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R&D Systems lcn2 recombinant protein
Lcn2 Recombinant Protein, supplied by R&D Systems, used in various techniques. Bioz Stars score: 94/100, based on 47 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/recombinant+mouse+lcn2+protein/Recombinant+Mouse+Lipocalin-2%2FNGAL+Protein%2C+CF/pm41436429-236-8-24
Average 94 stars, based on 47 article reviews

lcn2 recombinant protein - by Bioz Stars, 2026-10

94/100 stars

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Article Title: Atorvastatin Protects Against the Macrophage/Microglia-Related Neuroinflammation via Inhibiting Lipocalin-2 in Mouse Experimental Intracerebral Hemorrhage Model.
Article Snippet: The secondary antibodies used for immunofluorescent staining were donkey anti-rabbit IgG (H + L) Alexa Fluor 488 (Abcam, # ab150061, RRID:AB_2571722), donkey anti-rabbit IgG (H + L) Alexa Fluor 594 (Abcam, # ab150076, RRID:AB_2782993), donkey anti-mouse IgG (H + L) Alexa Fluor 488 (Abcam, # ab150105, RRID:AB_2732856), donkey anti-mouse IgG (H + L) Alexa Fluor 594 (Abcam, # ab150108, RRID:AB_2732073), donkey anti-goat IgG (H + L) Alexa Fluor 488(Abcam, # ab150137, RRID:AB_2892985), and donkey antigoat IgG (H + L) Alexa Fluor 594(Abcam, # ab150136, RRID:AB_2782994), all obtained from Abcam. .. In this study, atorvastatin was purchased from Pfizer (New York, NY, USA), and recombinant mouse LCN2 protein was obtained from R&D Systems (Minneapolis, MN, USA, #1857-LC). ..

Article Title: Astrocyte-secreted lipocalin-2 elicits bioenergetic failure-induced neuronal death that is causally related to high fatality in a mouse model of hepatic encephalopathy.
Article Snippet: Hepatic encephalopathy (HE) is a neurological complication arising from acute liver failure with poor prognosis and high mortality; the underlying cellular mechanisms are still wanting.. We previously found that neuronal death caused by mitochondrial dysfunction in rostral ventrolateral medulla (RVLM), which leads to baroreflex dysregulation, is related to high fatality in an animal model of HE.. Lipocalin-2 (Lcn2) is a secreted glycoprotein mainly released by astrocytes in the brain.

Article Title: Atorvastatin Protects Against the Macrophage/Microglia-Related Neuroinflammation via Inhibiting Lipocalin-2 in Mouse Experimental Intracerebral Hemorrhage Model
Article Snippet: The secondary antibodies used for immunofluorescent staining were donkey anti-rabbit IgG (H + L) Alexa Fluor 488 (Abcam, # ab150061, RRID:AB_2571722), donkey anti-rabbit IgG (H + L) Alexa Fluor 594 (Abcam, # ab150076, RRID:AB_2782993), donkey anti-mouse IgG (H + L) Alexa Fluor 488 (Abcam, # ab150105, RRID:AB_2732856), donkey anti-mouse IgG (H + L) Alexa Fluor 594 (Abcam, # ab150108, RRID:AB_2732073), donkey anti-goat IgG (H + L) Alexa Fluor 488(Abcam, # ab150137, RRID:AB_2892985), and donkey anti-goat IgG (H + L) Alexa Fluor 594(Abcam, # ab150136, RRID:AB_2782994), all obtained from Abcam. .. In this study, atorvastatin was purchased from Pfizer (New York, NY, USA), and recombinant mouse LCN2 protein was obtained from R&D Systems (Minneapolis, MN, USA, #1857-LC). ..

Article Title: Lipocalin-2 regulates adult neurogenesis and contextual discriminative behaviours.
Article Snippet: .. Cell treatments included the exposure of LCN2-nullderived neurospheres to recombinant mouse LCN2 protein (R&D Systems, Minneapolis, MN, USA), at a concentration of 100 ng ml− 1 for 24 or 72 h, and to 2 μM of DFO for 24 h. Also, Wt cultures were treated with 500 μM of ferric ammonium citrate (Sigma) for 24 h. For a more detailed description of the experimental procedures, please see Supplementary information. ..

Article Title: Lipocalin-2 inhibition alleviates neural injury by microglia ferroptosis suppression after experimental intracerebral hemorrhage in mice via enhancing ferritin light chain expression.
Article Snippet: Introduction: Microglia play pivotal roles in post-intracerebral hemorrhage (ICH) neural injury.. Iron metabolism, which is dysregulated after ICH, participates in microglial dysfunction.. Previous studies have shown that iron metabolism-related lipocalin-2 (LCN2) is involved in regulating microglial function following ICH.

Article Title: Neutralization of Lipocalin-2 Diminishes Stroke-Reperfusion Injury
Article Snippet: Dynabeads Protein G (10 μL, Cat# 10007D, Thermo) was incubated with 0.2 μg of LCN2 mAb (Cat# MAB18571, R&D) in 100 μL PBS for 60 min at room temperature [ ]. .. For the competitive interaction between LCN2 mAb and mouse LCN2 protein, LCN2 mAb bound on the Dynabeads was incubated with 0.06 μg of recombinant mouse LCN2 protein (Cat# 1857-LC, R&D) and mouse LCN2 mAb epitope peptide (YNVTSILVRDQDQGCRYWIRT, synthesized by AnaSpec, Fremont, CA, USA) in 10X, 100X, or 1000X molar ratio of LCN2 protein in 100 μL PBST for 16 h at 4 °C. .. For the competitive interaction between LCN2 mAb and human LCN2 protein, recombinant human LCN2 protein (Cat# 1757-LC, R&D) and human LCN2 mAb epitope peptide (YNVTSVLFRKKKCDYWIRT, synthesized by AnaSpec) were used.

