Journal: eLife

Article Title: A role for PVRL4-driven cell–cell interactions in tumorigenesis

doi: 10.7554/eLife.00358

Figure Lengend Snippet: ( A ) and ( B ) A series of PVRL4 deletion constructs were designed and their expression confirmed by Western blot. ( C ) PVRL4 mutants from ( A ) were tested for their ability to induce anchorage-independent colony formation in triplicate (error bars ± SD). ( D ) Cells with full-length PVRL4 or the cytoplasmic region deletion mutant were assayed for viability under conditions of anchorage deprivation by measuring total ATP content in cells cultured on ultra-low attachment plates for 72 hr. Values were normalized to an empty vector-transduced sample. Assays were performed in triplicate (error bars ± SD). ( E ) and ( F ) TL-HMECs expressing empty vector, full-length PVRL4 or cytoplasmic region deletion mutant containing cells were cultured on tissue culture-treated (adherent) or ultra-low attachment (suspension) dishes for 72 hr. RNA was isolated and mRNA levels for TGM1 ( E ) and KRT6A and IVL ( F ) were measured by RT-qPCR. Transcript levels were normalized to β-actin. qPCR was performed in quadruplicate (error bars ± SD). DOI: http://dx.doi.org/10.7554/eLife.00358.004

Article Snippet: For anchorage-independent colony formation assays in the presence of antibodies, the following antibodies were used: normal mouse IgG (MAB004; R&D Systems, Minneapolis, MN) and mouse anti-human PVRL4 IgG2B (MAB2659; R&D Systems) at 4 μg/ml.

Techniques: Construct, Expressing, Western Blot, Mutagenesis, Cell Culture, Plasmid Preparation, Suspension, Isolation, Quantitative RT-PCR

Journal: eLife

Article Title: A role for PVRL4-driven cell–cell interactions in tumorigenesis

doi: 10.7554/eLife.00358

Figure Lengend Snippet: ( A ) and ( B ) PVRL4 promotes cell clustering of TL-HMECs. Cells were dissociated off the tissue culture surface with trypsin-free cell dissociation buffer and kept in suspension for 1 hr. Small (3–5 cells) and large (>5 cells) cell clusters per field of view were counted, n = 3 (error bars ± SD). ( C ) GFP-labeled PVRL4-expressing TL-HMECs were allowed to aggregate with dsRed-labeled cells expressing either a PVRL1-targeting shRNA or a control shRNA. Representative phase-contrast and fluorescent images (red and green channels superimposed) are shown. ( D ) PVRL4 was co-expressed with the indicated shRNAs and anchorage-independent colony formation in TL-HMECs was assayed. Values were normalized to an empty vector-transduced sample. Assays were performed in triplicate (error bars ± SD). ( E ) PVRL4-expressing TL-HMECs were assayed for clustering in the presence of the indicated antibodies or isotype controls. Cell clusters were quantified as before. ( F ) Anchorage-independent growth induced by PVRL4 or an shRNA against PTEN was assayed in the presence of PVRL4-targeting antibody or control IgG. Colony numbers were normalized to the control sample. Anchorage-independent colony formation assays were performed in triplicate (error bars ± SD). DOI: http://dx.doi.org/10.7554/eLife.00358.005

Article Snippet: For anchorage-independent colony formation assays in the presence of antibodies, the following antibodies were used: normal mouse IgG (MAB004; R&D Systems, Minneapolis, MN) and mouse anti-human PVRL4 IgG2B (MAB2659; R&D Systems) at 4 μg/ml.

