down and fixed over-night at 4uC (with a 2.3 M sucrose solution on top of them). Cell pellets were cut on an ultramicrotome (Leica, Germany) at 70 nm thickness and sections were placed on carbon- coated hexagonal grids. The sections were incubated with rabbit polyclonal antibody against IGF1R overnight at 4uC, washed with PBS, incubated with ProteinA-Gold-10 nm antibody for 2 h at room temperature, and embedded in methyl-cellulose-0.3% uranyl acetate for 5 min. Photos were taken with a JEOL JEM1011 Transmission Electron Microscope connected to a digital video camera (MegaView III) or a Leica DM/RB microscope with an RCM module and a DFC-420C digital camera. 3–5 independent experiments were performed and the final data presented selected as the most representative of the results obtained. Western Blotting (WB) and Immunoprecipitation (IP) IP and WB studies were performed as previously reported [11,12]. The antibodies used were: anti-AKT, anti-p-AKT (Ser473), anti-MAPK42/44, anti-p-MAPK42/44 (Thr202/ Tyr204) (all from Cell Signaling, Danvers CA), anti-caveolin1 (Abcam), anti-clathrin (Affinity Bioreagents) and anti-GAPDH (Abcam). 3–6 independent experiments were performed and the final data presented selected as the most representative of the results obtained. Proliferation studies The dose-response proliferation levels of the cell lines subjected to drug treatment were analyzed to determine the IC50 of proliferation (control treatment performed using the vehicle for CPMZ and MCD). The percentage of proliferation inhibition induced by the specific CAV1/clathrin siRNAs was also evaluated. 4–6 independent experiments were performed and the final data are presented as averages of all replicates. To determine the proliferation rate, we used the MTT method, as previously described.[11] Apoptosis The apoptosis and necrosis indices were assessed by flow cytometry, as previously described.[11] 3–5 independent experi- ments were performed, the final data being presented as averages of all replicates. Briefly, cell pellets were washed twice with PBS and stained with Anexin V for 15 minutes, washed, and then stained with Propidium Iodide (IP) for 5 minutes. Cells were acquired in a FACS Calibur cytometer. Cells with doble staining were considered as Apoptotic and Necrotic (A/N) while cells with no staining were considered Alive. 4–6 independent experiments were performed and the final data are presented as averages of all replicates. Isobolographic Analysis The effects of the combination of AEW with CPMZ or MCD were analyzed using Loewe’s isobolographic analysis, revised by Steel and Peckman, as previously described [11]. Briefly, for isoeffective doses of a two-drug combination (dA + dB) and the individual drugs alone (DA and DB), combinations having a combination index (CI).1 were considered antagonistic; those with a combination index = 1 were additive, and those with a combination index ,1 were synergistic, the combination index being = dA/DA + dB/DB. The CI values for each condition were calculated using the IC50 of proliferation as the isoeffective point (using Origin 6.0 to plot the data). Isobolograms were acquired by plotting the IC50 of AEW on the Y-axis and the IC50 of CPMZ or MCD on the X-axis. IGF1R Endocytosis in Ewing Sarcoma Phospho-kinase array The effects of the AEW with CPMZ and/or MCD combina- tions were analyzed at the level of global kinase phosphorylation, using the dot-blot Proteome ProfilerTM Array Human Phospho- Kinase Array Kit (R&D Systems), according to manufacturer’s instructions. Briefly, cells were treated with 75 nM AEW, 7.5 mM CPMZ and/or 2.5 mM MCD for 1 hour, stimulated with IGF1 for 15 minutes, and lysates were prepared with the appropriate lysis buffer (PBS 1x, 1 mM Na2EDTA, 1 mM EGTA, 50 mM NaF, 1%Triton x-100, 5 mM Na3VO4 and protease inhibitor cocktail tablet (Roche). 400 mg of protein was used. The signals from the membranes were detected as described for WB and the levels of chemiluminescence of each spot were evaluated by 2D- densitometry using the AIDA . software (Fujifilm-Raytest, Straubenhardt, Germany). 3 independent experiments were performed and the final data are presented as averages of all replicates. Statistical Analyses One-Way ANOVA for independent samples was performed, using the VassarStats web site for Statistical Computation. Supporting Information Figure S1 siRNA knockdown efficiency studies. Ewing’s sarcoma cells were grown until they reached 30–40% confluence, treated with CAV1 or clathrin siRNAs, and then proteins were extracted and WB performed, as described in the Materials and Methods section. A) WB detection of clathrin and Cav1 protein levels. B) Densitometric analysis of the WBs. The siRNAs used effectively reduced CAV1/clathrin protein levels, with a 40–65% reduction, depending on the ES cell line treated. UTF: untransfected; mock: transfected without siRNA. 3 independent experiments were performed and the final data presented selected as the most representative of the results obtained -panel A and as averages of all replicates-panel B. (TIF) Figure S2 Densitometric analyses of WB shown in figure 2C and D of effects of CAV1 or clathrin inhibition/interference on the IGF1R signaling pathway. The inhibition of phosphorylation reached levels of over 60% reduction (p,0.05). The results obtained with the A673 and A4573 cell lines were identical to those described for TC71 (data not shown). Data presented is representative of 3–6 independent experiments. (TIF) Figure S3 Effects of CAV1 or clathrin inhibition/ interference on the IGF1R signaling pathway. Ewing’s sarcoma TC71 cells were starved overnight, stimulated with IGF1 (50 ng/ml) for 15 minutes, and then proteins were extracted and WB was performed, as described in the Material and Methods section. A) Effects of clathrin inhibition/interference. B) Effects of Cav1 inhibition/interference. The phosphorylation of AKT and MAPK proteins in siRNA- or drug-treated cells without IGF1 treatment showed that siRNA or drug treatment alone do not interfere with protein phosphorylation. 3 independent experiments were performed and the final data presented selected as the most representative of the results obtained. (TIF) Figure S4 Effects of CAV1 or clathrin inhibition on apoptosis in the ES cell lines A673 and A4573. Ewing’s sarcoma cells were grown until they reached 30–40% confluence, treated with MCD/CPMZ for 72 hours, and then apoptosis was measured by flow cytometry, as described in the Materials and PLoS ONE | www.plosone.org 10 May 2011 | Volume 6 | Issue 5 | e19846