Apoptosis and cell cycle are dependent on the Spi-1 expression level. (A) The cells were cultured in the presence (Dox+) or absence of dox (Dox−) for 3 and 4 days (d3, d4). The cells were then fixed and stained with propidium iodide, and the sub-G1 part of the cell population was assessed using a FACS. The figure illustrates the mean ± SD of 4 experiments (B, top) The cleavage of PARP and caspase 3 was detected by Western blot using antibodies against PARP and cleaved caspase 3 in A2B and C cells grown with dox (100 ng/mL) or without dox for 3 days. (Bottom) Representative image of A2B and control cells treated with dox for 3 days and stained by Hoechst. Gray arrows designate apoptotic cells. Scale bar equals 13 μm. (C) Cell-cycle analysis by quantitative flow cytometry. The cells were fixed and stained with propidium iodide after different times in culture with dox, and the DNA content was assessed using a FACS. Each phase was expressed as the percentage of cells in cycle after exclusion of the sub-G1 population. Debris derived from dead cells are shown in gray on the left part of G1 phase. Analysis was performed at least 4 times with similar results.