Techniques: Suspension, Labeling, Expressing, shRNA, Control, Plasmid Preparation

Journal: eLife

Article Title: A role for PVRL4-driven cell–cell interactions in tumorigenesis

doi: 10.7554/eLife.00358

Figure Lengend Snippet: PVRL4 expressing TL-HMECs were stably transduced with dsRed or GFP and mixed in equal proportions, followed by (i) immediate plating into semi-solid medium, or (ii) co-culturing on an adherent surface for 2 d, followed by plating into semi-solid medium. Resulting colonies were visualized under a fluorescent microscope and each colony was assessed for the presence of red and green fluorescence. Representative phase-contrast and fluorescent images (red and green channels) are shown. DOI: http://dx.doi.org/10.7554/eLife.00358.006

Article Snippet: For anchorage-independent colony formation assays in the presence of antibodies, the following antibodies were used: normal mouse IgG (MAB004; R&D Systems, Minneapolis, MN) and mouse anti-human PVRL4 IgG2B (MAB2659; R&D Systems) at 4 μg/ml.

Techniques: Expressing, Stable Transfection, Transduction, Microscopy, Fluorescence

Journal: eLife

Article Title: A role for PVRL4-driven cell–cell interactions in tumorigenesis

doi: 10.7554/eLife.00358

Figure Lengend Snippet: The colony formation efficiency of TL-HMECs transduced with empty vector or vector expressing PVRL4 was compared between unfiltered cell suspensions and cell suspensions that were filtered through a 35 μm nylon mesh strainer prior to plating into methylcellulose. Anchorage-independent colony formation assays were performed in triplicate (error bars ± SD). DOI: http://dx.doi.org/10.7554/eLife.00358.007

Article Snippet: For anchorage-independent colony formation assays in the presence of antibodies, the following antibodies were used: normal mouse IgG (MAB004; R&D Systems, Minneapolis, MN) and mouse anti-human PVRL4 IgG2B (MAB2659; R&D Systems) at 4 μg/ml.

Techniques: Transduction, Plasmid Preparation, Expressing

Journal: eLife

Article Title: A role for PVRL4-driven cell–cell interactions in tumorigenesis

doi: 10.7554/eLife.00358

Figure Lengend Snippet: PVRL4-expressing TL-HMECs were allowed to aggregate in the presence of the indicated antibodies or isotype controls. Representative images are shown. DOI: http://dx.doi.org/10.7554/eLife.00358.009

Article Snippet: For anchorage-independent colony formation assays in the presence of antibodies, the following antibodies were used: normal mouse IgG (MAB004; R&D Systems, Minneapolis, MN) and mouse anti-human PVRL4 IgG2B (MAB2659; R&D Systems) at 4 μg/ml.

Techniques: Expressing

Journal: eLife

Article Title: A role for PVRL4-driven cell–cell interactions in tumorigenesis

doi: 10.7554/eLife.00358

Figure Lengend Snippet: PVRL4-expressing TL-HMECs were assayed for clustering in the presence of the indicated antibodies or isotype controls. Cell clusters were quantified as before. DOI: http://dx.doi.org/10.7554/eLife.00358.010

Article Snippet: For anchorage-independent colony formation assays in the presence of antibodies, the following antibodies were used: normal mouse IgG (MAB004; R&D Systems, Minneapolis, MN) and mouse anti-human PVRL4 IgG2B (MAB2659; R&D Systems) at 4 μg/ml.

Techniques: Expressing

Journal: eLife

Article Title: A role for PVRL4-driven cell–cell interactions in tumorigenesis

doi: 10.7554/eLife.00358

Figure Lengend Snippet: ( A ) Schematics of chimeric constructs containing extracellular domains of PVRL4 or an shRNA-resistant version of PVRL1 fused to the transmembrane domain of CD8 (blue). ( B ) and ( C ) TL-HMECs were stably transduced with the indicated combinations of expression constructs and assayed for anchorage-independent growth ( B ) and clustering ( C ). Colony numbers were normalized to the control sample. Anchorage-independent colony formation assays were performed in triplicate (error bars ± SD). ( D ) Expression levels of endogenous and chimeric proteins were verified by Western blot. DOI: http://dx.doi.org/10.7554/eLife.00358.011

Article Snippet: For anchorage-independent colony formation assays in the presence of antibodies, the following antibodies were used: normal mouse IgG (MAB004; R&D Systems, Minneapolis, MN) and mouse anti-human PVRL4 IgG2B (MAB2659; R&D Systems) at 4 μg/ml.

