FIG. 3.

γ-IR triggers apoptosis in Myc-expressing MEFs and in Eμ-myc transgenic B cells. (A) FACS histograms of PE-annexin V binding. GFP-only- or Myc-ER-expressing MEFs were treated with 4-HT (1 μM) for 24 h and irradiated with 5 Gy, and after 24 h cells were stained with annexin-V-PE and propidium iodide (PI). The percentages of annexin V- and PI-negative and -positive cells are given in each quadrant. Results shown are representative of three independent experiments performed with two different MEF cell cultures. (B) γ-IR triggers caspase-9 and PARP cleavage, and the release of cytochrome c into the cytosol, in Myc-expressing MEFs. The indicated cells were treated for 24 h with 4-HT (1 μM) and treated with γ-IR, and lysates were analyzed at the indicated intervals by Western blotting for caspase-9 (left panel), the cytosolic fraction of cytochrome c (29) (middle panel), and PARP (right panel). The asterisk indicates the cleaved forms of caspase-9 and PARP. (C) γ-IR triggers apoptosis in B cells of 6-week-old (precancerous) Eμ-myc transgenic mice. Wild-type and Eμ-myc transgenic littermates were treated with 2 Gy of γ-IR. After 4 h bone marrow was harvested and sorted for B220⁺ cells by FACS, and cytospins were assessed for apoptotic cells by immunofluorescence using TUNEL assays. TUNEL-positive apoptotic cells appear bright green. Representative fields are shown.