Figure 10.

Investigation of activity dependence of the MDM2 degraders MD-222 and MD-224 and the MDM2 inhibitor MI-1061 on cereblon (CRBN)-binding, proteasome and neddylation. (A). Western blotting analysis of MDM2 and p53 proteins in RS4;11 cells. RS4;11 cells were treated with the MDM2 inhibitor MI-1061, the MDM2 degrader MD-222 or MD-224 for 2 h in the presence or absence of excess lenalidomide. MDM2, p53 and GAPDH (loading control) proteins were probed with specific antibodies. (B, C). Cell growth inhibition activity of the MDM2 inhibitor MI-1061 and the MDM2 degraders MD-222 and MD-224 in the absence or presence of lenalidomide in the RS4;11 cell line. Cells were treated for 4 days with MI-1061, MD-222 or MD-224 alone or in combination with indicated concentrations of lenalidomide for 4 days and cell viability was determined by a WST-8 assay. (D). Western blotting analysis of MDM2 and p53 proteins in RS4;11 cells. RS4;11 cells were treated with DMSO, MG-132 (10μM), PR-171 (1μM) or MLN4924 (3μM) for 4 hours, followed by treatment with DMSO control, MDM2 inhibitor MI-1061, or the MDM2 degrader MD-224 for additional 2 h. MDM2, p53 and GAPDH (loading control) proteins were probed with specific antibodies.