Journal: Oncogene

Article Title: H19 lncRNA identified as a master regulator of genes that drive uterine leiomyomas

doi: 10.1038/s41388-019-0808-4

Figure Lengend Snippet: TET3 affects DNA methylation and histone modifications of the MED12, TGFBR2, and TSP1 promoters. a UtLM cells were transfected with siCon or siTET3 for 48 h, followed by ChIP-qPCR analysis. Data are presented as mean relative TET3 enrichment over input. n = 3. Red numbers indicate nucleotide positions relative to the transcriptional start sites, with PCR products depicted as red-stripped bars. b Sequences of critical transcription regulatory regions (CTRR) of MED12 , TGFBR2 , and TSP1 . The differentially methylated cytosine residues are marked in red. The red numbers mark the positions of the indicated nucleotides relative to the transcriptional start sites. c UtLM cells were transfected with siCon or siTET3 for 48 h, followed by QMSP analysis. n = 3. d UtLM cells were transfected with siCon or siTET3 for 48 h, followed by ChIP-qPCR analysis. Data are presented as mean relative enrichment over input. n = 3. All data are representative of at least two independent experiments and are presented as mean ± SEM. * p < 0.05, ** p < 0.01

Article Snippet: Antibodies for TET3 (GeneTex, GTX121453; used at a dilution of 1/500), TGFBR2 (Abcam, ab184948; used at a dilution of 1/1000), TSP1 (Abcam, ab85762; used at a dilution of 1/500), MED12 (Novus Biological, NB100–2357; used at a dilution of 1/500), HMGA2 (Proteintech, 20795–1-AP; used at a dilution of 1/500), GRAF1 (Cell Signaling, 8802; used at a dilution of 1/500), SPARC (Cell Signaling, 8725; used at a dilution of 1/500), COL3A1 (LS-Bio, LS-C159386; used at a dilution of 1/1000), COL4A1 (LS-Bio, LS-C100552; used at a dilution of 1/500), COL5A2 (Origene, TA809611; used at a dilution of 1/500), and GAPDH (Abcam, ab128915; used at a dilution of 1/10000) were purchased.

Techniques: DNA Methylation Assay, Transfection, ChIP-qPCR, Methylation

Journal: Oncogene

Article Title: H19 lncRNA identified as a master regulator of genes that drive uterine leiomyomas

doi: 10.1038/s41388-019-0808-4

Figure Lengend Snippet: H19 and TET3 co-express with fibroid-promoting genes in vivo. a , c RT-qPCR analyses were performed on RNAs extracted from human fibroids and matched myometrium tissues. Spearman’s correlation showed positive correlations between expression of H19 and TET3 ( a , left panel), as well as TET3 and its target genes MED12 , TGFBR2 , and TSP1 ( c ) in a statistically significant manner. No correlation between expression of H19 and HMGA2 at the RNA level was detected ( a , right panel). Spearman’s correlation coefficient, p -values, and sample numbers are presented. b Results of western blotting analysis of HMGA2 in human fibroids and matched myometrium. n = 3. Data are representative of two independent experiments and are presented as mean ± SEM

Article Snippet: Antibodies for TET3 (GeneTex, GTX121453; used at a dilution of 1/500), TGFBR2 (Abcam, ab184948; used at a dilution of 1/1000), TSP1 (Abcam, ab85762; used at a dilution of 1/500), MED12 (Novus Biological, NB100–2357; used at a dilution of 1/500), HMGA2 (Proteintech, 20795–1-AP; used at a dilution of 1/500), GRAF1 (Cell Signaling, 8802; used at a dilution of 1/500), SPARC (Cell Signaling, 8725; used at a dilution of 1/500), COL3A1 (LS-Bio, LS-C159386; used at a dilution of 1/1000), COL4A1 (LS-Bio, LS-C100552; used at a dilution of 1/500), COL5A2 (Origene, TA809611; used at a dilution of 1/500), and GAPDH (Abcam, ab128915; used at a dilution of 1/10000) were purchased.

Techniques: In Vivo, Quantitative RT-PCR, Expressing, Western Blot

Journal: Stem Cell Research & Therapy

Article Title: SMYD1 modulates the proliferation of multipotent cardiac progenitor cells derived from human pluripotent stem cells during myocardial differentiation through GSK3β/β-catenin&ERK signaling

doi: 10.1186/s13287-024-03899-7

Figure Lengend Snippet: Primary and secondary antibodies

Article Snippet: , Anti- ERK1/2 , Cell Signaling Technology #4695 , Rabbit , Western blot , 1:1000.

