Gene expression analyses of SMAD4 knockdown in pancreatic cell line

The transforming growth factor β (TGF-β) signaling protein SMAD4 is lost in 60% of PDAC, and this has been associated with poorer prognosis. We expressed SMAD4 in human PDAC cell lines BxPC3, by selection of stable clones containing an inducible SMAD4 Tet-ON construct. After 24h of SMAD4 expression, TGF-β signaling-dependent G1-arrest was observed in BxPC3 cells with an increase in the G1-phase fraction from 48.9% to 71.5%. Microarray analysis of gene expression at 8h, 24h, and 48h after SMAD4 expression characterized the regulatory impact of SMAD4 expression in a SMAD4-null PDAC cell line and identified novel targets of TGF-β signaling. We used BxPC3 cells infected by pINDCUER-SMAD4-Puro virus. After 24h, 1µg/ml doxycycline was added to experimental wells. We profiled the gene expression after 8hr, 24hr and 48hr treatment with doxycycline as well as control at 8hr.