Intact dCyaA toxoid is endocytosed by a clathrin-dependent mechanism.

<p>(A) J774A.1 cells were incubated for 20, 40 and 60 minutes with 1 µg/ml of Alexa Fluor 488-labeled dCyaA or dCyaA-KP. For simultaneous visualization of early and recycling endocytic compartments, cells were co-incubated at 37°C for the last 20 minutes with Dyomics 547-labeled transferrin (10 µg/ml). Cells were washed in cold PBS, fixed with 4% PFA, and observed using a Leica confocal microscope TCS SP2 with a 63×/1.40 oil objective (HCX PL APO). (B) Co-localization of toxoids with transferrin was quantified using ImageJ software as described in details in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002580#s4" target="_blank">Materials and Methods</a>. (C) Murine RAW 264.7 macrophages transfected by dominant-negative mutant (DIII) of Eps-15 protein fused to GFP (blue) were incubated at 37°C for 30 min with the dCyaA or dCyaA-KP toxoids (1 µg/ml) labeled with Dyomics-647 (green). Dyomics 547-labeled transferrin (10 µg/ml) was added for the last 10 minutes of incubation (red), cells were washed with 0.1 M sodium acetate, 150 mM NaCl, pH 3.5 to remove surface-associated transferrin, fixed and observed with Olympus CellR microscope using a 100× oil immersion objective (N.A. 1.3). The calculated values of Pearson's correlation coefficients for transferrin (Dyomics 547) and toxoid (Dyomics 647) in non-transfected cells were 0.319 and 0.067 for dCyaA and dCyaA-KP, respectively.