Figure 3

Growth and cell cycle regulation during endothelial differentiation and rapamycin treatment. a) Rate of cell division. RoSH2 cells were labeled with CFDA, a cell-permeant fluorescent dye, cultured for 24 hours and re-plated on gelatin-coated plates to be maintained as undifferentiated cells (Undif) or on matrigel to induce differentiation (Dif). Cells were harvested at 0, 24, 48 and 72 hours. At 48 hours after replating, half of the remaining plates of cells under undifferentiating condition or differentiating condition were treated with 50 ηM rapamycin (R undif and Rdif, respectively). Median cellular fluorescence of the harvested cells was measured by flow cytometry and the number of cell divisions was calculated as a function of the loss in fluorescence; b) Cell cycle progression during endothelial differentiation. RoSH2 cells were plated on either gelatin-coated plate (self-renewing) or matrigel (differentiating) and labeled with BrdU for 16 hours. After removing BrdU, half of the gelatin-coated plates were treated with 50 ηM rapamycin. At 0, 6 and 12 hours, cells were harvested, stained with anti-BrdU and PI. DNA content of BrdU-labeled cells as measured by PI was analyzed by flow cytometry.