Panel A shows two bright field images of colony morphology following EGTA pre-treatment. The treated colony appears less compact with distinguishable individual cell boundaries. Scale bar is 20 micrometers. Panel B displays representative immunofluorescence images of hiPSCs treated and stained for Brachyury. Scale bar is 50 micrometers. Panel C is a bar graph quantifying Brachyury-positive nuclei following EGTA treatment, with mean and standard deviation displayed. Panel D shows representative immunofluorescence images of hiPSCs treated and stained for ppMLC2. Scale bar is 50 micrometers. Panel E is a bar graph quantifying the ppMLC2-positive area following EGTA treatment, with mean and standard deviation displayed. Panel F shows a representative immunoblot of control or CTNNA1 knockdown hiPSCs, probed for alpha-catenin, Nanog, Oct-4, and alpha-Tubulin as a loading control. Molecular weights are displayed on the right side. Panels G, H, and I are bar graphs quantifying alpha-catenin, Nanog, and Oct-4 expression obtained by densitometry, with mean and standard deviation displayed.
EGTA treatment and validation CTNNA1 KD hiPSCs (related to Fig. 4). (A) Bright-field picture showing the colony morphology following EGTA pretreatment. Colony appears less compact, and individual cell boundaries can be distinguished. Scale bar = 20 μm. (B and C) Representative immunofluorescence of hiPSCs treated as shown in Fig. 4 A and stained for Brachyury. Scale bar = 50 μm (B). Quantification of Brachyury-positive nuclei is shown following EGTA treatment. Mean and SD are displayed. n = 18 (Veh) and n = 20 fields of view across N = 3 independent biological repeats. A two-tailed unpaired t test was performed on biological repeats (C). (D and E) Representative immunofluorescence of hiPSC treated as shown in Fig. 4 A and stained for ppMLC2. Scale bar = 50 μm (D). Quantification of ppMLC2-positive area is shown following EGTA treatment. Mean and SD are displayed. n = 20 (Veh) and n = 25 fields of view across N = 4 independent biological repeats. A two-tailed unpaired t test was performed on biological repeats (E). (F–I) Representative immunoblot of control (Ctr) or CTNNA1 KD hiPSCs, probed for α-catenin, Nanog and Oct-4 (pluripotency), and α-tubulin as loading control. M.W. are displayed on the right side (F). Quantification of α-catenin (G), Nanog (H), and Oct-4 (I) expression was obtained by densitometry across N = 5–6 (α-catenin), N = 4 (Nanog), and N = 3 (Oct-4) independent biological repeats. Mean and SD are displayed. One-way ANOVA with Dunnett’s multiple comparisons posttest was performed. M.W., molecular weights; Veh, vehicle. ***P < 0.001, **P < 0.01, *P < 0.05. Source data are available for this figure: SourceDataFS5.