Cathepsin B contributes to the formation of the NPM-p20 fragment. (A) Human monocytes were treated with M-CSF for 6 days. MG132 (30μM) or clasto-lactacystin β-lactone (10μM) was added 4 hours before cell lysis and immunoblotting with indicated antibodies or measurement of proteasome activity with Suc-LLVY-AMC substrate (mean ± SD of triplicates; ****P < .001). AU indicates arbitrary units. (B) Human monocytes were treated with M-CSF for 6 days. MG132 (30μM) or cathepsin inhibitor (5μM) or cathepsin K inhibitor (5μM) was added 4 hours before cell lysis and immunoblotting with indicated antibodies (HSC, loading control) or measurement of cathepsin K activity with Ac-LR-AFC (mean ± SD of triplicates; ****P < .001). (C) Full-length NPM1 was produced in reticulocyte lysates in the presence of biotinylated lysine, then exposed to indicated recombinant active cathepsins at acidic pH for 2 hours at 37°C. Peptides were identified with streptavidin-HRP and revealed by chemiluminescence. (D) Human monocytes were treated with M-CSF for 6 days. MG132 (30μM), cathepsin inhibitor (5μM), or CA-074Me cathepsin B inhibitor (5μM) was added 4 hours before cell lysis and immunoblotting with indicated antibodies (HSC70, loading control) or measurement of cathepsin B activity with Z-RR-AMC substrate (mean ± SD of triplicates; ****P < .001). (E) Measurement of cathepsin B activity with Z-RR-AMC substrate in monocytes treated with M-CSF for 0, 3, or 6 days. (F) Immunoblot analysis of cathepsin B in monocytes exposed for indicated times to M-CSF. HSC70 was used as loading control. (G) Monocytes were collected from the bone marrow of wild-type (WT) or cathepsin B^−/− (CathB^−/−) mice and induced to differentiate into macrophages on exposure to murine M-CSF (100 ng/mL) for indicated times before immunoblot analysis of NPM1 expression (HSC70, loading control). Each panel is representative of at least 3 independent experiments.