Figure 3.
Detection of toxin intracellular trafficking to the ER in ARPE-19 cells treated with Stx1 or Stx2. (A) ARPE-19 cells were seeded in 12-well plates at a total cell density of approximately 1.0 × 105 cells/well. Cells were incubated with Alexa Fluor 488-conjugated Stx1 (100 ng/mL) or Alexa Fluor 594-conjugated Stx2 (10 ng/mL) for the time points indicated in the right upper corner of each panel. After washing, cells were fixed and DAPI reaction solution added to stain nuclei. Representative DAPI-positive cells visualized by fluorescence microscopy are shown. In each panel, the arrow depicts cells shown in higher magnification in the insert. Scale bar: 80 μm, insert: 30 μm; (B) To detect Stx trafficking to the ER, 1.0 × 105 ARPE-19 cells/well were seeded in 12-well plates. Subsequently, cells were stimulated with complete growth media containing Alexa Fluor 488-conjugated Stx1 (100 ng/mL) with 50 nM ER-tracker (red) live-cell staining dyes for detection of ER. After washing, cells were captured in time-lapse live imaging at the time points indicated in the right upper corner of each panel. Yellow fluorescence indicates co-localization of Stx1 with the ER-specific marker. The scale bars represent 30 μm. The bar graphs show the mean ± SEM of the Pearson’s correlation coefficient assessment of the co-localization of Stx1-Alexa Flour 488/ER tracker (lower panel). More than 30 cells were counted per condition (n = 3). Asterisks indicate significant differences between treated time at 10 min vs. indicated time points; ** = p < 0.01; *** = p < 0.001.