FIG. 8.

Late-phase inhibition of protein synthesis is signaled by the MRN DNA DSB complex. (A) MCF10A cells were transduced with lentiviruses expressing shRNAs to ATM, NBS1, or NS. Immunoblot analysis was conducted with antibodies as shown. (B) MCF10A cells were transduced with shRNA lentiviruses to ATM, NBS1, or NS and treated with 8 Gy IR, and protein-synthetic rates were measured over a 24-h period by [³⁵S]methionine/cysteine incorporation. (C) MCF10A cells silenced with shRNA-expressing lentiviruses as shown were subjected to 8 Gy IR and harvested at the times shown; equal amounts of protein extract were subjected to m⁷GTP-Sepharose cap chromatography, and retentates were recovered by elution with loading dye at elevated temperature, resolved by SDS-10% PAGE, and detected by immunoblot analysis with antibodies as indicated. (D) MCF10A cells were silenced with control NS or NBS1 shRNA lentivirus vectors, treated with 8 Gy IR, and analyzed by immunoblot analysis at the times shown. hyper, hyperphosphorylated; hypo, hypophosphorylated. (E) MCF10A cells were treated with the ATM inhibitor KU55933 at 10 μM added 30 min prior to treatment with 8 Gy IR and washed out 20 min following treatment and were analyzed by immunoblot analysis at the times shown post-IR treatment.