Smad-Responsive Luciferase Assay

Cells were plated at a density of 25,000 per well into 24-well plates and incubated overnight. Cells were transfected with 0.05 μg/well of an expression construct containing the Smad binding element promoter upstream of the luciferase gene using Mirus TransIT LT1 (Mirus Bio LLC, Madison, WI) according to the manufacturer's instructions. The Smad responsive element plasmid contains a CAGA sequence repeated twelve times upstream of the luciferase gene (gift from Dr. Aris Moustakas at Ludwig Institute for Cancer Research, Uppsala, Sweden). Plasmids for expression of wild-type p53 or mutant p53 R273H plasmids were transfected into cells at 0.05 μg/mL. Cells were transfected for 24 hr in serum-supplemented media. Cells were then washed with PBS and treated with TGFβ1 at 10 ng/mL (Sigma Aldrich) for 24 hr. SB-431542 (Selleck Chemicals, Houston, TX) was used at a concentration of 5 μM for all luciferase assays. The protocol and SBE-luciferase transfection efficiencies were normalized and run as previously described [28] (link). Normal cell luciferase activity was measured using a Synergy Mx (BioTek, Winooski, VT).