American Society of Hematology

Figure 2

$Figure 2. Cytoplasmic dislocation of NPM impairs caspase signaling and protects against TRAIL-induced cell death. (A) GST, GST-NPM wild-type, or GST-NPMc immobilized on glutathione-Sepharose were assayed by immunoblotting for binding to caspase-6, -8, and -3 in cytochrome c–induced HeLa S-100 cytosolic extracts. As a control, lysates (30-50 μg) were run directly in gels. (B) Western blot analysis for caspase activities on physiologic substrates. S-100 cytosolic fractions from HeLa cells incubated separately with either recombinant active caspase-3, -6, or -8 in the absence or presence of 5μM recombinant GST tag alone, GST-tagged NPM wild-type, or NPMc for 1 hour at 30°C. Cleavage of the respective caspase substrates were assayed in immunoblots. (C) GST or GST-NPMc immobilized on glutathione-Sepharose were assayed by immunoblotting for binding to caspase-3, -7, -6, or -8 in cytochrome c–treated protein extracts of blast cells from peripheral blood of AML patients. (D) Western blot analysis for caspase activation. S-100 cytosolic fractions from 3 hematopoietic cell lines treated with cytochrome c (300nM), dATP (900nM), in the presence or absence of 5μM recombinant NPMc at 30°C for 30 minutes. (E) Subcellular localization of NPM, caspase-6 and -8 in OCI/AML-2 or -3 cells. Scale bar represents 5 μm. (F) Apoptosis was scored by analyzing condensed nuclei in Hoechst-stained OCI/AML-2 or OCI/AML-3 cells subjected to 50 ng/mL TRAIL treatment for up to 24 hours at 37°C (line graph). In addition, OCI/AML-3 cells transfected with 6nM specific (siNPM) or control (siNEG) siRNAs and subjected to 50 ng/mL TRAIL treatment for up to 24 hours, before flow cytometric analysis by annexin V staining with propidium iodide counterstain (bar graph). Apoptotic cells are defined as cells that stain positively for annexin V but not propidium iodide. (G) Western blot analysis for caspase activation in OCI/AML-3 cells transfected with siNPM or siNEG for 48 hours. (H) Western blot analysis of caspase activity on physiologic substrates. Cytoplasmic fractions from OCI/AML-2, or OCI/AML-3 cells treated separately with either recombinant caspase-6 or -8. Portions of the extract from OCI/AML-3 cells were immunodepleted of NPM.$

Cytoplasmic dislocation of NPM impairs caspase signaling and protects against TRAIL-induced cell death. (A) GST, GST-NPM wild-type, or GST-NPMc immobilized on glutathione-Sepharose were assayed by immunoblotting for binding to caspase-6, -8, and -3 in cytochrome c–induced HeLa S-100 cytosolic extracts. As a control, lysates (30-50 μg) were run directly in gels. (B) Western blot analysis for caspase activities on physiologic substrates. S-100 cytosolic fractions from HeLa cells incubated separately with either recombinant active caspase-3, -6, or -8 in the absence or presence of 5μM recombinant GST tag alone, GST-tagged NPM wild-type, or NPMc for 1 hour at 30°C. Cleavage of the respective caspase substrates were assayed in immunoblots. (C) GST or GST-NPMc immobilized on glutathione-Sepharose were assayed by immunoblotting for binding to caspase-3, -7, -6, or -8 in cytochrome c–treated protein extracts of blast cells from peripheral blood of AML patients. (D) Western blot analysis for caspase activation. S-100 cytosolic fractions from 3 hematopoietic cell lines treated with cytochrome c (300nM), dATP (900nM), in the presence or absence of 5μM recombinant NPMc at 30°C for 30 minutes. (E) Subcellular localization of NPM, caspase-6 and -8 in OCI/AML-2 or -3 cells. Scale bar represents 5 μm. (F) Apoptosis was scored by analyzing condensed nuclei in Hoechst-stained OCI/AML-2 or OCI/AML-3 cells subjected to 50 ng/mL TRAIL treatment for up to 24 hours at 37°C (line graph). In addition, OCI/AML-3 cells transfected with 6nM specific (siNPM) or control (siNEG) siRNAs and subjected to 50 ng/mL TRAIL treatment for up to 24 hours, before flow cytometric analysis by annexin V staining with propidium iodide counterstain (bar graph). Apoptotic cells are defined as cells that stain positively for annexin V but not propidium iodide. (G) Western blot analysis for caspase activation in OCI/AML-3 cells transfected with siNPM or siNEG for 48 hours. (H) Western blot analysis of caspase activity on physiologic substrates. Cytoplasmic fractions from OCI/AML-2, or OCI/AML-3 cells treated separately with either recombinant caspase-6 or -8. Portions of the extract from OCI/AML-3 cells were immunodepleted of NPM.

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