G44 26

For sorting of CD44 high and CD44 low cells from spheroid cells, dissociated cells were filtered, incubated with DAPI for the exclusion of nonviable cells, and double-stained with a phycoerythrin (PE)-or allophycocyanin (APC)-conjugated monoclonal antibody against CD44 (G44-26; BD PharMingen). For evaluation of mTORC1 activation of CD44 high and CD44 low cells, the dissociated spheroid cells were double-stained with the conjugated anti-CD44 antibody and an anti-phospho (T37/46) 4EBP1 antibody (Cell Signaling Technology), followed by anti-rabbit IgG antibody conjugated with Alexa Fluor 488. For analyses of mouse xenograft tumors, additional staining with an APC-conjugated anti-human EpCAM antibody (BD Biosciences) was performed to select human epithelial tumor cells. To evaluate ROS levels, the cells were treated with the CellRox DeepRed reagent or H2DCFDA (Molecular Probes) according to the manufacturers' instructions. The stained cells were analyzed using the FACS Aria IIIu Cell Sorter (BD Biosciences) as previously described (Ohata et al., 2012) .