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. 2013 Mar 4;110(12):E1112–E1121. doi: 10.1073/pnas.1302184110

Fig. 8.

Fig. 8.

Gene expression profiling of Arf-null ES cells and EBs transduced with miR-205. Arf-null ES cells infected with a retroviral vector expressing GFP alone (Ctl) or with a vector coexpressing miR-205 and GFP were sorted for GFP expression, plated in the presence of LIF (designated ES), or induced for 4 d in the absence of LIF to form EBs (designated EB). Isolated RNAs labeled with fluorochromes were used to probe Affymetrix Mouse Gene Chips (Version 1.0). The data were normalized (Z score transformed) across samples so that the heat intensity scales shown in A, C, and D are in units of SD. (A) Indicated in the heat map are 632 probe sets that were significantly up-regulated (red) or down-regulated (green) with twofold or more changes. Control ES cells, ES cells expressing miR-205, control EBs, and EBs expressing miR-205 are indicated and are designated 1–4, respectively, at the top of the heat map. Although these experiments were not designed to identify genes directly targeted by miR-205, we selected the most probable miR-205 mouse target genes in each of three public databases (miRDB.org, TargetScan.org, and microRNA.org) and chose 26 genes for further analysis that were concordantly ranked in the “top 60.” None of the 144 probe sets corresponding to selected candidates exhibited >1.5-fold down-regulation in miR-205–expressing ES cells taken for comparative microarray analysis before LIF withdrawal. Six genes [Cdh11 (see also Fig. S2A), Cldn11, Plcb1, Ralyl, Zfp558, and Zfp758] were not expressed at significant levels in control GFP+ ES cells. The remaining candidates included Acsl1, Akna, BC030336, Cadm1, Ccny, Chn1, Ctps2, Dmxl2, Ezr, Lrch3, Lrp1, Mgrn1, Mllt4, Nacc2, Nfat5, Rfk, Sbf2, Sdha, Sorbs1, and Spata13. (B) qPCR analysis confirmed up-regulation of two ExEn markers in EBs transduced with miR-205. Error bars, mean ± SEM (n = 3 experiments). (C) Heat map of a subset of genes associated with pluripotency and differentiation toward germ layers other than ExEn (terms indicated at the right). Of note, Klf4, Tbx3, Lefty2, Tdgf1, Ncam1, and Fgf5 revealed less than twofold changes. (D) Heat map of genes involved in cell adhesion (i) and cell motility and migration (ii). Designation of lanes in C and D is identical to that in A. The data have been deposited in the Gene Expression Omnibus (GEO) under accession no. GSE42210.