Journal: Frontiers in Immunology

Article Title: Lipocalin 2 Protects Against Escherichia coli Infection by Modulating Neutrophil and Macrophage Function

doi: 10.3389/fimmu.2019.02594

Figure Lengend Snippet: Primer sequences for the real-time PCR amplification.

Article Snippet: Serum Lcn2 was quantified using Lcn2 Mouse ELISA kits (Boster, China) according to the manufacturer's instructions.

Techniques: Real-time Polymerase Chain Reaction, Amplification

Journal: Frontiers in Immunology

Article Title: Lipocalin 2 Protects Against Escherichia coli Infection by Modulating Neutrophil and Macrophage Function

doi: 10.3389/fimmu.2019.02594

Figure Lengend Snippet: Elevated lipocalin 2 (Lcn2) during E. coli O157:H7 infection. (A) Real-time PCR analysis of the levels of Lcn2 mRNA expression in indicated tissues of mice. (B) The mRNA levels of Lcn2 expression in indicated tissues of control and E. coli O157:H7-infected mice. (C) Serum levels of Lcn2 protein concentration in E. coli O157:H7-infected mice after challenge at different time points. (D–G) The mRNA expression levels of Lcn2 in indicated tissues from E. coli O157:H7-infected mice after challenge at different time points. (H) Protein levels of Lcn2 in mice detected on immunohistochemistry sections of liver and jejunum at 8 and 32 h after challenge with E. coli O157:H7. Original magnification was 200×. Values are average means of triplicate experiments. Error bars depict SEM ( n = 4 per time point). Results are expressed as means ± SEM. * P < 0.05, and ** P < 0.01.

Article Snippet: Serum Lcn2 was quantified using Lcn2 Mouse ELISA kits (Boster, China) according to the manufacturer's instructions.

Techniques: Infection, Real-time Polymerase Chain Reaction, Expressing, Control, Protein Concentration, Immunohistochemistry

Journal: Frontiers in Immunology

Article Title: Lipocalin 2 Protects Against Escherichia coli Infection by Modulating Neutrophil and Macrophage Function

doi: 10.3389/fimmu.2019.02594

Figure Lengend Snippet: The bacteriostatic characteristics of lipocalin 2 (Lcn2). (A,B) Bacterial loads in blood (CFU/ml) and livers (CFU/mg) of E. coli O157:H7-infected mice 32 hpi. (C,D) Serum levels of Lcn2 protein measured and growth of E. coli O157:H7 in RPMI with 20% acute-phase serum from wild-type (WT) or Lcn2 −/− mice. Values are average means of triplicate experiments with two mice per genotype per experiment. Error bars depict SEM ( n = 6 per group). Results are expressed as means ± SEM. P < 0.05 was considered statistically significant. * P < 0.05.

Article Snippet: Serum Lcn2 was quantified using Lcn2 Mouse ELISA kits (Boster, China) according to the manufacturer's instructions.

Techniques: Infection

Journal: Frontiers in Immunology

Article Title: Lipocalin 2 Protects Against Escherichia coli Infection by Modulating Neutrophil and Macrophage Function

doi: 10.3389/fimmu.2019.02594

Figure Lengend Snippet: Granulocyte abnormalities in Lcn2 −/− mice. (A) Hematological parameters of peripheral blood from wild-type (WT) and Lcn2 −/− mice. The data are presented as mean × 10 3 cells/μl. Error bars depict SEM ( n = 6 per group). (B) Flow cytometry analysis of neutrophils in the peripheral blood after intragastric administration with 2 × 10 8 CFU of E. coli O157:H7. Cells were stained with indicated clones of Gr-1 Ab, and positive cells were determined by flow cytometry. Values are average means of triplicate experiments with two mice per genotype per experiment. Error bars depict SEM. (C) Wright-Giemsa staining of peripheral blood smears from Lcn2 −/− mice identified atypical hyposegmented neutrophils in the peripheral blood. Original magnification ×63. In contrast, WT mice displayed normal neutrophil maturation. (D) Enumeration of the number of band neutrophils in the peripheral blood of Lcn2 −/− mice. The data are presented as mean band cell numbers per 100 leukocytes. Error bars depict SEM. Results are expressed as means ± SEM. P < 0.05 was considered statistically significant. * P < 0.05, and ** P < 0.01.

