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Addgene inc pcdna3 1
Pcdna3 1, supplied by Addgene inc, used in various techniques. Bioz Stars score: 92/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/pcdna3+1+prdm16/pcDNA3%2E1+PRDM16+(Plasmid+%2315503)/bio_rxiv__64898__2026__03__25__714341-52-7-11
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pcdna3 1 - by Bioz Stars, 2026-08

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( A ) Gene set enrichment analysis (GSEA) analysis of ventricular RNA-seq datasets at E15.5, P1, P7 and adulthood comparing homozygous Mybpc3 p.Pro109Serfs*12 and Mybpc3 +/+ ventricular transcriptome against Hallmark and Reactome gene sets. The first 4 columns show comparisons between mutant and control samples at E15.5, P1, P7 and adult stages. Most significant processes seem to occur at P1 and P7. The mutation causes similar direction changes at P1 and P7 on cell cycle terms (increased in the mutants), but opposite changes in metabolism, NMD and metabolism/proteostasis terms (oxphos, translation) which are decreased at P1 mutants compared to control but increased in the mutants at P7. The last two columns represent the longitudinal comparisons between P1 and P7 in both control and mutant samples. The WT (control) P7-P1 column indicates the developmental change in the transition from P1 to P7 (red, up in P7). NMD and proteostasis (translation etc.) terms are decreased in the controls at P7, but in the mutants, these same processes are increased. Cell cycle terms maintain the same enrichment direction in the mutants but seem to be even more exacerbated at P7. ( B ) Left, bubble plot of differentially expressed genes at P7 in homozygous Mybpc3 p.Pro109Serfs*12 and Mybpc3+/+ hearts highlights upregulation of several genes and downregulation of Prdm16 within a hypertrophy-associated gene signature. Right, ISH for Nppa and Ankrd1 in control and homozygous Mybpc3 p.Pro109Serfs*12 P7 hearts. 250 μm. lv, left ventricle; rv, right ventricle. (( C ) Bar plot summarizing the normalized enrichment score (NES) and –log(padj) values for Mybpc3 P109Sfs*12 mutant vs. WT comparisons at E15.5, P1, P7, and Adult. Dotted line: p-value = 0.25. Dashed line: p -value = 0.01. D ) Bar plot summarizing the NES and –log(padj) values for P7 vs. P1 comparisons in control (WT) and Mybpc3 p.Pro109Serfs*12 (P109S) mutant. Dotted line: p -value = 0.25. Dashed line: p-value = 0.01. The NES represents the enrichment score after normalization, with higher scores (red) indicating positive enrichment and lower scores (blue) indicating negative enrichment. The adjusted p-value (padj) corresponds to p-values corrected for multiple testing using the Benjamini–Hochberg procedure, which controls the false discovery rate. ( E-F’’ ) IF for Prdm16 (green), Endomucin (red), and DAPI (blue) in in ventricular sections of E18.5 wild type ( E-E ’’) and homozygous Prdm16 Δ8 mutant hearts. Arrowheads in E’’ point to nuclear Prdm16 expression in trabecular cardiomyocytes. Scale bar 250 μm. Magnified views are shown in ( ’ ). Scale bar 50 μm. ( G-J’ ) IF for Prdm16 in ventricular sections at E16.5 ( G-H’ ) and P7 ( I-J’ ) reveals expanded Prdm16 signal in E16.5 Mybpc3 p.Pro109Serfs*12 mutant trabeculae (arrowheads, H’ , inset), and reduced Prdm16 expression at P7 in subendocardial myocardium of mutants ( J’ ). Scale bar, 250 μm. ( K ) Quantification of the ratio of trabecular (TM) to compact myocardium (CM) Prdm16 expression in E16.5 hearts (n=3, ≥3 sections per heart), and the ratio of inner myocardium (IM) to outter myocardium (OM) in P7 hearts. (n=4 control and n=3 Mybpc3 p.Pro109Serfs*12, ≥3 sections per heart). Data are represented as means ± SD. Statistical significance was determined by unpaired Student’s t-test (* p -value < 0.05).

Journal: bioRxiv

Article Title: Abnormal ventricular wall patterning precedes and drives MYBPC3 hypertrophic cardiomyopathy

doi: 10.64898/2026.03.25.714341

Figure Lengend Snippet: ( A ) Gene set enrichment analysis (GSEA) analysis of ventricular RNA-seq datasets at E15.5, P1, P7 and adulthood comparing homozygous Mybpc3 p.Pro109Serfs*12 and Mybpc3 +/+ ventricular transcriptome against Hallmark and Reactome gene sets. The first 4 columns show comparisons between mutant and control samples at E15.5, P1, P7 and adult stages. Most significant processes seem to occur at P1 and P7. The mutation causes similar direction changes at P1 and P7 on cell cycle terms (increased in the mutants), but opposite changes in metabolism, NMD and metabolism/proteostasis terms (oxphos, translation) which are decreased at P1 mutants compared to control but increased in the mutants at P7. The last two columns represent the longitudinal comparisons between P1 and P7 in both control and mutant samples. The WT (control) P7-P1 column indicates the developmental change in the transition from P1 to P7 (red, up in P7). NMD and proteostasis (translation etc.) terms are decreased in the controls at P7, but in the mutants, these same processes are increased. Cell cycle terms maintain the same enrichment direction in the mutants but seem to be even more exacerbated at P7. ( B ) Left, bubble plot of differentially expressed genes at P7 in homozygous Mybpc3 p.Pro109Serfs*12 and Mybpc3+/+ hearts highlights upregulation of several genes and downregulation of Prdm16 within a hypertrophy-associated gene signature. Right, ISH for Nppa and Ankrd1 in control and homozygous Mybpc3 p.Pro109Serfs*12 P7 hearts. 250 μm. lv, left ventricle; rv, right ventricle. (( C ) Bar plot summarizing the normalized enrichment score (NES) and –log(padj) values for Mybpc3 P109Sfs*12 mutant vs. WT comparisons at E15.5, P1, P7, and Adult. Dotted line: p-value = 0.25. Dashed line: p -value = 0.01. D ) Bar plot summarizing the NES and –log(padj) values for P7 vs. P1 comparisons in control (WT) and Mybpc3 p.Pro109Serfs*12 (P109S) mutant. Dotted line: p -value = 0.25. Dashed line: p-value = 0.01. The NES represents the enrichment score after normalization, with higher scores (red) indicating positive enrichment and lower scores (blue) indicating negative enrichment. The adjusted p-value (padj) corresponds to p-values corrected for multiple testing using the Benjamini–Hochberg procedure, which controls the false discovery rate. ( E-F’’ ) IF for Prdm16 (green), Endomucin (red), and DAPI (blue) in in ventricular sections of E18.5 wild type ( E-E ’’) and homozygous Prdm16 Δ8 mutant hearts. Arrowheads in E’’ point to nuclear Prdm16 expression in trabecular cardiomyocytes. Scale bar 250 μm. Magnified views are shown in ( ’ ). Scale bar 50 μm. ( G-J’ ) IF for Prdm16 in ventricular sections at E16.5 ( G-H’ ) and P7 ( I-J’ ) reveals expanded Prdm16 signal in E16.5 Mybpc3 p.Pro109Serfs*12 mutant trabeculae (arrowheads, H’ , inset), and reduced Prdm16 expression at P7 in subendocardial myocardium of mutants ( J’ ). Scale bar, 250 μm. ( K ) Quantification of the ratio of trabecular (TM) to compact myocardium (CM) Prdm16 expression in E16.5 hearts (n=3, ≥3 sections per heart), and the ratio of inner myocardium (IM) to outter myocardium (OM) in P7 hearts. (n=4 control and n=3 Mybpc3 p.Pro109Serfs*12, ≥3 sections per heart). Data are represented as means ± SD. Statistical significance was determined by unpaired Student’s t-test (* p -value < 0.05).

Article Snippet: A full-length mouse Prdm16 cDNA cloned into pcDNA3.1 was obtained from addgene (#15503).

