American Society of Hematology

Figure 4

$Figure 4. HES1-induced activation and cleavage of PARP1. (A) Cell-specific cleavage of PARP1. A panel of 3 B-ALL and 3 T-ALL cell lines were transfected with HES1. At 48 hours, lysates were probed for PARP1 expression. Overall PARP1 levels were higher in B-ALL versus T-ALL, and HES1-mediated PARP1 cleavage was greater in B-ALL than in T-ALL. (B) HES1-induced PAR-ylation of PARP1. Cell free PAR-ylation assays were performed with recombinant HES1 (GST-HES1), PARP1, co-factor NAD+, and IP with anti-PARP1. Immunoblots for PAR and HES1 showed a > 12-fold increase in PAR-ylated PARP1 in triplicate experiments. (C) HES1-induced accumulation of cellular PAR. B-ALL (JM1) cells were transfected with FLAG-HES1 mRNA, and cellular PAR-ylated proteins were immunoprecipitated and quantitated with tetramethylbenzidine and an enzyme-linked immunoabsorbent assay reader. Intracellular PAR levels were increased and sustained from 2 to ≥ 6 days (mean ± SD). (D) HES1-induced depletion of ATP. Under similar conditions, total cellular ATP was determined with the use of the ATPlite kit as directed by the manufacturer. Luminescence was measured with a Molecular Devices luminometer. Raw data in counts are shown (n = 3). Relative ATP levels per cell decreased over time compared with vector control (P < .01, mean ± SD). (E) HES1-induced nuclear translocation of AIF in B-ALL. B-ALL (JM1) cells were transfected with FLAG-HES1 mRNA, and mitochondria and nuclear fractions were isolated at different time points. Anti-AIF immunoblot showed AIF translocation from mitochondria to nucleus. (F) HES1-induced AIF translocation can be rescued with shRNA to HES1. B-ALL (JM1) cells were transfected with HES1 mRNA, and mitochondria and nuclear fractions were isolated at 48 hours. HES1 mRNA, but not GFP control, induces nuclear translocation of AIF. Cotransfection with shRNA to HES1 inhibits effect of HES1. Lamin B (nuclear) and V-DAC (mitochondrial) confirm purity of fractions. WB indicates Western blotting.$

HES1-induced activation and cleavage of PARP1. (A) Cell-specific cleavage of PARP1. A panel of 3 B-ALL and 3 T-ALL cell lines were transfected with HES1. At 48 hours, lysates were probed for PARP1 expression. Overall PARP1 levels were higher in B-ALL versus T-ALL, and HES1-mediated PARP1 cleavage was greater in B-ALL than in T-ALL. (B) HES1-induced PAR-ylation of PARP1. Cell free PAR-ylation assays were performed with recombinant HES1 (GST-HES1), PARP1, co-factor NAD⁺, and IP with anti-PARP1. Immunoblots for PAR and HES1 showed a > 12-fold increase in PAR-ylated PARP1 in triplicate experiments. (C) HES1-induced accumulation of cellular PAR. B-ALL (JM1) cells were transfected with FLAG-HES1 mRNA, and cellular PAR-ylated proteins were immunoprecipitated and quantitated with tetramethylbenzidine and an enzyme-linked immunoabsorbent assay reader. Intracellular PAR levels were increased and sustained from 2 to ≥ 6 days (mean ± SD). (D) HES1-induced depletion of ATP. Under similar conditions, total cellular ATP was determined with the use of the ATPlite kit as directed by the manufacturer. Luminescence was measured with a Molecular Devices luminometer. Raw data in counts are shown (n = 3). Relative ATP levels per cell decreased over time compared with vector control (P < .01, mean ± SD). (E) HES1-induced nuclear translocation of AIF in B-ALL. B-ALL (JM1) cells were transfected with FLAG-HES1 mRNA, and mitochondria and nuclear fractions were isolated at different time points. Anti-AIF immunoblot showed AIF translocation from mitochondria to nucleus. (F) HES1-induced AIF translocation can be rescued with shRNA to HES1. B-ALL (JM1) cells were transfected with HES1 mRNA, and mitochondria and nuclear fractions were isolated at 48 hours. HES1 mRNA, but not GFP control, induces nuclear translocation of AIF. Cotransfection with shRNA to HES1 inhibits effect of HES1. Lamin B (nuclear) and V-DAC (mitochondrial) confirm purity of fractions. WB indicates Western blotting.

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