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    Millipore ucp1 promoter
    Knockdown of Irx3 repressed the differentiation of SVFs toward beige adipocytes in vitro. (A–B) (A) Relative mRNA and (B) protein levels of Irx3 in SVFs from IWAT were efficiently knocked down after a 3-day infection of mouse Irx3 lentiviral shRNA ( n = 3 for each group). (C–D) (C) Oil Red O staining and (D) TG concentration of the mature beige adipocytes under eight-day differentiation ( n = 6 for each group). (E) Relative mRNA expression of <t>Ucp1</t> , Cidea , Pgc-1α , C/ebpβ , and Prdm16 at different time points during beige adipocyte differentiation from IWAT SVFs ( n = 3–4 for different groups). (F–G) (F) Relative mRNA ( n = 4) and (G) protein expression of brown adipocyte marker genes of the induced beige adipocytes from mouse IWAT SVTs for eight days with the infection of mouse Irx3 lentiviral shRNA, with or without CL-316,243 activation. (H) Relative mRNA expression of adipocyte differentiation-related genes in beige adipocytes ( n = 4). (I) Relative mRNA expression of the indicated genes in the induced brown adipocytes from mouse BAT SVFs for eight days, with the infection of mouse Irx3 lentiviral shRNA ( n = 4). (J) Relative mRNA levels of IRX3 in SVFs from human sWAT after a 3-day infection of human IRX3 lentiviral shRNA (left), and the mRNA levels of brown adipocyte marker genes in the SVFs under 2-day differentiation ( n = 4). (K) OCR measurement of the beige adipocytes under 5-day differentiation, with the infection of mouse Irx3 lentiviral shRNA ( n = 5). (L–N) (L) Ucp1 promoter-luciferase reporter activity was measured in HEK293T cells transfected with pEGFP-C1 vector or IRX3 for 48 h. (M) Schematic diagram of the mutant mouse Ucp1 promoter deleting ACATGTGT among the − 3470 to − 3463 bp region proximal to the TSS of the Ucp1 gene. (N) Transcriptional activity of wild-type or mutant Ucp1 promoter was measured with IRX3 overexpression. (O) qPCR of Ucp1 promoter binding region which was recruited by Irx3 antibody in 8-day induced beige adipocytes, as analyzed by ChIP. Fold enrichment of Ucp1 promoter was given ( n = 3). For the measurement of luciferase activity, cells were seeded on 24-well plate and transfected with 800 ng pEGFP-C1 vector or IRX3, 200 ng Ucp1 promoter construct, and 1 ng pRL-SV40, followed by the harvest for luciferase activity assessment using a dual-luciferase reporter assay system (Promega). Luciferase activity was corrected for Renilla luciferase activity ( n = 3–4 for each group). For qPCR data, mRNA expression was normalized to 36b4 for mouse genes and βACTIN for human genes. SVF cells were isolated from IWAT and BAT of C57BL/6J mice or human sWAT wherever mentioned, and IRX3 shRNA were introduced to cells 24 h after seeding. Data were presented as mean ± s.e.m. * P
    Ucp1 Promoter, supplied by Millipore, used in various techniques. Bioz Stars score: 91/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "IRX3 Promotes the Browning of White Adipocytes and Its Rare Variants are Associated with Human Obesity Risk"

    Article Title: IRX3 Promotes the Browning of White Adipocytes and Its Rare Variants are Associated with Human Obesity Risk