Article Snippet: Serum Lcn2 was quantified using Lcn2 Mouse ELISA kits (Boster, China) according to the manufacturer's instructions.

Techniques: Flow Cytometry, Staining, Clone Assay

Journal: Frontiers in Immunology

Article Title: Lipocalin 2 Protects Against Escherichia coli Infection by Modulating Neutrophil and Macrophage Function

doi: 10.3389/fimmu.2019.02594

Figure Lengend Snippet: Reduced migration of lipocalin 2-deficient ( Lcn2 −/− ) neutrophils. (A,B) Flow cytometry analysis of peripheral blood and peritoneal exudates of heat-killed E. coli O157:H7-challenged mice following staining with a Gr-1 PE Ab. (C,D) ELISA analysis of TNF-α in the serum and peritoneal exudates of heat-killed E. coli O157:H7-challenged mice. (E,F) Quantitative determination of chemokines MCP-1 and MIP-2 mRNA expression in the liver of heat-killed E. coli O157:H7-challenged mice. Values are average means of triplicate experiments with two mice per genotype per experiment. Error bars depict SEM. Results are expressed as means ± SEM. P < 0.05 was considered statistically significant. * P < 0.05 and ** P < 0.01.

Article Snippet: Serum Lcn2 was quantified using Lcn2 Mouse ELISA kits (Boster, China) according to the manufacturer's instructions.

Techniques: Migration, Flow Cytometry, Staining, Enzyme-linked Immunosorbent Assay, Expressing

Journal: Frontiers in Immunology

Article Title: Lipocalin 2 Protects Against Escherichia coli Infection by Modulating Neutrophil and Macrophage Function

doi: 10.3389/fimmu.2019.02594

Figure Lengend Snippet: Decreased expression of inflammatory cytokines produced by lipocalin 2-deficient ( Lcn2 −/− ) macrophages. (A) Real-time PCR analysis of Lcn2 mRNA expression levels in the E. coli O157:H7-infected primary bone marrow-derived macrophages (BMDMs) from wild-type (WT) and Lcn2 −/− mice. (B–D) ELISA analysis of interleukin (IL)-6, IL-1β, and tumor necrosis factor (TNF)-α levels in the culture medium of the E. coli O157:H7-infected BMDMs from WT and Lcn2 −/− mice. (E–L) Real-time PCR analysis of cytokine mRNA expression levels in the E. coli O157:H7-infected pBMDMs from WT and Lcn2 −/− mice. (M) The infected BMDMs of WT and Lcn2 −/− mice were subjected to staining with rabbit monoclonal antibody iNOS, rat monoclonal antibody F4/80, Alexa Fluor 488 goat anti-rabbit IgG, and Alexa Fluor 647 goat anti-rat IgG in blocking buffer (1:200) and observed by fluorescence microscopy. Values are average means of triplicate experiments with two mice used for the isolation of BMDMs per genotype per experiment. Error bars depict SEM. Results are expressed as means ± SEM. P < 0.05 was considered statistically significant. * P < 0.05 and ** P < 0.01.

Article Snippet: Serum Lcn2 was quantified using Lcn2 Mouse ELISA kits (Boster, China) according to the manufacturer's instructions.

Techniques: Expressing, Produced, Real-time Polymerase Chain Reaction, Infection, Derivative Assay, Enzyme-linked Immunosorbent Assay, Staining, Blocking Assay, Fluorescence, Microscopy, Isolation

Journal: Frontiers in Immunology

Article Title: Lipocalin 2 Protects Against Escherichia coli Infection by Modulating Neutrophil and Macrophage Function

doi: 10.3389/fimmu.2019.02594

Figure Lengend Snippet: Lipocalin 2 (Lcn2) can promote migration and phagocytosis of macrophages. (A,B) Scratch wound healing assay of mouse RAW264.7 macrophages and quantification of the fold change of average migrated distance of cells was measured with microscope ( n = 3, mean ± SEM, scale bars, 100 μm). (C,D) ELISA analysis of monocyte chemoattractant protein (MCP)-1 and macrophage inflammatory protein (MIP)-2 levels in the culture medium of Lcn2-treated macrophages. (E) Flow cytometry analysis of mouse RAW264.7 macrophages incubated with FITC-dextran. Values are average means of triplicate experiments with two repetitions per treatment per experiment. Error bars depict SEM. * P < 0.05 and ** P < 0.01.

