Figure 5

P. aeruginosa infection did not up-regulate expression of PRXV in the human bronchial epithelial cells Calu-3, as it did in the cow primary tracheal epithelial cell cultures. A: Western blot results of PRXV expression of the Calu-3 cell lysates (CELLS) and the cell-conditioned medium (MEDIUM). The Calu-3 cells were stimulated with PAO1 bacteria either in the presence of FCS or without it. PRXV was not upregulated in these cells, and its secretion in the medium was not changed. To confirm that PAO1 induced alterations in Calu-3 layers, TER of epithelial layers was measured. B: Summary results of TER following exposure of the Calu-3 epithelial cells to PAO1 with and without FCS. PAO1 induced a significant decrease in TER, which was more pronounced in the presence of FCS. Open bar – initial TER, closed bar – TER after a 4-hour exposure to medium or bacteria. In the "control" condition, cells were exposed only to the medium (or the medium with FCS) without bacteria. * – p < 0.05 compared to the initial value, ⁺ – p < 0.05 compared to the control without bacteria, n = 6. C: P. aeruginosa infection up-regulated expression of PRXV protein in the primary cultures of the cow tracheal epithelial cells. C1-C2: Typical confocal microscopy images of the CTE cultures stained for PRXV (green fluorescence, FITC labeled secondary antibody) and co-stained with Propidium Iodine for DNA. C1 – control cultures (no infection), C2 – cultures infected with PAO1 for 12 hours. At the bottom of each image, a diagram of distribution of fluorescence intensity along the selected segment (blue bar) is given. Green line is PRXV fluorescence intensity, red line is DNA fluorescence intensity. Original magnification × 63. C3: MEAN data are given for fluorescence intensity in the control and PAO1-infected CTE cultures.