Techniques: Construct, shRNA, Stable Transfection, Transduction, Expressing, Control, Western Blot

Journal: eLife

Article Title: A role for PVRL4-driven cell–cell interactions in tumorigenesis

doi: 10.7554/eLife.00358

Figure Lengend Snippet: TL-HMEC cells infected with PVRL4-CD8tm or empty vector were fixed with methanol and stained with goat polyclonal anti-PVRL4 antibody followed by anti-goat Alexa Fluor 488 secondary antibody. DOI: http://dx.doi.org/10.7554/eLife.00358.012

Article Snippet: For anchorage-independent colony formation assays in the presence of antibodies, the following antibodies were used: normal mouse IgG (MAB004; R&D Systems, Minneapolis, MN) and mouse anti-human PVRL4 IgG2B (MAB2659; R&D Systems) at 4 μg/ml.

Techniques: Infection, Plasmid Preparation, Staining

Journal: eLife

Article Title: A role for PVRL4-driven cell–cell interactions in tumorigenesis

doi: 10.7554/eLife.00358

Figure Lengend Snippet: Clustering assays were performed with TL-HMECs expressing the following transgenes: (i) empty vector/control shRNA; (ii) PVRL4-CD8tm/control shRNA; (iii) PVRL4-CD8tm/anti-PVRL1 shRNA; and (iv) a 1:1 mixture of (iii) and (i). DOI: http://dx.doi.org/10.7554/eLife.00358.013

Article Snippet: For anchorage-independent colony formation assays in the presence of antibodies, the following antibodies were used: normal mouse IgG (MAB004; R&D Systems, Minneapolis, MN) and mouse anti-human PVRL4 IgG2B (MAB2659; R&D Systems) at 4 μg/ml.

Techniques: Expressing, Plasmid Preparation, Control, shRNA

Journal: eLife

Article Title: A role for PVRL4-driven cell–cell interactions in tumorigenesis

doi: 10.7554/eLife.00358

Figure Lengend Snippet: ( A ) Cell surface-localized proteins interacting with HA/FLAG-tagged PVRL4, but not with HA/FLAG-tagged GFP, as determined by mass spectrometry. ( B ) TL-HMECs expressing HA/FLAG tagged PVRL4 or HA/FLAG-tagged GFP were detached from the adherent surface with the enzyme-free cell dissociation buffer and incubated in suspension for 1 hr. Immunoprecipitations were performed with HA beads, followed by Western blot with FLAG and integrin β4 antibody. ( C ) TL-HMECs expressing vector control, PVRL4, or myr-PI3K were stably transduced with the indicated shRNA constructs and integrin β4 levels were assayed by Western blot. ( D ) TL-HMECs from ( C ) were assayed for anchorage-independent colony formation. Colony numbers were normalized to the vector control sample. Assays were performed in triplicate (error bars ± SD). ( E ) TL-HMECs stably transduced with the indicated constructs were detached from the adherent surface with enzyme-free cell dissociation buffer and incubated in 0.5% methylcellulose in suspension for 6 hr or cultured on an adherent surface for 48 hr. Levels of pY416-SFK (Src family kinases), total SFK, and vinculin loading control were measured by Western blot. Band intensity was measured with ImageJ software. ( F ) PVRL4-expressing TL-HMEC cells transduced with control or anti-PVRL1 shRNA were incubated in suspension in the conditions indicated. Levels of pY416-SFK (Src family kinases) and tubulin loading control were measured by Western blot. Band intensity was measured with ImageJ software. ( G ) TL-HMECs stably expressing PVRL4 or control vector were assayed for anchorage-independent colony formation in the presence of PP2 or vehicle control. Colony numbers were normalized to the vector sample. Assays were performed in triplicate (error bars ± SD). ( H ) TL-HMECs expressing PVRL4 were stably transduced with the indicated shRNA constructs and assayed for anchorage-independent colony formation. Colony numbers were normalized to the vector control sample. Assays were performed in triplicate (error bars ± SD). ( I ) SHP-2 levels were assayed by Western blot in TL-HMEC lysates from ( H ). DOI: http://dx.doi.org/10.7554/eLife.00358.019

Article Snippet: For anchorage-independent colony formation assays in the presence of antibodies, the following antibodies were used: normal mouse IgG (MAB004; R&D Systems, Minneapolis, MN) and mouse anti-human PVRL4 IgG2B (MAB2659; R&D Systems) at 4 μg/ml.

