Fig. 6. Inhibition of Raf/MEK/MAPK signaling leads to G2/M delay and downregulation of FOXM1 target genes. U0126 (25 µM) was added to synchronized BJ1 cells at 5.5 hours after aphidicolin release (black arrows). Cells were harvested for flow cytometric (A), RT-PCR (B) and western blot (C) analyses. Data in A and B were quantified using Modfit and TotalLab software, respectively. (A) After U0126 treatment, slower progression through G2/M was revealed by the delayed re-entry of cells into the subsequent G1 phase. (B) RT-PCR analysis indicated that U0126 treatment attenuated the increase in cyclin B1 mRNA levels associated with cell cycle progression through G2/M. GAPDH mRNA levels were analyzed in parallel as loading control. (C) Cell lysates were immunoblotted with anti-ERK, anti-phosphoERK, anti-cyclin B1, anti-Cdc25B and anti-tubulin antibodies. U0126 suppressed the phosphorylation of ERK, and the expression of cyclin B1 and Cdc25B, but had no effect on the expression of ERK and tubulin.