(A) Flow cytometric analysis of CSR to IgG1 in ATM^-/-, wild type (WT, littermate control for ATM), DNA-PKcs^-/--HL and WT-HL (littermate control for DNA-PKcs^-/--HL). DNA-PKcs^-/--HL and WT-HL are matched littermates expressing immunoglobulin heavy VB1-8 and light chain V3-83 genes that rescue B cell development in DNA-PKcs^-/-mice (Manis et al., 2002). B cells were labeled with CFSE to assess cell division and stimulated with LPS+IL4 for 3 days. Graphs show switching relative to matched wild type sample, each dot represents an individual experiment and the bar represents the average. (B) Flow cytometric analysis of CSR as in (A) in the presence or absence of PKi NU7026. Graphs as in (A) but show switching relative to the matched sample in the absence of Pki as indicated.

(A) Wild type and ATM^-/- B cells were either untreated or pretreated with PKi NU7026, stimulated with LPS+IL4 for 72 hours, and IgG1⁺ cells were sorted for further analysis. Genomic DNA was amplified by PCR, and Sμ/Sγ1 junctions were sequenced. Percentage of junctions with indicated nucleotide overlap or insertions is indicated. (B) Sμ/Sγ1 switch junctions with insertions greater than 2bp from ATM^-/- B cells in the presence or absence of PKi NU7026. The numbers below each sequence correspond to Sμ (Genbank J00440) and Sγ1 (Genbank D78344) germline sequences. Blue letters indicate sequences homologous to germline, red letters indicate mutations, insertions with no known homology or microhomology at junctions, -- indicates contiguous sequences that are not shown, / indicates a break in the sequence. Number of sequences analyzed: ATM^-/-=50; ATM^-/-+PKi=56; WT=44; WT+PKi=20.

(A) Frequency of IgH locus breaks (black), IgH locus translocations (grey, striped), and non-IgH breaks (white) in 3 independent cultures of ATM^-/- and wild type B cells mock or pre-treated with PKi NU7026. Metaphase spreads (>80 analyzed for each experiment) were prepared 72 hours after B cell stimulation with LPS and IL4. (B) c-myc/IgH translocations. Representative agarose gel (top) and Southern blots with c-myc (middle) and IgH (bottom) probes. Frequency of c-myc/IgH translocation per 10⁶ cells is plotted on the right p=0.0002 (ATM^-/- vs. ATM^-/- pretreated with PKi NU7026).

(A) Wild type, ATM^-/- and 53BP1^-/- B cells were cultured with LPS and IL4 with or without PKi NU7026 for 48h, and harvested 1 hour after 0 or 10 Gy irradiation as indicated. Western blots show 53BP1 phosphorylation (arrow) and tubulin loading controls. * indicates non-specific band. (B) 53BP1 focus formation in ATM^-/- B cells 6 hours after treatment with PKi (NU7026) or another potent and selective inhibitor of DNA-PKcs (NU7441) (Leahy et al., 2004) and 1Gy irradiation. (C) Western blots for Kap-1, SMC1, and Chk2 phosphorylation and tubulin loading controls in irradiated B cells treated as in (A). (D) Western blot shows p53 protein levels and p53 phosphorylation on Ser15, 4 hours after 0 or 5 Gy treatment. Cells were pretreated with PKi (NU7026) or an independent selective inhibitor of DNA-PKcs (NU7441). (E) DNAPKcs was depleted in cultured WT and ATM^-/- B cells using short hairpin RNAs. Five days after infection, cells were untreated or irradiated, sorted and blotted for DNA-PKcs, phosphorylated Kap-1, phosphorylated p53 (upper panel) or phosphorylated 53BP1 (lower panel). (F) B cells from WT, ATM^-/- and conditional ATM^c/cDNAPK^-/- mice were infected with a retrovirus expressing Cre-GFP (+) or GFP (-) for 5 days. Cells were sorted, irradiated and levels of Kap-1 and H2AX phosphorylation were assessed by Western blotting.

(A) Immortalized WT and ATM^-/- MEFs were preincubated for 1hour with or without PKi and levels of phosphorylated Kap-1 were determined 1hour after 5 or 20Gy irradiation. (B) Levels of DNAPKcs and IR induced phosphorylated Kap-1 in immortalized WT and ATM^-/- MEFs stably expressing lentiviruses harboring PKcs shRNA. Down regulation of DNA-PKcs in ATM^-/- MEFs was more efficient using PKsh62 compared to PKsh59. (C) Passage 2 primary MEFs derived from ATM^c/+, ATM^c/c or ATM^c/cDNAPKcs^-/- mice were infected with an adenovirus expressing Cre-GFP (+), irradiated with 20Gy and levels of Kap-1 and p53 phosphorylation were analyzed by Western blotting 1 hour post irradiation. (D) Decreased levels of DNA-PKcs in WT MEFs relative to B cells as determined by Western blotting.

(A) Wild type, ATM^-/-, and p53^-/- thymocytes were mock or pre-treated with ATMi (KU55933) or PKi (NU7026), irradiated with 5 Gy and the percentage of live cells was measured by flow-cytometry 24 hours post-IR using a caspase-3-substrate reagent (OncoImmunin Inc.). Percentage of live cells (Propidium Iodide (PI) and Caspase 3 negative) is indicated. Graph shows results of individual experiments (dots) normalized to unirradiated drug-treated samples of the same genotype. Bars indicate the average. (B) Quantitative RT-PCR analysis of p53-dependent induction of NOXA, PUMA, BAX and p21 mRNAs 4 hours after treatment with 5Gy plotted relative to un-irradiated controls of the same genotype. An average of two experiments performed in triplicate is shown. The experiments were repeated 6 times with similar results.