    Journal: EBioMedicine

    doi: 10.1016/j.ebiom.2017.09.010

    Knockdown of Irx3 repressed the differentiation of SVFs toward beige adipocytes in vitro. (A–B) (A) Relative mRNA and (B) protein levels of Irx3 in SVFs from IWAT were efficiently knocked down after a 3-day infection of mouse Irx3 lentiviral shRNA ( n = 3 for each group). (C–D) (C) Oil Red O staining and (D) TG concentration of the mature beige adipocytes under eight-day differentiation ( n = 6 for each group). (E) Relative mRNA expression of Ucp1 , Cidea , Pgc-1α , C/ebpβ , and Prdm16 at different time points during beige adipocyte differentiation from IWAT SVFs ( n = 3–4 for different groups). (F–G) (F) Relative mRNA ( n = 4) and (G) protein expression of brown adipocyte marker genes of the induced beige adipocytes from mouse IWAT SVTs for eight days with the infection of mouse Irx3 lentiviral shRNA, with or without CL-316,243 activation. (H) Relative mRNA expression of adipocyte differentiation-related genes in beige adipocytes ( n = 4). (I) Relative mRNA expression of the indicated genes in the induced brown adipocytes from mouse BAT SVFs for eight days, with the infection of mouse Irx3 lentiviral shRNA ( n = 4). (J) Relative mRNA levels of IRX3 in SVFs from human sWAT after a 3-day infection of human IRX3 lentiviral shRNA (left), and the mRNA levels of brown adipocyte marker genes in the SVFs under 2-day differentiation ( n = 4). (K) OCR measurement of the beige adipocytes under 5-day differentiation, with the infection of mouse Irx3 lentiviral shRNA ( n = 5). (L–N) (L) Ucp1 promoter-luciferase reporter activity was measured in HEK293T cells transfected with pEGFP-C1 vector or IRX3 for 48 h. (M) Schematic diagram of the mutant mouse Ucp1 promoter deleting ACATGTGT among the − 3470 to − 3463 bp region proximal to the TSS of the Ucp1 gene. (N) Transcriptional activity of wild-type or mutant Ucp1 promoter was measured with IRX3 overexpression. (O) qPCR of Ucp1 promoter binding region which was recruited by Irx3 antibody in 8-day induced beige adipocytes, as analyzed by ChIP. Fold enrichment of Ucp1 promoter was given ( n = 3). For the measurement of luciferase activity, cells were seeded on 24-well plate and transfected with 800 ng pEGFP-C1 vector or IRX3, 200 ng Ucp1 promoter construct, and 1 ng pRL-SV40, followed by the harvest for luciferase activity assessment using a dual-luciferase reporter assay system (Promega). Luciferase activity was corrected for Renilla luciferase activity ( n = 3–4 for each group). For qPCR data, mRNA expression was normalized to 36b4 for mouse genes and βACTIN for human genes. SVF cells were isolated from IWAT and BAT of C57BL/6J mice or human sWAT wherever mentioned, and IRX3 shRNA were introduced to cells 24 h after seeding. Data were presented as mean ± s.e.m. * P
    Figure Legend Snippet: Knockdown of Irx3 repressed the differentiation of SVFs toward beige adipocytes in vitro. (A–B) (A) Relative mRNA and (B) protein levels of Irx3 in SVFs from IWAT were efficiently knocked down after a 3-day infection of mouse Irx3 lentiviral shRNA ( n = 3 for each group). (C–D) (C) Oil Red O staining and (D) TG concentration of the mature beige adipocytes under eight-day differentiation ( n = 6 for each group). (E) Relative mRNA expression of Ucp1 , Cidea , Pgc-1α , C/ebpβ , and Prdm16 at different time points during beige adipocyte differentiation from IWAT SVFs ( n = 3–4 for different groups). (F–G) (F) Relative mRNA ( n = 4) and (G) protein expression of brown adipocyte marker genes of the induced beige adipocytes from mouse IWAT SVTs for eight days with the infection of mouse Irx3 lentiviral shRNA, with or without CL-316,243 activation. (H) Relative mRNA expression of adipocyte differentiation-related genes in beige adipocytes ( n = 4). (I) Relative mRNA expression of the indicated genes in the induced brown adipocytes from mouse BAT SVFs for eight days, with the infection of mouse Irx3 lentiviral shRNA ( n = 4). (J) Relative mRNA levels of IRX3 in SVFs from human sWAT after a 3-day infection of human IRX3 lentiviral shRNA (left), and the mRNA levels of brown adipocyte marker genes in the SVFs under 2-day differentiation ( n = 4). (K) OCR measurement of the beige adipocytes under 5-day differentiation, with the infection of mouse Irx3 lentiviral shRNA ( n = 5). (L–N) (L) Ucp1 promoter-luciferase reporter activity was measured in HEK293T cells transfected with pEGFP-C1 vector or IRX3 for 48 h. (M) Schematic diagram of the mutant mouse Ucp1 promoter deleting ACATGTGT among the − 3470 to − 3463 bp region proximal to the TSS of the Ucp1 gene. (N) Transcriptional activity of wild-type or mutant Ucp1 promoter was measured with IRX3 overexpression. (O) qPCR of Ucp1 promoter binding region which was recruited by Irx3 antibody in 8-day induced beige adipocytes, as analyzed by ChIP. Fold enrichment of Ucp1 promoter was given ( n = 3). For the measurement of luciferase activity, cells were seeded on 24-well plate and transfected with 800 ng pEGFP-C1 vector or IRX3, 200 ng Ucp1 promoter construct, and 1 ng pRL-SV40, followed by the harvest for luciferase activity assessment using a dual-luciferase reporter assay system (Promega). Luciferase activity was corrected for Renilla luciferase activity ( n = 3–4 for each group). For qPCR data, mRNA expression was normalized to 36b4 for mouse genes and βACTIN for human genes. SVF cells were isolated from IWAT and BAT of C57BL/6J mice or human sWAT wherever mentioned, and IRX3 shRNA were introduced to cells 24 h after seeding. Data were presented as mean ± s.e.m. * P

