Journal: Frontiers in Endocrinology

Article Title: In Utero Exposure to Metformin Reduces the Fertility of Male Offspring in Adulthood

doi: 10.3389/fendo.2021.750145

Figure Lengend Snippet: Analysis of epigenetic mark in spermatozoa. (A) Immunocytochemistry (green) against 5 methyl-cytosine in spermatozoa. Nucleus was stained with DAPI (blue). Quantification is shown on the right side of the micrographs (n=4 mice, at least 100 spermatozoa per individual were counted). Scale bar = 10 µm. (B) Percentage of 5mC in genomic DNA from spermatozoa was measured by ELISA assay and (C) percentage of genomic DNA methylation in epididymis cauda was analysed by LUMA. (D) Immunocytochemistry (green) against TET1 in spermatozoa. Nucleus was stained with DAPI (blue). Quantification is shown on the right side of the micrographs (n=4, at least 100 spermatozoa per individual were counted). Scale bar = 10 µm. (E) Immunocytochemistry (green) against phospho-Ser36-H2B in spermatozoa. Nucleus was stained with DAPI (blue). IgG was used as negative control. Quantification is shown on the right side of the micrographs (n=3-4, at least 100 spermatozoa per individual were counted). Scale bar = 10 µm. Values are expressed as mean ± SEM. *p < 0.05; ***p < 0.001.

Article Snippet: The global methylation level of each DNA sample was measured using Luminometric Methylation Assay (LUMA), a pyrosequencing-based method , in two independent experiments.

Techniques: Immunocytochemistry, Staining, Enzyme-linked Immunosorbent Assay, DNA Methylation Assay, Negative Control

Journal: Genome Biology

Article Title: Identification of X-chromosomal genes that drive sex differences in embryonic stem cells through a hierarchical CRISPR screening approach

doi: 10.1186/s13059-021-02321-2

Figure Lengend Snippet: Over-expression of Klhl13 and Dusp9 in male mESCs leads to an enhanced pluripotency state and slower differentiation kinetics. a Schematic representation of the dCas9-SunTag system used for gene activation. b–e To over-express Dusp9 (yellow) and Klhl13 (blue), male E14 mESCs, stably expressing the doxycycline-inducible SunTag system, were either transduced with one of two different sgRNAs targeting the respective promoter regions or with non-targeting control (NT) sgRNAs and were treated for 3 days with 1 μg/ml doxycycline as indicated. Protein levels of Dusp9 (left) and Klhl13 (right) were quantified via immunoblotting ( b ), expression levels of MAPK target genes Spry4 and Egr1 ( c ) and of naive pluripotency factors Nanog and Prdm14 ( e ) were assessed by qPCR and phosphorylation of Mek and Erk was quantified by immunoblotting ( d ). The immunoblot signals were normalized to Tubulin ( b ) or to total Mek/Erk ( d ) and to the mean of two doxycycline-treated non-targeting control sgRNAs. qPCR measurements were normalized to two housekeeping genes and to the respective untreated control (−Dox). Dots and triangles depict individual measurements of the two different sgRNAs, and thick bars show the mean of three biological replicates. f Dusp9- and Klhl13 over-expressing mESCs were treated with 1 μg/ml doxycycline 24 h before differentiation via LIF withdrawal for 4 days, and expression levels of pluripotency factors were measured by qPCR at different time points as indicated. Mean and standard deviation across 3 biological replicates is shown. g Global CpG methylation levels in cell lines over-expressing Dusp9 and Klhl13 via doxycycline treatment for 3 passages were assessed via pyrosequencing-based luminometric DNA methylation assay (LUMA). * p < 0.05 in a two-tailed paired Student’s t test comparing the Dusp9/Klhl13 over-expressing samples and the non-targeting controls (mean of sgRNA1 and sgRNA2)

Article Snippet: Mean and standard deviation across 3 biological replicates is shown. g Global CpG methylation levels in cell lines over-expressing Dusp9 and Klhl13 via doxycycline treatment for 3 passages were assessed via pyrosequencing-based luminometric DNA methylation assay (LUMA).

