Fig. 6.

Enhancement of actin rearrangement and toxin-mediated cytotoxicity by recombinant Srl (GST-Srl) in human colon carcinoma cells. (A) A Caco-2 cell monolayer grown on a glass coverslip was incubated for 3 h with 10 μg/ml GST-Srl in the presence of either 750 ng/ml toxin A or 300 ng/ml toxin B. Cells were fixed, stained with phalloidin (red), anti-Srl antibody (green), and DAPI (blue), and then visualized by confocal microscopy. Confocal images of F-actin only (panels a, b, and c) and x-y, x-z, and y-z sectional views of the merged images are shown (panels d, e, and f, respectively). Confocal images of GST-Srl only (panels j, k, and l) and x-y, x-z, and y-z sectional views of the merged images are shown (panels m, n, and o, respectively). Reconstructed 3D images are also shown (panels g, h, and i). Negative control denotes buffer-treated cells. Bar, 20 μm. Arrowheads in panels e and f point to the actin aggregate at the apical side of the cells. Arrowheads in panels n and o point to the sites of accumulation of GST-Srl. (B) TER was measured at hourly time intervals after apical treatment of Caco-2 monolayers with toxin A, toxin B, GST, or GST-Srl. Results are means ± SD of four independent measurements. (C) Caco-2 monolayers were incubated with 10 μg/ml GST-Srl or GST in the presence of 250 ng/ml toxin A in the apical compartment. Results are means ± SD of four independent measurements. Control denotes buffer-treated cells. (D) Results of an experiment similar to that in panel C but in the presence of 100 ng/ml toxin B. Results are means ± SD of four independent measurements.