FIGURE 7.

Suppression of JCV genome replication by abrogation of G₂ arrest. Effect of inhibition of G₂ checkpoint signaling on JCV DNA replication as determined by the DpnI replication assay. IMR-32 cells transfected with pBS-JCori and an expression vector for TAg were incubated for 24 h before addition of dimethyl sulfoxide (DMSO) (control), 50 nm ucn-01, or 2.5 mm caffeine (caf). The siRNA against non-targeting control (Ct) or Wee1 or/and Cdc2 were transfected 24 h before transfection of the plasmids. The collected cells were subjected to immunoblot analysis with antibodies to FLAG-TAg, Wee1, and Cdc2 (A), flow cytometric analysis of cell cycle profile for the TAg⁺ cell subset (B), and DpnI replication assay (C and D). Replicated DNA extracted from the cells was detected by Southern blot analysis with a DNA probe specific for pBS-JCori (C). The intensity of DNA bands was quantified and is indicated in the bar graph relative to the value of dimethyl sulfoxide (for ucn-01 and caffeine) or that of siCt (for siWee1, siCdc2, and siWee+siCdc2) (D); data are mean ± S.D. of triplicates from a representative experiment.