Article Title: Neutralization of Lipocalin-2 Diminishes Stroke-Reperfusion Injury
Article Snippet: After washing, the Dynabeads were incubated with different concentrations (0, 0.1, 0.5 and 2.5 μg) of LCN2 mAb (Cat# MAB18571, R&D) in 100 μL PBST for 30 min at room temperature. .. LCN2 mAb bound on the Dynabeads was incubated with 0.1 μg of recombinant mouse LCN2 protein (Cat# 1857-LC, R&D) in 100 μL PBST for 1 h at 4 °C. ..

Article Title: Lipocalin-2 induces macrophage/microglia pro-inflammatory phenotype after intracerebral hemorrhage via Nrf2 signaling inhibition in young and aged mice.
Article Snippet: The secondary antibodies were as follows donkey anti-rabbit IgG (H+L) Alexa Fluor 488 (Abcam, #ab150061, RRID: AB_2571722), donkey anti-rabbit IgG (H+L) Alexa Fluor 594 (Abcam, #ab150076, RRID:AB_2782993), donkey anti-mouse IgG (H+L) Alexa Fluor 488 (Abcam, #ab150105, RRID:AB_2732856), donkey anti-mouse IgG (H+L) Alexa Fluor 594 (Abcam, #ab150108, RRID:AB_2732073), donkey anti-goat IgG (H+L) Alexa Fluor 488 (Abcam, #ab150137, RRID:AB_2892985), and donkey anti-goat IgG (H+L) Alexa Fluor 594 (Abcam, #ab150136, RRID:AB_2782994). .. The brusatol used in this study was obtained from Sigma-Aldrich (#SML1868) and the recombinant mouse LCN2 protein was obtained from R&D Systems (Minneapolis, MN, USA, #1857-LC). ..

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Monomeric form of Ent can disseminate into systemic circulation. Serum was collected from male germ-free (GF) rats and GF rats co-housed (GFC) with conventional mice for 10 d (8 weeks old, n = 5–6) at Taconic Facility. Serum metabolites were extracted using standard procedures for targeted metabolomics profiling. The extracted samples were analyzed using HPLC coupled to Agilent 6495 QQQ mass spectrometry (LC–MS). Ent was quantified as its monomeric form 2, 3-dihydroxy benzoic acid (2, 3-DHBA). The data were normalized with internal standards and log2-transformed on a per-sample basis. (A) Ent spectra (B) Fold change in serum 2, 3-DHBA. Note: Trace amounts of 2, 3-DHBA in GF animal sera are likely to be from dead bacteria in the diet. (C) Fecal Lcn2. Results are expressed as mean ± SEM. *** p < 0.001.

Journal: Gut Microbes

Article Title: Microbial metabolite Enterobactin impairs mitochondrial respiration and alleviates colitis

doi: 10.1080/19490976.2026.2662638

Figure Lengend Snippet: Monomeric form of Ent can disseminate into systemic circulation. Serum was collected from male germ-free (GF) rats and GF rats co-housed (GFC) with conventional mice for 10 d (8 weeks old, n = 5–6) at Taconic Facility. Serum metabolites were extracted using standard procedures for targeted metabolomics profiling. The extracted samples were analyzed using HPLC coupled to Agilent 6495 QQQ mass spectrometry (LC–MS). Ent was quantified as its monomeric form 2, 3-dihydroxy benzoic acid (2, 3-DHBA). The data were normalized with internal standards and log2-transformed on a per-sample basis. (A) Ent spectra (B) Fold change in serum 2, 3-DHBA. Note: Trace amounts of 2, 3-DHBA in GF animal sera are likely to be from dead bacteria in the diet. (C) Fecal Lcn2. Results are expressed as mean ± SEM. *** p < 0.001.

Article Snippet: Mouse recombinant Lcn2 was purchased from Sino Biological Inc. (50060-MNAH).

Techniques: Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Transformation Assay, Bacteria

Ent exacerbates mitochondrial dysfunction in Lcn2 −/− BMDMs and is rescued by rec-Lcn2. BMDMs were isolated from wild-type (WT) and Lcn2-deficient (Lcn2 −/− ) mice to assess mitochondrial function using a Seahorse XF Analyzer. (A and B) Oxygen consumption rate (OCR) was measured (C) BMDMs were treated with Ent alone or in combination with ferric iron (1:1 ratio) for 24 h to assess the impact on mitochondrial respiration. (D) Quantification of key mitochondrial parameters revealed that Ent treatment significantly reduced basal respiration, ATP production, maximal respiration, proton leak, and spare respiratory capacity in Lcn2KO BMDMs (E) Mito-respiration was measured with Ent, rec-Lcn2 or both. (F) Cotreatment with rec-Lcn2 on basal respiration, maximal respiration, ATP production, and spare respiratory capacity indicating OCR. (G) Iron-chelating activity was assessed in the supernatants using the chrome azurol S (CAS) liquid assay. Control reactions included Ent alone, and rec-Lcn2 alone. (H) Mitochondrial reactive oxygen species (ROS) were measured using MitoSOX™ Red staining followed by flow cytometry analysis. Data represent mean ± SEM. Statistical significance was determined using unpaired Student’s t -test or one-way ANOVA where appropriate (* p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001).