    Techniques Used: In Vitro, Infection, shRNA, Staining, Concentration Assay, Expressing, Pyrolysis Gas Chromatography, Marker, Activation Assay, Luciferase, Activity Assay, Transfection, Plasmid Preparation, Mutagenesis, Over Expression, Real-time Polymerase Chain Reaction, Binding Assay, Chromatin Immunoprecipitation, Construct, Reporter Assay, Isolation, Mouse Assay

    The expression of Irx3 was correlated with the beige/brown adipocyte associated genes in vitro. (A–G) In fully differentiated beige/brown adipocytes from preadipocytes isolated from IWAT, EWAT, and BAT of C57BL/6J and 129/Sv mice, Ucp1 mRNA expression was correlated with Pgc-1α (A) and Dio2 (B). Irx3 mRNA expression showed positive correlation with mRNA levels of (C) Ucp1 , (D) Pgc-1α , (E) Dio2 , and (F) Cox7a1 . Irx3 mRNA expression showed no correlation with Leptin (G). Cells from C57BL/6J were shown in red, and from 129/Sv were in blue. Cells from IWAT, EWAT, and BAT were shown as dot, triangle, and square, respectively. The correlation was analyzed using ΔCT. (H-J) (H) Irx3 (I) Ucp1 and (J) Pparγ mRNA expression during the time course of beige adipocyte differentiation cultures ( n = 3). Quantitative amounts of gene expression were normalized to the housekeeping gene 36b4 . (K–L) Irx3 and Ucp1 protein levels at different time points during beige adipocyte differentiation from IWAT SVFs (K), during brown adipocyte differentiation from BAT SVFs (L). (M) Protein expression of Irx3 and Ap2 at different time points during white adipocyte differentiation from IWAT SVFs. (N) Intracellular location of Ucp1 and Irx3 protein in the fully differentiated beige adipocytes. Ucp1 protein, Irx3 protein and nucleus were indicated in green, red, and blue, respectively. Scar bar, 20 μm. Data were presented as mean ± s.e.m. * P
    Figure Legend Snippet: The expression of Irx3 was correlated with the beige/brown adipocyte associated genes in vitro. (A–G) In fully differentiated beige/brown adipocytes from preadipocytes isolated from IWAT, EWAT, and BAT of C57BL/6J and 129/Sv mice, Ucp1 mRNA expression was correlated with Pgc-1α (A) and Dio2 (B). Irx3 mRNA expression showed positive correlation with mRNA levels of (C) Ucp1 , (D) Pgc-1α , (E) Dio2 , and (F) Cox7a1 . Irx3 mRNA expression showed no correlation with Leptin (G). Cells from C57BL/6J were shown in red, and from 129/Sv were in blue. Cells from IWAT, EWAT, and BAT were shown as dot, triangle, and square, respectively. The correlation was analyzed using ΔCT. (H-J) (H) Irx3 (I) Ucp1 and (J) Pparγ mRNA expression during the time course of beige adipocyte differentiation cultures ( n = 3). Quantitative amounts of gene expression were normalized to the housekeeping gene 36b4 . (K–L) Irx3 and Ucp1 protein levels at different time points during beige adipocyte differentiation from IWAT SVFs (K), during brown adipocyte differentiation from BAT SVFs (L). (M) Protein expression of Irx3 and Ap2 at different time points during white adipocyte differentiation from IWAT SVFs. (N) Intracellular location of Ucp1 and Irx3 protein in the fully differentiated beige adipocytes. Ucp1 protein, Irx3 protein and nucleus were indicated in green, red, and blue, respectively. Scar bar, 20 μm. Data were presented as mean ± s.e.m. * P

