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A loxP site was placed between the <t>Uhrf2</t> and Gldc genes using CRISPR/Cas-9, and trans -allelic recombination was used as described in Methods to generate duplication alleles of Gldc alone (9R fragment; duplication=3c9R, triplication=4c9R) or of all the genes in the 9LR genomic segments (9L fragment; duplication=3cL, triplication=4cL). B. Representative western blots and quantification of GLDC protein in hippocampus of mice-with 4 copies of GLDC alone (4c9R), 4 copies of all other 14 genes of the 9p24.1 CNV (4c9L) and 4 copies of the entire 9p24.1 CNV(4c9LR) compared to wildtype (WT) (n=5 for each group, One-way ANOVA followed by Bonferroni test, F(3,37)=18.77; ***p<0.001, WT vs 4c9LR (t=6.07; ***p<0.001), WT vs 4c9R (t=5.24;***p<0.001). C. GLDC enzyme activity in hippocampal tissues of wild type (WT), 4c9LR, 4c9R and 3c9R (n=3-6, One-way ANOVA followed by Bonferroni test, F(3,16)=13.53; ***p=0.0001), WT vs 4c9LR (t=5.827; **p<0.01), WT vs 4c9R (t=3.366; *p<0.05). D, E. Qualitative fluorescence microscopy images of GLDC protein colocalization with S100beta (D) or Map2 (E) in CA1, CA3 and dentate gyrus regions of hippocampus, respectively, in wildtype (WT) and 4c9R mice. F. BDNF mRNA expression is reduced in 4c9R mice compared to WT (One-way ANOVA followed by Bonferroni test; F(2,17)=7.669; **p<0.01), WT vs 4c9R (t=2.729; *p<0.05). G. miR137 mRNA expression is increased in 4c9R mice compared to WT (One-way ANOVA followed by Bonferroni test, F(2,12)=5.683; *p<0.05, WT vs 4c9R (t=3.343, *p<0.05). H. Pyroxd2 mRNA expression is increased in hippocampus of 4c9LR and 4c9R mice (One-way ANOVA followed by Bonferroni test, F(2,12)=468.4; ***p<0.0001, 4c9LR vs WT (t=24.20; ***p<0.001) and WT vs 4c9R (t=28.33; ***p<0.001). Data are mean ± SEM, (n=5).
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Experiment validation of the nine hub 5mC regulators. (A–I) DNMT3B, MND3, NTHL1, SMUG1, TET1, TET3, UHRF1, <t>UHRF2,</t> <t>ZBTB33.</t> The results were presented as mean ± standard deviation ( t -testing). ** p < 0.01, (J) Western blot analysis of hub 5mC regulators.
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A loxP site was placed between the Uhrf2 and Gldc genes using CRISPR/Cas-9, and trans -allelic recombination was used as described in Methods to generate duplication alleles of Gldc alone (9R fragment; duplication=3c9R, triplication=4c9R) or of all the genes in the 9LR genomic segments (9L fragment; duplication=3cL, triplication=4cL). B. Representative western blots and quantification of GLDC protein in hippocampus of mice-with 4 copies of GLDC alone (4c9R), 4 copies of all other 14 genes of the 9p24.1 CNV (4c9L) and 4 copies of the entire 9p24.1 CNV(4c9LR) compared to wildtype (WT) (n=5 for each group, One-way ANOVA followed by Bonferroni test, F(3,37)=18.77; ***p<0.001, WT vs 4c9LR (t=6.07; ***p<0.001), WT vs 4c9R (t=5.24;***p<0.001). C. GLDC enzyme activity in hippocampal tissues of wild type (WT), 4c9LR, 4c9R and 3c9R (n=3-6, One-way ANOVA followed by Bonferroni test, F(3,16)=13.53; ***p=0.0001), WT vs 4c9LR (t=5.827; **p<0.01), WT vs 4c9R (t=3.366; *p<0.05). D, E. Qualitative fluorescence microscopy images of GLDC protein colocalization with S100beta (D) or Map2 (E) in CA1, CA3 and dentate gyrus regions of hippocampus, respectively, in wildtype (WT) and 4c9R mice. F. BDNF mRNA expression is reduced in 4c9R mice compared to WT (One-way ANOVA followed by Bonferroni test; F(2,17)=7.669; **p<0.01), WT vs 4c9R (t=2.729; *p<0.05). G. miR137 mRNA expression is increased in 4c9R mice compared to WT (One-way ANOVA followed by Bonferroni test, F(2,12)=5.683; *p<0.05, WT vs 4c9R (t=3.343, *p<0.05). H. Pyroxd2 mRNA expression is increased in hippocampus of 4c9LR and 4c9R mice (One-way ANOVA followed by Bonferroni test, F(2,12)=468.4; ***p<0.0001, 4c9LR vs WT (t=24.20; ***p<0.001) and WT vs 4c9R (t=28.33; ***p<0.001). Data are mean ± SEM, (n=5).

