Stimulation of the BCR induces tyrosine phosphorylation of STAT3 in CLL cells. (A) Time-dependent increase in pSTAT3 levels induced by incubation of CLL cells with anti-IgM antibodies. CLL cells were incubated without or with 10 μg/mL goat F(ab′)₂ anti-human IgM antibodies (upper panel) or with 20 ng/mL IL-6 (lower panel). Cells were harvested at several time points, lysed, and analyzed using western immunoblotting with anti-tyrosine pSTAT3, anti-serine pSTAT3, and anti-STAT3 antibodies. SET2 cells were used as positive controls. As shown, tyrosine pSTAT3 was detected 2 hours from exposure to anti-IgM antibodies (upper panel) but only after 15 minutes of exposure to IL-6. This experiment was repeated 3 times using samples from patients 4, 13, and 17 (supplemental Table 1). (B) Tyrosine pSTAT3 levels remained increased after prolonged (up to 48 hours) exposure to anti-IgM antibodies (upper panel) but diminished 1 hour and were no longer detected 2 hours after washout. As shown in the upper panel, CLL cells were incubated with 10 μg/mL anti-IgM antibodies for 1, 2, 4, 8, 16, and 48 hours; harvested; and analyzed by western immunoblotting using anti-tyrosine pSTAT3, anti-serine pSTAT3, anti-STAT3, and anti-actin antibodies. Cells from patient 2 (supplemental Table 1) were used in this experiment. Two additional experiments yielded similar results (data not shown). As depicted in the lower panel, IgM-induced tyrosine phosphorylation of STAT3 is short lived. CLL cells were incubated for 18 hours with or without (Cont.) 10 μg/mL anti-IgM antibodies. The antibodies were then washed out and the cells were harvested at different time points, and the cell lysates were analyzed using western immunoblotting with anti-tyrosine pSTAT3, anti-serine pSTAT3, and anti-STAT3 antibodies. This experiment was repeated 2 times using samples from patients 3 and 18 (supplemental Table 1). (C) IgM-induced tyrosine pSTAT3 is detected in the cytosol and nucleus of CLL cells. CLL cells were incubated for 2 hours with or without 10 μg/mL anti-IgM antibodies. The extract was fractionated, and the nuclear and cytoplasmic preparations were analyzed using western immunoblotting with anti-tyrosine pSTAT and anti-STAT3 antibodies. Anti-lamin B antibodies were used to detect the nuclear fractions, and anti-S6 antibodies to detect the cytoplasmic fractions. As shown, S6 was not detected in the nuclear fraction, and lamin B was not detected in the cytoplasmic fraction. Tyrosine pSTAT3 was detected both in the nuclear (lamin B–positive) and cytoplasm (S6-positive) fractions of CLL cells incubated with but not without anti-IgM antibodies. We intentionally loaded more cytosolic protein. This experiment was repeated 3 times using samples from patients 15, 16, and 18 (data obtained using cells from patient 18 are not shown) (supplemental Table 1). (D) Tyrosine pSTAT3 is detected in the nucleus and cytosol of IgM-stimulated but not unstimulated CLL cells. Cells were incubated for 2 hours without or with 10 μg/mL anti-IgM antibodies. The cells were cytospun, fixed on glass slides, and stained with the nuclear stain 4,6 diamidino-2-phenylindole, shown in blue (panels i, v); anti-S6 antibodies, shown in red (panels ii, vi); or anti-tyrosine pSTAT3 antibodies, shown in green (panels iii, vii). Tyrosine pSTAT3 was not detected in unstimulated CLL cells (left panel). However, following incubation with anti-IgM antibodies, tyrosine pSTAT3 was detected in the nucleus (panel vii) and also in the cytosol (merged panel viii). Cells from patient 7 (supplemental Table 1) were used in this experiment. (E) Anti-IgM antibodies increased STAT3-targeted gene levels. RNA was extracted from CLL cells incubated for 2 hours without or with 10 μg/mL anti-IgM antibodies. The left panel depicts agarose gel electrophoresis of RT-PCR, and the right panel depicts qRT-PCR assessed using the TakMan gene