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a Schematic overview of three distinct culture conditions used for mouse ES cell propagation: <t>FBS–LIF,</t> <t>2i-LIF</t> 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 .
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a Schematic overview of three distinct culture conditions used for mouse ES cell propagation: <t>FBS–LIF,</t> <t>2i-LIF</t> 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 .
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a Schematic overview of three distinct culture conditions used for mouse ES cell propagation: <t>FBS–LIF,</t> <t>2i-LIF</t> 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 .
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a Schematic overview of three distinct culture conditions used for mouse ES cell propagation: <t>FBS–LIF,</t> <t>2i-LIF</t> 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 .
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a Schematic overview of three distinct culture conditions used for mouse ES cell propagation: <t>FBS–LIF,</t> <t>2i-LIF</t> 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 .
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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 .

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