FIG 3.

E. coli proteins in exosomes were candidates as important factors in inflammatory responses. (a, b) Exosomes from live E. coli-infected macrophages (Mφ) were treated with proteinase K (150 μg/mL), DNase (100 μg/mL), or RNase (100 μg/mL) in the presence or absence of 0.01% Triton X-100. Mouse RAW264.7 Mφ was incubated with each exosome (0.1 μg/mL). (a) After incubation of cells for 24 h, the mRNA levels of inducible nitric oxide synthase (iNos) and cyclooxygenase-2 (Cox-2) were 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 < 0.01; ***, P < 0.005; ****, P < 0.001. (b) After incubation of cells for 24 h, the protein expressions of iNOS and COX-2 were measured by Western blotting. β-Actin was used as loading control. (c) Exosomes (none-exo, live-exo, or HI-exo) from Mφ uninfected or infected with live or heat-inactive (HI) E. coli were analyzed by mass spectrometry. Venn diagrams show the number of identified proteins.