Nucleophosmin cleavage by caspases in M-CSF–treated monocytes. Human peripheral blood monocytes from healthy donors were cultured with M-CSF (100 ng/mL) or GM-CSF (100 ng/mL) and IL-4 (10 ng/mL). (A) Immunoblot analysis of caspase-8, caspase-3, and NPM1 in cells exposed for indicated times to cytokines. HSC70 was used as loading control. (B) Immunoblot analysis of caspase-8, caspase-3, and NPM1 in monocytes exposed for indicated times to M-CSF in the absence or presence of 50μM qVD-OPH. HSC70 was used as loading control. (C) Phase contrast examination of monocytes exposed for 4 days to M-CSF in the absence or presence of qVD. (D) Flow cytometric analysis of indicated markers at the surface of monocytes exposed for 4 days to M-CSF in the absence or presence of qVD. Each panel is representative of at least 4 independent experiments. (E) NPM1 was produced in reticulocyte lysates and incubated for 8 hours with indicated recombinant active caspases. EV indicates empty vector, and −, controls (no DNA). (F) Immunoblot analysis of caspase-7 in monocytes exposed for indicated times to M-CSF. (G) Immunoblot analysis of NPM1 in caspase-3^−/− (C3^−/−) and wild-type (WT) monocytes exposed for 4 days to M-CSF in the absence or presence of 50μM qVD-OPH. (H) Immunoblot analysis of NPM1 in C3^−/−, C7^−/−, and wild-type (WT) monocytes exposed for 4 days to M-CSF (3 independent WT and 2 independent C7^−/− samples are shown). (I) NPM1 wild-type (WT) and mutants (Mut) were produced in reticulocyte lysates and incubated for 8 hours with recombinant active caspase-3. EV indicates empty vector; and −, controls (no DNA). (J) Immunoblot analysis of NPM1 in mouse peritoneal macrophages in the absence or presence of LPS. Each panel is representative of at least 3 independent experiments.