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: Oncotarget

Article Title: miR-101 reverses hypomethylation of the PRDM16 promoter to disrupt mitochondrial function in astrocytoma cells.

doi: 10.18632/oncotarget.6652

Figure Lengend Snippet: Figure 1: PRDM16 with a hypomethylated promoter is overexpressed and correlated with poor prognosis in astrocytoma patients. A. A schematic diagram of the CpG dinucleotides within the PRDM16 promoter. The nucleotide number is relative to the transcription start site of PRDM16. The red line indicates the region that was tested with BSP; the blue line indicates the region that was detected with MSP. B. The methylation status of PRDM16 in the astrocytoma cell lines was detected using MSP. U, unmethylated primer; M, methylated primer. C. BSP of the upstream regulatory region of PRDM16 was performed for each representative tissue sample (N, normal brain tissue; T, glioma sample). For each sample, at least five separate clones were sequenced, and the results are shown here. Unmethylated CpG sites are shown as open circles, whereas methylated CpG sites are represented by closed circles. For each row of circles, the sequence results for an individual clone of the bisulfite-PCR product are given. The number of methylated CpGs divided by the total number of true CpGs analyzed is given as a percentage to the right of each BSP result. D. The methylation status of PRDM16 was detected using BSP in astrocytoma (n=50) and normal brain tissue samples (n=10), and the results were verified using an independent samples t-test. *P< 0.05. E. Real-time PCR was used to detect the expression of PRDM16. PRDM16 expression levels in normal brain tissue samples were much lower than in the four astrocytoma cell lines, and this was verified via an independent samples t-test. **P<0.01. F. PRDM16 expression levels in normal brain tissue and astrocytoma tissue samples were tested using ISH. G. The correlation between PRDM16 methylation in tumor tissues and the OS values of astrocytoma patients. Patients with PRDM16 hypomethylation had shorter OS values than patients with normal levels of PRDM16 methylation. For this analysis, the Kaplan-Meier method was used. H. The correlation between tumor PRDM16 protein expression levels and OS values for astrocytoma patients. Patients with high PRDM16 expression levels had poor outcomes. The Kaplan-Meier method was also used for this analysis.

Article Snippet: The cell protein lysates, cytosol proteins and nuclear proteins were separated on 10% SDS-polyacrylamide gels, electrophoretically transferred to polyvinylidene difluoride membranes (Millipore, Danvers, MA, USA), and detected using rabbit polyclonal antibodies for PRDM16 (Santa Cruz Biotechnology, Santa Cruz County, CA, USA), mouse monoclonal antibodies for EZH2, EED (Santa Cruz Biotechnology) and GAPDH (Millipore), and rabbit polyclonal antibodies for H3K27me3, H3K9me3, H4K20me3 and H3K4me2 (Millipore) using a commercial ECL kit (Pierce, Rockford, IL, USA).

Techniques: Methylation, Clone Assay, Sequencing, Real-time Polymerase Chain Reaction, Expressing

Journal: Oncotarget

Article Title: miR-101 reverses hypomethylation of the PRDM16 promoter to disrupt mitochondrial function in astrocytoma cells.

doi: 10.18632/oncotarget.6652

Figure Lengend Snippet: Figure 3: The PRDM16 gene is a direct target of miR-101. A. The PRDM16 gene was predicted to be a target of miR-101 using the online software program TargetScan 5.1. B. The miR-101 targeting site in PRDM16 (3’-UTR) is shown. Wild-type and mutated PRDM16 3’-UTRs are shown. C. miR-101 regulates the expression of PRDM16 3’-UTR reporter constructs. The luciferase reporter assays were performed 48 h after transfection with the indicated pMIR-REPORT plasmid and a Renilla transfection control plasmid, which were co-transfected with miR-101 or a scrambled control. The data shown are means ± S.D.s of six replicates and are representative of three independent experiments. An independent samples t-test was used. *P<0.05. D. miR-101 inhibits the expression of PRDM16 mRNA. Real- time PCR analysis was performed 48 h after transfection with miR-101 and a scrambled control. An independent samples t-test was used. *P<0.05. E. miR-101 regulates the expression of the PRDM16 protein in four astrocytoma cells. Western blot analysis was performed 72 h after transfection with miR-101 and a scrambled control. GAPDH was used as an internal control.

