Journal: eLife

Article Title: Dynamic enhancer partitioning instructs activation of a growth-related gene during exit from naïve pluripotency

doi: 10.7554/eLife.44057

Figure Lengend Snippet: ( A ) Imprinted regulation of Liz and Zdbf2 in vivo. The Liz promoter is DNA methylated and silent on the maternal allele. Conversely, the paternal allele is expressed, leading to de novo DNA methylation at the paternal sDMR, and Zdbf2 activation. The epigenetic setting is programmed around the time of implantation in embryogenesis, but is then stably maintained throughout life. ( B ) Expression dynamics of promoter switch during protracted EpiLC differentiation. The pLiz -to- pZdbf2 promoter switch occurs at roughly D4. pLiz is shut down and pZdbf2 is active by D7. ( C ) Hierarchical clustering using correlation-based distance of WT RNA-seq data during EpiLC differentiation. Samples generated in this study are highlighted in bold and italic type. In comparison with publicly available data , D4 and D7 EpiLCs cluster more closely with EpiSCs, indicating a primed pluripotency state. ( D ) Plots of reads per kilobase million (RPKM) from RNA-seq of WT cells during EpiLC differentiation for selected markers. Upregulation of markers of primed pluripotency indicate that D7 EpiLCs have entered a primed pluripotency state.

Article Snippet: WGBS data from ESCs were previously generated ( ) and EpiLCs were prepared from 50 ng of bisulfite-converted genomic DNA using the EpiGnome/Truseq DNA Methylation Kit (Illumina) following the manufacturer instructions.

Techniques: In Vivo, Methylation, DNA Methylation Assay, Activation Assay, Stable Transfection, Expressing, RNA Sequencing, Generated, Comparison

Journal: eLife

Article Title: Dynamic enhancer partitioning instructs activation of a growth-related gene during exit from naïve pluripotency

doi: 10.7554/eLife.44057

Figure Lengend Snippet: ( A ) Chromatin and expression landscape at the Zdbf2 locus in ESCs (top) and D7 EpiLCs (bottom). In hypomethylated naïve ESCs, Zdbf2 initiates from the pLiz promoter while a ~ 25 kb block of H3K27me3 extends through pZdbf2 . Upon EpiLC differentiation, the H3K27me3 signal depletes while DNA methylation is gained; the pLiz ATAC-seq peak decreases concomitantly with decreased expression, while pZdbf2 becomes the main promoter. Four prominent ATAC-seq peaks of accessible chromatin (E1 to E4) lie between pLiz and pZdbf2 promoters. WGBS: Whole genome bisulfite sequencing. H3K27me3 ChIP-seq data is from . All other genomics data were generated for this study. One representative biological replicate is displayed for each RNA-seq track. ( B ) H3K27ac ChIP-qPCR shows enrichment for this mark at E1 to E3 in ESCs and D7 EpiLCs, while E4 only becomes enriched in EpiLCs. Data shown as ±s.e.m. from three biological replicates. ( C ) 4C-seq tracks from the pLiz (top) and pZdbf2 (bottom) viewpoints (VPs) in ESCs and D7 EpiLCs. ESC/EpiLC ratio between 4C-seq signals is indicated. pLiz interactions with E1-3 are more frequent in ESCS, but remain high in EpiLCs, likely due to proximity. pLiz seems less restricted in EpiLCs, with interactions spreading on the ‘right’ side of the locus. pZdbf2 exhibits increased interactions at E1 to E4 in EpiLCs over ESCs. Data from one representative biological replicate (two total). Statistical analyses were performed by two-tailed unpaired t-test: n.s = not significant, **p≤0.01, ***p≤0.001. 10.7554/eLife.44057.006 Figure 1—source data 1. Source data for .

Article Snippet: WGBS data from ESCs were previously generated ( ) and EpiLCs were prepared from 50 ng of bisulfite-converted genomic DNA using the EpiGnome/Truseq DNA Methylation Kit (Illumina) following the manufacturer instructions.