Techniques: Mass Spectrometry, Expressing, Incubation, Suspension, Western Blot, Plasmid Preparation, Control, Stable Transfection, Transduction, shRNA, Construct, Cell Culture, Software

Journal: eLife

Article Title: A role for PVRL4-driven cell–cell interactions in tumorigenesis

doi: 10.7554/eLife.00358

Figure Lengend Snippet: ( A ) Anti-HA beads were used to immunoprecipitate HA/FLAG-PVRL4 from the indicated lysates. Immunoprecipitates were blotted with anti-integrin β4 and anti-FLAG antibodies. ( B ) Immunoprecipitations with anti-integrin β4 antibodies or with a control IgG were performed from TL-HMEC lysates expressing either full-length PVRL4 or its cytoplasmic deletion mutant. Immunoprecipitates and input lysates were blotted with anti-integrin β4 and anti-PVRL4 antibodies. Asterisks denote heavy chains of control and anti-integrin β4 antibodies, which cross-react with an anti-goat secondary antibody. DOI: http://dx.doi.org/10.7554/eLife.00358.020

Article Snippet: For anchorage-independent colony formation assays in the presence of antibodies, the following antibodies were used: normal mouse IgG (MAB004; R&D Systems, Minneapolis, MN) and mouse anti-human PVRL4 IgG2B (MAB2659; R&D Systems) at 4 μg/ml.

Techniques: Control, Expressing, Mutagenesis

Journal: eLife

Article Title: A role for PVRL4-driven cell–cell interactions in tumorigenesis

doi: 10.7554/eLife.00358

Figure Lengend Snippet: ( A ) A view from the integrated Genome Viewer program showing focal amplification of the PVRL4 locus in SUM190 cells. The degree of amplification is denoted by the intensity of the color. ( B ) PVRL4 mRNA was stably depleted from SUM190 cells by four independent shRNAs. Transcript levels were measured by RT-qPCR and normalized to β-actin. qPCR was performed in quadruplicate (error bars ± SD). PVRL4-depleted and control cells were assayed for clonogenic survival and anchorage-independent colony formation. Assays were performed in triplicate (error bars ± SD). All values were normalized to the uninfected control sample. ECM: extracellular matrix. ( C ) The PVRL4-CD8 chimeric construct was used to rescue the defect in clonogenic survival observed with RNAi-mediated PVRL4 depletion. Assays were performed in triplicate (error bars ± SD). Colony numbers were normalized to the control shRNA sample. ( D ) Expression levels of endogenous and chimeric proteins were verified by Western blot. DOI: http://dx.doi.org/10.7554/eLife.00358.021

Article Snippet: For anchorage-independent colony formation assays in the presence of antibodies, the following antibodies were used: normal mouse IgG (MAB004; R&D Systems, Minneapolis, MN) and mouse anti-human PVRL4 IgG2B (MAB2659; R&D Systems) at 4 μg/ml.

Techniques: Amplification, Stable Transfection, Quantitative RT-PCR, Control, Construct, shRNA, Expressing, Western Blot

Journal: eLife

Article Title: A role for PVRL4-driven cell–cell interactions in tumorigenesis

doi: 10.7554/eLife.00358

Figure Lengend Snippet: PVRL4 transcript levels were measured by RT-qPCR and normalized to β-actin. qPCR was performed in quadruplicate (error bars ± SD). DOI: http://dx.doi.org/10.7554/eLife.00358.026

Article Snippet: For anchorage-independent colony formation assays in the presence of antibodies, the following antibodies were used: normal mouse IgG (MAB004; R&D Systems, Minneapolis, MN) and mouse anti-human PVRL4 IgG2B (MAB2659; R&D Systems) at 4 μg/ml.