Article Snippet: Serum Lcn2 was quantified using Lcn2 Mouse ELISA kits (Boster, China) according to the manufacturer's instructions.

Techniques: Migration, Wound Healing Assay, Microscopy, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Incubation

Journal: Cell reports

Article Title: Lipocalin-2 regulates astrocyte-oligodendrocyte interaction to drive post-stroke secondary demyelination.

doi: 10.1016/j.celrep.2025.115899

Figure Lengend Snippet: Figure 2. Contralateral LCN2 elevation arises from local astrocytes and trans-callosal spread (A and B) Representative images and quantifications showing time-course changes of astrocytic LCN2 in the contralateral corpus callosum (n = 5 mice; scale bar, 20 μm). (C and D) Immunoblotting and quantitative analysis of LCN2 (n = 5 mice).

Article Snippet: Serum LCN2 was evaluated according to the manufacturer’s directions of LCN2 ELISA kit (FEK0853, Boster Biological Technology, China).

Techniques: Western Blot

Journal: Cell reports

Article Title: Lipocalin-2 regulates astrocyte-oligodendrocyte interaction to drive post-stroke secondary demyelination.

doi: 10.1016/j.celrep.2025.115899

Figure Lengend Snippet: Figure 4. Extracellular LCN2 gradually accumulates within mature oligodendrocytes (A and B) Representative images and quantifications showing LCN2 (red) surrounded by and accumulated into mature oligodendrocytes (GST-pi + , green; Olig2 + , blue) in the contralateral corpus callosum after dMCAO (n = 5 mice; scale bar, 20 μm). (C) Experimental flowchart in vivo. (D and E) Representative images and quantitative analysis of GFP (green) reporter LV into astrocytes (GFAP, blue) with induced hLCN2 (red) in the corpus callosum of Lcn2 − /− mice. White arrows indicate transfected astrocytes (scale bar, 20 μm).

Article Snippet: Serum LCN2 was evaluated according to the manufacturer’s directions of LCN2 ELISA kit (FEK0853, Boster Biological Technology, China).

Techniques: In Vivo, Transfection

Journal: Cell reports

Article Title: Lipocalin-2 regulates astrocyte-oligodendrocyte interaction to drive post-stroke secondary demyelination.

doi: 10.1016/j.celrep.2025.115899

Figure Lengend Snippet: Figure 7. Oligodendrocyte Lrp2 knockdown suppresses LCN2-related white matter degeneration (A) Relative mRNA expression levels of LCN2 receptors (Lrp2, 24p3r, Lrp6, Mc1r, Mc3r, Mc4r) (n = 5 independent primary cell cultures). (B and C) Representative fluorescent images and quantification of LRP2 expression in cultured oligodendrocytes (n = 5 mice; scale bar, 20 μm).

Article Snippet: Serum LCN2 was evaluated according to the manufacturer’s directions of LCN2 ELISA kit (FEK0853, Boster Biological Technology, China).

Techniques: Knockdown, Expressing, Cell Culture

Journal: International Journal of Molecular Sciences

Article Title: Targeting Lipocalin-2 in Inflammatory Breast Cancer Cells with Small Interference RNA and Small Molecule Inhibitors

