FIG. 4.
(A to D) Impaired proliferation of Mdm4-null fibroblasts in cultures. (A) Growth curves for Mdm4+/+, Mdm4+/−, Mdm4−/−, and Mdm4−/− Trp53−/− MEF cultures at passage 2. A total of 2.5 × 10 4 cells were plated into 12-well plates. Cultures were harvested at daily intervals,and the total number of cells were determined and normalized to the number of cells at day 0 (20 h after plating) (relative number [Nb] of cells). The numbers refer to the mean values of two independently derived MEF cultures of each genotype. (B) Level of BrdU incorporation by asynchronously growing passage 2 Mdm4+/− Trp53+/− and Mdm4−/− Trp53+/− MEFs. (C) Quantitative analysis of the percentage of S-phase cells after serum stimulation for 24 h of G 0-synchronized Mdm4+/+, Mdm4+/−, and Mdm4−/− MEF cultures. The BrdU-labeled MEFs were analyzed by indirect immunofluorescence, and the S-phase cells were determined by staining with anti-BrdU antibody. (D) Photomicrographs of MEFs at passage 2. Mdm4-null MEFs were growth arrested; Mdm4+/+ MEFs were not growth arrested. (E) Constitutive high levels of p21 protein expression in Mdm4−/− MEFs at passage 1. Western blotting analysis from Mdm4+/+ cell lysates, two independent Mdm4+/−, and Mdm4−/− MEF cultures at passage 1. Vinculin serves as loading control. (F and G) Constitutive high levels of p53 protein expression in Mdm4−/− MEFs at passage 3. (F) p53 protein levels determined by Western blotting analysis using the sheep polyclonal antibody Ab-7 from Mdm4+/− and Mdm4−/− MEF cell lysates. Vinculin serves as a loading control. (G) p53 protein (red) levels examined by indirect immunofluorescence using the rabbit polyclonal antibody CM5 in Mdm4+/− and Mdm4−/− MEF cultures. The DNA (blue) is stained with 4′,6′-diamidino-2-phenylindole (Dapi). The absence of signal in Mdm4+/− Trp53−/− MEF cultures demonstrates the specificity of the antibody.