Transcriptional profile of day 3 EBs in the absence of endoglin. (A) Microarray heat map representing up- and down-regulated genes in day 3 eng^−/− (null) EBs compared with wild-type (WT). Results represent 3 biologic replicates. (B) Confirmatory real time PCR analyses for selected down-regulated genes. Transcripts are normalized to Gapdh. Error bars indicate SE from 3 independent experiments performed in duplicate. (C) Real time RT-PCR expression analysis for several TGF-β signaling molecules on day 3 WT and eng^−/− EBs. Transcripts are normalized to Gapdh. Error bars indicate SE from 3 independent experiments performed in duplicate. (D) Western blot analyses of day 3 WT and eng^−/− EBs for TGF-β signaling molecules, including Smad1, Smad2, phosphorylated Smad1/5/8 (pSmad1/5/8), and phosphorylated Smad2 (pSmad2). (E) Quantification of phosphorylated Smad1 and phosphorylated Smad2. After normalization to Gapdh levels, results were plotted as ratio between phosphorylated Smad and total Smad. (F) Real time RT-PCR expression analysis for 2 target genes of the ALK-5/Smad2 pathway on day 3.75 WT and eng^−/− EBs. Transcripts are normalized to Gapdh. Error bars indicate SE from 3 independent experiments performed in duplicate. *P < .05, **P < .01, ***P < .001.