Journal: Gut Pathogens
Article Title: Phenotypic characterization and complete genome of a tumorigenic pathobiont Escherichia coli LI60C3
doi: 10.1186/s13099-025-00732-1
Figure Lengend Snippet: Phenotypic characterization of the invasive feature and tumorigenic ability of E. coli strains. (A) Schematic diagram of microbial isolation from intestinal epithelial cells for in vitro and in vivo phenotypic assays. The invasive features, proliferation-inducing ability, and tumorigenic characteristics of the intraepithelial bacteria obtained from mouse colonocytes were evaluated in human cell lines and experimental murine models. (B) The intraepithelial E. coli strains (LI60C3, HM784C1, HM926C2, and HM936C4), adherent-invasive E. coli strains (LF82 and NC101), and nonpathogenic laboratory strains (MG1655, DH5α and JM109) were exposed to Caco-2 cells at MOI = 10 for 4 h. (a) Intracellular bacterial counts and (b) apical bacterial counts in the epithelial cultures are shown. (C) Cell cycle progression of Caco-2 cells after bacterial exposure for 24 h. The cell ratios in (a) S/G2/M to G1/G0 phase and (b) high to low Ki67 staining intensity are shown. Data are expressed as mean ± SEM ( N = 5–8/group). Each dot represents the data of one sample. * P < 0.05 vs. w/o, and # P < 0.05 vs. LI60C3. (D) A murine model with chemically induced colon cancer was inoculated with PBS vehicle, LI60C3, or LF82 at 10 9 CFU per mouse via the orogastric route. Representative images of intestinal tumors in each mouse group are displayed. Bar: 2 mm (macroscopic images) and 200 μm (histological images, H&E staining). (E) Increased tumor load was observed in mice inoculated with LI60C3, but not LF82. The tumor (a) number, (b) area, and (c) grade were determined for each mouse. The percentages of tumors with low- and high-grade dysplasia and carcinoma in each group are presented. Data are expressed as mean ± SEM ( N = 8–10/group). Each dot represents the data of one mouse. * P < 0.05 vs. PBS, and # P < 0.05 vs. LI60C3. The statistical significance of all panels is calculated by ANOVA followed by Tukey’s multiple comparison test, except those of (B-a), (C-a) and (E-a) are determined by the Kruskal–Wallis test. Experiments were repeated at least twice.
Article Snippet: The cells were then fixed with ice-cold 70% ethanol overnight, followed by washing with PBS containing 1% bovine serum albumin (BSA) (Cat. #A2153, Sigma-Aldrich) and centrifuged at 1500 × g for 5 min. After decanting the supernatant, the cells were stained with a primary antibody rabbit anti-human Ki67 (Cat. #9129) (1:400, Cell Signaling, Beverly, MA, USA) in PBS containing 0.5% TWEEN-20 (Cat. #TWN508, BioShop, Burlington, ON, Canada) and 1% BSA for 1 h at room temperature.
Techniques: Isolation, In Vitro, In Vivo, Bacteria, Staining, Comparison
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