Prl tk renilla control plasmid

To assess the ability of verteporfin to suppress YAP1/TAZ-TEAD complex formation and associated transcriptional activity, CME-1 cells were transfected with 8xGTIIC TEAD luciferase reporter plasmid DNA (Addgene #34615)^{22 (link)}. After 5 h, transfection medium was replaced with medium containing 0.075–0.15 µmol/L verteporfin and supplemented with 2% FBS. After incubation for 48 h, cells were lysed and luciferase activity was measured in triplicates using the Dual-Luciferase reporter assay system (Promega) as described previously^{13 (link)}. Firefly luciferase activity was normalized to the co-transfected Renilla pRL-TK control plasmid (Promega) to account for potential differences in transfection efficiency.

To assess YAP1‐mediated transcriptional activity, MLS cells were transfected with TEAD (8xGTIIC) luciferase reporter plasmid (Dupont et al, 2011). For extrinsic activation of YAP1, MLS cells were co‐transfected with a constitutively active YAP1^S127A mutant (Zhao et al, 2007). The amount of plasmid DNA in each transfection was kept constant by addition of the non‐coding plasmid backbone. Reporter assays were performed in triplicates using the Dual‐Luciferase Reporter Assay System (Promega) after 24 h. Firefly luciferase activity was normalized to a co‐transfected Renilla pRL‐TK control plasmid (Promega) to account for differences in transfection efficiency. For verteporfin treatment, medium containing transfection reagent was replaced after 6 h with medium containing 1 μM verteporfin and 2% FBS.

The 3´-UTR of SOCS1, with wild-type or mutant (Mut) binding sites for let-7e, was amplified and cloned into the pGL3 vector (Promega, Madison, WI, U.S.A.) to generate the plasmid pGL3-WT-SOCS1-3´-UTR or pGL3-Mut-SOCS1-3´-UTR, respectively. The dual-luciferase reporter assay was performed as described previously [21 (link)]. RAW264.7 cells were treated with let-7e mimics, Let-7e inhibitor and the luciferase reporter plasmids, pRL-TK-Renilla control plasmid (Promega) using Lipofectamine 2000 (Invitrogen). At 48 h post-transfection, luciferase activities were detected using the Dual Luciferase Reporter Kit (Beyotime Biotechnology, Jiangsu, China).