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Flow cytometry. Twenty-four hours after the treatment with ADR (1 μM), floating and attached cells were collected, washed in ice-cold PBS and fixed in 70% ethanol at ?20 °C. The cells were washed in ice-cold PBS and resuspended in phosphate-citrate buffer (4 mM citric APY29 and 200 mM Na2HPO4) and kept at room temperature for 15 min. Nuclear DNA was stained with propidium iodide (40 μg/ml) in the presence of RNase A (10 μg/ml) and the reaction mixture was incubated in the dark for 30 min. After the incubation with propidium iodide, DNA content of cells was examined by FACScan flow cytometer (Beckton–Dickinson) using CellQuest software.
Results
Down-regulation of NFBD1 as well as MDM2 in late phase of DNA damage response
Fig. 1.
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To further confirm this notion, COS7 cells were transiently transfected with the constant amount of the expression plasmid encoding MDM2 together with or without the increasing amounts of the expression plasmid for NFBD1. As shown in Fig. 1B, the amounts of MDM2 strongly increased in the presence of exogenously expressed NFBD1 in a dose-dependent manner. In contrast, NFBD1-mediated increase in the expression levels of MDM2 mRNA was undetectable ( Fig. 1C). These results suggest that NFBD1 has an ability to stabilize MDM2.