Effect of phorbol esters on IFN-signaling in CLL cells. (A). CLL cells were cultured for 24 hours with or without PDB (100 ng/mL) and/or IFN-α2b (1000 U/mL). pSTAT1, pSTAT3, and total STAT1 and STAT3 levels were then determined by immunoblotting. Immunoblots were stripped and reprobed with β-actin antibodies to assess loading. The results were similar for 21 additional samples. (B) CLL cells were cultured alone or with PDB for 48 hours (to allow abatement of cytokine production caused by PDB alone), washed, and recultured with IFN in the presence and absence of CHX (10 μg/mL) for 6 hours before determining pSTAT1 and pSTAT3 levels. The pSTAT3 levels were higher in cells treated with PDB and IFN than in cells treated with IFN alone and were not affected by the presence of CHX to block autocrine cytokine production. Similar results were obtained with 4 additional patient samples. (C-D) CLL cells were cultured alone or with PDB, IFN, or PDB and IFN with or without the classic PKC isozyme inhibitor, Go6976 (1μM) or the antioxidant (C), NAC (30μM) added 1 hour before stimulation (D). Cell lysates were collected 4 hours later and pSTAT1 and pSTAT3 levels determined by immunoblotting. Go6976 and NAC shortened STAT3 phosphorylation in CLL cells treated with PDB and IFN. This experiment was repeated with 8 different patient samples with similar results. (E) After 24 hours of culture, global phosphatase levels were determined using para-nitrophenyl phosphate as described in “Para-nitrophenyl phosphate assay for phosphatase activity.” This experiment was repeated with 8 different CLL patient samples and showed that global phosphatase activity was decreased in PDB-treated CLL cells, which could be reversed by blocking PKC and ROS. *P < .05.