Techniques: RNA Sequencing, Mutagenesis, Control, Expressing

( A ) Whole-mount views of P7 control, Mybpc3 +/+ ;R26 Prdm16/+ ;Myh6 MerCreMer/+ , homozygous Mybpc3 p.Pro109Serfs*12 and Mybpc3 p.Pro109Serfs*12;R26 Prdm16/+ ;Myh6 MerCreMer/+ hearts. Right, Cre-mediated transgenic expression was induced upon Tamoxifen administration at P1. Scale bar, 250 μm. ( B ) Quantification of Heart weight/Body weight ratio (n=25 Mybpc3+/+, n=16 Mybpc3+/+;R26 Prdm16/+ ;Myh6 MerCreMer/+ , n=13 homozygous Mybpc3 p.Pro109Serfs*12 and n=14 Mybpc3 p.Pro109Serfs*12;R26 Prdm16/+ ;Myh6 MerCreMer/+ ), Septum thickness (μm), Left Ventricular Free Wall (LVFW) thickness (μm)(n=4 Mybpc3+/+, n=3 Mybpc3+/+;R26 Prdm16/+ ;Myh6 MerCreMer/+ , n=3 homozygous Mybpc3 p.Pro109Serfs*12 and n=5 Mybpc3 p.Pro109Serfs*12;R26 Prdm16/+ ;Myh6 MerCreMer ≥3 sections per heart), compact myocardium leght (CM) and trabecular myocardium leght (TM) (μm) (n=3 hearts per genotype, ≥3 sections per heart). Data are represented as means ± SD. Statistical significance was determined by unpaired Student’s t-test (ns, not significant; * p -value < 0.05; *** p -value < 0.001; **** p -value < 0.0001). ( C ) H&E images of representative hearts of the different genotypes. Scale bar, 250 μm. ( D ) ISH for Nppa in the different genotypes. Scale bar, 250 μm. ( E ) WGA (green) and DAPI (blue) fluorescence staining of P7 control, Mybpc3 +/+ ;R26 Prdm16/+ ;Myh6 MerCreMer/+ , Mybpc3 p.Pro109Serfs*12 and Mybpc3 p.Pro109Serfs*12;R26 Prdm16/+ ;Myh6 MerCreMer/+ heart sections. Scale bar, 10 μm. ( F ) Cardiomyocyte cross sectional area quantification of P7 hearts (n=3, ≥6 sections per heart). Data are represented as means ± SD. Statistical significance was determined by unpaired Student’s t-test (ns, not significant; * p -value < 0.05; ** p -value < 0.005). ( G ) Ventricular wall mispatterning drives MYBPC3-associated HCM that is attenuated by Prdm16 expression. Top, wild type: During late gestation (E18.5), compact (CM, Hey2⁺) and trabecular cardiomyocytes (TM, Hey2 - ) are spatially segregated and exhibit balanced proliferation, enabling ventricular compaction progression at birth (P1) and subsequent maturation. Postnatally, Prdm16 promotes cardiomyocyte maturation and restrains hypertrophy, resulting in normal ventricular architecture by P7. Middle, Mybpc3 P109Sfs*12 mutant. Mybpc3 loss disrupts ventricular wall patterning, leading to loss of compact–trabecular segregation, increased trabecular proliferation, and impaired compaction. This is accompanied by peak transcriptional dysregulation from P1 to P7. Abnormally reduced Prdm16 expression impairs maturation and contributes to pathological cardiomyocyte hypertrophy. Bottom, Mybpc3 P109Sfs*12; R26 Prdm16/+ ; Myh6 MerCreMer model. Tamoxifen-mediated Prdm16 expression at P1 attenuates hypertrophy despite persistent early patterning defects, promoting cardiomyocyte maturation and partially normalizing ventricular structure.

Journal: bioRxiv

Article Title: Abnormal ventricular wall patterning precedes and drives MYBPC3 hypertrophic cardiomyopathy

doi: 10.64898/2026.03.25.714341

Figure Lengend Snippet: ( A ) Whole-mount views of P7 control, Mybpc3 +/+ ;R26 Prdm16/+ ;Myh6 MerCreMer/+ , homozygous Mybpc3 p.Pro109Serfs*12 and Mybpc3 p.Pro109Serfs*12;R26 Prdm16/+ ;Myh6 MerCreMer/+ hearts. Right, Cre-mediated transgenic expression was induced upon Tamoxifen administration at P1. Scale bar, 250 μm. ( B ) Quantification of Heart weight/Body weight ratio (n=25 Mybpc3+/+, n=16 Mybpc3+/+;R26 Prdm16/+ ;Myh6 MerCreMer/+ , n=13 homozygous Mybpc3 p.Pro109Serfs*12 and n=14 Mybpc3 p.Pro109Serfs*12;R26 Prdm16/+ ;Myh6 MerCreMer/+ ), Septum thickness (μm), Left Ventricular Free Wall (LVFW) thickness (μm)(n=4 Mybpc3+/+, n=3 Mybpc3+/+;R26 Prdm16/+ ;Myh6 MerCreMer/+ , n=3 homozygous Mybpc3 p.Pro109Serfs*12 and n=5 Mybpc3 p.Pro109Serfs*12;R26 Prdm16/+ ;Myh6 MerCreMer ≥3 sections per heart), compact myocardium leght (CM) and trabecular myocardium leght (TM) (μm) (n=3 hearts per genotype, ≥3 sections per heart). Data are represented as means ± SD. Statistical significance was determined by unpaired Student’s t-test (ns, not significant; * p -value < 0.05; *** p -value < 0.001; **** p -value < 0.0001). ( C ) H&E images of representative hearts of the different genotypes. Scale bar, 250 μm. ( D ) ISH for Nppa in the different genotypes. Scale bar, 250 μm. ( E ) WGA (green) and DAPI (blue) fluorescence staining of P7 control, Mybpc3 +/+ ;R26 Prdm16/+ ;Myh6 MerCreMer/+ , Mybpc3 p.Pro109Serfs*12 and Mybpc3 p.Pro109Serfs*12;R26 Prdm16/+ ;Myh6 MerCreMer/+ heart sections. Scale bar, 10 μm. ( F ) Cardiomyocyte cross sectional area quantification of P7 hearts (n=3, ≥6 sections per heart). Data are represented as means ± SD. Statistical significance was determined by unpaired Student’s t-test (ns, not significant; * p -value < 0.05; ** p -value < 0.005). ( G ) Ventricular wall mispatterning drives MYBPC3-associated HCM that is attenuated by Prdm16 expression. Top, wild type: During late gestation (E18.5), compact (CM, Hey2⁺) and trabecular cardiomyocytes (TM, Hey2 - ) are spatially segregated and exhibit balanced proliferation, enabling ventricular compaction progression at birth (P1) and subsequent maturation. Postnatally, Prdm16 promotes cardiomyocyte maturation and restrains hypertrophy, resulting in normal ventricular architecture by P7. Middle, Mybpc3 P109Sfs*12 mutant. Mybpc3 loss disrupts ventricular wall patterning, leading to loss of compact–trabecular segregation, increased trabecular proliferation, and impaired compaction. This is accompanied by peak transcriptional dysregulation from P1 to P7. Abnormally reduced Prdm16 expression impairs maturation and contributes to pathological cardiomyocyte hypertrophy. Bottom, Mybpc3 P109Sfs*12; R26 Prdm16/+ ; Myh6 MerCreMer model. Tamoxifen-mediated Prdm16 expression at P1 attenuates hypertrophy despite persistent early patterning defects, promoting cardiomyocyte maturation and partially normalizing ventricular structure.

Article Snippet: A full-length mouse Prdm16 cDNA cloned into pcDNA3.1 was obtained from addgene (#15503).

