Journal: Clinical Cancer Research

Article Title: Profiling of Chemonaive Osteosarcoma and Paired-Normal Cells Identifies EBF2 as a Mediator of Osteoprotegerin Inhibition to Tumor Necrosis Factor–Related Apoptosis-Inducing Ligand–Induced Apoptosis

doi: 10.1158/1078-0432.ccr-09-0300

Figure Lengend Snippet: Fig. 3. A, detailed diagram of the microarray hybridization signals for the probe-set (220392_at) corresponding to the EBF2 gene in osteosarcoma paired control samples and quantitative RT-PCR for EBF2 in an independent set of osteosarcoma and normal osteoblast samples (n = 12). B, top, quantitative RT-PCR results for OPG expression levels; bottom, OPG protein levels in the conditioned medium of independent samples (control: n = 12 and osteosarcoma: n = 12) were analyzed by ELISA. C, representative images of immunohistochemical analysis of EBF2 in osteosarcoma sections. b to d, high intensity of staining; a, almost complete absence of immunoreactivity. Bar, 100 μm.

Article Snippet: Sections were incubated with anti-EBF2 antibody (Genway) overnight at 4°C.

Techniques: Microarray, Hybridization, Control, Quantitative RT-PCR, Expressing, Enzyme-linked Immunosorbent Assay, Immunohistochemical staining, Staining

Journal: Journal of Lipid Research

Article Title: EBF2 regulates cardiolipin and phosphatidylethanolamine remodeling and mitochondrial dynamics in brown fat

doi: 10.1016/j.jlr.2025.100888

Figure Lengend Snippet: EBF2 directly regulates the expression of genes critical for lipid synthesis and metabolism in thermogenic fat. The genome-wide ChIP-sequencing data were analyzed for EBF2 binding sites for genes regulating lipid metabolism. A: EBF2 target genes are involved in phospholipid biosynthesis. B: Venn diagram depicting the number of EBF2-binding sites in each lipid gene. C: Lipid biosynthesis and metabolism pathways involving key genes regulated by EBF2. Genes upregulated and downregulated by EBF2 are highlighted in green and red, respectively. Genes indirectly regulated by EBF2 are highlighted in blue. D: Validation of EBF2 binding on regulatory sites of key lipid genes in BAT by EBF2 ChIP-qPCR analysis. Data are represented as mean ± SEM.

Article Snippet: Ebf2 flox/flox conditional null mice were generated by our laboratory using standard gene targeting techniques, where exon 3 of the Ebf2 gene was flanked by LoxP sites (Cyagen, Santa Clara, CA) in the C57BL/6 strain.

Techniques: Expressing, Genome Wide, ChIP-sequencing, Binding Assay, Biomarker Discovery, ChIP-qPCR

Journal: Journal of Lipid Research

Article Title: EBF2 regulates cardiolipin and phosphatidylethanolamine remodeling and mitochondrial dynamics in brown fat

doi: 10.1016/j.jlr.2025.100888

Figure Lengend Snippet: BAT-specific Ebf2 deletion perturbs the expression of genes regulating membrane lipid metabolism. BAT was collected from Ebf2-KO and WT P1-P3 neonates. A: Schematic representation and (B) Ebf2 KO validation in BAT of Ebf2 fl/fl and Ebf2 - KO neonates. Total RNA isolated from Ebf2 fl/fl and Ebf2-KO BAT was subjected to qPCR analysis and assessed the relative mRNA levels of genes involved in (C) thermogenic genes, (D) PPARγ and its target genes, (E) common targets of PPARγ and EBF2, (F and G) mRNA levels of CL remodeling and mitochondrial dynamics genes, (H and I) relative mRNA levels of genes involved in β-oxidation and phospholipid biosynthesis pathway in WT and Ebf2-KO BAT. n = 5–7 neonates/group. Results are represented as mean ± SEM. P values were determined by a two-tailed t- test. ∗ P ≤ 0.05, ∗∗ P < 0.01.

Article Snippet: Ebf2 flox/flox conditional null mice were generated by our laboratory using standard gene targeting techniques, where exon 3 of the Ebf2 gene was flanked by LoxP sites (Cyagen, Santa Clara, CA) in the C57BL/6 strain.

