Figure 2.

Epithelial and mesenchymal cells are sensitive to Stx2a. (A) Stx2a induces necrosis and cellular proliferation. HIOs were incubated for 27 hours with Stx2a (30 ng) by microinjection (luminal exposure) or addition to the medium (interstitial exposure). Cryosections were stained for necrosis (Sytox-green), proliferation (Ki-67, red), and DNA (4′,6-diamidino-2-phenylindole [DAPI], blue). Scale bar: 50 μm. (B and C) Fluorescence intensity was quantified using ImageJ software and plotted as means ± SD, n = 3. Statistical significance compared with saline control was assessed using 1-way analysis of variance, Dunnett’s posttest. ∗P = .01 to .05; ∗∗∗P < .001. (B) Cellular death by necrosis (Sytox-green fluorescence). (C) Cellular proliferation (Ki-67 fluorescence). (D) Time course of cellular death. Serial images of intact HIOs with mRuby2 fused to the promoter of E-cadherin grown in the presence of 5 μmol/L Sytox-green. Saline (control, top) or 30 ng Stx2a (bottom) were added to the medium at time zero and imaged at the indicated times (hours). Representative images of experiments performed in triplicate. Scale bar: 100 μm. (E and F) Fluorescence intensity quantified using ImageJ software, plotted as means ± SD, n = 3. Statistical significance at 148 hours compared with saline control was assessed using a 2-tailed, unpaired t test. (E) Stx2a causes loss of mRuby2 (E-cadherin expression). (F) Stx2a causes necrosis (Sytox-green). L, lumen; M, mesenchyme.