Journal: Cell metabolism

Article Title: A PRDM16-driven metabolic signal from adipocytes regulates precursor cell fate

doi: 10.1016/j.cmet.2019.05.005

Figure Lengend Snippet: (A) Western blot analysis of PRDM16, PPARγ, and α-Actin protein levels in whole iWAT (left) or in isolated adipocytes and stromal-vascular cells (SVC) from iWAT of young and aged mice acclimated to thermoneutrality. (B,C) Wildtype (Wt) and Prdm16+/− mutant mice were housed at thermoneutrality and treated with vehicle (Control) or CL for 4 days. n=3-4 mice per group. (B) Relative mRNA levels of Prdm16 and fibrosis marker genes in iWAT. (C) UCP1 immunofluorescence (top) and Picrosirius red staining of collagen fibers (bottom) in iWAT. Scale bar, 50 μM. (D,E) Thermoneutral-acclimated wildtype (Wt) and adipocyte-selective Prdm16-knockout (AdipoqCrePrdm16-KO) mice were treated with vehicle control or CL for 4 days. n=3-4 mice per group. (D) Picrosirius red staining of collagen fibers in iWAT. Scale bar, 50 μM. (E) Relative mRNA levels of fibrosis marker genes in iWAT. (F,G) young (2-month-old) and aged (12-month-old) wildtype (Ctl) and Fabp4-Prdm16 mice were acclimated to thermoneutrality and treated with CL for 4 days. n=3-5 mice per group. (F) Picrosirius red staining of collagen fibers. Scale bar, 50 μM. (G) Relative mRNA levels of fibrosis marker genes in iWAT. All data presented as mean ± s.e.m; * p<0.05, ** p<0.01, ***p<0.001 as analyzed by two-tailed Student’s t-test.

Article Snippet: Aged mice (+/− CL316,243) -Control (Puro) vs. PRDM16-expressing adipose cells GEO repository GSE129083 GSE129084 Experimental Models: Organisms/Strains R26 CreER The Jackson Laboratory 004847 Prdm16 flox The Jackson Laboratory 024992 Bdh1 flox Dr. Daniel Kelly NA Adipoq Cre The Jackson Laboratory 010803 Rosa26 tdTomato The Jackson Laboratory 007914 Pdgfrα creERT2 Dr. Brigid Hogan NA Adipoq rtTA Dr. Philipp Scherer NA Tre Cre The Jackson Laboratory 006234 Rosa26 mTmG The Jackson Laboratory 007676 Prdm16-deficient Dr. Patrick Seale NA Fabp4-Prdm16 Dr. Bruce Spiegelman NA Oligonucleotides See Table S1 for qPCR Primers Integrated DNA Technologies (IDT) NA Recombinant DNA PLKO.1-shRNA-Scramble Addgene, Sabatini, PMID15718470 1864 PLKO.1-shRNA-Bdh1 The RNAi Consortium (TRC)- Broad Institute via UPenn High-Throughput Screening Core TRCN0000041898 pMD2.G Addgene (Gift from Didier Trono) 12259 psPAX2 Addgene (Gift from Didier Trono) 12260 LentiCRISPR v2 Addgene (Gift from Feng Zhang) 52961 MSCV-GFP Dr. Patrick Seale NA MSCV-Prdm16 Dr. Patrick Seale NA Software and Algorithms GraphPad Prism 7 GraphPad Software https://www.graphpad.com/scientific-software/prism/ Image J NIH https://imagej.nih.gov/ij/ R Open source https://www.r-project.org/ GSEA Broad Institute http://software.broadinstitute.org/gsea/index.jsp Reactome (Pathway Browser) PMID: 29145629 https://reactome.org/PathwayBrowser/ Open in a separate window KEY RESOURCES TABLE.

Techniques: Western Blot, Isolation, Mutagenesis, Control, Marker, Immunofluorescence, Staining, Knock-Out, Two Tailed Test

Journal: Cell metabolism

Article Title: A PRDM16-driven metabolic signal from adipocytes regulates precursor cell fate

doi: 10.1016/j.cmet.2019.05.005

Figure Lengend Snippet: (A-D) iWAT precursor cells were transduced with control (CTL) or PRDM16-expressing retrovirus and treated with either vehicle control (CTL) or DMOG. After 2 days, cells were immunostained for ACTA2 or induced to differentiate into adipocytes for 5 days. (A) Experimental schema. (B) Western blot analysis of HIF1α, PRDM16 and α-Actin protein levels. (C) ACTA2 immunostaining (green) (top panel) and LipidTOX staining (red) of lipid droplets in differentiated adipocytes (bottom panel). Scale bar, 100 μM (top); 200 μM (bottom). (D) Relative mRNA levels of adipocyte genes. n=3 per group. (E-K) iWAT precursor cells were treated with: (1) control medium (DMEM), conditioned medium (CM) from GFP-expressing adipocytes (GFP-CM), or CM from PRDM16-expressing adipocytes (P-CM); and (2) vehicle, DMOG or recombinant TGFβ. After 24 h, cultures were immunostained for ACTA2 or induced to differentiate into adipocytes for 5 days. (E) Experimental schema. (F) ACTA2 immunostaining (green). Scale bar, 100 μm. (G) Violin plots showing relative fluorescent intensity per cell in indicated groups. The white solid boxes represent percentiles (bottom of box = 25th, horizontal line =50th, top of box = 75th). n=50-80 cells per condition. (H) Oil-Red-O staining (red) of adipocyte lipid droplets. (I) Relative mRNA levels of adipocyte markers in cultures from (H). n=3-5 per group. (J) ACTA2 immunostaining (top; green) and LipidTOX staining (bottom; red) of adipocyte lipid droplets in cultures treated with indicated CM and either vehicle or TGFβ. Scale bar, 100 μm. Violin plots showing relative fluorescent intensity per cell (right). (K) Relative mRNA levels of adipocyte marker genes in cultures from (J). n=3-5 per group. All data presented as mean ± sem. Analysis by two-way ANOVA with Tukey correction.

