FIG 4.

E. coli cirA-deleted mutant strain suppressed the exosome-mediated inflammatory responses. (a) Mouse RAW264.7 macrophages (Mφ) were incubated with OMVs (10 μg/7 mL) derived from single gene-deleted mutant strains (ΔompA, ΔompC, ΔfepA, ΔcirA, or ΔdegP) and wild type (WT) for 9 h. After culture media were replaced with fresh media containing antibiotics, cells were incubated for 1 h. After replacement with fresh media containing antibiotics, cells were incubated for 9 h. The protein expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in whole-cell lysates was measured by Western blotting. β-Actin was used as loading control. (b) RAW264.7 cells (recipient cells; 5 × 10⁵ cells) were incubated with each exosome from donor cells (5 × 10⁵ cells) for 24 h. The protein expression of iNOS and COX-2 in whole-cell lysates was measured by Western blotting. β-Actin was used as loading control. CS Analyzer 3.0 was used for image analysis software. Relative intensity is expressed as the mean ± SD (n = 3) of at least three independent biological replicates. (c) RAW264.7 cells (recipient cells; 5 × 10⁵ cells) were incubated with each exosome from donor cells (5 × 10⁵ cells) for 24 h. The mRNA expression of iNos and Cox-2 was analyzed by quantitative real-time PCR and normalized to 18S rRNA. Values are expressed as the mean ± SD (n = 4) of at least three independent biological replicates. One-way ANOVA and Tukey’s test were used for statistical analysis; *, P < 0.05; **, P < 0.01; ***, P < 0.005; ****, P < 0.001; ns, not significant.