Anti-IGF-1R Potentiate the Effect of AS602868 Figure 2. Anti-IGF-1 antibody enhances the cytotoxic effect of IKK2 inhibitors on RPMI8226 et LP1. 2.A Anti-IGF-1R antibody enhanced the cytotoxic effect of AS602868 on RPMI8226 and LP1 cell lines. Cells were plated in triplicate and exposed to various concentrations of AS602868 in the presence or absence of anti-IGF-1R antibody at a fixed concentration of 10 mg/mL. Cytotoxicity was evaluated using an MTT assay. Cells exposed to AS602868 alone were compared to controls cultured in serum alone. Cells exposed to AS602868 in the presence of Anti-IGF-1R antibody were compared to controls cultured in presence of Anti- IGF-1R antibody alone. 2.B Anti-IGF-1R antibody enhanced the cytotoxic effect of TPCA-1 on RPMI8226 and LP1 cell lines. Cells were plated in triplicate and exposed to various concentrations of TPCA-1 in the presence or absence of anti-IGF-1R antibody at a fixed concentration of 10 mg/mL. Cells exposed to TPCA-1 alone were compared to controls cultured in serum alone. Cells exposed to TPCA-1 in the presence of Anti-IGF-1R antibody were compared to controls cultured in presence of Anti-IGF-1R antibody alone. 2.C Anti-IGF-1 R potentiated the apoptotic effect of IKK2 inhibitors AS602868 and TPCA-1 on RPMI8226 and LP1 MM cell lines. Cells were exposed for 72 h with 10 mM AS602868 or 10 mM TPCA-1 in the presence or absence of anti-IGF-1R antibody at a fixed concentration of 10 mg/mL then analyzed by flow cytometry after annexin V staining. Mean6 SD of three experiments ** P,0.001: * P,0.05 versus control. 2.D Effect of anti-IGF-1R antibody+AS602868 on fresh human myeloma samples according to CD221 (IGF-1Ra) status. This combination induced more apoptosis on CD221hi plasma cells than in CD221lo cells. Cells were incubated with 10 mM AS602868 or/and anti-IGF-1R antibody for 24 h. Apoptosis was analyzed by flow cytometry. doi:10.1371/journal.pone.0022641.g002 Effect of AS602868 and anti-IGF-1R antibody on cell cycle progression in MM cell lines In order to determine the effect of AS602868 and/or anti-IGF- 1R on cell cycle progression, we exposed LP1, RPMI8226, MM.1S and U266 cells lines to 10 mM AS602868 and/or 10 mg/ mL anti-IGF-1R antibody. AS602868 alone caused a strong block of cells in the S phase of the cell cycle in the four cell lines, whereas anti-IGF-1R antibody blocked cells in G0–G1 phase only in the LP1 and RPMI8226 lines. When cells were exposed to both molecules, more than 70% of LP1 and RPMI8226 cells were blocked in G0–G1 (Figure 3A). Of note the degree and pattern of cell cycle blockage was quite similar in these two IGF-1R positive lines. Cdc2 p34 is essential for the G2 to M transition. In this study, the analysis of the expression level of cdc2 p34 showed that this protein became undetectable after exposure to AS602868 in the RPMI8226 cells, either alone or in combination with anti-IGF- 1R antibody (Figure 3B). We also analysed the level expression of cyclin A. AS602868 decreased the expression of cyclin A. This reduced content is compatible with the S phase block observed in cells exposed to AS602868. We also analysed the expression level of the cyclin D inhibitor, p21. As shown in Figure 3B, anti-IGF-1R alone or in combination with AS602868 enhanced the expression level of p21 and reduced the expression level of cyclin E which is in keeping with the G0/G1 block observed in cells exposed to anti- IGF-1R (Figure 3B). The expression level of the tumor supressor P53 increased in cells incubated with anti-IGF-1R or AS602868, as well as in cells exposed to the combination of anti-IGF-1R and AS602868 (Figure 3B). Effect of AS602868 and anti-IGF-1R antibody on mitochondrial membrane potential Apoptosis can be related to the release of pro-apoptotic proteins from mitochondria. In order to determine the apoptotic mechanisms triggered by AS602868 and anti-IGF-1R, we analysed the mitochondrial potential in MM cells by DiOC6 (3). In RPMI8226 and LP1, AS602868 induced depolarization of the mitochondrial membrane (Figure 4A). While anti-IGF-1R in itself did not cause depolarization of mitochondrial membrane its combination with AS602868 caused a greater dissipation of mitochondrial potential than did AS602868 alone. To determine whether these alterations in mitochondrial membrane potential were correlated with apoptotic signalling we studied the expression PLoS ONE | www.plosone.org 3 July 2011 | Volume 6 | Issue 7 | e22641