Journal: Gut Microbes

Article Title: Butyrate reduces adherent-invasive E. coli -evoked disruption of epithelial mitochondrial morphology and barrier function: involvement of free fatty acid receptor 3

doi: 10.1080/19490976.2023.2281011

Figure Lengend Snippet: An FFAR3 agonist improves mitochondrial network connectivity and barrier function in E. coli -LF82-infected T84 epithelial cells. Monolayers of the human colon-derived T84 epithelial cell line were treated with E. coli -LF82 (10 8 cfu, 4 h) ± a co-treatment with the FFAR3 agonist AR420626 (25 µM) and representative images collected in a random fashion by first identifying epithelia nuclei (blue, n) and then swapping the confocal laser channel to assess the mitochondrial network as defined by TOMM-20 immunostains. Twenty cells per monolayer were characterized by semi-quantitative assessment (a). (b) Mitochondrial membrane potential was assessed by TMRE fluorescence in a flow cytometer. A 10 min treatment with the metabolic toxin, FCCP (10 µM, n = 5 epithelial monolayers from separate experiments (indicated by different symbols)). Filter-grown T84 cell monolayers (starting transepithelial resistance (TER) range = 957–2155 Ohms.cm ) were cultured with E. coli -LF82 (10 8 cfu) ± AR420626 and TER and transcytosis of the bacteria assessed 24 h later. (c) TER is presented as the change over 24 h with each monolayer being its’ own control ( i.e ., pre-treatment value). (d) Bacterial transcytosis was assessed via serial dilution of culture-well basolateral medium on agar plates, with the data being converted to % transcytosis based on bacterial counts in the apical compartment and then E. coli -LF82 was normalized to 100 for comparison with E. coli +AR420626 in the same experiment (data are mean ± SEM; each data point is an individual experiment ( n = 6) in which measurements from 3 or 4 monolayers were averaged and are shown as a different symbol; * and #, p <.05 compared to control uninfected cells (con) and E. coli -LF82 only infected cells, respectively).

Article Snippet: Following the experimental treatment, protein was extracted from human organoids and epithelial cell lines and immunoblotting performed as previously described using mouse anti-human FFAR3 (1:1,000; #66811–1-1 g, Proteintech, Rosemount, IL) and GAPDH (1:1,000; ab8245, Abcam) antibodies, and a goat anti-mouse HRP-conjugated secondary antibody (1:5,000; sc-2031; Santa Cruz Biotech., Pasa Robles, CA).

Techniques: Infection, Derivative Assay, Membrane, Fluorescence, Flow Cytometry, Cell Culture, Bacteria, Control, Serial Dilution, Comparison

Journal: Gut Microbes

Article Title: Butyrate reduces adherent-invasive E. coli -evoked disruption of epithelial mitochondrial morphology and barrier function: involvement of free fatty acid receptor 3

doi: 10.1080/19490976.2023.2281011

Figure Lengend Snippet: An FFAR3 agonist improves mitochondrial network connectivity in E. coli -LF82-infected human organoids. Monolayers of colonic organoids derived from two healthy controls were treated with E. coli -LF82 (MOI = 100, 4 h) ± a co-treatment with the FFAR3 agonist AR420626 (AR: 25 µM). Representative confocal images of MitoTracker TM (red) and Hoescht (blue) co-stained organoids show the fused mitochondrial network of control organoid cells treated with the FFAR3 agonist alone (a), the puncta-like fragmented mitochondrial network of E. coli -LF82 infected organoid cells (b), and the intermediate fragments of the E. coli- LF82 infected organoids co-treated with the FFAR3 agonist (c) (n, nucleus; *, filamentous mitochondria; arrowhead, fragmented mitochondria). Twenty cells were assessed from four monolayers for semi-quantitative analysis (d). Data are mean ± SEM, * and #, p < .05 compared to control uninfected drug-treated cells (AR) and E. coli -LF82 only infected cells, respectively (two-way ANOVA followed by Tukey’s multiple comparison test) (frag., fragmented mitochondria).

Article Snippet: Following the experimental treatment, protein was extracted from human organoids and epithelial cell lines and immunoblotting performed as previously described using mouse anti-human FFAR3 (1:1,000; #66811–1-1 g, Proteintech, Rosemount, IL) and GAPDH (1:1,000; ab8245, Abcam) antibodies, and a goat anti-mouse HRP-conjugated secondary antibody (1:5,000; sc-2031; Santa Cruz Biotech., Pasa Robles, CA).

Techniques: Infection, Derivative Assay, Staining, Control, Comparison