Variations in IFN-mediated STAT3 phosphorylation and proliferation in CLL cells. (A) Schematic diagram of IFN-signaling. (B) Primary CLL cells were treated with IFN-α2b (1000 U/mL) for the indicated times, and whole cell lysates were immunoblotted with antibodies to phosphotyrosine 701-specific STAT1 or phosphotyrosine 705-specific STAT3, as well as total STAT1, STAT3, and β-actin as loading controls. Cell aliquots were also exposed to ionizing radiation (2.5 Gy) and p53 and p21 levels were determined by immunoblotting 18 hours later (bottom blots). Tumor cells from Pt. 31 and Pt. 92 had intact and absent p53 axes, respectively, according to this assay.¹⁹  (C) Tumor cells from 2 other patients (Pt. 81 bottom, and Pt. 44 top) were cultured for 4 days in the presence or absence of IFN. Cell size was determined by the forward scatter parameter of flow cytometry. (D) DNA content was also determined by flow cytometry at this time. The number on the right of the histogram represents the percentage of cells with DNA content greater than 2N and the number on the left represents the percentage with subdiploid DNA content.³⁹  (E) Immunoblots showing measurable pSTAT3 levels at 4 hours in CLL cells from Pt. 81 but not Pt. 44. F. Viable cells were counted in a hemocytometer after 4 days of culture. The results show that IFN induced a proliferative response when STAT3 phosphorylation persisted longer than 4 hours.