Figure 5

Role of ERK MAP kinase signaling in OE33 cell function. (A) Western blot of MMP-1, MMP-7 and phospho-ERK in the indicated oesophageal cell line extracts. ERK2 is used as a loading control. The positions of molecular weight markers are indicated. (B) Invasion assays of OE33 cells in the presence of U0126 or DMSO (-). Assays were performed for 48 hours and the number of invading cells was compared to the average number of invading cells treated with DMSO (taken as 1). Data are the mean and standard deviations of the relative number of invading cells from 2-3 experiments performed in duplicate. (C) Real time RT-PCR analysis of MMP-1 expression in untreated OE33 cells or Flo1 cells treated or untreated with PMA (average of duplicate samples in 2 experiments). (D) Comparative analysis of the relative number of adherent and OE33 cells grown for 96 hours in the presence of U0126 or DMSO (control). 2 × 10⁴ cells were seeded at day 0 (indicated as 1). The data are the mean relative cell numbers and standard deviations from two independent experiments. (E) Western blot of OE33 cell extracts for phospho-ERK. The cells were treated with U0126 at time 0 and allowed to grow for 12 hours. ERK2 was used as a loading control. (F) Real time RT-PCR analysis of PEA3, ER81, MMP1 and VEGF expression in OE33 cells treated with U0126 for the indicated time periods (0-12 hr). Average mRNA levels (from duplicate samples) were calculated relative to the respective mRNA levels of each gene in cells at time zero (taken as 1).