Figure 1

Cyclin D1 expression following inhibitor treatment: (A) MRC5 cells were left untreated (left) or were treated with 20 μM LY294002 (middle) to inhibit PI3K or 50 nM rapamycin (right) to inhibit mTOR. After 2 hrs cyclin D1, BrdU added as a final pulse, and DNA were stained and subjected to image analysis. Cyclin D1 levels for individual cells are plotted vs. DNA levels, with BrdU positive, S phase cells shown as small, solid circles; with cell cycle phases noted. (B) Serum was removed from NIH3T3 cultures for 4 hrs, after which cells were fixed and stained for total cyclin D1 and with an antibody able to recognize phospho-Thr-286 cyclin D1. The average cyclin D1 (left) or phospho-Thr-286 cyclin D1 (middle) for each cell cycle phase was determined and plotted (darker bars), together with the similar value determined from a parallel culture maintained in serum (lighter bars). Finally (right) the ratio of phospho-cyclin D1 to total cyclin D1 is presented for both the normal and serum deprived culture.