Techniques: Expressing, Blocking Assay, DNA Methylation Assay, Methylation Sequencing, ChIP-sequencing, Generated, RNA Sequencing, ChIP-qPCR, Two Tailed Test

Journal: eLife

Article Title: Dynamic enhancer partitioning instructs activation of a growth-related gene during exit from naïve pluripotency

doi: 10.7554/eLife.44057

Figure Lengend Snippet: ( A ) Model for enhancer regulation based on 4C-seq data. ( B ) Model for deletion of E1-3.. ( C ) RT-qPCR of Liz (left) and Zdbf2 (right) during EpiLC differentiation in WT and the ∆E1-3 mutant. The ∆E1-3 mutation significantly reduces both transcripts. Data are shown as ±s.e.m. from five and three biological replicates for WT and mutant, respectively. ( D ) DNA methylation of pLiz (left) and the sDMR (right) during EpiLC differentiation as measured by bisulfite conversion followed by pyrosequencing (BS-pyro) in WT and the ∆E1-3 mutant. When Liz fails to activate, DNA methylation is acquired faster at pLiz , and fails to properly accumulate at the sDMR. Data shown as ±s.e.m. from five and three biological replicates for WT and mutant, respectively. ( E ) Model for deletion of E4. ( F ) RT-qPCR of Liz (left) and Zdbf2 (right) during EpiLC differentiation in WT and the ∆E4 mutant. There is no effect on Liz expression dynamics, but Zdbf2 does not properly activate. Data shown as ±s.e.m. from four biological replicates for each genotype. ( G ) DNA methylation of pLiz (left) and the sDMR (right) during EpiLC differentiation as measured by BS-pyro in WT and the ∆E4 mutant. DNA methylation is unperturbed in the ∆E4 mutant. Data are shown as ±s.e.m. from four biological replicates for each genotype. ( H ) H3K27me3 ChIP-qPCR in ESCs (left) and EpiLCs (right). There is no significant effect on polycomb dynamics in ∆E4 mutation, except mild ectopic spreading upstream of the sDMR region. pPax5 and pOct4 are positive and negative controls, respectively. Data shown as ±s.e.m. from three biological replicates for each genotype. Statistical analyses were performed by two-tailed unpaired t-test: n.s = not significant, *p≤0.05, **p≤0.01, ***p≤0.001. 10.7554/eLife.44057.009 Figure 2—source data 1. Source data for .

Article Snippet: WGBS data from ESCs were previously generated ( ) and EpiLCs were prepared from 50 ng of bisulfite-converted genomic DNA using the EpiGnome/Truseq DNA Methylation Kit (Illumina) following the manufacturer instructions.

Techniques: Quantitative RT-PCR, Mutagenesis, DNA Methylation Assay, Expressing, ChIP-qPCR, Two Tailed Test

Journal: eLife

Article Title: Dynamic enhancer partitioning instructs activation of a growth-related gene during exit from naïve pluripotency

doi: 10.7554/eLife.44057

Figure Lengend Snippet: ( A ) Alleles generated by CRISPR/Cas9 mediated deletions of enhancer elements. ( B ) RT-qPCR of Liz (left) and Zdbf2 (right) during EpiLC differentiation in WT and the ∆E3 mutant. There is no effect on Liz or Zdbf2 expression. Data shown as ±s.e.m. from at least four biological replicates for each genotype. ( C ) DNA methylation of pLiz (left) and the sDMR (right) during EpiLC differentiation as measured by BS-pyro in WT and the ∆E3 mutant. DNA methylation is unperturbed. Data shown as ±s.e.m. from four biological replicates for each genotype. ( D ) H3K27me2 ChIP-qPCR at E4 in WT ESCs and EpiLCs. E4 is depleted for the mark in both cell types. Data shown as ±s.e.m. from three biological replicates. Ctsh and Oct4 are positive and negative controls, respectively.

Article Snippet: WGBS data from ESCs were previously generated ( ) and EpiLCs were prepared from 50 ng of bisulfite-converted genomic DNA using the EpiGnome/Truseq DNA Methylation Kit (Illumina) following the manufacturer instructions.

