CpG ODN-induced apoptosis of B-CLL cells depends on NF-κB and JAK/STAT signaling pathways and tyrosine phosphorylation of STAT1. (A) Cleavage of caspase-9, caspase-3, and PARP in B-CLL cells cultured with CpG 2006 were determined by Western blots at the indicated time points. Data are representative results from 2 of 3 independent experiments with B-CLL cells from different CLL patients. (B) B-CLL cells were cultured with or without CpG 2006 for 5 days in the presence or absence of pan-caspase, caspase-3, or caspase-9 inhibitor. Data are the mean ± SD from 3 independent experiments. *P < .01, B-CLL cell numbers versus CpG 2006 only group. (C) B-CLL cells with or without CAPE and/or AG490 pretreatment were cultured in media with or without CpG 2006 for 5 days. *P < .01, the number of viable B-CLL cells in CAPE and/or AG490 pretreated group versus B-CLL cells cultured with CpG 2006 only. Data are the mean ± SD from 5 independent experiments. Western blots of AG490 pretreatment on CpG 2006-induced activation and cleavage of caspase-9, caspase-3, and PARP in B-CLL cells at day 5 cultures. (D[b]) Western blot time course of CpG 2006-induced tyrosine- or serine-phosphorylated forms of STAT1 expression in B-CLL cells from 5 patients. Data are densitometrically assessed and presented as the mean ± SD in the adjacent bar diagram on the right. *P < .01, STAT1 expression in B-CLL cells before versus at each time point after CpG 2006 stimulation. (E) Western blots of tyrosine- or serine-phosphorylated forms of STAT1 expression in B-CLL cells with or without CAPE or AG490 pretreatment and cultured in media with or without CpG 2006 stimulation were determined at the indicated time points. Data are representative results of 3 independent experiments. (F) Western blots of STAT1 antisense ODN on total, tyrosine-, or serine-phosphorylated forms of STAT1 expression in B-CLL cells cultured with or without CpG 2006 for 2 days. Data are representative results of 3 independent experiments. (G) B-CLL cells with or without STAT1 antisense ODN or STAT1 sense ODN pretreatment were cultured in the presence or absence of CpG 2006 for 5 days. *P < .01, the number of viable B-CLL cells in STAT1 antisense ODN or STAT1 sense ODN pretreated group versus B-CLL cells cultured with CpG 2006 only. Data are the mean ± SD from 3 independent experiments. (H) B-CLL cells with or without STAT1 antisense ODN or STAT1 sense ODN pretreatment were cultured with CpG 2006 for 3 days. Expression of surface markers (shaded histogram) on fresh or cultured B-CLL cells was analyzed by flow cytometry, indicated with MFI number, and overlaid with isotype control (unshaded histogram). Data are representative results from 1 of 5 independent experiments. The adjacent diagrams on the right are MFI fold increases of the indicated surface molecules on day 3 cultured versus uncultured B-CLL cells from 5 CLL patients. *P < .01. The horizontal bar represents the median level with the indicated fold increase number.