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. 2021 Oct 15;17(10):e1010005. doi: 10.1371/journal.ppat.1010005

Fig 2. c3564 and c3565 promote host cell death during infection in cell culture models.

Fig 2

(A) Prevalence of c3564 and c3565 in various E. coli pathotypes. A duplex PCR method was used to detect the presence of the c3564 and c3565 genes in a laboratory collection of E. coli isolates. APEC, avian pathogenic E. coli; EHEC, enterohemorrhagic E. coli; ETEC, enterotoxigenic E. coli; UPEC, uropathogenic E. coli. (B) c3564 and c3565 affected the morphology of kidney epithelial cells. A498 kidney epithelial cells were infected with CFT073 and its derivatives for the indicated amounts of time and then subjected to phase-contrast microscopy (magnification, 20×). All images are representative of three independent experiments. Scale bar, 50 μm. (C), (D), and (E) Cytotoxicity assays on different cell types. A498 kidney epithelial cells (C), THP-1 human macrophages (D), and J774A.1 murine macrophage cells (E) were infected with various bacterial strains at a multiplicity of infection (MOI) of 10 for ~2.5 h, and the cell culture supernatants were then subjected to LDH release measurement. Cytotoxicity (%) was determined by comparing the LDH in culture supernatants to the total cellular LDH (the amount of LDH released upon cell lysis with 0.1% Triton X-100) according to the formula [(experimental − target spontaneous)/(target maximum − target spontaneous)] × 100. The data are the mean ± SD of three replicates from three independent experiments. *, P < 0.05; ***, P < 0.001 by one-way ANOVA followed by Dunnett’s multiple comparisons test against wild-type CFT073.