Sbe4 luc

SBE4-Luc was from Addgene, and pRL-TK was from Promega Corporation (Madison, WI). A549 cells were cotransfected with SBE4-Luc, pRL-TK and control siRNA/SHP2 siRNA. Luciferase activity was detected according to the protocols for Dual-Luciferase Reporter Assay System (Promega) and measured by SpectraMax M5 plate reader (Molecular Devices, Sunnyvale, CA).

For transfection of Wilms cells with the TOP- or FOPflash-reporter (firefly luciferase) plasmids (Upstate Biotechnology, now supplied by ThermoFisher, Ottawa, ON, Canada) cells were plated in six-well plates at a concentration of 1.5 × 10⁵ cells/well in MSCGM without antibiotics. At a confluence of 50–80%, the cells were cotransfected with TOP- or FOPflash-plasmids and as internal control for transfection efficiency with pRL-TK (Wilms1) or pRL-CMV (Wilms3) (Renilla luciferase) plasmids (Promega, Walldorf, Germany) using Lipofectamine LTX Reagent according to the protocol of the supplier (Invitrogen). All assays were done at least in duplicates or in triplicates and the firefly and Renilla luciferase activities were measured using the Dual-Luciferase Reporter Assay System (Promega) 24 h following transfection. The Renilla luciferase activity was used to normalize the transfection efficiency and the relative luciferase activity was used to calculate the TOP to FOP ratio.
Luciferase reporter containing four copies of the Smad binding site (SBE4-Luc; Addgene, Watertown, MA, USA) [18 (link)]) was used to test for the activity of the TGFβ pathway. Transfection was performed in triplicates and the standard error was determined.

For overexpression experiments, p65 and p65-S536A plasmids were kindly provided by Carl Sasaki^{55 (link)} and pIKKb-ON plasmid was obtained from Addgene (Ref. 11105). Lx2 cells were seeded 24 h prior transfection at 170,000 cells/well in six-well plates. Transfection was done with 0.6 μg total DNA mixed with 1.2 μl Lipofectamine 3000 following the manufacturer’s instructions (Invitrogen, New York, NY, USA). The media was replaced 24 h later and the overexpression was assessed by quantitative real-time polymerase chain reaction (qRT-PCR) and western blot. For luciferase reporter assays, SBE4-Luc (Addgene, 16495) and pRL-CMV (Promega) were used. Lx2 cells were seeded 24 h prior transfection at 75,000 cells/well in 12-well plates. Transfection was done with 0.5 μl Lipofectamine 3000 mixed with 0.05 μg CMV-Renilla and 0.2 μg pSBE-Luc. One day later cells were treated as described (DMSO, 30 μM DHA, 7.5 ng/ml TGFβ) for 24 h, and Renilla and Firefly luciferase activities were measured using the Dual Luciferase System (Promega) according to the manufacturer’s instructions in a Berthold Luminometer (Lumat LB 9507). The values obtained for Firefly luciferase were corrected for equal transfection efficiency with those for Renilla luciferase.

The SMAD-binding element (SBE) luciferase reporter SBE4-Luc (Addgene, #16495) and TopFlash was used for detecting the effect EZH2 knockdown on target gene transcription of TGFβ and Wnt signaling, respectively. HepG2 cells were first infected with lentivirus carrying shEZH2 or empty vector (shCtrl) and selected with puromycin at 1 μg/ml for 2 days. Afterward, when cells were grown at ∼80% confluency, 300 ng of SBE4-Luc reporter and 1 ng of Renilla luciferase reporter plasmids were co-transfected to HepG2 cells for 48 h. In parallel, an empty vector pGL3-basic (Promega) and was also transfected in the same way. Similarly, the Wnt-responsive reporter TopFlash and its negative control FopFlash were transfected to HepG2 cells, for monitoring Wnt target transcription after knockdown. Luciferase activity was measured using the Dual-Luciferase Assay System (Promega, #E1960). Data are shown as the mean ± SEM of SBE4-Luc/vector or TopFlash/FopFlash ratios that were obtained from at least three independent experiments. Significance was calculated using Student’s t-test.