Techniques: Immunofluorescence, Western Blot, Flow Cytometry, ChIP-chip

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: CHD8 dosage regulates transcription in pluripotency and early murine neural differentiation

doi: 10.1073/pnas.1921963117

Figure Lengend Snippet: Dosage-sensitive effect of CHD8 deletion on transcription in ESCs. (A) The gnomAD human gene constraint scores (missense and probability of intolerance to heterozygous pLoF variation [pLI] for all genes with CHD8 labeled). (B) Schematic showing the CHD8 CRISPR-mediated deletion approach and Western blot of CHD8 levels in Chd8+/− and Chd8−/− ESC lines. (C) Doubling time of WT, Chd8+/−, and Chd8−/− cells. Asterisks denote P < 0.05 (*). Values are expressed as the mean ± SE, n = 3. (D) Alkaline phosphatase staining of WT, Chd8+/−, and Chd8−/− ESCs in 2i media. Magnification: 200×. (E) Venn diagrams depicting overlap of DEGs in Chd8+/− and Chd8−/− ESCs. Genes with increased expression are on top, and genes with decreased expression are on the bottom (FDR < 0.05). (F) Heatmap showing dosage-sensitive transcriptional response for genes that are differentially expressed in Chd8−/−. (G) GO terms for DEGs (padj < 0.05).

Article Snippet: Antibodies used in this study are mouse a-FLAG M2 (1:1,000, Sigma-Aldrich), rabbit a-CHD8 (1:2,500, Novus Biologicals #NB100-60417; 1:2,000, Novus Biologicals #NB100-60418), mouse a-TBP (1:2,000, Abcam #ab818), rabbit a-DCX (1:200, CST #4604S), mouse a-Nestin (1:200, #MAB353), mouse a-Tuj1 (1:200, BioLegend #801201), rabbit a-Pax6 (1:200, BioLegend #901301), rabbit a-NeuroD1 (1:200, Proteintech #12081-1-AP), and mouse a-Map2a (1:200, M9942).

Techniques: Labeling, CRISPR, Western Blot, Staining, Expressing

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: CHD8 dosage regulates transcription in pluripotency and early murine neural differentiation

doi: 10.1073/pnas.1921963117

Figure Lengend Snippet: CHD8 deletion results in up-regulation of neuronal genes and Sox TFs during differentiation into NPCs. (A) Schematic showing the differentiation of WT, Chd8+/−, and Chd8−/− ESCs into NPCs. (B) Venn diagrams depicting overlap of DEGs in Chd8+/− and Chd8−/− NPCs. Genes with increased gene expression are on top, and genes with decreased expression are on bottom (FDR < 0.05). (C) Heatmap showing dosage sensitive transcriptional response for genes that are differentially expressed in Chd8−/−. (D) GO terms for DEGs. (E and F) Volcano plots of RNA-seq data for CHD8+/− and CHD8−/−. DEGs are highlighted in red (FDR < 0.05), and genes involved with mature neuronal development and Sox TFs are labeled. Genes labeled with yellow background were stained by IF in G. (G) Representative IF staining of day 8 NPCs, with antibodies listed on left side and respective genotypes on top (n = 2 biological replicates with similar results). Magnification: 200×.

Article Snippet: Antibodies used in this study are mouse a-FLAG M2 (1:1,000, Sigma-Aldrich), rabbit a-CHD8 (1:2,500, Novus Biologicals #NB100-60417; 1:2,000, Novus Biologicals #NB100-60418), mouse a-TBP (1:2,000, Abcam #ab818), rabbit a-DCX (1:200, CST #4604S), mouse a-Nestin (1:200, #MAB353), mouse a-Tuj1 (1:200, BioLegend #801201), rabbit a-Pax6 (1:200, BioLegend #901301), rabbit a-NeuroD1 (1:200, Proteintech #12081-1-AP), and mouse a-Map2a (1:200, M9942).

Techniques: Gene Expression, Expressing, RNA Sequencing, Labeling, Staining

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: CHD8 dosage regulates transcription in pluripotency and early murine neural differentiation

doi: 10.1073/pnas.1921963117

Figure Lengend Snippet: CHD8 regulates accessibility at key regions in ESCs and during differentiation to NPCs. (A) Percentage of increased (blue) and decreased (orange) peaks for Chd8−/− ESCs. (B) Genomic annotation enrichment for increased and decreased sites for Chd8−/− ESC ATAC-seq datasets. (C) Percentage of increased (blue) and decreased (orange) peaks for Chd8−/− NPCs. (D) Genomic annotation enrichment for increased and decreased sites for Chd8+/−and Chd8−/− NPC ATAC-seq datasets. (E) Genome tracks showing gain of accessibility at Grb10 locus in ESCs. (F) Genome tracks showing loss of accessibility at Basp1 locus in NPCs.

Article Snippet: Antibodies used in this study are mouse a-FLAG M2 (1:1,000, Sigma-Aldrich), rabbit a-CHD8 (1:2,500, Novus Biologicals #NB100-60417; 1:2,000, Novus Biologicals #NB100-60418), mouse a-TBP (1:2,000, Abcam #ab818), rabbit a-DCX (1:200, CST #4604S), mouse a-Nestin (1:200, #MAB353), mouse a-Tuj1 (1:200, BioLegend #801201), rabbit a-Pax6 (1:200, BioLegend #901301), rabbit a-NeuroD1 (1:200, Proteintech #12081-1-AP), and mouse a-Map2a (1:200, M9942).