Techniques: Control, Transgenic Assay, Expressing, Fluorescence, Staining, Mutagenesis

A. PRDM16 complex was immunopurified from differentiated brown adipocytes. Endogenous E4bp4 was detected by immunoblotting. B. ChIP-seq for PRDM16 and PRDM16 knockout (PRDM16-KO) on the Cers6 gene in BAT. C. Breeding strategy for AdipoQ_SPH mice and littermate control mice. Primary brown adipocytes were used in the experiments. The cartoon was created using a Biorender. D. Relative mRNA levels of indicate genes in primary brown adipocytes of the Control and PRDM16-OE groups. n = 3 per group. E. Relative mRNA levels of Cers6 in primary brown adipocytes of the Control and PRDM16-OE groups. n = 3 per group. F. C16:0 quantification by mass spectrometry in primary brown adipocytes of the Control and PRDM16-OE groups. C16:0 levels were normalized to total protein (µg). n = 3 per group. G. Western blotting for E4bp4 in immortalized brown adipocytes with knockdown for E4bpa (sh-E4BP4) or control. Representative results are shown from two independent experiments. Gel source data are presented in the Supplementary data. H. Representative images of Oil Red O staining in control and sh-E4BP4 brown adipocytes after seven days of differentiation. I. Relative mRNA levels of adipogenic genes in immortalized brown adipocytes of the Control and sh-E4BP4. n = 3 per group. J. Relative mRNA levels of Cers6 of brown adipocytes sh-E4BP4 overexpressing GFP or Prdm16. n=3. K. C16:0 quantification by mass spectrometry of brown adipocytes sh-E4BP4 overexpressing GFP or Prdm16. n=3. L. A proposed mechanism by which E4BP4-PRDM16 complex controls Cers6 expression. For D-F, and I-K data are presented as mean±SEM. Two-sided P-values were calculated using an unpaired Student’s t-test (D, E, F, I, J, and K). *P < 0.05, **P < 0.01, ***P < 0.001.

Journal: bioRxiv

Article Title: E4BP4 Safeguards Brown Fat Mitochondria from Obesity-Induced Fragmentation via Ceramide Repression

doi: 10.1101/2025.05.19.652826

Figure Lengend Snippet: A. PRDM16 complex was immunopurified from differentiated brown adipocytes. Endogenous E4bp4 was detected by immunoblotting. B. ChIP-seq for PRDM16 and PRDM16 knockout (PRDM16-KO) on the Cers6 gene in BAT. C. Breeding strategy for AdipoQ_SPH mice and littermate control mice. Primary brown adipocytes were used in the experiments. The cartoon was created using a Biorender. D. Relative mRNA levels of indicate genes in primary brown adipocytes of the Control and PRDM16-OE groups. n = 3 per group. E. Relative mRNA levels of Cers6 in primary brown adipocytes of the Control and PRDM16-OE groups. n = 3 per group. F. C16:0 quantification by mass spectrometry in primary brown adipocytes of the Control and PRDM16-OE groups. C16:0 levels were normalized to total protein (µg). n = 3 per group. G. Western blotting for E4bp4 in immortalized brown adipocytes with knockdown for E4bpa (sh-E4BP4) or control. Representative results are shown from two independent experiments. Gel source data are presented in the Supplementary data. H. Representative images of Oil Red O staining in control and sh-E4BP4 brown adipocytes after seven days of differentiation. I. Relative mRNA levels of adipogenic genes in immortalized brown adipocytes of the Control and sh-E4BP4. n = 3 per group. J. Relative mRNA levels of Cers6 of brown adipocytes sh-E4BP4 overexpressing GFP or Prdm16. n=3. K. C16:0 quantification by mass spectrometry of brown adipocytes sh-E4BP4 overexpressing GFP or Prdm16. n=3. L. A proposed mechanism by which E4BP4-PRDM16 complex controls Cers6 expression. For D-F, and I-K data are presented as mean±SEM. Two-sided P-values were calculated using an unpaired Student’s t-test (D, E, F, I, J, and K). *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: E4bp4 (Addgene, catalog no. 34572) and Prdm16 (Addgene, catalog no. 15503) plasmids were transfected using Lipofectamine 3000 (Thermo Fisher Scientific) according to the manufacturer’s protocol.

Techniques: Western Blot, ChIP-sequencing, Knock-Out, Control, Mass Spectrometry, Knockdown, Staining, Expressing

P0 cochlear epithelium cell densities in Prdm16 cKO compared with control

Journal: The Journal of Neuroscience

Article Title: Kölliker's Organ Functions as a Developmental Hub in Mouse Cochlea Regulating Spiral Limbus and Tectorial Membrane Development

doi: 10.1523/JNEUROSCI.0721-24.2025

Figure Lengend Snippet: P0 cochlear epithelium cell densities in Prdm16 cKO compared with control

Article Snippet: E14.5 embryos from WT C57BL/6 pregnant mice were dissected, and the cochleae were electroporated with either Prdm16 overexpressing plasmid (Addgene pcDNA3.1 PRDM16; 15503) or control backbone plasmid (Addgene pcDNA3.1; 172604) as previously described ( ).

Techniques: Control

P21 cochlear hair cell densities in Prdm16 cKO compared with control

Journal: The Journal of Neuroscience

Article Title: Kölliker's Organ Functions as a Developmental Hub in Mouse Cochlea Regulating Spiral Limbus and Tectorial Membrane Development

doi: 10.1523/JNEUROSCI.0721-24.2025

Figure Lengend Snippet: P21 cochlear hair cell densities in Prdm16 cKO compared with control

Techniques: Control

P28 cochlear morphometric measurements in Prdm16 cKO compared with control

Journal: The Journal of Neuroscience

Article Title: Kölliker's Organ Functions as a Developmental Hub in Mouse Cochlea Regulating Spiral Limbus and Tectorial Membrane Development

doi: 10.1523/JNEUROSCI.0721-24.2025

Figure Lengend Snippet: P28 cochlear morphometric measurements in Prdm16 cKO compared with control

Techniques: Control, Membrane

Differentially expressed genes in Kölliker's organ cells comparing Prdm16 cKO to control samples at E16.5 ( n = 3/group, Log2 FC > 1, p value < 0.05)

Journal: The Journal of Neuroscience

Article Title: Kölliker's Organ Functions as a Developmental Hub in Mouse Cochlea Regulating Spiral Limbus and Tectorial Membrane Development

doi: 10.1523/JNEUROSCI.0721-24.2025

Figure Lengend Snippet: Differentially expressed genes in Kölliker's organ cells comparing Prdm16 cKO to control samples at E16.5 ( n = 3/group, Log2 FC > 1, p value < 0.05)

Techniques: Control

Prdm16 regulates cochlear lengthening and restricts sensory fates in developing Kölliker's organ. A , Immunostained whole mount cochlear epithelium from P0 Prdm16 cKO and littermate heterozygote controls showing shortening of the cochlear duct. Arrowheads refer to the beginning and end of the cochlear duct length measurement. B , Quantification of cochlear duct length ( n = 6/group, mean ± SD, multiple unpaired two-tailed Student's t test, p value as indicated). C , Immunostained whole mount cochlear epithelium stained with phalloidin for F-actin enrichment in hair bundles, MYO6 for hair cells, and SOX2 for supporting cells showing the presence of ectopic islands of sensory epithelia within the Kölliker's organ (white arrows), increased density of epithelial cells in Prdm16 cKO apical turn compared with littermate heterozygote controls including inner hair cells, outer hair cells, and supporting cells. D , Quantification and statistical analysis ( n = 6/group, mean ± SD, multiple unpaired two-tailed Student's t test or Mann–Whitney U test, p value as indicated). OHCs, outer hair cells; IHCs, inner hair cells; SC, supporting cells. Scale bar = 100 µm.

Journal: The Journal of Neuroscience

Article Title: Kölliker's Organ Functions as a Developmental Hub in Mouse Cochlea Regulating Spiral Limbus and Tectorial Membrane Development

doi: 10.1523/JNEUROSCI.0721-24.2025

Figure Lengend Snippet: Prdm16 regulates cochlear lengthening and restricts sensory fates in developing Kölliker's organ. A , Immunostained whole mount cochlear epithelium from P0 Prdm16 cKO and littermate heterozygote controls showing shortening of the cochlear duct. Arrowheads refer to the beginning and end of the cochlear duct length measurement. B , Quantification of cochlear duct length ( n = 6/group, mean ± SD, multiple unpaired two-tailed Student's t test, p value as indicated). C , Immunostained whole mount cochlear epithelium stained with phalloidin for F-actin enrichment in hair bundles, MYO6 for hair cells, and SOX2 for supporting cells showing the presence of ectopic islands of sensory epithelia within the Kölliker's organ (white arrows), increased density of epithelial cells in Prdm16 cKO apical turn compared with littermate heterozygote controls including inner hair cells, outer hair cells, and supporting cells. D , Quantification and statistical analysis ( n = 6/group, mean ± SD, multiple unpaired two-tailed Student's t test or Mann–Whitney U test, p value as indicated). OHCs, outer hair cells; IHCs, inner hair cells; SC, supporting cells. Scale bar = 100 µm.