doi: 10.3390/ijms22168581

Figure Lengend Snippet: siRNA-based silencing in LCN2-overexpressing IBC cells. ( A ) Western blot analysis for LCN2 and β-actin (as loading control) in a panel of IBC (SUM149, SUM190, and MDA-IBC3) and non-IBC (MDA-MB-231, SKBR3, and MCF7) cells. ( B ) Densitometric analysis of band intensities was performed, and values were calculated relative to non-IBC cells, MCF7. Results are shown as Mean ± SEM of triplicate experiments, **** p < 0.001). Two different siRNAs targeting exon 3 and exon 5 of the human LCN2 sequence (NC_000009.12) were used. Western blot analysis of ( C ) MDA-IBC3 cells and ( D ) SUM149 cells were performed after transiently transfected with LCN2-siRNA-1, LCN2-siRNA-2, and negative control-siRNA (NC-siRNA) at 100 nmol/L concentration, as described in materials and methods. Non-treated (NT) cells were treated with the transfection reagent. Densitometric analysis of band intensities of ( E ) MDA-IBC3 and ( F ) SUM149 cells was calculated relative to the NC-siRNA. Results are shown as Mean ± SEM of triplicate experiments (** p < 0.01).

Article Snippet: Our study used a structure-based computational approach to identify potential LCN2 inhibitors in the ZINC database of the Asinex library.

Techniques: Western Blot, Control, Sequencing, Transfection, Negative Control, Concentration Assay

Journal: International Journal of Molecular Sciences

Article Title: Targeting Lipocalin-2 in Inflammatory Breast Cancer Cells with Small Interference RNA and Small Molecule Inhibitors

doi: 10.3390/ijms22168581

Figure Lengend Snippet: LCN2-siRNA-based silencing inhibits colony formation, migration, and invasion of IBC cells. Colony formation assay was performed after LCN2-siRNA-based silencing in MDA-IBC3 and SUM149 cells. Cell proliferation was performed in ( A ) MDA-IBC3 cells and ( B ) SUM149 cells. Results are shown as Mean ± SEM of triplicate experiments (* p < 0.05, ** p < 0.01, *** p < 0.001). ( C ) Migration assay was performed after LCN2-siRNA transfection (100 nM siRNA, final concentration) in SUM149. ( D ) NC-siRNA cells represent 100% migration. Images of migrated cells were taken at 20× magnification, scale bar = 100 µm. Results are shown as Mean ± SEM of triplicate experiments (**** p < 0.0001). ( E ) Invasion assay was performed after LCN2-siRNA transfection (100 nM siRNA, final concentration) in SUM149 cells. ( F ) NC-siRNA cells represent 100% invasion. Images of invaded cells were acquired with a light microscope 20× magnification, Scale bar = 100 µm. Results are shown as Mean ± SEM of triplicate experiments (**** p < 0.0001).

Article Snippet: Our study used a structure-based computational approach to identify potential LCN2 inhibitors in the ZINC database of the Asinex library.

Techniques: Migration, Colony Assay, Transfection, Concentration Assay, Invasion Assay, Light Microscopy

Journal: International Journal of Molecular Sciences

Article Title: Targeting Lipocalin-2 in Inflammatory Breast Cancer Cells with Small Interference RNA and Small Molecule Inhibitors

doi: 10.3390/ijms22168581

Figure Lengend Snippet: LCN2-siRNA-based silencing induces apoptosis and cell cycle arrest in IBC cells. SUM149 cells were transfected with 100 mM of negative control (NC-siRNA) or LCN2 siRNA (siRNA-2). ( A ) Caspase-3 fluorometric activity assay in SUM149 cells 72 h after LCN2-siRNA-2 and NC-siRNA transfection. Docetaxel (0.5 nM final concentration) was used as a positive control. ( B ) Western blot analysis of apoptotic-related proteins. ( C ) Histogram showing cell cycle arrest at G0/G1 to S phase transition after LCN2-siRNA-2 transfection in SUM149 cells compared with NC-siRNA. ( D ) Quantification of the flow cytometry data showed an increase in SUM149-LCN2-siRNA-2 transfected cells at G0/G1 to S phase transition. ( E ) Western blot analysis of cell cycle-related proteins 72 h after siRNAs transfection. ( F , G ) Densitometric analysis of the band intensities showed in E. Results are shown as Mean ± SEM of triplicate experiments (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001).

Article Snippet: Our study used a structure-based computational approach to identify potential LCN2 inhibitors in the ZINC database of the Asinex library.