Techniques: Expressing, Membrane, Biomarker Discovery, Isolation, Two Tailed Test

Journal: Journal of Lipid Research

Article Title: EBF2 regulates cardiolipin and phosphatidylethanolamine remodeling and mitochondrial dynamics in brown fat

doi: 10.1016/j.jlr.2025.100888

Figure Lengend Snippet: Myf5 Cre -driven Ebf2 deletion accumulates PS and SM in brown fat mitochondria. BAT was collected from Ebf2 fl/fl and Ebf2 KO P1-P3 neonates. Mitochondrial fraction was isolated by pooling 3–4 neonatal BAT to generate each biological replicate. Lipids isolated from the mitochondrial fraction of BAT were subjected to shotgun lipidome analyses. A: Schematic representation of isolation of BAT mitochondrial fraction followed by lipid isolation and analysis from P1-P3 Ebf2 fl/fl and Ebf2 - KO pups. B: Analysis of total polar lipids and (C–D) distribution of individual lipid classes in Ebf2 fl/fl and Ebf2 - KO mitochondria. E: The volcano plot shows the differential abundance of BAT lipid species between Ebf2 fl/fl and Ebf2 KO mitochondria. F and G: A score plot was obtained after PCA based on the levels of SM and PS species. H: A heatmap with hierarchical clustering shows the changes in the concentration of individual lipid species. I and J: Quantification of mitochondrial PS and SM species. Results are presented as mean ± SEM. Two-tailed Student’s t- test was performed to test differences between two independent groups. n = 4 biological replicates, where each replicate contains BAT of 3–4 neonates. ∗ P ≤ 0.05, ∗∗ P < 0.01, and ∗∗∗ P < 0.001.

Article Snippet: Ebf2 flox/flox conditional null mice were generated by our laboratory using standard gene targeting techniques, where exon 3 of the Ebf2 gene was flanked by LoxP sites (Cyagen, Santa Clara, CA) in the C57BL/6 strain.

Techniques: Isolation, Concentration Assay, Two Tailed Test

Journal: Journal of Lipid Research

Article Title: EBF2 regulates cardiolipin and phosphatidylethanolamine remodeling and mitochondrial dynamics in brown fat

doi: 10.1016/j.jlr.2025.100888

Figure Lengend Snippet: Ebf2 deletion diminishes the abundance of CL species in brown fat mitochondria. Heatmap representing the differential abundance of mitochondrial CL molecular species (A). B: Score plot obtained after PCA based on lipid class abundance, including 95% confidence regions for Ebf2 fl/fl and Ebf2 KO mitochondria. C: Altered levels of individual CL species. n = 4 biological replicates/group, where each replicate was generated by pooling BAT of 3–4 neonates. D: Immunoblot analysis of UCP1 and OXPHOS complex proteins with vinculin as a loading control. E: Immunoblot quantification (n = 4/group). F: Oxygen consumption rate of mitochondria isolated from WT and Ebf2-KO mature brown adipocytes (n = 3). Results are presented as mean ± SEM. Two-tailed t- test was performed to test differences between two independent groups. ∗ P ≤ 0.05, ∗∗ P < 0.01, and ∗∗∗ P < 0.001.

Article Snippet: Ebf2 flox/flox conditional null mice were generated by our laboratory using standard gene targeting techniques, where exon 3 of the Ebf2 gene was flanked by LoxP sites (Cyagen, Santa Clara, CA) in the C57BL/6 strain.

Techniques: Generated, Western Blot, Control, Isolation, Two Tailed Test

Journal: Journal of Lipid Research

Article Title: EBF2 regulates cardiolipin and phosphatidylethanolamine remodeling and mitochondrial dynamics in brown fat

doi: 10.1016/j.jlr.2025.100888

Figure Lengend Snippet: Ebf2 deficiency alters PC levels in brown fat mitochondria. Heatmap representing the differential abundance of mitochondrial LPC and PC molecular species (A). B and C: Score plot obtained after PCA based on LPC and PC abundance, including 95% confidence regions. D and E: Abundance of molecular species of LPC and PC levels in Ebf2 fl/fl and Ebf2 - KO BAT mitochondria. Results are presented as mean ± SEM. Two-tailed t- test was performed to test differences between two independent groups, n = 4 biological replicates/group, where each replicate was generated by pooling BAT of 3–4 neonates. ∗ P ≤ 0.05, ∗∗ P < 0.01, and ∗∗∗ P < 0.001.

