Figure 4. VGLL4 Overexpression Decreased TEAD1 Stability.

(A) VGLL4 overexpression decreased TEAD1 protein level. Different doses of TEAD1 plasmids (indicated in μg) were co-transfected with 1.6 μg of VGLL4-GFP plasmid. Cells were collected for western blot 24 hr after transfection.

(B and C) Generation and validation of TEAD1-Dendra2 construct. Tead1-Dendra2 plasmid was transfected into 293T cells. Western blot confirmed expression of TEAD1-Dendra2 fusion protein (B). TEAD1-Dendra2 merge fusion protein was green before illumination with 405 nm light. After 30 s of illumination, a fraction of TEAD1-Dendra2 exhibited red fluorescence (C).

(D and E) Doxycycline (Dox)-inducible expression of VGLL4 caused TEAD1-Dendra2 degradation. pTEAD1-Dendra2 and pEF1a-rtTA were co-transfected into 293T cells along with pTetO empty vector (upper panel) or pTetO:HA-VGLL4 (lower panel). Twenty-four hours after transfection, Dox was added. Cells were analyzed at the indicated time points (D). Quantification of TEAD1-Dendra2 protein level is shown in (E). *p < 0.05, n = 3.

(F) Time-lapse imaging of TEAD1-Dendra2 or Dendra2 proteins. Indicated plasmids were transfected into 293T cells. Twenty-four hours later, cells were treated with Dox. Four hours later, Dendra2 was photoconverted with 405-nm light. Relative red fluorescence intensity (RFI) was monitored for 3 hr by taking one image per minute. RFI was normalized to the value immediately after photoconversion. Plot shows average RFI signal over 10 min. n = 10. Experiment is representative of three independent repeats.

(G) Dual luciferase assay of YAP-TEAD1 transcriptional activity. 293T cells were co-transfected with YAP[S127A], EF1a:rtTA, 8xGIITC-luciferase, pRL-TK internal control, and either TetO empty vector or TetO-VGLL4 as indicated. E64 was added as indicated. Twenty-four hours after transfection, cells were treated with Dox for the indicated number of hours, when cell extracts were analyzed for Firefly and Renilla luciferase activity. Relative luciferase activity was the ratio of Firefly to Renilla luciferase, normalized to empty vector at time 0. *p < 0.05, n = 4.

(H) Model of VGLL4 regulation of YAP-TEAD1 activity. In the absence of VGLL4, YAP binds to TEAD1 to activate target gene expression. VGLL4 overexpression suppressed YAP-TEAD1 activity by both inhibiting TEAD1 transcriptional activity (i) and promoting TEAD1 degradation (ii).

All error bars represent the SEM.