5 NF-κB and AKT signaling prevent DNA damage by suppressing RAG1/2 | 127 fluorescence-activated cell sorter–purified IkBaSR-YFP+ cells compared with IkBaSR-YFP– cells (SUPPL Figure 5). Treatment with IKKβi and AKTi increased RAG activity in the control cell population (increasing from a mean of 2% to 32% GFP+ cells), reaching a level similar to that in IkBaSR-transduced cells (Figure 5B), showing limited increase of RAG activity upon treatment (∼1.5-fold). We assessed whether NF-kB is also involved in suppressing RAG expression in untransformed primary mouse pro-B cells. In vitro expanded primary Figure 4. Inhibition of AKT and NF-kB signaling induces RAG-dependent DNA damage in mouse Abl pre-B cells. (A) Mean fluorescence intensity (MFI) of intracellular gH2AX staining in mouse Abl pre-B cells treated as indicated on x-axis. Red symbols represent gH2AX MFI in WT cells, and blue symbols represent gH2AX MFI in RAG2−/− cells. MFIs of untreated controls were subtracted to correct for background staining. Three independent experiments were performed, and horizontal lines represent means. Statistical significances between groups were determined by ANOVA using a Bonferroni’s posttest. (B) Overlay FACS histograms of intracellular gH2AX staining in IKKbi- and AKTi-treated cells. Small B cells (FSC low; left gate) vs large B cells (FSC high; right gate) are shown. (C) Real-time RT-PCR of Cyclin-A, Cdk2, and Cyclin-D1 mRNA expression in WT mouse Abl pre-B cells. Cells were treated with 2.5 mM IKKbi, 2.5 mM AKTi, or 10 mM STI571 for 48 hours. Real-time RT-PCR results are presented relative to the expression of the 18S rRNA housekeeping gene. Three independent experiments were performed, PCRs were performed at least in duplicate, and error bars represent means ± SD of 3 independent experiments. (D) Jk2-RSS DNA breaks are detected by LM-PCR in cell-cycle sorted (G1 and S phase) WT mouse Abl pre-B cells that were treated with 2.5 mM IKKbi and 2.5 mM AKTi. Threefold dilutions of linker-ligated DNA were amplified with a Jk2-specific forward primer and an LM-PCR linker-specific reverse primer. PCR products indicated with an asterisk were excised from the gel, cloned, and sequenced. The middle section of panel D shows the alignments of sequences obtained from G1 and S phase cells to the germline Jk2 sequence. Jk2-ko5 primer site, Jk2-RSS, and the Jk2 coding region are indicated by colored boxes. Sequencing results are listed at the bottom of panel D. *P < .05; **P < .01; ***P < .001. ns, not significant.
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