Journal: Gut Microbes

Article Title: Microbial metabolite Enterobactin impairs mitochondrial respiration and alleviates colitis

doi: 10.1080/19490976.2026.2662638

Figure Lengend Snippet: Ent exacerbates mitochondrial dysfunction in Lcn2 −/− BMDMs and is rescued by rec-Lcn2. BMDMs were isolated from wild-type (WT) and Lcn2-deficient (Lcn2 −/− ) mice to assess mitochondrial function using a Seahorse XF Analyzer. (A and B) Oxygen consumption rate (OCR) was measured (C) BMDMs were treated with Ent alone or in combination with ferric iron (1:1 ratio) for 24 h to assess the impact on mitochondrial respiration. (D) Quantification of key mitochondrial parameters revealed that Ent treatment significantly reduced basal respiration, ATP production, maximal respiration, proton leak, and spare respiratory capacity in Lcn2KO BMDMs (E) Mito-respiration was measured with Ent, rec-Lcn2 or both. (F) Cotreatment with rec-Lcn2 on basal respiration, maximal respiration, ATP production, and spare respiratory capacity indicating OCR. (G) Iron-chelating activity was assessed in the supernatants using the chrome azurol S (CAS) liquid assay. Control reactions included Ent alone, and rec-Lcn2 alone. (H) Mitochondrial reactive oxygen species (ROS) were measured using MitoSOX™ Red staining followed by flow cytometry analysis. Data represent mean ± SEM. Statistical significance was determined using unpaired Student’s t -test or one-way ANOVA where appropriate (* p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001).

Article Snippet: Mouse recombinant Lcn2 was purchased from Sino Biological Inc. (50060-MNAH).

Techniques: Isolation, Activity Assay, Control, Staining, Flow Cytometry

Ent impairs mitochondrial respiration in model intestinal epithelia. Caco-2, intestinal epithelial cells (IEC), were treated with iron-free Ent (25 μM) or iron-bound Ent-Fe³⁺ (25 μM; 1:1 ratio) or Fe +3 alone and assessed mitochondrial function. (A) Mitochondrial respiration was evaluated using the Seahorse XF Analyzer by measuring oxygen consumption rate (OCR). (B) Basal and maximal respiration, ATP-linked respiration, proton leak and spare respiratory capacity (C) Mito-respiration was measured with Ent, rec-Lcn2, or both. (D) IEC were treated with 25 µM Ent for 24 h and total cellular proteins were analyzed by immunoblotting using antibodies against key subunits of the mitochondrial oxidative phosphorylation (OXPHOS) complexes I–V of electron transport chain. Densitometry scanning of complex II (E) Immunoblots of Hif1α, p -AMPK, total AMPK, and loading control β-actin (F and G) IEC were treated with 25 µM enterobactin for 24 h, and mitochondrial ROS levels were assessed using MitoSOX™ Red dye followed by flow cytometry analysis. Data represent mean ± SEM from at least three independent experiments. Statistical significance was determined using one-way ANOVA where appropriate (* p < 0.05 and ** p < 0.01).

Journal: Gut Microbes

Article Title: Microbial metabolite Enterobactin impairs mitochondrial respiration and alleviates colitis

doi: 10.1080/19490976.2026.2662638

Figure Lengend Snippet: Ent impairs mitochondrial respiration in model intestinal epithelia. Caco-2, intestinal epithelial cells (IEC), were treated with iron-free Ent (25 μM) or iron-bound Ent-Fe³⁺ (25 μM; 1:1 ratio) or Fe +3 alone and assessed mitochondrial function. (A) Mitochondrial respiration was evaluated using the Seahorse XF Analyzer by measuring oxygen consumption rate (OCR). (B) Basal and maximal respiration, ATP-linked respiration, proton leak and spare respiratory capacity (C) Mito-respiration was measured with Ent, rec-Lcn2, or both. (D) IEC were treated with 25 µM Ent for 24 h and total cellular proteins were analyzed by immunoblotting using antibodies against key subunits of the mitochondrial oxidative phosphorylation (OXPHOS) complexes I–V of electron transport chain. Densitometry scanning of complex II (E) Immunoblots of Hif1α, p -AMPK, total AMPK, and loading control β-actin (F and G) IEC were treated with 25 µM enterobactin for 24 h, and mitochondrial ROS levels were assessed using MitoSOX™ Red dye followed by flow cytometry analysis. Data represent mean ± SEM from at least three independent experiments. Statistical significance was determined using one-way ANOVA where appropriate (* p < 0.05 and ** p < 0.01).

Article Snippet: Mouse recombinant Lcn2 was purchased from Sino Biological Inc. (50060-MNAH).

Techniques: Western Blot, Phospho-proteomics, Control, Flow Cytometry

Therapeutic oral administration of 2, 3-DHBA is effective in protecting against DSS-induced pathology than 2, 5-DHBA. Eight-weeks-old male C57BL/6J mice (WT, n = 3–5) were administered with 2% DSS in drinking water for 7 d, confirmed that mice were rectally bleeding. DSS water was replaced with regular water. Control mice were given regular water. After the cessation of DSS (i.e., during recovery phase), mice were administered with either 2, 3-dihydroxybenzoic acid (2, 3-DHBA) or 2, 5-dihydroxybenzoic acid (2, 5-DHBA: 500 µg/mouse, orally) or PBS for 7 d. Feces and blood were collected for the isolation of hemolysis-free serum. Intestinal barrier function was assessed via FITC-dextran administered 4 h before euthanasia. (A) Schematic representation of the experiment (B) Percent body weight loss/gain; (C) Gross colon images (D) Colon length (E) % Spleen weight (F) % Cecum weight (G) Serum fluorescence (H) Serum Lcn2 (I) Fecal Lcn2 (J) Serum KC (K) Colonic MPO. Data are presented as mean ± SEM from at least three independent experiments or biological replicates. Statistical comparisons were performed using one-way ANOVA with Tukey’s post hoc test (* p < 0.05, ** p < 0.01, and *** p < 0.001).