    Techniques Used: Expressing, In Vitro, Isolation, Mouse Assay, Pyrolysis Gas Chromatography

    The association of IRX3 and human obesity. (A) Relative mRNA levels of IRX3 in oWAT or sWAT from normal weight ( n = 9) and obese subjects ( n = 21) measured by qPCR. Gene expression was normalized to βACTIN . (B) Comparison of the frequency of the IRX3 rare variants in normal weight subjects and obese patients. (C) Sequence conservation analysis of the IRX3 orthologs related to variants identified in obese subjects and controls. Mutant sites are shown with a red box. Dark gray represents residues that are completely conserved, mild gray partially conserved, and light gray shows similar residues. (D) Protein expression levels of WT and mutant IRX3 in HEK293T cells. GFP antibody was used to indicate IRX3, and actin antibody served as a loading control. (E) Ucp1 promoter-luciferase reporter activity was measured in HEK293T cells transfected with pEGFP-C1 vector, WT or mutant IRX3 for 48 h ( n = 3). Normalized luciferase activities are shown as fold change and compared to WT IRX3 ( n = 4). oWAT, omental white adipose tissue. sWAT, subcutaneous white adipose tissue. Data were presented as mean ± s.e.m. * P
    Figure Legend Snippet: The association of IRX3 and human obesity. (A) Relative mRNA levels of IRX3 in oWAT or sWAT from normal weight ( n = 9) and obese subjects ( n = 21) measured by qPCR. Gene expression was normalized to βACTIN . (B) Comparison of the frequency of the IRX3 rare variants in normal weight subjects and obese patients. (C) Sequence conservation analysis of the IRX3 orthologs related to variants identified in obese subjects and controls. Mutant sites are shown with a red box. Dark gray represents residues that are completely conserved, mild gray partially conserved, and light gray shows similar residues. (D) Protein expression levels of WT and mutant IRX3 in HEK293T cells. GFP antibody was used to indicate IRX3, and actin antibody served as a loading control. (E) Ucp1 promoter-luciferase reporter activity was measured in HEK293T cells transfected with pEGFP-C1 vector, WT or mutant IRX3 for 48 h ( n = 3). Normalized luciferase activities are shown as fold change and compared to WT IRX3 ( n = 4). oWAT, omental white adipose tissue. sWAT, subcutaneous white adipose tissue. Data were presented as mean ± s.e.m. * P

    Techniques Used: Real-time Polymerase Chain Reaction, Expressing, Sequencing, Mutagenesis, Luciferase, Activity Assay, Transfection, Plasmid Preparation

