SMAD4 and breast carcinoma: Using a highly metastatic variant of the triple-negative MDA-MB-231 human breast cancer line, MDA-MB-231-HM (231-HM) [30], we reduced SMAD4 expression using two short-hairpin RNAs (shRNAs), SMAD4sh4 and SMAD4sh5, that target different regions of the SMAD4 gene (Fig. 1b).