Techniques:

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: CHD8 dosage regulates transcription in pluripotency and early murine neural differentiation

doi: 10.1073/pnas.1921963117

Figure Lengend Snippet: CHD8 cooperates with Sox TFs to regulate accessibility and transcription. (A) Heatmap representation of ATAC-seq chromVAR bias-corrected deviations in the 50 most variable TF motifs across ESC WT and Chd8−/− ATAC-seq replicates. (B) Heatmap representation of ATAC-seq chromVAR bias-corrected deviations in the 50 most variable TF motifs across NPC WT and Chd8−/− ATAC-seq replicates. (C) Western blot showing co-IP of CHD8 with SOX2 in ESCs (antibodies used are the following: SOX2 A: sc-365823; SOX2 B: AB5603). (D) Heat map displaying CHD8 ChIP-seq at SOX2 peaks (41) (Left) and SOX2 ChIP-seq at CHD8 peaks (Right).

Article Snippet: Antibodies used in this study are mouse a-FLAG M2 (1:1,000, Sigma-Aldrich), rabbit a-CHD8 (1:2,500, Novus Biologicals #NB100-60417; 1:2,000, Novus Biologicals #NB100-60418), mouse a-TBP (1:2,000, Abcam #ab818), rabbit a-DCX (1:200, CST #4604S), mouse a-Nestin (1:200, #MAB353), mouse a-Tuj1 (1:200, BioLegend #801201), rabbit a-Pax6 (1:200, BioLegend #901301), rabbit a-NeuroD1 (1:200, Proteintech #12081-1-AP), and mouse a-Map2a (1:200, M9942).

Techniques: Western Blot, Co-Immunoprecipitation Assay, ChIP-sequencing

Journal: PLoS ONE

Article Title: The Impact of Genome-Wide Supported Schizophrenia Risk Variants in the Neurogranin Gene on Brain Structure and Function

doi: 10.1371/journal.pone.0076815

Figure Lengend Snippet: Basic demographics according to NRGN rs12541 and rs12807809 genotype.

Article Snippet: No allele frequency differences were observed for rs12807809 (Chi-Square test Hapmap χ 2 =1.333, p=0.248; Chi-Square test MCIC χ 2 =2.667, p=0.102).

Techniques:

Journal: PLoS ONE

Article Title: The Impact of Genome-Wide Supported Schizophrenia Risk Variants in the Neurogranin Gene on Brain Structure and Function

doi: 10.1371/journal.pone.0076815

Figure Lengend Snippet: a) Cortical statistical map illustrating reduced cortical thickness for rs12807809 C carriers compared to TT homozygotes. The -log(CWP-value) is represented according to the color code. b) Boxplot showing mean and two standard errors of the standardized residuals for the effects of NRGN rs12807809 genotype on left rostal ACC volume controlled for intracranial volume, age, gender, diagnosis and scanner field strength.

Article Snippet: No allele frequency differences were observed for rs12807809 (Chi-Square test Hapmap χ 2 =1.333, p=0.248; Chi-Square test MCIC χ 2 =2.667, p=0.102).

Techniques: Biomarker Discovery

Journal: Cell stem cell

Article Title: Lipid Deprivation Induces a Stable, Naive-to-Primed Intermediate State of Pluripotency in Human PSCs

doi: 10.1016/j.stem.2019.05.001

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit anti-p44/42 MAPK (Phospho-ERK1/2) , Cell Signaling , 9101; RRID:AB_331646.

Techniques: Recombinant, Knock-Out, Reverse Transcription, Membrane, Methylation, Staining, Bisulfite Sequencing, RNA Sequencing, ChIP-sequencing, ChIP-qPCR, Control, Software, Magnetic Cell Separation, Protease Inhibitor

Journal: Science translational medicine

Article Title: NEDD9 targets COL3A1 to promote endothelial fibrosis and pulmonary arterial hypertension

doi: 10.1126/scitranslmed.aap7294

Figure Lengend Snippet: (A) Protein-protein interaction network analysis was used to clarify the molecular pathways that regulate two functionally distinct fibrosis subtypes: adaptive and pathogenic. Genes related to dermal and vascular fibrosis were collected from the curated literature, focusing on pulmonary arterial hypertension (PAH) as a human disease correlate to these findings in silico. Genes specifically associated with lung fibrosis (n = 362) were excluded from the analysis to limit the probability of analyzing pathways responsible for lung parenchymal fibrosis rather than pulmonary vascular fibrosis. (B) The gene products (proteins) associated with adaptive fibrosis (blue), pathogenic fibrosis (red), or both adaptive and pathogenic fibrosis (blue with red border) were mapped to the consolidated human protein-protein interactome (12), resulting in the fibrosome. (C) An aldosterone (ALDO)-fibrosome protein-protein interactome subnetwork resulting from associations involving fibrosis genes connected to ALDO-regulated genes. Arrow points to the SMAD3 target and Cas protein NEDD9. (D) Betweenness centrality (BC), a measure of importance in information transfer across the network of a node (protein) based on the shortest paths, was used to identify NEDD9 as a critical node in the phenotype transition from adaptive to pathogenic fibrosis. BC of an ALDO-regulated gene (ai) in connecting adaptive and pathogenic fibrosis (F1 and F2, respectively) is the sum of the fraction of all fibrosis gene pairs’ shortest paths in the interactome that pass through this ALDO-regulated gene (t) in the interactome, and σ(s,t|ai) is the number of those shortest paths in the interactome that pass through ALDO-regulated gene ai. The BC score for NEDD9 was ranked 6th of 86 proteins.

