American Society of Hematology

Figure 5

$Figure 5. Notch ligand peptide DSL induces cell type–specific PARP1 activation, AIF translocation, and apoptosis. (A) The DSL peptide induces cell type–specific growth inhibition and cell death. T-ALL (SupT1) and B-ALL (JM1) cells were incubated with 100μM DSL peptide and were harvested at different points for Trypan blue exclusion viable cell counts. Cell counts were normalized to day 1 (control) (mean ± SD). (B) DSL induces HES1 up-regulation. B-ALL (JM1) cells were incubated with 100μM DSL peptide for 6 hours. Immunoblot for HES1 shows a 5-fold increase over baseline HES1 levels. For comparison, T-ALL (SupT1) and control HEK293 cells are shown. DSL-induced HES1 expression in B-ALL cells is far below constitutive HES1 levels in T-ALL and comparable to the nonmalignant control cells. β-actin used as a loading control. (C) DSL-induced PAR-ylation of PARP1. B-ALL (JM1) cells were incubated with 100μM DSL or scrambled peptide for 48 hours. Cell lysates were immunoprecipitated with anti-PARP1 antibody and immunoblotted with anti-PAR antibody. Increase (15-fold) in PAR-ylated PARP1 was seen after DSL exposure, whereas scrambled peptide had no effect. Transfection with shRNA to HES1 reduced PAR-ylation of PARP1 by 56%, whereas control shRNA did not. Immunoblot probed for IgG to show equal loading of immunoprecipitated protein. (D) DSL induces cell type–specific AIF translocation in B-ALL cells. T-ALL (SupT1) and B-ALL (JM1) cells were exposed to 100μM DSL peptide for 48 hours. Mitochondria and nuclear fractions were probed for AIF, showing near complete translocation of AIF. Lamin B (nuclear) and V-DAC (mitochondrial) confirm purity of fractions. (E) DSL peptide induces apoptosis in B-ALL which depends on HES1, PARP1, and AIF. B-ALL (JM1) cells were transfected with shRNAs against HES1, PARP1, AIF, and controls that coexpress either GFP or RFP, named shControl-G, shControl-R. Transfected cells were incubated with 100μM DSL or scrambled peptide for 72 hours, stained with annexin V, and flow cytometry was performed for GFP/RFP and annexin V. Percentage of events displayed in each quadrant. Nuc indicates nuclear; mito, mitochondrial.$

Notch ligand peptide DSL induces cell type–specific PARP1 activation, AIF translocation, and apoptosis. (A) The DSL peptide induces cell type–specific growth inhibition and cell death. T-ALL (SupT1) and B-ALL (JM1) cells were incubated with 100μM DSL peptide and were harvested at different points for Trypan blue exclusion viable cell counts. Cell counts were normalized to day 1 (control) (mean ± SD). (B) DSL induces HES1 up-regulation. B-ALL (JM1) cells were incubated with 100μM DSL peptide for 6 hours. Immunoblot for HES1 shows a 5-fold increase over baseline HES1 levels. For comparison, T-ALL (SupT1) and control HEK293 cells are shown. DSL-induced HES1 expression in B-ALL cells is far below constitutive HES1 levels in T-ALL and comparable to the nonmalignant control cells. β-actin used as a loading control. (C) DSL-induced PAR-ylation of PARP1. B-ALL (JM1) cells were incubated with 100μM DSL or scrambled peptide for 48 hours. Cell lysates were immunoprecipitated with anti-PARP1 antibody and immunoblotted with anti-PAR antibody. Increase (15-fold) in PAR-ylated PARP1 was seen after DSL exposure, whereas scrambled peptide had no effect. Transfection with shRNA to HES1 reduced PAR-ylation of PARP1 by 56%, whereas control shRNA did not. Immunoblot probed for IgG to show equal loading of immunoprecipitated protein. (D) DSL induces cell type–specific AIF translocation in B-ALL cells. T-ALL (SupT1) and B-ALL (JM1) cells were exposed to 100μM DSL peptide for 48 hours. Mitochondria and nuclear fractions were probed for AIF, showing near complete translocation of AIF. Lamin B (nuclear) and V-DAC (mitochondrial) confirm purity of fractions. (E) DSL peptide induces apoptosis in B-ALL which depends on HES1, PARP1, and AIF. B-ALL (JM1) cells were transfected with shRNAs against HES1, PARP1, AIF, and controls that coexpress either GFP or RFP, named shControl-G, shControl-R. Transfected cells were incubated with 100μM DSL or scrambled peptide for 72 hours, stained with annexin V, and flow cytometry was performed for GFP/RFP and annexin V. Percentage of events displayed in each quadrant. Nuc indicates nuclear; mito, mitochondrial.

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