Sample Collection and Peripheral Blood Mononuclear Cell (PBMC) Isolation and Cell Culture

8 ml of peripheral venous blood was collected in heparin from the patients and controls for PBMC isolation using density gradient centrifugation method. Cell viability was checked by trypan blue dye (Sigma Aldrich, USA) exclusion test and more than 98% viability was obtained. PBMCs (1 × 10⁶) were suspended in RPMI 1640 without fetal bovine serum (FBS) and incubated with or without IL-6 (Peprotech, Rocky Hill, NJ, USA) at a dose of 30 ng/ml for 2 h. For STAT3 inhibition, PBMCs were incubated in the presence of 10 µM of the stattic (Sigma Aldrich, USA), a specific STAT3 inhibitor, for 1 h followed by treatment with 30 ng/ml of IL-6 for 2 h in 5% CO₂. Cells were subsequently harvested for RNA extraction. Two prostate cancer cell lines viz. STAT3^−/− PC-3 (25, 26) and STAT3^+/+ LNCaP (25, 27) were procured from National Center for Cell Science, Pune, India. The cells were maintained in RPMI 1640 and Ham’s F-12 (Sigma Aldrich, USA) media, respectively, supplemented with 10% FBS and antibiotics. Cells were grown as monolayers at 37°C in 5% CO₂. Confluent cultures were trypsinized (0.25% trypsin + 0.02% EDTA), re-suspended in complete medium, and seeded at a concentration of 1–2 × 10⁵ cells/well/ml into 12-well tissue culture plates and incubated for 48 h at 37°C to obtain 70–80% confluency. Once confluent, cells were serum starved for 4 h before stimulation with IL-6 for 2 h, harvested, and RNA was extracted.