IGF1R Endocytosis in Ewing Sarcoma Figure 4. Effects of CAV1 or clathrin inhibition/interference in ES cell line proliferation and apoptosis. A) CAV1 or clathrin inhibition reduced ES cell line proliferation in a dose-dependent manner with an IC50 of proliferation similar in all ES cell lines tested (between 10–15 mM for CPMZ and 4–4.5 mM for MCD). B) The results obtained regarding the induction of apoptosis were similar to those obtained with the inhibition of proliferation in all cell lines (A673 and A4573 data shown in SD figure S3). A/N: Apoptotic and Necrotic cells. C) CAV1 or clathrin siRNAs induced around 40% of the inhibition of proliferation and 40–45% of apoptosis induction. Data presented as the average of 4–6 independent replicates. doi:10.1371/journal.pone.0019846.g004 other tumor types [29,30,31]. The observation of IGF1R internalization through caveolin-dependent mechanisms clearly confirms previously published data [9] and provides new insight into the regulation of RTKs throughout internalization in ES. Moreover, Notario’s group has recently shown that CAV1 is a target of EWS/FLI1 and that it is determinant for the oncogenic phenotype and tumorigenicity of ES cells. In fact, this particular study [33] shows that CAV1 knockdown reduces the growth of ES cell-derived tumors in nude mice xenografts. Also in that study the reactivation of CAV1 expression in ES cells enabled the rescue of the original oncogenic phenotype. Furthermore, Toretsky’s data [34] showed that IGF1R is required for the EWS/FLI-1 transformation of fibroblasts. Taken together, all these findings lead to the hypothesis that these molecular hits could be promoting ES tumorigenicity. Our results suggest that IGF1R internalization in ES is under a highly controlled spatial and temporal regulation, combining both clathrin and CAV1 mechanisms and underscoring the role of CAV1 in RTK endocytosis. In keeping with the observations discussed previously, together with Podar’s studies in multiple myeloma [35] and our results from the phospho-array assay, we suggest that IGF1R clathrin- PLoS ONE | www.plosone.org 6 May 2011 | Volume 6 | Issue 5 | e19846