Article Snippet: The cell protein lysates, cytosol proteins and nuclear proteins were separated on 10% SDS-polyacrylamide gels, electrophoretically transferred to polyvinylidene difluoride membranes (Millipore, Danvers, MA, USA), and detected using rabbit polyclonal antibodies for PRDM16 (Santa Cruz Biotechnology, Santa Cruz County, CA, USA), mouse monoclonal antibodies for EZH2, EED (Santa Cruz Biotechnology) and GAPDH (Millipore), and rabbit polyclonal antibodies for H3K27me3, H3K9me3, H4K20me3 and H3K4me2 (Millipore) using a commercial ECL kit (Pierce, Rockford, IL, USA).

Techniques: Software, Expressing, Construct, Luciferase, Transfection, Plasmid Preparation, Control, Real-time Polymerase Chain Reaction, Western Blot

Journal: Oncotarget

Article Title: miR-101 reverses hypomethylation of the PRDM16 promoter to disrupt mitochondrial function in astrocytoma cells.

doi: 10.18632/oncotarget.6652

Figure Lengend Snippet: Figure 4: miR-101 reversed the hypomethylation status of the PRDM16 promoter. A. PRDM16 methylation levels were increased by miR-101 in U251 cells. The unmethylated and methylated CpG sites are indicated by opened and closed circles, respectively. Each row indicates the sequencing result of one clone of the bisulfite-PCR product. The number of methylated CpGs was divided by the total number of true CpGs analyzed and is given as a percentage to the right of each BSP result. B. Analysis of promoter activity of the PRDM16 core promoter constructs via luciferase reporter assays. The construct containing the sequence spanning the region from -506 to -256 was sufficient to mediate maximal promoter activity. The core promoter ranged from -506 to -256. PGL3-control is the positive control, and pGL3- enhancer is the negative control. An independent samples t-test was used. *P<0.05. C. The histones occupancy of the PRDM16 promoter was affected by miR-101. A ChIP assay was used to detect the H3K4me2, H3K27me3, H3K9me3 and H4K20me3 occupancy the PRDM16 core promoter. U251 cells were transfected with miR-101 or a scrambled control. An independent samples t-test was used. *P<0.05. D. PRDM16 expression was regulated by EZH2, EED and DNMT3A. Real-time PCR analysis was performed 48 h after transfection with EZH2 siRNA, EED siRNA, DNMT3A siRNA or a scrambled control. An independent samples t-test was used. *P<0.05. E. The histones occupancy of the PRDM16 promoter was affected by EZH2 siRNA, EED siRNA and DNMT3A siRNA. A ChIP assay was performed to detect the H3K4me2, H3K27me3, H3K9me3 and H4K20me3 occupancy of the PRDM16 core promoter. U251 cells transfected with EZH2 siRNA, EED siRNA and DNMT3A siRNA were analyzed. An independent samples t-test was used. *P<0.05. F. Left: Real-time PCR was used to detect DNMT3A expression after transfection with a vector or pcDNA3.1-DNMT3A. Right: a ChIP assay was used to detect the H3K4me2 occupancy of the PRDM16 core promoter. U251 cells transfected with miR-101 and pcDNA3.1-DNMT3A were analyzed.

Article Snippet: The cell protein lysates, cytosol proteins and nuclear proteins were separated on 10% SDS-polyacrylamide gels, electrophoretically transferred to polyvinylidene difluoride membranes (Millipore, Danvers, MA, USA), and detected using rabbit polyclonal antibodies for PRDM16 (Santa Cruz Biotechnology, Santa Cruz County, CA, USA), mouse monoclonal antibodies for EZH2, EED (Santa Cruz Biotechnology) and GAPDH (Millipore), and rabbit polyclonal antibodies for H3K27me3, H3K9me3, H4K20me3 and H3K4me2 (Millipore) using a commercial ECL kit (Pierce, Rockford, IL, USA).

