Conclusions Here we demonstrate that the aberrant expression of an oncogenic ETS transcription factor in prostate cells can switch the regulation of a cell migration gene expression program from RASERK to PI3KAKT sellectchem control. This pro vides a mechanistic rationale for the correlation between PI3K signaling and ERG expression in prostate tumors and identifies a novel mode of ETS regulation that might be exploited by future therapeutics. Methods Cell culture and viral transduction All cell lines were authenticated by the University of Arizona Genetics Core using PowerPlex 16HS Assay with 80% match to eight core STR loci, with the exception of LNCaP, which was obtained from ATCC immediately prior to use. Cell lines were cultured according to ATCC recommendations as fol lows.
RWPE and RWPE KRAS Keratinocyte SFM, LNCaP and CWR22Rv1 RPMI 1640 with 10% fetal bovine serum, PC3 F12K medium with 10% FBS. 293 EBNA, HEK 293 T, DU145 and VCaP Dulbeccos modification Eagle with 10% FBS, MDA PCa 2b BRFF HPC1 with 20% FBS. All media Inhibitors,Modulators,Libraries were supplemented with 1% PenicillinStreptomycin. ETS proteins with N terminal 3xFlag tags were stably expressed Inhibitors,Modulators,Libraries in RWPE via retrovirus as described previ ously. Plasmids for lentiviral shRNA knockdowns were obtained from AddGene, mTOR, Raptor and Rictor, are from Sarbassov et al. Lentivirus was produced by co transfection of pLKO. 1 constructs in HEK293T cells with pMDLg pRRE, pRSV Rev and pMD2. G envelope plasmids from Dull et al. and AddGene. Transwell migration and In vitro scratch assays Transwell migration assays were done as described pre viously.
In brief, 5��104 Inhibitors,Modulators,Libraries cells were introduced to the transwell and incubated for 63 h, except for RWPE KRAS cells summarized in Figure 2C, which were incubated for 54 h. Migrated cells are reported as the mean of four representative fields per membrane, and the mean of two technical replicates per biological replicate. For in vitro scratch assays, cells were plated in 35 mm Inhibitors,Modulators,Libraries plates and grown to full confluence, and the cultures were scratched by pipette tip. Migration into the open area was documented at 40 h post scratching by micros copy. Free area was measured using TScratch software. Measuring protein and RNA RNA levels Inhibitors,Modulators,Libraries were measured by quantitative reverse transcription PCR as described previously, using primers in Additional file 4 Table S1.
Whole cell extracts of equivalent cell number were separated by SDS PAGE and blotted to nitrocellulose. Antibodies for immunoblotting add to your list were ERK and pERK from Santa Cruz Biotechnology, ETV5 and ETV1 from Abcam, pAKT and pMEK from Cell Signaling, Tubulin from Sigma, ETV4 from Aviva Systems Biology, and ERG from Biocare Medical. Purification of His tagged ETS proteins for antibody validation was as described previously. DNA bind ing activity was verified by EMSA. Concentration was calculated by comparison to BSA standards on Coomas sie stained 10% SDS PAGE gels.