Fig 2: RUNX2 rescues Vgll4 deletion–mediated inhibition of osteogenesis differentiation.(A) Coimmunoprecipitation experiments of RUNX2, TEAD1, VGLL4, and VGLL4 HF4A in HEK-293T cells. The arrow indicated IgG heavy chain. (B) 6xOSE2-luciferase activity was determined in C3H10T1/2 cells cotransfected with RUNX2, VGLL4, VGLL4 HF4A, and TEAD1 (n = 3). (C) RT-PCR analysis of Alp expression in calvarial cells. Cells isolated from WT mice were infected with GFP, TEAD1, VGLL4, or VGLL4 HF4A lentivirus. (D) Coimmunoprecipitation experiments of RUNX2, TEAD1, and an increasing amount of VGLL4 or VGLL4 HF4A in HEK-293T cells. The arrow indicated IgG heavy chain. (E) 6xOSE2-luciferase activity was determined in C3H10T1/2 cells cotransfected with RUNX2, TEAD1, and an increasing amount of VGLL4 or VGLL4 HF4A. (F) Competitive GST pull-down assay to detect the effect of VGLL4 Super-TDU on the interaction between RUNX2 and TEAD4. (G) Cells isolated from Vgll4fl/fl and Vgll4prx1 mice were infected with GFP and RUNX2 lentivirus. Osteoblast differentiation was evaluated by Alp staining and Alizarin red staining after culture in osteoblast differentiation medium for 7 days (top) and 14 days (bottom). Data are representative of three independent experiments. Scale bars, 3 mm. (H) Alp activity quantification was measured by phosphatase substrate assay (n = 3). (I) Relative mRNA levels of Vgll4, Runx2, Alp, Col1a1, and Osterix were quantified by RT-PCR. (J) Schematic model of VGLL4/TEADs/RUNX2 in regulating osteogenic differentiation. In (B), (C), (E), (H), and (I), data were presented as means ± SEM; *P < 0.05, **P < 0.01, and ***P < 0.001; ns, no significance; unpaired Student’s t test.

Fig 3: SKP2 and K63-linked ubiquitination enhance the YAP-TEAD interaction. a HEK293A cells were stably transfected with SFB-YAP and then subjected to a pulldown assay with S-protein beads and immunoblotting with antibodies against YAP-interacting proteins. H: high density; L: low density. b The HEK293A SFB-YAP stable cell line was transfected with MYC-SKP2 and then subjected to a pulldown assay with S-protein beads and immunoblotting with antibodies against YAP-interacting proteins. c The HEK293A SFB-YAP stable cell line was transfected with MYC-SKP2. SFB-YAP was pulled down with S-protein beads and then incubated with His-TEAD1 protein purified from bacteria. After in vitro binding, the proteins bound to S-protein beads were eluted by boiling in Laemmli sample buffer and immunoblotted with antibodies against His and FLAG tags. d HEK293T cells were transfected with SFB-YAP (WT or the 2KR mutant). SFB-YAP was pulled down with S-protein beads and then incubated with His-TEAD1 protein purified from bacteria. After in vitro binding, the proteins bound to S-protein beads were eluted by boiling in Laemmli sample buffer and immunoblotted with antibodies against His and FLAG tags. e SKP2 forms a complex with YAP and TEAD1. HA-TEAD1 and MYC-SKP2 were co-transfected with or without SFB-YAP into HEK293T cells. SFB-YAP was pulled down with streptavidin beads and then eluted with biotin buffer. The eluded proteins were subjected to immunoprecipitation with a MYC-specific antibody to pull down MYC-SKP2 and immunoblotting with antibodies against FLAG, HA, and MYC

Fig 4: YAP and TAZ are complexed to the Z immediate-early promoter.HONE-Akata cells were transfected with either A) a vector control or co-transfected with a FLAG-tagged YAP(5SA) expression vector and TEAD1 vector, or B) a vector control or a FLAG-tagged TAZ(S89A) expression vector and TEAD1 vector. ChIP assays were performed 24 hours later using anti-FLAG antibody as described in the materials and methods. Binding of FLAG-tagged YAP(5SA) and TAZ(S89A) to various parts of the EBV genome, including the lytic Z, R and BMRF1 promoters and the latent Cp promoter was determined by qPCR. C) HONE-Akata cells were transfected with a vector control or FLAG-tagged YAP(5SA) expression vector and myc-tagged TEAD1 vector. ChIP assays were subsequently done 24 hours post transfection with a myc antibody as described in the materials and methods. Binding of myc-TEAD1 protein to the Z promoter, or negative control (NC) sites on the EBV genome was determined by qPCR. All experiments are representative of two independent biological replicates, and statistical significance was determined by the two-sample t-test.

Fig 5: Pancreatic cancer cells selected for extracellular protein-dependent growth exhibit increased macropinocytosis and a unique gene expression program. (A) Schematic representation of experimental strategy to measure global changes in gene expression and chromatin accessibility after short-term (16 h) or long-term (3 wk) culture of PDA cells in complete (+Leu) or leucine-free (-Leu) medium with or without supplementation of 3% bovine serum albumin (BSA). (B) Uptake of fluorescent dextran (left panel) and albumin (right panel) by parental KRPC and KRPCS cells in complete medium as measured by fluorescence confocal microscopy. Bars represent mean ±SEM fluorescence intensity per cell, across 12 fields of view. (***) P < 0.0001, unpaired two-tailed t-test. (C) Venn diagram depicting total number of unique and shared called peaks from ATAC-seq of KRPC cells cultured for 3 wk in ±Leu + 3% BSA. (D) Box plot of mean log2 RPKM expression values of genes within 5 kb of unique ATAC-seq peaks identified in KRPCS cells. (***) P < 0.0001, one-way ANOVA. (E) Top de novo transcription factor motifs enriched within ATAC-seq peaks identified in KPRCS cells. (F) Average ATAC-seq read coverage across genomic regions bound by Tead1 ±5 kb in KRPC cells cultured for 3 wk in ±Leu + 3% BSA. (G) Tracks showing ATAC-seq and RNA-seq read coverage as well as called Tead1 ChIP-seq peaks at the Ctgf locus.