Cells were grown in a water saturated environment in 5% CO2 and 95% air at 37 C. FTI treatment assay PC Cl3 rat thyrocytes stably transfected with RP3 and vector control at mid log phase growth were trypsinized and live cells were counted using a trypan blue stain. Cells were plated on a 12 well polysty KPT-330 mw rene culture dish at a cell density of 2. 5 105 cells per well suspended in 2 ml culture medium. Cells were allowed to adhere and stabilize for 18 hours. At that time, culture supernatants were removed, cells were washed using room temperature 1�� PBS, and 2 ml of cul ture medium containing FTI or 2 ml of control culture medium was added to each well. DMSO effects were controlled for all culture conditions including control maintained a 0. 05% DMSO concentration.

Cells were allowed to grow in the presence of FTI containing media for 24 hours. RT PCR analysis Total cell RNA was extracted using the TRIzol method of RNA isolation. Precip itated DNA was digested with RNAse free DNAse. Total RNA was dena tured and reverse transcribed using SuperScript III Reverse Transcriptase in a reaction mix containing random primers, Oligo dT, and 0. 1 M DTT for 90 minutes at 42 C. Reverse transcription was confirmed, and the pres ence of genomic DNA excluded, with PCR of cDNA and similarly incubated but non transcribed RNA using prim ers for glyceraldehyde 3 phosphate dehydrogenase. cDNA was amplified by PCR using primers spe cific for rat G3pdh, rat Ccl2, rat Cxcl1, and human RP3. Amplified products for chemokine specific primers and h RP3 were normalized using the amplified product for G3pdh.

PCR cycling conditions for all reactions were denaturation at 94 C for 4 min for 1 cycle. 20 cycles of denaturation at 94 C for 30 sec, primer annealing at 60 C for 30 se, and extension at 72 C for 1 min. and final single extension cycle of 72 C for 7 min. The PCR product GSK-3 sizes were trichostatin a mechanism of action as follows r G3pdh, 227 bp. r Ccl2, 320 bp. r Cxcl1, 215 bp. and h RP3, 302 bp. PCR products were visualized using gel electrophoresis and quantified using the BioRad Gel Doc and Quantity One program. ELISA CCL2 and CXCL1 secretion were measured using an ELISA according to the manufacturers protocol. PC Cl3pMV7 and PC Cl3RP3 thyrocytes at mid log phase growth were co cultured with FTI according to described protocol. Culture media were collected and filtered before use in ELISA. All values refer to the average of duplicate samples from triplicate experiments s. e. m. Western blot analysis Immunoblotting was performed to assess protein levels of the tyrosine kinase domain of RP3 using Ret goat polyclonal IgG and phosphorylated Ret rabbit IgG in PC Cl3RP3 cells following the FTI co culture assay described herein. Protein content was quantified with the BioRad DC Protein Assay.