Techniques: Two Tailed Test, Staining, MANN-WHITNEY

Prdm16 is required for spiral limbus and tectorial membrane structure as evident by hematoxylin and eosin (H&E) staining. H&E-stained cochlear sections from P7 ( A ), P14 ( B ), and P21 ( C ) Prdm16 cKO and littermate heterozygote controls ( n = 3/group). Images show hypoplastic spiral limbus (SL), lack of inner sulcus (IS), lack of interdental cells (IDCs), and abnormal attachment of tectorial membrane (TM) to the Reissner's membrane (RM) in the middle and apical turns in Prdm16 cKO cochleae. Dashed boxes indicate magnified insets. OC, Organ of Corti; KO, Kölliker's organ; PD, prosensory domain; SV, stria vascularis; TM, tectorial membrane; SL, spiral limbus; RM, Reissner's membrane; OSL, osseous spiral lamina; IS, inner sulcus; IDCS, interdental cells. Scale bar, 200 µm.

Journal: The Journal of Neuroscience

Article Title: Kölliker's Organ Functions as a Developmental Hub in Mouse Cochlea Regulating Spiral Limbus and Tectorial Membrane Development

doi: 10.1523/JNEUROSCI.0721-24.2025

Figure Lengend Snippet: Prdm16 is required for spiral limbus and tectorial membrane structure as evident by hematoxylin and eosin (H&E) staining. H&E-stained cochlear sections from P7 ( A ), P14 ( B ), and P21 ( C ) Prdm16 cKO and littermate heterozygote controls ( n = 3/group). Images show hypoplastic spiral limbus (SL), lack of inner sulcus (IS), lack of interdental cells (IDCs), and abnormal attachment of tectorial membrane (TM) to the Reissner's membrane (RM) in the middle and apical turns in Prdm16 cKO cochleae. Dashed boxes indicate magnified insets. OC, Organ of Corti; KO, Kölliker's organ; PD, prosensory domain; SV, stria vascularis; TM, tectorial membrane; SL, spiral limbus; RM, Reissner's membrane; OSL, osseous spiral lamina; IS, inner sulcus; IDCS, interdental cells. Scale bar, 200 µm.

Techniques: Membrane, Staining

Prdm16 is required for tectorial membrane anchorage and collagen. A , Trichrome staining of cochlear sections from P21 Prdm16 cKO and littermate heterozygote controls showing collagen fibers (blue), nuclei (black), and cytoplasm (red) demonstrating faint collagen staining in the tectorial membrane in Prdm16 cKO cochlea as well as thinning of the marginal zone (black arrowheads). B , C , Immunostained cochlear sections from P21 Prdm16 cKO and littermate heterozygote controls showing deficient COLLAGEN II and COLLAGEN IX immunostaining in the tectorial membrane in the apical turn of Prdm16 cKO (tectorial membrane outlined with dashed lines). D , Quantification of mean corrected fluorescence intensity (MCFI) of Collagen II and Collagen IX within the tectorial membrane in Prdm16 cKO versus controls ( n = 4/group, mean ± SD, multiple unpaired two-tailed Student's t tests, p value as indicated). E , F , Fluorescence in situ hybridization probing for Otoa and Tecta mRNAs in P0 and P7 cochlear sections from Prdm16 cKO and littermate heterozygote controls showing loss of Otoa signal (a marker for interdental cells) from the middle and apical turns of Prdm16 cKO cochlea (yellow arrowheads). SL, spiral limbus; TM, tectorial membrane; and OC, organ of Corti. Scale bar, 100 µm.

Journal: The Journal of Neuroscience

Article Title: Kölliker's Organ Functions as a Developmental Hub in Mouse Cochlea Regulating Spiral Limbus and Tectorial Membrane Development

doi: 10.1523/JNEUROSCI.0721-24.2025

Figure Lengend Snippet: Prdm16 is required for tectorial membrane anchorage and collagen. A , Trichrome staining of cochlear sections from P21 Prdm16 cKO and littermate heterozygote controls showing collagen fibers (blue), nuclei (black), and cytoplasm (red) demonstrating faint collagen staining in the tectorial membrane in Prdm16 cKO cochlea as well as thinning of the marginal zone (black arrowheads). B , C , Immunostained cochlear sections from P21 Prdm16 cKO and littermate heterozygote controls showing deficient COLLAGEN II and COLLAGEN IX immunostaining in the tectorial membrane in the apical turn of Prdm16 cKO (tectorial membrane outlined with dashed lines). D , Quantification of mean corrected fluorescence intensity (MCFI) of Collagen II and Collagen IX within the tectorial membrane in Prdm16 cKO versus controls ( n = 4/group, mean ± SD, multiple unpaired two-tailed Student's t tests, p value as indicated). E , F , Fluorescence in situ hybridization probing for Otoa and Tecta mRNAs in P0 and P7 cochlear sections from Prdm16 cKO and littermate heterozygote controls showing loss of Otoa signal (a marker for interdental cells) from the middle and apical turns of Prdm16 cKO cochlea (yellow arrowheads). SL, spiral limbus; TM, tectorial membrane; and OC, organ of Corti. Scale bar, 100 µm.

Techniques: Membrane, Staining, Immunostaining, Fluorescence, Two Tailed Test, In Situ Hybridization, Marker

Validating differentially expressed genes within Kölliker's organ. Fluorescence in situ hybridization probing for Calb1 mRNAs ( A ) and Ctgf mRNA ( B ) in E16.5 cochlear sections from Prdm16 cKO and littermate heterozygote controls showing reduced Calb1 and Ctgf signals within Kölliker's organ (yellow brackets) from the middle and apical turns of Prdm16 cKO cochlea ( n = 3–4/group). C , Quantification of Calb1 and Ctgf mRNA expression levels from microdissected cochlear ducts at E16.5 showing downregulation in Prdm16 cKO compared with littermate heterozygote controls using qRT-PCR ( n = 3/group, multiple unpaired two-tailed Student's t test, p value as indicated). D , Fluorescence in situ hybridization probing for Hes1 mRNAs in E16.5 cochlear sections from Prdm16 cKO and littermate heterozygote controls showing no signal detection within Kölliker's organ in either group. Scale bar, 100 µm.

Journal: The Journal of Neuroscience

Article Title: Kölliker's Organ Functions as a Developmental Hub in Mouse Cochlea Regulating Spiral Limbus and Tectorial Membrane Development

doi: 10.1523/JNEUROSCI.0721-24.2025

Figure Lengend Snippet: Validating differentially expressed genes within Kölliker's organ. Fluorescence in situ hybridization probing for Calb1 mRNAs ( A ) and Ctgf mRNA ( B ) in E16.5 cochlear sections from Prdm16 cKO and littermate heterozygote controls showing reduced Calb1 and Ctgf signals within Kölliker's organ (yellow brackets) from the middle and apical turns of Prdm16 cKO cochlea ( n = 3–4/group). C , Quantification of Calb1 and Ctgf mRNA expression levels from microdissected cochlear ducts at E16.5 showing downregulation in Prdm16 cKO compared with littermate heterozygote controls using qRT-PCR ( n = 3/group, multiple unpaired two-tailed Student's t test, p value as indicated). D , Fluorescence in situ hybridization probing for Hes1 mRNAs in E16.5 cochlear sections from Prdm16 cKO and littermate heterozygote controls showing no signal detection within Kölliker's organ in either group. Scale bar, 100 µm.