Journal: Gut Microbes

Article Title: Microbial metabolite Enterobactin impairs mitochondrial respiration and alleviates colitis

doi: 10.1080/19490976.2026.2662638

Figure Lengend Snippet: Therapeutic oral administration of 2, 3-DHBA is effective in protecting against DSS-induced pathology than 2, 5-DHBA. Eight-weeks-old male C57BL/6J mice (WT, n = 3–5) were administered with 2% DSS in drinking water for 7 d, confirmed that mice were rectally bleeding. DSS water was replaced with regular water. Control mice were given regular water. After the cessation of DSS (i.e., during recovery phase), mice were administered with either 2, 3-dihydroxybenzoic acid (2, 3-DHBA) or 2, 5-dihydroxybenzoic acid (2, 5-DHBA: 500 µg/mouse, orally) or PBS for 7 d. Feces and blood were collected for the isolation of hemolysis-free serum. Intestinal barrier function was assessed via FITC-dextran administered 4 h before euthanasia. (A) Schematic representation of the experiment (B) Percent body weight loss/gain; (C) Gross colon images (D) Colon length (E) % Spleen weight (F) % Cecum weight (G) Serum fluorescence (H) Serum Lcn2 (I) Fecal Lcn2 (J) Serum KC (K) Colonic MPO. Data are presented as mean ± SEM from at least three independent experiments or biological replicates. Statistical comparisons were performed using one-way ANOVA with Tukey’s post hoc test (* p < 0.05, ** p < 0.01, and *** p < 0.001).

Article Snippet: Mouse recombinant Lcn2 was purchased from Sino Biological Inc. (50060-MNAH).

Techniques: Control, Isolation, Fluorescence

Fig. 3 Downregulation of LCN2 alleviated LPS-related neuronal damage. A Representative immunoﬂuorescence images show astrocyte activation and LCN2 ﬂuorescence in primary hippocampal astrocytes. B Quantitative analysis of LCN2 ﬂuorescence in primary hippocampal astrocytes (n = 9–10). C LCN2 protein levels were detected in primary hippocampal astrocytes by immunoblotting, actin was used as a loading control, and D quantitative analysis of the LCN2, (n = 4). E ELISA kit was used to measure the LCN2 levels in ACM, (n = 4). F Primary hippocampal neurons were treated with Control-ACM, LPS-ACM, AAV-Sh-LCN2-ACM, and the MAP2 was measured by immunoﬂuorescence. Sholl analysis (Scale bar: 20 μm), (n = 10) (G) and quantitative analyses of dendritic length (n = 15) (H) were performed. Data are presented as Mean ± SEM. one-way ANOVA was used for statistical analysis. *p < 0.05, **p < 0.01, ****p < 0.0001, versus LPS group.

Journal: Cell death & disease

Article Title: LCN2 induces neuronal loss and facilitates sepsis-associated cognitive impairments.

doi: 10.1038/s41419-025-07469-4

Figure Lengend Snippet: Fig. 3 Downregulation of LCN2 alleviated LPS-related neuronal damage. A Representative immunoﬂuorescence images show astrocyte activation and LCN2 ﬂuorescence in primary hippocampal astrocytes. B Quantitative analysis of LCN2 ﬂuorescence in primary hippocampal astrocytes (n = 9–10). C LCN2 protein levels were detected in primary hippocampal astrocytes by immunoblotting, actin was used as a loading control, and D quantitative analysis of the LCN2, (n = 4). E ELISA kit was used to measure the LCN2 levels in ACM, (n = 4). F Primary hippocampal neurons were treated with Control-ACM, LPS-ACM, AAV-Sh-LCN2-ACM, and the MAP2 was measured by immunoﬂuorescence. Sholl analysis (Scale bar: 20 μm), (n = 10) (G) and quantitative analyses of dendritic length (n = 15) (H) were performed. Data are presented as Mean ± SEM. one-way ANOVA was used for statistical analysis. *p < 0.05, **p < 0.01, ****p < 0.0001, versus LPS group.

Article Snippet: Recombinant LCN2 protein was procured from MCE Company (Product No. HY-P70658A).

Techniques: Activation Assay, Western Blot, Control, Enzyme-linked Immunosorbent Assay

Fig. 7 Downregulation of LCN2 resisted mitochondrial dysfunction. A Primary hippocampal neurons were treated with ACM. colocalization of Lamp2 (lysosome marker) and Tomm20 (mitochondrial marker) by immunoﬂuorescence. Quantitative analysis of colocalization of Lamp2 and Tomm20/Lamp2 ﬂuorescence in primary hippocampal neurons (n = 6–7). B The mitochondrial portions were isolated, and Pink1 protein levels were detected by immunoblotting in primary hippocampal neurons, COX IV was used as a loading control. Quantitative analysis of Pink1 expression was conducted (n = 4). Data are presented as Mean ± SEM. One-way ANOVA was used for the three groups. **p < 0.01, ***p < 0.001, versus LPS group.