Article Snippet: Aged mice (+/− CL316,243) -Control (Puro) vs. PRDM16-expressing adipose cells GEO repository GSE129083 GSE129084 Experimental Models: Organisms/Strains R26 CreER The Jackson Laboratory 004847 Prdm16 flox The Jackson Laboratory 024992 Bdh1 flox Dr. Daniel Kelly NA Adipoq Cre The Jackson Laboratory 010803 Rosa26 tdTomato The Jackson Laboratory 007914 Pdgfrα creERT2 Dr. Brigid Hogan NA Adipoq rtTA Dr. Philipp Scherer NA Tre Cre The Jackson Laboratory 006234 Rosa26 mTmG The Jackson Laboratory 007676 Prdm16-deficient Dr. Patrick Seale NA Fabp4-Prdm16 Dr. Bruce Spiegelman NA Oligonucleotides See Table S1 for qPCR Primers Integrated DNA Technologies (IDT) NA Recombinant DNA PLKO.1-shRNA-Scramble Addgene, Sabatini, PMID15718470 1864 PLKO.1-shRNA-Bdh1 The RNAi Consortium (TRC)- Broad Institute via UPenn High-Throughput Screening Core TRCN0000041898 pMD2.G Addgene (Gift from Didier Trono) 12259 psPAX2 Addgene (Gift from Didier Trono) 12260 LentiCRISPR v2 Addgene (Gift from Feng Zhang) 52961 MSCV-GFP Dr. Patrick Seale NA MSCV-Prdm16 Dr. Patrick Seale NA Software and Algorithms GraphPad Prism 7 GraphPad Software https://www.graphpad.com/scientific-software/prism/ Image J NIH https://imagej.nih.gov/ij/ R Open source https://www.r-project.org/ GSEA Broad Institute http://software.broadinstitute.org/gsea/index.jsp Reactome (Pathway Browser) PMID: 29145629 https://reactome.org/PathwayBrowser/ Open in a separate window KEY RESOURCES TABLE.

Techniques: Transduction, Control, Expressing, Western Blot, Immunostaining, Staining, Recombinant, Marker

Journal: Cell metabolism

Article Title: A PRDM16-driven metabolic signal from adipocytes regulates precursor cell fate

doi: 10.1016/j.cmet.2019.05.005

Figure Lengend Snippet: (A) Gene Set Enrichment Analysis (GSEA) of genes upregulated in PRDM16-expressing vs. control cells. Enriched pathways were defined by a normalized enrichment score > 1.3 and FDR < 0.05. (B) Expression heat map of “fatty acid oxidation (FAO) and ketone body metabolism” genes in PRDM16-expressing vs. control cells. (C) Quantification of FAO. Adipocytes were cultured in medium containing U-13C-palmitate for 6 h prior to metabolite analysis by LC-MS. Relative molar quantity of 13C2-labeled acetyl-CoA in GFP- and PRDM16-expressing adipocytes was calculated by multiplying % molar enrichment of 13C2-labeled acetyl-CoA by relative total acetyl-CoA quantitation. n=3-4 per group. (D) Heat map showing standardized amounts of indicated metabolites in GFP- and PRDM16-expressing adipocytes, across samples (Z score). n=3 per group (E-G) PRDM16-expressing adipocytes (donor) were treated with vehicle or Etomoxir (Eto) for 24 hr prior to collecting conditioned medium (CM). HIF1α-expressing (DMOG-treated) precursors were treated with CM and induced to undergo adipocyte differentiation. To control for effects of Eto carried over in CM, a subset of recipient cells were directly exposed to Eto [Eto(recipient)] by mixing Eto with P-CM prior to treatment. (E) Experimental schema (F) Oil-Red-O staining of cultures after adipogenic induction. (G) Relative mRNA levels of adipocyte genes in cultures from (F). n=4-5 per group. (H,I) AdipoChaser (AdipoqrtTA;TRECre;Rosa26mTmG) mice were pulse labelled by feeding them with doxycyxline (dox) chow (600 mg/kg) for 9 days. During the 3-day washout period, mice were treated with Eto or vehicle for 3 days, followed by treatment with CL and either Eto or vehicle for another 4 days (chase). (I) Immunofluorescence staining of PLIN1 (red) in adipocytes and GFP reporter (green). Quantification of adipogenesis (% GFP (−)/total PLIN1+ cells) (right). All data presented as mean ± s.e.m; * p <0.05, **p<0.01, ***p < 0.001 as analyzed by two-tailed Student’s t-test.