Techniques: Generated, CRISPR, Quantitative RT-PCR, Mutagenesis, Expressing, DNA Methylation Assay, ChIP-qPCR

Journal: eLife

Article Title: Dynamic enhancer partitioning instructs activation of a growth-related gene during exit from naïve pluripotency

doi: 10.7554/eLife.44057

Figure Lengend Snippet: ( A ) 4C-seq tracks from the CTCF_PS VP in WT ESCs and EpiLCs. Ratios between 4C-seq signals is indicated between the samples, and gene and CTCF binding tracks are below. The CTCF_PS forms a loop with two CTCF sites downstream of the Gpr1 gene. The looping diminishes in EpiLCs. ( B ) CTCF ChIP-qPCR in WT ESCs and EpiLCs and Eed-/- ESCs. CTCF binding remains unchanged in all conditions on the two sites downstream of Gpr1 (termed CTCF Left_1 and Left_2). At the CTCF_PS, CTCF binding reduces from WT ESCs to EpiLCs, which is correlated with decreased interactions between CTCF_PS and the two upstream CTCF sites. CTCF binding remains enriched in Eed-/- ESCs, consistent with the maintained loop structure. Data shown as ±s.e.m. from three biological replicates. ( C ) CTCF ChIP-qPCR in WT, Dnmt tKO and ∆Liz ESCs and EpiLCs. CTCF binding is depleted at the CTCF_PS in EpiLCs in all three contexts. Data shown as ±s.e.m. from three biological replicates. ( D ) Model for CTCF-mediated partitioning of the locus. In ESCs, pLiz and E1-3 are active, and physically separated from the silent E4 and pZdbf2 . During differentiation, the partition is diminished, allowing E1-3 to bolster pZdbf2 activation, while pLiz has become silent. ( E ) RT-qPCR of Liz (left) and Zdbf2 (right) during EpiLC differentiation in WT and the ∆CTCF_PS mutant. Liz is less expressed in mutant ESCs, but reaches WT levels of expression during differentiation. Nevertheless, Zdbf2 does not properly activate in the mutant. Data are shown as ±s.e.m. from four biological replicates for each genotype. ( F ) DNA methylation of pLiz (left) and the sDMR (right) during EpiLC differentiation as measured by BS-pyro in WT and the ∆ mutant. DNA methylation is unperturbed in the mutant at pLiz , but is reduced at the sDMR. Data shown as ±s.e.m. from four biological replicates for each genotype. ( G ) RT-qPCR of Zdbf2 in ESCs in absence of CTCF partition and/or PRC2. While Zdbf2 is already upregulated in the Eed mutant, this effect is exacerbated in the absence of the partition, likely because pZdbf2 is less restrained from interacting with E1-3. Data shown as ±s.e.m. from three biological replicates for each genotype. Statistical analyses were performed by two-tailed unpaired t-test: n.s. = not significant, *p≤0.05, **p≤0.01, ***p≤0.001. 10.7554/eLife.44057.017 Figure 4—source data 1. Source data for .

Article Snippet: WGBS data from ESCs were previously generated ( ) and EpiLCs were prepared from 50 ng of bisulfite-converted genomic DNA using the EpiGnome/Truseq DNA Methylation Kit (Illumina) following the manufacturer instructions.

Techniques: Binding Assay, ChIP-qPCR, Activation Assay, Quantitative RT-PCR, Mutagenesis, Expressing, DNA Methylation Assay, Two Tailed Test

Journal: Proceedings of the Japan Academy. Series B, Physical and Biological Sciences

Article Title: Efficient induction of transgene-free human pluripotent stem cells using a vector based on Sendai virus, an RNA virus that does not integrate into the host genome

doi: 10.2183/pjab.85.348

Figure Lengend Snippet: Expression of exogenous genes in human fibroblasts by SeV vectors. A. Efficient induction of GFP cDNA by TSΔF/SeV in BJ and HDF at an MOI of 3. BC: bright contrast. B. Schematic presentation of SeV vector genomes. Reprogramming genes were inserted at 18+, PM, HN, HNL and Leis (L), respectively. The expression levels of inserted genes decreased depending on the inserted site (polar effect: Refs. ) as shown by Western blotting on day 3 after infection. Anti-SeV blot was performed to confirm equal infection efficiency of the vectors.

Article Snippet: Human fibroblast BJ from neonatal foreskin (ATCC, USA), and HDF from facial dermis of 36-year femail (Cell Applications, Inc., USA), were maintained in fibroblast growth medium (Cell Applications, Inc., USA) or DMEM (Invitrogen, USA) supplemented with 10% FBS.