    IRX3/Irx3 expression is induced in browning adipose tissues. (A) Relative mRNA levels of Ucp1 and Irx3 in IWAT from mice under cold stress (4 °C) for one week ( n = 6) and at 25 °C ( n = 8). (B–C) Relative mRNA levels of Ucp1 and Irx3 in IWAT (B) and BAT (C) from mice subjected to PBS or CL-316,243 for 10 days. For A–C, gene expression was normalized to 36b4 . (D–E) Protein levels of Ucp1 and Irx3 (left) and the quantification value of Irx3 relative to Hsp90 (right) in IWAT (D) and BAT (E) in the mice subjected to PBS or CL-316,243 treatment. (F) Representative images of immunohistochemical staining in IWAT from mice subjected to PBS or CL-316,243 treatment (scale bar, 50 μm for the upper and middle panels, and 100 μm for the bottom panel). (G) PET-CT, gross image, HE staining (scale bar, 20 μm), and electron microscopy (scale bar, 5 μm) of the browning WAT from pheochromocytoma patients. (H) Representative images of immunohistochemical staining in oWAT from pheochromocytoma patients (scale bar, 100 μm for the upper panel, and 50 μm for the bottom panel). Data were presented as mean ± s.e.m. * P
    Figure Legend Snippet: IRX3/Irx3 expression is induced in browning adipose tissues. (A) Relative mRNA levels of Ucp1 and Irx3 in IWAT from mice under cold stress (4 °C) for one week ( n = 6) and at 25 °C ( n = 8). (B–C) Relative mRNA levels of Ucp1 and Irx3 in IWAT (B) and BAT (C) from mice subjected to PBS or CL-316,243 for 10 days. For A–C, gene expression was normalized to 36b4 . (D–E) Protein levels of Ucp1 and Irx3 (left) and the quantification value of Irx3 relative to Hsp90 (right) in IWAT (D) and BAT (E) in the mice subjected to PBS or CL-316,243 treatment. (F) Representative images of immunohistochemical staining in IWAT from mice subjected to PBS or CL-316,243 treatment (scale bar, 50 μm for the upper and middle panels, and 100 μm for the bottom panel). (G) PET-CT, gross image, HE staining (scale bar, 20 μm), and electron microscopy (scale bar, 5 μm) of the browning WAT from pheochromocytoma patients. (H) Representative images of immunohistochemical staining in oWAT from pheochromocytoma patients (scale bar, 100 μm for the upper panel, and 50 μm for the bottom panel). Data were presented as mean ± s.e.m. * P

    Techniques Used: Expressing, Mouse Assay, Immunohistochemistry, Staining, Positron Emission Tomography, Electron Microscopy

    Related Articles

    Chromatin Immunoprecipitation:

    Article Title: IRX3 Promotes the Browning of White Adipocytes and Its Rare Variants are Associated with Human Obesity Risk
    Article Snippet: .. 2.14 Chromatin Immunoprecipitation Assay Chromatin immunoprecipitation (ChIP) assay was carried out to investigate the interaction between IRX3 and the Ucp1 promoter using a commercial kit (Millipore, 17–371) in accordance with the manufacturer's instructions. .. Nuclei were extracted from the mature beige adipocytes induced from preadipocytes within IWAT of C57BL/6J mice.

    Article Title: IRX3 Promotes the Browning of White Adipocytes and Its Rare Variants are Associated with Human Obesity Risk
    Article Snippet: .. Chromatin immunoprecipitation (ChIP) assay was carried out to investigate the interaction between IRX3 and the Ucp1 promoter using a commercial kit (Millipore, 17–371) in accordance with the manufacturer's instructions. .. Nuclei were extracted from the mature beige adipocytes induced from preadipocytes within IWAT of C57BL/6J mice.

