aving migrated to the lower face 20567609 were fixed in methanol and stained with Hoechst 33258. Ten images of each filter were randomly captured with an Axioplan2 microscope and an Axiocam HRc camera. Cells present on each image were counted with the Colony1.1H software. Reverse-transcription Quantitative Real-time PCR RNAs were isolated with the NucleospinH kit. Reverse transcription was carried out for 1 h at 55uC with 1 mg total RNA, oligo dT primers, dNTPs and Superscript II reverse transcriptase. Primers 
for PCR were designed with the NCBI-Primer-BLAST software. The PCR protocol was as recommended for the Mx3005P Real-time PCR SystemH. Accumulation of PCR products was measured by SYBR greenH fluorescence. Raw data analysis was performed with the MxProH software. For each sample, Ct – Ct was calculated, and 9570468 this value was used to calculate the ratio of test gene mRNA to gapdh internal control mRNA. ELISA Conditioned culture medium from 500,000 young or senescent NHDFs was harvested after 48 h of culture, filtered through a 0.45-mm-pore-size filter unit, and concentrated by ultrafiltration with a cut-off of 3 kDa. Quantification of HGF/SF was performed with the QuantikineH ELISA human HGF immunoassay according to the manufacturer’s instructions. Migration Assays Assays were performed either with young NHEKs or PSENHEKs. Cells were starved in basal keratinocyte medium for 16 h and then seeded in KBM onto TranswellH cell culture inserts with 8-mm pore size at a density of 30,000 cells per well. Different LY-411575 biological activity attractant media were placed in the lower chambers: 100% KBM or 90% KBM-10% FGM, 90% KBM-10% YF-CM, 90% KBM-10% SF-CM, or 90% KBM-10%, CM harvested from NHDFs treated with 12.5 mM GM6001 or transfected with siRNA. Alternatively, activated recombinant MMP-1 or MMP-2, dissolved at 50 mM in FGM, or recombinant HGF-SF or recombinant TGF-b1, dissolved at 10 ng/ml in In-gel Zymography Assays Latent and active forms of MMP-1 and -2 secreted into the culture medium were assayed on the basis of their ability to digest gelatin, as described previously. Briefly, proteins of fibroblastconditioned media were resolved by SDS-PAGE under nonreducing conditions in 10% polyacrylamide gels containing 0.1% gelatin. Following electrophoresis, the gels were treated with zincand calcium-containing buffer to allow enzyme refolding. Gelatin digestion was then allowed to proceed for 24 h and the gels were finally stained with R-250 Coomassie blue. It should be noted that under these conditions of enzyme refolding and digestion of denatured gelatin, both the active and MMP-PAR-1 in Senescence and Early Carcinogenesis latent proteinase isoforms show activity. MMPs are identified on the basis of their respective molecular weights. Immunohistochemical Detection of PAR-1 in Tissue Sections Deparaffinized, rehydrated sections of skin biopsies from healthy young or aged human donors, obtained from the Bonn University tumor library, were treated with 1% H2O2 in PBS to block endogenous peroxidases. Nonspecific binding was prevented by incubation in PBS+5% BSA and 10% rabbit non-immune serum. Endogenous avidin and biotin were inhibited with the specific Avidin/Biotin Blocking Kit of Vector Labs. Primary antibodies and secondary antibodies were diluted, respectively, in PBS+5% BSA+10% rabbit non-immune serum or PBS+2% BSA. An isotype-specific control immunoglobulin was used on an adjacent section. Detection was done with streptavidin-peroxidase and the DAB Peroxidase Substrate Ki
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