Clonal human derived renal proximal tubule cells were grown in DMEM F12 supplemented with 10% fetal bovine serum, 2 mM L glutamine, penicillin and streptomycin and maintained at 37 C in a 5% CO2 water saturated atmosphere. A stably HPSE silenced HK 2 cell line was obtained leave a message by transfection with shRNA plasmid targeting human HPSE purchased from OriGene, as previously described. HPSE silenced HK 2 cells were grown in the same medium of wild type HK 2 cells. Cells were grown to sub confluence, starved in serum free medium for 24 hours and then cul tured in serum free medium with 10, 100, 200 and 500 nM EVE for 6 hours. Fibroblast growth factor 2, a growth factor that induces EMT was used as positive con trol. Control cultures were incubated with DMSO alone.

AKT12 small interfering RNA has been used to specifically silence AKT1 and AKT2. HK2 WT cells were seeded into 6 well plates at a density of 1. 5 105 cells per well in 2 ml complete Inhibitors,Modulators,Libraries growth medium. After 24 h, the siRNA was added in serum free medium. After 24 h the medium was replaced with fresh complete growth medium. Cells were incubated for an additional 24 h and then starved, Inhibitors,Modulators,Libraries treated with EVE and assayed for gene expression. RNA expression analysis of HPSE, SMA, FN, VIM and MMP 9 Total RNA was extracted from the cell monolayer using the GenElute Mammalian Total RNA Miniprep kit including DNase treatment. Yield and purity were assessed using Nanodrop and Agilent 2100 Bioanalyzer, respectively. Total RNA from each sample was reverse transcribed into cDNA using SuperScript II reverse transcriptase.

Real time PCR were performed on an ABI Prism 7500 using Power SYBR Green Master Mix 2. A quantitative analysis was performed to eval uate the expression of HPSE, Inhibitors,Modulators,Libraries MMP 9, SMA, VIM, FN, TGFB2 and EGFR normalized to GAPDH. The com parative Ct method was used to quantify gene expression, and the relative quantification Inhibitors,Modulators,Libraries was calcu lated as 2 Ct. Melting curve analysis was performed to check for any presence of non specific amplification products. Immunofluorescence for SMA, VIM and FN WT and HPSE silenced cells were seeded in 22 mm glass dishes and cultured to subconfluence, serum starved for 24 h, and then incubated with or without EVE for 24 h to analyze SMA, VIM and FN protein expression. Cells were fixed in 4% paraformaldehyde and permeabilized in phosphate buffered saline 0. 2% Triton 100.

Cells were incubated Inhibitors,Modulators,Libraries with primary antibodies for SMA, VIM and FN overnight at 4 C in PBS with 1% BSA, then washed three times for screening library 5 min with PBS before incubating them for 1 h at 37 C with the secondary antibody in PBS with 1% BSA. Nuclei were counter stained with Hoechst 33258. Zymography for MMP9 Gelatin substrate zymography was used to assess MMP9 activity in WT and shHPSE HK 2 cell conditioned media. Conditioned media were prepared by incubating sub confluent cells in serum free medium for 24 h, then with EVE at different dosages for a further 24 h.