Techniques: Expressing, Plasmid Preparation, Western Blot, Infection

Journal: Proceedings of the Japan Academy. Series B, Physical and Biological Sciences

Article Title: Efficient induction of transgene-free human pluripotent stem cells using a vector based on Sendai virus, an RNA virus that does not integrate into the host genome

doi: 10.2183/pjab.85.348

Figure Lengend Snippet: Genomic Southern blot, karyotyping and fingerprinting of SeV-iPSC. A. DNA fingerprinting of SeV-iPS clones. PCR analysis of three variable number of tandem repeats (VNTR) loci of D17S1290, MCT118 and ApoB-100 using genomic DNA from the SeV-iPS clones confirmed that these clones were originated from human fibroblasts BJ or HDF. B1, HNL1 and HNL5 were derived from BJ; XH1, 7H5, 7H8 and 7H10 were from HDF. B. Viral transgenes were not detected from the host genome as analyzed by genomic Southern blot. C. Karyotyping of SeV-iPSC. Viral-free SeV-iPSC HNLs at passage 34 were used for karyotyping.

Article Snippet: Human fibroblast BJ from neonatal foreskin (ATCC, USA), and HDF from facial dermis of 36-year femail (Cell Applications, Inc., USA), were maintained in fibroblast growth medium (Cell Applications, Inc., USA) or DMEM (Invitrogen, USA) supplemented with 10% FBS.

Techniques: Southern Blot, DNA Profiling, Clone Assay, Derivative Assay

Journal: Proceedings of the Japan Academy. Series B, Physical and Biological Sciences

Article Title: Efficient induction of transgene-free human pluripotent stem cells using a vector based on Sendai virus, an RNA virus that does not integrate into the host genome

doi: 10.2183/pjab.85.348

Figure Lengend Snippet: In vivo pluripotency of transgenes-free SeV-iPSC. Hematoxylin and eosin staining of teratoma sections of SeV-iPS clones (6 weeks post-injection into SCID mice). Tissues were differentiated from Tg-free human neonatal fibroblast BJ-derived HNLs (A to C), HNL1 (D to I), and adult fibroblast HDF-derived XH1 (J to L) containing multiple tissues derived from three germ layers: glandular structures (A, G, K), cartilage (B, J), bone (C, F, white arrows) and bone marrow-like structure (F), epithelium (J, D), transitional epithelium (E), population of secreting-like cells (B, D, indicated by black arrows), muscle (C, I, K, L), and glomerulus of kidney-like tissue (H).

Article Snippet: Human fibroblast BJ from neonatal foreskin (ATCC, USA), and HDF from facial dermis of 36-year femail (Cell Applications, Inc., USA), were maintained in fibroblast growth medium (Cell Applications, Inc., USA) or DMEM (Invitrogen, USA) supplemented with 10% FBS.

Techniques: In Vivo, Staining, Clone Assay, Injection, Derivative Assay

Journal: Cell Proliferation

Article Title: Crotonylation of MCM6 enhances chemotherapeutics sensitivity of breast cancer via inducing DNA replication stress

doi: 10.1111/cpr.13759

Figure Lengend Snippet: Knockdown of minichromosome maintenance 6 (MCM6) inhibited cells growth and induced cells apoptosis by inducing DNA replication stress. (A) MCF7 and 4 T1 cells transiently transfected with control siRNA (siNC) or MCM6 siRNA (siMCM6#1, siMCM6#2) were subjected to western blot analysis. (B) Cell growth curves were measured by cell proliferation assay. (C–D) Cell proliferation was detected by colony formation (C) and BrdU staining (D). Quantitative analysis of colony numbers is shown in the right panel. For colony formation assays, colonies containing more than 50 cells were counted and plotted. (E) The cell cycle of MCF7 and 4 T1 cells was analysed by flow cytometry. (F) DNA synthesis in MCF7 cells was determined by a DNA fibre assay. Cells were labelled with CldU (20 min) and IdU (40 min). (G) Representative images of neutral comet assay in siNC and siMCM6 cells. Quantification of DNA damage was determined by tail moment using CASP software in the right panel. (H) MCM6‐knockdown MCF7 cells were treated with 5 mM HU, followed by western blotting to confirm γH2AX expression. (I) MCF7 cells were transduced with siRNA against MCM6. The repression of BRCA1 and Rad51 was measured by western blotting. (J) AnnexinV‐PE and 7‐AAD flow‐cytometric analysis of siMCM6 MCF7 cells. (K) Viability of MCF7 and 4T1cells was analysed by MTT 48 h after treatment with different concentrations of paclitaxel. Mean ± SD, n = 3. * p < 0.05, ** p < 0.01, *** p < 0.001, versus control. BRCA, breast invasive carcinoma.