Techniques: Methylation, Sequencing, Activity Assay, Construct, Luciferase, Control, Positive Control, Negative Control, Transfection, Expressing, Real-time Polymerase Chain Reaction, Plasmid Preparation

Journal: Oncotarget

Article Title: miR-101 reverses hypomethylation of the PRDM16 promoter to disrupt mitochondrial function in astrocytoma cells.

doi: 10.18632/oncotarget.6652

Figure Lengend Snippet: Figure 6: PRDM16 knockdown reversed the anti-apoptotic effect of miR-101. A. Mitochondrial membrane potentials were monitored by using JC-1 staining. U251 cells were transfected with anti-miR-NC or siNC, anti-miR-101 or siNC, anti-miR-NC or siPRDM16, and anti- miR-101 or siPRDM16, respectively. Red fluorescence indicates normal U251 cells, and green fluorescence indicates cells with mitochondrial dysfunction. B, C and D. Mitochondrial ATP levels, ADP/ATP ratios and ROS production levels were detected 48 h after transfection with anti- miR-NC, anti-miR-101, anti-miR-NC or siNC, anti-miR-101 or siNC, anti-miR-NC or siPRDM16, and anti-miR-101 or siPRDM16, respectively. E. Flow cytometry analysis was performed 48 h after transfection of U251 cells with anti-miR-NC or siNC, anti-miR-101 or siNC, anti-miR-NC or siPRDM16, and anti-miR-101 or siPRDM16, respectively. F. Western blot analysis of Bax, Bcl-2 and Smac expression levels after transfection with anti-miR-NC or siNC, anti-miR-101 or siNC, anti-miR-NC or siPRDM16, and anti-miR-101 or siPRDM16, respectively. GAPDH and VDAC were used as loading controls for cytoplasmic protein and mitochondrial protein levels, respectively.

Article Snippet: The cell protein lysates, cytosol proteins and nuclear proteins were separated on 10% SDS-polyacrylamide gels, electrophoretically transferred to polyvinylidene difluoride membranes (Millipore, Danvers, MA, USA), and detected using rabbit polyclonal antibodies for PRDM16 (Santa Cruz Biotechnology, Santa Cruz County, CA, USA), mouse monoclonal antibodies for EZH2, EED (Santa Cruz Biotechnology) and GAPDH (Millipore), and rabbit polyclonal antibodies for H3K27me3, H3K9me3, H4K20me3 and H3K4me2 (Millipore) using a commercial ECL kit (Pierce, Rockford, IL, USA).

Techniques: Knockdown, Membrane, Staining, Transfection, Fluorescence, Flow Cytometry, Western Blot, Expressing

Journal: Oncotarget

Article Title: miR-101 reverses hypomethylation of the PRDM16 promoter to disrupt mitochondrial function in astrocytoma cells.

doi: 10.18632/oncotarget.6652

Figure Lengend Snippet: Figure 7: A schematic diagram of the working model for the miR-101 suppressing PRDM16 directly and epigenetically in astrocytoma cells.

Article Snippet: The cell protein lysates, cytosol proteins and nuclear proteins were separated on 10% SDS-polyacrylamide gels, electrophoretically transferred to polyvinylidene difluoride membranes (Millipore, Danvers, MA, USA), and detected using rabbit polyclonal antibodies for PRDM16 (Santa Cruz Biotechnology, Santa Cruz County, CA, USA), mouse monoclonal antibodies for EZH2, EED (Santa Cruz Biotechnology) and GAPDH (Millipore), and rabbit polyclonal antibodies for H3K27me3, H3K9me3, H4K20me3 and H3K4me2 (Millipore) using a commercial ECL kit (Pierce, Rockford, IL, USA).

Techniques:

Journal: Development (Cambridge, England)

Article Title: PRDM16 regulates a temporal transcriptional program to promote progression of cortical neural progenitors

doi: 10.1242/dev.194670

Figure Lengend Snippet: PRDM16 regulates cortical laminar organization. (A) Immunostaining images of P0 control and Prdm16 KO cortices stained with the upper-layer marker SATB2 in red (top and bottom) and the mid-layer marker CTIP2 in green (top). Cortical layers are highlighted according to expression of SATB2 and CTIP2. White arrowheads point to some retained SATB2+ cells in the deep layers. (B) Quantification of the marker-positive cells in an 80 µm-wide column across the cortex ( n =3). (C) Measurement of layer marker gene expression and Prdm16 by RT-qPCR from E15.5 control and Prdm16 KO cortices. (D) Images of E15.5 control and Emx1-Cre:: Prdm16 cKO cortices show reduction of the SATB2+ layer (blue) and expansion of the CTIP2+ (green) and FEZF2+ (red) layer. (E) Quantification of the marker-positive cells in a 100 µm width column across the cortex ( n =3). (F) Images of P15 control and Prdm16 cKO cortices stained with SATB2 (red), CTIP2 (green) and TBR1 (blue). Dorsal lateral and medial areas are highlighted with pink and orange rectangles, respectively. (G) Higher magnification images of rectangles in F show reciprocal effects on CTIP2+ and SATB2+ layers in cKO cortices. Heterotopia tissue is highlighted by the white dashed line. (H) Quantification of the numbers of the three cell types in a 300 µm-wide column in each area ( n =3). All data are shown as mean±s.d.; * P <0.05; ** P <0.01; *** P <0.001 (two-tailed unpaired Student's t -test). n.s., not significant. Scale bars: 50 µm (A,D,G); 100 µm (F).