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    Millipore ucp1 promoter
    Knockdown of Irx3 repressed the differentiation of SVFs toward beige adipocytes in vitro. (A–B) (A) Relative mRNA and (B) protein levels of Irx3 in SVFs from IWAT were efficiently knocked down after a 3-day infection of mouse Irx3 lentiviral shRNA ( n = 3 for each group). (C–D) (C) Oil Red O staining and (D) TG concentration of the mature beige adipocytes under eight-day differentiation ( n = 6 for each group). (E) Relative mRNA expression of <t>Ucp1</t> , Cidea , Pgc-1α , C/ebpβ , and Prdm16 at different time points during beige adipocyte differentiation from IWAT SVFs ( n = 3–4 for different groups). (F–G) (F) Relative mRNA ( n = 4) and (G) protein expression of brown adipocyte marker genes of the induced beige adipocytes from mouse IWAT SVTs for eight days with the infection of mouse Irx3 lentiviral shRNA, with or without CL-316,243 activation. (H) Relative mRNA expression of adipocyte differentiation-related genes in beige adipocytes ( n = 4). (I) Relative mRNA expression of the indicated genes in the induced brown adipocytes from mouse BAT SVFs for eight days, with the infection of mouse Irx3 lentiviral shRNA ( n = 4). (J) Relative mRNA levels of IRX3 in SVFs from human sWAT after a 3-day infection of human IRX3 lentiviral shRNA (left), and the mRNA levels of brown adipocyte marker genes in the SVFs under 2-day differentiation ( n = 4). (K) OCR measurement of the beige adipocytes under 5-day differentiation, with the infection of mouse Irx3 lentiviral shRNA ( n = 5). (L–N) (L) Ucp1 promoter-luciferase reporter activity was measured in HEK293T cells transfected with pEGFP-C1 vector or IRX3 for 48 h. (M) Schematic diagram of the mutant mouse Ucp1 promoter deleting ACATGTGT among the − 3470 to − 3463 bp region proximal to the TSS of the Ucp1 gene. (N) Transcriptional activity of wild-type or mutant Ucp1 promoter was measured with IRX3 overexpression. (O) qPCR of Ucp1 promoter binding region which was recruited by Irx3 antibody in 8-day induced beige adipocytes, as analyzed by ChIP. Fold enrichment of Ucp1 promoter was given ( n = 3). For the measurement of luciferase activity, cells were seeded on 24-well plate and transfected with 800 ng pEGFP-C1 vector or IRX3, 200 ng Ucp1 promoter construct, and 1 ng pRL-SV40, followed by the harvest for luciferase activity assessment using a dual-luciferase reporter assay system (Promega). Luciferase activity was corrected for Renilla luciferase activity ( n = 3–4 for each group). For qPCR data, mRNA expression was normalized to 36b4 for mouse genes and βACTIN for human genes. SVF cells were isolated from IWAT and BAT of C57BL/6J mice or human sWAT wherever mentioned, and IRX3 shRNA were introduced to cells 24 h after seeding. Data were presented as mean ± s.e.m. * P
    Ucp1 Promoter, supplied by Millipore, used in various techniques. Bioz Stars score: 91/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ucp1 promoter/product/Millipore
    Average 91 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    ucp1 promoter - by Bioz Stars, 2020-08
    91/100 stars
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    Knockdown of Irx3 repressed the differentiation of SVFs toward beige adipocytes in vitro. (A–B) (A) Relative mRNA and (B) protein levels of Irx3 in SVFs from IWAT were efficiently knocked down after a 3-day infection of mouse Irx3 lentiviral shRNA ( n = 3 for each group). (C–D) (C) Oil Red O staining and (D) TG concentration of the mature beige adipocytes under eight-day differentiation ( n = 6 for each group). (E) Relative mRNA expression of Ucp1 , Cidea , Pgc-1α , C/ebpβ , and Prdm16 at different time points during beige adipocyte differentiation from IWAT SVFs ( n = 3–4 for different groups). (F–G) (F) Relative mRNA ( n = 4) and (G) protein expression of brown adipocyte marker genes of the induced beige adipocytes from mouse IWAT SVTs for eight days with the infection of mouse Irx3 lentiviral shRNA, with or without CL-316,243 activation. (H) Relative mRNA expression of adipocyte differentiation-related genes in beige adipocytes ( n = 4). (I) Relative mRNA expression of the indicated genes in the induced brown adipocytes from mouse BAT SVFs for eight days, with the infection of mouse Irx3 lentiviral shRNA ( n = 4). (J) Relative mRNA levels of IRX3 in SVFs from human sWAT after a 3-day infection of human IRX3 lentiviral shRNA (left), and the mRNA levels of brown adipocyte marker genes in the SVFs under 2-day differentiation ( n = 4). (K) OCR measurement of the beige adipocytes under 5-day differentiation, with the infection of mouse Irx3 lentiviral shRNA ( n = 5). (L–N) (L) Ucp1 promoter-luciferase reporter activity was measured in HEK293T cells transfected with pEGFP-C1 vector or IRX3 for 48 h. (M) Schematic diagram of the mutant mouse Ucp1 promoter deleting ACATGTGT among the − 3470 to − 3463 bp region proximal to the TSS of the Ucp1 gene. (N) Transcriptional activity of wild-type or mutant Ucp1 promoter was measured with IRX3 overexpression. (O) qPCR of Ucp1 promoter binding region which was recruited by Irx3 antibody in 8-day induced beige adipocytes, as analyzed by ChIP. Fold enrichment of Ucp1 promoter was given ( n = 3). For the measurement of luciferase activity, cells were seeded on 24-well plate and transfected with 800 ng pEGFP-C1 vector or IRX3, 200 ng Ucp1 promoter construct, and 1 ng pRL-SV40, followed by the harvest for luciferase activity assessment using a dual-luciferase reporter assay system (Promega). Luciferase activity was corrected for Renilla luciferase activity ( n = 3–4 for each group). For qPCR data, mRNA expression was normalized to 36b4 for mouse genes and βACTIN for human genes. SVF cells were isolated from IWAT and BAT of C57BL/6J mice or human sWAT wherever mentioned, and IRX3 shRNA were introduced to cells 24 h after seeding. Data were presented as mean ± s.e.m. * P

