FIGURE 8.

siRNA-mediated depletion of hChlR1, Fen1, or Scc1 leads to defects in sister chromatid pairing. A, HeLa cells were transfected with control (aspecific siRNA) or hChlR1-, Fen1-, or Scc1-specific siRNAs and metaphase spreads prepared 48 h following transfection. DNA was stained with 4′,6-diamidino-2-phenylindole, whereas the inset shown to the right represents a higher magnification of a sister chromatid pair; the size reference bar shown is 3 μm long. B (top), the centromeric region of chromosome 9 was probed using the FISH procedure (33), DNA was stained with 4′,6-diamidino-2-phenylindole, and chromosomes were visualized by microscopy; the size reference bar shown is 1 μm. Bottom, the distance between chromatid pairs of at least 100 chromosomes in each case was measured using MetaMorph software and grouped together by the distance separating the sister chromatid as shown in the lower right side of the figure. The results are expressed as the mean percentage of cells ± S.D. of the experiment performed in triplicate. C, an aliquot of cells from each siRNA experiment was harvested and subjected to Western blot analysis to determine the expression of hChlR1, Fen1, and Scc1 proteins using antibodies specific for each protein.α-Tubulin was included as loading control.