Article Snippet: Sodium crotonate (NaCR, Absin, abs42074236), hycamptothecin (HCPT, Solarbio, IH2120), hydroxycarbamide (HU, Solarbio, H8420) and paclitaxel (PTX, Solarbio, YZ‐100382) were dissolved in DMSO and used at the specified concentrations and times as indicated.

Techniques: Knockdown, Transfection, Control, Western Blot, Proliferation Assay, BrdU Staining, Flow Cytometry, DNA Synthesis, Neutral Comet Assay, Software, Expressing, Transduction

Journal: Cell Proliferation

Article Title: Crotonylation of MCM6 enhances chemotherapeutics sensitivity of breast cancer via inducing DNA replication stress

doi: 10.1111/cpr.13759

Figure Lengend Snippet: MCM6‐K599cr induced DNA replication stress and caused cells apoptosis. C Cell proliferation was detected by proliferation assay (A), BrdU staining (B) and colony formation (C). Quantitative analysis of colony numbers is shown in the right panel. For colony formation assays, colonies containing more than 50 cells were counted and plotted. (D) The cell cycle of MCF7 and 4 T1 cells was analysed by flow cytometry. (E) DNA synthesis in MCF7 cells was determined by a DNA fibre assay. Cells were labelled with CldU (20 min) and IdU (40 min). (F) Representative images of neutral comet assay in control (CTRL), MCM6‐K599Q (K599Q), and MCM6‐K599A (K599A) cells. Quantification of DNA damage was determined by tail moment using CASP software in the right panel. (G) MCM6‐mutant cells were treated with 5 mM HU, followed by western blotting to confirm γH2AX, BRCA1 and Rad51 expression. (H) AnnexinV‐PE and 7‐AAD flow‐cytometric analysis of all MCF7 cells. (I) Viability of MCF7 cells was analysed by MTT 48 h after treatment with different concentrations of HU and paclitaxel. Mean ± SD, n = 3. * p < 0.05, ** p < 0.01, *** p < 0.001, versus control. BRCA, breast invasive carcinoma.

Article Snippet: Sodium crotonate (NaCR, Absin, abs42074236), hycamptothecin (HCPT, Solarbio, IH2120), hydroxycarbamide (HU, Solarbio, H8420) and paclitaxel (PTX, Solarbio, YZ‐100382) were dissolved in DMSO and used at the specified concentrations and times as indicated.

Techniques: Proliferation Assay, BrdU Staining, Flow Cytometry, DNA Synthesis, Neutral Comet Assay, Control, Software, Mutagenesis, Western Blot, Expressing

Journal: Cell Proliferation

Article Title: Crotonylation of MCM6 enhances chemotherapeutics sensitivity of breast cancer via inducing DNA replication stress

doi: 10.1111/cpr.13759

Figure Lengend Snippet: Kaempferol inhibited tumour growth via enhancing paclitaxel (PTX) sensitivity. (A) Cell proliferation was detected by proliferation assay after treatment with 50 μM kaempferol or 10 nM paclitaxel alone, 50 μM paclitaxel and 10 nM kaempferol combination. (B) Schematic diagram of 4 T1‐bearing mice modelling and in vivo experimental procedure. (C) Representative images of the tumour tissues in each group. (D–E) Representative images of the tumour weight (D) and volume (E) in each group. (F) Body weight changes in each group. (G) Immunohistochemistry images of SIRT7 and Kcr expression in each group. (H) Extraction the proteins from each group tissues, then analysed by immunoprecipitation using the indicated antibodies. Mean ± SD, n = 3. * p < 0.05, ** p < 0.01, *** p < 0.001, versus control.

Article Snippet: Sodium crotonate (NaCR, Absin, abs42074236), hycamptothecin (HCPT, Solarbio, IH2120), hydroxycarbamide (HU, Solarbio, H8420) and paclitaxel (PTX, Solarbio, YZ‐100382) were dissolved in DMSO and used at the specified concentrations and times as indicated.

