Figure 4. Analysis of myosin isoform... | Rockefeller University Press

Figure 4.

$Analysis of myosin isoform expression in replated hPSC-CMs. hESC-CMs or hiPSC-CMs were cultivated for indicated days on laminin-coated glass coverslips or replated on day 35 or day 18, respectively, and further cultivated for indicated days. (A) Myosin isoform expression was analyzed in hESC-CMs by single-cell immunofluorescence (IF) using specific antibodies against α- (green) and β-MyHC (red). Nuclei were stained with DAPI (blue). Scale bars: 10 μm. (B) Single-cell IF analysis showing replated hESC-CMs from 35 + 4 d with mixed α- and β-MyHC expression with insets (white squares) demonstrating a detailed view of sarcomeric staining as relevant for classification (see below). Scale bars: 10 μm. (C) Single-cell IF analysis of myosin expression in hESC-CMs. The fractions of cells in the different categories (see Materials and methods) are shown as the percentage of the total number of cells analyzed (n, set to 100%) for each time point. Mean ± SD; indicated number of cells (n) were obtained from six individual coverslips derived from three differentiations; P values: exclusively β-MyHC expressing cells. (D) MyHC isoforms were separated by SDS-PAGE and the ratio of α-MyHC/β-MyHC expression in hESC-CMs was determined densitometrically. Mean ± SEM; n = 3 individual coverslips from three differentiations. Representative SDS-PAGE gel with samples from human right atrium (AD) as indicated; molecular weight marker: 200 kD. (E) Myosin isoform expression was analyzed in hiPSC-CMs by single-cell IF using specific antibodies against α- and β-MyHC. 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%) for each time point. Mean; two coverslips derived from one differentiation. (F) MyHC isoforms were separated by SDS-PAGE and the ratio of α-MyHC/β-MyHC expression in hiPSC-CMs was determined densitometrically. Mean ± SEM; n = 3 or 4 individual coverslips from one differentiation. Representative SDS-PAGE gel, with samples from the human right atrium (AD) and left ventricle (LV) as indicated; molecular weight marker: 200 kD. Source data are available for this figure: SourceData F4.$

Analysis of myosin isoform expression in replated hPSC-CMs. hESC-CMs or hiPSC-CMs were cultivated for indicated days on laminin-coated glass coverslips or replated on day 35 or day 18, respectively, and further cultivated for indicated days. (A) Myosin isoform expression was analyzed in hESC-CMs by single-cell immunofluorescence (IF) using specific antibodies against α- (green) and β-MyHC (red). Nuclei were stained with DAPI (blue). Scale bars: 10 μm. (B) Single-cell IF analysis showing replated hESC-CMs from 35 + 4 d with mixed α- and β-MyHC expression with insets (white squares) demonstrating a detailed view of sarcomeric staining as relevant for classification (see below). Scale bars: 10 μm. (C) Single-cell IF analysis of myosin expression in hESC-CMs. The fractions of cells in the different categories (see Materials and methods) are shown as the percentage of the total number of cells analyzed (n, set to 100%) for each time point. Mean ± SD; indicated number of cells (n) were obtained from six individual coverslips derived from three differentiations; P values: exclusively β-MyHC expressing cells. (D) MyHC isoforms were separated by SDS-PAGE and the ratio of α-MyHC/β-MyHC expression in hESC-CMs was determined densitometrically. Mean ± SEM; n = 3 individual coverslips from three differentiations. Representative SDS-PAGE gel with samples from human right atrium (AD) as indicated; molecular weight marker: 200 kD. (E) Myosin isoform expression was analyzed in hiPSC-CMs by single-cell IF using specific antibodies against α- and β-MyHC. 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%) for each time point. Mean; two coverslips derived from one differentiation. (F) MyHC isoforms were separated by SDS-PAGE and the ratio of α-MyHC/β-MyHC expression in hiPSC-CMs was determined densitometrically. Mean ± SEM; n = 3 or 4 individual coverslips from one differentiation. Representative SDS-PAGE gel, with samples from the human right atrium (AD) and left ventricle (LV) as indicated; molecular weight marker: 200 kD. Source data are available for this figure: SourceData F4.