In order to investigate the involvement of IGF 1R in the augmented PI3K Akt signaling pathway in the InsR silenced cells, we performed the following two experiments. First, the cells were under incubated with vehicle or an IGF 1R specific antagonist, AG538 for 12 hours, and the cell lysates were subject to in vitro immunoprecipitation lipid kinase assay to mea sure PI3K activity. Pharmacological blockade of IGF 1R effectively reversed the effect of InsR silencing on redu cing PI3K activity. The finding suggests that the elevated PI3K activity in InsR silenced cells was mediated by IGF 1R. Next, we investigated sensitivity of the cells to exogen ous IGF 1. The cells were stimulated with vehicle or a recombinant IGF 1 for 5mintes, and the cell lysates were subject to in vitro immuno precipitation lipid kinase assay to measure PI3K activity.

The ratio of PI3K activity in rIGF 1 treated cells and vehicle treated cells were used as an indicator for cell sensitivity to IGF 1. In SC transfected Inhibitors,Modulators,Libraries cell, PI3K activity was significantly increased by rIGF 1 pretreatment. In InsR silenced cells, however, the increase in PI3K activity in response to rIGF 1 was even more significant. These results Inhibitors,Modulators,Libraries suggest an increase in sensitivity to IGF 1 in InsR silenced cells. antibody followed by immunoblotting with anti IGF 1RB for co immunoprecipitation of hybrid receptors. Sup pression of InsR resulted Inhibitors,Modulators,Libraries in 1 decreased IGF 1R co precipitation, consistent with reduction in hybrid receptor formation. 2 increased formation of IGF 1R homodimers. The latter was confirmed by immunoblotting the supernatants Inhibitors,Modulators,Libraries with anti IGF 1RB.

Detection of IGF 1R was significantly increased in the anti InsR immuno precipitated supernatant from InsR silenced cells, indicat ing increased formation of IGF 1R homodimers. Taken together, InsR silencing resulted in reduced formation in IGF 1R InsR hybrid receptor but increased Inhibitors,Modulators,Libraries formation of IGF 1R homodimer in MES 13 cells. These alterations in the balance of InsR and IGF 1R homodimer and hybrid receptor might contribute to the observed changes in signaling pathways in the InsR silenced cells. CREB 1 mediated MMP 9 expression leads to increased degradation of cellular FN in the InsR silenced cells In the process for exploring the downstream factors which are involved in altered FN accumulation, a couple of unique phenotypes in InsR silenced cells were indenti InsR silencing induces alteration in InsR IGF 1R hybrid formation InsR and IGF 1R are known to be originated from a common ancestral gene and sharing structures which are similar enough to form a hybrid receptor.

To determine whether or not formation of hybrid receptors play a role in the observed activation of IGF 1R PI3K phase 3 Akt signaling pathway in the InsR silenced cells, two set of experiments were performed. First, co immunoprecipitation experi ments were carried out in wild type MES 13 cells. The results verified that IGF 1R and InsR can be reciprocally co immunoprecipitated.