Figure 6

Neither the Erk pathway nor the PI3K pathway is involved in PKCε-mediated migration of SK-N-BE(2)C cells. (A) Cells were allowed to migrate for 6 h towards the lower chambers which were supplemented with DMSO (Ctrl) or 16 nM TPA in the absence or presence of the PI3K inhibitor LY294002 (20 μM) or the MEK inhibitor PD98059 (50 μM). Data, the number of migrated cells expressed as percent of the number of migrated cells after TPA treatment in the absence of inhibitors, are mean ± SEM of three independent experiments. (B) Confluent monolayers of SK-N-BE(2)C cells were scraped with a pipette tip. Cells were incubated in serum-containing medium (ctrl) and 16 nM TPA with or without 20 μM LY294002 or 50 μM PD98059. Data, migration into the scratch area, are presented as relative values where DMSO at the 24 h timepoint is 100% and are the mean ± SEM of three independent experiments. (C) Cells were transfected with 50 nM of siRNA oligonucleotide against PKCε (ε) or an equal amount of water (0) before stimulation with 16 nM TPA for different time periods. Thereafter cells were lysed and analysed by Western blotting using antibodies against phosphorylated Erk (p-Erk) and total Erk (tot-Erk).