Article Snippet: The gene products (proteins) of the remaining fibrosis genes were mapped to the consolidated human protein-protein interactome (CHI) ( 12 ) to generate a network of fibrosis protein-protein interactions.

Techniques: In Silico

Journal: Bone Research

Article Title: MEF2C regulates osteoclastogenesis and pathologic bone resorption via c-FOS

doi: 10.1038/s41413-020-00120-2

Figure Lengend Snippet: MEF2C binds to the upstream region of FOS gene and regulates NFATC1 expression. a A schematic view of FOS upstream regions with putative MEF2C binding sites predicted based on the analysis of MEF2C ChIP-sequencing data. b ChIP-qPCR analysis of the three putative MEF2C binding sites in the FOS upstream region. MEF2C binding was assessed in human OCPs transduced with adenoviral particles encoding GFP or MEF2C-FLAG and immunoprecipitated with FLAG antibodies. Dotted line represents enrichment level in the negative control HBB region in GFP transduced samples. ( n = 4) Mouse osteoclast precursor cells from MEF2C ΔMX KO or littermate control WT mice were transduced with retroviral particles encoding GFP or FOS and cultured with M-CSF and RANKL. c Representative image of TRAP staining of mouse osteoclasts. Scale bar, 100 μm. d Cumulative data showing numbers of osteoclasts from four independent experiments. Human osteoclast precursor cells were nucleofected with control or MEF2C #2 siRNAs. e RT-qPCR analysis of human NFATC1 mRNA after 48 h of culture with or without RANKL normalized relative to TBP mRNA (control samples with RANKL set at 1.0). n = 5. f Representative immunoblot of human NFATc1. p38 was used as a loading control. Right panel, densitometric quantitation of band intensity from three donors. Mouse osteoclast precursor cells from MEF2C ΔMX mice or littermate control WT mice were cultured with M-CSF and RANKL. g RT-qPCR analysis of mouse Nfatc1 mRNA after 24 h of culture with or without RANKL (50 ng·mL −1 ) normalized relative to Hprt mRNA (control samples without RANKL set at 1.0). WT; n = 11, KO; n = 12. h Representative images of immunoblotting analysis of mouse NFATc1 expression. Right panel, densitometric quantitation of band intensity from 4 samples of each genotype. Data are shown as mean ± SD. Statistics used: e repeated measurement two-way ANOVA, f paired t -test, g two-way ANOVA, h Welch’s t -test. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: The antibodies used for immunoblotting are as follows: NFATc1 (#sc-7294), RANK (#sc-37436) (Santa Cruz Biotechnology, Dallas, TX, USA); MEF2C (#5030), c-Fos (#2250), IkBα (#9242), p-p38 (#9215), p-ERK1/2 (#9101), ERK1/2 (#9102), p65 (#4764), p105/p50 (#3035), and p38 (#9212) (Cell Signaling Technology, Danvers, MA, USA); Lamin B1 (Abcam #16048, Cambridge, UK); α-tubulin (Sigma-Aldrich #T9026, St. Louis, MO, USA); FLAG (for immunoblotting: Biolegend #637301, San Diego, CA, USA.

Techniques: Expressing, Binding Assay, ChIP-sequencing, ChIP-qPCR, Transduction, Immunoprecipitation, Negative Control, Control, Retroviral, Cell Culture, Staining, Quantitative RT-PCR, Western Blot, Quantitation Assay

Journal: Scientific Reports

Article Title: Histone H3K9 demethylase JMJD2B induces hepatic steatosis through upregulation of PPARγ2

doi: 10.1038/s41598-018-31953-x

Figure Lengend Snippet: JMJD2B expression increases in hepatic steatotic cell and animal models. ( A ) HepG2 cells were incubated with a mixture of palmitic acid (PA) and oleic acid (OA) (1:2 ratio) at 800 μM concentrations for 24 h, and intracellular triglyceride (TG) levels were analyzed by a TG assay kit. JMJD2B mRNA and protein levels were examined by qPCR and western blotting, respectively. Data represent means ± SEM of three independent experiments performed in triplicate. *p < 0.05 vs. no treatment. The full-length western blots corresponding to truncated blots are presented in Supplementary Figure . ( B ) HepG2 cells were treated with T0901317 (10 μM) for 24 h, and intracellular triglyceride (TG) levels were measured by a TG assay kit. JMJD2B mRNA and protein levels were examined by qPCR and western blotting, respectively. Data represent means ± SEM of three independent experiments performed in triplicate. *p < 0.05 vs. no treatment. The full-length western blots are presented in Supplementary Figure . ( C ) Total RNAs were isolated from the livers of HFD-induced obese mice. The JMJD2B mRNA levels were assessed by qPCR. Data represent means ± SEM of 5 mice. * p < 0.05 vs. ND mice. ND: normal diet. HFD: high fat diet.