Article Snippet: Ebf2 flox/flox conditional null mice were generated by our laboratory using standard gene targeting techniques, where exon 3 of the Ebf2 gene was flanked by LoxP sites (Cyagen, Santa Clara, CA) in the C57BL/6 strain.

Techniques: Two Tailed Test, Generated

Journal: Journal of Lipid Research

Article Title: EBF2 regulates cardiolipin and phosphatidylethanolamine remodeling and mitochondrial dynamics in brown fat

doi: 10.1016/j.jlr.2025.100888

Figure Lengend Snippet: Ebf2 deletion elevates ROS levels in mature brown adipocytes. Brown preadipocytes of control and Ebf2-KO were differentiated until day 7 of differentiation, and the lipid droplets were stained with BODIPY (A). Morphological differences between WT and Ebf2-KO mature brown adipocytes and nuclear fraction subjected to immunoblot analysis of EBF2, with TATA-binding protein (TBP) as loading control (A and B). Relative mRNA levels of brown fat-specific and membrane lipid biosynthesis genes in WT and Ebf2-KO brown adipocytes. n = 3 biological replicates (C and D). E and F: Evaluation of mitochondrial ROS levels using dihydroethidium (DHE) staining in WT and Ebf2 - KO brown adipocytes with quantification (scale represents 50 μm, n = 4/group). Results are presented as mean ± SEM. Two-tailed t- test was performed to test differences between two independent groups. ∗ P ≤ 0.05, ∗∗ P < 0.01, and ∗∗∗ P < 0.001.

Article Snippet: Ebf2 flox/flox conditional null mice were generated by our laboratory using standard gene targeting techniques, where exon 3 of the Ebf2 gene was flanked by LoxP sites (Cyagen, Santa Clara, CA) in the C57BL/6 strain.

Techniques: Control, Staining, Western Blot, Binding Assay, Membrane, Two Tailed Test

Journal: Journal of Lipid Research

Article Title: EBF2 regulates cardiolipin and phosphatidylethanolamine remodeling and mitochondrial dynamics in brown fat

doi: 10.1016/j.jlr.2025.100888

Figure Lengend Snippet: Deletion of Ebf2 impairs mitochondrial PE remodeling and dynamics. A: Heatmap depicting altered levels of mitochondrial PE species in BAT of Ebf2 fl/fl and Ebf2-KO neonates. B and C: PCA score plot obtained based on LPE and PE abundance, including 95% confidence regions for Ebf2 fl/fl and Ebf2 - KO BAT mitochondria. D and E: Quantification of major mitochondrial LPE and PE molecular species. n = 4 biological replicates, where each replicate contains 3–4 neonates. F and H: Double immunofluorescence staining to evaluate DRP1 levels in WT and Ebf2 - KO mature brown adipocytes (scale represents 40 μm). Results are presented as mean ± SEM. Immunoblot analysis of mitochondrial fission and fusion proteins with β-actin as a loading control and respective quantification (G and H) in BAT of Ebf2 fl/fl and Ebf2 - KO pups. Two-tailed t- test was performed to test differences between two independent groups. n = 4–5/group. ∗ P ≤ 0.05, ∗∗ P < 0.01, and ∗∗∗ P < 0.001.

Article Snippet: Ebf2 flox/flox conditional null mice were generated by our laboratory using standard gene targeting techniques, where exon 3 of the Ebf2 gene was flanked by LoxP sites (Cyagen, Santa Clara, CA) in the C57BL/6 strain.

Techniques: Double Immunofluorescence Staining, Western Blot, Control, Two Tailed Test

Journal: Histochemistry and Cell Biology

Article Title: EBF1 is expressed in pericytes and contributes to pericyte cell commitment

doi: 10.1007/s00418-021-02015-7

Figure Lengend Snippet: Primary antibodies used in the study

Article Snippet: Rabbit polyclonal anti-EBF2 , Merck Millipore , AB10524.

