Effect of DAC and IL-6 treatment on SOCS3, STAT3, P-STAT3, and Mcl-1 expression and apoptosis of leukemic LGLs. Results were obtained by LGL cultures treated for 96 hours with or without DAC and then stimulated with IL-6 for 1 hour. (A) SOCS3 expression levels in LGLs obtained by RT-PCR and normalized on GAPDH. The results of 4 representative patients are reported. (B) Western blot analysis of LGL extracts for P-STAT3, total STAT3, and SOCS3. β-Actin served as a loading control. Two representative cases of patients are shown. (C) Mcl-1 expression levels obtained by western blot analysis (D) and by RT-PCR of LGLs from a representative patient with LGLL. (E) Annexin V/PI assay measuring LGL mortality percentage (± SE) in PBMC culture after DAC treatment (4 days of incubation) or with AG490, inhibiting STAT3 signaling. A staining with anti-CD57-FITC was used to identify leukemic LGLs from PBMCs.