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Fig. 1 | Stem Cell Research & Therapy

Fig. 1

From: The non-homologous end-joining activity is required for Fanconi anemia fetal HSC maintenance

Fig. 1

Inhibition of NHEJ sensitizes Fanca−/− HSPCs to PARPi-induced cell death and genomic instability. a Gating strategy for sorting HSPCs (LinSca1+c-kit+; LSK). b Inhibition of DNA-PKcs further sensitizes Fanca−/− HSPCs to PARPi-induced cell death. BM LSK cells isolated from wild-type (WT) or Fanca−/− mice were treated with increasing doses of DNA-PKcs inhibitor NU7026 in the presence of PARP inhibitor KU58948 (1 μM; Axon Medchem) for 36 h. Cell viability was determined by trypan blue assay. Percentages of viable cells were normalized to that of WT control at dose 0 μM. *p < 0.05, **p < 0.01, or ***p < 0.001 vs WT control at dose 0 μM. c Inhibition of DNA-PKcs exacerbates genomic instability in Fanca−/− LSK cells. BM LSK cells isolated from WT or Fanca−/− mice were treated with DNA-PKcs inhibitor NU7026 (10 μM), or vehicle control, in the presence of PARP inhibitor KU58948 (1 μM) for 36 h. The cells were subjected to chromosomal breakage analysis. Quantification of chromosomal aberrations in 50 cells in random fields is shown. **p < 0.01 vs WT vehicle control. d Knockdown of Ku70 increases cell death in Fanca−/− HSPCs. BM LSK cells from WT or Fanca−/− mice were transduced with lentiviruses co-expressing eGFP and scramble shRNA or shRNA targeting Ku70. Transduced cells were sorted for eGFP expression and treated with PARP inhibitor KU58948 (1 μM) for 36 h. Cell viability was determined by trypan blue assay. Percentages of viable cells were normalized to that of WT cells transduced with the scramble shRNA control. Insert: Ku70 expression in cells expressing Ku70 shRNAs. *p < 0.05 or **p < 0.01 vs WT scramble shRNA control. e Knockdown of Ku70 exacerbates genomic instability in Fanca−/− LSK cells. The cells described in c were subjected to chromosomal breakage analysis. Quantification of chromosomal aberrations in 50 cells in random fields is shown. *p < 0.05 or **p < 0.01 vs WT scramble shRNA control. f Knockdown of Ku70 increases MMC-induced cell death in Fanca−/− HSPCs. WT and Fanca−/− or Fanca−/− LSK cells expressing Ku70 shRNA (Fanca−/−/Ku70) were treated with increasing doses of MMC (0–1.0 μM) for 36 h. Cell viability was determined by trypan blue assay. Percentages of viable cells were normalized to that of WT cells transduced with the scramble shRNA control. *p < 0.05, **p < 0.01, or **p < 0.001 vs WT scramble shRNA control. g Knockdown of Ku70 exacerbates MMC-induced genomic instability in Fanca−/− HSPCs. The cells described in f were treated with MMC (0.2 μM) for 36 h and then subjected to chromosomal breakage analysis. Quantification of chromosomal aberrations in 50 cells in random fields is shown. **p < 0.01 or ***p < 0.001 vs WT scramble shRNA control

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