Macropinocytosis is not involved in enhanced Stx2 translocation during microaerobic STEC infection.
A. HRP was added to T84 cells incubated with EDL933 or purified Stx2 for 5 h under AE (■) or MA (□) conditions. HRP concentrations in apical and basal media were determined by HRP activity assay. Translocation rates are expressed as percentage of apical HRP recovered in basal compartments. Data are shown as means ± SEM from four independent experiments performed in duplicate.
B. Polarized T84 cells were incubated with EDL933, NEM or left non-infected (NI) for 8 h under aerobic (A) or microaerobic conditions (M). After separation of cell lysates by SDS-PAGE, phosphorylated (p-Src) and total Src kinase (Src) were detected by Western blotting. Shown are representative images from two independent experiments performed in duplicate.
C and D. Polarized T84 cells were incubated with EDL933 for 5 h under MA conditions. Apical sides were either left untreated (NT) or incubated with cytochalasin D (cyt), blebbistatin (ble) or ML141. TER (C) and Stx2 translocation (D) was determined after infection. Data are shown as means ± SEM from four independent experiments performed in triplicate. ***P < 0.001 versus NT or ML 141.