Techniques: Over Expression, Activation Assay, Stable Transfection, Expressing, Transduction, Control, Western Blot, Phospho-proteomics, Standard Deviation, CpG Methylation Assay, DNA Methylation Assay, Two Tailed Test

Journal: Genome Biology

Article Title: Identification of X-chromosomal genes that drive sex differences in embryonic stem cells through a hierarchical CRISPR screening approach

doi: 10.1186/s13059-021-02321-2

Figure Lengend Snippet: Over-expression of Klhl13 and Dusp9 in male mESCs leads to an enhanced pluripotency state and slower differentiation kinetics. a Schematic representation of the dCas9-SunTag system used for gene activation. b–e To over-express Dusp9 (yellow) and Klhl13 (blue), male E14 mESCs, stably expressing the doxycycline-inducible SunTag system, were either transduced with one of two different sgRNAs targeting the respective promoter regions or with non-targeting control (NT) sgRNAs and were treated for 3 days with 1 μg/ml doxycycline as indicated. Protein levels of Dusp9 (left) and Klhl13 (right) were quantified via immunoblotting ( b ), expression levels of MAPK target genes Spry4 and Egr1 ( c ) and of naive pluripotency factors Nanog and Prdm14 ( e ) were assessed by qPCR and phosphorylation of Mek and Erk was quantified by immunoblotting ( d ). The immunoblot signals were normalized to Tubulin ( b ) or to total Mek/Erk ( d ) and to the mean of two doxycycline-treated non-targeting control sgRNAs. qPCR measurements were normalized to two housekeeping genes and to the respective untreated control (−Dox). Dots and triangles depict individual measurements of the two different sgRNAs, and thick bars show the mean of three biological replicates. f Dusp9- and Klhl13 over-expressing mESCs were treated with 1 μg/ml doxycycline 24 h before differentiation via LIF withdrawal for 4 days, and expression levels of pluripotency factors were measured by qPCR at different time points as indicated. Mean and standard deviation across 3 biological replicates is shown. g Global CpG methylation levels in cell lines over-expressing Dusp9 and Klhl13 via doxycycline treatment for 3 passages were assessed via pyrosequencing-based luminometric DNA methylation assay (LUMA). * p < 0.05 in a two-tailed paired Student’s t test comparing the Dusp9/Klhl13 over-expressing samples and the non-targeting controls (mean of sgRNA1 and sgRNA2)

Article Snippet: Since Dusp9 has been suggested to be responsible for the reduction of global CpG methylation levels typically observed in female mESCs (20–30% compared to 60–80% in male mESCs) [ , , ], we analyzed how over-expression of Dusp9 and Klhl13 affected global DNA methylation through the pyrosequencing-based luminometric DNA methylation assay (LUMA; Fig. g).

Techniques: Over Expression, Activation Assay, Stable Transfection, Expressing, Transduction, Control, Western Blot, Phospho-proteomics, Standard Deviation, CpG Methylation Assay, DNA Methylation Assay, Two Tailed Test

Journal: BioMed Research International

Article Title: Environmental Impact on DNA Methylation in the Germline: State of the Art and Gaps of Knowledge

doi: 10.1155/2015/123484

Figure Lengend Snippet: Selected epidemiological studies on the effects of environmental chemical exposures on human DNA methylation.

Article Snippet: , 113 mother-child pairs, Bangladesh , PBL (maternal and umbilical cord samples) , Global LINE-1 , Alu , Methyl incorporation assay; bisulfite PCR pyrosequencing; LUMA (Luminometric Methylation Assay) , Hypermethylation (sex-dependent) , [ ] .

Techniques: Methylation, DNA Methylation Assay, Sequencing, Combined Bisulfite Restriction Analysis Assay, Enzyme-linked Immunosorbent Assay, Control, Flow Cytometry, Immunodetection

Journal: BioMed Research International

Article Title: Environmental Impact on DNA Methylation in the Germline: State of the Art and Gaps of Knowledge

doi: 10.1155/2015/123484

Figure Lengend Snippet: Synopsis of papers reporting effects of experimental treatments on DNA methylation of rodent male or female germ cells (when additional tissues were analyzed, they are specified).

Article Snippet: , 113 mother-child pairs, Bangladesh , PBL (maternal and umbilical cord samples) , Global LINE-1 , Alu , Methyl incorporation assay; bisulfite PCR pyrosequencing; LUMA (Luminometric Methylation Assay) , Hypermethylation (sex-dependent) , [ ] .

Techniques: DNA Methylation Assay, In Utero, Injection, Sequencing, Methylation, Mass Spectrometry, Control, Biomarker Discovery, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Immunostaining, Sampling, Immunohistochemistry, Combined Bisulfite Restriction Analysis Assay, Immunocytochemistry, In Vitro