Figure 7. β-MyHC expression is reduced... | Rockefeller University Press

Figure 7.

$β-MyHC expression is reduced by inhibition of FAK. (A) Western blot analysis of FAK and ERK1/2-MAPK expression and phosphorylation in hESC-CMs grown for indicated days on laminin-coated glass coverslips. Representative blot, with β-tubulin serving as a loading control. (B) Western blot analysis of ERK1/2-MAPK phosphorylation in 35 d hESC-CMs grown on Matrigel-coated PDMS treated with or without 5 μM FAK-inhibitor 14 (FAK-Inh) or 10 μM MEK1/2 inhibitor U0126 (MEK1/2-Inh) for 2 d. The ratio of phospho/total ERK1/2-MAPK expression was determined densitometrically. Mean ± SEM; n = 3 individual coverslips from one differentiation. Representative blot, with β-tubulin serving as a loading control. (C) Myosin isoform expression was analyzed by single-cell IF using specific antibodies against α-MyHC and β-MyHC in hESC-CMs grown on laminin-coated glass coverslips treated with or without 5 μM FAK-inhibitor 14 (FAK-Inh) or 10 μM MEK1/2 inhibitor U0126 (MEK1/2-Inh) from day 7 on for 2 or 4 d. The fractions of cells in the different indicated categories (see Materials and methods) are shown as a percentage of the total number of cells analyzed (n, set to 100%, from three coverslips derived from one differentiation; P values: exclusively β-MyHC expressing cells. (D) hESC-CMs were grown for 35 d on laminin-coated glass coverslips or on Matrigel-coated PDMS and treated with or without 5 µM FAK-inhibitor 14 (FAK-Inh) or 10 μM MEK1/2 inhibitor U0126 (MEK1/2-Inh) for 2 d as indicated. MLC2 isoform expression was analyzed by single-cell IF using specific antibodies against MLC2v and MLC2a. The fractions of cells in the different categories (see Materials and methods) are shown as a percentage of the total number of cells analyzed (n, set to 100%, from three coverslips derived from one differentiation; P values: exclusively MLC2v expressing cells). Source data are available for this figure: SourceData F7.$

β-MyHC expression is reduced by inhibition of FAK. (A) Western blot analysis of FAK and ERK1/2-MAPK expression and phosphorylation in hESC-CMs grown for indicated days on laminin-coated glass coverslips. Representative blot, with β-tubulin serving as a loading control. (B) Western blot analysis of ERK1/2-MAPK phosphorylation in 35 d hESC-CMs grown on Matrigel-coated PDMS treated with or without 5 μM FAK-inhibitor 14 (FAK-Inh) or 10 μM MEK1/2 inhibitor U0126 (MEK1/2-Inh) for 2 d. The ratio of phospho/total ERK1/2-MAPK expression was determined densitometrically. Mean ± SEM; n = 3 individual coverslips from one differentiation. Representative blot, with β-tubulin serving as a loading control. (C) Myosin isoform expression was analyzed by single-cell IF using specific antibodies against α-MyHC and β-MyHC in hESC-CMs grown on laminin-coated glass coverslips treated with or without 5 μM FAK-inhibitor 14 (FAK-Inh) or 10 μM MEK1/2 inhibitor U0126 (MEK1/2-Inh) from day 7 on for 2 or 4 d. The fractions of cells in the different indicated categories (see Materials and methods) are shown as a percentage of the total number of cells analyzed (n, set to 100%, from three coverslips derived from one differentiation; P values: exclusively β-MyHC expressing cells. (D) hESC-CMs were grown for 35 d on laminin-coated glass coverslips or on Matrigel-coated PDMS and treated with or without 5 µM FAK-inhibitor 14 (FAK-Inh) or 10 μM MEK1/2 inhibitor U0126 (MEK1/2-Inh) for 2 d as indicated. MLC2 isoform expression was analyzed by single-cell IF using specific antibodies against MLC2v and MLC2a. The fractions of cells in the different categories (see Materials and methods) are shown as a percentage of the total number of cells analyzed (n, set to 100%, from three coverslips derived from one differentiation; P values: exclusively MLC2v expressing cells). Source data are available for this figure: SourceData F7.