IGF1R Endocytosis in Ewing Sarcoma Figure 1. Confocal microscopy studies of IGF1R internalization in TC71 cells by Cav1-and clathrin-dependent mechanisms. Ewing’s sarcoma TC71 cells were treated as described in the Material and Methods section. A) Cav1-dependent endocytosis studies. B) Clathrin-dependent endocytosis studies. Under basal conditions (non-stimulated with IGF1, see ‘control’ rows), IGF1R is confined to the membrane surface, while after IGF1 treatment it is internalized by clathrin- and caveolin1-dependent mechanisms, thus co-localizing with both clathrin/Cav1 inside the endocytic vesicles (see asterisks). The far right side panels show high magnifications for merge images. The bottom panels show additional images for IGF1R localization in the cytoplasm after IGF1-driven internalization. After ligand stimulation, IGF1R localizes both in ES cells membrane and cytoplasm, co- localizing with clathrin within the clathrin-coated pits (see arrows). The results obtained with CAV1 were similar to those shown for clathrin (data not shown). Scale bars represent 10–20 mm, left and right hand panels, respectively. Data presented is representative of 4–6 independent experiments. doi:10.1371/journal.pone.0019846.g001 PLoS ONE | www.plosone.org 3 May 2011 | Volume 6 | Issue 5 | e19846