Journal: Experimental & Molecular Medicine

Article Title: Uncovering the acetylation sites of Dnmt3L that regulate protein stability and differentiation potency in embryonic stem cells

doi: 10.1038/s12276-026-01655-w

Figure Lengend Snippet: a Schematic overview of three distinct culture conditions used for mouse ES cell propagation: FBS–LIF, 2i-LIF and 2i-LIF-KSR. b Western blot analysis of Dnmt proteins and cofactors in R1 mouse ES cells maintained under the indicated conditions at early and late passages. Molecular weight marker sizes (kDa) are indicated on the left. c Temporal dynamics of Dnmt3L and Dnmt3A expression at the transcript (bars) and protein (lines) levels during long-term propagation in 2i-LIF (left) and 2i-LIF-KSR (right) conditions ( n = 4). Outlier values falling outside the plotted range are indicated as numbers above the corresponding passages. d Reactivation of Dnmt3L and other de novo Dnmts at the transcript and protein levels upon re-exposure to FBS–LIF after extended 2i-LIF or 2i-LIF-KSR culture ( n = 4). e Heat map illustrating the expression changes of transcripts associated with pluripotency, germline development, genomic imprinting and DNA methylation under distinct culture conditions. Expression values were calculated from four independent biological replicates. f CHX-chase assay to assess the protein stabilities of Dnmt3L and Dnmt3A2 in mouse ES cells at p6 under the 2i-LIF condition. Short (s.e) and long (l.e) exposures are shown. g Quantitative analysis of Dnmt3L (red), Dnmt3A2 (black) and Dnmt3B (blue) protein levels from the CHX-chase assay ( n = 4). Protein expression was normalized to β-actin. Quantitative data are represented as mean ± standard error of the mean (s.e.m.). Statistical analysis was performed using a two-way ( c and g ) or one-way ( d ) ANOVA with the Bonferroni post hoc test. * P < 0.05, ** P < 0.01, *** P < 0.001 compared with control groups. ### P < 0.001 compared with Dnmt3A2. Exact P values and replicate numbers are provided in the . See also Supplementary Figs. and .

Article Snippet: To maintain naive pluripotency, mouse ES cells were maintained in 2i-LIF medium containing 3 μM CHIR99021 (BioGems) and 1 μM PD0325901 (BioGems) under the following two conditions on a 0.1% gelatin-coated tissue culture dish: FBS free or supplemented with knockout serum replacement (KSR) (Thermo Fisher Scientific).

Techniques: Western Blot, Molecular Weight, Marker, Expressing, DNA Methylation Assay, Control

Journal: Nature Cell Biology

Article Title: The nuclear periphery confers repression on H3K9me2-marked genes and transposons to shape cell fate

doi: 10.1038/s41556-025-01703-z

Figure Lengend Snippet: ( a ) Dendrogram and heatmap of individual LAP2β CUT & RUN replicates (3 per condition) showing similarity of replicates for each genotype. ( b ) Akaike Information Criterion (AIC) and Bayesian Information Criterion (BIC) values ( b ) and differences ( c ) between numbers of LAP2β domain states used in HMM domain calling. ( d ) Spike-in-controlled LAP2β tracks across a 50 Mb segment of chromosome 12 and corresponding 2-state HMM domain calls for 3 replicates of LAP2β CUT & RUN in WT mESCs. ( e ) Overlap between individual replicate LAP2β domains called by 2-state HMM in WT mESCs determined by multiinter package (Bedtools). ( f ) Comparison of LAP2β and LB1 domain protein-coding genes defined in this study versus previously defined LAD-resident protein-coding genes (determined by LB1 ChIP-seq) in mESCs in 2i + LIF culture conditions (PMID 29033129). ( g ) Number of LAP2β and LB1 domains identified in WT mESCs. ( h ) Genomic length of LAP2β and LB1 domains in WT mESCs.

Article Snippet: mES cells were cultured at 37 °C in 5% CO 2 under normoxic conditions in serum-free 2i + LIF medium (N2B27 basal medium, 3 μM CHIR-99021 (Selleckchem, S1263), 1 μM PD0325901 (Selleckchem, S1036), 10 3 U ml −1 LIF (Millipore Sigma, ESG1107), 55 μM β-mercaptoethanol (Gibco, 21985023) and 1× penicillin–streptomycin (GenClone, 25-512)).

Techniques: Comparison, ChIP-sequencing

Journal: Nature Cell Biology

Article Title: The nuclear periphery confers repression on H3K9me2-marked genes and transposons to shape cell fate

doi: 10.1038/s41556-025-01703-z

Figure Lengend Snippet: ( a ) Dendrogram and heatmap of individual H3K9me2 CUT & RUN replicates (3 per condition) showing similarity of replicates for each genotype. ( b ) Akaike Information Criterion (AIC) and Bayesian Information Criterion (BIC) values ( b ) and differences ( c ) between numbers of H3K9me2 domain states used in HMM domain calling. ( d ) Spike-in-controlled H3K9me2 tracks across a 50 Mb segment of chromosome 12 and corresponding 2-state HMM domain calls for 3 replicates of H3K9me2 CUT & RUN in WT mESCs. ( e ) Overlap between individual replicate H3K9me2 domains called by 2-state HMM in WT mESCs determined by multiinter package (Bedtools). ( f ) Breakdown of WT mESC H3K9me2 domain genes, defined as those genes at least 90% within an H3K9me2 domain, by gene class. # indicates that lncRNAs are significantly depleted compared to the mouse genome (two-sided χ 2 test, p < 0.0001) while * indicates that pseudogenes are significantly enriched in H3K9me2 domains compared to the mouse genome (two-sided χ 2 test, p < 0.0001). ( g ) Overlap of H3K9me2 domain protein-coding genes defined in this study versus in a previous list of protein-coding genes within H3K9me2 domains identified by ChiP-seq in mESCs in 2i + LIF culture conditions (PMID 29033129). ( h ) Number of H3K9me2 domains identified across genotypes. ( i ) Genomic length of H3K9me2 domains across genotypes. ( j ) Kernel density plot showing distribution of spike-in-controlled H3K8me2 signal across genotypes.

