Soft Agar Colony-forming Assay Cells exhibiting exponential growth were suspended in complete growth medium containing 0.33% Bacto-agar (A-6013 Type 1 Low EEO; Sigma-Aldrich) and overlaid on 0.5% agarose gel in 30-mm dishes (104 cells/dish). Medium containing IL-19 (200 ng/mL) was overlaid on the top agar. The dishes were maintained at 37��C in a humidified www.selleckchem.com/products/MDV3100.html incubator (5% CO2, 95% O2) for two weeks. During this period, the medium was changed every 3 days. The number of visible colonies (>50 ��m) were counted under a microscope. All experiments were done in triplicate. Real-time Migration Assays CE81T cells were seeded at 1��105 cells/ml in 6-well dishes and allowed to attach for 18 h. The cells were then exposed to medium containing hIL-19 (200 ng/ml).

Cell migration kinetics was recorded using a JuLI Smart fluorescent cell analyzer instrument (JuLI Smart; Montreal Biotechnologies Inc. (MBI), Dorval, PQ, Canada) for approximately 12 h. The result then analyzed using ImageJ Software (http://rsbweb.nih.gov/ij/). FBS (10%) was used as the positive control. Western Blotting CE81T cells (1��106) were plated in 6-cm dishes, starved for 16 h, and then stimulated with IL-19 (200 ng/ml) for the indicated times. Total cell lysate was collect by adding 1�� RIPA buffer containing phenylmethanesulfonyl fluoride (PSMF) (RIPA:PSMF=101) and centrifuged at 13000 rpm at 4��C to collect the supernatant. Western blotting with antibody specific for phosphor-STAT3, phosphor-P-38, phosphor-Jun N-terminal protein kinase (JNK), phosphor-extracellular signal-regulated kinase (ERK), phosphor-nuclear factor-kappa B (NF-��B), and phosphor-Akt and ��-actin (Cell Signaling Technology, Beverly, MA, USA) was used following the manufacturer��s instructions.

��-actin was used as an internal control. Real-time Quantitative Polymerase Chain Reaction (Real-time QPCR) CE81T cells were stimulated with hIL-19 (200 ng/ml) for the indicated time. Total RNA was extracted as described above. The amplified template was detected using Maxima? SYBR Green/ROX qPCR Master Mix (2X) (Fermentas, Burlington, Ontario, Canada) with a real-time PCR system (LightCycler? 480; Roche, Basel, Switzerland) with gene-specific primer (Table 2). ��-actin was used as an internal control. Tumorigenicity in BALB/c Scid Mice Eight-week-old male BALB/c Scid mice were acquired from the NCKU Laboratory Animal Center.

The mice were housed under strict pathogen-free conditions and given sterile food and water. The Committee on Animal Research at NCKU approved all procedures (No. 099072). In vitro cultured CE81T cells (1��106 cells/0.2 ml DMEM with 10% v/v FBS) (Gibco BRL) were subcutaneously injected into the abdominal region of each mouse. Tumor development Brefeldin_A was assessed every 2 days one week after the injection of tumor cells. Thirty-two days after the injection, the mice were killed and their tumors were harvested for further measurement. Statistical Analysis Statistical analysis was done using SPSS 14.0 (SPSS Inc.