Techniques:

Journal: Molecular Metabolism

Article Title: EBF2 promotes the recruitment of beige adipocytes in white adipose tissue

doi: 10.1016/j.molmet.2015.11.001

Figure Lengend Snippet: Ebf2 is required for in vivo beiging of ingWAT. (A) Confocal immunofluorescence imaging of whole-mount ingWAT tissue. EBF2 protein is present in wild type (WT) but not Ebf2 KO ingWAT. (B) H&E staining of ingWAT from WT and Ebf2 KO mice before (left panels) or after (right panels) injection with CL-316,243 to induce beiging. (C) Confocal immunofluorescence imaging of whole-mount ingWAT tissue. UCP1 protein is highly expressed in WT but not Ebf2 KO IngWAT following CL 316,243 injection. (D – G) Relative mRNA levels from in WT or Ebf2 knockout ingWAT before and after CL 316,243 injection as measured by RT-qPCR. Levels of (D) Ebf2 , (E) the general adipose genes Pparϒ, AdipoQ and Fabp4 , (F) the brown selective genes Ucp1, Cidea, Pgc1α, Pparα and Prdm16 , and (G) the mitochondrial genes Cox7a1, Cox8b, Cycs . (n = 3). (H) Relative oxygen consumption of ingWAT from WT and Ebf2 KO mice. (n = 4). P-values are represented with asterisks (*, p-value ≤ .05; **, p-value ≤ .01; ***, p-value ≤ .001) and error bars represent SEM. Scale bars = 10 microns.

Article Snippet: Ebf2 whole body knockout animals were obtained from R. Reed (Johns Hopkins, Baltimore, MD, USA) and have been described previously .

Techniques: In Vivo, Immunofluorescence, Imaging, Staining, Injection, Knock-Out, Quantitative RT-PCR

Journal: Molecular Metabolism

Article Title: EBF2 promotes the recruitment of beige adipocytes in white adipose tissue

doi: 10.1016/j.molmet.2015.11.001

Figure Lengend Snippet: Ebf2 is required for beiging. (A) Relative EBF2 protein levels from WT primary ingWAT stromal vascular cells plus or minus rosi treatment as assayed by western blot. (B) Relative Ebf2 mRNA levels from WT or Ebf2 KO primary ingWAT stromal vascular cells before and after rosi treatment as measured by RT-qPCR. ( C) Oil Red O staining of differentiated and rosi treated WT or Ebf2 KO primary ingWAT stromal vascular cells. (D – G) Relative mRNA levels from WT or Ebf2 KO primary ingWAT stromal vascular cells plus or minus rosi treatment as measured by RT-qPCR. Levels of (D) the general adipose genes Pparϒ and Fabp4 , (E) the brown selective genes Ucp1 and Cidea, (F) the brown selective genes Pgc1α, Pparα and Prdm16 , and (G) the mitochondrial genes Cox5b, Cox7a1, Cox8b . (H) Western blot to measure protein levels of UCP1 in differentiated and rosi treated WT or Ebf2 knockout primary ingWAT stromal vascular cells. NS = non-specific band loading control. (I) Relative Ucp1 and Pgc1α mRNA levels from WT or Ebf2 knockout primary ingWAT stromal vascular cells before and after Iso treatment as measured by RT-qPCR. P-values are represented with asterisks (*, p-value ≤ .05; **, p-value ≤ .01; ***, p-value ≤ .001) and error bars represent standard deviation.

Article Snippet: Ebf2 whole body knockout animals were obtained from R. Reed (Johns Hopkins, Baltimore, MD, USA) and have been described previously .

Techniques: Western Blot, Quantitative RT-PCR, Staining, Knock-Out, Control, Standard Deviation

Journal: Molecular Metabolism

Article Title: EBF2 promotes the recruitment of beige adipocytes in white adipose tissue

doi: 10.1016/j.molmet.2015.11.001

Figure Lengend Snippet: EBF2-expression drives a brown fat/thermogenic profile in primary adipocytes. (A – C) Relative mRNA levels from control or retroviral EBF2-expressing primary ingWAT stromal vascular cells with or without rosi treatment as measured by RT-qPCR. Levels of (A) Ebf2, (B) the general adipose genes Pparϒ, AdipoQ and Fabp4 , and (C) the brown selective genes Ucp1, Cidea, Pgc1α, Pparα and Prdm16 , and (D) the mitochondrial genes Cox7a1, Cox8b, Cycs . (E) Western blot to measure protein levels of EBF2 and UCP1 levels in control or retroviral EBF2-expressing primary ingWAT stromal vascular cells following rosi treatment. P-values are represented with asterisks (*, p-value ≤ .05; **, p-value ≤ .01; ***, p-value ≤ .001) and error bars represent standard deviation.