Article Snippet: The antibody against JMJD2B (NB100-74605) was purchased from Novus Biologicals (Littelton, CO, USA).

Techniques: Expressing, Incubation, Western Blot, Isolation

Journal: Scientific Reports

Article Title: Histone H3K9 demethylase JMJD2B induces hepatic steatosis through upregulation of PPARγ2

doi: 10.1038/s41598-018-31953-x

Figure Lengend Snippet: Overexpression of JMJD2B increases intracellular TG levels and stimulates PPARγ2 expression in HepG2 cells. HepG2 cells in 6-well plates were infected with Ad-GFP or Ad-JMJD2B. ( A ) The levels of JMJD2B, H3K9me, H3K9me2, and H3K9me3 were measured by western blotting. The full-length western blots corresponding to truncated blots are provided in Supplementary Figure . ( B ) Intracellular TG levels were measured by TG assay kit. ( C ) PPARγ2 expression was assessed by qPCR and western blotting. The full-length western blots corresponding to truncated blots are presented in Supplementary Figure . ( D , E ) The expression of PPARγ2 steatosis target genes was evaluated by qPCR. Data represent means ± SEM of three independent experiments performed in triplicate. * p < 0.05, ** p < 0.01 vs. Ad-GFP.

Article Snippet: The antibody against JMJD2B (NB100-74605) was purchased from Novus Biologicals (Littelton, CO, USA).

Techniques: Over Expression, Expressing, Infection, Western Blot

Journal: Scientific Reports

Article Title: Histone H3K9 demethylase JMJD2B induces hepatic steatosis through upregulation of PPARγ2

doi: 10.1038/s41598-018-31953-x

Figure Lengend Snippet: Knockdown of JMJD2B reduces the intracellular TG level and represses PPARγ2 expression in HepG2 cells. HepG2 cells in 6-well plates were transfected with scramble RNA or JMJD2B siRNA. ( A ) The levels of JMJD2B, H3K9me, H3K9me2, and H3K9me3 were measured by western blotting. The full-length western blots corresponding to truncated blots are presented in Supplementary Figure . ( B ) Intracellular TG levels were measured by a TG assay kit. ( C ) PPARγ2 expression was assessed by qPCR and western blotting. The full-length western blots corresponding to truncated blots are presented in Supplementary Figure . ( D , E ) The expression of PPARγ2 steatosis target genes was evaluated by qPCR. Data represent means ± SEM of three independent experiments performed in triplicate. * p < 0.05 vs. scramble RNA.

Article Snippet: The antibody against JMJD2B (NB100-74605) was purchased from Novus Biologicals (Littelton, CO, USA).

Techniques: Knockdown, Expressing, Transfection, Western Blot

Journal: Scientific Reports

Article Title: Histone H3K9 demethylase JMJD2B induces hepatic steatosis through upregulation of PPARγ2

doi: 10.1038/s41598-018-31953-x

Figure Lengend Snippet: JMJD2B lessens the enrichment of H3K9me2 and H3K9me3 on PPARγ2 promoters and increases the binding of PPARγ2 to PPREs on promoters of CD36 and PLIN2 . HepG2 cells were infected with adenovirus Ad-GFP or Ad-JMJD2B. ( A ) The recruitment of JMJD2B to the PPARγ2 promoter was analyzed by ChIP-qPCR. ( B ) The enrichment of H3K9me2 and H3K9me3 on promoters of PPARγ2 was analyzed by ChIP-qPCR. ( C ) The recruitment of PPARγ2 to PPRE on promoters of CD36 and PLIN2 was assessed by ChIP-qPCR. Data represent means ± SEM of three independent experiments performed in triplicate. * p < 0.05, ** p < 0.01 vs. Ad-GFP.

Article Snippet: The antibody against JMJD2B (NB100-74605) was purchased from Novus Biologicals (Littelton, CO, USA).