Article Snippet: Aged mice (+/− CL316,243) -Control (Puro) vs. PRDM16-expressing adipose cells GEO repository GSE129083 GSE129084 Experimental Models: Organisms/Strains R26 CreER The Jackson Laboratory 004847 Prdm16 flox The Jackson Laboratory 024992 Bdh1 flox Dr. Daniel Kelly NA Adipoq Cre The Jackson Laboratory 010803 Rosa26 tdTomato The Jackson Laboratory 007914 Pdgfrα creERT2 Dr. Brigid Hogan NA Adipoq rtTA Dr. Philipp Scherer NA Tre Cre The Jackson Laboratory 006234 Rosa26 mTmG The Jackson Laboratory 007676 Prdm16-deficient Dr. Patrick Seale NA Fabp4-Prdm16 Dr. Bruce Spiegelman NA Oligonucleotides See Table S1 for qPCR Primers Integrated DNA Technologies (IDT) NA Recombinant DNA PLKO.1-shRNA-Scramble Addgene, Sabatini, PMID15718470 1864 PLKO.1-shRNA-Bdh1 The RNAi Consortium (TRC)- Broad Institute via UPenn High-Throughput Screening Core TRCN0000041898 pMD2.G Addgene (Gift from Didier Trono) 12259 psPAX2 Addgene (Gift from Didier Trono) 12260 LentiCRISPR v2 Addgene (Gift from Feng Zhang) 52961 MSCV-GFP Dr. Patrick Seale NA MSCV-Prdm16 Dr. Patrick Seale NA Software and Algorithms GraphPad Prism 7 GraphPad Software https://www.graphpad.com/scientific-software/prism/ Image J NIH https://imagej.nih.gov/ij/ R Open source https://www.r-project.org/ GSEA Broad Institute http://software.broadinstitute.org/gsea/index.jsp Reactome (Pathway Browser) PMID: 29145629 https://reactome.org/PathwayBrowser/ Open in a separate window KEY RESOURCES TABLE.

Techniques: Expressing, Control, Cell Culture, Liquid Chromatography with Mass Spectroscopy, Labeling, Quantitation Assay, Staining, Immunofluorescence, Two Tailed Test

Journal: Cell metabolism

Article Title: A PRDM16-driven metabolic signal from adipocytes regulates precursor cell fate

doi: 10.1016/j.cmet.2019.05.005

Figure Lengend Snippet: (A) BHB concentration in control (GFP) and adipocyte conditioned medium in the presence or absence of Eto-treatment, as determined by LC-MS. (B) Absolute quantity of BHB in GFP-CM and P-CM collected at 1h and 6h time points. n=4 per group. (C) Relative quantity of HMG-CoA in GFP- and PRDM16-expressing adipocytes at 1h and 6h time points. AUC, area under the curve. AUC for HMG-CoA was normalized to 13C315N1HMG-CoA internal standard (STD) and total protein in each sample. n = 4 per group. (D) BHB efflux rate in GFP- and PRDM16-expressing adipocytes. n=4 per group. (E) GFP- and PRDM16-expressing adipocytes were incubated with 13C-palmitate. The absolute amount of 13C-labeled BHB in CM was measured at 1h and 6h time points. n=4 per group (F) Relative BHB levels in iWAT from mice housed at thermoneutrality or exposed to 5°C cold for indicated times. n=5 per group. (G) BHB levels in IWAT of wildtype (wt) control and Prdm16+/− mice housed at thermoneutrality or exposed to cold for 8 d. n= 3-6 per group. (H-K) Adipose precursors were treated with: (1) vehicle or BHB (250 μM); and (2) vehicle, DMOG, or TGFβ. After 2 days, cells were either immunostained for ACTA2 or induced to differentiate into adipocytes for 5 days. (H) Experimental schema. (I) Immunofluorescence staining of ACTA2. Scale bar, 100 μM. (J) Violin plots showing relative fluorescent intensity per cell in indicated groups. The white solid boxes represent percentiles (bottom of box = 25th, horizontal line =50th, top of box = 75th). n=50-80 cells per condition. (K) LipidTOX staining of adipocyte lipid droplets. Scale bar, 200 μM.

Article Snippet: Aged mice (+/− CL316,243) -Control (Puro) vs. PRDM16-expressing adipose cells GEO repository GSE129083 GSE129084 Experimental Models: Organisms/Strains R26 CreER The Jackson Laboratory 004847 Prdm16 flox The Jackson Laboratory 024992 Bdh1 flox Dr. Daniel Kelly NA Adipoq Cre The Jackson Laboratory 010803 Rosa26 tdTomato The Jackson Laboratory 007914 Pdgfrα creERT2 Dr. Brigid Hogan NA Adipoq rtTA Dr. Philipp Scherer NA Tre Cre The Jackson Laboratory 006234 Rosa26 mTmG The Jackson Laboratory 007676 Prdm16-deficient Dr. Patrick Seale NA Fabp4-Prdm16 Dr. Bruce Spiegelman NA Oligonucleotides See Table S1 for qPCR Primers Integrated DNA Technologies (IDT) NA Recombinant DNA PLKO.1-shRNA-Scramble Addgene, Sabatini, PMID15718470 1864 PLKO.1-shRNA-Bdh1 The RNAi Consortium (TRC)- Broad Institute via UPenn High-Throughput Screening Core TRCN0000041898 pMD2.G Addgene (Gift from Didier Trono) 12259 psPAX2 Addgene (Gift from Didier Trono) 12260 LentiCRISPR v2 Addgene (Gift from Feng Zhang) 52961 MSCV-GFP Dr. Patrick Seale NA MSCV-Prdm16 Dr. Patrick Seale NA Software and Algorithms GraphPad Prism 7 GraphPad Software https://www.graphpad.com/scientific-software/prism/ Image J NIH https://imagej.nih.gov/ij/ R Open source https://www.r-project.org/ GSEA Broad Institute http://software.broadinstitute.org/gsea/index.jsp Reactome (Pathway Browser) PMID: 29145629 https://reactome.org/PathwayBrowser/ Open in a separate window KEY RESOURCES TABLE.