Article Snippet: Ebf2 whole body knockout animals were obtained from R. Reed (Johns Hopkins, Baltimore, MD, USA) and have been described previously .

Techniques: Expressing, Control, Retroviral, Quantitative RT-PCR, Western Blot, Standard Deviation

Journal: Molecular Metabolism

Article Title: EBF2 promotes the recruitment of beige adipocytes in white adipose tissue

doi: 10.1016/j.molmet.2015.11.001

Figure Lengend Snippet: Ectopic ingWAT expression of EBF2 induces beiging in vivo . (A) qPCR of relative Ebf2 mRNA levels from WT (white bar) and Fabp4-Ebf2 (black bar) mice in BAT, ingWAT and epiWAT (mice housed at room temperature). (n = 3). (B) Western blot to measure EBF2 protein levels in WT or Fabp4-Ebf2 (TG) mice in ingWAT, BAT and epiWAT (mice housed at room temperature). (C) H&E staining of ingWAT from WT and Fabp4-Ebf2 mice. (D) Confocal immunofluorescence imaging of UCP1 protein levels in ingWAT from WT or Fabp4-Ebf2 mice. (E – G) ) Relative mRNA levels from WT and Fabp4-Ebf2 ingWAT as measured by RT-qPCR. Levels of (E) the general adipose genes Pparϒ, AdipoQ and Fabp4 , (F) the brown selective genes Cidea, Pgc1α, Pparα and Prdm16 and (G) mitochondrial genes Cox7a1, Cox8b, Cycs . (H) Heat map of the top up-regulated and down-regulated genes in Fabp4-Ebf2 ingWAT compared to WT, with corresponding GO analysis. (I) Cross comparison between RNAseq data comparing WT and Fabp4-Ebf2 ingWAT gene expression and microarray comparing thermoneutral and cold exposed ingWAT. The majority of overlapping upregulated genes are related to mitochondrial function. (J) Relative UCP1 mRNA levels of WT (white bars) and Fabp4-Ebf2 (black bars) ingWAT from mice housed at thermoneutrality (TN, 30 °C) or room temperature (RT) as measured by RT-qPCR. (K) Relative oxygen consumption of ingWAT from WT and TG Fabp4-Ebf2 mice housed at room temperature, n = 4. (L) Body weight of wild type (square) and TG Fabp4-Ebf2 (circle) for mice housed at thermoneutrality on HFD beginning at 5 weeks of age. (n = 5). (M) Fat mass and lean weight mass of WT or TG Fabp4-Ebf2 (n = 5) mice after 25 weeks on HFD at TN. (N) Gross histology of ingWAT fat pads from WT or TG Fabp4-Ebf2 mice after 25 weeks on HFD at TN. P-values are represented with asterisks (*, p-value ≤ .05; **, p-value ≤ .01; ***, p-value ≤ .001) and error bars denote SEM. Scale bars = 10 microns.

Article Snippet: Ebf2 whole body knockout animals were obtained from R. Reed (Johns Hopkins, Baltimore, MD, USA) and have been described previously .

Techniques: Expressing, In Vivo, Western Blot, Staining, Immunofluorescence, Imaging, Quantitative RT-PCR, Comparison, Gene Expression, Microarray

Journal: Genes & Development

Article Title: EBF2 transcriptionally regulates brown adipogenesis via the histone reader DPF3 and the BAF chromatin remodeling complex

doi: 10.1101/gad.294405.116

Figure Lengend Snippet: EBF2 binds and regulates the chromatin state at brown fat-specific genes. ( A ) ChIP-seq profiles in reads per million total reads (RPM) for EBF2 (dark blue), H3K27ac (red), RNA polymerase II (Pol II; light blue), and PPARγ (gray) in Ebf2 wild-type and knockout BAT at Ucp1 and Ppar α. ( B ) Box plot showing changes in Pol II levels within the gene body and H3K27ac levels within 100 kb of BAT-selective (BAT-sel.), common, and WAT-selective (WAT-sel.) genes in Ebf2 knockout/wild-type BAT. Wilcoxon rank sum test, (***) P < 10 −16 . ( C ) Correlation analysis between EBF2 occupancy and differentially regulated H3K27ac peaks in Ebf2 knockout relative to wild-type BAT. ( D ) Scatter plot analysis of differentially expressed genes in Ebf2 wild-type versus knockout BAT. Fold change >1.5; false discovery rate <0.01. ( E ) Gene ontology analysis of down-regulated genes in Ebf2 knockout relative to wild-type BAT with at least one proximal EBF2-binding site within a 50-kb window around the transcription start site.

