e saturation density. Whereas AXTshLUC cells continued to grow past confluence, resulting in the formation of large piles and high saturation density, AXT-sh1 and AXT-sh2 cells manifested contact inhibition and lower saturation density. Aberrant Imp3 expression in tumor cells may thus promote anchorage-independent growth and loss of contact inhibition. Knockdown of Imp3 in AXT Cells Suppresses Tumorigenic Activity in vivo We next examined whether knockdown of Imp3 in AXT cells might affect tumorigenic activity in vivo. Whereas all mice injected subcutaneously with Imp3-depleted AXT cells developed palpable tumors, the weight of these tumors was significantly smaller than those derived from AXT-shLUC cells. None of the mice injected with Imp3-depleted AXT cells manifested lung or liver metastasis, whereas all mice injected with AXT-shLUC cells developed metastases at both sites. We also evaluated tumorigenic activity after intraperitoneal injection of osteosarcoma cells, which resulted in earlier death from primary tumors. Whereas AXT-sh2 cells did not generate lethal tumors within.140 days, AXT-shLUC cells did so within Imp3 Activates Osteosarcoma Tumorigenesis In Vivo 6 Imp3 Activates Osteosarcoma Tumorigenesis In Vivo 33 days. One mouse injected subcutaneously with AXT-sh1 cells developed lethal osteosarcoma tumors at 49 days after cell injection, and the Imp3 expression in these tumors was markedly increased compared with the parental AXT-sh1 cells. Furthermore, Imp3 expression in a lethal tumor derived from AXT-sh2 cells at 141 days after intraperitoneal cell injection was also increased compared with the parental cells. The efficiency of Imp3 knockdown by shRNA might be reduced during tumor development with a long latency. Together, these findings indicated that upregulation of Imp3 in osteosarcoma cells plays an important role in tumorigenesis in vivo. Imp3 Regulates Tumorigenic Activity Independently of Igf2 Possessing six RNA binding motifs, including two RNA recognition motifs and four KH domains, Imp3 is implicated in the regulation of target mRNAs. To Imp3 Activates Osteosarcoma Tumorigenesis In Vivo examine the role of Imp3 in translational regulation in AXT cells, we further determined its intracellular localization by centrifugation of cell lysates on a sucrose gradient. Immunoblot analysis revealed that ribosomal protein S6 was present in fractions 3 to 7, corresponding to ribosome subunits as well as TG100 115 individual ribosomes, and in fractions 8 to 16, corresponding to polysomes. Imp3 was found to colocalize largely with rpS6 in the gradient fractions. In contrast, Ago2 was present mostly in fractions 1 to 3, consistent with previous observations. The distribution pattern for Imp3 suggested that the 
protein localizes to ribosomes and polysomes. Igf2 mRNA has been implicated as a main target of Imp3, and activation of Igf2 mRNA translation driven by Imp3 was previously found resulting in modulation of cellular functions such as proliferation and tumorigenic activity as well as tumor cell invasion. We therefore examined whether the effects of Imp3 on cell behavior observed in this study might be mediated by Igf2. We first evaluated whether exogenous Igf2 might recapitulate the enhancement of cell proliferation induced by Imp3. The rate of proliferation of AX and Imp3-depleted AXT cells under non-adherent conditions was increased by Igf2 in a concentrationdependent manner, with this effect reaching a plateau at concentra- tion o