Figure 3.
Pharmaceutical inhibition of actomyosin contractility promotes hiPSC and hESC conversion to the mesoderm lineage. (A) Experimental design. WT hiPSCs or hESCs were pretreated overnight with small molecules to lower contractility, followed by differentiation. (B) Immunoblot of Brachyury during hiPSC-to-mesoderm commitment (0–48 h) ±10 μM ROCK inhibitor Y-27632, as shown in A. M.W. are displayed on the right side. (C) Brachyury expression was quantified by densitometry and normalized to α-tubulin as a loading control across N = 3 independent biological repeats. Mean and SD are displayed. A two-tailed unpaired t test was performed. (D–G) Relative expression of mesoderm markers (MESP1, TBX6) (D and E) and EMT markers (SNAI1, SNAI2) (F and G) 48 h after CHIR treatment ±10 μM ROCK inhibitor as shown in A. N = 3 independent biological repeats except for MESP1, N = 4 independent biological repeats. Mean and SD are displayed. A two-tailed unpaired t test was performed. (H) Representative MaxIP immunofluorescences during hiPSC-to-mesoderm commitment (0–48 h) ±10 μM ROCK inhibitor as shown in A. Cells were stained with a nuclear marker (DNA), EMT marker (ZO-1—inverted LUT), and mesoderm marker (T/Bra—inverted LUT). Scale bar = 50 µm. (I and J) Quantification of Brachyury expression (I) and ZO-1 intactness (J) is represented as violin plots with individual measurements (small dots) averaged for each biological repeat (large dots). Median (plain red line) and quartiles (dotted black lines) are displayed. n = 10–15 technical repeats across N = 3 independent biological repeats. One-way ANOVA with Tukey’s multiple comparisons posttest was performed on biological repeats. (K and L) Analysis of Brachyury expression by flow cytometry following 24-h CHIR treatment ±10 μM ROCK inhibitor, as shown in A, using Bra-mNG knock-in H9 hESC line (K). Quantification of the percentage of mNG-positive cells from N = 4 independent biological repeats. Mean and SD are displayed. A two-tailed unpaired t test was performed (L). (M and N) Representative MaxIP immunofluorescence of hiPSCs treated with a MLCK inhibitor (3 μM ML-7) or Veh (DMSO), as shown in A. Cells were stained for nuclei (DNA) and ppMLC2 (inverted LUT). Scale bar = 50 µm. Magnified views of the yellow dotted ROI are shown for ppMLC2. Scale bar = 20 µm (M). Percentage of cellular area positive for ppMLC2 following Veh or ML-7 treatment is 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 (N). (O and P) Immunoblot of Brachyury (mesoderm) and PARP (cell death marker) using hiPSCs in the presence (+ ML-7) or absence (+ Veh) of 3 μM of MLCK inhibitor at the basal state (0 h) and during CHIR treatment (24, 48 h), as shown in A. Cells treated with CHIR and Q-VD (caspase inhibitor) for 48 h were used as a control for PARP immunoblot. M.W. are displayed on the right side (O). Brachyury expression was quantified by densitometry and normalized to α-tubulin as a loading control across N = 3 independent biological repeats. Mean and SD are displayed. The Mann–Whitney test was performed for the 24-h time points, and a two-tailed unpaired t test was performed for the 48-h time points (P). (Q) Experimental design. Effects of Y-27632 were tested at different time points along the mesoderm commitment. 10 μM Y-27632 was added 12 h before differentiation (−12 h), at the time of differentiation (0 h), or 12 h after differentiation (+12 h). All conditions were collected 24 h after differentiation. (R and S) Representative immunoblot for Brachyury following the addition of Veh or 10 μM Y-27632 as shown in Q. M.W. are displayed on the right side (R). Brachyury expression was quantified by densitometry and normalized to α-tubulin as a loading control across N = 3 independent biological repeats. Mean and SD are displayed. Two-way ANOVA with Šidák’s multiple comparisons posttest was performed (S). M.W., molecular weights; Veh, vehicle. ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05. Source data are available for this figure: SourceDataF3. Refer to the image caption for details. Panel A: A schematic diagram shows the experimental design where hiPSCs or hESCs are pre-treated overnight with small molecules to lower contractility, followed by differentiation. Panel B: An immunoblot displays Brachyury expression during hiPSC-to-mesoderm commitment at 0, 24, and 48 hours with and without the ROCK inhibitor Y-27632. Panel C: A bar graph quantifies Brachyury expression normalized to alpha-Tubulin, showing mean and standard deviation across three independent biological repeats. Panel D to G: Bar graphs show relative expression of mesoderm markers (MESP1, TBX6) and EMT markers (SNAI1, SNAI2) 48 hours post CHIR treatment with and without Y-27632. Panel H: Representative MaxIP immunofluorescences show cells stained with nuclear, EMT, and mesoderm markers at 24 and 48 hours with and without Y-27632. Panel I to J: Violin plots quantify Brachyury expression and ZO-1 intactness, with individual measurements and median values. Panel K to L: Flow cytometry analysis and quantification of Brachyury expression in Bra-mNeonGreen knock-in H9 hESC line after 24 hours of CHIR treatment with and without Y-27632. Panel M to N: MaxIP immunofluorescence images and violin plots show hiPSCs treated with MLCK inhibitor (ML-7) or vehicle, stained for nuclei and phospho T18 slash S19 Myosin Light Chain 2. Panel O to P: Immunoblot and bar graph show Brachyury and PARP expression in hiPSCs with and without ML-7 during CHIR treatment. Panel Q: A schematic diagram shows the experimental design testing the effects of Y-27632 at different times along mesoderm commitment. Panel R to S: Immunoblot and bar graph show Brachyury expression following addition of vehicle or Y-27632 at different times.

