Ectopic activation of ARHGAP8 in melanoma promotes cell motility

ARHGAP8 was expressed at negligible levels in normal melanocytes but was ectopically activated in approximately 10% of melanoma specimens and melanoma cell lines. ARHGAP8 was ectopically expressed in ARHGAP8-negative M14 melanoma cells using the pIRES2 vector (Clontech), producing a bicistronic ARHGAP8-ires-eGFP mRNA. M14 cells stably transfected with vector control or N-terminally FLAG-tagged ARHGAP8 were cultured in 2D or 3D embedded growth conditions and then subjected to gene expression profiling using Affymetrix U133_Plus_2 gene chips. M14 cells stably transfected with pIRES2 alone or pIRES2 containing N-terminally FLAG-tagged ARHGAP8 were cultured in 2D or 3D conditions for 4 d as described. Total RNA was isolated using Trizol reagent (Invitrogen) and processed for application to Affymetrix gene chips.