Cells were cultured and treated as previously described. Primary astrocyte cultures were prepared from the cortex of 6 day old Sprague Dawley rat pups as previously described. The purity of primary EPZ-5676 astrocyte cultures was assessed with the astrocyte specific marker, GFAP, showing over 95% GFAP positive astrocytes. The cells were plated on 12 well plates and 10 cm culture dishes for MMP gelatin zymography and RT PCR, respectively. The culture medium was changed every 3 days. MMP gelatin zymography After TGF b1 treatment, the culture medium was collected, mixed with equal amounts of non reduced sample buffer, and electrophoresed on 10% SDS polya crylamide gels containing 1 mgml gelatin as a protease substrate. Following electrophoresis, gelatinolytic activity was determined Inhibitors,Modulators,Libraries as previously described.

Mixed human MMP 2 and MMP 9 standards were used as positive controls. Because cleaved MMPs were not reliably detectable, only proform zymogens were quantified. When inhibi Inhibitors,Modulators,Libraries tors were used, they were added 1 h prior to the appli cation of TGF b1. Treatment of RBA 1 cells with the pharmacological inhibitors alone had no significant effect on cell viability determined by an XTT assay. Total RNA extraction and RT PCR analysis For RT PCR analysis of MMP 9 mRNA expression, total RNA was extracted from RBA 1 cells stimulated by TGF b1 as previously described. The cDNA obtained from 1 ug total RNA was used as a template for PCR amplification. Oligonucleotide primers were designed based on Genbank entries for rat MMP 9 and b actin. The PCR amplification was performed in 30 cycles at 55 C, 30 s.

72 C, 1 min. 94 C, 30 s. PCR fragments were analyzed on 2% agarose 1X TAE gel containing ethidium bromide and their size was compared Inhibitors,Modulators,Libraries to a molecular weight markers. Amplifi cation of b actin, a relatively invariant internal reference RNA, was performed in parallel, and cDNA amounts were standardized to equivalent b actin mRNA levels. These primer sets specifically recognize only Inhibitors,Modulators,Libraries the genes of interest as indicated by amplification of a single band of the expected size and direct sequence analysis of the PCR product. Cell migration assay RBA 1 cells were grown to confluence in 6 well plates and starved with serum free DMEMF 12 medium for 24 h. The monolayer cells were manually scratched with a pipette tip to create extended and definite scratches in the center of the dishes with a bright and clear field.

Inhibitors,Modulators,Libraries The detached cells were removed by washing the cells once with PBS. Serum free DMEMF 12 medium with or without TGF b1 was added to each dish as indicated after pretreatment with the inhibitors for 1 h. Images of migratory cells from the scratched boundary were observed and acquired at 48 h with a digital camera and a light microscope. The images shown represent check this one of three individual experiments. Preparation of cell extracts and western blot analysis Growth arrested RBA 1 cells were incubated with TGF b1 at 37 C for the indicated time intervals.