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

Journal: Cell Death & Disease

Article Title: LincRNA-EPS alleviates osteoclastogenesis under inflammatory microenvironment through preventing excessive iron metabolism

doi: 10.1038/s41419-026-08716-y

Figure Lengend Snippet: A Heatmap of significantly differently expressed genes (DEGs) between WT and KO osteoclast precursors ( | log2FC | > 1.3, FDR < 0.01). B Volcano plot of DEGs (red: up-regulated in KO; blue: down-regulated in KO). C KEGG pathway enrichment of DEGs. D Reactome pathway enrichment of DEGs. E Venn diagram of overlapped genes from top 10 pathways of KEGG and Reactome enrichment. F Heatmap of core dysregulated genes. G Lcn2 mRNA expression after RANKL-primed. ( n = 3). H Lcn2 mRNA expression after LPS-induced. ( n = 3). Two-way ANOVA with Tukey-Kramer test was used in G and H . Data were presented as mean ± SD. ** P < 0.01. **** P < 0.0001.

Article Snippet: Following lentivirus were purchased from OBiO Technology (Shanghai) Corp., Ltd: lincRNA-EPS overexpression lentivirus (pASLenti-pA-Ttc39aos1-CMV-EF1-EGFP-WPRE) and negative control (pASLenti-pA-MCS-CMV-EF1-EGFP-WPRE), Lcn2 knockdown lentivirus (pCLenti-U6-shRNA(Lcn2)-CMV-EGFP-WPRE) and negative control (pCLenti-U6-shRNA(NC)-CMV-EGFP-WPRE).

Techniques: Expressing

Journal: Cell Death & Disease

Article Title: LincRNA-EPS alleviates osteoclastogenesis under inflammatory microenvironment through preventing excessive iron metabolism

doi: 10.1038/s41419-026-08716-y

Figure Lengend Snippet: A Immunohistochemistry (IHC) of Lcn2 in periodontal tissues. Scale bar: 100 μm. B ELISA quantification of secreted Lcn2 protein in OCP supernatants ( n = 3). C Volcano plot of differentially expressed proteins in WT vs. KO osteoclasts. D KEGG pathway enrichment of differentially expressed proteins. E Gene Set Enrichment Analysis (GSEA) of differentially expressed proteins. F GSEA of “transport of small molecules” pathway with core enrichment proteins. G Western blot analysis of proteins related to iron metabolism during inflammatory osteoclastogenesis. Protein expression levels were quantified by grayscale analysis using ImageJ software ( n = 3). Two-way ANOVA with Tukey-Kramer test was used in B and G . Data were presented as mean ± SD. Significance levels were denoted as follows: * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: Following lentivirus were purchased from OBiO Technology (Shanghai) Corp., Ltd: lincRNA-EPS overexpression lentivirus (pASLenti-pA-Ttc39aos1-CMV-EF1-EGFP-WPRE) and negative control (pASLenti-pA-MCS-CMV-EF1-EGFP-WPRE), Lcn2 knockdown lentivirus (pCLenti-U6-shRNA(Lcn2)-CMV-EGFP-WPRE) and negative control (pCLenti-U6-shRNA(NC)-CMV-EGFP-WPRE).

Techniques: Immunohistochemistry, Enzyme-linked Immunosorbent Assay, Western Blot, Expressing, Software

Journal: Cell Death & Disease

Article Title: LincRNA-EPS alleviates osteoclastogenesis under inflammatory microenvironment through preventing excessive iron metabolism

doi: 10.1038/s41419-026-08716-y

Figure Lengend Snippet: A Lcn2 knockdown efficiency in WT and KO OCPs ( n = 3). B Iron metabolism genes expression of OCPs upon LPS stimulus after Lcn2 knockdown ( n = 3). C Western blot of iron metabolism proteins in WT and KO cells under RANKL-primed and LPS stimulation after Lcn2 knockdown ( n = 3). D Fe 2+ levels in WT and KO cells with Lcn2 knockdown or recombinant Lcn2 supplementation. Scale bar: 5 μm. Quantification of Fe 2+ intensity was compared ( n = 3). E TRAP staining showing the osteoclast differentiation after Lcn2 knockdown and recombinant Lcn2 supplementation. Scale bar: 20um. The number of osteoclasts, the average and sum of nuclei count, and the maximum diameter of osteoclasts were analyzed ( n = 3). Two-way ANOVA with Tukey-Kramer test was used in A – C . One-way ANOVA with Tukey–Kramer test was used in D and E . Data were presented as mean ± SD. Significance levels were denoted as follows: * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, ns refers to no significant difference.

