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sc-12-0761_sm_SupplFigure1.pdf297KFigure S1 Generation of transgenic murine embryonic stem cells overexpressing FHL2. Schematic representation of the analysis of positive clones in transfected double transgenic αMHC-GFP/αMHCNeoR ESCs. Selected clones (8, 7 and 6) were analyzed by southern blot, qPCR and Western blot. (A): Restriction enzymes used for the generation of the antisense riboprobes using the pCS-MT-FHL2 expressing plasmid for Southern blot analysis. (B): Southern blot detection using the riboprobes described in A. Note the presence of multiple copies in the clone 8, which was used in this study as FHL2-overexpressing ESC line. (C): QPCR showing significant up-regulation of Fhl2 transcripts level in the transgenic cell line. Relative mRNA levels were normalized to Gapdh (D): Western blot analysis confirming up-regulation of FHL2 expression in the transgenic line. GAPDH was used as loading control.
sc-12-0761_sm_SupplFigure2.pdf199KFigure S2 FHL2 accelerates reduction in gene expression of undifferentiated cardiogenic cells in ESC-EBs. FHL2 gain-of-function in ESC-EBs resulted in a reduction of the pluripotent gene Oct4, the panmesodermal, and cardiac mesodermal markers Brachyury and Mesp1, respectively, following 7 days of differentiation in comparison to wild-type cells as shown by qPCR. Relative mRNA levels were normalized to Gapdh. Data represent mean ± SEM; n=6, ** p<0.01, *** p<0.001 (two-tailed Student's t-test).
sc-12-0761_sm_SupplFigure3.pdf211KFigure S3 Inhibition of β-catenin-dependent transcription rescues cardiac differentiation in FHL2-ESCEBs. Quercetin (QC) treatment from day 9 of differentiation completely reversed the decrease in αMHC-GFP expression in differentiating FHL2-ESC-EBs (red triangles and discontinuous lines) in comparison to untreated FHL2-ESC-CMs (red dots and continuous lines). αMHC-GFP expression in FHL2-ESC-EBs upon quercetin reaches similar levels of expression as in untreated control ESC-EBs (black dots and continuous lines). αMHC-GFP expression was calculated as percentages of total EBs. Data represent mean ± SEM; n=10, ** p<0.01, *** p<0.001 (two-tailed Student's t-test).
sc-12-0761_sm_SupplFigure4.pdf349KFigure S4 Transient overexpression of FHL2 enhances β-catenin transcriptional activity while promoting cardiogenesis in P19 cells. (A): FHL2 expression in transiently transfected (FHL2), empty vector transfected (CT) as well as undifferentiated (undiff) pluripotent P19 cells shown by Western blot. (B): Nuclear β-catenin accumulation upon FHL2 transient overexpression. Immunoblot of β-catenin in cytosolic and nuclear fractions showing β-catenin nuclear accumulation in FHL2-P19 cells. Semiquantification via densitometric analysis in 3 independent experiments. Normalization was performed with α.tubulin for the cytosolic fraction and Histone H1 for the nuclear fraction. (C): qPCR analysis of an early cardiogenic specification marker Alpk3, and an early cardiogenic marker Mef2a showing increased expression in FHL2-P19 transient-expressing cells at 2 days of differentiation. (D): Up-regulation of the cycling gene Cyclin D1 normalized to Gapdh expression at 2 days of differentiation in FHL2-P19 transiently expressing cells in comparison to CT P19 cells. (E): KI67 (red) expression analyzed by confocal immunofluorescence (percentage of total DAPI positive cells) following 2 days of differentiation demonstrating increased KI67 cell number in FHL2-P19 cells. Representative pictures are depicted. (F): Significantly decreased expression of Brachyury, Flk1 and Mesp1 as determined by qPCR analysis normalized to Gapdh expression at 10 days of differentiation in FHL2-P19 cells. Data represent mean ± SEM; A and B: n=3, C-F: n=6, two-tailed Student's t-test; * p<0.05; ** p<0.01; *** p<0.001. Scale bar: 10 μm.
sc-12-0761_sm_SupplTable1.pdf302KTable S1: List of primers used in this study. NCBI reference sequence, primer positions, generated fragment length and annealing temperatures are provided.

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