Techniques: Fluorescence, In Situ Hybridization, Expressing, Quantitative RT-PCR, Two Tailed Test

Prdm16 cKO cochlear phenotype persists through postnatal maturation. A , Immunostained cochlear sections from P7 Prdm16 cKO and littermate heterozygote controls showing MYO6 + ectopic hair cells (EcHCs) in the region of interdental cells (IDCs) as well as multiple rows of outer hair cells in the middle and apical turns consistent with P0 phenotype ( n = 4/group). B , Schematic diagram recapitulating the phenotype described in A . C , Immunostained whole mount cochlear epithelium from P21 Prdm16 cKO and littermate heterozygote controls showing increased OHC density in middle and apical turns, as well as increased IHC density in apical turn in Prdm16 cKO cochlea. D , Scanning electron microscopic images from P21 Prdm16 cKO and littermate heterozygote control cochleae showing immature bundle morphology in the apical turn of Prdm16 cKO ( n = 3/group). E , Quantification and statistical analysis of OHC and IHC densities ( n = 4/group, mean ± SD, Mann–Whitney U test, p value as indicated). OHCs, outer hair cells; IHCs, inner hair cells; EcHCs, Ectopic hair cells; SCs, supporting cells; IDCs, interdental cells; KO, Kölliker's organ; SL, spiral limbus. Scale bar: 100 µm unless otherwise specified.

Journal: The Journal of Neuroscience

Article Title: Kölliker's Organ Functions as a Developmental Hub in Mouse Cochlea Regulating Spiral Limbus and Tectorial Membrane Development

doi: 10.1523/JNEUROSCI.0721-24.2025

Figure Lengend Snippet: Prdm16 cKO cochlear phenotype persists through postnatal maturation. A , Immunostained cochlear sections from P7 Prdm16 cKO and littermate heterozygote controls showing MYO6 + ectopic hair cells (EcHCs) in the region of interdental cells (IDCs) as well as multiple rows of outer hair cells in the middle and apical turns consistent with P0 phenotype ( n = 4/group). B , Schematic diagram recapitulating the phenotype described in A . C , Immunostained whole mount cochlear epithelium from P21 Prdm16 cKO and littermate heterozygote controls showing increased OHC density in middle and apical turns, as well as increased IHC density in apical turn in Prdm16 cKO cochlea. D , Scanning electron microscopic images from P21 Prdm16 cKO and littermate heterozygote control cochleae showing immature bundle morphology in the apical turn of Prdm16 cKO ( n = 3/group). E , Quantification and statistical analysis of OHC and IHC densities ( n = 4/group, mean ± SD, Mann–Whitney U test, p value as indicated). OHCs, outer hair cells; IHCs, inner hair cells; EcHCs, Ectopic hair cells; SCs, supporting cells; IDCs, interdental cells; KO, Kölliker's organ; SL, spiral limbus. Scale bar: 100 µm unless otherwise specified.

Techniques: Control, MANN-WHITNEY

Prdm16 overexpression is not sufficient to inhibit sensory fates in vitro. A , E14.5 WT cochlear explants 4 d post electroporation with Prdm16 -expressing or control plasmids. Cultures are immunostained with phalloidin for F-actin enrichment in hair cell bundles (green), MYO6 for hair cells (white), and TUJ1 for neurons (red). No significant changes are noticed in the sensory epithelium. B , Quantification of MYO6+ cells in apical and middle turns of the explants ( n = 6–8/group, mean ± SD, multiple unpaired two-tailed Student's t test, p value as indicated). C , Immunostained explants stained with FLAG antibody for plasmid expression, and SOX2 for supporting cells showing colocalization. Scale bar, 100 µm.

Journal: The Journal of Neuroscience

Article Title: Kölliker's Organ Functions as a Developmental Hub in Mouse Cochlea Regulating Spiral Limbus and Tectorial Membrane Development

doi: 10.1523/JNEUROSCI.0721-24.2025

Figure Lengend Snippet: Prdm16 overexpression is not sufficient to inhibit sensory fates in vitro. A , E14.5 WT cochlear explants 4 d post electroporation with Prdm16 -expressing or control plasmids. Cultures are immunostained with phalloidin for F-actin enrichment in hair cell bundles (green), MYO6 for hair cells (white), and TUJ1 for neurons (red). No significant changes are noticed in the sensory epithelium. B , Quantification of MYO6+ cells in apical and middle turns of the explants ( n = 6–8/group, mean ± SD, multiple unpaired two-tailed Student's t test, p value as indicated). C , Immunostained explants stained with FLAG antibody for plasmid expression, and SOX2 for supporting cells showing colocalization. Scale bar, 100 µm.

Techniques: Over Expression, In Vitro, Electroporation, Expressing, Control, Two Tailed Test, Staining, Plasmid Preparation

Schematic representation of plasmid map pAAV-FoxP4, pAAV-PRDM16, and pAAV-FST.

Journal: International Journal of Molecular Sciences

Article Title: Gene Therapy Approach for Treatment of Obese Agouti Mice

doi: 10.3390/ijms252212144

Figure Lengend Snippet: Schematic representation of plasmid map pAAV-FoxP4, pAAV-PRDM16, and pAAV-FST.

Article Snippet: The mouse PRDM16 gene was amplified from a commercially available plasmid, pcDNA3.1 PRDM16 (Addgene plasmid #15503; RRID: Addgene_15503).

Techniques: Plasmid Preparation

Progression of body weight change relative to weight before AAV administration in agouti mice treated at 12 weeks of age with an intra-WAT injection of empty AAV (Control), AAV-FoxP4, AAV-PRDM16, or AAV-FST vectors. Data are presented as mean ± S.D. values of three mice for each group. * p < 0.1 control group vs. AAV-FoxP4 group, # p < 0.1 control group vs. AAV-PRDM16 group, † p < 0.1 control group vs. AAV-FST group.

Journal: International Journal of Molecular Sciences

Article Title: Gene Therapy Approach for Treatment of Obese Agouti Mice

doi: 10.3390/ijms252212144

Figure Lengend Snippet: Progression of body weight change relative to weight before AAV administration in agouti mice treated at 12 weeks of age with an intra-WAT injection of empty AAV (Control), AAV-FoxP4, AAV-PRDM16, or AAV-FST vectors. Data are presented as mean ± S.D. values of three mice for each group. * p < 0.1 control group vs. AAV-FoxP4 group, # p < 0.1 control group vs. AAV-PRDM16 group, † p < 0.1 control group vs. AAV-FST group.

Article Snippet: The mouse PRDM16 gene was amplified from a commercially available plasmid, pcDNA3.1 PRDM16 (Addgene plasmid #15503; RRID: Addgene_15503).

Techniques: Injection, Control

The fold changes of the identified lipids between the AAV9-FST, AAV8-FoxP4, and AAV8-PRDM16 groups against the control (empty AAV). Fold change and p -value were calculated in Metaboanalyst 5.0 [ <xref ref-type=

15 ]. Lipids showing p -value > 0.05 are marked «Ns» (non-significant)." width="100%" height="100%">

Journal: International Journal of Molecular Sciences

Article Title: Gene Therapy Approach for Treatment of Obese Agouti Mice

doi: 10.3390/ijms252212144

Figure Lengend Snippet: The fold changes of the identified lipids between the AAV9-FST, AAV8-FoxP4, and AAV8-PRDM16 groups against the control (empty AAV). Fold change and p -value were calculated in Metaboanalyst 5.0 [ 15 ]. Lipids showing p -value > 0.05 are marked «Ns» (non-significant).

Article Snippet: The mouse PRDM16 gene was amplified from a commercially available plasmid, pcDNA3.1 PRDM16 (Addgene plasmid #15503; RRID: Addgene_15503).

