In vitro analysis of the <i>β-catenin</i>-<i>Gata3</i>-<i>Ret</i> molecular cascade.

<p>(A, B) Bioinformatic analysis of <i>Gata3</i> and <i>Ret</i> genomic loci 50 kb upstream of the translational start site. Conserved regions between mouse and human sequences are depicted as thick black lines. Conserved TCF/Lef binding sites (consensus sequence (C/G)TTG(A/T)(A/T)) <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1000316#pgen.1000316-Waterman1" target="_blank">[69]</a> (A) and Gata3 binding sites (consensus sequence (A/T)GATA(A/T)) <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1000316#pgen.1000316-Orkin1" target="_blank">[70]</a> (B), are represented as asterisks. (B) A putative Gata3 binding site located in a previously reported <i>RET</i> regulatory element <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1000316#pgen.1000316-Fisher1" target="_blank">[50]</a> and the mutated version used in (D) are indicated in brackets. (C) Quantitative RT-PCR analysis of <i>Gata3</i> expression in mIMCD3 reveals a marked increase in endogenous <i>Gata3</i> expression upon treatment with 3 µM BIO versus DMSO control. Results are expressed as ratios relative to control (n = 3, ** P<0.01, Student's t-test). (D) β-Galactosidase reporter activity is decreased in Gata3-expressing mIMCD3 cells transfected with an expression construct containing the mutated Gata3 binding sequence (as indicated in B), when compared to the wild-type control. Results are expressed as ratios relative to control. (n = 3, * P<0.05, Student's t-test).