Techniques: Binding Assay, Infection, ChIP-qPCR

Journal: Scientific Reports

Article Title: Histone H3K9 demethylase JMJD2B induces hepatic steatosis through upregulation of PPARγ2

doi: 10.1038/s41598-018-31953-x

Figure Lengend Snippet: Adenovirus-mediated JMJD2B overexpression stimulated hepatic steatosis in vivo . C57BL/6 mice (8 weeks old) were injected with adenovirus Ad-GFP or Ad-JMJD2B. After injection, Ad-injected mice were fed a HFD for 2 weeks. ( A ) JMJD2B expression was determined by qPCR ( B ) Immunostaining of JMJD2B (scale bar = 50 μm). Representative photographs are shown. JMJD2B positive cells were counted in ten random areas at 400× magnification and analyzed by using image J. Quantification of immunostaining assay is represented as percentage of JMJD2B positive cells. ( C ) Hepatic triglyceride (TG) levels were measured by a TG assay kit. ( D ) Liver morphology, ORO and H&E staining (scale bar = 100 μm). Representative images are presented. ( E ) Serum total TG levels. ( F ) Serum total cholesterol levels. Data represent means ± SEM from 5 mice. * p < 0.05 vs. Ad-GFP-infected mice.

Article Snippet: The antibody against JMJD2B (NB100-74605) was purchased from Novus Biologicals (Littelton, CO, USA).

Techniques: Over Expression, In Vivo, Injection, Expressing, Immunostaining, Staining, Infection

Journal: Scientific Reports

Article Title: Histone H3K9 demethylase JMJD2B induces hepatic steatosis through upregulation of PPARγ2

doi: 10.1038/s41598-018-31953-x

Figure Lengend Snippet: Adenovirus-mediated JMJD2B overexpression promotes the expression of PPARγ2 and its steatosis target genes in mouse liver. C57BL/6 mice (8 weeks old) were injected with adenovirus Ad-GFP or Ad-JMJD2B. After injection, Ad-injected mice were fed a HFD for 2 weeks. ( A ) PPARγ2 expression was measured by qPCR. ( B , C ) The expression of PPARγ2 steatosis target genes was measured by qPCR. Data represent means ± SEM from 5 mice. * p < 0.05, ** p < 0.01 vs. Ad-GFP-infected mice.

Article Snippet: The antibody against JMJD2B (NB100-74605) was purchased from Novus Biologicals (Littelton, CO, USA).

Techniques: Over Expression, Expressing, Injection, Infection

Journal: Scientific Reports

Article Title: Histone H3K9 demethylase JMJD2B induces hepatic steatosis through upregulation of PPARγ2

doi: 10.1038/s41598-018-31953-x

Figure Lengend Snippet: Gomisin N (GN) represses the expression of JMJD2B, PPARγ2, and PPARγ2 steatosis target genes in HepG2 cells. HepG2 cells were treated with GN (100 μM) for 12 h. ( A ) JMJD2B expression was assessed by qPCR and western blotting. The full-length western blots corresponding to truncated blots are given in Supplementary Figure . ( B ) PPARγ2 expression was measured by qPCR. ( C , D ) The expression of PPARγ2 steatosis target genes was assessed by qPCR. Data represent means ± SEM of three independent experiments performed in triplicate. * p < 0.05 vs. no treatment.

Article Snippet: The antibody against JMJD2B (NB100-74605) was purchased from Novus Biologicals (Littelton, CO, USA).

Techniques: Expressing, Western Blot

Journal: bioRxiv

Article Title: Differential effects of pre-mRNA splicing inhibitors on RNA polymerase II transcription

doi: 10.1101/2023.01.16.524270

Figure Lengend Snippet: (A) Representative images of immunofluorescence analysis or 5’EU incorporation in HeLa cells treated with DMSO, 100 µM DRB, a CDK9 inhibitor, or 90 µM Madrasin for 1h. EU (green), DAPI (blue), scale bars: 50 µm. (B) Quantification of 5’EU intensity per nucleus for DMSO (blue), DRB (purple), and Madrasin (red). Boxplot settings are: min to max values with the box showing 25-75 percentile range. > 10,000 nuclei were quantified per condition. Statistical test: Kruskal-Wallis test. P-value: **** < 0.0001. (C) qRT-PCR with primers amplifying a region from the pol I transcribed pre-rRNA. HeLa cells were treated with DMSO or 90 µM Madrasin for 30 minutes (grey) or 60 minutes (white). cDNA was generated with random hexamers. Values are normalised to the 7SK snRNA and shown as relative to DMSO, mean ± SEM, n = 3 biological replicates. Statistical test: two-tailed unpaired t-test. P-value: n.s. not significant. (D) qRT-PCR with primers amplifying different protein-coding genes. HeLa cells were treated with DMSO or 90 µM Madrasin for 30 minutes (grey) or 60 minutes (white). cDNA was generated with random hexamers. Values are normalised to the 7SK snRNA and shown as relative to DMSO, mean ± SEM, n = 3 biological replicates. Statistical test: two-tailed unpaired t-test. P-value: * < 0.05, ** < 0.01, *** < 0.001. (E) Metagene profile of total pol II mNET-seq performed in HeLa cells treated with DMSO (blue) or 90 µM Madrasin for 30 minutes (red) on scaled expressed protein-coding genes. (F) Screenshot of the genome browser total pol II mNET-seq DMSO (blue) and Madrasin (red) tracks of the protein-coding gene KPNB1 . The arrow indicates the sense of transcription. (G) Total pol II ChIP-qPCR of the KPNB1 protein-coding gene in HeLa cells treated with DMSO (black) or 90 µM Madrasin for 30 minutes (white). Mean ± SEM, n = 3 biological replicates. Statistical test: two-tailed unpaired t-test. P-value: * < 0.05, *** < 0.001.