Article Snippet: Ebf2 fl/fl conditional null mice were generated by our laboratory using standard gene targeting techniques to insert loxP sites flanking exon 3 of the Ebf2 gene (Cyagen) in the C57BL/6 strain.

Techniques: ChIP-sequencing, Knock-Out, Binding Assay

Journal: Genes & Development

Article Title: EBF2 transcriptionally regulates brown adipogenesis via the histone reader DPF3 and the BAF chromatin remodeling complex

doi: 10.1101/gad.294405.116

Figure Lengend Snippet: EBF2 interacts with the BAF complex, which incorporates the subunit DPF3 in brown adipocytes. ( A ) Coimmunoprecipitation experiment in 293T cells transfected with pcDNA3.1-Flag-EBF2 ± pMX-BRG1 followed by Flag immunoprecipitation. ( B ) Endogenous coimmunoprecipitation in differentiated mature brown adipocytes. Sheep IgG was used as a negative control. ( C ) RT-qPCR analysis of BAF subunit expression in BAT and WAT from 6-wk-old male mice. Mean ± SE. n = 3. Two-sample Student's t -test, (*) P < 0.05; (**) P < 0.01. ( D ) RT-qPCR analysis of Dpf and Ucp1 expression in inguinal white adipose following 2 wk of cold exposure at 4°C. Mean ± SE. n = 5. Two-sample Student's t -test, (**) P < 0.01. ( E ) Western blot analysis of DPF3, UCP1, and Tubulin (loading control) in inguinal WAT (iWAT) and BAT.

Article Snippet: Ebf2 fl/fl conditional null mice were generated by our laboratory using standard gene targeting techniques to insert loxP sites flanking exon 3 of the Ebf2 gene (Cyagen) in the C57BL/6 strain.

Techniques: Transfection, Immunoprecipitation, Negative Control, Quantitative RT-PCR, Expressing, Western Blot, Control

Journal: Genes & Development

Article Title: EBF2 transcriptionally regulates brown adipogenesis via the histone reader DPF3 and the BAF chromatin remodeling complex

doi: 10.1101/gad.294405.116

Figure Lengend Snippet: DPF3 is required for activation of the brown fat program and mitochondrial function. ( A ) RT-qPCR analysis of day 7 mature brown adipocytes following shRNA-mediated Dpf3 depletion. ( B ) Oil-Red-O staining of shScr (control) and shDpf3 brown adipocytes. ( C ) RT-qPCR analysis of pan-adipogenic gene expression in control and DPF3-depleted mature brown adipocytes. ( D ) RT-qPCR analysis of brown fat-specific gene expression in control and DPF3-depleted mature brown adipocytes basally or following 3 h of stimulation with 1 µM iso. Mean ± SD. n = 3. Two-way ANOVA with Holm-Šídák multiple tests correction comparing shScr versus shDpf3 under basal or iso-stimlated conditions, (*) P < 0.05; (**) P < 0.01. ( E ) Western blot analysis of DPF3, EBF2, UCP1, and Actin (loading control) in control and DPF3-depleted brown adipocytes. ( F ) Oxygen consumption rate (OCR) in control and DPF3-depleted brown adipocytes; OCR was normalized to protein concentration. Mean ± SE. n = 23. Two-sample Student's t -test with Holm-Šídák multiple tests correction, (***) P < 0.001. ( G ) Uncoupled respiration in control and DPF3-depleted cells; OCR was normalized to protein concentration. Mean ± SE. n = 23. Two-sample Student's t -test, (***) P < 0.001 ( H ) OCR after acute iso stimulation in control and DPF3-depleted cells; OCR was normalized to protein concentration. Mean ± SE. n = 23. Two-sample Student's t -test, (***) P < 0.001. ( I ) Western blot analysis of DPF3, MTCO1, and Actin (loading control) in control and DPF3-depleted brown adipocytes. ( J ) Quantification of complex IV activity in control and DPF3-depleted brown adipocytes. Mean ± SE. n = 6. Two-sample Student's t -test, (***) P < 0.001. ( K ) Mean TMRE fluorescence assessed by flow cytometry in control and DPF3-depleted brown adipocytes. n = 3; 200,000 events recorded per sample. One-way ANOVA with Holm-Šídák correction for multiple comparisons, (**) P < 0.01.

