IGF1R Endocytosis in Ewing Sarcoma Figure 6. Effects of IGF1R tyrosine kinase inhibition combined with CAV1 and/or clathrin inhibition on the ES cell phospho- proteome. A) AKT and MAPK42/44 protein levels and B) densitometric evaluation. The reduction in the phosphorylation of AKT and MAPK42/44 was over 60%. C) Graphic representation of the most relevant changes in protein phosphorylation. Some of them underwent the strongest reduction in phosphorylation simply with CPMZ/MCD treatment (p38, ERK1/2, MEK1/2, or TOR). However, others needed the triple combination in order for the lowest levels of activation to be obtained (such as AKT, HSP27, or AMPKa1). A: AEW, C: CPMZ and M: MCD treatments; + control: IGF1 stimulation without drug treatment; - control: basal conditions, without IGF1 stimulation. Data presented is representative of 3–5 independent experiments for the WB studies and 3 independent experiments for the phospho kinase array. doi:10.1371/journal.pone.0019846.g006 In conclusion, here we show that blockade of IGF1R internalization, either by clathrin or caveolin1, inhibits IGF1R signalling in ES cells. Accordingly, we provide new evidence regarding the role of cav1/clathrin endocytosis in IGF1R internalization. Finally, we consider the combination of TK inhibitors with endocytosis inhibitors as a major and plau- sible strategy to reduce ES cell proliferation through IGF1R signaling. The results shown here are focused on Ewing Sarcoma, but it is extremely likely that they may find a more general application in sarcomas, and in several subsets of other more prevalent neoplasms that depend largely upon IGF1R survival signals (i.e. subsets of lung or breast carcinoma), therefore increasing the potential impact of our findings for sarcoma therapy. Materials and Methods Cell lines The A673, TC-71, SK-ES-1, A4573 and TTC466 cell lines were obtained and maintained as previously described [11,12,37]. All cells were free of Mycoplasma, as screened with the MycoAlert Mycoplasma Detection kit (Lonza, Rockland, ME). Drugs AEW541 (AEW) was kindly provided by Novartis Pharma AG, Basel, Switzerland. Chlorpromazine (CPMZ) and Methyl-b- cyclodextrin (MCD) were purchased from Sigma-Aldrich (St Louis, MO). siRNA Caveolin1 pre-designed siRNA, clathrin-validated siRNAs and Silencer negative control siRNA were purchased from Ambion (Austin, TX). Cell lines were treated with 100 nM siRNAs, as previously described,[12] using lipofectamine 2000 (Invitrogen, Carlsbad, CA), according to the manufacturer’s instructions. Immunofluorescence (IF) and Confocal Microscopy (CM) Cells were grown overnight on sterile glass slides (covered with gelatine 0.1%), with serum-free medium and then stimulated with IGF1 for 15 minutes. The glass slides were washed with PBS and PLoS ONE | www.plosone.org 8 May 2011 | Volume 6 | Issue 5 | e19846