    Journal: EBioMedicine

    Article Title: IRX3 Promotes the Browning of White Adipocytes and Its Rare Variants are Associated with Human Obesity Risk

    doi: 10.1016/j.ebiom.2017.09.010

    Figure Lengend Snippet: Knockdown of Irx3 repressed the differentiation of SVFs toward beige adipocytes in vitro. (A–B) (A) Relative mRNA and (B) protein levels of Irx3 in SVFs from IWAT were efficiently knocked down after a 3-day infection of mouse Irx3 lentiviral shRNA ( n = 3 for each group). (C–D) (C) Oil Red O staining and (D) TG concentration of the mature beige adipocytes under eight-day differentiation ( n = 6 for each group). (E) Relative mRNA expression of Ucp1 , Cidea , Pgc-1α , C/ebpβ , and Prdm16 at different time points during beige adipocyte differentiation from IWAT SVFs ( n = 3–4 for different groups). (F–G) (F) Relative mRNA ( n = 4) and (G) protein expression of brown adipocyte marker genes of the induced beige adipocytes from mouse IWAT SVTs for eight days with the infection of mouse Irx3 lentiviral shRNA, with or without CL-316,243 activation. (H) Relative mRNA expression of adipocyte differentiation-related genes in beige adipocytes ( n = 4). (I) Relative mRNA expression of the indicated genes in the induced brown adipocytes from mouse BAT SVFs for eight days, with the infection of mouse Irx3 lentiviral shRNA ( n = 4). (J) Relative mRNA levels of IRX3 in SVFs from human sWAT after a 3-day infection of human IRX3 lentiviral shRNA (left), and the mRNA levels of brown adipocyte marker genes in the SVFs under 2-day differentiation ( n = 4). (K) OCR measurement of the beige adipocytes under 5-day differentiation, with the infection of mouse Irx3 lentiviral shRNA ( n = 5). (L–N) (L) Ucp1 promoter-luciferase reporter activity was measured in HEK293T cells transfected with pEGFP-C1 vector or IRX3 for 48 h. (M) Schematic diagram of the mutant mouse Ucp1 promoter deleting ACATGTGT among the − 3470 to − 3463 bp region proximal to the TSS of the Ucp1 gene. (N) Transcriptional activity of wild-type or mutant Ucp1 promoter was measured with IRX3 overexpression. (O) qPCR of Ucp1 promoter binding region which was recruited by Irx3 antibody in 8-day induced beige adipocytes, as analyzed by ChIP. Fold enrichment of Ucp1 promoter was given ( n = 3). For the measurement of luciferase activity, cells were seeded on 24-well plate and transfected with 800 ng pEGFP-C1 vector or IRX3, 200 ng Ucp1 promoter construct, and 1 ng pRL-SV40, followed by the harvest for luciferase activity assessment using a dual-luciferase reporter assay system (Promega). Luciferase activity was corrected for Renilla luciferase activity ( n = 3–4 for each group). For qPCR data, mRNA expression was normalized to 36b4 for mouse genes and βACTIN for human genes. SVF cells were isolated from IWAT and BAT of C57BL/6J mice or human sWAT wherever mentioned, and IRX3 shRNA were introduced to cells 24 h after seeding. Data were presented as mean ± s.e.m. * P

    Article Snippet: 2.14 Chromatin Immunoprecipitation Assay Chromatin immunoprecipitation (ChIP) assay was carried out to investigate the interaction between IRX3 and the Ucp1 promoter using a commercial kit (Millipore, 17–371) in accordance with the manufacturer's instructions.