Journal: Cell death & disease

Article Title: LCN2 induces neuronal loss and facilitates sepsis-associated cognitive impairments.

doi: 10.1038/s41419-025-07469-4

Figure Lengend Snippet: Fig. 7 Downregulation of LCN2 resisted mitochondrial dysfunction. A Primary hippocampal neurons were treated with ACM. colocalization of Lamp2 (lysosome marker) and Tomm20 (mitochondrial marker) by immunoﬂuorescence. Quantitative analysis of colocalization of Lamp2 and Tomm20/Lamp2 ﬂuorescence in primary hippocampal neurons (n = 6–7). B The mitochondrial portions were isolated, and Pink1 protein levels were detected by immunoblotting in primary hippocampal neurons, COX IV was used as a loading control. Quantitative analysis of Pink1 expression was conducted (n = 4). Data are presented as Mean ± SEM. One-way ANOVA was used for the three groups. **p < 0.01, ***p < 0.001, versus LPS group.

Article Snippet: Recombinant LCN2 protein was procured from MCE Company (Product No. HY-P70658A).

Techniques: Marker, Isolation, Western Blot, Control, Expressing

Fig. 8 Schematic illustration of the effects of LCN2 in the sepsis brain. Sepsis upregulates LCN2 triggering oxidative stress, mitochondrial dysfunction, and neuronal loss, thus leading to synaptic damage and cognitive impairments.

Journal: Cell death & disease

Article Title: LCN2 induces neuronal loss and facilitates sepsis-associated cognitive impairments.

doi: 10.1038/s41419-025-07469-4

Figure Lengend Snippet: Fig. 8 Schematic illustration of the effects of LCN2 in the sepsis brain. Sepsis upregulates LCN2 triggering oxidative stress, mitochondrial dysfunction, and neuronal loss, thus leading to synaptic damage and cognitive impairments.

Article Snippet: Recombinant LCN2 protein was procured from MCE Company (Product No. HY-P70658A).

Techniques:

FIGURE 3. Inhibiting ER stress with 4PBA alleviates inflammation in BV2 cells in vitro. (A) RT-qPCR analysis of Tnfa, Il6, and Nos2 mRNA levels in TUN-treated BV2 cells reveals ER stress–induced proinflammatory activation versus untreated controls (NC). Data: mean ± SEM (n = 6). P < 0.05, **P < 0.001 (Student’s t-test). (B) Volcano plot illustrating downregulation of mRNA expression associated with proinflammatory responses in BV2 cells treated with 4PBA under LPS-induced inflammatory conditions, as assessed through bulk RNA-seq analysis. (C) GO analysis of the downregulated differentially expressed genes (DEGs) between the LPS group and LPS + 4PBA group in BV2 cells. (D) KEGG pathway analysis confirms suppression of LPS-induced inflammatory cascades, including NOD-like receptor and NF-κB signaling (top 15 pathways shown). (E) Intersection analysis identifies 24 shared genes between ER stress–associated genes (GeneCards) and LPS_4PBA DEGs, including key mediators Nos2, Lcn2, Il1b, and Il6. (F, G) Western blot quantification demonstrates 4PBA-mediated downregulation of LCN2, pro–IL-1β, cleaved IL-1β, and iNOS protein levels versus LPS controls. Data: mean ± SEM (n = 4). P < 0.05, **P < 0.001 (one-way ANOVA).

Journal: Investigative ophthalmology & visual science

Article Title: Endoplasmic Reticulum Stress Drives Neuroinflammation Through Lipocalin 2 Upregulation in Retinal Microglia After Optic Nerve Injury.

doi: 10.1167/iovs.66.5.12

Figure Lengend Snippet: FIGURE 3. Inhibiting ER stress with 4PBA alleviates inflammation in BV2 cells in vitro. (A) RT-qPCR analysis of Tnfa, Il6, and Nos2 mRNA levels in TUN-treated BV2 cells reveals ER stress–induced proinflammatory activation versus untreated controls (NC). Data: mean ± SEM (n = 6). P < 0.05, **P < 0.001 (Student’s t-test). (B) Volcano plot illustrating downregulation of mRNA expression associated with proinflammatory responses in BV2 cells treated with 4PBA under LPS-induced inflammatory conditions, as assessed through bulk RNA-seq analysis. (C) GO analysis of the downregulated differentially expressed genes (DEGs) between the LPS group and LPS + 4PBA group in BV2 cells. (D) KEGG pathway analysis confirms suppression of LPS-induced inflammatory cascades, including NOD-like receptor and NF-κB signaling (top 15 pathways shown). (E) Intersection analysis identifies 24 shared genes between ER stress–associated genes (GeneCards) and LPS_4PBA DEGs, including key mediators Nos2, Lcn2, Il1b, and Il6. (F, G) Western blot quantification demonstrates 4PBA-mediated downregulation of LCN2, pro–IL-1β, cleaved IL-1β, and iNOS protein levels versus LPS controls. Data: mean ± SEM (n = 4). P < 0.05, **P < 0.001 (one-way ANOVA).

Article Snippet: For recombinant LCN2 (rLCN2, 1857-LC-050; R&D Systems, Minneapolis, MN, USA) treatment, the left eye of each mouse received 1 μg/mL rLCN2 in 1 μL of 0.01 M PBS immediately after ONC, while the right eye served as the control and received an equivalent volume of PBS alone.

