Fig. 2.

Stx1 taken up by MPC is transcytosed across the T84 cells in holotoxin form. A: XZ projection through an EHEC-infected T84 cell shows apical F-actin bleb (red) that takes up Stx1 (green). B: XZ projection through EHEC-infected T84 cells with internalized Stx1 shows that Stx1 (green) is transported to the basolateral side of the cells inside the F-actin-coated macropinosomes (red). F-actin, red by phalloidin-AlexaFluor 568; Stx1, green by conjugation to AlexaFluor 488; nuclei, blue by Hoechst. White arrows in A and B indicate the green Stx1-containing vesicles coated with red F-actin. C: SDS-PAGE shows the relative distribution of Stx1A and cleaved Stx1A1 in total cell lysates (endocytosis) and in basal conditioned media (transcytosis) from T84 cells incubated with Stx1 over indicated time in the presence of 50 μM furin inhibitor decanoyl-RVKR-CMK or in EHEC-infected cells. M indicates the corresponding molecular mass marker on a gel. Stx1 taken up by MPC is not cleaved inside the T84 cells 4 h later. Transcytosed toxin collected from basal conditioned media 4 h after EHEC infection is also not cleaved.