Figure S6.
Contractility affects β-catenin localization at AJs (related to Fig. 5). (A) Immunoblot assessing the effects of CN03 treatment at 4 and 16 h. Membrane was probed for total and phospho-MLC2 (MLC2 and ppMLC2, respectively), Nanog and Oct-4, and α-tubulin as a loading control. M.W. are displayed on the right side. (B) Independent technical repeats from Fig. 5, A and B. Repeats across the 3 biological repeats were averaged and shown as a dot with error bars (SD). (C) Still picture from overnight confocal imaging of mEGFP-β-catenin knock-in hiPSCs treated with Veh, 4 μg/ml CN03, and 10 μM Y-27632 at the basal state. Pixels are color-coded by intensity using the Red Fire LUT. Scale bar = 20 µm (related to Video 2). (D) Experimental design. Cells were pretreated overnight with Veh or 10 μM Y-27632 or 4 μg/ml CN03 in mTeSR1 and differentiated using CHIR media complemented with Veh or 10 μM Y-27632 or 4 μg/ml CN03 for 24 h before collecting protein lysates. (E and F) Representative immunoblot for total β-catenin. The membrane was also probed for Brachyury to show effect of each drug and α-tubulin as a loading control. M.W. are displayed on the right side (E). β-Catenin expression was quantified across N = 6 (Veh and Y-27632) and N = 4 (CN03) independent biological repeats. Mean and SD are displayed. One-way ANOVA with Šidák’s multiple comparisons posttest was performed (F). (G–K) FRAP experiment was performed on EGFP-β-catenin knock-in hiPSCs pretreated with Veh or 10 μM Y-27632 for 16 h in mTeSR1 (0-h CHIR) or pretreated and differentiated with CHIR supplemented with Veh or 10 μM Y-27632 for 24 h (24-h CHIR). Representative still images at 0 h (G) and 24 h after CHIR (I), before and after bleaching. FRAP area (ROI) is marked as a dotted circle. Scale bar = 2 μm. Fluorescence within the ROI was recorded prior and following bleaching at 0 h (H) and 24 h (J) after CHIR. Half-recovery time was calculated and reported as column graphs. Mean and SD are displayed. A Mann–Whitney test was performed (K). n = 19–25 cells across N = 2 independent biological repeats. M.W., molecular weights; Veh, vehicle. Source data are available for this figure: SourceDataFS6. Refer to the image caption for details. Panel A shows an immunoblot, lanes represent treatments and time points, bands show phosphorylated myosin light chain 2, total myosin light chain 2, Nanog, Oct4 and tubulin levels. Panel B shows scatter plot, x-axis shows phosphorylated myosin light chain 2 percentage area and y-axis shows junctional beta catenin signal. Panel C shows fluorescence microscopy images of enhanced green fluorescent protein beta catenin localization under different treatments. Panel D shows schematic diagram of experimental design for pre-treatment and differentiation conditions. Panel E shows an immunoblot of total beta catenin and Brachyury under treatment conditions. Panel F shows bar graph, x-axis shows treatment conditions and time points and y-axis shows normalized beta catenin levels. Panel G shows fluorescence recovery after photobleaching images at zero hours CHIR, regions of interest marked. Panel H shows line graph, x-axis shows time in seconds and y-axis shows fluorescence recovery percentage. Panel I shows fluorescence recovery after photobleaching images at 24 hours CHIR. Panel J shows line graph, x-axis shows time in seconds and y-axis shows fluorescence recovery percentage. Panel K shows bar graph, x-axis shows treatment conditions and y-axis shows half recovery time percentage.

Contractility affects β-catenin localization at AJs (related to Fig. 5). (A) Immunoblot assessing the effects of CN03 treatment at 4 and 16 h. Membrane was probed for total and phospho-MLC2 (MLC2 and ppMLC2, respectively), Nanog and Oct-4, and α-tubulin as a loading control. M.W. are displayed on the right side. (B) Independent technical repeats from Fig. 5, A and B. Repeats across the 3 biological repeats were averaged and shown as a dot with error bars (SD). (C) Still picture from overnight confocal imaging of mEGFP-β-catenin knock-in hiPSCs treated with Veh, 4 μg/ml CN03, and 10 μM Y-27632 at the basal state. Pixels are color-coded by intensity using the Red Fire LUT. Scale bar = 20 µm (related to Video 2). (D) Experimental design. Cells were pretreated overnight with Veh or 10 μM Y-27632 or 4 μg/ml CN03 in mTeSR1 and differentiated using CHIR media complemented with Veh or 10 μM Y-27632 or 4 μg/ml CN03 for 24 h before collecting protein lysates. (E and F) Representative immunoblot for total β-catenin. The membrane was also probed for Brachyury to show effect of each drug and α-tubulin as a loading control. M.W. are displayed on the right side (E). β-Catenin expression was quantified across N = 6 (Veh and Y-27632) and N = 4 (CN03) independent biological repeats. Mean and SD are displayed. One-way ANOVA with Šidák’s multiple comparisons posttest was performed (F). (G–K) FRAP experiment was performed on EGFP-β-catenin knock-in hiPSCs pretreated with Veh or 10 μM Y-27632 for 16 h in mTeSR1 (0-h CHIR) or pretreated and differentiated with CHIR supplemented with Veh or 10 μM Y-27632 for 24 h (24-h CHIR). Representative still images at 0 h (G) and 24 h after CHIR (I), before and after bleaching. FRAP area (ROI) is marked as a dotted circle. Scale bar = 2 μm. Fluorescence within the ROI was recorded prior and following bleaching at 0 h (H) and 24 h (J) after CHIR. Half-recovery time was calculated and reported as column graphs. Mean and SD are displayed. A Mann–Whitney test was performed (K). n = 19–25 cells across N = 2 independent biological repeats. M.W., molecular weights; Veh, vehicle. Source data are available for this figure: SourceDataFS6.

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