Generation of core TGF-β/Smad3 tumor suppressor signature. (A) Unsupervised hierarchical clustering of differentially expressed Smad3 target genes in M3 and M4 cells treated with TGF-β in vitro for 6 hours. The 38 genes induced by TGF-β in vitro in M3 only were taken forward for further analysis. (B) RTQ-PCR validation of the 26 genes out of the original 38 genes that microarray analysis showed to be also regulated by TGF-β in M3 tumors in vivo. M3 tumors transduced with the dnTβRII represents the low TGF-β signal condition in vivo, while M3 tumors transduced with control lentivirus represents the high TGF-β signal condition in vivo. Expression was normalized to the low signal condition for each gene. Results are the mean +/−SEM for three tumors/experimental group. The difference between the high and low TGF-β signaling conditions is statistically significant (P <0.05; unpaired t test) for all genes shown. Note the six genes, marked by arrows, which are downregulated by TGF-β in vivo whereas they were upregulated in vitro. (C) Smad3 dependence of TGF-β regulation of select target genes in vivo. Further RT-QPCR quantitation was performed for six representative genes under the following conditions: (i) M3 cells treated with 5 ng/ml TGF-β (high TGF-β signal condition) or vehicle (low signal condition) in vitro; (ii) M3 tumors in vivo following transduction with a dnTβRII to block all TGF-β responses (low signal condition) or LacZ control lentivirus (high signal condition); (iii) M3 tumors in vivo following transduction with shSmad3 to block Smad3-mediated responses (low signal condition) or shGFP control lentivirus (high signal condition). Results are mean +/−SEM for three to six independent samples/group, normalized to low signaling condition. *statistically significant (P <0.05) for high vs. low signaling condition, unpaired t test. dnTβRII, dominant-negative type II TGF-β receptor; TGF-β, transforming growth factor beta.