Overexpression of LIP in MCF10A cells was accomplished download catalog using a pEIZ lentiviral construct driven by the EF alpha 1 promoter. Overexpression of LIP led to decreases in apoptosis as evidenced by the number of Annexin V positive cells and the accumula tion of cells in sub G1 at both 48 hr and 96 hr of anoi kis. These data suggest that the LIP isoform has an anti apoptotic action and plays a role in cellular survival of anoikis. Thus the biological consequence of IGF 1R mediated increases in LIP expression may include the actions of LIP to participate in the regula tion of cell survival. Our data demonstrate that treat ment of cells with IGF 1 or overexpression of LIP leads to decreases in the percentage of cells in sub G1, and Inhibitors,Modulators,Libraries decreases in the number of cells positive for Annexin V, thus representing a decrease in apoptosis.

Taken together, the data in Figure 6 demonstrate that C/EBPb knockdown leads to increased cell death and an accumulation of cells Inhibitors,Modulators,Libraries in sub G1 and suggest that C/ EBPb expression is important for survival and resistance to anoikis. Furthermore, we showed that IGF 1R treat ment can partially rescue control cells from anoikis. however, cells with reduced C/EBPb expression, are not successfully rescued from anoikis. This is most clearly observed in clonogenic outgrowth assays of C/EBPb knock down cells. Suspension culture of vector control and C/EBPb knock down cells, Inhibitors,Modulators,Libraries in the presence of IGF 1 for 24 hr, followed by harvest and subsequent plating for adherent growth revealed a dra matic reduction in the survival and Inhibitors,Modulators,Libraries clonogenic activity of cells with knocked down C/EBPb expression.

Similarly, overexpression of LIP reduced anoikis, as evidenced by the decreased number of Annexin V posi tive cells and the decreased number of sub G1 cells. In summary, C/EBPb expression appears to play an impor tant role in protection from anoikis and may be an Inhibitors,Modulators,Libraries inte gral downstream mediator of the protective effects of IGF 1R signaling. In summary, our data demonstrate that IGF 1 stimula tion of mammary epithelial cells leads to increased expression of LIP and an elevation in the LIP/LAP ratio. We additionally demonstrate that IGF 1R induced LIP expression is biologically active as determined on a C/ EBP responsive promoter construct. Although IGF 1R signaling can crosstalk with EGFR signaling to regulate Erk1/2 activity in our study, IGF 1R induced LIP expres sion is independent of EGFR signaling. We demonstrate that Akt activity is a critical determinant in the regula tion of IGF 1R induced LIP expression and that EGFR dependent, Erk1/2 activity is not necessary for IGF 1R induced LIP expression. Lastly we show that LIP plays a role to increase the survival of cells from so anoikis and may participate in IGF 1R mediated suppression of anoikis.