Plasmid rescue The repaired NHEJ reporter pEGFP-Pem1-Ad2 substrates were rescued from human cells using a Qiagen mini-preparation protocol, transformed into E. coli (TOP10) and colonies carrying the repaired plasmids were selected on LB plates containing 30 mg/ml of kanamycin. The fidelity of NHEJ repair events was examined by digesting the plasmid DNA from individual colonies with the restriction enzyme HindIII prior to agarose gel electrophoresis. Precise junctional information was obtained by DNA sequencing using a variety of primers (sequences available upon request) located upstream and downstream of the Ad2 exon sequence. Those events that had not restored the original restriction site were always analyzed by sequencing. For I-SceI- digested substrate, all the repair products were directly sequenced, as incompatible I-SceI sites should not restore the original restriction site(s). Microhomology assay The microhomology assay was performed as described [26]. In brief, 2.5 mg of EcoRV- (NEB) and AfeI- (NEB) digested plasmid pDVG94 were transfected into cells that were ,60% confluent, in 6 well plates, using Lipofectamine 2000 (Invitrogen) according to manufacturer’s instruction. The transfection efficiencies of wild- type HCT116 and the derivative mutant cell lines were determined using the plasmid pEGFP-Pem1 as described above. After transfection (48 hr), plasmid DNA was recovered using a modified Qiagen mini-preparation protocol. Repaired pDVG94 plasmid was PCR amplified using primer FM30 and a 59- radiolabeled primer DAR5 [26]. The PCR product was digested with BstXI (NEB). Restriction fragments were separated by electrophoresis along with undigested PCR product in a 6% polyacrylamide gel in TBE buffer. The gel was subsequently dried and exposed to film. The bands representing the undigested (180 bp) or digested (120 bp) PCR products were quantified using ImageQuant software. RNA silencing All RNA interference reagents were purchased from Dharma- con, including SmartPool siRNA pools against Ku70. Ku70 or scrambled control siRNAs (0.5 mM) were combined in a 1:1 fashion with Dharmafect-1 reagent in a total volume of 400 mL. After a 20 min incubation at room temperature the siRNA solution was diluted to 2.0 mL with complete growth media and then added to the target cells. This procedure was repeated at least one additional time at ,24 hr intervals. Supporting Information Figure S1 The indicated cell lines were transfected with HindIII- (Top panels) or I-SceI- (Bottom panels) linearized pEGFP-Pem1- Ad2 together with a supercoiled pCherry plasmid (to monitor transfection efficiency). The number in the top right corner corresponds to the percentage of cells that turned green after 24 hr as a percentage of the cells productively transfected. Found at: doi:10.1371/journal.pgen.1000855.s001 (6.55 MB TIF) Figure S2 The data presented individually in Tables S1, S3, S5, and S7 using the HindIII-linearized pEGFP-Pem1-Ad2-lenearized plasmid was consolidated into 4 categories: perfect joins (dark rectangles), imperfect joins (light gray rectangles), microhomology (white rectangles), and insertions (dark gray rectangles) and is presented as the percentage of total events for each of the indicated cell lines. Found at: doi:10.1371/journal.pgen.1000855.s002 (8.38 MB TIF) Ku86 Regulates Pathway Choice in Human Cells Figure S3 The data presented individually in Tables S2, S4, S6, and S8 using the I-SceI-linearized pEGFP-Pem1-Ad2-lenearized plasmid was consolidated into 3 categories: imperfect joins (light gray rectangles), microhomology (white rectangles) and insertions (dark gray rectangles) and is presented as the percentage of total events for each of the indicated cell lines. N.B. Perfect joining is not possible with this substrate. Found at: doi:10.1371/journal.pgen.1000855.s003 (8.70 MB TIF) Figure S4 The data presented individually in Tables S1, S3, S5, and S7 using the HindIII-linearized pEGFP-Pem1-Ad2-lenearized plasmid was analyzed only for deletions. Each dot represents an individual data point and some dots overlap. The mean (dark rectangle) and the median (gray rectangle) are shown for each of the indicated cell lines. Found at: doi:10.1371/journal.pgen.1000855.s004 (8.41 MB TIF) Figure S5 The data presented individually in Tables S2, S4, S6, and S8 using the I-SceI-linearized pEGFP-Pem1-Ad2-lenearized plasmid was analyzed only for deletions. Each dot represents an individual data point and some dots overlap. The mean (dark rectangle) and the median (gray rectangle) are shown for each of the indicated cell lines. Found at: doi:10.1371/journal.pgen.1000855.s005 (8.55 MB TIF) Table S1 Sequence analysis for HindIII-linearized pEGFP- Pem1-Ad2 plasmids recovered from Ku-deficient cells. Found at: doi:10.1371/journal.pgen.1000855.s006 (10.54 MB TIF) Table S2 Sequence analysis for I-SceI-linearized pEGFP-Pem1- Ad2 plasmids recovered from Ku-deficient cells. Found at: doi:10.1371/journal.pgen.1000855.s007 (10.06 MB TIF) Table S3 Sequence analysis for HindIII-linearized pEGFP- Pem1-Ad2 plasmids recovered from DNA-PKCS-deficient cells. Found at: doi:10.1371/journal.pgen.1000855.s008 (7.79 MB TIF) Table S4 Sequence analysis for I-SceI-linearized pEGFP-Pem1- Ad2 plasmids recovered from DNA-PKCS-deficient cells. Found at: doi:10.1371/journal.pgen.1000855.s009 (9.39 MB TIF) Table S5 Sequence analysis for HindIII-linearized pEGFP- Pem1-Ad2 plasmids recovered from XLF-deficient cells. Found at: doi:10.1371/journal.pgen.1000855.s010 (7.26 MB TIF) Table S6 Sequence analysis for I-SceI-linearized pEGFP-Pem1- Ad2 plasmids recovered from XLF-deficient cells. Found at: doi:10.1371/journal.pgen.1000855.s011 (8.34 MB TIF) Table S7 Sequence analysis for HindIII-linearized pEGFP- Pem1-Ad2 plasmids recovered from LIGIV-deficient cells. Found at: doi:10.1371/journal.pgen.1000855.s012 (7.31 MB TIF) Table S8 Sequence analysis for I-SceI-linearized pEGFP-Pem1- Ad2 plasmids recovered from LIGIV-deficient cells. Found at: doi:10.1371/journal.pgen.1000855.s013 (8.11 MB TIF) Acknowledgments The authors are indebted to Dr. V. Gorbunova (University of Rochester, NY), Dr. G. Illiakis (University of Duisburg-Essen Medical School, Germany), and Dr. D. van Gent (Erasmus University, Netherlands), and members of their laboratories who were extremely generous with their reagents and advice. We thank Drs. Anja-Katrin Bielinsky (University of Minnesota, MN) and Katheryn Meek (Michigan State University, MI) for their helpful comments on the manuscript. We are indebted to Brian Ruis and Sehyun Oh (University of Minnesota, MN) for access to their DNA- PKcs and LIGIV mutant cell lines, respectively. PLoS Genetics | www.plosgenetics.org 12 February 2010 | Volume 6 | Issue 2 | e1000855