Panel A: A flowchart depicts the experimental design involving MYPT1CA-NES-mNG cells treated with vehicle (Veh) or doxycycline (Dox) and subsequently treated with CHIR for 24 hours. Panel B: A schematic diagram shows the quantification pipeline for nuclear beta-catenin, involving staining with Hoechst and active non-phospho S45 beta-catenin, and measuring nuclear mean intensity. Panel C: A bar graph quantifies the mean intensity of active non-phospho S45 beta-catenin in MYPT1CA cells treated with Veh or Dox before and after 24 hours of CHIR treatment. The y-axis represents nuclear active beta-catenin mean intensity, and the x-axis shows different treatment conditions. Panel D: A bar graph displays the expression levels of TCF genes (transcripts per million, tpm) from parental WTC hiPSC, with the y-axis indicating gene expression and the x-axis listing different TCF genes. Panel E: Two bar graphs show the normalized binding enrichment of IgG and beta-catenin to WNT target genes AXIN2 and SP5 at 0 hours and 24 hours of CHIR treatment. The y-axis represents normalized binding enrichment, and the x-axis lists the genes and treatment times. Panel F: Two bar graphs illustrate the relative expression of WNT target genes LEF1 and AXIN2 with increasing concentrations of CHIR in the presence or absence of the ROCK inhibitor Y-27632. The y-axis shows normalized mRNA expression, and the x-axis indicates different treatment conditions.
Reduced cell contractility promotes nuclear accumulation of β-catenin (related to Fig. 6). (A) Experimental design. (B) Schematic representation of the quantification pipeline for nuclear β-catenin. (C) Quantification of active non–phospho-S45 β-catenin mean intensity in MYPT1CA cells as depicted in C. Veh and Dox were added 16 h prior to CHIR, and cells were fixed before (0 h) or after 24-h CHIR treatment supplemented with Veh or Dox. n = 10–12 technical repeats across N = 3 independent biological repeats. Mean and SD are displayed. One-way ANOVA with Šidák’s multiple comparisons posttest was performed. (D) Expression of TCF genes (tpm) from parental WTC hiPSC obtained from the Allen Institute transcriptomic data. N = 6 independent biological repeats. (E) Positive control for the CUT&RUN assay using IgG and β-catenin binding to WNT target genes (AXIN2 and SP5) between 0 and 24 h of CHIR treatment. Mean and SD are displayed. N = 3 independent biological repeats. One-way ANOVA with Dunnett’s multiple comparisons posttest was performed. (F) Relative expression of WNT target genes (LEF1 and AXIN2) with increasing concentrations of CHIR in the presence (magenta) or absence (cyan) of 10 μM of ROCK inhibitor Y-27632. N = 7 independent biological repeats. Mean and SEM are displayed. One-way ANOVA with Šidák’s multiple comparisons posttest was performed. Veh, vehicle; tpm, transcripts per million.