Figure 3. Cell cycle arrest by glucocorticoids requires p27 induction. (A) p27 knockdown in CEM cells. CEM cells were transduced with control (scramble) or p27 shRNA (p27–30) expressing constructs by lentiviral infection and analyzed for p27 expression 5 d after infection. Additionally, cells were treated with Dex (100 nM) for 24 h. Fifty µg of protein extracts were resolved by SDS-PAGE and p27 levels determined by immunoblotting using the Odyssey infrared imaging system. (B) Parental CEM cells and single clones derived from p27 shRNA (p27–30) pools were tested for p27 and cyclin D3 expression in the absence and presence of Dex (100 nM, 24 h). Protein extracts from cells were subjected to SDS-PAGE and immunoblotting using specific antibodies. α-tubulin was used as a loading control. A representative immunoblot is shown. (C) Reduced cell cycle arrest in p27-knockdown cells. Proliferation and cell cycle distribution of cells used in (B) were determined by BrdU incorporation and PI staining. BrdU-positive S-phase cells were determined by anti BrdU labeling (see “Materials and Methods”; Fig. S4). Changes in the number of S-phase (BrdU-positive) cells by Dex after 24 h were calculated and expressed as % reduction of S-phase cells compared with control (0.07% ethanol) treated cells. Data show mean values and SD from at least 3 independent experiments; statistical analysis was performed by unpaired Student t test (*P < 0.05; **P < 0.01). (D) Endogenous Skp2 is downregulated by Dex and Skp2 overexpression reduces p27 levels and glucocorticoid-induced cell cycle arrest. Parental control CEM cells and lentivirally HA-Skp2 transduced cells were analyzed for glucocorticoid response (24 h Dex) by immunoblotting and FACS analyses as described in (C). Skp2 and p27 expression was determined by using specific antibodies. α-tubulin levels indicate equal loading. Diagram shows mean values and SD from 3 independent experiments; statistical analysis was performed by unpaired Student t test (*P < 0.05). (E) Inverse regulation of Skp2 and p27 protein expression by Dex. S49.1 and CEM cells were treated for the indicated time periods with Dex (100 nM) and 50 µg of protein extracts subjected to immunoblot analysis. Images and quantitative data were obtained by using the ImageQuant LAS 4000 digital imaging system. Data are expressed as fold-induction relative to the control; error bars indicate SD and are derived from n > 3 independent experiments analyzed in triplicates; statistical analysis was performed by unpaired Student t test from consecutive time points (p27) or relative to control (Skp2) (*P < 0.05; **P < 0.01). (F) Regulation of Skp2 mRNA by Dex in S49.1 and CEM cells. Procedure is identical to the one described in Figure 2A. Data are expressed as fold-induction relative to the control, error bars indicate SD and are derived from n = 3 independent experiments analyzed in triplicates; statistical analysis was performed by unpaired Student t test (**P < 0.01).