f nucleatum strain atcc 23726  (ATCC)

Journal: bioRxiv

Article Title: Fusobacterium nucleatum produces previously unappreciated AHR-activating metabolites and promotes CRC cell proliferation via the AHR-TERT axis

doi: 10.1101/2025.09.25.678601

Figure Lengend Snippet: Identification of Fusobacterium nucleatum metabolites as AHR agonists (A) Induction of Cyp1a1 promoter activity by gut bacterial culture extracts ( n = 3). HepG2 cells transfected with a luciferase-based AHR-reporter system were treated with organic extracts of respective culture supernatants and 3MC (a known AHR agonist as a positive control, 3 μM) for 24 h. (B) Chemical structures of three Fn metabolites identified. (C) Induction of Cyp1a1 promoter activity by Fn culture extracts and respective Fn metabolites alone or in combination ( n = 3). FTIN (7.8 nM), STIN (1.2 nM), TIN (2.7 nM), and indole (25 µM). The Fn culture extract and the Fn metabolite concentrations tested were equivalent to 10% of the Fn culture. (D) Correlation between FTIN levels in the culture of Fn strains and Cyp1a1 promoter activity induced by the culture extracts of Fn strains. Overnight cultures of respective strains grown in BHIc medium were used for FTIN measurement and the AHR reporter assays. FTIN measurement, with a limit of quantification (LOQ) of 0.2 ng/ml, was from two independent experiments ( n = 2), and the AHR reporter assay was performed in triplicate with extract samples from a single experiment ( , left). The correlation coefficient and associated p -value were obtained by Pearson correlation analysis ( , right). (E) Induction of Cyp1a1 promoter activity in human hepatoma HepG2 cells or murine hepatoma Hepa1c1c7 cells by synthesized Fn metabolites ( n = 3). Respective cell lines transfected with the AHR reporter system were treated with varying concentrations of synthetic FTIN, TIN, STIN, or the positive control TCDD (a known AHR agonist) for 4 h. (F) Induction of CYP1A1 mRNA expression in HepG2 cells and Cyp1a1 mRNA expression in Hepa1c1c7 cells treated with synthetic FTIN, TIN, STIN, or TCDD (positive control) for 4 h ( n = 3). (G) Time-course measurement of CYP1A enzymes’ ethoxyresorufin- O -deethylase (EROD) activity in HepG2 cells treated with FTIN (1 µM), TIN (1 µM), STIN (1 µM), 3-MC (a transient AHR agonist, 1 µM), or TCDD (a sustained AHR agonist, 3 nM). Shown is a representative of two independent experiments ( n = 2) performed in triplicate with similar results. (H) The binding of Fn metabolites to the AHR was determined using an AHR photoaffinity ligand (PAL) competition assay in human quamous cell carcinoma HN30 ( n = 3). HN30 cells were treated with respective Fn metabolites (FTIN, STIN, and TIN at 10 µM). Indirubin (a known AHR ligand, 100 nM) and estradiol (10 µM) were used as positive and negative controls, respectively. Data (right) represent mean percent PAL binding ± SD (normalized to AHR) relative to vehicle-treated controls. Data are represented as mean ± standard deviation (SD), where appropriate. Statistical significance was assessed by ordinary one-way ANOVA with Dunnett’s multiple comparisons test (G) and with Šídák’s multiple comparisons test (H): **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; ns, not significant.

Article Snippet: For other experiments involving strain F. nucleatum ATCC 23726 ( Fn ), extracts were prepared with cultures grown overnight in M9CT at 37°C under anaerobic conditions unless otherwise mentioned.

Techniques: Activity Assay, Transfection, Luciferase, Positive Control, Reporter Assay, Synthesized, Expressing, Binding Assay, Competitive Binding Assay, Standard Deviation