Il 24

Human PCa LNCaP and PC3 cells were plated at 6,000 per well in 96-well plates and incubated overnight at 37°C in a humidified incubator containing 5% CO2. On the following day, IL-3, 6, 10 and 11(Invitrogen, USA) and IL-24(R&D, USA) up to 100 ng/ml (0.01, 0.1, 1, 2,5,10, 50, 100 ng/ml) in complete medium were added to different wells and cultivated for additional 48 hrs. Control wells were added with complete medium. Cell viability was determined using the Sulforhodamine B assay (SRB) (Sigma, USA) according to the manufacturer's instructions. Briefly, culture medium was aspirated and the cells were fixed by addition of 100 μl cold 10% trichloroacetic acid (TCA) at 4°C for 1 h, washed five times with deionized water and left to dry at room temperature. The cells were then stained with 100 μl SRB dye 0.4% (w/v) dissolved in 1% acetic acid (v/v) for at least 15 minutes, washed four times with 1% acetic acid to remove unbound dye and left to dry at room temperature. The dye bound protein was solubilized with 150 μl 10 mM unbuffered tris base and examined with Multi-Mode Microplate Reader (Biotek Synergy2, USA) for optical density reading at 560nm.

CCD-18Co human colon fibroblast cell line (ATCC, Manassas, VA, USA) was cultured in Eagle's Minimum Essential Medium (EMEM) (ATCC) supplemented with L-glutamine, 10% heat-inactivated FBS (Invitrogen) and 1% streptomycin and penicillin (Sigma-Aldrich) at 37 °C and 5% CO₂. To perform mRNA expression analysis and Annexin/PI staining, CCD-18Co cells were seeded in 6-well plates (n = 6 well/treatment group) at a density of 5 × 10⁵ cells/well, for fibroblast migration, MTT and SiriusRed assays, cells were seeded into 96-well plates at a density of 10⁴ cells/well (n = 5 well/treatment group). After plating, cells were treated with recombinant IL-24 (100 ng/ml, R&D), TGF-β1 (1 nM, R&D) or PDGF-B (10 ng/ml, R&D). Control cells were treated with corresponding solvents (IL-24 and PDGF-B: PBS; TGF-β1: 4 mM HCl) alone.