Panel A shows the experimental design diagram. Panel B displays a series of immunoblots for various markers (Brachyury, Snail, Slug, Pax6, Otx1 slash 2) with treatments indicated (Veh, EGTA) and time points (0h, 24h, 48h). Panel C, D, and E are bar graphs showing the quantification of Brachyury, Snail, and Slug expression normalized to alpha-Tubulin, respectively, with error bars representing standard deviation. Panel F shows an immunoblot for phospho-MLC2 and MLC2 in control and CTNNA1 knockdown lines. Panel G is a bar graph showing the normalized phospho slash total MLC2 ratio with error bars. Panel H displays immunoblots for alpha-catenin, Brachyury, and alpha-Tubulin in control and CTNNA1 knockdown lines treated with Veh or Y-27. Panel I is a bar graph showing normalized Brachyury expression with error bars.
AJ disengagement promotes mesoderm fate. (A) Experimental design. hiPSCs were pretreated with 1 μM EGTA to disrupt calcium-dependent E-cadherin junctions. Following pretreatment, hiPSCs were differentiated in the presence of a lower concentration of EGTA. (B–E) Representative immunoblot of hiPSCs treated as shown in A and probed for Brachyury (mesoderm marker), Snail and Slug (EMT markers), and Pax6 and Otx1/2 (neuroectoderm markers as negative controls). The NSC lysate was used as a positive control for Pax6 and Otx1/2. The nonspecific band is designated by an asterisk. M.W. are displayed on the right side (B). The expression of Brachyury (C), Snail (D), and Slug (E) was quantified by densitometry and normalized to α-tubulin across N = 3 independent biological repeats. Mean and SD are displayed. One-way ANOVA with Šidák’s multiple comparisons posttest was performed. (F and G) Representative immunoblot of control (Ctr) and CTNNA1 KD hiPSC lines at the basal state and probed for total and phospho-MLC2. M.W. are displayed on the right side (F). Quantification of active MLC2 (ppMLC2/MLC2) was measured by densitometry across N = 3 independent biological repeats. Mean and SD are displayed. One-way ANOVA with Dunnett’s multiple comparisons posttest was performed (G). (H and I) Representative immunoblot of control (Ctr) and CTNNA1 KD #1 and #2 hiPSC lines, differentiated for up to 48 h in the presence or absence of 10 μM of ROCK inhibitor Y-27632 and probed for α-catenin, Brachyury, and α-tubulin as a loading control. M.W. are displayed on the right side (H). Normalized Brachyury expression 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 (I). M.W., molecular weights; NSC, neural stem cell. ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05. Source data are available for this figure: SourceDataF4.