Article Snippet: Ebf2 fl/fl conditional null mice were generated by our laboratory using standard gene targeting techniques to insert loxP sites flanking exon 3 of the Ebf2 gene (Cyagen) in the C57BL/6 strain.

Techniques: Activation Assay, Quantitative RT-PCR, shRNA, Staining, Control, Gene Expression, Western Blot, Protein Concentration, Activity Assay, Fluorescence, Flow Cytometry

Journal: Genes & Development

Article Title: EBF2 transcriptionally regulates brown adipogenesis via the histone reader DPF3 and the BAF chromatin remodeling complex

doi: 10.1101/gad.294405.116

Figure Lengend Snippet: DPF3 regulates the chromatin state at brown fat-specific genes. ( A ) FAIRE-qPCR analysis of control and DPF3-depleted mature brown adipocytes basally or following 3 h of stimulation with 1 µM iso. The insulin and β-globin promoters serve as negative controls; enrichment was analyzed as percentage of input. ( B , C ) ChIP-qPCR analysis of BRG1 ( B ) and EBF2 ( C ) binding in brown adipocytes ± iso; chromatin enrichment was analyzed as percentage of input recovery and normalized to 18S percentage of input to produce a fold enrichment. The insulin promoter served as a negative control. All data show mean ± SD. n = 3. Two-way ANOVA with Holm-Šídák multiple tests correction comparing shScr versus shDpf3 under basal or iso-stimulated conditions, (*) P < 0.05; (**) P < 0.01.

Article Snippet: Ebf2 fl/fl conditional null mice were generated by our laboratory using standard gene targeting techniques to insert loxP sites flanking exon 3 of the Ebf2 gene (Cyagen) in the C57BL/6 strain.

Techniques: Control, ChIP-qPCR, Binding Assay, Negative Control

Journal: Genes & Development

Article Title: EBF2 transcriptionally regulates brown adipogenesis via the histone reader DPF3 and the BAF chromatin remodeling complex

doi: 10.1101/gad.294405.116

Figure Lengend Snippet: EBF2 transcriptionally regulates Dpf3 expression. ( A ) RT-qPCR analysis of Ebf2 −/− brown adipocytes following retroviral-mediated control (Puro) or EBF2 overexpression. ( B ) RT-qPCR analysis in primary inguinal adipocytes following retroviral-mediated control (Puro) or EBF2 overexpression with and without rosiglitazone included throughout differentiation. Mean ± SD. n = 3. To compare all groups in a pairwise fashion, data were analyzed using a two-way ANOVA with Holm-Šídák multiple tests correction, (*) P < 0.05; (**) P < 0.01. ( C ) RT-qPCR analysis following CRISPR-mediated gene knockout in control ( Rosa26 ) and Ebf2 knockout mature brown adipocytes basally or following 3 h of stimulation with 1 µM iso. Two-way ANOVA with Holm-Šídák multiple tests correction comparing shScr versus shDpf3 under basal or iso-stimulated conditions, (*) P < 0.05; (**) P < 0.01. ( D ) RT-qPCR analysis of Dpf3 expression in control and Ebf2 knockout mature brown adipocytes basally or following 3 h of stimulation with 1 µM iso. Mean ± SD. n = 3. Two-way ANOVA with Holm-Šídák multiple tests correction comparing shScr versus shDpf3 under basal or iso-stimulated conditions, (**) P < 0.01. ( E ) Western blot analysis for EBF2, DPF3, and Actin (loading control) in control and Ebf2 knockout brown adipocytes. ( F ) Western blot analysis for EBF2, DPF3, UCP1, and Tubulin (loading control) in wild-type ( Ebf2 fl/fl ) or knockout ( Myf5 Cre /+ ; Ebf2 fl/fl ) BAT and iWAT. ( G ) RT-qPCR analysis of the mature adipocyte fraction from wild-type ( Ebf2 fl/fl ) or knockout ( Myf5 Cre /+ ; Ebf2 fl/fl ) animals. n = 3 animals pooled per genotype. Error bars show SD of technical replicates. Two-sample Student's t -test, (*) P < 0.05, (**) P < 0.01.

