Increased actomyosin contractility is sufficient to block mesoderm commitment and EMT (related to Fig. 1). (A) Experimental design. hiPSCs were pretreated with 4 µg/ml CN03 or Veh for 3 h. Differentiation was initiated with CHIR supplemented with Veh or 4 µg/ml CN03 for 24 h before fixation and staining. (B) Representative MaxIP immunofluorescence of hiPSCs treated as explained in A and stained for phospho-MLC2 (inverted LUT). Scale bar = 50 µm. (C and D) Representative MaxIP immunofluorescence of hiPSCs treated as explained in A and stained for Brachyury (inverted LUT), nuclei, and ZO-1. Scale bar = 50 µm (C). Violin plot (median, quartiles) reporting the percentage of Brachyury-positive nuclei after 24 h of differentiation and quantified across N = 4 independent biological repeats (representing n = 20 technical repeats). An unpaired t test was performed on biological replicates (D). (E) Representative MaxIP immunofluorescence image of Dox-induced ROCK2^CA hiPSCs (mVenus-positive) mixed with WT hiPSCs (mVenus-negative). Cells were stained for DNA (nuclei), F-actin (top—inverted LUT), and ppMLC2 (bottom—inverted LUT). Scale bar = 50 µm. Magnified views comparing mVenus-positive (green dotted ROI) and mVenus-negative (black dotted ROI) areas are shown for F-actin and ppMLC2. Scale bar = 10 µm. (F) Percentage of the ROI area positive for mVenus based on cluster considered as mVenus-negative and mVenus-positive using the ROCK2^CA cell line. On average, mVenus-negative clusters have <15% of their area positive for mVenus. Mean and SEM are displayed. n = 7 (no Dox) and n = 15 fields of view (+Dox) across N = 3 independent biological experiments. (G) Representative MaxIP immunofluorescence of Dox-induced MLCK^CA hiPSCs (mVenus-positive) mixed with WT hiPSCs (mVenus-negative). Cells were stained for DNA (nuclei), F-actin (top—inverted LUT), and ppMLC2 (bottom—inverted LUT). Scale bar = 50 µm. Magnified views comparing mVenus-positive (green dotted ROI) and mVenus-negative (black dotted ROI) areas are shown for F-actin and ppMLC2. Scale bar = 10 µm. (H) Percentage of the ROI area positive for mVenus based on clusters considered as mVenus-negative and mVenus-positive using the MLCK^CA cell line. On average, mVenus-negative clusters have <15% of their area positive for mVenus. Mean and SEM are displayed. n = 7 (no Dox) and n = 18 fields of view (+Dox) across N = 3 independent biological experiments. (I and J) Representative MaxIP immunofluorescence for Brachyury in +Veh and Dox-induced MLCK^CA coculture. mVenus-positive cell clusters are highlighted by an orange dotted line. Individual channels are inverted LUT. Scale bar = 50 µm (I). Percentage of Brachyury-positive nuclei is reported as a violin plot, comparing mVenus-positive vs mVenus-negative cluster in the presence or absence of Dox. Median (plain red line) and quartiles (dotted black lines) are displayed. n = 7 (+Veh) and n = 18 (+ Dox) fields of view (small dots) across N = 3 independent biological repeats (large dots). One-way ANOVA with Tukey’s multiple comparisons posttest was performed (J). (K) Representative MaxIP immunofluorescence for EOMES, Slug, and ZO-1 in Dox-induced MLCK^CA coculture. mVenus-positive cell clusters are highlighted by an orange dotted line. Individual channels are inverted LUT. Scale bar = 50 µm. Veh, vehicle. ****P < 0.0001, **P < 0.01.