Figure 2: Effects of Pseudomonas fluorescens on zonulin expression in Caco-2 cells.

(A) Human Caco-2 cell monolayer was exposed to Pseudomonas fluorescens and fixed with paraformaldehyde. Zonulin expression was determined by immunostaining with anti-zonulin antibodies (red), nucleus was stained by DAPI (blue). At 0 hour, the uninfected Caco-2 monolayer showed less zonulin staining in the cell cytoplasma. (B) One hour after Pseudomonas fluorescens exposure, zonulin staining can be visualized in the cytoplasma. (C) After 5 hrs of Pseudomonas fluorescens exposure, the area with zonulin staining full filled the entire cell cytoplasm indicate significant increase in zonulin expression. (D) Zonulin staining in cell cytoplasm gradually decreased 24 hrs after Pseudomonas fluorescens exposure. (E) Secreted form of zonulin released from Caco2 monolayers exposed to Pseudomonas fluoresce. The zonulin levels 0–5 hrs post-incubation in the cell culture supernatants were lower than the detection limit. Pseudomonas fluorescens induced significant zonulin release 5 hrs post-incubation and gradually increase at 19 hrs after bacterial exposure. The zonulin level decreased after 24 hrs of Pseudomonas fluoresce exposure. Data are expressed as mean ± SD (n = 3, *P < 0.05 versus 0 hour).