Journal: Nature Communications

Article Title: Transcription factor Hlx controls a systematic switch from white to brown fat through Prdm16-mediated co-activation

doi: 10.1038/s41467-017-00098-2

Figure Lengend Snippet: Prdm16 is a co-activator of Hlx and their thermogenic functions are inter-dependent. a mRNA levels of iWAT injected with overexpression adenoviruses ( n = 4 mice per group). b iWAT isolated from mice in ( a ) was immunostained with a Tom20 antibody. Green , Tom20; blue , DAPI. Scale bar , 200 µm. c Flag-Hlx and HA-Prdm16 or HA-Pgc1α were transfected into HEK293 cells. Extracts were immunoblotted with an HA antibody after immunoprecipitation with a Flag antibody. d Cell extracts of brown adipocytes were immunoprecipitated with an antibody against Hlx and immunoblotted with an antibody against Prdm16. e Transcriptional activity of Gal4-Hlx fusion protein on Gal4 promoter in the presence of increased amounts of Prdm16 ( n = 3). f Transcriptional activity of Hlx on the 3.1-kb Ucp1 promoter in the presence of Prdm16 ( n = 3). g Top , Cyan bars depict Prdm16 peaks containing putative Hlx-binding motifs at the promoters of indicated genes, and the positions (in kb) of these peaks relative to transcription start sites are indicated. Black bars depict the location of control sites used in ChIP-qPCR assay. Middle , ChIP-qPCR analysis of Hlx association with the corresponding Prdm16 peaks in brown adipocytes with or without Hlx knockdown. Data represent average of two experiments. Bottom , ChIP-qPCR analysis of Prdm16 association with the corresponding Prdm16 peaks in brown adipocytes with or without Hlx knockdown. Data represent average of two experiments. h Brown preadipocytes were infected with Prdm16 shRNA lentivirus and differentiated. Mature adipocytes were infected with Hlx expression adenovirus, and gene expression was analyzed ( n = 3). i Mature adipocytes generated as in ( h ) were stained with MitoTracker Red. Scale bar , 200 µm. j Brown preadipocytes infected with Hlx shRNA lentivirus were differentiated and infected with Prdm16 adenoviruses at day 2 and day 4. Gene expression was analyzed at day 6 ( n = 3). k Mature adipocytes generated as in ( j ) were stained with MitoTracker Red. Scale bar , 200 µm. l Mature brown adipocytes generated as in ( j ) were measured for oxygen consumption without or with Oligomycin A (1 µ M ) ( n = 3). All images shown are representative of three experiments. All error bars represent s.e.m. Two-tailed unpaired Student’s t-test was performed. * p < 0.05; ** p < 0.01; *** p < 0.001

Article Snippet: Standard ChIP assays were performed with antibodies against Hlx (Millipore, Cat# 09-084), Prdm16 (R&D Systems, Cat# AF6295), or normal rabbit IgG.

Techniques: Injection, Over Expression, Isolation, Transfection, Immunoprecipitation, Activity Assay, Binding Assay, Control, ChIP-qPCR, Knockdown, Infection, shRNA, Expressing, Gene Expression, Generated, Staining, Two Tailed Test

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: Journal of Translational Medicine

Article Title: Type 2 diabetes is associated with decreased PGC1α expression in epicardial adipose tissue of patients with coronary artery disease

doi: 10.1186/s12967-016-0999-1

Figure Lengend Snippet: PGC1α, UCP1 and PRDM16 mRNA expression levels in EAT and SAT. TaqMan ® real time PCR for PGC1α, UCP1 and PRDM16 was performed on human EAT ( a – c ) and thoracic SAT ( d – f ) from coronary artery disease (CAD) patients with type 2 diabetes (CAD-DM2) (n = 22) and without it (CAD-NDM2) (n = 22) and non-CAD patients (NCAD) (n = 23). Results were expressed as the mean ± SEM of the completed experiment in duplicate. *P < 0.05 vs CAD-NDM2 and # P < 0.05 vs NCAD. PGC1α peroxisome proliferator-activated receptor gamma coactivator-1 alpha; UCP1 uncoupling protein 1; PRDM16 PR domain containing 16; EAT epicardial adipose tissue; SAT subcutaneous adipose tissue

Article Snippet: The gene expression levels in the adipose tissue were determined by real time quantitative polymerase chain reaction (PCR) using a predesigned and validated Taqman primer/probe sets [UCP1 (Hs00222453_m1, RefSeq NM_021833.4), PGC1α (Hs01016719_m1, RefSeq NM_013261.3), PRDM16 (Hs00922674_m1, RefSeq NM_022114.3) and cyclophilin A (Hs99999904_m1, RefSeq NM_021130.3)].

Techniques: Expressing, Real-time Polymerase Chain Reaction

Journal: Journal of Translational Medicine

Article Title: Type 2 diabetes is associated with decreased PGC1α expression in epicardial adipose tissue of patients with coronary artery disease

doi: 10.1186/s12967-016-0999-1

Figure Lengend Snippet: Comparison of PGC1α, UCP1 and PRDM16 mRNAs in EAT and SAT. TaqMan ® real-time PCR analysis for mRNA expressions of PGC1α ( a ), UCP1 ( b ) and PRDM16 ( c ) in human adipose tissues (EAT and SAT) from coronary artery disease (CAD) patients. mRNA expression in thoracic SAT were compared as a fraction of epicardial fat, which was arbitrarily assigned the value of 1. Results were expressed as the mean ± SEM of the completed experiment in duplicate (n = 36). PGC1α peroxisome proliferator-activated receptor gamma coactivator-1 alpha; UCP1 uncoupling protein 1; PRDM16 PR domain containing 16; EAT epicardial adipose tissue; SAT subcutaneous adipose tissue

Article Snippet: The gene expression levels in the adipose tissue were determined by real time quantitative polymerase chain reaction (PCR) using a predesigned and validated Taqman primer/probe sets [UCP1 (Hs00222453_m1, RefSeq NM_021833.4), PGC1α (Hs01016719_m1, RefSeq NM_013261.3), PRDM16 (Hs00922674_m1, RefSeq NM_022114.3) and cyclophilin A (Hs99999904_m1, RefSeq NM_021130.3)].

Techniques: Comparison, Real-time Polymerase Chain Reaction, Expressing