Fig. 2. Inhibition of U12-1 cell growth by OLIG2. (A) Growth curves of U12-1 cells incubated in Tet-on or Tet-off medium. The cell cycle was synchronized by serum deprivation for 24 hours before transfer of the cells to Tet-on or Tet-off medium for the indicated time. Data represent the number of viable cells and are means ± s.d. of values from four independent experiments. ^*P<0.05, ^**P<0.01 (Student's t-test) versus the corresponding value for Tet-on cells. (B) OLIG2-induced inhibition of DNA synthesis in U12-1 cells. Cells were incubated for 72 hours in Tet-on or Tet-off medium and then assayed for BrdU incorporation. BrdU-incorporating cells were detected with antibodies for BrdU (green). The nuclei were counterstained with PI (red). Bars, 100 µm. Cell proliferation was determined by counting BrdU-positive nuclei per total nuclei from three random fields. Data are means ± s.d. of values from three independent experiments. ^***P<0.001 (Student's t-test). (C) Inhibition of anchorage-independent growth of U12-1 cells by OLIG2. Cells were grown on soft agar plates containing Tet-on or Tet-off medium for 3 weeks, after which the number of colonies with a diameter of 125-250 µm or >250 µm was counted. Representative micrographs of colonies formed by Tet-on cells are shown in the lower panels. Quantitative data (upper panel) are means ± s.d. of values from three independent experiments. ^*P<0.05 (Student's t-test).