Figure 4
Loss of Casp9 or Apaf1 results in decreased apoptosis and increased DNA damage after ENU treatment. (A) FLCs from Casp9−/− or Apaf1−/− pups (or WT littermates) were treated with ENU or an equivalent concentration of vehicle only (0.1% DMSO) for 1 hour and then allowed to recover for 24 hours. Cells were then stained with Annexin V to measure apoptosis. Casp9−/− and Apaf1−/− FLCs had reduced apoptosis in response to ENU. Each bar represents the mean ± SEM for 3 to 9 mice. (B) To determine whether the reduction in apoptosis detected in Casp9−/− and Apaf1−/− FLCs was associated with an increase in DNA damage in the surviving cells, the alkaline comet assay was used to measure DNA damage in cells exposed to varying doses of ENU with varying lengths of recovery time after exposure. Casp9−/− cells showed increased DNA damage at all ENU doses tested at 72 hours post-ENU treatment. DNA damage decreased over time in ENU-treated Casp9+/+ FLCs as apoptotic cells were cleared, but was sustained at high levels for up to 120 hours after ENU exposure (300 μg/mL) in Casp9−/− FLCs. Each bar represents the mean ± SEM of at least 140 nuclei from 2 to 3 (Casp9+/−) or 3 to 5 (Casp9−/− and Casp9+/+) FLC samples. (C) Comets from Casp9−/− and Apaf1−/− nuclei (300 μg/mL ENU, 72 hours after treatment) had significantly higher levels of DNA damage (% of DNA in tail) compared with other genotypes. Bars show the mean of at least 200 nuclei from at least 3 mice, with vertical lines extending to the first and third quartiles. (D) Lethally-irradiated WT congenic mice were transplanted with Casp9+/+, Casp9−/−, Apaf1+/+, or Apaf1−/− FLCs and treated with ENU (200 mg/kg IP) or vehicle only (10% DMSO) at 12 weeks posttransplant. Seventy-two hours after injection, mice were sacrificed and bone marrow nuclei were analyzed for DNA damage by comet assay. Consistent with in vitro results, Casp9−/− and Apaf1−/− bone marrow nuclei showed increased levels of DNA damage compared with WT nuclei after ENU treatment (n = 3-4 mice, at least 270 nuclei from each). *P < .05, ***P < .0005.

Loss of Casp9 or Apaf1 results in decreased apoptosis and increased DNA damage after ENU treatment. (A) FLCs from Casp9−/− or Apaf1−/− pups (or WT littermates) were treated with ENU or an equivalent concentration of vehicle only (0.1% DMSO) for 1 hour and then allowed to recover for 24 hours. Cells were then stained with Annexin V to measure apoptosis. Casp9−/− and Apaf1−/− FLCs had reduced apoptosis in response to ENU. Each bar represents the mean ± SEM for 3 to 9 mice. (B) To determine whether the reduction in apoptosis detected in Casp9−/− and Apaf1−/− FLCs was associated with an increase in DNA damage in the surviving cells, the alkaline comet assay was used to measure DNA damage in cells exposed to varying doses of ENU with varying lengths of recovery time after exposure. Casp9−/− cells showed increased DNA damage at all ENU doses tested at 72 hours post-ENU treatment. DNA damage decreased over time in ENU-treated Casp9+/+ FLCs as apoptotic cells were cleared, but was sustained at high levels for up to 120 hours after ENU exposure (300 μg/mL) in Casp9−/− FLCs. Each bar represents the mean ± SEM of at least 140 nuclei from 2 to 3 (Casp9+/−) or 3 to 5 (Casp9−/− and Casp9+/+) FLC samples. (C) Comets from Casp9−/− and Apaf1−/− nuclei (300 μg/mL ENU, 72 hours after treatment) had significantly higher levels of DNA damage (% of DNA in tail) compared with other genotypes. Bars show the mean of at least 200 nuclei from at least 3 mice, with vertical lines extending to the first and third quartiles. (D) Lethally-irradiated WT congenic mice were transplanted with Casp9+/+, Casp9−/−, Apaf1+/+, or Apaf1−/− FLCs and treated with ENU (200 mg/kg IP) or vehicle only (10% DMSO) at 12 weeks posttransplant. Seventy-two hours after injection, mice were sacrificed and bone marrow nuclei were analyzed for DNA damage by comet assay. Consistent with in vitro results, Casp9−/− and Apaf1−/− bone marrow nuclei showed increased levels of DNA damage compared with WT nuclei after ENU treatment (n = 3-4 mice, at least 270 nuclei from each). *P < .05, ***P < .0005.

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