(D) Measurement of initial (CldU+) tract size from protocol while shown in panel C. addition to the previously characterized part of 53BP1 in DNA double-strand break restoration. Rabbit Polyclonal to FGFR1 (phospho-Tyr766) for 24 h and then added hydroxyurea (HU) for 3 h to induce replication fork stalling. This duration of HU exposure was selected because it Manitimus causes replication fork stalling, but fork collapse and the common appearance of double-strand breaks take place only after HU treatments of 12 h or more or with inactivation of ATR (9, 31). We measured cell viability 18 h and 24 h after removal of HU (Fig. 1A and ?andB).B). WT cells showed a small decrease in viability following HU treatment, but 53BP1?/? cells showed a significantly higher Manitimus decrease in viability. We also measured the viability of WT and 53BP1?/? B cells following short-term exposure to the DNA polymerase Manitimus inhibitor aphidicolin or the replication chain terminator gemcitabine (Fig. 1C to ?feet).E). In each case, 53BP1?/? cells showed increased death relative to that of the WT cells, consistent with a role for 53BP1 in protecting cells from the effects of replication stress. Open in a separate windows FIG 1 53BP1 is required for survival of B lymphocytes following transient replication stress. (A) Circulation cytometry analysis of splenic B cells cultured 24 h and either not treated (NT) or treated with 4 mM hydroxyurea (HU) for 3 h. Cell death was assayed 24 h after removal of HU by quantifying the percentage of cells staining for propidium iodide (PI). Numbers in gated areas indicate percentage of the cell populace that remained viable. FSC, ahead scatter of analyzed cells. (B) Quantification of data from panel A. The graph shows percentages of WT and 53BP1?/? cells that became inviable 18 h or 24 h after HU treatment (= 3). Manitimus Error bars display SDs. values were determined with Student’s test. (C) Circulation cytometry analysis of B cells cultured for panel A and then either not treated or treated with 40 M aphidicolin (APH) for 2 h. PI staining shows cells that became inviable measured 18 h post-APH treatment. Numbers in gated areas indicate percentages of the cell populations that remained viable. (D) Circulation cytometry analysis of B cells cultured as for panel A and then either not treated or treated with 250 nM gemcitabine (GEM) for 2 h. PI staining shows cells that became inviable measured 18 h post-GEM treatment. Numbers in gated areas indicate percentages of the cell populations that remained viable. (E) Quantification of data from panels C and D. The graph shows percentages of WT and 53BP1?/? cells that became inviable 18 h after APH or GEM treatment (= 5). Error bars display SDs. values were determined with Student’s test. (F) Colony assay showing survival of mouse embryonic fibroblasts (MEFs) after HU treatment. Cells used were 53BP1?/? MEFs stably transduced having a 53BP1BRCT construct or GFP vector only. Colony figures were normalized to the untreated sample. The chart shows means from 3 experiments. Error bars display SDs. (G) Colony assay showing survival of MEFs stably transduced with either shGFP or sh53BP1 shRNA constructs. Colony figures were normalized to the untreated sample. The chart Manitimus shows means from 2 experiments. Error bars display SDs. To test if 53BP1 deficiency also causes improved cell death following replication stress in immortalized cell lines, we performed clonogenic colony formation assays to measure cell growth following hydroxyurea treatment. First we launched constructs containing either a 53BP1 cDNA (53BP1BRCT) (32) or perhaps a green fluorescent protein (GFP)-only vector into 53BP1?/? mouse embryonic fibroblasts (MEFs) (Fig. 1F). MEFs complemented with the 53BP1 manifestation construct grew at a rate equivalent to that of the control vector, indicating that the presence of 53BP1 does not have a powerful effect on growth after replication stress in immortalized cell lines. As a second test, we knocked down 53BP1 in WT MEFs using short hairpin RNA (shRNA) (Fig. 1G). Cells expressing sh53BP1.