Fig. 2.

LGG induces ROS that oxidizes cysteines. (A) Intestinal epithelial cells (SKCO15) were contacted with 10⁸ CFU/mL of E. coli or LGG for 15 min, and then with 15 µM HydroCy3 for 30 min before confocal microscopic analysis at 555 nm, (scale bars 20 µm). (B) SKCO15 were contacted with 10⁸ CFU/mL of E. coli or LGG for 15 min, labeled for 30 min with a thiol-reactive, Thiol Tracker™ fluorescence probe, and then analyzed by fluorescence microscopy at 405 nm (scale bars 200 µm). Mean image intensity is shown at bottom left for A and B. (C) Biotinylated-iodoacetamide (BIAM) labeling of cysteine residues in lysates of LGG or E. coli contacted SKCO15 cells, followed by pull-down of labeled residues with streptavidin conjugated agarose and detected by Western blot using HRP conjugated streptavidin as a probe. The relative intensity of each lane of the blot is shown in arbitrary units to the left. Each value was normalized to calnexin that served as a loading control to give the relative oxidation amounts. (D) Dual labeling of LGG or E. coli contacted (15 mins) SKCO15 cultured cells with HydroCy3 (red) and Lysotracker (green). Note co-localization of lysotracker and hydro-Cy3 in LGG contacted cells (bars 10 µm).