Figure 5

hMDMX overexpression in HER cells is not sufficient to prevent the Nutlin-3 induced p53-activation, which resembles retinoblastoma cell lines. A. The various HER cell lines were treated with 10 μM Nutlin-3 for 24 hours, and protein levels were analyzed with immunoblotting using the indicated antibodies. B. qRT-PCR analysis of cells treated as in A. Expression levels of hMDM2-p2, p21 and PUMA are shown as the fold induction relative to untreated wild-type HER cells. C. The various HER cell lines were continuously treated with 10 μM Nutlin-3 and proliferation was measured after 120 hours using a WST-1 assay. Relative cell numbers are displayed as survival relative to untreated cells. D. Cells were treated with 10 μM Nutlin-3 for 24 hours and analyzed by flow cytometry. Percentages of cells in S-phase are displayed as indicative for cell proliferation. E. Parental and shRB-HRasV12-HA-hMDMX (RRHX) transformed VH10 and HER cells were treated with 10 μM Nutlin-3 for 24 hours and analyzed with immunoblotting using the indicated antibodies. F. Quantification of the indicated protein levels using Odyssey 2.1 analysis software (LI-COR Biosciences) for at least two different exposures. Relative protein levels were calculated using Vinculin expression as an internal control and indicated as fold induction relative to untreated cells. G. Parental and RRHX transformed HER cells and the retinoblastoma cell lines Y79 and Weri1 were treated with 10 μM Nutlin-3 for 24 hours, and analyzed with immunoblotting using the indicated antibodies.