Figure 7
Effect of PE on p27kip1 level and effect of p27kip1 antisense oligonucleotides on PE-induced inhibition of DNA synthesis. A, Preconfluent rat-1 cells were stimulated with 2, 5, and 10 μM PE for 18 h. Total cell lysates were subjected to Western blot analysis as described in Methods. Percentages representing the quantitation of protein bands by densitometric analysis of blots obtained from three different experiments are indicated on top of each immunoblot. B, Representative Western blot showing the effect of p27kip1 antisense and sense oligonucleotides treatment on PE-induced increase in p27kip1 protein level. Cells were transiently transfected with p27kip antisense or sense oligonucleotide for 48 h using Oligofectamine™ Reagent according to the manufacturer's instruction (Invitrogen). C, Transfected cells as in B were serum-starved for 24 h and then treated with 5 μM PE. Thymidine uptake was measured as described in Methods 48 h post-transfection. Data are expressed as the percent decrease in [3H]thymidine uptake above the basal value obtained in unstimulated cells. Values are the mean ± S.E of three independent experiments performed in different batches of cells. * Value significantly different from the corresponding vehicle, p < .05.