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Fig. 4. Knockdown of p27^Kip1 expression by siRNA abrogated OLIG2-mediated growth inhibition and DNA synthesis in U12-1 cells. (A) Suppression of p27^Kip1 by siRNA in U12-1 Tet-off cells. U12-1 cells seeded on 3.5-cm dishes were serum starved for 18 hours and transfected with either p27^Kip1 siRNA or negative control siRNA for 6 hours. Thereafter, 1x10⁵ cells were reseeded in Tet-off medium and were lysed at the indicated days. (B) Growth curves of U12-1 cells in the presence of p27^Kip1 siRNA. Cells were cultured in Tet-on or Tet-off medium at the indicated days. Data represent the number of viable cells and are means ± s.d. of values from three independent experiments. ^*P<0.05, ^**P<0.01, ^***P<0.001 (Student's t-test) versus the corresponding value for control siRNA-transfected cells. (C) DNA synthesis of U12-1 cells in the presence of p27^Kip1 siRNA. Cells were incubated for 72 hours in Tet-on or Tet-off medium, thereafter assayed for BrdU incorporation. DNA synthesis was examined by immunostaining with anti-BrdU antibody 3 days after transfection of siRNA against p27^Kip1 in the presence or absence of OLIG2. BrdU-incorporating cells were detected with antibody 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<0.01, ^***P<0.001 (Student's t-test). (D) Effect of cell density on the OLIG2-mediated growth inhibition in U12-1 cells. U12-1 cells were seeded at a density of 700 cells/cm² in 6-cm dishes (low density) and 50,000 cells/cm² in 12-well plates (high density), respectively, and cultured for 3 days in either Tet-on or Tet-off medium. At intervals of 24 hours, cells in three dishes were separately harvested and their number and viability were determined. Data are means ± s.d. of values from at least three independent experiments. ^*P<0.05, ^**P<0.01, ^***P<0.001 (Student's t-test).

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