Figure S4.
Key mechanosensitive pathways are not involved in mesoderm specification enhancement following decreased contractility. (A and B) Immunofluorescence for YAP in MYPT1CA-NES-mNG cells treated with Dox to promote cell relaxation. Cells were stained for nuclei and YAP. Scale bar = 50 μm (A). N/C YAP ratio was quantified from n = 106 cells (Veh) and n = 80 cells (Dox) across N = 3 independent biological repeats. Mean (red plain line), and first and fourth quartiles (black dotted lines) are displayed on the violin plot. A Mann–Whitney test was performed (B). (C and D) Immunoblot for pYAP and total YAP following differentiation (0- to 48-h CHIR) in the presence or absence of 10 μM of ROCK inhibitor Y-27632. Brachyury expression was also probed as an internal positive control, and α-tubulin was used as a loading control. M.W. are displayed on the right side (C). Ratio of phospho/total YAP was quantified across N = 4 independent biological repeats. Mean and SD are displayed. One-way ANOVA with Šidák’s multiple comparisons posttest was performed (D). (E) Relative expression of YAP target genes CTGF and CYR61 during differentiation ±10 μM Y-27632. N = 4 independent biological repeats. Mean and SD are displayed. One-way ANOVA with Šidák’s multiple comparisons posttest was performed. (F) Relative expression of canonical WNT (WNT3A, WNT8A), noncanonical WNT (WNT4), and mesoderm marker (TBX6) in MYPT1CA-NES-mNG cells treated with CHIR (0–48 h) in the presence of Veh or Dox. N = 3 independent biological repeats. Mean and SD are displayed. One-way ANOVA with Šidák’s multiple comparisons posttest was performed. (G) Relative expression of TBX6 and MESP1 (mesoderm markers) following CHIR treatment ±10 μM Y-27632 complemented or not with 7.5 µM porcupine inhibitor (IWP-2 or DMSO, respectively). N = 3 independent biological repeats. Mean and SD are displayed. One-way ANOVA with Tukey’s multiple comparisons posttest was performed. M.W., molecular weights; N/C, nuclear-to-cytosolic; Veh, vehicle; pYAP, phospho-S127 YAP. ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05. Source data are available for this figure: SourceDataFS4. Refer to the image caption for details. Panel A shows fluorescence microscopy images of nuclei and Yes-associated protein localization in cells with or without doxycycline treatment. Panel B shows violin plot, x-axis shows treatment conditions vehicle and doxycycline and y-axis shows Yes-associated protein nuclear to cytosolic ratio. Panel C shows western blot of phosphorylated Yes-associated protein at serine 127, total Yes-associated protein, Brachyury, and tubulin across time points. Panel D shows bar graph, x-axis shows time after CHIR treatment with or without Y-27632 and y-axis shows ratio of phosphorylated to total Yes-associated protein. Panel E shows bar graphs, x-axis shows time and treatment conditions and y-axis shows relative expression of connective tissue growth factor and cysteine rich angiogenic inducer 61. Panel F shows bar graphs, x-axis shows time and treatment conditions and y-axis shows normalized messenger ribonucleic acid expression of WNT3A, WNT8A, WNT4, and TBX6. Panel G shows bar graphs, x-axis shows treatment conditions and y-axis shows relative expression of TBX6 and mesoderm posterior 1.

Key mechanosensitive pathways are not involved in mesoderm specification enhancement following decreased contractility. (A and B) Immunofluorescence for YAP in MYPT1CA-NES-mNG cells treated with Dox to promote cell relaxation. Cells were stained for nuclei and YAP. Scale bar = 50 μm (A). N/C YAP ratio was quantified from n = 106 cells (Veh) and n = 80 cells (Dox) across N = 3 independent biological repeats. Mean (red plain line), and first and fourth quartiles (black dotted lines) are displayed on the violin plot. A Mann–Whitney test was performed (B). (C and D) Immunoblot for pYAP and total YAP following differentiation (0- to 48-h CHIR) in the presence or absence of 10 μM of ROCK inhibitor Y-27632. Brachyury expression was also probed as an internal positive control, and α-tubulin was used as a loading control. M.W. are displayed on the right side (C). Ratio of phospho/total YAP was quantified across N = 4 independent biological repeats. Mean and SD are displayed. One-way ANOVA with Šidák’s multiple comparisons posttest was performed (D). (E) Relative expression of YAP target genes CTGF and CYR61 during differentiation ±10 μM Y-27632. N = 4 independent biological repeats. Mean and SD are displayed. One-way ANOVA with Šidák’s multiple comparisons posttest was performed. (F) Relative expression of canonical WNT (WNT3A, WNT8A), noncanonical WNT (WNT4), and mesoderm marker (TBX6) in MYPT1CA-NES-mNG cells treated with CHIR (0–48 h) in the presence of Veh or Dox. N = 3 independent biological repeats. Mean and SD are displayed. One-way ANOVA with Šidák’s multiple comparisons posttest was performed. (G) Relative expression of TBX6 and MESP1 (mesoderm markers) following CHIR treatment ±10 μM Y-27632 complemented or not with 7.5 µM porcupine inhibitor (IWP-2 or DMSO, respectively). N = 3 independent biological repeats. Mean and SD are displayed. One-way ANOVA with Tukey’s multiple comparisons posttest was performed. M.W., molecular weights; N/C, nuclear-to-cytosolic; Veh, vehicle; pYAP, phospho-S127 YAP. ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05. Source data are available for this figure: SourceDataFS4.

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