Techniques: Control

A RRBS profiling of DNA methylation level at MyoD1 promoter in iBAT of D1KO and fl/fl mice. B , C Ucp1 ( B , n = 4/group) and Myod1 ( C , n = 4/group) expression in iBAT of mice during late embryonic and postnatal development. *Indicates statistical significance vs. 17E with one-way ANOVA followed by Fisher’s LSD multiple comparisons test; in ( B ), F (7,24) = 48.31, p < 0.0001, and in ( C ), F (7,24) = 10.54, p < 0.0001. D , E Ucp1 ( D ) and Myod1 ( E ) expression in iBAT of mice during cold exposure ( n = 3/group). *indicates statistical significance vs. Time 0 with one-way ANOVA followed by Fisher’s LSD multiple comparisons test; in ( D ), F (3,8) = 6.406, p = 0.016, and in ( E ), F (3,8) = 25.096, p < 0.0001. F Ucp1 , Prdm16 and myogenic marker gene expression in iBAT and gastrocnemius (GAS) muscle ( n = 4/group). *Indicates statistical significance between iBAT and GAS as analyzed by two-tailed unpaired Student’s t -test, except for Myod1 and Atp2a1 , which were analyzed by Mann–Whitney’s nonparametric U test. G Pyrosequencing analysis of DNA methylation level at Myod1 promoter in iBAT and GAS muscle ( n = 4/group). *Indicates statistical significance between iBAT and GAS as analyzed by Mann–Whitney’s nonparametric U test. H ChIP assay of DNMT1 binding to Myod1 promoter in undifferentiated BAT1 preadipocytes and differentiated BAT1 brown adipocytes ( n = 4/group). *indicates statistical significance by two-tailed unpaired Student’s t -test. I ChIP assay of DNMT1 binding to Myod1 promoter in iBAT from HFD- or LFD-fed mice ( n = 6/group). *Indicates statistical significance by two-tailed unpaired Student’s t -test. J Pyrosequencing analysis of DNA methylation levels at Myod1 promoter in BAT1 brown adipocytes transfected with scramble or Dnmt1 siRNA ( n = 6/group). *Indicates statistical significance between iBAT and GAS as analyzed by Mann–Whitney’s nonparametric U test. K Quantitative RT-PCR analysis of myogenic marker gene and BAT gene expression in BAT1 brown adipocytes transfected with scramble, Dnmt1 , Myod1 , or Dnmt1 + Myod1 siRNA ( n = 4/group). *Indicates statistical significance among groups. For Dnmt1 and Myod1, statistical significance was analyzed by Kruskal–Wallis non-parametric ANOVA H test by rank followed by Pairwise Comparisons test between groups, H(3) = 13.560, p = 0.004 for Dnmt1 , and H(3) = 13.097, p = 0.004 for Myod1 . For Ucp1 , Pgc1α , Myog and Acta1 , statistical significance was analyzed by one-way ANOVA followed by Fisher’s LSD multiple comparisons test: for Ucp1 , F (3,12) = 45.139, p < 0.0001; for Pgc1α , F (3,12) = 51.81, p < 0.0001; for Myog , F (3,12) = 33.178, p < 0.0001; for Acta1 , F (3,12) = 20.045, p < 0.0001. L , M Myod1 ( L ) and BAT-specific gene expression ( M ) in Myod1 -overexpressed BAT1 brown adipocytes treated with PBS or isoproterenol (Iso). n = 6/group. *indicates statistical significance analyzed by Kruskal–Wallis non-parametric ANOVA H test by rank followed by Pairwise Comparisons test between groups. In ( L ), H(3) = 17.613, p = 0.001. In ( M ), for Ucp1 , H(3) = 21.6, p < 0.0001; for Prdm16 , H(3) = 17.553, p = 0.001; for Pgc1α , H(3) = 20.309, p < 0.0001; for Elovl3 , H(3) = 19.62, p < 0.0001; for Cpt1b , H(3) = 18.033, p < 0.0001; for Cidea , H(3) = 18.023, p < 0.0001; for pgc1β , H(3) = 21.367, p < 0.0001; for Acox1 , H(3) = 16.847, p = 0.001; for Cox1 , H(3) = 19.807, p = 0.0009. For ( J – M ), BAT1 cells were differentiated into brown adipocytes as described under Methods. Scramble or targeting siRNAs, or control or Myod1 overexpressing plasmids were transfected into day 4 differentiated BAT1 cells using Amaxa Nucleofector II Electroporator with an Amaxa cell line nucleofector kit L. Cells were harvested 2 days after for pyrosequencing or gene expression analysis. All data are expressed as mean ± SEM.

Journal: Nature Communications

Article Title: Epigenetic interaction between UTX and DNMT1 regulates diet-induced myogenic remodeling in brown fat

doi: 10.1038/s41467-021-27141-7

Figure Lengend Snippet: A RRBS profiling of DNA methylation level at MyoD1 promoter in iBAT of D1KO and fl/fl mice. B , C Ucp1 ( B , n = 4/group) and Myod1 ( C , n = 4/group) expression in iBAT of mice during late embryonic and postnatal development. *Indicates statistical significance vs. 17E with one-way ANOVA followed by Fisher’s LSD multiple comparisons test; in ( B ), F (7,24) = 48.31, p < 0.0001, and in ( C ), F (7,24) = 10.54, p < 0.0001. D , E Ucp1 ( D ) and Myod1 ( E ) expression in iBAT of mice during cold exposure ( n = 3/group). *indicates statistical significance vs. Time 0 with one-way ANOVA followed by Fisher’s LSD multiple comparisons test; in ( D ), F (3,8) = 6.406, p = 0.016, and in ( E ), F (3,8) = 25.096, p < 0.0001. F Ucp1 , Prdm16 and myogenic marker gene expression in iBAT and gastrocnemius (GAS) muscle ( n = 4/group). *Indicates statistical significance between iBAT and GAS as analyzed by two-tailed unpaired Student’s t -test, except for Myod1 and Atp2a1 , which were analyzed by Mann–Whitney’s nonparametric U test. G Pyrosequencing analysis of DNA methylation level at Myod1 promoter in iBAT and GAS muscle ( n = 4/group). *Indicates statistical significance between iBAT and GAS as analyzed by Mann–Whitney’s nonparametric U test. H ChIP assay of DNMT1 binding to Myod1 promoter in undifferentiated BAT1 preadipocytes and differentiated BAT1 brown adipocytes ( n = 4/group). *indicates statistical significance by two-tailed unpaired Student’s t -test. I ChIP assay of DNMT1 binding to Myod1 promoter in iBAT from HFD- or LFD-fed mice ( n = 6/group). *Indicates statistical significance by two-tailed unpaired Student’s t -test. J Pyrosequencing analysis of DNA methylation levels at Myod1 promoter in BAT1 brown adipocytes transfected with scramble or Dnmt1 siRNA ( n = 6/group). *Indicates statistical significance between iBAT and GAS as analyzed by Mann–Whitney’s nonparametric U test. K Quantitative RT-PCR analysis of myogenic marker gene and BAT gene expression in BAT1 brown adipocytes transfected with scramble, Dnmt1 , Myod1 , or Dnmt1 + Myod1 siRNA ( n = 4/group). *Indicates statistical significance among groups. For Dnmt1 and Myod1, statistical significance was analyzed by Kruskal–Wallis non-parametric ANOVA H test by rank followed by Pairwise Comparisons test between groups, H(3) = 13.560, p = 0.004 for Dnmt1 , and H(3) = 13.097, p = 0.004 for Myod1 . For Ucp1 , Pgc1α , Myog and Acta1 , statistical significance was analyzed by one-way ANOVA followed by Fisher’s LSD multiple comparisons test: for Ucp1 , F (3,12) = 45.139, p < 0.0001; for Pgc1α , F (3,12) = 51.81, p < 0.0001; for Myog , F (3,12) = 33.178, p < 0.0001; for Acta1 , F (3,12) = 20.045, p < 0.0001. L , M Myod1 ( L ) and BAT-specific gene expression ( M ) in Myod1 -overexpressed BAT1 brown adipocytes treated with PBS or isoproterenol (Iso). n = 6/group. *indicates statistical significance analyzed by Kruskal–Wallis non-parametric ANOVA H test by rank followed by Pairwise Comparisons test between groups. In ( L ), H(3) = 17.613, p = 0.001. In ( M ), for Ucp1 , H(3) = 21.6, p < 0.0001; for Prdm16 , H(3) = 17.553, p = 0.001; for Pgc1α , H(3) = 20.309, p < 0.0001; for Elovl3 , H(3) = 19.62, p < 0.0001; for Cpt1b , H(3) = 18.033, p < 0.0001; for Cidea , H(3) = 18.023, p < 0.0001; for pgc1β , H(3) = 21.367, p < 0.0001; for Acox1 , H(3) = 16.847, p = 0.001; for Cox1 , H(3) = 19.807, p = 0.0009. For ( J – M ), BAT1 cells were differentiated into brown adipocytes as described under Methods. Scramble or targeting siRNAs, or control or Myod1 overexpressing plasmids were transfected into day 4 differentiated BAT1 cells using Amaxa Nucleofector II Electroporator with an Amaxa cell line nucleofector kit L. Cells were harvested 2 days after for pyrosequencing or gene expression analysis. All data are expressed as mean ± SEM.

