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Details about these methodologies can be found in Supplemental Experimental Procedures. Ninety-six transwell plates were used for the 3D Matrigel invasion assay. Cells (1?�� 103) were suspended in a mixture of culture MI-773 cell line medium and Matrigel (Becton Dickinson) (1:1, v/v) and layered onto solidified Matrigel. For 6�C8?days after culturing, colonies were observed under a light microscope or confocal microscope, representative pictures of each experiment were taken, and cell numbers morphologically exhibiting the invasive outgrowth phenotype were counted. Cells (2?�� 105) were plated in 6-well plates 1?day before assay, and cell images under a light microscope were obtained every 15?min for 16?hr. Velocity of the?cell movements was analyzed using AxioVision Version 4.8 Cell tracking software (Carl Zeiss). Cells (5?�� 104) in suspension were incubated with 10?��M DCFH-DA (CELL BIOLABS) for 10?min at 37��C, washed with PBS, and treated with or without 500?��M H2O2. The fluorescence corresponding to the cellular ROS level was monitored in a black plate using a fluorescence spectrophotometer at excitation of 480?nm and emission of 530?nm. Cells stably expressing a luciferase gene were inoculated intravenously through the tail vein of 7-week-old female NOD-scid, IL-2R��-null (NOG) mice. NOG mice were obtained from the Central Institute for Experimental Animals (Kawasaki, Japan). Tumor growth in the lungs was monitored using the IVIS imaging system (Xenogen), Ponatinib manufacturer and tissue Oxalosuccinic acid samples were stained with hematoxylin and eosin. Details about these methodologies can be found in Supplemental Experimental Procedures. All animal care followed the Guideline for Animal Experiments at the National Cancer Center and was approved by the institutional committee for Ethics of Animal Experimentation. Data are represented as the means �� SD of three or more independent experiments. Statistical analysis was performed by Student��s t test and unpaired t?test at significance levels of p?