Article Snippet: Following lentivirus were purchased from OBiO Technology (Shanghai) Corp., Ltd: lincRNA-EPS overexpression lentivirus (pASLenti-pA-Ttc39aos1-CMV-EF1-EGFP-WPRE) and negative control (pASLenti-pA-MCS-CMV-EF1-EGFP-WPRE), Lcn2 knockdown lentivirus (pCLenti-U6-shRNA(Lcn2)-CMV-EGFP-WPRE) and negative control (pCLenti-U6-shRNA(NC)-CMV-EGFP-WPRE).

Techniques: Knockdown, Expressing, Western Blot, Recombinant, Staining

Journal: Cell Death & Disease

Article Title: LincRNA-EPS alleviates osteoclastogenesis under inflammatory microenvironment through preventing excessive iron metabolism

doi: 10.1038/s41419-026-08716-y

Figure Lengend Snippet: A Efficiency of lincRNA-EPS overexpression in WT OCPs ( n = 3). B Lcn2 gene expression (left panel), Lcn2 protein expression (middle panel) and secretion (right panel) post-overexpression after LPS stimulation ( n = 3). C Intracellular Fe 2+ levels detected by FerroOrange probe ( n = 3). Scale bar: 10 μm. D Expression of iron metabolism-related genes post-overexpression after LPS stimulation ( n = 3). E Expression of iron metabolism proteins and osteoclastogenesis key proteins after lincRNA-EPS overexpression( n = 3). F TRAP staining showing the osteoclast differentiation after lincRNA-EPS overexpression. Scale bar: 20 um. The number of osteoclasts, the maximal nuclei count and the maximum diameter were analyzed ( n = 3). G The expression of osteoclastogenesis genes after lincRNA-EPS overexpression ( n = 3). Two-way ANOVA with Tukey-Kramer test was used in A , B , D , E , G . Unpaired t -test was used in C and F . Data were presented as mean ± SD. Significance levels were denoted as follows: * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, ns refers to no significant difference. In D , G , significance levels were also denoted as a ( P < 0.05), b ( P < 0.01), c ( P < 0.001) and d ( P < 0.0001) when compared with the same group at “LPS-” timepoint.

Article Snippet: Following lentivirus were purchased from OBiO Technology (Shanghai) Corp., Ltd: lincRNA-EPS overexpression lentivirus (pASLenti-pA-Ttc39aos1-CMV-EF1-EGFP-WPRE) and negative control (pASLenti-pA-MCS-CMV-EF1-EGFP-WPRE), Lcn2 knockdown lentivirus (pCLenti-U6-shRNA(Lcn2)-CMV-EGFP-WPRE) and negative control (pCLenti-U6-shRNA(NC)-CMV-EGFP-WPRE).

Techniques: Over Expression, Gene Expression, Expressing, Staining

Journal: Cell Death & Disease

Article Title: LincRNA-EPS alleviates osteoclastogenesis under inflammatory microenvironment through preventing excessive iron metabolism

doi: 10.1038/s41419-026-08716-y

Figure Lengend Snippet: A WT or KO osteoclasts were induced by RANKL and different concentrations of Lcn2 ( n = 3). Scale bar: 20 um. B and F IHC staining of Lcn2 of periodontal tissues ( n = 6). Scale bar: 100 μm. C and G Micro-CT images of alveolar bone resorption in Lcn2 -knockdown WT mice and KO mice ( n = 8). Scale bar: 1 mm. D and H TRAP staining of periodontal tissues. Scale bar: 100 μm. E and I IF staining of ferritin heavy chain and Ctsk. The inset in the upper-left corner shows a 2× magnified view ( n = 6). Scale bar: 50 μm. Data were presented as mean ± SD. Two-way ANOVA with Tukey–Kramer test was used in A . Unpaired t -test was used in B , E , F , and I . Paired t -test was used in C and G . Significance levels were denoted as follows: * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, ns refers to no significant difference. In A , significance levels were also denoted as a ( P < 0.05), b ( P < 0.01), c ( P < 0.001) and d ( P < 0.0001) when compared with the data in “0 ng/mL” group in the same cell type.

Article Snippet: Following lentivirus were purchased from OBiO Technology (Shanghai) Corp., Ltd: lincRNA-EPS overexpression lentivirus (pASLenti-pA-Ttc39aos1-CMV-EF1-EGFP-WPRE) and negative control (pASLenti-pA-MCS-CMV-EF1-EGFP-WPRE), Lcn2 knockdown lentivirus (pCLenti-U6-shRNA(Lcn2)-CMV-EGFP-WPRE) and negative control (pCLenti-U6-shRNA(NC)-CMV-EGFP-WPRE).

Techniques: Immunohistochemistry, Micro-CT, Knockdown, Staining