Techniques: Transfection, Negative Control, Activity Assay, Concentration Assay, Positive Control, Western Blot, Sublimation, Flow Cytometry

Journal: International Journal of Molecular Sciences

Article Title: Targeting Lipocalin-2 in Inflammatory Breast Cancer Cells with Small Interference RNA and Small Molecule Inhibitors

doi: 10.3390/ijms22168581

Figure Lengend Snippet: Molecular model and docking of ZINC00784494 and ZINC00640089 ligands into LCN2-calyx pocket. ( A ) Surface model representation of LCN2-calyx pockets. Pockets #1, #2, and #3 (dotted circles) are represented with key amino acid residues in yellow color (right panel). ( B ) Cartoon docking representation and predicted binding interactions of ligands with key residues of LCN2-calyx pocket. Interactions are represented with yellow dotted lines. Residues are displayed with a three-letter code and numbers representing the position in the polypeptide. ( C ) Surface docking representation of ligands (represented as sticks) ZINC00784494 (magenta) and ZINC00640089 (yellow) into the LCN2-calyx pocket.

Article Snippet: Our study used a structure-based computational approach to identify potential LCN2 inhibitors in the ZINC database of the Asinex library.

Techniques: Binding Assay

Journal: International Journal of Molecular Sciences

Article Title: Targeting Lipocalin-2 in Inflammatory Breast Cancer Cells with Small Interference RNA and Small Molecule Inhibitors

doi: 10.3390/ijms22168581

Figure Lengend Snippet: LCN2 inhibitors reduce cell proliferation and cell viability in IBC cells. ( A ) For colony formation assays SUM149 cells were treated with LCN2 inhibitors at 10 µM, 1 µM, and 0.1 µM concentration. ( B ) The percentage of clonogenicity was calculated relative to DMSO. Results are shown as Mean ± SEM of triplicate experiments (** p < 0.01, *** p < 0.001). ( C ) Representative plate showing a colony formation assay of SUM149 cells treated with the LCN2 inhibitor. ( D ) Cell viability was assessed in SUM149 cells with Alamar Blue dye 72 h after LCN2 inhibitor treatment. The percentage of cell viability was calculated relative to DMSO. Results are shown as Mean ± SEM of triplicate experiments (**** p < 0.0001).

Article Snippet: Our study used a structure-based computational approach to identify potential LCN2 inhibitors in the ZINC database of the Asinex library.

Techniques: Concentration Assay, Colony Assay

Journal: International Journal of Molecular Sciences

Article Title: Targeting Lipocalin-2 in Inflammatory Breast Cancer Cells with Small Interference RNA and Small Molecule Inhibitors

doi: 10.3390/ijms22168581

Figure Lengend Snippet: LCN2 inhibitors ZINC00784494 and ZINC00640089 reduced p-Akt in a dose-dependent manner in SUM149 cells. SUM149 cells were incubated with each inhibitor as described in the “materials and methods” section. Changes in AKT and p-AKT protein levels were measured by Western blot with specific antibodies against these protein forms. ( A ) ZINC00784494, ( B ) ZINC00640089.

Article Snippet: Our study used a structure-based computational approach to identify potential LCN2 inhibitors in the ZINC database of the Asinex library.

Techniques: Incubation, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Targeting Lipocalin-2 in Inflammatory Breast Cancer Cells with Small Interference RNA and Small Molecule Inhibitors

doi: 10.3390/ijms22168581

Figure Lengend Snippet: LCN2-inhibitors ZINC00784494 and ZINC00640089 showed specificity toward LCN2-calyx. Cell proliferation in MCF7, MCF7-EV and, MCF7-LCN2 cells after treatment with ( A ) ZINC00784494 inhibitor and ( B ) ZINC00640089 inhibitor. ( C ) A representative clonogenic assay of MCF7, MCF7-EV, and MCF7-LCN2 treated with ZINC00784494 and ZINC00640089. Results are shown as Mean ± SEM of triplicate experiments (** p < 0.01, *** p < 0.001, **** p < 0.0001).

Article Snippet: Our study used a structure-based computational approach to identify potential LCN2 inhibitors in the ZINC database of the Asinex library.

Techniques: Clonogenic Assay