Techniques: Concentration Assay, Control, Liquid Chromatography with Mass Spectroscopy, Expressing, Incubation, Labeling, Immunofluorescence, Staining

Journal: Cell metabolism

Article Title: A PRDM16-driven metabolic signal from adipocytes regulates precursor cell fate

doi: 10.1016/j.cmet.2019.05.005

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Aged mice (+/− CL316,243) -Control (Puro) vs. PRDM16-expressing adipose cells GEO repository GSE129083 GSE129084 Experimental Models: Organisms/Strains R26 CreER The Jackson Laboratory 004847 Prdm16 flox The Jackson Laboratory 024992 Bdh1 flox Dr. Daniel Kelly NA Adipoq Cre The Jackson Laboratory 010803 Rosa26 tdTomato The Jackson Laboratory 007914 Pdgfrα creERT2 Dr. Brigid Hogan NA Adipoq rtTA Dr. Philipp Scherer NA Tre Cre The Jackson Laboratory 006234 Rosa26 mTmG The Jackson Laboratory 007676 Prdm16-deficient Dr. Patrick Seale NA Fabp4-Prdm16 Dr. Bruce Spiegelman NA Oligonucleotides See Table S1 for qPCR Primers Integrated DNA Technologies (IDT) NA Recombinant DNA PLKO.1-shRNA-Scramble Addgene, Sabatini, PMID15718470 1864 PLKO.1-shRNA-Bdh1 The RNAi Consortium (TRC)- Broad Institute via UPenn High-Throughput Screening Core TRCN0000041898 pMD2.G Addgene (Gift from Didier Trono) 12259 psPAX2 Addgene (Gift from Didier Trono) 12260 LentiCRISPR v2 Addgene (Gift from Feng Zhang) 52961 MSCV-GFP Dr. Patrick Seale NA MSCV-Prdm16 Dr. Patrick Seale NA Software and Algorithms GraphPad Prism 7 GraphPad Software https://www.graphpad.com/scientific-software/prism/ Image J NIH https://imagej.nih.gov/ij/ R Open source https://www.r-project.org/ GSEA Broad Institute http://software.broadinstitute.org/gsea/index.jsp Reactome (Pathway Browser) PMID: 29145629 https://reactome.org/PathwayBrowser/ Open in a separate window KEY RESOURCES TABLE.

Techniques: Generated, Recombinant, Sample Prep, SYBR Green Assay, RNA Sequencing, Control, Software

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

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

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

Journal: Cell reports

Article Title: Defective brown adipose tissue thermogenesis and impaired glucose metabolism in mice lacking Letmd1

doi: 10.1016/j.celrep.2021.110104

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Primary antibodies used in this study were anti-Letmd1 (LSBio; LS-C335200), anti-Brg1 (Bethyl; A300-813A-T or Cell Signaling; 49360S), anti-Ucp1 (R&D systems; MAB6158), anti-Prdm16 (R&D systems; AF6295), anti-Ebf2 (R&D systems; AF7006), anti-OXPHOS (MitoSciences; MS604), anti-lamin (Santa Cruz Biotechnology; sc-376248), anti-VDAC (Cell Signaling; 4661T), anti-FABP4 (Santa Cruz; sc-271529), anti-Flag (GenScript; A00187), anti-V5 (ThermoFisher; R96025), anti-β-actin (Santa Cruz; sc-47778), anti-Gapdh (Santa Cruz; sc-25778), and anti-vinculin (Sigma, V9131).

Techniques: Recombinant, Cell Culture, Reverse Transcription, SYBR Green Assay, Chromatin Immunoprecipitation, Plasmid Preparation, Software

Journal: The Journal of Physiology

Article Title: NAD replenishment restores mitochondrial function and thermogenesis in the brown adipose tissue of mice with obesity

doi: 10.1113/jp288453

Figure Lengend Snippet: Figure 2. High-fat diet impact on NAD-related enzymes and thermogenic markers Tissue from C57BL/6J mice treated with a high-fat diet (HFD) for 8 weeks. A, experimental design. B, BAT protein homogenates were used to evaluate Nampt, NMNat3, and TFAM protein levels (n = 4 Control vs. 5 Obese). C–E, protein quantification of Nampt (C), NMNat3 (D), and TFAM (E). F, imaging by transmission electron micro- scopy of BAT Control vs. Obese. G, TEM mitochondria area quantification in μm2 (n = 14 Control vs. 15 Obese, mitochondria). H, BAT protein homogenates of animals treated with HFD for 3, 7, and 14 days, as well as 8 weeks, were used to evaluate phosphorylation of DRP1 in serine 616 (n = 3 per group). I, protein quantification of pDRP1616. J, thermographic imaging of C57BL/6J mice after 8 weeks of intervention. K, quantification of BAT temperature obtained from the thermographic camera using the FLIR software (n = 9 Control vs. 10 Obese). L, BAT protein homogenates were used to evaluate thermogenic markers, UCP1, PRDM16, CPT1A, and β-3 adrenergic receptors (n = 4 Control vs. 5 Obese). M–P, protein quantification of UCP1 (M), PRDM16 (N), CPT1A (O), and β-3 adrenergic receptor (P). All values are expressed as the mean ± SD. The asterisk indicates statistically significant differences vs. the control group at P < 0.05.

Article Snippet: Proteins were detected using the following antibodies: Phospho-DRP1 (Ser616) (Cell Signaling Technology, Danvers, MA, USA; #3455; dilution 1:500), DRP1 (D6C7) (Cell Signaling Technology; #8570s; dilution 1:1000), UCP1 (Cell Signaling Technology; #14670S; dilution 1:1000), PRDM16 (Rockland, Limerick, PA, USA; #600-401-H12; dilution 1:1000), carnitine palmitoyltransferase I (CPT1A) (Cell Signaling © 2025 The Author(s).

