IKKβ inhibition by ML120B induces apoptosis in bone marrow and spleen in vivo. (A) Histologic evaluation and nuclear fragmentation detection in situ with C57BL/6 bone marrow cytospins 6 hours after a single dose of vehicle alone or ML120B (300 mg/kg). Arrow indicates area of apoptotic foci. (B) Total cell count and percent viable pre–B cells (B220⁺/IgM^–/annexin V^–) in the bone marrow 6 hours after a single oral dose of ML120B. Mice were given a single oral dose as indicated (n = 4/group) and 6 hours later cell counts per femur (× 10⁶) and flow cytometry were performed. Percent B220⁺/IgM^–/annexin V^– cells were calculated. (C) Nuclear fragmentation detection in situ with C57BL/6 bone marrow cytospins 6 hours after single oral dose with ML120B or vehicle control as indicated. One hundred to 200 cells were scored from cytospins from bone marrow of individual mice (n = 4/group). Histology and detection of nuclear fragmentation in spleens (D) and thymi (E) 6 hours after a single oral dose of ML120B. Arrow indicates area of apoptotic foci. B220 staining of spleen sections was performed 18 hours after a single oral dose, and percent viable B cells (B220⁺/annexin V^–) in the spleen (n = 4 per group) 6 hours after a single dose are shown. For quantitation of cells positive for nuclear fragmentation, 3 to 4 spleens and 4 thymi were scored per dose of ML120B, 3 to 4 fields within 3 follicles were scored per spleen, and 3 fields were scored per thymus in the cortex region. Spleen and thymus data show average number of positive cells per field. Error bars indicate SD. Original magnifications: (A, C) 400 × (objective 40 ×/0.75 numeric aperture [NA]); (D, H&E and apoptotic) 200 × (objective 20 ×/0.50 NA); (D, B220) 40 × (objective 4 ×/0.13 NA); and (E) 100 × (objective 10 ×/0.30 NA).