Figure 2.

Loss of Ku80 in human fibroblasts leads to nondisjunction and telomere abnormalities. (A) Nuclear morphology of control and Ku80-depleted WI38 cells. WI38 fibroblasts were infected with Ku80 shRNA or a scrambled control shRNA for 24 hours and selected in puromycin for 72 hours. Seven days after the treatment with shRNA, cells were stained with DAPI. Images were captured at 20X magnification (first 3 parts) or 40X magnification (last part). Non-infected WI38 fibroblasts were included as an additional control. (B and C) Representative chromosomes from telomere FISH analysis of metaphase spreads from control (B) and Ku80-depleted (C) cultures seven days after infection. Images were captured at 100X magnification and enlarged for clarity. Control indicates non-transfected WI38 cells. The percentage of cells containing telomere abnormalities for each treatment is as follows: non-treated controls, 17% (n = 35); scrambled controls, 10% (n = 30); Ku80-depleted, 62.5% (n = 24). Fisher's exact test was performed to determine significance and the differences between Ku80-depleted and both scrambled and untreated controls were significant (p = 0.004 and p = 0.015, respectively). (D) Total genomic DNA from control (1) and Ku80-depleted (2) fibroblasts was subjected to telomere restriction fragment (TRF) analysis seven days after infection as described in Materials and Methods. Representative Southern blot is shown. Molecular weight standards are provided on the side of the blot.