Journal: bioRxiv

Article Title: A marker chromosome in psychosis identifies glycine decarboxylase (GLDC) as a novel regulator of neuronal and synaptic function in the hippocampus

doi: 10.1101/2023.05.29.542745

Figure Lengend Snippet: A loxP site was placed between the Uhrf2 and Gldc genes using CRISPR/Cas-9, and trans -allelic recombination was used as described in Methods to generate duplication alleles of Gldc alone (9R fragment; duplication=3c9R, triplication=4c9R) or of all the genes in the 9LR genomic segments (9L fragment; duplication=3cL, triplication=4cL). B. Representative western blots and quantification of GLDC protein in hippocampus of mice-with 4 copies of GLDC alone (4c9R), 4 copies of all other 14 genes of the 9p24.1 CNV (4c9L) and 4 copies of the entire 9p24.1 CNV(4c9LR) compared to wildtype (WT) (n=5 for each group, One-way ANOVA followed by Bonferroni test, F(3,37)=18.77; ***p<0.001, WT vs 4c9LR (t=6.07; ***p<0.001), WT vs 4c9R (t=5.24;***p<0.001). C. GLDC enzyme activity in hippocampal tissues of wild type (WT), 4c9LR, 4c9R and 3c9R (n=3-6, One-way ANOVA followed by Bonferroni test, F(3,16)=13.53; ***p=0.0001), WT vs 4c9LR (t=5.827; **p<0.01), WT vs 4c9R (t=3.366; *p<0.05). D, E. Qualitative fluorescence microscopy images of GLDC protein colocalization with S100beta (D) or Map2 (E) in CA1, CA3 and dentate gyrus regions of hippocampus, respectively, in wildtype (WT) and 4c9R mice. F. BDNF mRNA expression is reduced in 4c9R mice compared to WT (One-way ANOVA followed by Bonferroni test; F(2,17)=7.669; **p<0.01), WT vs 4c9R (t=2.729; *p<0.05). G. miR137 mRNA expression is increased in 4c9R mice compared to WT (One-way ANOVA followed by Bonferroni test, F(2,12)=5.683; *p<0.05, WT vs 4c9R (t=3.343, *p<0.05). H. Pyroxd2 mRNA expression is increased in hippocampus of 4c9LR and 4c9R mice (One-way ANOVA followed by Bonferroni test, F(2,12)=468.4; ***p<0.0001, 4c9LR vs WT (t=24.20; ***p<0.001) and WT vs 4c9R (t=28.33; ***p<0.001). Data are mean ± SEM, (n=5).

Article Snippet: Copy number PCRs were performed using commercial kits designed to detect the Gldc and Uhrf2 genes (Mm00496811_cn GLDC (#4400291), Mm00735356_cn Uhrf2 (#4400291) and TaqManTM Copy Number Reference Assay, mouse, Tfrc (#4458366) Thermo Fisher Scientific, MA).

Techniques: CRISPR, Western Blot, Activity Assay, Fluorescence, Microscopy, Expressing

Experiment validation of the nine hub 5mC regulators. (A–I) DNMT3B, MND3, NTHL1, SMUG1, TET1, TET3, UHRF1, UHRF2, ZBTB33. The results were presented as mean ± standard deviation ( t -testing). ** p < 0.01, (J) Western blot analysis of hub 5mC regulators.

Journal: Frontiers in Cardiovascular Medicine

Article Title: 5mC modification patterns provide novel direction for early acute myocardial infarction detection and personalized therapy

doi: 10.3389/fcvm.2022.1053697

Figure Lengend Snippet: Experiment validation of the nine hub 5mC regulators. (A–I) DNMT3B, MND3, NTHL1, SMUG1, TET1, TET3, UHRF1, UHRF2, ZBTB33. The results were presented as mean ± standard deviation ( t -testing). ** p < 0.01, (J) Western blot analysis of hub 5mC regulators.

Article Snippet: Primary antibodies targeting to beta actin (ab8226, Abcam), UHRF2 (ABE1028, MilliporeSigma), TET3 (ab153724, Abcam), UHRF2 (ZBTB33, MilliporeSigma), TET1 (ab19198, Abcam), DNMT3B (ab2851, Abcam), NTHL1 (ab191413, Abcam), UHRF1 (ab213223, Abcam), MBD3 (ab157464, Abcam), and SMUG1 (ab192240, Abcam), were incubated with targeted proteins at 4°C overnight, followed by incubating with appropriate horseradish peroxidase-conjugated secondary antibodies.

Techniques: Biomarker Discovery, Standard Deviation, Western Blot

Nine 5mC regulators expression level at four time points of AMI patients in GSE59867. (A–I) MND3, NTHL1, SMUG1, UHRF1, UHRF2, ZBTB33, DNMT3B, TET1, TET3. * p < 0.05; ** p < 0.01; *** p < 0.001; ns, not significant.

Journal: Frontiers in Cardiovascular Medicine

Article Title: 5mC modification patterns provide novel direction for early acute myocardial infarction detection and personalized therapy

doi: 10.3389/fcvm.2022.1053697

Figure Lengend Snippet: Nine 5mC regulators expression level at four time points of AMI patients in GSE59867. (A–I) MND3, NTHL1, SMUG1, UHRF1, UHRF2, ZBTB33, DNMT3B, TET1, TET3. * p < 0.05; ** p < 0.01; *** p < 0.001; ns, not significant.

Article Snippet: Primary antibodies targeting to beta actin (ab8226, Abcam), UHRF2 (ABE1028, MilliporeSigma), TET3 (ab153724, Abcam), UHRF2 (ZBTB33, MilliporeSigma), TET1 (ab19198, Abcam), DNMT3B (ab2851, Abcam), NTHL1 (ab191413, Abcam), UHRF1 (ab213223, Abcam), MBD3 (ab157464, Abcam), and SMUG1 (ab192240, Abcam), were incubated with targeted proteins at 4°C overnight, followed by incubating with appropriate horseradish peroxidase-conjugated secondary antibodies.

Techniques: Expressing