Panel A: The experimental design diagram shows the treatment of wild-type hiPSCs with either a vehicle, Rho activator CN03, or ROCK inhibitor Y-27632 for 3 hours, followed by fixation, staining, and imaging. Panel B: Immunofluorescence images display hiPSCs treated with vehicle, CN03, or Y-27632, stained for phosphorylated myosin light chain 2 (ppMLC2) and beta-catenin. The scale bar is 50 micrometers. Panel C: A scatter plot with a linear regression line shows the relationship between junctional beta-catenin and cellular ppMLC2 across three independent biological repeats. The R-squared value is 0.9592. Panel D: Another experimental design diagram illustrates the treatment of EGFP-beta-catenin knock-in hiPSCs with vehicle, CN03, or Y-27632 for 3 hours, followed by imaging. Panel E: Representative still images from EGFP-beta-catenin knock-in hiPSCs treated with vehicle, CN03, or Y-27632. The scale bar is 50 micrometers. Panel F: A violin plot quantifies junctional beta-catenin for different treatments across four independent biological repeats, showing median and quartiles. Statistical analysis using one-way ANOVA with Dunnett's multiple comparison post-test was performed.
Contractility differentially regulates β-catenin localization at AJs. (A) Experimental design. (B) Representative immunofluorescence of fixed hiPSCs treated with Veh, 4 µg/ml CN03, or 10 µM Y-27632 for 3 h and stained for ppMLC2 and β-catenin (inverted LUT), as shown in A. Scale bar = 50 µm. (C) Linear regression analysis of junctional β-catenin and cellular ppMLC2 across N = 3 independent biological repeats (representing n = 16 for Veh, n = 19 for CN03, and n = 17 technical repeats for Y-27632). R2 value is reported. (D) Experimental design. (E and F) Representative still pictures from mEGFP-β-catenin KI hiPSCs treated with Veh, 4 µg/ml CN03, or 10 µM Y-27632 for 3 h, as shown in D. Scale bar = 50 µm (E). Quantification of junctional β-catenin is reported for the different treatments across N = 4 independent biological repeats (representing n = 25 technical repeats). Violin plots representing median and quartiles. One-way ANOVA with Dunnett’s multiple comparisons posttest was performed on biological repeats (F). KI, knock-in; Veh, vehicle. **P < 0.01, *P < 0.05.