Fig 1.
E. granulosus AgB uptake by mammalian cells in culture.
Immunofluorescence assay was performed on NIH-3T3, A549, J774 and RH cells exposed to 40 μg/ml AgB for 4 h, and mock treated cells (Control). AgB was labeled with polyclonal antibodies against AgB8/1, 2 and 4 subunits and an Alexa Fluor 488-conjugated secondary antibody (green). Nuclei and cytoskeleton were stained with DAPI (blue) and Alexa Fluor 594-conjugated phalloidin (red), respectively. Images are median optical sections from z-stacks obtained by confocal microscopy. Inserts correspond to two-fold digital magnification of boxed areas. Arrows indicate vesicular-like distribution of internalized AgB. Scale bar, 10 μm.
Fig 2.
Caveolae/raft-mediated endocytosis is the main route involved in E. granulosus AgB internalization by A549 and RH cells.
Indicated cells were incubated in medium alone (Ctrl), with 100 μg/ml genistein (GNT), or with 5 μg/ml chlorpromazine (CPZ) for 30 min and then exposed to 40 μg/ml AgB for 1.5 h. AgB was detected using anti-AgB polyclonal antibodies followed by anti-rabbit IgG Alexa Fluor 488 conjugated antibody (green). Cell nuclei were labelled with DAPI (blue). A and B, Inhibition of caveolae/raft-mediated endocytosis by genistein reduces AgB internalization. C and D, inhibition of clathrin-mediated endocytosis pathway by chlorpromazine does not cause a significant decrease in uptake of AgB. The quantitative data presented in B and D are measurements from three experiments with RH cells and two with A549 cells. Arbitrary units correspond to immunofluorescence intensity calculated as total immunofluorescence in the cell divided by the area of the cell. Error bars indicate SEM. *p = 0.037, ***p = 0.0004 according to Student’s t-test.
Fig 3.
Internalized E. granulosus AgB colocalizes with protein endocytic markers in RH cells.
Alexa Fluor 633-conjugated Tfn was used as a marker of clathrin-mediated endocytosis and Alexa Fluor 555-conjugated Ctx-B was used as a marker of caveolae/raft-mediated endocytosis. Confocal microscopy images of RH cells incubated with 40 μg/ml AgB and 1 μg/ml Ctx-B (A) or 50 μg/ml Tfn (B) are presented. AgB was detected using polyclonal antibodies against AgB8/1, 2 and 4 subunits and a secondary anti-rabbit IgG Alexa Fluor 488 conjugated antibody (green). Arrows indicate colocalization points. Cell nuclei were labeled with DAPI (blue). The endocytic markers are shown in red.
Fig 4.
E. granulsous AgB reaches the endolysosomal system after endocytosis by RH cells.
DiI-labelled AgB (red) at 40 μg/ml and Lysosensor DND-189 (green) at 1.5 μM were added to the cell culture media and left incubate for 1.5 h. Median sections from z-stacks of confocal images are shown. Arrows indicate colocalization points.
Fig 5.
A549 (black bars) and RH (gray bars) cells were treated for 24 h with the indicated concentrations of E. granulosus AgB. Cell viability is expressed as percentage of MTT reduction measured for untreated cells and assumed as 100% (horizontal dashed line). Error bars correspond to the SEM values of five independent experiments.