Supplemental Material

This article contains the following supporting material:

• Supplemental Figure 1 - Kinetics of ERK inhibition and recovery. A. Serum-starved MEFs were trypsinized, reseeded (~10⁶ cells per 10 ml) in 100-mm dishes and stimulated with 10% FBS for 2 h to maximally activate ERK1/2. DMSO (vehicle) or 50 μM U0126 was then added, and cells were collected after 5, 15, or 30 min. Lysates (20 μg protein) from collected cells were analyzed for ERK activation by western blotting with antibodies to ERK (ERK gel-shifts) and pERK. The specificity of U0126 was assessed by western blotting the filters with antibodies to total and phosphoSer⁴⁷³-AKT. B. Serum-starved MEFs were trypsinized, suspended (~10⁶ cells per 10 ml) in DMEM, 1 mg/ml fatty-acid free BSA, preincubated with DMSO (-) or 50 μM U0126 (+), reseeded into 100-mm culture dishes, and stimulated with 10% FBS for 2 h in the continuous presence of DMSO or 50 μM U0126 prior to collection and lysis. At the same time, replicate cultures incubated with 10% FBS in presence of DMSO or U0126 were washed twice with cold DMEM and then re-stimulated with fresh medium containing 10% FBS, with (+) or without (-) 50 μM U0126. Collected cells were analyzed by immunoblotting with antibodies for ERK, pERK, AKT1, and phospho-Ser⁴⁷³ AKT. The culture conditions were: i) FBS/DMSO-treated cells that were washed and re-fed fresh medium lacking U0126 (-/-); ii) FBS/U0126-treated cells that were washed and then re-fed fresh medium containing FBS/DMSO (+/-); and iii) FBS/U0126-treated cells that were washed and then re-fed fresh medium containing FBS/U0126 (+/+). The thin vertical space indicates removal of extraneous information.
• Supplemental Figure 2 - Effect of ERK inhibition in early passage human fibroblasts. A. Quiescent human fibroblasts were treated as described for Figure 2A except that 1.5 x 10⁶ cells were plated in 20 ml medium in 150-mm dishes and the incubations times were optimized for the G1-S phase interval of human fibroblasts. The results show the mean ± SD for two experiments. B. Quiescent human fibroblasts were treated as in Fig. 1F and analyzed by western blotting with antibodies to cyclin D1, ERK and cdk4 (loading control). ERK activation is shown by gel-shift (indicated by the upper arrow for ERK2).