Figure 2.

Early G1 phase ERK activity is dispensable for S phase entry. (A) Quiescent MEFs were preincubated with DMSO (vehicle) or 50 μM U0126 (U0) before being reseeded in 100-mm dishes containing coverslips. The cells were stimulated with 10% FBS with BrdU in the continued presence of U0126. U0126 was washed-out (wo) at the indicated times, and the cells were refed DMEM-10% FBS and BrdU without U0126. Coverslips were collected after 9 and 21 h of total incubation. Entry into S phase was measured by incorporation of BrdU using epifluorescence microscopy. Results show the mean ± SD of three experiments. (B) Quiescent MEFs preincubated with DMSO or U0126 as in A, were reseeded onto 35-mm dishes containing coverslips and stimulated with 10% FBS in the presence of BrdU. U0126 was either maintained throughout the mitogenic stimulation (dotted line) or washed-out after 3 h of mitogenic stimulation (dashed line). In the latter case, the cells were refed DMEM-10% FBS and BrdU without U0126. Coverslips were collected at the indicated times, and S phase entry was determined by BrdU incorporation.