FIG. 2.

Apoptosis is induced in transformed C/EBPβ-deficient macrophages following M-CSF withdrawal. (A) J2-KO1 cells (2 × 10⁶) were plated and cultured with or without M-CSF (NF, no factor). DNA was isolated at 0, 4, and 8 h after M-CSF withdrawal. Three micrograms of each DNA sample, or positive control DNA from apoptotic U937 cells (lane 7), was analyzed by agarose gel electrophoresis. (B) J2-WT1 or J2-KO1 macrophages (10⁶) were incubated in the presence or absence of M-CSF for 24 h. Adherent and nonadherent cells were collected and stained with annexin V-FITC and PI to assess apoptosis. Dot plots of flow cytometry data from a representative experiment are shown, with PI staining on the y axis and annexin V staining on the x axis. The percentages of each subset are indicated. Cells in the lower right quadrant (annexin V positive) and the upper right quadrant (annexin V-PI double positive) are considered apoptotic. In the right panel, the results are graphed as the mean percentage of apoptotic cells (± standard error of the mean) from three to five independent experiments. (C) GF dependence of primary macrophages. Untransformed BMDM from wt and C/EBPβ^−/− mice were cultured in the presence or absence of 100 ng of M-CSF/ml. Cell viability was measured at 0, 24, and 48 h by neutral red dye uptake. Data are the average of three independent experiments, with standard errors indicated. Statistical analysis of the data in panels B and C was performed using the unpaired Student's t test (*, P < 0.05; **, P < 0.01).