Figure 6
HO53 treatment counteracts the disruptive effect of P. aeruginosa PAO1 conditioned medium in ALI differentiated BCi cells. Differentiated BCi cells (trans epithelial electrical resistance, TEER ≥ 1000 Ω × cm2) were treated with 75 μM HO53 in the lower chamber of transwell insert for 3 days and challenged with PAO1 conditioned medium (PAO1 cond medium) applied on the apical surface of the cells for the next 24 h. (a) TEER measurement after every hour from 0 to 7 h and after 24 h post PAO1 culture medium challenge. Data shown is representative of two experiment and showing mean values of four independent ALI filters at each time point ± SEM. Samples for confocal microscopy and Western blot were collected at selected time points in order to analyze tight junctions (TJ) integrity. (b) Confocal images showing disruption of occludin (red) and ZO-1 (green) after 6 h post PAO1 culture medium challenge of HO53 treated cells as compared to control cells (Ctrl), Bar = 10 µm. (c) Western blot analyses of occludin and claudin-1 after 0, 3, 6 and 24 h exposure to PAO1 culture medium. GAPDH was used as a loading control. The full-length blot for occludin is presented in Supplementary Figure S9 and for claudin-1 and GAPDH in Supplementary Figure S10d.
