FIG. 1.

Depletion of DDB1 activates cell cycle checkpoints. (A) The cell cycle distribution of HeLa cells transfected with control or DDB1 siRNA oligonucleotides was analyzed by flow cytometry detection of DNA content following propidium iodide staining 3 days after transfection. The percentage of mitotic cells in each population was determined by immunostaining with a phosphopeptide-specific antibody to histone H3 S10. (B) HeLa cells transfected with DDB1 or control siRNA oligonucleotides were processed for flow cytometry 4 days after transfection. Where indicated, the cells were cotransfected with ATM- or ATR-specific siRNA or treated with 8 mM caffeine for 24 h prior to analysis. The depletion efficiency of the ATR and ATM siRNA was monitored by immunoblot analysis. (C to E) Checkpoint signaling was examined in HeLa (C), U2OS (D), and RPE-hTERT (E) cells 3 days after transfection with control or DDB1 siRNA oligonucleotides. Cell lysates were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotted with the indicated antibodies. (F and G) U2OS cells were infected with retroviruses encoding an empty vector or an siRNA-immune Flag-DDB1 cDNA (DDB1*). Following selection the cells were transfected with control or DDB1 siRNA oligonucleotides. Checkpoint signaling was examined 3 days after transfection by immunoblotting with the indicated antibodies (F) or monitoring DNA content by flow cytometry of propidium iodide-stained cells (G).