Techniques: Proliferation Assay, In Vivo, Immunohistochemistry, Expressing, Extraction, Immunoprecipitation, Control

Journal: Current biology : CB

Article Title: Synaptic-like Vesicles Facilitate Pioneer Axon Invasion.

doi: 10.1016/j.cub.2019.06.078

Figure Lengend Snippet: Figure 7. Modulation of Actin Structures Alters Vesicles at the DREZ (A, C, and E) Confocal z-projections of pioneer growth cone actin organization in Tg(sox10:syn-gfp) in animals treated with DMSO (A), SU6656 (C), and paclitaxel (E) from a 24-h time lapse starting at 48 hpf. White arrows denote actin concentrations at DREZ. White box denotes insets on right. Dashed blue lines denote DRG tracings. Note: fluorescence outside of white box in (E) is a spinal oligodendrocyte. (B, D, and F) Growth cone Syn-GFP intensity tracings throughout navigation in DMSO (B; n = 6), SU6656 (D; n = 7), and paclitaxel (F; n = 7) DRG. Inhibition of actin- based invasion abrogates Syn-GFP accumulation, and promotion of actin-based invasion generates robust Syn-GFP accumulation. Green line denotes representative graph. Dashed black line denotes mean ± SEM. Blue box denotes DREZ. (G) Percent of axons that demonstrate vesicle accumulations at DREZ (n = 6 DMSO; n = 7 SU6656; n = 7 paclitaxel). (H and I) Duration (H) and time of initiation (I) of vesicle concentrates at the DREZ showing changes in vesicles when actin invasion is altered (n = 6 DMSO; n = 7 SU6656; n = 7 paclitaxel). (J) Model of actin and synaptic-like vesicle coordinated invasion of the spinal cord by DRG pioneer axons. SEM is shown. Tukey’s HSD was used for (H) and (I). Scale bars, 10 mm. See also Video S6.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Rabbit anti-Synapsin 1/2 Synaptic Systems Cat #: 106 002; RRID: AB_2622240 Anti-Rab8a mAb produced in mouse Abnova Cat #: H00004218-M02; RRID: AB_519014 Anti-MMP14 antibody produced in rabbit Sigma Cat #: SAB4501901; RRID: AB_10744760 Znp-1 (anti-synaptotagmin 2) DHSB RRID: AB_10013783 SV2 (synaptic vesicle glycoprotein 2A) DHSB RRID: AB_2315387 Alexa Fluor 594 goat anti-mouse Thermo Fisher Cat #: A-11032; RRID: AB_2535767 Alexa Fluor 594 goat anti-rabbit Thermo Fisher Cat #: R37117; RRID: AB_2534116 Alexa Flour 647 goat anti-mouse Thermo Fisher Cat #: A-21235; RRID: AB_2535806 Alexa Flour 647 goat anti-rabbit Thermo Fisher Cat #: A-20991; RRID: AB_2535814 Alexa Flour 488 goat anti-rabbit Thermo Fisher Cat #: A-11034; RRID: AB_2576217 Bacterial Strains Mix & Go Competent Cells Zymo Research Cat #: T3007 Chemicals, Peptides, and Recombinant Proteins Paclitaxel Acros Cat #: 328420050; CAS: 33069-62-4 GM6001 Santa Cruz Biotechnology Cat #: sc-203979; CAS: 142880-36-2 SU6656 Santa Cruz Biotechnology Cat #: sc-203286; CAS: 330161-87-0 LR Clonease II Plus Thermo Fisher Cat #: 12538120 TRIzol Thermo Fisher Cat #: 15596026 Chloroform Sigma-Aldrich Cat #: C2432; CAS: 67-66-2 Isopropanol Sigma-Aldrich Cat #: W292907; CAS: 67-63-0 ProtoScript II First Strand cDNA Synthesis Kit New England Biolabs Cat # E6300S BseRI New England Biolabs Cat #: R5081S Critical Commercial Assays RNAscope Multiplex Fluorescent Reagent Kit v2 Advanced Cell Diagnostics Cat #: 323100 RNAscope Probe-Dr-mmp14a Advanced Cell Diagnostics Cat #: 575081 RNAscope Probe-Dr-polr2 Advanced Cell Diagnostics Cat #: 559921 RNAscope 3-plex Negative Control Probe Advanced Cell Diagnostics Cat #: 320871 Opal 650 Fluorophore Akoya Biosciences Cat #: FP1496001KT Experimental Models: Organisms/Strains