Techniques: Control, Imaging, Transmission Assay, Phospho-proteomics, Software

Journal: The Journal of Physiology

Article Title: NAD replenishment restores mitochondrial function and thermogenesis in the brown adipose tissue of mice with obesity

doi: 10.1113/jp288453

Figure Lengend Snippet: Figure 4. Nicotinamide riboside effect on body weight and thermogenic parameters Animals were treated with HFD and NR supplementation for 8 weeks. A, body weight gain curve (n = 12 Control vs. 12 Obese vs. 13 Obese+NR). B, fat mass weight of mesenteric, subcutaneous, and epididymal fats (as percentage of control). C, accumulated food intake over the 8 weeks of experiment (kcal). D. respirometry exchange ratio (RER) curve of 24 h (n = 5 Control vs. 5 Obese vs. 6 Obese+NR). E, area under the curve (AUC) quantification of the light cycle of RER. F, AUC quantification of the dark cycle of RER. G. Whole body heat production curve of 24 h (n = 5 Control vs. 5 Obese vs. 6 Obese+NR). H, AUC body heat quantification of the light cycle. I, AUC body heat quantification of the dark cycle. J, ambulatory activity curve of 24 h (n = 5 Control vs. 5 Obese vs. 6 Obese+NR). K, AUC ambulatory activity quantification of the light cycle. L, AUC ambulatory activity quantification of the dark cycle. M, BAT protein homogenates were used to evaluate UCP1, CPT1A, PRDM16, and β-3 adrenergic receptor protein levels (n = 4 Control vs. 5 Obese vs. 5 Obese+NR). N–Q, protein quantification of UCP1 (N), CPT1A (O), PRDM16 (P), and β-3 adrenergic receptor (Q). R, images of BAT from C57BL/6J treated with HFD and NR through 8 weeks. S, BAT weight (percentage of control). T, imaging by transmission electron microscopy of BAT Control vs. Obese. U, TEM adipocytes area quantification in μm2 related to the control group (n = 5 Control vs. 9 Obese vs. 5 Obese+NR). All values are expressed as the mean ± SD. The asterisk indicates statistically significant differences vs. the control group at P < 0.05.

Article Snippet: Proteins were detected using the following antibodies: Phospho-DRP1 (Ser616) (Cell Signaling Technology, Danvers, MA, USA; #3455; dilution 1:500), DRP1 (D6C7) (Cell Signaling Technology; #8570s; dilution 1:1000), UCP1 (Cell Signaling Technology; #14670S; dilution 1:1000), PRDM16 (Rockland, Limerick, PA, USA; #600-401-H12; dilution 1:1000), carnitine palmitoyltransferase I (CPT1A) (Cell Signaling © 2025 The Author(s).

Techniques: Control, Activity Assay, Imaging, Transmission Assay, Electron Microscopy

Journal: International Journal of Molecular Sciences

Article Title: Interleukin-4 Improves Metabolic Abnormalities in Leptin-Deficient and High-Fat Diet Mice

doi: 10.3390/ijms21124451

Figure Lengend Snippet: IL-4 promoted white adipocyte browning. ( A ) Wild-type C57BL/6 (WT) and leptin-deficient 145E mice were fed the normal diet (ND) for 8 weeks. Simultaneously, normal saline and IL-4 (1 μg/mouse) were intraperitoneally administrated twice a week. Proteins were extracted from the epididymal fat tissues and subjected to Western blot analysis with indicated antibodies. * p < 0.05 vs. WT/saline group, and # p < 0.05 vs. 145E/saline group, n = 6. ( B ) C57BL/6 mice were fed the normal diet (ND) or high-fat diet (HFD) for 12 weeks. Simultaneously, normal saline and IL-4 (1 μg/mouse) were intraperitoneally administrated twice a week for the last 8 weeks. Proteins were extracted from the epididymal fat tissues and subjected to Western blot analysis with indicated antibodies. * p < 0.05 vs. ND/saline group and # p < 0.05 vs. HFD/saline group, n = 6. ( C ) Postconfluent 3T3-L1 cells differentiated into mature adipocytes, as described in the methods. The undifferentiated control and differentiated cells were stained with Oil Red O. Postconfluent 3T3-L1 cells were differentiated into mature adipocytes in the presence of IL-4 (0 and 10 ng/mL), as described in the methods. Total RNA were extracted and mRNA expression measured with qRT-PCR for PRDM16 ( D ), PGC-1α ( E ), and UCP-1 ( F ). * p < 0.05 vs. saline group, n = 4.

Article Snippet: Primary antibodies used were: Akt, phospho-Akt, signal transducers and activators of transcription 3 (STAT3), phospho-STAT3, STAT6, phospho-STAT6, PRDM16, PGC-1α, UCP-1, CD68, CD206, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: Saline, Western Blot, Control, Staining, Expressing, Quantitative RT-PCR

Journal: Advanced Science

Article Title: PRDM16 Reduces Cellular Senescence by Upregulating GSTM1

doi: 10.1002/advs.202501233

Figure Lengend Snippet: PRDM16 was decreased in aged organs and SnCs. a) qPCR analysis of PRDM family members in the kidneys, lungs, heart and stomach of young (2 months old) and aged (24 months old) mice (n = 3). b) qPCR analysis of Prdm16 in multiple organs of 2 months mice (n = 6). c) Analysis of one published transcriptomic dataset showed that PRDM16 decreased with age in human normal renal cortex (n = 71). d–f) Tests for linear trend were conducted between age and PRDM16 mRNA level in the heart (d) (n = 384), brain hippocampus (e) (n = 187) and lung (f) (n = 345) of humans based on transcriptomes from ADEIP. g) Correlational analysis of PRDM16 mRNA level (log 10 (2 −(ΔΔCt) )) and age in human normal lung samples (n = 13). h) Correlational analysis of the mRNA levels (log 10 (2 −(ΔΔCt) )) of PRDM16 and CDKN1A in human normal lung samples (n = 13). i) qPCR analysis of Prdm16 in the kidneys, lungs, brain hippocampus and heart of 2 months old (2 M) and 24 months old (24 M) mice (n = 6). j) Representative immunohistochemical (IHC) staining images of PRDM16 in the kidney of 2 months and 24 months mice. Scale bar: 50 µm. k) Diagram detailing X‐ray radiation, BLM and DOX induced cellular senescence. l) qPCR analysis of Prdm16 in senescent HK‐2, Beas‐2B and H9C2 cells (n = 3). Data are mean ± SEM. * p < 0.05, ** p < 0.01 and *** p < 0.001. Spearman's correlations (c, g and h), Test for linear trend (d–f), Two‐tailed Student's unpaired t ‐test analysis (i and l).

