FIG. 10.

HP1 tethering represses VP16-induced transgene activation in a Dicer-independent manner. (A) Real-time RT-PCR analysis of CFP mRNA levels. Cells consecutively transfected with control (black bars) or Dicer (gray bars) siRNA were cotransfected with plasmids as indicated. The relative CFP mRNA levels were determined by normalizing the ratios of CFP mRNA to β-actin mRNA against those of cells cotransfected with control siRNA, phTet-On-FLAG-NLS-VP16, and pBluescriptII KS(−). The remaining levels of Dicer mRNA and protein in Dicer-depleted cells relative to that of control siRNA-transfected cells are shown in parentheses (left, mRNA; right, protein). The mean values and standard deviations from three independent experiments are shown. (B) Western blot analysis of FLAG-tagged fusion proteins in control siRNA-transfected cells. Total cell lysates were resolved by 10% SDS-PAGE and detected with an anti-FLAG antibody. pBlue, pBluescriptII KS(−); VP16, hTet-On-FLAG-NLS-VP16 (bands indicated by arrows in lanes 1 to 5); ΔVP, hTet-On-FLAG-NLS-ΔVP (band indicated by open triangle in lane 2); HP1, hTet-On-FLAG-HP1 (bands indicated by closed triangles in lanes 3 to 5).