Techniques: Quantitative RT-PCR

Journal: eLife

Article Title: A role for PVRL4-driven cell–cell interactions in tumorigenesis

doi: 10.7554/eLife.00358

Figure Lengend Snippet: The clonogenic potential of the indicated cell lines in the presence of control or PVRL4 shRNA constructs was assessed. Assays were performed in triplicate (error bars ± SD). Colony numbers were normalized to the control shRNA sample. DOI: http://dx.doi.org/10.7554/eLife.00358.027

Article Snippet: For anchorage-independent colony formation assays in the presence of antibodies, the following antibodies were used: normal mouse IgG (MAB004; R&D Systems, Minneapolis, MN) and mouse anti-human PVRL4 IgG2B (MAB2659; R&D Systems) at 4 μg/ml.

Techniques: Control, shRNA, Construct

Journal: eLife

Article Title: A role for PVRL4-driven cell–cell interactions in tumorigenesis

doi: 10.7554/eLife.00358

Figure Lengend Snippet: Cell line growth in methylcellulose-containing media on an ultra-low attachment surface was assessed in the presence of control or PVRL4 shRNA constructs. Representative images are shown. DOI: http://dx.doi.org/10.7554/eLife.00358.028

Article Snippet: For anchorage-independent colony formation assays in the presence of antibodies, the following antibodies were used: normal mouse IgG (MAB004; R&D Systems, Minneapolis, MN) and mouse anti-human PVRL4 IgG2B (MAB2659; R&D Systems) at 4 μg/ml.

Techniques: Control, shRNA, Construct

Journal: eLife

Article Title: A role for PVRL4-driven cell–cell interactions in tumorigenesis

doi: 10.7554/eLife.00358

Figure Lengend Snippet: ( A ) and ( B ) Female nude mice were injected into their mammary fat pads with SUM190 cells expressing PVRL4-targeted or control shRNA (n = 10 per group, error bars ± SEM). The resulting tumors were excised, scaled ( A ), and photographed ( B ). ( C ) SUM185 cells were stably transduced with PVRL4-targeted or control shRNA and injected into the mammary fat pads of female nude mice (n = 10 per group, error bars ± SEM). Tumor volume was measured with calipers at the indicated time points. ( D ) Female nude mice with ∼50 mm 3 SUM190-eGFP xenografts were randomized into two cohorts (n = 7 per group) and injected with anti-PVRL4 monoclonal antibodies or control IgG on the indicated days. Tumor volume was measured with calipers (error bars ± SEM). ( E ) Levels of PVRL4 protein were measured in tumor lysates from anti-PVRL4 antibody or control-treated mice, 7 days after the last treatment. ( F ) Tumor sections from control IgG ( A – C ) or anti-PVRL4 antibody-treated ( D – F ) mice were stained with hematoxylin/eosin and photographed. Representative images are shown. DOI: http://dx.doi.org/10.7554/eLife.00358.029

Article Snippet: For anchorage-independent colony formation assays in the presence of antibodies, the following antibodies were used: normal mouse IgG (MAB004; R&D Systems, Minneapolis, MN) and mouse anti-human PVRL4 IgG2B (MAB2659; R&D Systems) at 4 μg/ml.

Techniques: Injection, Expressing, Control, shRNA, Stable Transfection, Transduction, Bioprocessing, Staining

Journal: eLife

Article Title: A role for PVRL4-driven cell–cell interactions in tumorigenesis

doi: 10.7554/eLife.00358

Figure Lengend Snippet: Freshly explanted tumors from control IgG or anti-PVRL4 antibody-treated mice (N = 3 per group) were visualized using a two-photon confocal microscope. Representative images are shown. DOI: http://dx.doi.org/10.7554/eLife.00358.031

Article Snippet: For anchorage-independent colony formation assays in the presence of antibodies, the following antibodies were used: normal mouse IgG (MAB004; R&D Systems, Minneapolis, MN) and mouse anti-human PVRL4 IgG2B (MAB2659; R&D Systems) at 4 μg/ml.

