Journal: Nucleic Acids Research

Article Title: Regulation of YAP activity by nuclear G-actin binding

doi: 10.1093/nar/gkag248

Figure Lengend Snippet: Nuclear actin-YAP binding plays a pivotal role in cell proliferation. ( A–B ) AlphaFold 3 prediction model of TEAD4 (magenta) interaction with (A) YAP (cyan) or (B) YAP (cyan) and G-actin (green) in the presence of M-CAT DNA. The black box indicated the predicted four-chain α-helical interface (4α) interacts with TEAD4 DNA binding domain (DBD). ( C ) Immunoblot analysis of Lysate and IP fractions from GFP-trap co-immunoprecipitation experiment in HeLa wt cells or HeLa cells stably expressing FLAG-3X NLS-EGFP-YAP WT and transiently transfected with FLAG-NLS-actin and myc-TEAD4. ( D ) ChIP-qPCR analysis of YAP–TEAD4 recruitment to target genes in HeLa cells transfected with NLS-EGFP (pink, triangle), NLS-EGFP-YAP WT (blue, circle) and NLS-EGFP-YAP DDY (green, square). ZFP37 served as a negative control. Data are from three independent experiments. Statistical analysis was done using a two-tailed unpaired Student’s t-test ( P < 0.05, P < 0.01). ( E ) Quantitative PCR (qPCR) analysis of YAP target gene expression in HeLa cells transfected with NLS-BFP (Mock) or NLS-actin in the absence or presence of myc-YAP WT or myc-YAP DDY . Data are normalized to the NLS-BFP Mock control mRNA transcripts. Each dot represents an independent experiment. Statistical analysis was done using One-way ANOVA with Tukey´s multiple comparison test ( P < 0.05, P < 0.01, P < 0.0001). ( F ) Dose response curves of purified YAP WT (blue) and YAP DDY (green) over TEAD4-coated CM5 chip, indicating the dissociation constant (K d ). ( G ) Representative images of Click-iT® Plus EdU assay of HeLa cells transfected with empty plasmid (Mock), myc-YAP WT and myc -YAP DDY and stained for anti-myc (green), DAPI (blue) and EdU (magenta). Images are shown as MIPs, Scale bar = 20 μm. ( H ) Quantification of the ratio of EdU + cells. Data are shown as a violin plot with median, quartiles and individual data points from three independent biological replicates (green, magenta, cyan). Each dot represents the percentage of EdU + cells per field of view. Statistical analysis was done using One-way ANOVA with Tukey´s multiple comparison test ( P < 0.0001). n = 26, 24, and 24.

Article Snippet: The following primary were used: mouse monoclonal anti-FLAG® M2 antibody (1:1000, Merck, F1804), YAP (D8H1X) XP® Rabbit mAb (1:500, Cell Signaling Technology, 14 074), rabbit polyclonal anti-Myc Tag (1:1000, Cell Signaling Technology, 2272), mCherry (E5D8F) Rabbit mAb (1:1000, Cell Signaling Technology, 43 590), anti-Tubulin (11H10) Rabbit mAb (1:1000, Cell Signaling Technology, 2125), mouse monoclonal anti-Actin antibody (Merck, A4700), eGFP mouse monoclonal antibody (F56-6A1.2.3) (1:1000, Invitrogen, MA1-952), anti-GAPDH mouse mAb (6C5) (1:2000, Millipore, CB1001).

Techniques: Binding Assay, Western Blot, Immunoprecipitation, Stable Transfection, Expressing, Transfection, ChIP-qPCR, Negative Control, Two Tailed Test, Real-time Polymerase Chain Reaction, Targeted Gene Expression, Control, Comparison, Purification, EdU Assay, Plasmid Preparation, Staining