Article Snippet: For each IP, 60 μg of chromatin was incubated overnight on a rotating wheel at 4°C with the following antibodies: normal rabbit IgG (2729S, Cell Signaling), RNA polymerase II antibody (NBP2-32080, Novus Biologicals), CDC73 (A300-170A, Bethyl Laboratories), SUPT5H antibody (A300-868A, Bethyl Laboratories), CPSF73 (A301-091A, Bethyl Laboratories), and CPSF100 (A301-581A, Bethyl Laboratories).

Techniques: Immunofluorescence, Quantitative RT-PCR, Generated, Two Tailed Test, ChIP-qPCR

Journal: bioRxiv

Article Title: Differential effects of pre-mRNA splicing inhibitors on RNA polymerase II transcription

doi: 10.1101/2023.01.16.524270

Figure Lengend Snippet: (A) Metagene profiles of total pol II mNET-seq performed in HeLa cells treated with DMSO (blue) or 90 µM Madrasin for 30 minutes (red) on scaled expressed intronless genes. (B) Screenshot of the genome browser total pol II mNET-seq DMSO (blue) and Madrasin (red) tracks of the intronless gene JUN . The arrow indicates the sense of transcription. (C) Total pol II ChIP-qPCR of the JUN intronless gene in HeLa cells treated with DMSO (black) or 90 µM Madrasin for 30 minutes (white). Mean ± SEM, n = 3 biological replicates. Statistical test: two-tailed unpaired t-test. P-value: * < 0.05, *** < 0.001, **** < 0.0001. (D) Metagene profiles of total pol II mNET-seq performed in HeLa cells treated with DMSO (blue) or 90 µM Madrasin for 30 minutes (red) on scaled expressed histone genes. (E) Screenshot of the genome browser total pol II mNET-seq DMSO (blue) and Madrasin (red) tracks of the histone gene HIST1H1C , also known as H1-2 . The arrow indicates the sense of transcription. (F) Total pol II ChIP-qPCR of the HIST1H1C histone gene in HeLa cells treated with DMSO (black) or 90 µM Madrasin for 30 minutes (white). Mean ± SEM, n = 3 biological replicates. Statistical test: two-tailed unpaired t-test. P-value: * < 0.05.

Article Snippet: For each IP, 60 μg of chromatin was incubated overnight on a rotating wheel at 4°C with the following antibodies: normal rabbit IgG (2729S, Cell Signaling), RNA polymerase II antibody (NBP2-32080, Novus Biologicals), CDC73 (A300-170A, Bethyl Laboratories), SUPT5H antibody (A300-868A, Bethyl Laboratories), CPSF73 (A301-091A, Bethyl Laboratories), and CPSF100 (A301-581A, Bethyl Laboratories).

Techniques: ChIP-qPCR, Two Tailed Test

Journal: bioRxiv

Article Title: Differential effects of pre-mRNA splicing inhibitors on RNA polymerase II transcription

doi: 10.1101/2023.01.16.524270

Figure Lengend Snippet: (A) Ratios of SPT5 / total pol II, CDC73 / total pol II, or CPSF73 / total pol II from ChIP-qPCR on the KPNB1 intron-containing gene in HeLa cells treated with DMSO (black) or 90 µM Madrasin for 30 minutes (white). Mean ± SEM, n = 3 biological replicates. Statistical test: two-tailed unpaired t-test. P-value: * < 0.05, ** < 0.01, *** < 0.001, **** < 0.0001. (B) Ratios of SPT5 / total pol II, CDC73 / total pol II, or CPSF73 / total pol II from ChIP-qPCR on the JUN intronless gene in HeLa cells treated with DMSO (black) or 90 µM Madrasin for 30 minutes (white). Mean ± SEM, n = 3 biological replicates. Statistical test: two-tailed unpaired t-test. P-value: * < 0.05. (C) Ratios of SPT5 / total pol II, CDC73 / total pol II, or CPSF73 / total pol II from ChIP-qPCR on the HIST1H1C histone gene in HeLa cells treated with DMSO (black) or 90 µM Madrasin for 30 minutes (white). Mean ± SEM, n = 3 biological replicates. Statistical test: two-tailed unpaired t-test. P-value: * < 0.05, *** < 0.001.

Article Snippet: For each IP, 60 μg of chromatin was incubated overnight on a rotating wheel at 4°C with the following antibodies: normal rabbit IgG (2729S, Cell Signaling), RNA polymerase II antibody (NBP2-32080, Novus Biologicals), CDC73 (A300-170A, Bethyl Laboratories), SUPT5H antibody (A300-868A, Bethyl Laboratories), CPSF73 (A301-091A, Bethyl Laboratories), and CPSF100 (A301-581A, Bethyl Laboratories).