Article Snippet: The replicated Prdm16 KO (×3) and control (×3) ChIP-seq samples, after the adaptor trimming by Trimmomatic, were mapped to the University of California, Santa Cruz (UCSC) Mus musculus (mm10) genome assembly using Bowtie2 with the default parameters.

Techniques: Immunostaining, Control, Staining, Marker, Expressing, Gene Expression, Quantitative RT-PCR, Two Tailed Test

Journal: Development (Cambridge, England)

Article Title: PRDM16 regulates a temporal transcriptional program to promote progression of cortical neural progenitors

doi: 10.1242/dev.194670

Figure Lengend Snippet: Forebrain-specific depletion of PRDM16 delayed mid-to-late neurogenesis transition. (A) Images of P5 control and Prdm16 cKO cortices, stained with CTIP2, BrdU and EdU. White, green and red arrowheads point to BrdU+CTIP2+ cells. (B) Quantification of the numbers of the CTIP2+BrdU− and CTIP2+BrdU+ cells in a 300 µm-wide column across the cortex ( n =3). All data are shown as mean±s.d.; * P <0.05; ** P <0.01; *** P <0.001 (two-tailed unpaired Student's t -test). n.s., not significant. (C) Illustration of the progression delay: mutant RG produce CTIP2+ neurons at E14.5. Scale bar: 100 µm.

Article Snippet: The replicated Prdm16 KO (×3) and control (×3) ChIP-seq samples, after the adaptor trimming by Trimmomatic, were mapped to the University of California, Santa Cruz (UCSC) Mus musculus (mm10) genome assembly using Bowtie2 with the default parameters.

Techniques: Control, Staining, Two Tailed Test, Mutagenesis

Journal: Development (Cambridge, England)

Article Title: PRDM16 regulates a temporal transcriptional program to promote progression of cortical neural progenitors

doi: 10.1242/dev.194670

Figure Lengend Snippet: PRDM16 promotes IP cell production during late cortical neurogenesis. (A) Images of E15.5 control and Prdm16 cKO cortices, stained with the IP marker TBR2 (green), the RG marker PAX6 (red) and DAPI (blue). (B) Quantification of the marker-positive cells in a 200 µm-wide column across the cortex ( n =3). (C) Images of E15.5 cortices injected with the shcontrol-GFP vector or shPrdm16 -GFP by in utero electroporation, showing GFP (green), TBR2 (red) and DAPI (blue). (D) Quantification of the fraction of TBR2+GFP+ cells among GFP+ cells. Control: n =4; shPrdm16 : n =3. (E) Images of E15.5 control and Prdm16 cKO cortices, stained with EdU (blue), TBR2 (green) and KI67 (red). White dashed lines highlight the SVZ. (F) Quantification of the fraction of EdU+Ki67− over EdU+ cells ( n =3) within a 250 µm-wide column. (G) Quantification of KI67+ cells within a 250 µm-wide column ( n =3). (H) Images of E15.5 control and Prdm16 cKO cortices following a 2 h EdU pulse labeling, stained with EdU (green) and TBR2 (red) antibodies. (I) Quantification of the fraction of EdU+TBR2+ over TBR2+ cells within a 250 µm-wide column. (J) Images of E15.5 control and Prdm16 cKO cortices, stained with PH3 (green), TBR2 (red) and DAPI (blue). (K) Quantification of the fraction of PH3+ at the apical surface and the basal side of the ventricle within a 250 µm-wide column. All data are shown as mean±s.d.; * P <0.05; ** P <0.01; *** P <0.001 (two-tailed unpaired Student's t -test). n.s., not significant. Scale bars: 50 µm.

