Figure 4

Butyrate reduced cell damage by limiting replication of cytosolicS. Infantis Caco-2 cells. A Observation of the apical surface of Caco-2 cell monolayer ultrastructure with scanning electron microscopy. SI(a), SI(b) and SI(c) showed that S. Infantis infected cell was bacteria-laden and these bacteria were extruded out of the monolayer. B Western blot analysis for the expression levels of claudin-1, occludin, and ZO-1 proteins. The right panel shows the quantitation analysis by Image J software (n = 3, **P < 0.01, ***P < 0.001). C Detection of the distribution and expression of ZO-1 (Green) in Caco-2 cells by immunofluorescence staining. Red: Salmonella. Blue: DAPI. Scale bar, 10 µm. D Calcein-AM/PI cell mortality test for S. Infantis-induced cell death rate. E Effect of butyrate on intracellular bacteria as assessed by the gentamicin protection assay. F The intracellular bacteria were assessed by the gentamicin protection assay, without (total intracellular bacteria, black curve) or with (cytosolic bacteria, gray curve) chloroquine. G The number of the infected cells containing 1 to 10, 10 to 20, 20 to 50, or more than 50 bacteria per cell at 8 hpi was measured using a fluorescence microscope. At least 100 cells were counted for each group and the percentage of cells containing the different numbers of bacteria is shown (mean ± SEM, n = 3), **P < 0.01, ***P < 0.001. H Caco-2 cells were infected with S. Infantis-pFPV (Green). Cells at 8 hpi were stained with LAMP-1 (Red) to label S. Infantis in SCV, while the cytosolic S. Infantis were green. “Zoom” denotes a magnified image of the white dotted area. Blue: DAPI. Scale bar, 10 µm.