mouse lipocalin  (R&D Systems)

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

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

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

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