We observed that under basal conditions (without IGF1 stimulation), IGF1R was present in the plasma membrane. However, upon ligand stimulation IGF1R was internalized by clathrin-dependent mechanisms (figure 1B) and by CAV1- dependent mechanisms, although to a lesser extent (figure 1A). Finally, we observed IGF1R co-localization with both clathrin and CAV1 in the endocytic vesicles (figure1 A and B). Moreover, since ES cells have scant cytoplasm, we were interested in confirming IGF1R signaling there, distinguishing this signaling from that performed by active IGF1R present in the cell membrane. As shown in figure 2, RCM/EM confirmed our previous results, clearly demonstrating that IGF1R was located in the cytoplasm (figure 2A top panel) and in vesicles budding off the cell membrane (figure 2A lower panel). In order to confirm IGF1R internalization mediated by clathrin and CAV1, we performed co-IP studies. The results showed that after stimulation with IGF1, IGF1R co-precipitated with both clathrin and CAV1 (figure 2B). Again, co-precipitation occurred mainly with clathrin. Finally, we were able to confirm our initial hypothesis: in ES, IGF1R is indeed internalized by clathrin- and CAV1-dependent mechanisms. CAV1 and clathrin interference/inhibition leads to a blockade of IGF1R internalization Here we tested the impact of clathrin or CAV1 interference and inhibition on IGF1R internalization and signaling using two different approaches: 1) specific siRNAs and 2) a drug approach, using MCD and CPMZ. MCD is known to deplete cholesterol, disrupting caveolae,[13] while CPMZ is able to inhibit clathrin- heavy-chain conformations.[14] Regarding clathrin and CAV1 interference, siRNAs effectively reduced CAV1 and clathrin protein levels (40–65% reduction) (see Figure S1 and figure 3A). Regarding clathrin and CAV1 inhibition through the use of MCD and CPMZ, we observed that the disruption of caveolae and the inhibiton of clathrin-heavy-chain conformations led to a decrease in IGF1R internalization (figure 3B). Overall, the interference or inhibition of endocytosis significantly decreased IGF1R levels of internalization, even after IGF1 stimulation, IGF1R being retained in the cell membrane. This shows that clathrin and CAV1 are key partners in IGF1R internalization. CAV1 and clathrin inhibition/interference impairs the IGF1R signaling pathway in ES cells Several groups have shown that RTKs are frequently activated in endosomes, and so we were interested in determining the impact of the inhibition of clathrin and CAV1 endocytosis on IGF1R signalling. Accordingly, we also studied the consequent effects on cell growth and survival. In order to accomplish this, we compared the effects of CAV1 and clathrin inhibition and interference in 3 ES cell lines (TC71, A673 and A4573) with different levels of sensitivity to IGF1R inhibition, the A673 cell line being less sensitive, as reported previously by our group.[11] Globally, we observed that inhibition with MCD and CPMZ was slightly more efficient than interference by siRNAs in all 3 cell lines tested. Nevertheless, both approaches led to the inhibition of IGF1R signaling, with a reduction in AKT and MAPK phosphorylation of more than 60% (p,0.05) (figures 2C and D and Figure S2 and Figure S3). No changes regarding IGF1R phosphorylation were detected. The reduction in MAPK was less evident in the case of CAV1 inhibition. Regarding CAV1 or clathrin inhibition (treatment with MCD and CPMZ), we detected a clear reduction in proliferation, with a dramatic induction of IGF1R Endocytosis in Ewing Sarcoma apoptosis in a dose-dependent manner. The IC50 of proliferation was similar in all cell lines (between 10–15 mM for CPMZ and 4– 4.5 mM for MCD) (figure 4A). Similar results were obtained with the induction of apoptosis (figure 4B, and Figure S4). Additionally, another ES cell line quite independent of IGF1 showed higher IC50 proliferation values, showing that CPMZ and MCD toxicity is actually related to IGF1R endocytosis inhibition and not due to non-specific secondary effects of these drugs (Figure S5). Regarding the siRNA approach, we obtained around 40% of proliferation inhibition and 40–45% of apoptosis induction (figure 4C) for CAV1/clathrin interference at 72 hours of treatment (figure S4 for A673 and A4573 apoptosis data). Interestingly, both treatments mainly induced late apoptosis, few cells undergoing necrosis. CAV1 or clathrin inhibition improves drug-induced IGF1R tyrosine kinase inhibition Previous observations obtained in our own work indicated that IGF1R signaling can be enchanced after clathrin- and/or caveolin-dependent internalization. Accordingly we were interest- ed in testing a new drug combination that has never been assayed before in ES. Here we tested the effect of the combination of the commonly used tyrosine kinase inhibitor, AEW541(AEW), with the previously evaluated endocytosis inhibitors CPMZ and MCD on the levels of proliferation (figure 5A and B) and the induction of apoptosis (figure 5C). As illustrated in the isobolograms shown in figure 5A, AEW541 in combination with MCD exerted additive effects on the cell lines tested (figure 5A). Regarding the combination of AEW with CPMZ, the effects were synergistic in two ES cell lines (A4573 and TC71) (with CI between 0.73–0.86, as shown in the table of figure 5B), and additive in the case of the A673 cell line, which is resistant to IGF1R inhibition and consequently less dependent on the IGF1R signaling pathway, as previously shown by our group [11,12]. As expected, we observed a marked reduction in AKT and ERK1/2(MAPK) phosphorylation (figure 6A y B and Figure S6), in accordance with our data on the effects of CPMZ/MCD on IGF1R signaling (figure 2C and D). AKT phosphorylation elicited a reduction of around 50% when treated with AEW, and 35–40% when treated with CPMZ/MCD. The triple combination almost completely abolished AKT phosphorylation. Regarding MAPK, its phosphorylation was only reduced with AEW or CPMZ treatment (45 and 65% reduction, respectively), undergoing a higher reduction with the AEW+CPMZ combination. This suggests that CAV1 internalization probably does not mediate MAPK signaling. In this study we detected changes in the phosphorylation of several other proteins (figure 6C), such as p38, TOR, MEK1/2, among others. Interestingly, 37% of all proteins underwent the strongest reduction in phosphorylation with CPMZ or MCD treatment and only 10% did so with AEW, while 24% underwent the highest reduction in phosphorylation with the triple treatment. These results suggest that AEW alone is able to inhibit IGF1R signaling but that this process may be enhanced by the combination with endocytosis inhibitors such as CPMZ and/or MCD. Discussion It has been described that IGF1R upregulation promotes cell survival and proliferation in many types of cancer, including carcinomas such as prostate [15], breast [16] and lung carcinomas [17,18], and also in sarcomas [11,19]. Moreover, IGF1R plays an important role in the transformation, motility and metastasis of PLoS ONE | www.plosone.org 2 May 2011 | Volume 6 | Issue 5 | e19846