Techniques: Inhibition

Journal: Acta Neuropathologica

Article Title: “De novo replication repair deficient glioblastoma, IDH-wildtype” is a distinct glioblastoma subtype in adults that may benefit from immune checkpoint blockade

doi: 10.1007/s00401-023-02654-1

Figure Lengend Snippet: “De novo replication repair deficient glioblastoma, IDH-wildtype” has a distinct global hypomethylation epigenetic signature compared to conventional glioblastoma, IDH-wildtype. a Violin plot of DNA methylation data showing the mean beta-value for each of approximately 850,000 CpG sites across 7 de novo RRD glioblastomas and 98 conventional glioblastomas. b Heatmap of DNA methylation profiles for 7 de novo RRD glioblastoma alongside 98 conventional glioblastoma. Shown are the 5000 most differentially methylated probes amongst the 105 glioblastomas revealing extensive hypomethylation of CpG sites in the de novo RRD glioblastoma compared to the cohort of conventional glioblastomas. See Supplementary Table S5 for detailed annotations of these 5000 most differentially methylated CpG sites

Article Snippet: In addition to the chromosomal copy number data obtained from the targeted next-generation DNA sequencing platform, chromosomal copy number profiles were derived from the Infinium EPIC DNA methylation array data using the conumee package (v.1.26.0) [ ].

Techniques: DNA Methylation Assay, Methylation

Journal: Acta Neuropathologica

Article Title: “De novo replication repair deficient glioblastoma, IDH-wildtype” is a distinct glioblastoma subtype in adults that may benefit from immune checkpoint blockade

doi: 10.1007/s00401-023-02654-1

Figure Lengend Snippet: “De novo replication repair deficient glioblastoma, IDH-wildtype” has a unique epigenetic signature distinct from all established reference methylation classes of IDH-wildtype glioblastomas in adults. a tSNE dimensionality reduction plot of genome-wide DNA methylation profiles for 7 de novo RRD glioblastomas alongside 1143 reference samples spanning 25 CNS tumor methylation groups and 3 control tissue methylation groups. See Supplementary Table S7 for sample manifest. b Results of DNA methylation-based classification for the 7 de novo RRD glioblastoma samples using version 12.7 of the DKFZ Molecular Neuropathology classifier. See Supplementary Table S8 for further details

Article Snippet: In addition to the chromosomal copy number data obtained from the targeted next-generation DNA sequencing platform, chromosomal copy number profiles were derived from the Infinium EPIC DNA methylation array data using the conumee package (v.1.26.0) [ ].

Techniques: Methylation, Genome Wide, DNA Methylation Assay, Control

Journal: Acta Neuropathologica

Article Title: “De novo replication repair deficient glioblastoma, IDH-wildtype” is a distinct glioblastoma subtype in adults that may benefit from immune checkpoint blockade

doi: 10.1007/s00401-023-02654-1

Figure Lengend Snippet: Deconvolution analysis reveals a unique cellular composition of “De novo replication repair deficient glioblastoma, IDH-wildtype” compared to conventional glioblastoma, IDH-wildtype. a Cellular composition of 7 de novo RRD glioblastoma and 98 conventional glioblastoma was estimated by methyCIBERSORT deconvolution of Infinium EPIC DNA methylation profiles at time of initial diagnostic surgery before any adjuvant therapy or immune checkpoint blockade. The de novo RRD glioblastomas demonstrated a greater proportion of microglia and CD8 + T-cells, and lower proportion of regulatory T-cells (Tregs) compared to conventional glioblastoma. See Supplementary Table S9 for source data. b Representative photomicrographs of immunohistochemistry for CD8 and CD163 on a de novo RRD glioblastoma and a conventional glioblastoma for comparison

Article Snippet: In addition to the chromosomal copy number data obtained from the targeted next-generation DNA sequencing platform, chromosomal copy number profiles were derived from the Infinium EPIC DNA methylation array data using the conumee package (v.1.26.0) [ ].

Techniques: DNA Methylation Assay, Diagnostic Assay, Adjuvant, Immunohistochemistry, Comparison