Figure 2.
Genetic inhibition of actomyosin contractility promotes hiPSC conversion to the mesoderm lineage. (A) Summary of the main regulators of MLC phosphorylation. (B) Schematic representation of full-length human MYPT1 (1030 aa), interacting with PP1c phosphatase and a protein of unknown function M20. Truncated MYPT1 Nterm 1–300 (containing the MLC-binding domain) leads to constitutive recruitment to and activation of PP1C. Truncated MYPT1 was fused with NES-mNG as a marker, and is referred to as MYPT1CA-NES-mNG. (C) Immunoblot of ppMLC2 and total MLC2 in WT hiPSCs treated with Dox or hiPSCs expressing MYPT1CA-NES-mNG in the presence or absence of 1 μg/ml Dox. α-Tubulin was used as a loading control. M.W. are displayed on the right side. (D and E) Representative MaxIP immunofluorescence of hiPSCs expressing MYPT1CA-NES-mNG and treated or not with Dox overnight. Cells were fixed and stained for ppMLC2 (magenta), DNA (blue), and tight junction marker ZO-1 (orange). Scale bar = 50 µm (D). Fraction of the cellular area positive for ppMLC2 following Dox induction of MYPT1CA-NES-mNG hiPSC and reported as violin plots. Median (plain red line) and quartiles (dotted black lines) are displayed. n = 32 technical repeats across N = 3 independent biological repeats. A two-tailed unpaired t test was performed on the biological repeats (E). (F) Representative MaxIP immunofluorescence of hiPSCs expressing MYPT1CA-NES-mNG and treated or not with Dox overnight. Cells were stained for F-actin using phalloidin. (G) Experimental design. hiPSCs were transduced with pInducer20-MYPT1CA-NES-mNG, and stable population was selected with puromycin. MYPT1CA cells were treated or not with Dox for 16 h and treated with CHIR supplemented or not with Dox. Parental WT line was differentiated with Dox. (H and I) Representative immunoblot for Brachyury (mesoderm marker), and Snail and Slug (EMT markers) using WT and MYPT1CA-NES-mNG hiPSCs, following CHIR treatment (0–48 h) in the presence or absence of Dox, as shown in G. M.W. are displayed on the right side (H). Brachyury expression was quantified by densitometry and normalized to α-tubulin as a loading control across N = 3–4 independent biological repeats. Mean and SD are displayed. A one-way ANOVA with Tukey’s multiple comparisons posttest was performed (I). (J) Immunoblot of PARP (cell death marker) and Brachyury (mesoderm marker) during mesoderm commitment (0-h to 48-h CHIR) using MYPT1CA-NES-mNG hiPSCs induced +/Dox, as shown in G. M.W. are displayed on the right side. (K and L) Representative MaxIP immunofluorescences of WT and MYPT1CA-NES-mNG hiPSCs treated with CHIR for 24 h +/− Dox as shown in G. Cells were stained for nuclei (DNA) and mesoderm marker (T/Bra—inverted LUT). Scale bar = 100 µm (K). Quantification of Brachyury expression is presented as violin plots. Median (plain red line) and quartiles (dotted black lines) are displayed. n = 15–16 technical repeats across N = 3 independent biological repeats. One-way ANOVA with Šidák’s multiple comparisons posttest was performed on biological repeats (L). M.W., molecular weights. ***P < 0.001, **P < 0.01, *P < 0.05. Source data are available for this figure: SourceDataF2. Refer to the image caption for details. Panel A shows MLC phosphorylation. The illustration (Panel B) shows the structure of full-length human MYPT1 and a truncated version fused with NES-mNeonGreen. The immunoblots (Panels C, H, J) display protein expression levels under different conditions. The immunofluorescence images (Panels D, F, K) show staining for various markers in hiPSCs treated with doxycycline and slash or CHIR. The violin plots (Panels E, L) present quantitative data on the fraction of cellular area positive for ppMLC2 and Brachyury expression. The experimental design (Panel G) outlines the treatment conditions for hiPSCs. The images and data collectively demonstrate the effects of genetic inhibition of actomyosin contractility on mesoderm differentiation in hiPSCs. The bar graph (Panel I) shows normalized Bra expression (A.U.).

Genetic inhibition of actomyosin contractility promotes hiPSC conversion to the mesoderm lineage. (A) Summary of the main regulators of MLC phosphorylation. (B) Schematic representation of full-length human MYPT1 (1030 aa), interacting with PP1c phosphatase and a protein of unknown function M20. Truncated MYPT1 Nterm 1–300 (containing the MLC-binding domain) leads to constitutive recruitment to and activation of PP1C. Truncated MYPT1 was fused with NES-mNG as a marker, and is referred to as MYPT1CA-NES-mNG. (C) Immunoblot of ppMLC2 and total MLC2 in WT hiPSCs treated with Dox or hiPSCs expressing MYPT1CA-NES-mNG in the presence or absence of 1 μg/ml Dox. α-Tubulin was used as a loading control. M.W. are displayed on the right side. (D and E) Representative MaxIP immunofluorescence of hiPSCs expressing MYPT1CA-NES-mNG and treated or not with Dox overnight. Cells were fixed and stained for ppMLC2 (magenta), DNA (blue), and tight junction marker ZO-1 (orange). Scale bar = 50 µm (D). Fraction of the cellular area positive for ppMLC2 following Dox induction of MYPT1CA-NES-mNG hiPSC and reported as violin plots. Median (plain red line) and quartiles (dotted black lines) are displayed. n = 32 technical repeats across N = 3 independent biological repeats. A two-tailed unpaired t test was performed on the biological repeats (E). (F) Representative MaxIP immunofluorescence of hiPSCs expressing MYPT1CA-NES-mNG and treated or not with Dox overnight. Cells were stained for F-actin using phalloidin. (G) Experimental design. hiPSCs were transduced with pInducer20-MYPT1CA-NES-mNG, and stable population was selected with puromycin. MYPT1CA cells were treated or not with Dox for 16 h and treated with CHIR supplemented or not with Dox. Parental WT line was differentiated with Dox. (H and I) Representative immunoblot for Brachyury (mesoderm marker), and Snail and Slug (EMT markers) using WT and MYPT1CA-NES-mNG hiPSCs, following CHIR treatment (0–48 h) in the presence or absence of Dox, as shown in G. M.W. are displayed on the right side (H). Brachyury expression was quantified by densitometry and normalized to α-tubulin as a loading control across N = 3–4 independent biological repeats. Mean and SD are displayed. A one-way ANOVA with Tukey’s multiple comparisons posttest was performed (I). (J) Immunoblot of PARP (cell death marker) and Brachyury (mesoderm marker) during mesoderm commitment (0-h to 48-h CHIR) using MYPT1CA-NES-mNG hiPSCs induced +/Dox, as shown in G. M.W. are displayed on the right side. (K and L) Representative MaxIP immunofluorescences of WT and MYPT1CA-NES-mNG hiPSCs treated with CHIR for 24 h +/− Dox as shown in G. Cells were stained for nuclei (DNA) and mesoderm marker (T/Bra—inverted LUT). Scale bar = 100 µm (K). Quantification of Brachyury expression is presented as violin plots. Median (plain red line) and quartiles (dotted black lines) are displayed. n = 15–16 technical repeats across N = 3 independent biological repeats. One-way ANOVA with Šidák’s multiple comparisons posttest was performed on biological repeats (L). M.W., molecular weights. ***P < 0.001, **P < 0.01, *P < 0.05. Source data are available for this figure: SourceDataF2.

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