Article Snippet: The replicated Prdm16 KO (×3) and control (×3) ChIP-seq samples, after the adaptor trimming by Trimmomatic, were mapped to the University of California, Santa Cruz (UCSC) Mus musculus (mm10) genome assembly using Bowtie2 with the default parameters.

Techniques: Control, Staining, Marker, Injection, Plasmid Preparation, In Utero, Electroporation, Labeling, Two Tailed Test

Journal: Development (Cambridge, England)

Article Title: PRDM16 regulates a temporal transcriptional program to promote progression of cortical neural progenitors

doi: 10.1242/dev.194670

Figure Lengend Snippet: Depletion of PRDM16 led to defective gene expression in the developing cortex. (A) Schematic of the RNA-seq tissue and a list of layer marker genes described in the study. (B) Log2 fold-change in KO versus wild-type (WT) of upper-, mid- and deep-layer genes. A gene set test shows the upper-layer markers are significantly downregulated. (C,D) In situ hybridization for Pou3f2, Unc5D, Pcp4 and Fezf2 in control and Prdm16 cKO mutant cortices at E13.5 (C) and E15.5 (D). Black arrows indicate ventricle surface. Scale bars: 100 µm.

Article Snippet: The replicated Prdm16 KO (×3) and control (×3) ChIP-seq samples, after the adaptor trimming by Trimmomatic, were mapped to the University of California, Santa Cruz (UCSC) Mus musculus (mm10) genome assembly using Bowtie2 with the default parameters.

Techniques: Gene Expression, RNA Sequencing, Marker, In Situ Hybridization, Control, Mutagenesis

Journal: Development (Cambridge, England)

Article Title: PRDM16 regulates a temporal transcriptional program to promote progression of cortical neural progenitors

doi: 10.1242/dev.194670

Figure Lengend Snippet: PRDM16 represses gene expression through binding to permissive enhancers. (A) Genomic distribution of PRDM16 ChIP-seq peaks. (B) PRDM16 ChIP-seq peaks in correlation with the ATAC-seq signal in wild-type and mutant cortices. Both types of signals are centered at ChIP-seq peak summits. Top: metaplots showing distribution of signal intensity (reads per million; RPM); bottom: heatmaps showing each ChIP-seq peak in correlation with its ATAC-seq signal. (C,D) Volcano plots showing significantly increased (light pink) or decreased (light green) ATAC-seq signal in mutant versus control at PRDM16-bound loci (FDR≤0.2). The associated genes that had expression change in the mutant FB (C) or mutant RG (D) are indicated in purple (upregulated) or dark-green (downregulated). The bar plots on the right measure the fraction of genes that changed expression level and ATAC-seq intensity at the PRDM16-bound sites, showing that the upregulated genes are associated with increased ATAC-seq signal.

Article Snippet: The replicated Prdm16 KO (×3) and control (×3) ChIP-seq samples, after the adaptor trimming by Trimmomatic, were mapped to the University of California, Santa Cruz (UCSC) Mus musculus (mm10) genome assembly using Bowtie2 with the default parameters.

Techniques: Gene Expression, Binding Assay, ChIP-sequencing, Mutagenesis, Control, Expressing

Journal: Development (Cambridge, England)

Article Title: PRDM16 regulates a temporal transcriptional program to promote progression of cortical neural progenitors

doi: 10.1242/dev.194670

Figure Lengend Snippet: PRDM16 represses mid-layer neuronal genes including Fezf2. (A) Re-analysis of the cortex scRNA-seq data shows that PRDM16 targets are enriched in the RG, mid- and deep-layer neuron clusters at E13.5. The y -axis plots the percentage of cells that have summed expression of PRDM16 targets per cell >200 total normalized log2counts. (B) Screenshot of the Fezf2 locus with a PRDM16 peak in the RG enhancer of Fezf2 . (C) RT-qPCR from primary neural stem cell culture of control and Prdm16 mutant cortical cells. Three pairs of control and Prdm16 KO embryos were used. (D) Luciferase assays in N2A cells. PRDM16-FL-VP64 induced significantly higher expression of the Fezf2 reporter than the empty pGL3-luc alone. Four biological replicates were used. (E) RT-qPCR from the N2A cells expressing pCDH-Puro (empty vector control), pCDH-PRDM16-FL or pCDH-PRDM16-PRdeletion constructs. Two independent stable lines for each construct and three technical replicates of each stable line were used. All data are shown as mean±s.d.; * P <0.05; ** P <0.01; *** P <0.001 (two-tailed unpaired Student's t -test). n.s., not significant.

