Thus, ana lysis of the EMT status may help to predict TKI 258 re sponsiveness independent of molecular analysis of RTK signaling. Methods Cell culture Human bladder cancer cell lines T24, HT1376, BFTC 905, 5637, HU456, UMUC3, RT4, RT112, TCC SUP, MGHU4 were cultured in RPMI1640 medium supple mented with 10% fetal bovine serum, 1% stable glutam ine and 1% Penicillin/Streptomycin solutions at 37 C with 5% CO2 in humidified air. Dovitinib was kindly provided by Novartis Pharma AG. RT4 and RT112 cells are known to be wild type for FGFR3 and T24 and UMUC3 have activating RAS mutations acting downstream of RTKs. RNA and protein extraction RNA and protein extraction was performed with Trifast according to the manufac turers protocol.

Quantitative real time RT PCR 1 ug RNA was used as template for cDNA synthesis after digest of genomic DNA with RNase free DNase. Realtime RT PCR was performed with SYBR Green Fluorescein Mix. Cycling conditions were, 95 C for 15 min, followed by 45 cycles of 95 C for 15 s, 60 C for 15 s, 72 C for 30 s. Rela tive levels of mRNA are displayed as Ct values with the mean of B actin and porphobilinogen deaminase as reference mRNA Western blot After determination of protein concentration, 40 ug of each sample was subjected to sodium dodecyl sulphate polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membrane by electrophor esis. The membranes were blocked at room temperature for 1. 5 h. Primary antibodies for vimentin, E cadherin, N cadherin, and for B actin were added and incubated overnight at 4 C in tris buffered saline with 0.

1% tween containing 5% dry milk. Then, secondary horseradish peroxidase coupled anti rabbit or anti mouse immunoglobulin was added for band detection with enhanced chemiluminescent lu ciferase kit by an image system allowing measurement of band intensity for determination of relative protein abundance. Proliferation/viability assay TACS XTT Kit with a long term protocol was used to assess the effects of TKI 258 on cell viability, an assay that closely correlates with proliferation. Cells were seeded into 96 well plates with 150 ul medium and TKI 258 was added one day later in a dose range as indicated. Medium and TKI 258 was replaced once after 2 d Carfilzomib and incubation continued for further 3 d. Then, XTT solu tion was added and the optical density was measured at 490 nm. The IC50 values were calculated by non linear regression analysis with the equation of a sigmoidal dose response with variable slope Y 1. Colony formation assay This assay measures cell proliferation in a cell contact independent way. Cells were plated in pre tested appro priate densities yielding 100 500 cells per plate. The plates were cultured for 8 12 days in the presence or absence of TKI 258.