Article Snippet: For sub-cloning of Prdm16 and Dnmt1 fragments for co-immunoprecipitation experiments, Fragments of Prdm16 (1–223, 224–454, 455–680, 681–880, 881–1038, and 1039–1176) were PCR-amplified using the full-length Prdm16 plasmids (Addgene #15503) and sub-cloned into XbaI/EcoRI sites of Flag-HA-pcDNA3.1 vector (Addgene #52535).

Techniques: DNA Methylation Assay, Expressing, Marker, Gene Expression, Two Tailed Test, Binding Assay, Transfection, Quantitative RT-PCR, Control

A Comparison of genome-wide alterations in chromatin accessibility landscape assessed by ATAC-seq with the corresponding gene expression assessed by RNA-seq in iBAT of UTXKO and fl/Y mice fed HFD for 12 weeks ( n = 3 replicates per group). B ATAC-seq analysis of chromatin accessibility at Prdm16 gene locus in iBAT of UTXKO and fl/Y mice fed HFD for 12 weeks ( n = 3 replicates per group). C Quantitative RT-PCR analysis of Prdm16 mRNA in iBAT of LFD- or HFD-fed mice ( n = 8/Group). *Indicates statistical significance between the two groups by two-tailed unpaired Student’s t -test. D , E ChIP assay of UTX binding to Prdm16 promoter ( D , n = 4/group) and ChIP assay of H3K27me3 levels at Prdm16 promoter ( E , n = 4/group) in iBAT of LFD- or HFD-fed mice. *Indicates statistical significance between the two groups by Mann–Whitney’s nonparametric U test in ( D ) and two-tailed unpaired Student’s t -test in ( E ). F ChIP assay of H3K27me3 levels at Prdm16 promoter in control or Utx knockdown BAT1 brown adipocytes treated with isoproterenol ( n = 4/Group). *Indicates statistical significance between the two groups by Mann–Whitney’s nonparametric U test. G – H Pyrosequencing analysis of DNA methylation at Myod1 promoter ( G , n = 6/group) and Myod1 expression ( H , n = 8/group) in BAT1 brown adipocytes transfected with scramble or Utx siRNA. *Indicates statistical significance between the two groups by Mann–Whitney’s nonparametric U test in ( G ) and two-tailed unpaired Student’s t -test in ( H ). I , J Pyrosequencing analysis of DNA methylation at Myod1 promoter ( I , n = 4/group) and Myod1 expression ( J , n = 8 for Scramble and 7 for Prdm16 siRNA) in BAT1 brown adipocytes transfected with scramble or Prdm16 siRNA. *Indicates statistical significance between the two groups by Mann–Whitney’s nonparametric U test in ( I ) and two-tailed unpaired Student’s t -test in ( J ). K Pyrosequencing analysis of DNA methylation at Myod1 promoter in BAT1 brown adipocytes transfected with pSPORT6 or pSPORT6 encoding Prdm16 overexpressing plasmids ( n = 4/group). *indicates statistical significance between the two groups by Mann–Whitney’s nonparametric U test. All data are expressed as mean ± SEM.

Journal: Nature Communications

Article Title: Epigenetic interaction between UTX and DNMT1 regulates diet-induced myogenic remodeling in brown fat

doi: 10.1038/s41467-021-27141-7

Figure Lengend Snippet: A Comparison of genome-wide alterations in chromatin accessibility landscape assessed by ATAC-seq with the corresponding gene expression assessed by RNA-seq in iBAT of UTXKO and fl/Y mice fed HFD for 12 weeks ( n = 3 replicates per group). B ATAC-seq analysis of chromatin accessibility at Prdm16 gene locus in iBAT of UTXKO and fl/Y mice fed HFD for 12 weeks ( n = 3 replicates per group). C Quantitative RT-PCR analysis of Prdm16 mRNA in iBAT of LFD- or HFD-fed mice ( n = 8/Group). *Indicates statistical significance between the two groups by two-tailed unpaired Student’s t -test. D , E ChIP assay of UTX binding to Prdm16 promoter ( D , n = 4/group) and ChIP assay of H3K27me3 levels at Prdm16 promoter ( E , n = 4/group) in iBAT of LFD- or HFD-fed mice. *Indicates statistical significance between the two groups by Mann–Whitney’s nonparametric U test in ( D ) and two-tailed unpaired Student’s t -test in ( E ). F ChIP assay of H3K27me3 levels at Prdm16 promoter in control or Utx knockdown BAT1 brown adipocytes treated with isoproterenol ( n = 4/Group). *Indicates statistical significance between the two groups by Mann–Whitney’s nonparametric U test. G – H Pyrosequencing analysis of DNA methylation at Myod1 promoter ( G , n = 6/group) and Myod1 expression ( H , n = 8/group) in BAT1 brown adipocytes transfected with scramble or Utx siRNA. *Indicates statistical significance between the two groups by Mann–Whitney’s nonparametric U test in ( G ) and two-tailed unpaired Student’s t -test in ( H ). I , J Pyrosequencing analysis of DNA methylation at Myod1 promoter ( I , n = 4/group) and Myod1 expression ( J , n = 8 for Scramble and 7 for Prdm16 siRNA) in BAT1 brown adipocytes transfected with scramble or Prdm16 siRNA. *Indicates statistical significance between the two groups by Mann–Whitney’s nonparametric U test in ( I ) and two-tailed unpaired Student’s t -test in ( J ). K Pyrosequencing analysis of DNA methylation at Myod1 promoter in BAT1 brown adipocytes transfected with pSPORT6 or pSPORT6 encoding Prdm16 overexpressing plasmids ( n = 4/group). *indicates statistical significance between the two groups by Mann–Whitney’s nonparametric U test. All data are expressed as mean ± SEM.

Techniques: Comparison, Genome Wide, Gene Expression, RNA Sequencing, Quantitative RT-PCR, Two Tailed Test, Binding Assay, Control, Knockdown, DNA Methylation Assay, Expressing, Transfection

