Effects of dasatinib on cell cycle were assessed using Nim-Dapi staining. Cells were plated evenly in control and experimental wells and allowed to grow to log-phase then treated with 100 nM dasatinib for 24 hours. To perform cell cycle analysis, cells were washed with PBS and trypsin was applied to release cells, which were then centrifuged at 3,000 rpm for 5 minutes. Supernatant was aspirated and cells were then resuspended in 100 μL of Nim-Dapi (NPE Systems, Pembroke Pines, FL) and gently vortexed. Cells were analyzed Selleck Midostaurin with UV using a Cell Lab Quanta SC flow cytometer (Beckman-Coulter). Apoptosis assays were performed using

an Annexin V-FITC apoptosis detection kit (MBL, Woburn, MA) and flow cytometry. Cells were plated and treated as for cell cycle studies and exposed to 100 nM dasatinib for 5 days. After incubation, cells were processed as directed in the kit and analyzed using an FITC signal detector and propidium iodide (PI) detector using a Cell Lab Quanta SC flow cytometer. A one-sided t test was performed measuring significance of the increase in G0/G1 and the decrease in living cells in cell cycle and apoptosis assays, respectively. Lentivirus transduction of a Src short hairpin RNA (shRNA)

was used to knockdown Src expression. HLE and SNU 423 cells were plated in a six-well plate at a density of 2-2.5 × 105 cells/well and incubated overnight at 37°C and 5% CO2 in their respective media. Lentiviral particles containing this website c-SRC shRNA (Santa Cruz Biotechnology) was diluted with transduction media consisting

of serum-free, antibiotic-free media and 5 μg/mL of Polybrene (Santa Cruz Biotechnology) to a multiplicity of infection (MOI) 7 and 13. Cells were washed with PBS and then 1 mL of diluted virus was added. Cells transfected with lentiviral particles containing scramble shRNA were used as a negative control and cop-GFP lentivirus was used as a control for transduction efficiency. After 18 hours, lentiviral shRNA media was removed and replaced with media containing serum and antibiotics. After overnight incubation the cells were trypsinized and placed in T-25 flasks with media containing 5 μg/mL puromycin (Santa Cruz Biotechnology). Cells were detached 72 hours posttransduction and analyzed by flow cytometry for green fluorescence protein why (GFP) expression. All noninfected cells were killed by puromycin, and remaining cells had GFP expression, indicating 90%-100% transduction efficiency. Western blot was performed for total Src and phospho-Src as described above. Growth effects of shRNA were analyzed as described above comparing a vector control with the respective shRNA clone. EGFR, epidermal growth factor receptor; GEO, Gene Expression Omnibus; HB, hepatoblast; HC, hepatocyte; HCC, hepatocellular carcinoma; PI, propidium iodide; SFK, Src-family of tyrosine kinases. A total of 20 human HCC cell lines were used in the study. Available clinical data from the respective repositories are in Table 1.