Techniques: ChIP-qPCR, Two Tailed Test

Journal: bioRxiv

Article Title: Differential effects of pre-mRNA splicing inhibitors on RNA polymerase II transcription

doi: 10.1101/2023.01.16.524270

Figure Lengend Snippet: (A) Representative images of immunofluorescence analysis or 5’EU incorporation in HeLa cells treated with DMSO or 30 µM Isoginkgetin for 1h to 6h. EU (green), DAPI (blue), scale bars: 50 µm. (B) Quantification of 5’EU intensity per nucleus for DMSO (blue) and Isoginkgetin (other colours). Boxplot settings are: min to max values with the box showing 25-75 percentile range. 8,466 nuclei were quantified per condition. Statistical test: Kruskal-Wallis test. P-value: **** < 0.0001. (C) qRT-PCR with primers amplifying a region from the pol I transcribed pre-rRNA. HeLa cells were treated with DMSO or 30 µM Madrasin for 1h to 6h. cDNA was generated with random hexamers. Values are normalised to the 7SK snRNA and shown as relative to DMSO, mean ± SEM, n = 3 biological replicates. Statistical test: two-tailed unpaired t-test. P-value: n* < 0.05. (D) qRT-PCR with primers amplifying different protein-coding genes. HeLa cells were treated with DMSO or 30 µM Isoginkgetin for 1h to 6h. cDNA was generated with oligo(dT). Values are normalised to the GAPDH protein-coding gene and shown as relative to DMSO, mean ± SEM, n = 3 biological replicates. Statistical test: two-tailed unpaired t-test. P-value: * < 0.05, ** < 0.01, *** < 0.001, **** < 0.0001. (E) Total pol II ChIP-qPCR of the KPNB1 intron-containing gene in HeLa cells treated with DMSO (black) or 30 µM Isoginkgetin for 1h to 6h. Mean ± SEM, n = 3 biological replicates. Statistical test: two-tailed unpaired t-test. P-value: * < 0.05, ** < 0.01. (F) Total pol II ChIP-qPCR of the JUN intronless gene in HeLa cells treated with DMSO (black) or 30 µM Isoginkgetin for 1h to 6h. Mean ± SEM, n = 3 biological replicates. Statistical test: two-tailed unpaired t-test. P-value: * < 0.05. (G) Total pol II ChIP-qPCR of the HIST1H1C histone gene in HeLa cells treated with DMSO (black) or 30 µM Isoginkgetin for 1h to 6h. Mean ± SEM, n = 3 biological replicates. Statistical test: two-tailed unpaired t-test. P-value: * < 0.05.

Article Snippet: For each IP, 60 μg of chromatin was incubated overnight on a rotating wheel at 4°C with the following antibodies: normal rabbit IgG (2729S, Cell Signaling), RNA polymerase II antibody (NBP2-32080, Novus Biologicals), CDC73 (A300-170A, Bethyl Laboratories), SUPT5H antibody (A300-868A, Bethyl Laboratories), CPSF73 (A301-091A, Bethyl Laboratories), and CPSF100 (A301-581A, Bethyl Laboratories).

Techniques: Immunofluorescence, Quantitative RT-PCR, Generated, Two Tailed Test, ChIP-qPCR

Journal: bioRxiv

Article Title: Differential effects of pre-mRNA splicing inhibitors on RNA polymerase II transcription

doi: 10.1101/2023.01.16.524270

Figure Lengend Snippet: (A) Western blots on the chromatin fraction of HeLa cells treated with DMSO or with Isoginkgetin for 1h, 2h, or 4h. The antibodies are indicated on the right, with histone H3 used as a loading control. Quantifications of the western blots are shown below each panel. Each quantification has been normalised to histone H3 signal and then to the DMSO condition. (B) ChIP-qPCR of total pol II, Ser2P, Ser5P, and CPSF100 across the KPNB1 intron-containing gene in HeLa cells treated with DMSO (black) or 30 µM Isoginkgetin for 1h, 1h30, or 2h. Mean ± SEM, n = 3 biological replicates. Statistical test: two-tailed unpaired t-test. P-value: * < 0.05, ** < 0.01, *** < 0.001, **** < 0.0001. (C) Western blot on whole cell extract of HeLa cells treated with DMSO or with Isoginkgetin for 1h, 2h, or 4h. The antibodies are indicated at the bottom, with Ponceau S staining used as loading control.

Article Snippet: For each IP, 60 μg of chromatin was incubated overnight on a rotating wheel at 4°C with the following antibodies: normal rabbit IgG (2729S, Cell Signaling), RNA polymerase II antibody (NBP2-32080, Novus Biologicals), CDC73 (A300-170A, Bethyl Laboratories), SUPT5H antibody (A300-868A, Bethyl Laboratories), CPSF73 (A301-091A, Bethyl Laboratories), and CPSF100 (A301-581A, Bethyl Laboratories).

Techniques: Western Blot, Control, ChIP-qPCR, Two Tailed Test, Staining