Panel A shows immunofluorescent images of hiPSCs treated with a ROCK inhibitor (Y-27632) or vehicle, stained for nuclei and ppMLC2. Magnified views highlight ppMLC2. Panel B presents a violin plot of the cellular area positive for ppMLC2, comparing Y-27632 treatment to vehicle. Panel C displays bar graphs of relative mRNA expression for unrelated genes at 0h and 48h post CHIR treatment with and without Y-27632. Panel D shows immunofluorescent images of hiPSCs treated with CHIR and Y-27632 or vehicle, stained for nuclei, ZO-1, and Slug. Panel E presents immunofluorescent images of hiPSCs treated with another ROCK inhibitor (H-1152) or vehicle, stained for nuclei and ppMLC2, with magnified views. Panel F shows a violin plot of the cellular area positive for ppMLC2, comparing H-1152 treatment to vehicle. Panels G to K display bar graphs of relative expression of mesoderm and EMT markers at 48h post CHIR treatment with and without H-1152. Panel L shows an immunoblot of mesoderm and cell death markers during hiPSC-to-mesoderm commitment with H-1152 and Q-VD-OPH. Panel M presents immunofluorescent images for Brachyury and nuclei at 24h post-CHIR treatment with and without H-1152. Panel N shows an immunoblot of cell death, mesoderm, and pluripotency markers using hESC H9 with and without Y-27632. Panels O to Q present bar graphs quantifying Brachyury expression, PARP cleavage, and Nanog expression normalized to Tubulin. Panel R shows an immunoblot of T slash Bra expression following treatment with BMP4 and bFGF with and without Y-27632.
Pharmaceutical inhibition of actomyosin contractility promotes hiPSC and hESC conversion to the mesoderm lineage (related to Fig. 3). (A and B) Representative MaxIP immunofluorescences of hiPSCs treated with a ROCK inhibitor (10 μM Y-27632) or Veh (H2O) and stained for nucleus marker (DNA) and ppMLC2 (inverted LUT). Scale bar = 50 µm. Magnified views of the yellow dotted ROI are shown for ppMLC2. Scale bar = 20 µm (A). Fraction of the cellular area positive for ppMLC2 following Y-27632 treatment and reported as violin plots. Median (plain red line) and quartiles (dotted black lines) are displayed. n = 30 technical repeats across N = 3 independent biological repeats. A two-tailed unpaired t test was performed on the biological repeat (B). (C) Relative expression of genes not directly related to mesoderm commitment at 0 and 48 h after CHIR treatment in the presence (+Y-27) or absence (+Veh) of 10 μM of ROCK inhibitor. N = 4 independent biological repeats. Mean and SD are displayed. A two-tailed unpaired t test was performed. (D) Representative MaxIP immunofluorescences of hiPSCs treated 48 h with CHIR in the presence (+ Y-27632) or absence (+ Veh) of 10 μM of ROCK inhibitor. Cells were stained for nuclear marker (DNA), EMT markers (ZO-1 and Slug—inverted LUT). Scale bar = 50 µm. (E and F) Representative MaxIP immunofluorescences of hiPSCs treated with a ROCK inhibitor (1 μM H-1152) or Veh (H2O) and stained for nuclear marker (DNA) and ppMLC2 (inverted LUT). Scale bar = 50 µm. Magnified views of the yellow dotted ROI are shown for ppMLC2. Scale bar = 20 µm (E). Fraction of the cellular area positive for ppMLC2 following H-1152 treatment and reported as violin plots. Median (plain red line) and quartiles (dotted black lines) are displayed. n = 15 technical repeats across N = 3 independent biological repeats. A two-tailed unpaired t test was performed on the biological repeats (F). (G–K) Relative expression of mesoderm markers (TBXT, MESP1, TBX6) (G–I) and EMT markers (SNAI1, SNAI2) (J and K) 48 h after CHIR treatment in the presence (+H-1152) or absence (+Veh) of 1 μM of ROCK inhibitor. N = 3 independent biological repeats. Mean and SD are displayed. A two-tailed unpaired t test was performed. (L) Immunoblot probing for mesoderm marker expression (T/Bra) and cell death marker (PARP cleavage) during hiPSC-to-mesoderm commitment (0- to 48-h CHIR) in the presence of 1 μM of ROCK inhibitor (+H-1152). The addition of the pan-caspase inhibitor Q-VD-OPH (+ Q-VD) to the differentiation medium totally blocks mesoderm commitment and was used as a control. α-Tubulin was used as a loading control. M.W. are displayed on the right side. (M) Representative MaxIP immunofluorescences for mesoderm marker Brachyury (T/Bra—inverted LUT) and nuclei. Cells were fixed 24 h after CHIR treatment in the presence or absence of ROCK inhibitor H-1152. Scale bar = 50 µm. (N) Representative immunoblot of PARP (cell death), T/Bra (mesoderm), and Oct-4 and Nanog (pluripotency markers) using hESC H9 in the presence (+ Y-27632) or absence (+ Veh) of 10 μM of ROCK inhibitor, as shown in Fig. 3 A. M.W. are displayed on the right side. (O–Q) Brachyury expression (O), PARP cleavage (P), and Nanog expression (Q) were quantified by densitometry and normalized to α-tubulin as a loading control across N = 4 (PARP) and N = 3 (Brachyury and Nanog) independent biological repeats. Mean and SD are displayed. A two-tailed unpaired t test was performed. (R) Expression of T/Bra was analyzed by western blot following treatment with a combination of 50 ng/ml BMP4 and 100 ng/ml bFGF, in the presence or absence of 10 μM Y-27632. pERK and total ERK were used as a positive control for FGF pathway activity. M.W. are shown on the right-hand side. M.W., molecular weights; Veh, vehicle. ***P < 0.001, **P < 0.01, *P < 0.05. Source data are available for this figure: SourceDataFS2.