Article Snippet: Global Prdm16 knockout mice were generated by using CRISPR‐Cas9 technology at Cyagen Biosciences Inc.

Techniques: Immunohistochemical staining, Immunohistochemistry, Two Tailed Test

Journal: Advanced Science

Article Title: PRDM16 Reduces Cellular Senescence by Upregulating GSTM1

doi: 10.1002/advs.202501233

Figure Lengend Snippet: Genetic deletion of Prdm16 contributed to senescence in multiple organs. a) qPCR analysis of Prdm16 , senescence and senescence‐associated secretory phenotype (SASP) markers in multiple organs including kidney, heart, lung, brain hippocampus, stomach, and gut of 3 weeks wide type (WT) and global Prdm16 knockout (KO) mice (n = 6). b) Serum SASP cytokines were measured in 9‐month‐old (9 M) WT and KO mice (n = 6). c) Single‐cell RNA sequencing was conducted with the kidney tissues obtained from 9‐month‐old WT and KO mice (n = 3). A bubble diagram illustrating the enrichment of gene ontology (GO) terms derived from differentially expressed genes in DCT, endothelial cells, macrophages, and stromal cells. d) Representative images of senescence‐associated‐β‐galactosidase (SA‐β‐gal) staining in the kidney. The percentage of SA‐β‐gal positive area was calculated (n = 6). Scale bar: 50 µm. e) Representative IHC staining images of p16 in the lung. The percentage of p16 positive nuclei is calculated (n = 6). Scale bar: 50 µm. Data are mean ± SEM. * p < 0.05, ** p < 0.01 and *** p < 0.001. n.s, not significant. Two‐tailed Student's unpaired t ‐test analysis (a, b, d and e).

Article Snippet: Global Prdm16 knockout mice were generated by using CRISPR‐Cas9 technology at Cyagen Biosciences Inc.

Techniques: Knock-Out, RNA Sequencing, Derivative Assay, Staining, Immunohistochemistry, Two Tailed Test

Journal: Advanced Science

Article Title: PRDM16 Reduces Cellular Senescence by Upregulating GSTM1

doi: 10.1002/advs.202501233

Figure Lengend Snippet: Global knockout of Prdm16 mice manifested as progressive organ injury at 9 months old. a) Gross appearance of wide type (WT) and Prdm16 knockout (KO) mice (9 months old). b) Body weight curve from the first month to the ninth month (n = 6). c) Single‐cell RNA sequencing (scRNA‐seq) analysis was performed using renal cortex of WT and Prdm16 KO mice. Bar‐plot was generated to show the proportion of annotated cell types (n = 3). d) Heatmaps of representative differentially expressed genes in proximal convoluted tubule (PCT), endothelial cells and macrophages based on scRNA‐seq analysis (n = 3). e) Representative IHC staining images of Fibronectin in the kidney and transmission electron microscopy (TEM) images in the tubular epithelial cells of mice. Scale bar: 50 µm (Fibronectin), 5 µm (TEM 2.5k) and 2 µm (TEM 5k). f) The percentage of Fibronectin positive area was calculated (n = 6). g) Mitochondrial density in tubular epithelial cells of mice was calculated (n = 4). h,i) Representative IHC staining images of Collagen IV in the lung (h). Scale bar: 100 µm. The percentage of Collagen IV positive area was calculated (i) (n = 6). j) Representative images of M‐mode echocardiograms. k) Quantitative analysis of ejection fraction (EF), fractional shortening (FS), cardiac output, and stroke volume (n = 6). l) Heart weight to body weight (HW/BW) was calculated (n = 6). m) Representative IF images of wheat germ agglutinin (WGA) staining in the heart. The relative cross‐sectional area of cardiomyocytes was calculated (n = 6). Scale bar: 25 µm. n) Strength analysis of the forelimb grip strength test (n = 8). o) Latency analysis of the rotarod test (n = 8). p) Swimming track in the Morris water maze (MWM) test. q) Analysis of average speed, duration on platform and the number of times crossing platform in mice (n = 9 for WT group, n = 8 for KO group). Data are mean ± SEM. * p < 0.05, ** p < 0.01 and *** p < 0.001. Two‐tailed Student's unpaired it‐test analysis (b, f, g, i, k–o and q).

Article Snippet: Global Prdm16 knockout mice were generated by using CRISPR‐Cas9 technology at Cyagen Biosciences Inc.

Techniques: Knock-Out, RNA Sequencing, Generated, Immunohistochemistry, Transmission Assay, Electron Microscopy, Staining, Two Tailed Test

