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. 2009 Oct 2;284(48):33320–33332. doi: 10.1074/jbc.M109.050427

FIGURE 5.

FIGURE 5.

CUL4B down-regulation inhibits cell proliferation. A and B, validation of the effect and specificity of RNAi against CUL4B in stably transfected cells. A, RNA was extracted from wild-type HeLa cells and each of the stably transfected cell lines. The expression of CUL4A and CUL4B mRNA was quantified by real-time PCR. The assay was performed in triplicate, and relative means ± S.E. are shown. B, equal amounts (60 μg) of protein lysates were subjected to SDS-PAGE (12%) and then detected using the antibodies against CUL4B and β-actin, respectively. C, RNAi of CUL4B inhibits cell proliferation. Equal numbers of the indicated cells were plated on 96-well plates, and cell proliferation was determined by MTT assay at different time points. The data in each time point are the mean results ± S.D. of the averaged values from 10 replicates. These experiments were performed in triplicate. *, p < 0.001 versus miNeg. D, effects of CUL4B RNAi on DNA synthesis. The indicated cells were synchronized by serum starvation and then stimulated to progress through the cell cycle by the addition of fresh medium containing 10% fetal bovine serum. After 20 h of treatment, before being fixed, cells were labeled with 50 μm BrdUrd for 45 min. Shown were the percentages of BrdUrd-labeled cells. Results represent means ± S.D. *, p < 0.01 versus miNeg. E, effect of CUL4B RNAi on cell cycle distribution of HeLa cells. Cells were synchronized and restimulated to enter the cell cycle as described above. Cell cycle distribution was determined by flow cytometric analysis of DNA content. The x and y axes show DNA content and cell number, respectively. The results shown are representative of three independent experiments with similar results. Bars, means ± S.D. *, p < 0.05 versus miNeg. F, effects of CUL4B RNAi on S phase progression. The indicated cells were synchronized by a double thymidine block and then released to progress through the cell cycle by replating in fresh drug-free medium. For each time point, the cells were labeled with BrdUrd for 45 min. The percentage of BrdUrd-labeled cells is shown.