ABSTRACT: The present study was designed to identify Mkl1 target genes whose expression requires either the B1 site of Mkl1 and serum response factor (SRF), respectively, or the SAP domain of Mkl1. For this purpose, we obtained the transcriptomes of four stable 4T1 cell lines that either overexpress full length Mkl1-RFP (4T1-FL), Mkl1-RFP with a mutated SRF-interaction site (4T1-mutB1), Mkl1-RFP with a deletion of the SAP domain (4T1-ÎSAP) or an empty vector encoding RFP alone (4T1 control). Stable 4T1 cell lines were grown in triplicates in 0.03% FCS/DMEM medium for 48 h before total RNA was extracted. RNA was converted into labeled cDNA and hybridized to Affymetrix GeneChip Mouse Gene 1.0 ST arrays. RMA normalized expression values were calculated with the affy package from the Bioconductor 2.4 release (1) and differentially expressed genes were identified using moderated t-statistics calculated with the empirical Bayes method as implemented in the Bioconductor limma package (2). To be considered as differentially expressed between 4T1-FL and 4T1-mutB1 or 4T1-âSAP cells, genes had to pass the filters: adjusted P-value ⤠0.01 (with Benjamin-Hochberg false discovery correction), a minimum absolute linear fold change difference of 2.0 and a minimum average expression value of 4.0 (log2). References: (1) Gentleman, R., Carey, V., Bates, D., Bolstad, B., Dettling, M., Dudoit, S., Ellis, B., Gautier, L., Ge, Y., Gentry, J., Hornik, K., Hothorn, T., Huber, W., Iacus, S., Irizarry, R., Leisch, F., Li, C., Maechler, M., Rossini, A., Sawitzki, G., Smith, C., Smyth, G., Tierney, L., Yang, J., Zhang, J. (2004) Bioconductor: open software development for computational biology and bioinformatics. Genome Biology 5, R80 (2) Smyth, G. K., Speed, T. (2003) Normalization of cDNA microarray data. Methods 31, 265-273
