PCR and FISH localization of the DRneo probe in K562DRneo and NC10DRneo cells. (Ai) Detection of integrated DRneo in K562 and NC10 cell clones stably transfected with DRneo (K562DRneo and NC10DRneo, respectively) by PCR. A vertical black line has been inserted to indicate a repositioned gel lane. (Aii) The DRneo construct labeled with spectrum red was used for FISH analysis of metaphase spreads prepared from K562DRneo cells. The DRneo was integrated at a telomeric position on the chromosomes as shown in DAPI-stained (left, red arrow) and G-banded (middle, red arrow) images. Two-color FISH was used to localize DRneo to chromosome 4 in K562DRneo cells (right image). Chromosomes from 2 different metaphase cells are shown with yellow signal indicating chromosome 4 alpha satellite probe, localized to the centromere (black arrow) and red signal localizing the DRneo probe to the telomeric region of the chromosome (red arrow). (B) Colocalization of DRneo and γH2AX, Ku86, DNA ligase IV, DNA ligase IIIα, and WRN in K562DRneo cells by FISH. Images show colocalization of these proteins (TRITC, red signal) with DRneo (FITC, green signal) in K562DRneo cells transfected with ISce-1. Nuclei are stained with DAPI (blue). Right-hand panels are merged images of FITC and TRITC showing colocalization.