Strain-Dependent Induction of Human Enterocyte Apoptosis by Blastocystis Disrupts Epithelial Barrier and ZO-1 Organization in a Caspase 3- and 9-Dependent Manner

Figure 8

(a) Representative confocal micrographs illustrating ZO-1 distribution in Caco-2 monolayers. Monolayers were grown to confluence on poly-L-lysine treated coverslips. Caco-2 cells were then coincubated for 6 h with either Blastocystis ST-4 or ST-7. Some monolayers were treated with broad-spectrum caspase inhibitor z-VAD-fmk before coincubation with ST-7. Normal culture media and 0.5 μM staurosporine were used as negative and positive controls, respectively. Compared to negative control, ST-7 treatment resulted in obvious reduction in ZO-1 apical localization in Caco-2 cell line. ST-4 did not alter ZO-1 integrity. Pretreatment with z-VAD-fmk rescued ST-7-induced ZO-1 changes in the epithelium. (b) Quantification of ZO-1 staining is shown as graphs. Each cell layer (1–25) corresponds to series of images from Z-stack sections taken at 1 μm thickness through the cell monolayer shown in Figure 8. 𝑋-axis illustrates cell layers from apical to basolateral. 𝑌-axis illustrates the number of pixels present over the entire area of image. Monolayers interacting with ST-7 and treated with staurosporine resulted in marked reduction in number of pixels in cell layers representing apical region, compared to ST-4 treated epithelium and normal control. Pretreatment of epithelium with broad-spectrum caspase inhibitor, z-VAD-fmk, resulted in inhibition of ST-7-induced ZO-1 changes in the monolayer. Results shown are mean of 4 separate Z-stacks for each treatment. (magnification: 600x).