Nu7026

OVCA429 cells were isolated from a patient with late stage, cisplatin-resistant ovarian carcinoma. OVCA429/pCEG cells carrying a green fluorescence protein empty vector and OVCA429/NICD3 cells constitutively expressing green fluorescence protein tagged with NICD3 were sorted by a fluorescence-activated cell sorter and maintained in RPMI 1640 medium (Mediatech Inc, Herndon, VA) supplemented with 10% heat-inactivated fetal bovine serum and 100 U/mL penicillin and streptomycin at 37°C in a 5% CO₂ incubator [34] (link). N-acetyl cysteine (NAC) and MSeA (Sigma-Aldrich, St. Louis, MO) were dissolved in phosphate-buffered saline. NAC is an antioxidant that mainly abolishes hydrogen peroxide. Carboplatin (Enzo Life Sciences, Farmingdale, NY) was dissolved in water. KU 60019 and NU 7026 (Tocris, Ellisville, MO) were dissolved in dimethyl sulfoxide, and were ATP-competitive, selective chemical inhibitors of the kinase activity of ATM and DNA-PK_cs[35] (link), respectively.

B cells were isolated from mouse spleens (n = 6 per genotype) and stimulated for class-switching in culture for 72 hr. Where indicated, cultures were incubated with DNA-PKcs inhibitor 20 µM NU7026 (Tocris, Bristol, UK) dissolved in DMSO, or mock-treated. The stimulation procedure and flow-sorting for CSR analysis was as described [31] (link), [67] (link). Prior to this analysis, cells were counted; numbers and viability were similar for all groups. Sμ-Sγ1 CSR junctions were amplified by PCR using the following conditions for 25 cycles at 95°C (30 s), 55°C (30 s), 68°C (180 s) using the primers (FWD 5′-AATGGATACCTCAGTGGTTTTTAATGGTGGGTTTA-3′; REV 5′ CAATTAGCTCCTGCTCTTCTGTGG-3′) and Pfu Turbo (Stratagene, La Jolla, CA). To the PCR reaction, 5 U of Taq polymerase (Promega, Madison, WI) was added and incubated at 72°C for 10 min. The resulting product was TOPO TA cloned and transformed into Top10 E. coli cells (Life Technologies, Carlsbad, CA) and plasmids were purified and sent for sequencing using M13 FWD and REV primers in addition to the amplification primers for sequencing. 100 clones for each group were analyzed for mutations, deletions, insertions, and sequence overlaps at the junction and both 30 nt upstream and downstream of the junction. p-values were determined by using two-tailed Fisher's exact test.

B cells were isolated from mouse spleens, purified by negative selection with anti-CD43 depletion (Miltenyi) and stimulated with IL-4 and Lipopolysaccharide and IL-4 (Sigma) for 72 hr. Where indicated, cultures were incubated with DNA-PKcs inhibitor 20 μM NU7026 (Tocris) dissolved in DMSO, or mock-treated. Three mice were analyzed in triplicate and cell count numbers and viability were similar for all groups. The culture, flow-cytometric analysis for CSR analysis and junction analysis has been described [32 (link), 50 (link)]. Sμ-Sγ1 CSR junctions were amplified by PCR using the following conditions for 25 cycles at 95°C (30 s), 55°C (30 s), 68°C (180 s) using the primers (FWD 5′-AATGGATACCTCAGTGGTTTTTAATGGTGGGTTTA-3′; REV 5′ CAATTAGCTCCTGCTCTTCTGTGG-3′) and Pfu Turbo (Stratagene). To the PCR reaction, 5 U of Taq polymerase (Promega) was added and incubated at 72°C for 10 min. The resulting product was TOPO TA cloned and transformed into Top10 E. coli cells (Life Technologies) and plasmids were purified and sent for sequencing using M13 FWD and REV primers in addition to the amplification primers for sequencing. 100 clones for each group were analyzed for mutations, deletions, insertions, and sequence overlaps at the junction and both 30 nt upstream and downstream of the junction.

MEK1/2 inhibitor U0126 was purchased from Cell Signaling (Danvers, MA). DNA-PKc inhibitor Nu7026 was purchased from Tocris (Bristol, UK). ATM inhibitor Ku55933 was purchased from Millipore (Billerica, MA). Pierce LDH Cytotoxicity Assay Kit was purchased from Life Technologies (Grand Island, NY), and LDH release was measured according to the manufacturer's instructions.