Journal: Advanced Science

Article Title: PRDM16 Reduces Cellular Senescence by Upregulating GSTM1

doi: 10.1002/advs.202501233

Figure Lengend Snippet: Tubule‐specific Prdm16 deletion aggravated aging‐related kidney diseases. a) Diagram detailing irradiation (IR) in Prdm16 fl/fl mice and Ksp‐Cre / Prdm16 fl/fl mice. b) qPCR analysis of Cdkn1a and Fibronectin in the kidney cortex (n = 5 for Prdm16 fl/fl group, n = 6 for the other 3 groups). c,d) Representative western blot images (c) and quantification (d) of senescence and fibrotic markers in the kidney cortex (n = 5 for Prdm16 fl/fl group, n = 6 for the other 3 groups). e) Representative images of Masson's trichrome staining (MTS) in the kidney. The percentage of fibrotic area was calculated (n = 5 for Prdm16 fl/fl group, n = 6 for the other 3 groups). Scale bar: 50 µm. f) Diagram detailing unilateral ischemia reperfusion injury (UIRI) surgery in irradiated mice. g) Serum creatinine in mice (n = 6). h,j) Representative western blot images (h) and quantification (j) of senescence and fibrotic markers in the kidney cortex (n = 6). Numbers (1–3) represent different animals in a given group. i) Representative images of SA‐β‐gal staining and MTS in the kidney. Scale bar: 50 µm. k,l) The percentages of SA‐β‐gal positive area (k) and fibrotic area (l) were calculated (n = 6). Data are mean ± SEM. * p < 0.05, ** p < 0.01 and *** p < 0.001. One‐way ANOVA followed by Tukey's post‐test (b, d and e), two‐tailed Student's unpaired it‐test analysis (g and j–l).

Article Snippet: Global Prdm16 knockout mice were generated by using CRISPR‐Cas9 technology at Cyagen Biosciences Inc.

Techniques: Irradiation, Western Blot, Staining, Two Tailed Test

Journal: Advanced Science

Article Title: PRDM16 Reduces Cellular Senescence by Upregulating GSTM1

doi: 10.1002/advs.202501233

Figure Lengend Snippet: Lentivirus‐mediated delivery of exogenous PRDM16 gene mitigated cellular senescence and ameliorated renal aging. a) Diagram detailing X‐ray irradiation (IR) induced cellular senescence in HK‐2 cells transfected with negative control lentivirus (NC‐LV) or PRDM16 overexpression lentivirus (PR‐LV). b) qPCR analysis of tubular injury, senescence and SASP related genes displayed by a heatmap (n = 3). c) Microscopic images depicted morphological changes of HK‐2 cells. Representative images of SA‐β‐gal staining and IF images of γ‐H2AX. Scale bar: 200 µm (left panel), 50 µm (middle panel) and 20 µm (right panel). The arrows pointed to positive cells. d) The percentages of morphologically altered senescent cells, SA‐β‐gal positive cells and γ‐H2AX positive cells in total cells were calculated (n = 3). e) Diagram detailing renal cortex lentivirus injection in old mice. f) qPCR analysis of Prdm16 and Cdkn1a in the kidney cortex (n = 6). g) Representative images of SA‐β‐gal staining and IF images of γ‐H2AX in the kidney. Scale bar: 50 µm (top panel) and 10 µm (bottom panel). h,i) The percentages of SA‐β‐gal (h) and γ‐H2AX (i) positive area were calculated (n = 6). j) Representative IF images of α‐SMA and images of MTS in the kidney. Scale bar: 50 µm. k,l) The percentages of α‐SMA positive area (k) and fibrotic area (l) were calculated (n = 6). Data are mean ± SEM. * p < 0.05, ** p < 0.01 and *** p < 0.001. One‐way ANOVA followed by Tukey's post‐test (d, f, h, i, k and l).

Article Snippet: Global Prdm16 knockout mice were generated by using CRISPR‐Cas9 technology at Cyagen Biosciences Inc.

Techniques: Irradiation, Transfection, Negative Control, Over Expression, Staining, Injection

Journal: Advanced Science

Article Title: PRDM16 Reduces Cellular Senescence by Upregulating GSTM1

doi: 10.1002/advs.202501233

Figure Lengend Snippet: PRDM16 inhibited oxidative DNA damage and improved glutathione metabolism. a) Diagram detailing combined analysis of kidney scRNA‐seq data from 9‐month‐old WT and Prdm16 KO mice (n = 3) and bulk RNA sequencing (bulk RNA‐seq) data from irradiated PRDM16‐overexpressing and control HK‐2 cells (n = 3). Gene Ontology (GO) enrichment analysis was performed to differentially expressed genes of proximal convoluted tubule (PCT) and distal convoluted tubule (DCT) from scRNA‐seq, as well as differentially expressed genes of HK‐2 cells from RNA‐seq. Common GO terms were identified by intersection. b) Representative IF images of 8‐oxo‐dG in the kidney. The ratio of 8‐oxo‐dG positive cells to total cells was calculated (n = 5 for Prdm16 fl/fl group, n = 6 for the other 3 groups). Scale bar: 20 µm. c) Representative IF images of 8‐oxo‐dG and Dihydroethidium (DHE) in HK‐2 cells. The percentage of 8‐oxo‐dG positive cells in total cells and relative fold change of DHE intensity were calculated. Scale bar: 20 µm. d) Enrichment plot from the GSEA results based on scRNA‐seq data of PCT. e) Diagram detailing the enzymes involved in glutathione metabolism. qPCR analysis of Gclc , Gclm and Gss in the kidney of aging mice treated with negative control lentivirus (NC‐LV) or PRDM16 overexpression lentivirus (PR‐LV). GSH/GSSG ratio and GST activity of irradiated HK‐2 cells and the kidney of aging mice were detected and calculated. (n = 3 for HK‐2 cells and n = 6 for mice). Data are mean ± SEM. * p < 0.05, ** p < 0.01 and *** p < 0.001. n.s: not significant. One‐way ANOVA followed by Tukey's post‐test (b, c and e).

Article Snippet: Global Prdm16 knockout mice were generated by using CRISPR‐Cas9 technology at Cyagen Biosciences Inc.

