Impact of overexpression of Mcl-1 on B lymphoid, myeloid cells, and stem cells. (A-C) Mcl-1 enhances survival of B cells. (A) B220+ cells isolated from LNs of individual mice were incubated in vitro with anti-IgM F(ab′)2 (0.5 μg/mL), and viability was determined over 72 hours relative to cells in untreated cultures. WT mice (black, n = 3); Mcl-1(33) mice (red, n = 4 mice); BCL-2(69) mice (blue, n = 4). Data are mean ± SEM. (B-C) B220+ cells were isolated from spleens of WT, Mcl-1(33), and Bcl-2 mice, labeled with carboxyfluorescein diacetate succinimidyl ester and incubated with CpG. Fluorescence intensity (B) and viable cell number (C) were monitored over time. Data are representative of 3 independent experiments performed for a single mouse of each genotype. Supplemental figure 3 contains further analyses performed on B cells. (D-E) Mcl-1 enhances survival of granulocytes. Granulocytes (Mac1+Gr1+) sorted from BM of individual mice were cultured in vitro for 4 days in medium alone (D) or medium containing 10 μg/mL etoposide (E). WT mice (black, n = 5-7); Mcl-1(33) mice (red, n = 4-6); BCL-2(69) mice (blue, n = 3 or 4). Data are mean ± SEM. (F) Mcl-1 enhances resistance of multipotential stem cells to γ-irradiation. BM cells were subjected to 1.5 Gy γ-irradiation or mock-treated before transplantation (7.5-15 × 104 cells/mouse) into lethally irradiated recipients (2 × 5.5 Gy, 3 hours apart). Spleen colonies were counted on day 12, and results are plotted as mean spleen colony-forming cells (CFU-s) for triplicate recipient mice per 7.5 × 104 transplanted cells. Bars represent mean ± SEM. ***P < .001 (Student t test). ns indicates not significant. For each genotype, BM cells from 4 donor mice were each transplanted into 6 recipients (3 receiving unirradiated cells and 3 receiving irradiated cells). WT donor mice (+/+, black); Mcl-1(33) donor mice (T/+, red).
