Effects of combined treatment with E4031 and dexamethasone in vivo. (A) NOD-SCID mice (n = 16, 4 for each group) were inoculated with REH cells on day 0, and 1 week after inoculation were treated with: (i) saline, (ii) E4031 (20 mg/kg), (iii) dexamethasone (15 mg/kg), or intravenous dexamethasone (15 mg/kg) plus E4031 (20 mg/kg) for 2 weeks. At the end of treatment, mice were killed and bone marrow, spleens, and livers were collected. Left panel, bone marrow engraftment in control, dexamethasone-, dexamethasone + E4031-, and E4031-treated mice, quantified by determining the percentage of hCD45⁺ versus mCD45⁺ cells by flow cytometry. Right panel, FragEL staining of bone marrow from mice inoculated with REH cells and treated as in panel A. Results show the number of TUNEL-positive leukemic cells per microscopic field (epiphysis or diaphysis, 40× magnification). The data are means ± SD of triplicate experiments. (B) Peripheral blood invasion in control, dexamethasone-, dexamethasone + E4031-, and E4031-treated mice, quantified by determining the percentage of hCD45⁺ versus mCD45⁺ cells by flow cytometry. (C) Extent of leukemic cell invasion in the spleen (left) and in the liver (right) of mice inoculated with REH cells and treated as in panel A. The extent of leukemic cell infiltration was calculated by anti-hMHCI staining measured with Leica DC Viewer software at 40×. Images were acquired on a Leica DM 4000B microscope with a Leica DFC 320 camera (Leica Microsystems; PL Fluotar 40×/0.70 objective) *P < .05. (D) Immunohistochemical staining with an anti-hMHCI antibody of the spleens of mice inoculated with the REH cell line and treated as in panel A. Scale bar = 50 μm. Inset, magnification of images reported on the left. *P < .05; **P < .01; ***P < .001