GILZ controls cyclin D1 and p21 expression. (A) Left: cyclin D1, phosphorylated retinoblastoma (p-Rb) and p21 were measured by western blot in CTRL and in pGILZ clones. β-actin was used as a loading control. Right: the steady-state levels of cyclin D1 and p21 mRNAs were assayed by semi-quantitative RT-PCR in CTRL and in pGILZ clones; β-actin was used as a loading control. (B) p21 and cyclin D1 gene products were measured by western blot (left) or by semi-quantitative RT-PCR (right) 48 h after BG-1 cells were transfected with 4 μg of control (siCT) or GILZ-specific (siGILZ) siRNA; β-actin was used as a loading control. Blots: one representative experiment of three. (C) Top: CTRL and pGILZ clones were synchronized by double thymidine block. Following removal of the block, cells were analyzed for DNA content by PI staining and cell cycle distribution was analyzed by flow cytometry at various time points; the percentages of cells in different cycle phases were determined by ModFit Cell Cycle Analysis software. Data are representative of three independent experiments. Bottom:Percentage of cells in each phase of the cell cycle. Results from three independent experiments (mean ± SE). Unpaired Student's t test was used for comparisons.