Techniques: In Vitro, Quantitative RT-PCR, Activation Assay, Expressing, RNA Sequencing, Western Blot

FIGURE 4. Mutual promotion between LCN2 and ER stress. (A) RT-qPCR results indicate elevated mRNA levels of Lcn2 in the TUN group compared to the NC group. Data are shown as mean ± SEM with n = 6 biological repeats. ***P < 0.001, calculated by Student’s t-test. (B, C) Immunoblot analysis demonstrates a significant decrease in LCN2 protein expression in the TUN + 4PBA group compared to the TUN group in BV2 cells. Data are shown as mean ± SEM with n = 4. *P < 0.05, ***P < 0.001, calculated by Student’s t-test. (D, E) Immunoblot analysis shows a significant upregulation of GRP78 and CHOP protein expression in the TUN group compared to the negative control group in BV2 cells, which was alleviated by Lcn2 knockdown using siRNA. Data are shown as mean ± SEM with n = 5. *P < 0.05, **P < 0.01, ***P < 0.001, calculated by one-way ANOVA. (F, G) Immunoblot analysis shows a significant upregulation of GRP78 and CHOP protein expression in the LPS group compared to the negative control group in BV2 cells, which was alleviated by Lcn2 knockdown using siRNA. Data are shown as mean ± SEM with n = 4. **P < 0.01, ***P < 0.001, calculated by one-way ANOVA.

Journal: Investigative ophthalmology & visual science

Article Title: Endoplasmic Reticulum Stress Drives Neuroinflammation Through Lipocalin 2 Upregulation in Retinal Microglia After Optic Nerve Injury.

doi: 10.1167/iovs.66.5.12

Figure Lengend Snippet: FIGURE 4. Mutual promotion between LCN2 and ER stress. (A) RT-qPCR results indicate elevated mRNA levels of Lcn2 in the TUN group compared to the NC group. Data are shown as mean ± SEM with n = 6 biological repeats. ***P < 0.001, calculated by Student’s t-test. (B, C) Immunoblot analysis demonstrates a significant decrease in LCN2 protein expression in the TUN + 4PBA group compared to the TUN group in BV2 cells. Data are shown as mean ± SEM with n = 4. *P < 0.05, ***P < 0.001, calculated by Student’s t-test. (D, E) Immunoblot analysis shows a significant upregulation of GRP78 and CHOP protein expression in the TUN group compared to the negative control group in BV2 cells, which was alleviated by Lcn2 knockdown using siRNA. Data are shown as mean ± SEM with n = 5. *P < 0.05, **P < 0.01, ***P < 0.001, calculated by one-way ANOVA. (F, G) Immunoblot analysis shows a significant upregulation of GRP78 and CHOP protein expression in the LPS group compared to the negative control group in BV2 cells, which was alleviated by Lcn2 knockdown using siRNA. Data are shown as mean ± SEM with n = 4. **P < 0.01, ***P < 0.001, calculated by one-way ANOVA.

Techniques: Quantitative RT-PCR, Western Blot, Expressing, Negative Control, Knockdown

FIGURE 5. Upregulation of LCN2 expression in the retina post-ONC. (A) RT-qPCR results indicate elevated mRNA levels of Lcn2 in the ONC group compared to the sham group. Data are shown as mean ± SEM with n = 5 biological repeats. ***P < 0.001, calculated by Student’s t-test. (B, C) Immunoblot analysis demonstrates a significant increase in LCN2 expression in the retina in the ONC group compared to the sham group. Data are shown as mean ± SEM with n = 3. **P < 0.01, calculated by Student’s t-test. (D, E) Immunohistochemical analysis shows an increase in LCN2 expression in the retina following ONC. Data are shown as mean ± SEM with n = 4–5 biological repeats. **P < 0.01, calculated by Student’s t-test. (F) Immunofluorescence analysis reveals LCN2 (red) predominantly localized to Tuj1+ RGCs in sham retinas. Following ONC injury, prominent LCN2 accumulation is observed in Iba1+ microglia and GFAP+ astrocytes. Scale bar: 50 μm.

Journal: Investigative ophthalmology & visual science

Article Title: Endoplasmic Reticulum Stress Drives Neuroinflammation Through Lipocalin 2 Upregulation in Retinal Microglia After Optic Nerve Injury.

doi: 10.1167/iovs.66.5.12

Figure Lengend Snippet: FIGURE 5. Upregulation of LCN2 expression in the retina post-ONC. (A) RT-qPCR results indicate elevated mRNA levels of Lcn2 in the ONC group compared to the sham group. Data are shown as mean ± SEM with n = 5 biological repeats. ***P < 0.001, calculated by Student’s t-test. (B, C) Immunoblot analysis demonstrates a significant increase in LCN2 expression in the retina in the ONC group compared to the sham group. Data are shown as mean ± SEM with n = 3. **P < 0.01, calculated by Student’s t-test. (D, E) Immunohistochemical analysis shows an increase in LCN2 expression in the retina following ONC. Data are shown as mean ± SEM with n = 4–5 biological repeats. **P < 0.01, calculated by Student’s t-test. (F) Immunofluorescence analysis reveals LCN2 (red) predominantly localized to Tuj1+ RGCs in sham retinas. Following ONC injury, prominent LCN2 accumulation is observed in Iba1+ microglia and GFAP+ astrocytes. Scale bar: 50 μm.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Immunohistochemical staining, Immunofluorescence

FIGURE 6. LCN2 exacerbates neuroinflammation and retinal ganglion cell degeneration post-ONC. (A–C) Microglial phagocytic activation in retinal flat mounts visualized by CD68+/Iba1+ dual immunofluorescence across experimental groups at 7 days post-ONC. Scale bar: 50 μm. Quantification of (B) Iba1+ cell density and (C) CD68+/Iba1+ activation index. Data represent mean ± SEM; n = 4 biological replicates; *P < 0.05, ***P < 0.001 (one-way ANOVA with Tukey’s post hoc test). (D) Transcriptional profiling of microglial-associated inflammatory mediators in retinal tissues. RT-qPCR analysis of Fcgr3, Tnfa, Il1b, and Il6 mRNA levels normalized to Gapdh. Data represent mean ± SEM; n = 6; **P < 0.01, ***P < 0.001 (one-way ANOVA with Tukey’s post hoc test). (E, F) RGC degeneration quantification by RBPMS+ cell counting in retinal whole mounts. (E) Representative images showing RGC loss patterns. (F) Survival rates normalized to sham controls. Scale bars: 50 μm. Data represent mean ± SEM; n = 4–6; *P < 0.05, ***P < 0.001 (one-way ANOVA with Tukey’s post hoc test). (G, H) Therapeutic modulation of LCN2 by 4PBA treatment. (G) Quantification of LCN2 protein levels normalized to β-tubulin and (H) representative immunoblots. Data represent mean ± SEM; *P < 0.05, calculated by one-way ANOVA.

