Figure 5

ADAMTS1 expression is downregulated in shNFAT1-4T1 cells. (a) Microarray analysis of three independent cultures of control (pLKO-transduced 4T1 cells) and shNFAT1-transduced 4T1 cells. Hierarchical clustering of the indicated 4T1 cells (top) in their control (pLKO) and NFAT1-silenced versions (shNFAT1) was performed as described in Materials and Methods. The heatmap representation highlights upregulated genes in red and downregulated genes in green. (b) ADAMTS1 is downregulated in shNFAT1-4T1 cells (Java Tree View extract of data shown in a, top panel). Semi-quantitative reverse transcription-PCR analysis of ADAMTS1 expression (middle panel) and quantification in lower panel are shown. HPRT was used to normalize the experiments. Quantification was made of three independent experiments (n=3, data are represented as mean±S.E.M.). (c) ChIP/PCR detection of ADAMTS1 promoter in chromatin obtained from 4T1 control (pLKO) and NFAT1-silenced (shNFAT1) 4T1 cells maintained either under steady state (upper panels) or stimulated with PMA/Ionomycin for 6 h to acutely activate NFAT (lower panels). Note the absence of detection of ADAMTS1 promoter co-immunoprecipitated with the anti-NFAT1 antibody in shNFAT1-4T1 cells. The histogram shows the quantification of the co-immunoprecipitated DNA in the different conditions used (n=5, data are represented as mean±S.E.M.). (d) NFAT1-silenced 4T1 cells cultured for 24 h on FITC-gelatin show decreased in situ protease activity compared with control 4T1 (pLKO) cells. (e) Quantification of experiments shown in d, as described in Materials and Methods. Data are represented as normalized degradation (degradation index), which was calculated as the area of degraded matrix per cell relative to control pLKO-4T1 cells (n=4, data are represented as mean±S.E.M.)