2.3. Luciferase assay

TEAD-dependent luciferase assays were performed by transfecting sub-confluent 12-well plates of HEK293T cells with 0.1 µg pGT₄Tluc, which harbours the firefly luciferase gene driven by four tandem copies of the GTIIC site 30-mer containing the TEF-1 DNA binding sites found in the polyomavirus F101 enhancer [29] (Fig. 2A), 0.3 µg pF-5xUAS-MCS-W–SV40puro [32], [33] expressing full-length (entire coding sequence) wild-type (WT) or mutant YAP constructs, 0.05 µg pRL-TK Renilla luciferase (internal control), with or without 0.05 µg pCI-HA-TEAD-2, which can associate with full-length mYAP and hYAP isoforms via their TEAD-binding domains, and pUC13 to a total of 0.5 µg DNA per well using Effectene (Qiagen #301425).

YAP transactivation, PDZ-binding, and TEAD-binding domains are required for TEAD-mediated transcriptional activity. (A) Schematic illustration of the pGT₄Tluc luciferase TEAD reporter. HEK293T cells were transfected with pGT₄Tluc (Control), FLAG-tagged wild-type (WT), ΔTAD, ΔPDZ or ΔTEAD mYAP constructs (B), and pCI-HA-TEAD-2 (HA-TEAD2) as indicated. (C) After 24 h cells were harvested and luciferase activity was determined. Firefly luciferase activity was normalised to Renilla luciferase activity. The average luciferase activities were normalised to WT mYAP without HA-TEAD2, which was set to 100%. Data is presented as mean+SEM from three independent experiments. *p<0.05, **p<0.01, ***p<0.001. (D) Cell lysates from (C) were separated by SDS-PAGE, transferred to membrane and immunoblotted for FLAG and β-Actin as indicated. Size markers are shown in kilodaltons. (E) HEK293T cells were transfected with pGT₄Tluc (Control), mYAP or hYAP1-2α constructs, and pCI-HA-TEAD-2 (HA-TEAD2) as indicated. After 24 h cells were harvested and processed as in (B). Cell lysates were immunoblotted for YAP and β-Actin as indicated. Size markers are shown in kilodaltons. Vertical line indicates the samples were blotted on separate gels.

For GAL4 fusion experiments, sub-confluent 12-well plates of HEK293T cells were transfected with 0.1 µg pFR-Luc (Stratagene) [31], 0.05 µg pFA-CMV-GAL4-FLAG YAP-TAD fusion constructs (Fig. 4A), 0.1 µg pRL-TK, and pUC13 to a total of 0.5 µg DNA per well using Effectene. For HeLa and D645 cell lines, cells were transfected with 0.4 µg pFR-Luc, 0.2 µg pFA-CMV-FLAG YAP-TAD fusion constructs, and 0.4 µg pRL-TK per well using ViaFect (Promega).

Luciferase activity was measured after 24 h using the Dual Luciferase Reporter Assay (Promega). Statistical significance was calculated by performing a one-way ANOVA with Tukey’s multiple comparison test from at least three independent experiments.