Journal: Investigative ophthalmology & visual science

Article Title: Endoplasmic Reticulum Stress Drives Neuroinflammation Through Lipocalin 2 Upregulation in Retinal Microglia After Optic Nerve Injury.

doi: 10.1167/iovs.66.5.12

Figure Lengend Snippet: FIGURE 6. LCN2 exacerbates neuroinflammation and retinal ganglion cell degeneration post-ONC. (A–C) Microglial phagocytic activation in retinal flat mounts visualized by CD68+/Iba1+ dual immunofluorescence across experimental groups at 7 days post-ONC. Scale bar: 50 μm. Quantification of (B) Iba1+ cell density and (C) CD68+/Iba1+ activation index. Data represent mean ± SEM; n = 4 biological replicates; *P < 0.05, ***P < 0.001 (one-way ANOVA with Tukey’s post hoc test). (D) Transcriptional profiling of microglial-associated inflammatory mediators in retinal tissues. RT-qPCR analysis of Fcgr3, Tnfa, Il1b, and Il6 mRNA levels normalized to Gapdh. Data represent mean ± SEM; n = 6; **P < 0.01, ***P < 0.001 (one-way ANOVA with Tukey’s post hoc test). (E, F) RGC degeneration quantification by RBPMS+ cell counting in retinal whole mounts. (E) Representative images showing RGC loss patterns. (F) Survival rates normalized to sham controls. Scale bars: 50 μm. Data represent mean ± SEM; n = 4–6; *P < 0.05, ***P < 0.001 (one-way ANOVA with Tukey’s post hoc test). (G, H) Therapeutic modulation of LCN2 by 4PBA treatment. (G) Quantification of LCN2 protein levels normalized to β-tubulin and (H) representative immunoblots. Data represent mean ± SEM; *P < 0.05, calculated by one-way ANOVA.

Techniques: Activation Assay, Immunofluorescence, Quantitative RT-PCR, Cell Counting, Western Blot

FIGURE 7. AAV-mediated LCN2 gene silencing reduces microglial activation and enhances RGC survival following ONC. (A, B) Retinal whole mounts at 3 weeks after intravitreal injection of AAV2-U6-shLcn2-CMV-EGFP (AAV-Lcn2) demonstrate EGFP fluorescence (green) colocalized with RBPMS+ RGCs (red) and Iba1+ microglia (red), confirming targeted delivery to both RGCs and microglia. Scale bars: 50 μm. (C) Western blot shows markedly reduced LCN2 protein levels in ONC-injured retinas treated with AAV-Lcn2 versus AAV-null controls. (D, E) AAV-Lcn2 treatment reduces microglial density by 35.2% in ONC-injured retinas, accompanied by a morphologic shift from amoeboid to ramified states. Scale bars: 50 μm. Data represent mean ± SEM; n = 4; ***P < 0.001 (one-way ANOVA with Tukey’s post hoc test). (F, G) Lcn2 silencing elevates RGC survival to 53.5% ± 3.1% post-ONC, significantly exceeding AAV-null controls (40.5% ± 2.8%; P < 0.001). Scale bars: 50 μm. Data: mean ± SEM; n = 4; *P < 0.001 (one-way ANOVA, Tukey’s post hoc test).

Journal: Investigative ophthalmology & visual science

Article Title: Endoplasmic Reticulum Stress Drives Neuroinflammation Through Lipocalin 2 Upregulation in Retinal Microglia After Optic Nerve Injury.

doi: 10.1167/iovs.66.5.12

Figure Lengend Snippet: FIGURE 7. AAV-mediated LCN2 gene silencing reduces microglial activation and enhances RGC survival following ONC. (A, B) Retinal whole mounts at 3 weeks after intravitreal injection of AAV2-U6-shLcn2-CMV-EGFP (AAV-Lcn2) demonstrate EGFP fluorescence (green) colocalized with RBPMS+ RGCs (red) and Iba1+ microglia (red), confirming targeted delivery to both RGCs and microglia. Scale bars: 50 μm. (C) Western blot shows markedly reduced LCN2 protein levels in ONC-injured retinas treated with AAV-Lcn2 versus AAV-null controls. (D, E) AAV-Lcn2 treatment reduces microglial density by 35.2% in ONC-injured retinas, accompanied by a morphologic shift from amoeboid to ramified states. Scale bars: 50 μm. Data represent mean ± SEM; n = 4; ***P < 0.001 (one-way ANOVA with Tukey’s post hoc test). (F, G) Lcn2 silencing elevates RGC survival to 53.5% ± 3.1% post-ONC, significantly exceeding AAV-null controls (40.5% ± 2.8%; P < 0.001). Scale bars: 50 μm. Data: mean ± SEM; n = 4; *P < 0.001 (one-way ANOVA, Tukey’s post hoc test).

