Es assessed by Sanger sequencing. (A) Chromatograms representing the genomic region around the 23388095 polymorphic site rs1056719 in Granta-519 cells after seven days of control treatment with the solvent PBS. (B) Rebalancing after seven days of pulsed treatment with 1.5 mM DNA methyltransferase inhibitor 5-aza-29-deoxycytidine (DAC). (C) Re-constitution of the allelic imbalance after 1.5 mM DAC treatment for seven days and consecutive withdrawal of the compound for 33 days. (TIF)Figure S7 Allelic DNA methylation in lymphoid cell lines with allele-specific expression of the DAPK1 gene. (A) Scheme of the DAPK1 promoter region and the associated CpG island. Grey boxes display the first two exons of DAPK1. Nucleotide positions are given relative to the DAPK1 transcriptional start site. The dashed line represents the amplicon purchase Licochalcone-A Analyzed by bisulfite sequencing. This region exhibited extensive DAPK1 allele-specific DNA methylation in Granta-519 cells. (B, C) Bisulfite-sequencing of the DAPK1 59 region in JVM-2 (no ASE) and EHEB (monoallelic expression) cells. As a heterozygous SNP could be detected in neither cell line 80-49-9 site between 220 and +600 bp, a clear allelic separation is not possible. Red boxes represent singleCpG methylation, blue boxes represent unmethylated CpGs, white boxes stand for missing data. Methylation levels are calculated in percent for each CpG dinucleotide. (TIF) Table S1 Oligonucleotides and primers.(DOC)AcknowledgmentsThe authors would like to thank Yoon Jung Park and Dieter Weichenhan for helpful discussion and Oliver Mucke for excellent technical support. ?Author ContributionsConceived and designed the experiments: QW RC Ar AdC CP. Performed the experiments: QW RC CO. Analyzed the data: QW RC TH DM AR SMT. Contributed reagents/materials/analysis tools: JCB SS. Wrote the paper: RC CP.Sanger sequencing. (A) Sequences from 12 single clones of
Excitation-contraction (E-C) coupling in the adult mammalian heart is governed by the Ca2+-induced Ca2+ release (CICR) mechanism. The process involves entry of Ca2 through L-type Ca2+ channel that activates the ryanodine receptors (RyRs)mediated Ca2+ release from sarcoplasmic reticulum (SR) and resulting in intracellular Ca2+ transients [1]. Ca2+ sparks, a local and transient Ca2+ release originating from a single RyR or a cluster of RyRs, constitute the elementary events of cardiac E-C coupling [2]. Whole cell Ca2+ transients are believed to represent the recruitment and summation of many Ca2+ sparks after an increase in opened L-type Ca2+ channels [3]. RyR Ca2+ release channel is tightly linked to the gating of L-type Ca2+ channel 1662274 and plays a key role in the intracellular Ca2+-handling in cardiac myocytes [4]. Such property of Ca2+ sparks may reflect the organizational maturity of RyRs in the cardiomyocytes [5]. Human induced pluripotent stem cells (hiPSCs) can be generated by somatic reprogramming of fibroblasts with a set oftranscription factors and differentiated into multiple cell lineages, including cardiomyocytes [6]. In contrast to human embryonic stem cells (hESCs), hiPSCs are capable of giving rise to a renewable source of cardiomyocytes (CMs) from individuals. These hiPSCderived cardiomyocytes (hiPSC-CMs) offer immensely valuable tool in personalized pharmaceutical evaluation of therapeutic agents such as anti-arrhythmics and provide a tenable means for cardiomyocytes replacement therapy. Therefore, it is important to understand the functional characteristics of such hiPSC-CMs, especi.Es assessed by Sanger sequencing. (A) Chromatograms representing the genomic region around the 23388095 polymorphic site rs1056719 in Granta-519 cells after seven days of control treatment with the solvent PBS. (B) Rebalancing after seven days of pulsed treatment with 1.5 mM DNA methyltransferase inhibitor 5-aza-29-deoxycytidine (DAC). (C) Re-constitution of the allelic imbalance after 1.5 mM DAC treatment for seven days and consecutive withdrawal of the compound for 33 days. (TIF)Figure S7 Allelic DNA methylation in lymphoid cell lines with allele-specific expression of the DAPK1 gene. (A) Scheme of the DAPK1 promoter region and the associated CpG island. Grey boxes display the first two exons of DAPK1. Nucleotide positions are given relative to the DAPK1 transcriptional start site. The dashed line represents the amplicon analyzed by bisulfite sequencing. This region exhibited extensive DAPK1 allele-specific DNA methylation in Granta-519 cells. (B, C) Bisulfite-sequencing of the DAPK1 59 region in JVM-2 (no ASE) and EHEB (monoallelic expression) cells. As a heterozygous SNP could be detected in neither cell line between 220 and +600 bp, a clear allelic separation is not possible. Red boxes represent singleCpG methylation, blue boxes represent unmethylated CpGs, white boxes stand for missing data. Methylation levels are calculated in percent for each CpG dinucleotide. (TIF) Table S1 Oligonucleotides and primers.(DOC)AcknowledgmentsThe authors would like to thank Yoon Jung Park and Dieter Weichenhan for helpful discussion and Oliver Mucke for excellent technical support. ?Author ContributionsConceived and designed the experiments: QW RC Ar AdC CP. Performed the experiments: QW RC CO. Analyzed the data: QW RC TH DM AR SMT. Contributed reagents/materials/analysis tools: JCB SS. Wrote the paper: RC CP.Sanger sequencing. (A) Sequences from 12 single clones of
Excitation-contraction (E-C) coupling in the adult mammalian heart is governed by the Ca2+-induced Ca2+ release (CICR) mechanism. The process involves entry of Ca2 through L-type Ca2+ channel that activates the ryanodine receptors (RyRs)mediated Ca2+ release from sarcoplasmic reticulum (SR) and resulting in intracellular Ca2+ transients [1]. Ca2+ sparks, a local and transient Ca2+ release originating from a single RyR or a cluster of RyRs, constitute the elementary events of cardiac E-C coupling [2]. Whole cell Ca2+ transients are believed to represent the recruitment and summation of many Ca2+ sparks after an increase in opened L-type Ca2+ channels [3]. RyR Ca2+ release channel is tightly linked to the gating of L-type Ca2+ channel 1662274 and plays a key role in the intracellular Ca2+-handling in cardiac myocytes [4]. Such property of Ca2+ sparks may reflect the organizational maturity of RyRs in the cardiomyocytes [5]. Human induced pluripotent stem cells (hiPSCs) can be generated by somatic reprogramming of fibroblasts with a set oftranscription factors and differentiated into multiple cell lineages, including cardiomyocytes [6]. In contrast to human embryonic stem cells (hESCs), hiPSCs are capable of giving rise to a renewable source of cardiomyocytes (CMs) from individuals. These hiPSCderived cardiomyocytes (hiPSC-CMs) offer immensely valuable tool in personalized pharmaceutical evaluation of therapeutic agents such as anti-arrhythmics and provide a tenable means for cardiomyocytes replacement therapy. Therefore, it is important to understand the functional characteristics of such hiPSC-CMs, especi.