Techniques: RNA Sequencing, Irradiation, Control, Negative Control, Over Expression, Activity Assay

Journal: Advanced Science

Article Title: PRDM16 Reduces Cellular Senescence by Upregulating GSTM1

doi: 10.1002/advs.202501233

Figure Lengend Snippet: PRDM16 acted as a transcription activator of GSTM1. a) Radiation plot depicting the predicted scores of PRDM16 binding to the promoter region of GST family members based on the chromatin immunoprecipitation sequencing (ChIP‐Seq) data of PRDM16 deposited in the Cistrome DB database. b) qPCR analysis of Gstt2 and Gstm1 in the kidney and heart of 2‐month‐old and 24‐month‐old mice (n = 6). c,d) A representative western blot (c) and quantification (d) of PRDM16 and GSTM1 in HK‐2 cells (n = 3). Numbers (1‐2) represent different wells of cells. e,f) A representative western blot (e) and quantification (f) of PRDM16 and GSTM1 in renal cortex (n = 6). Numbers (1–3) represent different animals in the given group. g) qPCR analysis of GSTM1 in HK‐2 cells with different treatments (n = 3). h) HEK‐239T cells were co‐transfected with firefly luciferase reporter plasmid of GSTM1 and Renilla reniformis luciferase reporter plasmid, either negative control siRNA or PRDM16 siRNA. Relative luciferase activity of different groups was measured. i) Two reported PRDM16 binding sites were found in the promoter region of GSTM1. Three primers were designed for each binding site. Representative images of ChIP‐PCR conducted in HK‐2 cells using anti‐PRDM16 and anti‐IgG antibody. Data are mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001. n.s: not significant. Two‐tailed Student's unpaired t ‐test analysis (b and h), one‐way ANOVA followed by Tukey's post‐test (d, f and g).

Article Snippet: Global Prdm16 knockout mice were generated by using CRISPR‐Cas9 technology at Cyagen Biosciences Inc.

Techniques: Binding Assay, ChIP-sequencing, Western Blot, Transfection, Luciferase, Plasmid Preparation, Negative Control, Activity Assay, Two Tailed Test

Journal: Advanced Science

Article Title: PRDM16 Reduces Cellular Senescence by Upregulating GSTM1

doi: 10.1002/advs.202501233

Figure Lengend Snippet: Restoration of GSTM1 alleviated cellular senescence and renal aging induced by PRDM16 deficiency in vitro and in vivo. a,b) A representative western blot (a) and quantification (b) of senescence and fibrotic markers in HK‐2 cells with different treatments (n = 3). NC‐LV, negative control lentivirus. GSTM1‐LV, GSTM1 overexpression lentivirus. NC‐si, negative control siRNA. PR‐si, PRDM16 siRNA. Numbers (1‐2) represent different wells of cells in a given group. c) Representative images of SA‐β‐gal staining, IF images of γ‐H2AX and DHE staining. Scale bar: 50 µm (top panels) and 20 µm (middle and bottom panel). d) The percentages of SA‐β‐gal positive cells and γ‐H2AX positive cells in total cells were calculated (n = 3). The relative fold change of DHE intensity was quantified (n = 3). e) Diagram detailing kidney lentivirus injection in irradiated mice. f) Representative IHC staining images of GSTM1, IF images of 8‐oxo‐dG and α‐SMA and images of MTS in the kidney. Scale bar: 20 µm (8‐oxo‐dG) and 50 µm (the others). g,h) A representative western blot (g) and quantification (h) of GSTM1, senescence and fibrosis markers in the kidney cortex. (i) qPCR analysis of Cdkn1a in the kidney cortex (n = 5 for Prdm16 fl/fl + NC‐LV group and Ksp‐Cre/Prdm16 fl/fl + NC‐LV + IR group, n = 6 for Prdm16 fl/fl + NC‐LV + IR group and Ksp‐Cre/Prdm16 fl/fl + GSTM1‐LV + IR group). Data are mean ± SEM. * p < 0.05, ** p < 0.01 and *** p < 0.001. One‐way ANOVA followed by Tukey's post‐test (b, d, h and i).

Article Snippet: Global Prdm16 knockout mice were generated by using CRISPR‐Cas9 technology at Cyagen Biosciences Inc.

Techniques: In Vitro, In Vivo, Western Blot, Negative Control, Over Expression, Staining, Injection, Irradiation, Immunohistochemistry

Journal: BMC Pharmacology & Toxicology

Article Title: A novel 11β-hydroxysteroid dehydrogenase type1 inhibitor CNX-010-49 improves hyperglycemia, lipid profile and reduces body weight in diet induced obese C57B6/J mice with a potential to provide cardio protective benefits

doi: 10.1186/2050-6511-15-43

Figure Lengend Snippet: Effect of CNX-010-49 on thermogenesis and body weight in DIO mice. Body weight was measured weekly (A) . Non-shivering thermogenesis was measured as described in the methods (B) . Immunohistochemistry of PRDM16 and UCP1 (C) in subcutaneous adipose tissue sections was performed as mentioned in the Methods. Subcutaneous adipocytes size (D) and serum leptin (E) levels were measured at the end of treatment. Statistical comparison was conducted by One-way ANOVA followed by Dunnett’s test or repeated measures ANOVA followed by Bonferroni correction (A) (n = 8 mice/group). Two-way repeated measures ANOVA indicated that CNX-010-49 significantly reduced body weight (p = 0.001; F = 14.02; Df = 22). (# - significance of HFD control against lean control, *- significance of CNX-010-49 treatment against HFD control) (*P < 0.05, **P < 0.01 and ***P < 0.001).

Article Snippet: For PRDM16 and UCP1 immuno staining, subcutaneous adipose sections were de-paraffinized and blocked in 1% BSA in PBS for 30 min at RT and incubated with PRDM16 (ProSci, USA) or UCP1(Abcam, USA) primary antibodies for 90 min at RT, followed by washing with PBS.

Techniques: Immunohistochemistry, Comparison, Control