cy7 1:200 (H1.2F3, 104511), CD8a-PE 1:200 (53-6.7, 100707), CD80-PE/Cy7 1:250 (16-10A1, 104733), F4/80-APC/Cy7 1:200 (BM8, 123118), Galectin9-PE/Cy7 1:250 (108A2, 137913), Ly-6G/Ly-6C (Gr-1)-PE/Cy7 1:250 (RB6-8C5, 108415), Ly-6C-PE/Cy7 1:250 (HK1.4, 128017), Ly-6G-APC 1:250 (1A8, 127613), NK-1.1-PE 1:200 (PK136, 108707), TIGIT (Vstm3)-PE/Cy7 1:250 (1G9, 142107); from BD Bioscience, CD11b-PE 1:250 (M1/70, 557397); from eBioscience, CD206-APC 1:200 (MR6F3, 17-2061-80), CD326 (EpCAM)-APC-eF780 1:400 (G8.8, 47-5791-82); from TONBO, CD45-PE 1:350 (30-F11, 50-0451); from R&D Systems, CD112 (Nectin-2)-APC 1:200 (829038, FAB3869A).

Techniques: Immunofluorescence, Staining, Comparison

Journal: iScience

Article Title: Heterogeneity of perivascular astrocyte endfeet depending on vascular regions in the mouse brain

doi: 10.1016/j.isci.2023.108010

Figure Lengend Snippet: Localization of nectin-2 δ in the mouse hippocampus (A) Localization of nectin-2δ (N2δ) in stratum lacunosum-moleculare of the hippocampus. a, artery/arteriole; b, vein/venule; and c, capillary. Arrowheads, arteries/arterioles; arrows, veins/venules; ∗, capillaries. Scale bars, 20 μm. (B) The ratio of nectin-2δ-positive blood vessels in the hippocampus is shown as a bar graph. (C) Co-localization of nectin-2δ and GFAP at artery/arteriole in stratum lacunosum-moleculare of the hippocampus. Scale bar, 20 μm. Line scan of fluorescence intensity of the white line (a–b). AU, arbitrary units. These images are representative of three independent experiments. See also Figures S9 and .

Article Snippet: Rat Anti-Mouse Nectin 2 Monoclonal Antibody, Unconjugated, Clone 502-57 , MBL International , Cat# D083-3, RRID: AB_590848.

Techniques: Fluorescence

Journal: iScience

Article Title: Heterogeneity of perivascular astrocyte endfeet depending on vascular regions in the mouse brain

doi: 10.1016/j.isci.2023.108010

Figure Lengend Snippet: Localization of nectin-2α/δ in the purified PV-AEF (A and B) Immunofluorescence images of the purified PV-AEF immunostained with the indicated Abs. The anti-nectin-2 Ab, which recognizes nectin-2α/δ, was used. The purified PV-AEF were identified by AQP4-positive and DAPI-negative cell fragments and vesicles. The areas encircled by the cyan dotted line indicate the areas of the purified PV-AEF. Scale bars, 5 μm. (C and D) Mean fluorescence intensity of nectin-2 and AQP4 was shown in the bar graphs. The area of PV-AEF was measured by the AQP4 signal and divided into two groups according to the median value. Data are presented as mean and SD (n = 28, 28), and dot shows each data. The Mann-Whitney U test was performed. These images are representative of three independent experiments.

Article Snippet: Rat Anti-Mouse Nectin 2 Monoclonal Antibody, Unconjugated, Clone 502-57 , MBL International , Cat# D083-3, RRID: AB_590848.

Techniques: Purification, Immunofluorescence, Fluorescence, MANN-WHITNEY

Journal: iScience

Article Title: Heterogeneity of perivascular astrocyte endfeet depending on vascular regions in the mouse brain

doi: 10.1016/j.isci.2023.108010

Figure Lengend Snippet:

Article Snippet: Rat Anti-Mouse Nectin 2 Monoclonal Antibody, Unconjugated, Clone 502-57 , MBL International , Cat# D083-3, RRID: AB_590848.

Techniques: Produced, Recombinant, Affinity Purification, Protease Inhibitor, Western Blot, Fluorsave, Bicinchoninic Acid Protein Assay, Double Staining, TUNEL Assay, Mass Spectrometry, Plasmid Preparation, Software