Reduced cell contractility promotes β-catenin nuclear activity and enhances binding to the mesodermal gene. (A) Representative immunofluorescence images for active non–phospho-S45 β-catenin (inverted LUT—confocal slice) and mNG (MaxIP). MYPT1^CA-NES-mNG hiPSCs were pretreated with Veh or Dox overnight and fixed at 0 and 24 h after CHIR treatment ± Dox as shown in Fig. S7 A. Scale bar = 50 µm. Insets represent a magnified view of the active non–phospho-S45 β-catenin with an example of the nuclei binary mask overlaid (pink). Scale bar = 10 µm. (B and C) Still confocal time-lapse imaging of mEGFP-β-catenin knock-in hiPSCs treated with CHIR ±10 μM Y-26732 (related to Video 3). Time is shown in hours. Scale bar = 10 μm (B). mEGFP-β-catenin intensity is reported as a scan line plot across a cell at 0 and 20 h (C). (D) Experiment design for GFP-Trap immunoprecipitation. mEGFP-β-catenin knock-in hiPSCs were pretreated with Veh or 10 μM Y-27632 and differentiated for 24 h. Nuclei were isolated, and nuclear lysates were mixed with GFP-Trap beads. NE buffer = Nuclei Extraction buffer. LSB = lysate sample buffer. (E and F) Representative GFP-Trap immunoprecipitation of mEGFP-β-catenin knock-in hiPSCs, probed for TCF7L2/TCF4 and α-tubulin as a loading control. M.W. are displayed on the right side (E). Quantification of TCF4 (Prey) binding to mEGFP-β-catenin (bait) was measured by densitometry across N = 3 independent biological repeats. Mean and SD are displayed. One-way ANOVA with Šidák’s multiple comparisons posttest was performed (F). (G) CUT&RUN assay for IgG or β-catenin binding to TBXT promoter in WT hiPSCs pretreated with Veh or 10 μM Y-27632 (0-h CHIR) or following 24 h of CHIR treatment in the presence of absence of ROCK inhibitor. N = 3 independent biological repeats. Mean and SD are displayed. One-way ANOVA with Šidák’s multiple comparisons posttest was performed. M.W., molecular weights; Veh, vehicle. *P < 0.05. Source data are available for this figure: SourceDataF6.