Techniques: Control, Microscopy

Journal: eLife

Article Title: A role for PVRL4-driven cell–cell interactions in tumorigenesis

doi: 10.7554/eLife.00358

Figure Lengend Snippet: Europium-labeled SUM190 cells were incubated with fresh human NK cells in the presence of an isotype control or anti-PVRL4 antibody, and the degree of lysis was measured by the DELFIA europium assay. The maximum signal was determined by a complete lysis of labeled SUM190 cells in DELFIA lysis buffer. As a positive control, hMB humanized mouse lymphoma cells were mixed with effector cells in the presence of ADCC-competent anti-CD52 antibody. ADCC: antibody-dependent cytotoxicity. DOI: http://dx.doi.org/10.7554/eLife.00358.032

Article Snippet: For anchorage-independent colony formation assays in the presence of antibodies, the following antibodies were used: normal mouse IgG (MAB004; R&D Systems, Minneapolis, MN) and mouse anti-human PVRL4 IgG2B (MAB2659; R&D Systems) at 4 μg/ml.

Techniques: Labeling, Incubation, Control, Lysis, Positive Control

Journal: eLife

Article Title: A role for PVRL4-driven cell–cell interactions in tumorigenesis

doi: 10.7554/eLife.00358

Figure Lengend Snippet: Paraffin-embedded sections of SUM190 xenografts from mice treated with either control IgG or anti-PVRL4 antibody were stained with anti-mouse F4/80 antibody. Representative images are shown. DOI: http://dx.doi.org/10.7554/eLife.00358.033

Article Snippet: For anchorage-independent colony formation assays in the presence of antibodies, the following antibodies were used: normal mouse IgG (MAB004; R&D Systems, Minneapolis, MN) and mouse anti-human PVRL4 IgG2B (MAB2659; R&D Systems) at 4 μg/ml.

Techniques: Control, Staining

Journal: eLife

Article Title: A role for PVRL4-driven cell–cell interactions in tumorigenesis

doi: 10.7554/eLife.00358

Figure Lengend Snippet: ( A ) Tumor lysates from control antibody- or anti-PVRL4 antibody-treated mice were blotted for E-cadherin and vimentin. ( B ) PVRL4 was stably depleted by two independent shRNAs in SUM190 cells and lysates were blotted for E-cadherin and vimentin. DOI: http://dx.doi.org/10.7554/eLife.00358.034

Article Snippet: For anchorage-independent colony formation assays in the presence of antibodies, the following antibodies were used: normal mouse IgG (MAB004; R&D Systems, Minneapolis, MN) and mouse anti-human PVRL4 IgG2B (MAB2659; R&D Systems) at 4 μg/ml.

Techniques: Control, Stable Transfection

Journal: eLife

Article Title: A role for PVRL4-driven cell–cell interactions in tumorigenesis

doi: 10.7554/eLife.00358

Figure Lengend Snippet: 293T cells were transfected with empty pQCXIN (green line), pQCXIN-human PVRL4 (blue line), or pCMV-SPORT6-mouse PVRL4 (magenta line). Live-cell FACS was performed with mouse anti-human PVRL4 antibody followed by anti-mouse secondary antibody conjugated to Alexa Fluor 488 fluorophore. The FITC-A fluorescent signal for three labeled cell populations is shown. DOI: http://dx.doi.org/10.7554/eLife.00358.035

Article Snippet: For anchorage-independent colony formation assays in the presence of antibodies, the following antibodies were used: normal mouse IgG (MAB004; R&D Systems, Minneapolis, MN) and mouse anti-human PVRL4 IgG2B (MAB2659; R&D Systems) at 4 μg/ml.

Techniques: Transfection, Labeling

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