Article Snippet: mES cells were cultured at 37 °C in 5% CO 2 under normoxic conditions in serum-free 2i + LIF medium (N2B27 basal medium, 3 μM CHIR-99021 (Selleckchem, S1263), 1 μM PD0325901 (Selleckchem, S1036), 10 3 U ml −1 LIF (Millipore Sigma, ESG1107), 55 μM β-mercaptoethanol (Gibco, 21985023) and 1× penicillin–streptomycin (GenClone, 25-512)).

Techniques: ChIP-sequencing

Journal: Life Science Alliance

Article Title: Distinctive features of lincRNA gene expression suggest widespread RNA-independent functions

doi: 10.26508/lsa.201800124

Figure Lengend Snippet: (A) Bulk RNA-seq heat map of differentially expressed mRNAs and lincRNAs in mESCs grown in 2i + LIF medium, mESCs grown in serum + LIF medium, or derived NPCs. Black bars on the left indicate lincRNAs, and lincRNAs explored in this study are labelled on the right. The 2,000 genes with highest variance were selected. (B) An example of a lincRNA with strong differential expression (bulk RNA-seq) during mESC to NPC differentiation. (C) LincRNA and mRNA expression distributions from bulk RNA-seq analysis of mESCs (serum + LIF). (D) Two-dimensional t-SNE (t-Distributed Stochastic Neighbor Embedding) projection of single cells during mESC to NPC differentiation, coloured by sample (“cell annotations”) or the expression of marker genes. (E) Heat map showing the most variably expressed mRNAs and lincRNAs during mESC to NPC differentiation (rows), across single cells (columns). Protein-coding genes encoding stem cell, neuronal, or cell cycle factors are highlighted (green, blue, and violet bars), as are lincRNAs (black bars). LincRNAs selected for further study are indicated by red bars. (F) Magnified view of the highlighted region (red rectangle) from (E), revealing subpopulations of serum + LIF mESCs with characteristic expression of pluripotency markers (rows indicated with orange labels) and lincRNAs (rows indicated with black bars). Previously reported subpopulations are indicated (columns highlighted by green bars underneath). See also and Table S1A.

Article Snippet: Where indicated, mESCs were grown in 2i medium (NDiff 227; StemCells, with 0.33 vol/vol LIF-conditioned medium, 1 μM MEK inhibitor PD 0325901 [Tocris], and 3 μM GSK3 inhibitor CHIR99021 [Calbiochem]) on gelatin-coated dishes.

Techniques: RNA Sequencing, Derivative Assay, Quantitative Proteomics, Expressing, Marker

Journal: Life Science Alliance

Article Title: Distinctive features of lincRNA gene expression suggest widespread RNA-independent functions

doi: 10.26508/lsa.201800124

Figure Lengend Snippet: (A) Expression of highly variable lincRNAs (rows; listed in Table S1B) in single cells (columns), clustered by lincRNA expression. Cell types (mESCs in 2i + LIF or serum + LIF, and day 6 and 8 NPCs) and cell cycle stages (assigned by clustering on cell cycle marker gene expression) are indicated at the top. (B) Cell-to-cell variation in expression for mRNAs (grey) and lincRNAs (red), across mESCs (top), or NPCs (bottom), quantified using the DM method . The median DMs for lincRNAs and mRNAs are listed, and the distributions tested for similarity using the Wilcoxon rank sum test. (C) Scatter plot comparing the 90th expression percentile across single cells versus the median expression, for each lincRNA (red) and mRNA (grey). (D, E) Single cell expression pattern of a highly variable mESC lincRNA (D, Pvt1 ) and a uniformly expressed mESC lincRNA (E, linc1333 ), overlaid on the t-SNE projection. (F) An example of correlated lincRNA-mRNA single cell expression across mESC to NPC differentiation, with cells ordered by pseudotime determined using Monocle . (G) Top panels: cell cycle stage assignments for day 8 NPCs based on single cell expression of marker genes . Bottom panels: examples of three lincRNAs expressed during specific cell cycle stages ( XLOC_071380 = G1/S enriched, and Miat and Rmst = G2/M enriched). For each lincRNA, the heat bars show expression levels across the 67 G1/S cells (top bar) and 48 G2/M cells (bottom bar). In (B, C), key lincRNAs selected for detailed exploration are highlighted in green. See also and Tables S1A, B, and S3.

Article Snippet: Where indicated, mESCs were grown in 2i medium (NDiff 227; StemCells, with 0.33 vol/vol LIF-conditioned medium, 1 μM MEK inhibitor PD 0325901 [Tocris], and 3 μM GSK3 inhibitor CHIR99021 [Calbiochem]) on gelatin-coated dishes.

Techniques: Expressing, Marker, Gene Expression