Journal: Science Advances

Article Title: Hypermethylation of DNA impairs megakaryogenesis in delayed platelet recovery after allogeneic hematopoietic stem cell transplantation

doi: 10.1126/sciadv.ads3630

Figure Lengend Snippet: ( A ) Methylated CG of each sample. Left, all genes; right, megakaryocyte-regulated genes. GH1–3 indicate HSCs from three patients when they had GGF; DH1–3 indicate HSCs from three patients when they had DPR; GM1–3 refer to MKPs from three patients when they had GGF; DM1–3 refer to MKPs from three patients when they had DPR. ( B ) Mean methylation rate across CG islands of HSCs and MKPs in patients with DPR and GGF. ( C ) Chromosomal distribution of DMRs. X axis refers to 23 pairs of chromosomes and mitochondrial chromosomes; y axis refers to the count of hypomethylated and hypermethylated DMRs of each chromosome. Left, GGFH versus PTH; right, GGFM versus PTM. GGFH, HSCs from patients with GGF; PTH, HSCs from patients with DPR; GGFM, MKPs from patients with GGF; PTM, MKPs from patients with DPR.

Article Snippet: End repair, ligation of methylated adaptors (#L2V11DR-BC 1-96 adaptor plate, NuGEN, Tecan Genomics Inc., Redwood City, CA) and final repair were performed according to the manufacturer’s instructions (Ovation Ultralow Methyl-Seq Library System, NuGEN).

Techniques: Methylation

Journal: Science Advances

Article Title: Hypermethylation of DNA impairs megakaryogenesis in delayed platelet recovery after allogeneic hematopoietic stem cell transplantation

doi: 10.1126/sciadv.ads3630

Figure Lengend Snippet: ( A ) Platelets and polyploid megakaryocytes (≥8N) were elevated in mice receiving decitabine (top and middle), and no difference was observed in MKPs (bottom). ( B ) Meg01 cells were cultured in the presence of decitabine, and higher percentage of CD41 + cells were observed in those treated with decitabine compared to DMSO. ( C ) The bisulfite conversion rate of each sample (top). Modification rate of mCG, mCH, hmCG, and hmCH of megakaryocytes (middle) and MKPs (bottom) from decitabine and DMSO group. ( D ) Principal components analysis of mCG modification profiles (Red, decitabine group; blue, control group. From top to bottom, megakaryocytes-CpG, megakaryocytes-DMRs, MKPs-CpG, MKP-DMRs). ( E ) Profile of combination of differentially methylated cytosine (DMC) methylation difference and differentially expressed genes (left), and the summary of genes that were hypo- or hypermethylated and up- or down-regulated (right). # B, Baseline; *BS, bisulfite sequencing; △ oxBS, oxidative bisulfite sequencing.

Article Snippet: End repair, ligation of methylated adaptors (#L2V11DR-BC 1-96 adaptor plate, NuGEN, Tecan Genomics Inc., Redwood City, CA) and final repair were performed according to the manufacturer’s instructions (Ovation Ultralow Methyl-Seq Library System, NuGEN).

Techniques: Cell Culture, Modification, Control, Methylation, Methylation Sequencing, Oxidative Bisulfite Sequencing

Journal: Circulation

Article Title: Therapeutic Modulation of the Immune Response in Arrhythmogenic Cardiomyopathy

doi: 10.1161/CIRCULATIONAHA.119.040676

Figure Lengend Snippet: A. Representative confocal immunofluorescence images from cultures of cardiac myocytes (CMs) derived from a control hiPSC cell line and a line from an ACM patient with a pathogenic variant in plakophilin-2 (PKP2) grown in the absence or presence of Bay 11–7082. Virtually all PKP2 cells are positive for cardiac troponin-I (cTnI) indicating they are cardiac myocytes. Control hiPSC-CMs showed normal prominent cell surface staining for plakoglobin. Marked nuclear accumulation of immunoreactive signal for phospho-RelA/p65 (pRelA/p65) is seen in PKP2 hiPSC- CMs but not in control cells. Treatment of PKP2 hiPSC-CMs with Bay 11–7082 prevented nuclear accumulation of phospho-RelA/p65 signal. Scale bar = 20μm. B. Representative cytokine arrays prepared from cultures of cardiac myocytes derived from a control hiPSC cell line and a line from a patient with a disease causing variant in plakophillin-2 (PKP2). Arrays are shown for cells grown in the absence (untreated) or presence of Bay 11–7082. The spots in the upper right and left and lower left corners are reference markers (RBs) to compare overall exposure levels. C. Quantitative data (mean ± SEM; n=3 for each cohort and condition) for expression of selected cytokines in control and PKP2 cells with or without Bay 11–7082. * P<0.05 for any cohort vs. control cardiac myocytes; † P<0.05 for Bay 11–7082-treated PKP2 cardiac myocytes vs. untreated PKP2 cardiac myocytes. D. Representative cytokine arrays prepared from culture media (supernatant) from cardiac myocytes derived from a control hiPSC cell line and a line from a patient with a disease causing variant in PKP2. Arrays are shown for media isolated from cells grown in the absence or presence of Bay 11–7082. The spots in the upper right and left and lower left corners are reference markers (RBs) to compare overall exposure levels. E. Quantitative data (mean ± SEM; n=3 for each cohort andcondition) for expression of selected cytokines in media from control and PKP2 cells with or without Bay 11–7082. * P<0.05 for any cohort vs. control cardiac myocytes; † P<0.05 for Bay 11–7082 treated PKP2 cardiac myocytes vs. untreated PKP2 cardiac myocytes. Statistical analyses in panels C and E were performed using one-way ANOVA with Tukey’s multiple comparisons test.

Article Snippet: Following completion of functional studies, mice were euthanized and hearts were excised and processed for additional studies either by freezing fresh tissue (−80C) or fixing tissue in 10% buffered formalin and embedding in paraffin. hiPSC-cardiac myocyte differentiation and Bay11–7082 treatment These studies involved a previously characterized hiPSC line from an ACM patient with a c.2013delC (p.Lys672Argfs*12) mutation in plakophilin-2 ( PKP2 ) originally produced by Joseph Wu at Stanford and described previously, 8 and an unrelated healthy control hiPSC line previously derived at Johns Hopkins by Gordon Tomaselli and colleagues.

Techniques: Immunofluorescence, Derivative Assay, Control, Variant Assay, Staining, Expressing, Isolation