Panel A shows the experimental design for co-culture of hiPSCs transduced with lentivector expressing ROCK2CA or MLCKCA. hiPSCs are mixed with mVenus-negative WT hiPSCs, and ROCK2CA slash MLCKCA expression is induced by doxycycline. Panel B shows immunofluorescence images for Brachyury in vehicle and doxycycline-induced ROCK2CA co-culture, treated for 72 hours with CHIR. mVenus-positive cell clusters are highlighted by an orange dotted line. Individual channels are presented as inverted LUT. Panel C shows a violin plot quantifying Brachyury-positive cells, comparing mVenus-positive vs mVenus-negative clusters in the presence or absence of doxycycline. Panel D shows immunofluorescence images for EOMES in vehicle and doxycycline-induced ROCK2CA co-culture, treated for 72 hours with CHIR. mVenus-positive cell clusters are highlighted by an orange dotted line. Individual channels are presented as inverted LUT. Panel E shows a violin plot quantifying EOMES-positive cells, comparing mVenus-positive vs mVenus-negative clusters in the presence or absence of doxycycline. Panel F shows immunofluorescence images for Slug in vehicle and doxycycline-induced ROCK2CA co-culture, treated for 72 hours with CHIR. mVenus-positive cell clusters are highlighted by an orange dotted line. Individual channels are presented as inverted LUT. Panel G shows a violin plot quantifying Slug-positive cells, comparing mVenus-positive vs mVenus-negative clusters in the presence or absence of doxycycline. Panel H shows immunofluorescence images for ZO-1 in vehicle and doxycycline-induced ROCK2CA co-culture, treated for 72 hours with CHIR. mVenus-positive cell clusters are highlighted by an orange dotted line. Individual channels are presented as inverted LUT. Magnified mVenus-positive and mVenus-negative areas are shown as insets.
Increased actomyosin contractility is sufficient to block mesoderm commitment and EMT. (A) Experimental design for coculture experiment. hiPSCs were transduced with lentivector expressing ROCK2CA or MLCKCA. mVenus positive–transduced cells were mixed with mVenus-negative WT hiPSCs. ROCK2CA/MLCK CA expression is induced by the addition of Dox 24 h prior to initiating mesoderm differentiation. Dox induction was maintained during the 72 h of differentiation using CHIR before fixing and staining. Note that mVenus expression is constitutive and used as a marker for transduction, while the expression of ROCK2/MLCK is Dox-inducible. (B–H) Representative MaxIP immunofluorescence for Brachyury (B), EOMES (D), Slug (F), and ZO-1 (H) in Veh and Dox-induced ROCK2CA coculture, treated for 72 h with CHIR. mVenus-positive cell clusters are highlighted by an orange dotted line. Individual channels are presented as inverted LUT. Scale bar = 50 µm. For H, magnified mVenus-positive and mVenus-negative areas are shown as insets. Scale bar = 20 µm. Quantification of Brachyury (C), EOMES (E), and Slug-positive cells (G) is reported as a violin plot, comparing mVenus-positive vs mVenus-negative clusters in the presence or absence of Dox. Median (plain red line) and quartiles (dotted black lines) are displayed. For Brachyury, n = 7 (+ Veh) and n = 15 (+ Dox) across N = 3 independent biological repeats. For EOMES, n = 9 across N = 3 independent biological repeats. For Slug, n = 9 (+ Veh) and n = 10 (+ Dox) across N = 3 independent biological repeats. One-way ANOVA with Tukey’s multiple comparisons posttest was performed. Veh, vehicle. **** P < 0.0001, *** P < 0.001, ** P < 0.01.