Article Snippet: Ebf2 fl/fl conditional null mice were generated by our laboratory using standard gene targeting techniques to insert loxP sites flanking exon 3 of the Ebf2 gene (Cyagen) in the C57BL/6 strain.

Techniques: Expressing, Quantitative RT-PCR, Retroviral, Control, Over Expression, CRISPR, Gene Knockout, Knock-Out, Western Blot

Journal: Genes & Development

Article Title: EBF2 transcriptionally regulates brown adipogenesis via the histone reader DPF3 and the BAF chromatin remodeling complex

doi: 10.1101/gad.294405.116

Figure Lengend Snippet: EBF2 directly regulates Dpf3 expression via an intronic enhancer. ( A ) ChIP-seq profiles at Dpf3 in RPM for EBF2 (dark blue), H3K27ac (red), RNA Pol II (light blue), and PPARγ (gray) in Ebf2 wild-type and knockout BAT. ( B ) ChIP-qPCR analysis of EBF2 binding at the Dpf3 +20-kb site over the course of brown adipocyte differentiation. The insulin promoter served as a negative control. Mean ± SD. n = 3. ( C ) RT-qPCR analysis of Dpf3 expression over the course of brown adipocyte differentiation. Mean ± SD. n = 3. ( D ) CRISPR–Cas9-mediated genomic editing strategy at the EBF motif in the Dpf3 +20-kb enhancer. ( E ) ChIP-qPCR for EBF2 in control ( Rosa26 ) and EBF gRNA-expressing pooled brown adipocytes. The insulin promoter served as a negative control. Mean ± SD. n = 3. Two-sample Student's t -test, (**) P < 0.01. ( F ) Gene expression analysis in control and EBF gRNA-expressing brown adipocytes. Mean ± SD. n = 3. Two-sample Student's t -test, (**) P < 0.01. ( G ) Western blot analysis of DPF3 and Actin (loading control) expression in control and EBF gRNA-expressing brown adipocytes.

Article Snippet: Ebf2 fl/fl conditional null mice were generated by our laboratory using standard gene targeting techniques to insert loxP sites flanking exon 3 of the Ebf2 gene (Cyagen) in the C57BL/6 strain.

Techniques: Expressing, ChIP-sequencing, Knock-Out, ChIP-qPCR, Binding Assay, Negative Control, Quantitative RT-PCR, CRISPR, Control, Gene Expression, Western Blot

Journal: Genes & Development

Article Title: EBF2 transcriptionally regulates brown adipogenesis via the histone reader DPF3 and the BAF chromatin remodeling complex

doi: 10.1101/gad.294405.116

Figure Lengend Snippet: Critical role for the histone-binding activity of DPF3 in brown adipocytes. ( A ) Schematic of the DPF3A and DPF3 domain structures. (NLS) Nuclear localization sequence; (NID) nuclear receptor interaction domain. ( B ) Flag immunoprecipitation of ectopically expressed control vector (Puro), DPF3A, or DPF3B followed by blotting for endogenous BRG1 in mature brown adipocytes. ( C ) Gene expression analysis in control (Puro), DPFA-expressing, and DPF3B-expressing mature brown adipocytes. Mean ± SD. n = 3. Two-sample Student's t -test, (*) P < 0.05; (**) P < 0.01. ( D ) Western blot analysis of control or EBF2-expressing C3H-10T1/2 cells infected with control (Puro), DPF3A, or DPF3B. ( E , F ) Gene expression analysis of common adipogenic genes ( E ) or Ucp1 ( F ) in C3H-10T1/2 cells. Mean ± SD. n = 3. Two-sample Student's t -test, (**) P < 0.01. ( G ) Model for EBF2-mediated regulation at brown fat genes.

Article Snippet: Ebf2 fl/fl conditional null mice were generated by our laboratory using standard gene targeting techniques to insert loxP sites flanking exon 3 of the Ebf2 gene (Cyagen) in the C57BL/6 strain.

Techniques: Binding Assay, Activity Assay, Sequencing, Immunoprecipitation, Control, Plasmid Preparation, Gene Expression, Expressing, Western Blot, Infection