Experimental setup for proteomic analyses and characterization of Giardia ESPs.

(A) The collection of Giardia intestinalis, isolates WB and GS, excretory-secretory products (ESPs) in a serum-free medium from axenic culture and co-culture with differentiated intestinal epithelial cells (IECs), Caco-2, for 2h and 6h. Samples were processed as described in Materials and Methods followed by liquid chromatography/mass spectrometry to identify secreted proteins. (B) Studies of IECs exposed to G. intestinalis ESPs using differential gene expression analyses by RNA sequencing, quantitative real time PCR, Western blots, ELISA and confocal microscopy. The panel illustrates the experimental setup (left to right) showing IECs exposed to ESPs of either Giardia isolates through a Transwell insert (direct exposure) or different amounts of ESPs collected from axenic culture (i.e. the absence of IECs, 1 μg, 5 μg or 10 μg per ml of medium). Abbreviation: Dulbecco’s Modified Eagles Medium (DMEM).