A ChIP assay of DNMT1 binding to Myod1 promoter in control or Prdm16 knockdown BAT1 brown adipocytes treated with or without isoproterenol ( n = 4/group). Data are expressed as mean ± SEM. Indicates statistical significance between different treatments analyzed by Kruskal–Wallis non-parametric ANOVA H test by rank followed by Pairwise Comparisons test between groups, H(3) = 9.816, p = 0.020. B Co-IP of DNMT1 and FLAG-PRDM16 in HEK293T cells. Data are representative from two independent experiments. C Co-IP of DNMT1 and various fragments of PRDM16. HA-tagged fragments of PRDM16 were expressed along with full-length DNMT1 in HEK293T cells. Cell lysates were immunoprecipitated with anti-DNMT1 antibodies followed by immunoblotting with HA or DNMT1 antibodies. Color-coded domain architecture of PRDM16 shows a PR/SET domain (PR), an N-terminal zinc-finger domain containing seven C2H2 zinc finger motifs (ZF1), a proline rich domain (PRR), a repression domain (RD), a second C-terminal zinc-finger domain containing three C2H2 zinc finger motifs (ZF2), and an acidic activation domain (AD). Data are representative from two independent experiments. D Co-IP of PRDM16 and various fragments of DNMT1. HA-tagged fragments of DNMT1 were expressed along with full-length PRDM16 in HEK293T cells. Cell lysates were immunoprecipitated with anti-HA antibodies followed by immunoblotting with HA or PRDM16 antibodies. Color-coded domain architecture of DNMT1 shows the N-terminal independently folded domain (NTD), replication foci-targeting sequence (RFTS) domain, a Zn-finger like CXXC motif, two bromo adjacent homology (BAH1 and BAH2) domains, and the catalytic domain. Data are representative from two independent experiments. E DNMT1 protein levels in Prdm16-overexpressed HEK293T cells treated with cycloheximide (CHX) (60 µg/ml) for various time. Data are representative from two independent experiments. F The interaction between PRDM16 and DNMT1 on Myod1 promoter in Prdm16 overexpressed BAT1 brown adipocytes measured by ChIP and Re-ChIP assay via sequential immunoprecipitation of PRDM16 and then DNMT1 ( n = 4/group). Data are expressed as mean ± SEM. *Indicates statistical significance between two groups by Mann–Whitney’s nonparametric U test. G , H Expression of miR-133a , miR133b , miR-206 and miR-1 in iBAT of female D1KO and fl/fl mice fed with a regular chow diet ( G , n = 4 for fl/fl and 6 for D1KO) and in BAT1 brown adipocytes with Myod1 overexpression ( H , n = 6/group). Data are expressed as mean ± SEM. *indicates statistical significance between the two groups by Mann–Whitney’s nonparametric U test in ( G ) and by two-tailed unpaired student’s t -test in ( H ). I , J Dnmt1 and miR-133a expression ( I ) and BAT-specific gene expression ( J ) in BAT1 brown adipocytes transfected with Dnmt1 siRNA, miR-133a inhibitor or both ( J ) ( n = 3/group). Data are expressed as mean ± SEM. *Indicates statistical significance analyzed by one-way ANOVA followed by Fisher’s LSD multiple comparisons test. In ( I ), for Dnm1 expression, F = (3,8) = 4.62, p = 0.037; for miR-133 expression, F (3,8) = 21.370, p < 0.0001. In ( J ), for Ucp1 expression, F = (3,8) = 4.827, p = 0.033; for Prdm16 expression, F = (3,8) = 10.863, p = 0.003; for Pgc1β expression, F (3,8) = 11.213, p = 0.003. K Schematic illustration of the interaction between UTX-regulated PRDM16 and DNMT1 in the maintenance of brown fat identity and suppression of myogenic remodeling in mature brown adipocytes. In brief, in mature brown adipocytes, UTX maintains the persistent demethylation of the repressive mark H3K27me3 at Prdm16 promoter, leading to high expression of Prdm16 ; PRDM16 then recruits the DNA methyltransferase DNMT1 to Myod1 promoter, causing Myod1 promoter hypermethylation, and suppressing Myod1 expression. In addition, reduced Myod1 expression relieves the inhibition on Prdm16 by miR-133, further increasing Prdm16 expression. The interaction between PRDM16 and DNMT1 coordinately serves to maintain brown adipocyte identity while repressing myogenic remodeling in mature brown adipocytes, thus promoting their active brown adipocyte thermogenic function. Suppressing this interaction by HFD feeding induces brown adipocyte-to-myocyte remodeling, which limits brown adipocyte thermogenic capacity and compromises diet-induced thermogenesis, leading to the development of obesity.

Journal: Nature Communications

Article Title: Epigenetic interaction between UTX and DNMT1 regulates diet-induced myogenic remodeling in brown fat

doi: 10.1038/s41467-021-27141-7

Figure Lengend Snippet: A ChIP assay of DNMT1 binding to Myod1 promoter in control or Prdm16 knockdown BAT1 brown adipocytes treated with or without isoproterenol ( n = 4/group). Data are expressed as mean ± SEM. Indicates statistical significance between different treatments analyzed by Kruskal–Wallis non-parametric ANOVA H test by rank followed by Pairwise Comparisons test between groups, H(3) = 9.816, p = 0.020. B Co-IP of DNMT1 and FLAG-PRDM16 in HEK293T cells. Data are representative from two independent experiments. C Co-IP of DNMT1 and various fragments of PRDM16. HA-tagged fragments of PRDM16 were expressed along with full-length DNMT1 in HEK293T cells. Cell lysates were immunoprecipitated with anti-DNMT1 antibodies followed by immunoblotting with HA or DNMT1 antibodies. Color-coded domain architecture of PRDM16 shows a PR/SET domain (PR), an N-terminal zinc-finger domain containing seven C2H2 zinc finger motifs (ZF1), a proline rich domain (PRR), a repression domain (RD), a second C-terminal zinc-finger domain containing three C2H2 zinc finger motifs (ZF2), and an acidic activation domain (AD). Data are representative from two independent experiments. D Co-IP of PRDM16 and various fragments of DNMT1. HA-tagged fragments of DNMT1 were expressed along with full-length PRDM16 in HEK293T cells. Cell lysates were immunoprecipitated with anti-HA antibodies followed by immunoblotting with HA or PRDM16 antibodies. Color-coded domain architecture of DNMT1 shows the N-terminal independently folded domain (NTD), replication foci-targeting sequence (RFTS) domain, a Zn-finger like CXXC motif, two bromo adjacent homology (BAH1 and BAH2) domains, and the catalytic domain. Data are representative from two independent experiments. E DNMT1 protein levels in Prdm16-overexpressed HEK293T cells treated with cycloheximide (CHX) (60 µg/ml) for various time. Data are representative from two independent experiments. F The interaction between PRDM16 and DNMT1 on Myod1 promoter in Prdm16 overexpressed BAT1 brown adipocytes measured by ChIP and Re-ChIP assay via sequential immunoprecipitation of PRDM16 and then DNMT1 ( n = 4/group). Data are expressed as mean ± SEM. *Indicates statistical significance between two groups by Mann–Whitney’s nonparametric U test. G , H Expression of miR-133a , miR133b , miR-206 and miR-1 in iBAT of female D1KO and fl/fl mice fed with a regular chow diet ( G , n = 4 for fl/fl and 6 for D1KO) and in BAT1 brown adipocytes with Myod1 overexpression ( H , n = 6/group). Data are expressed as mean ± SEM. *indicates statistical significance between the two groups by Mann–Whitney’s nonparametric U test in ( G ) and by two-tailed unpaired student’s t -test in ( H ). I , J Dnmt1 and miR-133a expression ( I ) and BAT-specific gene expression ( J ) in BAT1 brown adipocytes transfected with Dnmt1 siRNA, miR-133a inhibitor or both ( J ) ( n = 3/group). Data are expressed as mean ± SEM. *Indicates statistical significance analyzed by one-way ANOVA followed by Fisher’s LSD multiple comparisons test. In ( I ), for Dnm1 expression, F = (3,8) = 4.62, p = 0.037; for miR-133 expression, F (3,8) = 21.370, p < 0.0001. In ( J ), for Ucp1 expression, F = (3,8) = 4.827, p = 0.033; for Prdm16 expression, F = (3,8) = 10.863, p = 0.003; for Pgc1β expression, F (3,8) = 11.213, p = 0.003. K Schematic illustration of the interaction between UTX-regulated PRDM16 and DNMT1 in the maintenance of brown fat identity and suppression of myogenic remodeling in mature brown adipocytes. In brief, in mature brown adipocytes, UTX maintains the persistent demethylation of the repressive mark H3K27me3 at Prdm16 promoter, leading to high expression of Prdm16 ; PRDM16 then recruits the DNA methyltransferase DNMT1 to Myod1 promoter, causing Myod1 promoter hypermethylation, and suppressing Myod1 expression. In addition, reduced Myod1 expression relieves the inhibition on Prdm16 by miR-133, further increasing Prdm16 expression. The interaction between PRDM16 and DNMT1 coordinately serves to maintain brown adipocyte identity while repressing myogenic remodeling in mature brown adipocytes, thus promoting their active brown adipocyte thermogenic function. Suppressing this interaction by HFD feeding induces brown adipocyte-to-myocyte remodeling, which limits brown adipocyte thermogenic capacity and compromises diet-induced thermogenesis, leading to the development of obesity.

Techniques: Binding Assay, Control, Knockdown, Co-Immunoprecipitation Assay, Immunoprecipitation, Western Blot, Activation Assay, Sequencing, Expressing, Over Expression, Two Tailed Test, Gene Expression, Transfection, Inhibition

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