Pharmaceutical inhibition of actomyosin contractility promotes hiPSC and hESC conversion to the mesoderm lineage. (A) Experimental design. WT hiPSCs or hESCs were pretreated overnight with small molecules to lower contractility, followed by differentiation. (B) Immunoblot of Brachyury during hiPSC-to-mesoderm commitment (0–48 h) ±10 μM ROCK inhibitor Y-27632, as shown in A. M.W. are displayed on the right side. (C) Brachyury expression was quantified by densitometry and normalized to α-tubulin as a loading control across N = 3 independent biological repeats. Mean and SD are displayed. A two-tailed unpaired t test was performed. (D–G) Relative expression of mesoderm markers (MESP1, TBX6) (D and E) and EMT markers (SNAI1, SNAI2) (F and G) 48 h after CHIR treatment ±10 μM ROCK inhibitor as shown in A. N = 3 independent biological repeats except for MESP1, N = 4 independent biological repeats. Mean and SD are displayed. A two-tailed unpaired t test was performed. (H) Representative MaxIP immunofluorescences during hiPSC-to-mesoderm commitment (0–48 h) ±10 μM ROCK inhibitor as shown in A. Cells were stained with a nuclear marker (DNA), EMT marker (ZO-1—inverted LUT), and mesoderm marker (T/Bra—inverted LUT). Scale bar = 50 µm. (I and J) Quantification of Brachyury expression (I) and ZO-1 intactness (J) is represented as violin plots with individual measurements (small dots) averaged for each biological repeat (large dots). Median (plain red line) and quartiles (dotted black lines) are displayed. n = 10–15 technical repeats across N = 3 independent biological repeats. One-way ANOVA with Tukey’s multiple comparisons posttest was performed on biological repeats. (K and L) Analysis of Brachyury expression by flow cytometry following 24-h CHIR treatment ±10 μM ROCK inhibitor, as shown in A, using Bra-mNG knock-in H9 hESC line (K). Quantification of the percentage of mNG-positive cells from N = 4 independent biological repeats. Mean and SD are displayed. A two-tailed unpaired t test was performed (L). (M and N) Representative MaxIP immunofluorescence of hiPSCs treated with a MLCK inhibitor (3 μM ML-7) or Veh (DMSO), as shown in A. Cells were stained for nuclei (DNA) and ppMLC2 (inverted LUT). Scale bar = 50 µm. Magnified views of the yellow dotted ROI are shown for ppMLC2. Scale bar = 20 µm (M). Percentage of cellular area positive for ppMLC2 following Veh or ML-7 treatment is 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 (N). (O and P) Immunoblot of Brachyury (mesoderm) and PARP (cell death marker) using hiPSCs in the presence (+ ML-7) or absence (+ Veh) of 3 μM of MLCK inhibitor at the basal state (0 h) and during CHIR treatment (24, 48 h), as shown in A. Cells treated with CHIR and Q-VD (caspase inhibitor) for 48 h were used as a control for PARP immunoblot. M.W. are displayed on the right side (O). Brachyury expression was quantified by densitometry and normalized to α-tubulin as a loading control across N = 3 independent biological repeats. Mean and SD are displayed. The Mann–Whitney test was performed for the 24-h time points, and a two-tailed unpaired t test was performed for the 48-h time points (P). (Q) Experimental design. Effects of Y-27632 were tested at different time points along the mesoderm commitment. 10 μM Y-27632 was added 12 h before differentiation (−12 h), at the time of differentiation (0 h), or 12 h after differentiation (+12 h). All conditions were collected 24 h after differentiation. (R and S) Representative immunoblot for Brachyury following the addition of Veh or 10 μM Y-27632 as shown in Q. M.W. are displayed on the right side (R). Brachyury expression was quantified by densitometry and normalized to α-tubulin as a loading control across N = 3 independent biological repeats. Mean and SD are displayed. Two-way ANOVA with Šidák’s multiple comparisons posttest was performed (S). M.W., molecular weights; Veh, vehicle. ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05. Source data are available for this figure: SourceDataF3.

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