Fig. 2. Activity of the Raf/MEK/MAPK pathway is required for nuclear translocation of FOXM1. (A) Immunoblot analysis of total ERK and phosphoERK expression to monitor the effect of U0126 and ATA treatment on Raf/MEK/MAPK activity. (B) Scheme for drug treatment to inhibit MEK1/2. Synchronized BJ1 cells were incubated with U0126 (10 µM) or the inactive analog U0124 (10 µM) from 7 hours to 8 hours after aphidicolin release. Cells were harvested 1 hour later (i.e. at 8 hours after release) and immunostained for FOXM1. (C) Without drug treatment, FOXM1 was predominantly cytoplasmic at 7 hours after release. (D) 1 hour later, FOXM1 became mainly nuclear. (E) Treatment with U0126 abolished FOXM1 nuclear translocation at 8 hours after release. (F) FOXM1 nuclear translocation was not affected by U0124 treatment. (G-K) ATA promotes nuclear translocation of FOXM1. (G) Scheme for ATA treatment and U0126/U0124 pre-treatment. (H) In cells treated with solvent (DMSO), FOXM1 was predominantly expressed in the cytoplasm. (I) After incubation with 200 µM ATA for 1 hour, FOXM1 became localized mainly to the nucleus. (J) ATA stimulation of FOXM1 nuclear translocation was abrogated by pre-incubation with 10 µM U0126 for 1 hour. (K) U0124 pre-treatment could not counteract the stimulatory effect of ATA.