Pgl2 vector

TCam-2 cells (3 × 10⁵ cells per well) were seeded into a 96-well plate a day before transfection. Cells were co-transfected with 80 ng of pGL2 vector (Promega) bearing NF-κB enhancer region and TATA box upstream of firefly luciferase gene,^{43 (link)}Renilla vector (Promega) and shPEG3-1 vector or miR-514a-3p mimic using lipofectamine 2000 (Life Technologies). After 48 h post transfection, firefly and Renilla activity was measured using Dual-Glo Luciferase Assay (E2940; Promega). Firefly luciferase activity was divided by the Renilla luciferase activity in each condition and normalized to the shControl or NC transfectants. All experiments were performed in triplicate independently.

The YAP/TEAD binding elements were predicted using the JASPAR database (http://jaspar.genereg.net/). An NMU promoter including the YAP/TEAD binding site was obtained with NMU upstream regions generated by PCR using primers including the KpnI recognition site 5′-CCGGTACCCACCAGACGGACAAAGG-3′ and XhoI recognition site 5′-CCCTCGAGCACCTC TGTGGAAGCAC-3′, and ligated into the pGL2 vector (Promega, Madison, USA) directly. For mutagenesis of the NMU promoter, site-directed mutagenesis was performed following standard protocols as follows: mutation 1, 5′-CATTCATGGTTGGTGGGAATGTAAATTGGTATAAC-3′ to 5′-CATTCATGGTTGGTGGTGGCGTAAATTGGTATAAC-3′, and mutation 2, 5′-ATGTACAAGAACATTCCAAGCAGCCTGGTTCATG-3′ to 5′-ATGTACAAGAACTCGTCAAGCAGCCTGGTTCATG-3′. Successful incorporation of the mutations was confirmed by DNA sequencing. For luciferase assays, 293FT cells were seeded in 24-well plates, and reporter constructs with or without the YAP1 construct and Renilla were co-transfected (Figure 6C). After 24 h, the medium was removed, and the cells were harvested for luciferase assays (Dual Luciferase Assay kit, Promega). Luciferase activity was normalized to Renilla luciferase activity.

The tri-reporter was constructed by linking the coding sequences of firefly luciferase, eGFP, and HSV1Δtk, which has a truncation at the HSV1tk N-terminus. As previously described^{28 (link)}, the HSV1tk (GenBank: CAA23742) coding sequence (a.a. 1–376) was inserted into the C-terminus of an eGFP expression vector (EGFP-C2, Clontech) at the EcoR1/BamH1 sites. The first 135 bp of HSV1tk in the eGFP-tk vector was removed by PCR-directed mutagenesis, resulting in the eGFP-Δtk vector. The F1B promoter, which was isolated from an F1B-GFP vector^{12 (link),26 (link)} as a 540-bp ApaL1/Age1 fragment, was used to replace the CMV promoter in the eGFP-Δtk vector, resulting in the F1B-eGFP-Δtk vector. The coding sequence of firefly luciferase (Luc) was isolated from the pGL2 vector (Promega) by PCR and inserted into the F1B-eGFP-Δtk vector at the Age1 site to generate the F1B-Luc-eGFP-Δtk vector (F1B-TMIR). Luc was similarly inserted into the eGFP-Δtk vector to generate the CMV-TMIR vector. All vectors were verified by DNA sequencing.