Article Snippet: The replicated Prdm16 KO (×3) and control (×3) ChIP-seq samples, after the adaptor trimming by Trimmomatic, were mapped to the University of California, Santa Cruz (UCSC) Mus musculus (mm10) genome assembly using Bowtie2 with the default parameters.

Techniques: Expressing, Quantitative RT-PCR, Stem Cell Culture, Control, Mutagenesis, Luciferase, Plasmid Preparation, Construct, Two Tailed Test

Journal: Development (Cambridge, England)

Article Title: PRDM16 regulates a temporal transcriptional program to promote progression of cortical neural progenitors

doi: 10.1242/dev.194670

Figure Lengend Snippet: PRDM16 regulates temporal dynamics of RG gene expression. (A) Volcano plot showing differentially expressed genes between E13.5 and E15.5 RG (FDR<0.2 and FC>1.4-fold): 248 (light pink) higher at E15.5 and 120 (light green) higher at E13.5. The 25 significantly up- and downregulated genes in Prdm16 cKO/wild-type (WT) RG (FDR<0.05) are highlighted in purple and dark green, respectively. (B) Expression changes of the 25 genes in Prdm16 cKO/WT RG. The genes containing PRDM16 binding are highlighted in orange. (C) In situ hybridization of Cdkn1c and Flrt3 in the control and cKO brains. Black arrows indicate the VZ surface; white arrowhead points to an increased expression in IP cells. (D,E) Genome browser view of the Cdkn1c (D) and the Flrt3 (E) loci. Enhancer regions responsive to PRDM16-VP64 are highlighted. (F) Luciferase assay testing the highlighted enhancers in the Cdkn1c and the Flrt3 loci. Activity in response to PRDM16-VP64 is normalized to CAGIG and is compared between tested enhancers and the pGL3-promoter vector. The data was from three biological replicates, each of which has four technical replicates. (G,H) Knockdown tests by shPrdm16 and shCdkn1c using in utero electroporation. G shows images from E16.5 cortices injected with the shRNA constructs and EdU, stained with EdU (blue), GFP (green) and TBR2 (red). Arrowheads highlight TBR2+GFP+EdU+ triple-positive cells; arrows highlight GFP+TBR2+ double-positive cells. H shows quantification of the fraction of GFP+EdU+TBR2+ in GFP+TBR2+ cells ( n =3). (I,J) Knockdown tests of shPrdm16 and shFlrt3 using in utero electroporation. I shows images from P5 cortices injected with the shRNA constructs. Dashed white lines indicate white matter (WM). J shows quantification of the ratio of GFP+ cells in the white matter versus cortical plate. shControl: n =7; shPrdm16 +shcontrol: n =4; shPrdm16 + shFlrt3 : n =11. All data are shown as mean±s.d.; * P <0.05; ** P <0.01; *** P <0.001 (two-tailed unpaired Student's t -test). n.s., not significant. Scale bars: 100 µm.

Article Snippet: The replicated Prdm16 KO (×3) and control (×3) ChIP-seq samples, after the adaptor trimming by Trimmomatic, were mapped to the University of California, Santa Cruz (UCSC) Mus musculus (mm10) genome assembly using Bowtie2 with the default parameters.

Techniques: Gene Expression, Expressing, Binding Assay, In Situ Hybridization, Control, Luciferase, Activity Assay, Plasmid Preparation, Knockdown, In Utero, Electroporation, Injection, shRNA, Construct, Staining, Two Tailed Test

Journal: Development (Cambridge, England)

Article Title: PRDM16 regulates a temporal transcriptional program to promote progression of cortical neural progenitors

doi: 10.1242/dev.194670

Figure Lengend Snippet: A model summarizing PRDM16 regulation on temporal dynamics of RG. PRDM16 regulates three classes of genes encoding mid-layer determinants, stage-specific cell-cycle regulators and migration cues. The solid black and the dashed red lines denote direct and indirect regulation by PRDM16, respectively.

Article Snippet: The replicated Prdm16 KO (×3) and control (×3) ChIP-seq samples, after the adaptor trimming by Trimmomatic, were mapped to the University of California, Santa Cruz (UCSC) Mus musculus (mm10) genome assembly using Bowtie2 with the default parameters.

Techniques: Migration

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