Plates were stained with crystal violet and cell colonies were counted. Plating efficiency was calculated as the percentage of seeded tumor cells forming macro scopic colonies. Cell migration Cell migration was determined using both wound healing and transwell assays. For the wound healing assay, cells were seeded in a 6 well plate and grown for 48 h to allow them to reach confluency. Prior to the treatment, a 2 mm wide scratch was made in the mono layer using a sterilized 1 ml pipette tip. Cell migration was assessed 24 h after treatment. For the transwell assay, cells were seeded into a commercial transwell insert and incubated with desired agents. Migrated cells on the bottom of the filter were stained and counted under a light microscope 24 h after treatment.

Cell invasion Invasive ability of cells was tested using a transwell insert pre loaded with Matrigel. Inserts were incubated with serum free medium at 37 C for 2 h to allow rehydration of Matrigel. Agents to be tested were added into both upper and lower chambers at equal concentrations. Cells suspended in serum free medium were then loaded onto the top chamber. Complete medium was used in the lower chamber as a chemo attractant. After 24 h of incubation, the Matrigel was removed and the inserts were stained with crystal violet. Invaded cells on the underside of the filter were counted. Anoikis Cells were seeded into a 6 well plate coated with poly HEMA at a density of 105/well and continuously incubated with the compounds for 72 h. The suspended cells were har vested and incubated with trypsin EDTA at 37 C for 20 min to dissociate cell clumps.

Single cell suspen sions were stained with the trypan blue and cells were counted using a hemocytometer. Cell death was cal culated from the ratio of positive stained to total cells. Western blot Cells were harvested and disrupted in a radioimmuno precipitation assay lysis buffer buffer. Equal amounts of whole cell lysates were resolved by SDS PAGE, electrotransferred to a nitrocellu lose membrane, probed with relevant primary antibodies at 4 C overnight, incubated with horseradish peroxidase conjugated secondary antibodies and detected with an enhanced chemiluminescence substrate. Quantitative real time PCR qPCR was performed as described previously. Briefly, total RNA was extracted using TRIzol Anacetrapib and reverse transcription was conducted following the instructions of the TaqMan Reverse Transcription Kit.

All reactions were performed on the ABI7500 Fast Real Time PCR System. mRNA levels of tested genes were normalized to Actin according to the follow ing formula 2^ , where CT is the threshold cycle. Fold of gene expression of PC 3 cells was defined as 1 . Background Receptor tyrosine kinase signaling is altered in urothelial cancer. Namely, FGFR dependent signaling is affected.