Ku86 Regulates Pathway Choice in Human Cells Figure 6. The reduction of Ku results in elevated levels of end joining in C-NHEJ mutant cell lines. (A) FACS profiles using the pEGFP- Pem1-Ad2 reporter substrate are shown for the indicated cell lines. The profiles for the Top and Bottom panels were generated using HindIII- and I- SceI-linearized plasmids, respectively. The percent of the substrate that was repaired is shown in the right-hand corner of each profile. (B) Western blot analyses demonstrate a reduction in Ku protein levels. Western blots for extracts derived from the indicated cell lines are shown using antibodies against either Ku86, Ku70 or (as a loading control) tubulin (Tub). Each of the blots was quantitated using a phosphoimager and the level of a particular Ku subunit relative to the amount expressed in the parental cell line is indicated below each blot. (C) Four independent experiments comparable to those depicted in (A) were performed and the average percent repair is shown with the standard deviation. doi:10.1371/journal.pgen.1000855.g006 microhomology-mediated end joining was virtually 100%. Impor- tantly, the re-introduction of a wild-type DNA-PKcs or XLF cDNA, into their respective cell line, partially and completely, respectively, reverted the repair events to a C-NHEJ spectrum. Again, the degree of complementation was directly related to the degree of complementing protein expression achieved in these cell lines (data not shown). PLoS Genetics | www.plosgenetics.org 7 February 2010 | Volume 6 | Issue 2 | e1000855