Techniques: Activation Assay, Injection, Fluorescence, Western Blot

FIGURE 8. LCN2 knockdown attenuates the inflammatory response by suppressing NLRP3 inflammasome activation through the TLR4/NF- κL pathway. (A) Heatmap depicting the differential gene expression between the control and LPS (1 μg/mL) treated groups in BV2 cells based on bulk RNA-seq. (B) Immunoblot analysis of LCN2 levels in BV2 cells treated with varying concentrations of LPS (0–3 μg/mL) and exposed to 1 μg/mL LPS for 1 to 3 days. (C) Representative microscopy images of resting and activated microglia following LPS treatment. Increased presence of activated microglia exhibiting a ramified morphology and enlarged soma (indicated by yellow arrowheads) observed after treatment with rLCN2 for 24 hours. Data are shown as mean ± SEM with n = 6. *P < 0.05, ***P < 0.001, calculated by one-way ANOVA. Scale bar: 50 μm. (D) mRNA expression of proinflammatory cytokines secreted by microglia in vitro after treatment with LPS and/or siLCN2 measured via RT-qPCR. Gene expressions were normalized to Gapdh. Data are presented as mean ± SEM with n = 6. *P < 0.05, ***P < 0.001, calculated using one-way ANOVA. (E, F) Immunoblot analysis of iNOS, pro–IL-1β, cleaved IL-1β, and LCN2 in BV2 cells treated with LPS (1 μg/mL) and siLcn2. Data are presented as mean ± SEM with n = 4. **P < 0.01, ***P < 0.001, calculated using one-way ANOVA. (G) Gene set enrichment analysis profiles of downregulated pathways, including NF-κB and Toll-like receptor signaling pathways, following Lcn2 knockdown in an LPS-induced environment. (H, I) Immunoblot detection of NLPR3, TLR4, p-NF-κB, and t-NF-κB in BV2 cells or the nucleus treated with LPS (1 μg/mL) and siLcn2. Data are presented as mean ± SEM with n = 4. **P < 0.01, ***P < 0.001, calculated using one-way ANOVA. (J) Representative immunofluorescence images of BV2 cells staining with NF-κB showing a decrease in phosphorylated NF-κB in the cell nucleus in the LPS + siLcn2 group compared to the LPS group.

Journal: Investigative ophthalmology & visual science

Article Title: Endoplasmic Reticulum Stress Drives Neuroinflammation Through Lipocalin 2 Upregulation in Retinal Microglia After Optic Nerve Injury.

doi: 10.1167/iovs.66.5.12

Figure Lengend Snippet: FIGURE 8. LCN2 knockdown attenuates the inflammatory response by suppressing NLRP3 inflammasome activation through the TLR4/NF- κL pathway. (A) Heatmap depicting the differential gene expression between the control and LPS (1 μg/mL) treated groups in BV2 cells based on bulk RNA-seq. (B) Immunoblot analysis of LCN2 levels in BV2 cells treated with varying concentrations of LPS (0–3 μg/mL) and exposed to 1 μg/mL LPS for 1 to 3 days. (C) Representative microscopy images of resting and activated microglia following LPS treatment. Increased presence of activated microglia exhibiting a ramified morphology and enlarged soma (indicated by yellow arrowheads) observed after treatment with rLCN2 for 24 hours. Data are shown as mean ± SEM with n = 6. *P < 0.05, ***P < 0.001, calculated by one-way ANOVA. Scale bar: 50 μm. (D) mRNA expression of proinflammatory cytokines secreted by microglia in vitro after treatment with LPS and/or siLCN2 measured via RT-qPCR. Gene expressions were normalized to Gapdh. Data are presented as mean ± SEM with n = 6. *P < 0.05, ***P < 0.001, calculated using one-way ANOVA. (E, F) Immunoblot analysis of iNOS, pro–IL-1β, cleaved IL-1β, and LCN2 in BV2 cells treated with LPS (1 μg/mL) and siLcn2. Data are presented as mean ± SEM with n = 4. **P < 0.01, ***P < 0.001, calculated using one-way ANOVA. (G) Gene set enrichment analysis profiles of downregulated pathways, including NF-κB and Toll-like receptor signaling pathways, following Lcn2 knockdown in an LPS-induced environment. (H, I) Immunoblot detection of NLPR3, TLR4, p-NF-κB, and t-NF-κB in BV2 cells or the nucleus treated with LPS (1 μg/mL) and siLcn2. Data are presented as mean ± SEM with n = 4. **P < 0.01, ***P < 0.001, calculated using one-way ANOVA. (J) Representative immunofluorescence images of BV2 cells staining with NF-κB showing a decrease in phosphorylated NF-κB in the cell nucleus in the LPS + siLcn2 group compared to the LPS group.

Techniques: Knockdown, Activation Assay, Gene Expression, Control, RNA Sequencing, Western Blot, Microscopy, Expressing, In Vitro, Quantitative RT-PCR, Protein-Protein interactions, Immunofluorescence, Staining

FIGURE 9. Schematic illustrating the proposed molecular mecha- nism by which LCN2 connects ER stress and inflammation following optic nerve injury.

Journal: Investigative ophthalmology & visual science

Article Title: Endoplasmic Reticulum Stress Drives Neuroinflammation Through Lipocalin 2 Upregulation in Retinal Microglia After Optic Nerve Injury.

doi: 10.1167/iovs.66.5.12

Figure Lengend Snippet: FIGURE 9. Schematic illustrating the proposed molecular mecha- nism by which LCN2 connects ER stress and inflammation following optic nerve injury.

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