    Techniques: In Vitro, Infection, shRNA, Staining, Concentration Assay, Expressing, Pyrolysis Gas Chromatography, Marker, Activation Assay, Luciferase, Activity Assay, Transfection, Plasmid Preparation, Mutagenesis, Over Expression, Real-time Polymerase Chain Reaction, Binding Assay, Chromatin Immunoprecipitation, Construct, Reporter Assay, Isolation, Mouse Assay

    The expression of Irx3 was correlated with the beige/brown adipocyte associated genes in vitro. (A–G) In fully differentiated beige/brown adipocytes from preadipocytes isolated from IWAT, EWAT, and BAT of C57BL/6J and 129/Sv mice, Ucp1 mRNA expression was correlated with Pgc-1α (A) and Dio2 (B). Irx3 mRNA expression showed positive correlation with mRNA levels of (C) Ucp1 , (D) Pgc-1α , (E) Dio2 , and (F) Cox7a1 . Irx3 mRNA expression showed no correlation with Leptin (G). Cells from C57BL/6J were shown in red, and from 129/Sv were in blue. Cells from IWAT, EWAT, and BAT were shown as dot, triangle, and square, respectively. The correlation was analyzed using ΔCT. (H-J) (H) Irx3 (I) Ucp1 and (J) Pparγ mRNA expression during the time course of beige adipocyte differentiation cultures ( n = 3). Quantitative amounts of gene expression were normalized to the housekeeping gene 36b4 . (K–L) Irx3 and Ucp1 protein levels at different time points during beige adipocyte differentiation from IWAT SVFs (K), during brown adipocyte differentiation from BAT SVFs (L). (M) Protein expression of Irx3 and Ap2 at different time points during white adipocyte differentiation from IWAT SVFs. (N) Intracellular location of Ucp1 and Irx3 protein in the fully differentiated beige adipocytes. Ucp1 protein, Irx3 protein and nucleus were indicated in green, red, and blue, respectively. Scar bar, 20 μm. Data were presented as mean ± s.e.m. * P

    Journal: EBioMedicine

    Article Title: IRX3 Promotes the Browning of White Adipocytes and Its Rare Variants are Associated with Human Obesity Risk

    doi: 10.1016/j.ebiom.2017.09.010

    Figure Lengend Snippet: The expression of Irx3 was correlated with the beige/brown adipocyte associated genes in vitro. (A–G) In fully differentiated beige/brown adipocytes from preadipocytes isolated from IWAT, EWAT, and BAT of C57BL/6J and 129/Sv mice, Ucp1 mRNA expression was correlated with Pgc-1α (A) and Dio2 (B). Irx3 mRNA expression showed positive correlation with mRNA levels of (C) Ucp1 , (D) Pgc-1α , (E) Dio2 , and (F) Cox7a1 . Irx3 mRNA expression showed no correlation with Leptin (G). Cells from C57BL/6J were shown in red, and from 129/Sv were in blue. Cells from IWAT, EWAT, and BAT were shown as dot, triangle, and square, respectively. The correlation was analyzed using ΔCT. (H-J) (H) Irx3 (I) Ucp1 and (J) Pparγ mRNA expression during the time course of beige adipocyte differentiation cultures ( n = 3). Quantitative amounts of gene expression were normalized to the housekeeping gene 36b4 . (K–L) Irx3 and Ucp1 protein levels at different time points during beige adipocyte differentiation from IWAT SVFs (K), during brown adipocyte differentiation from BAT SVFs (L). (M) Protein expression of Irx3 and Ap2 at different time points during white adipocyte differentiation from IWAT SVFs. (N) Intracellular location of Ucp1 and Irx3 protein in the fully differentiated beige adipocytes. Ucp1 protein, Irx3 protein and nucleus were indicated in green, red, and blue, respectively. Scar bar, 20 μm. Data were presented as mean ± s.e.m. * P

    Article Snippet: 2.14 Chromatin Immunoprecipitation Assay Chromatin immunoprecipitation (ChIP) assay was carried out to investigate the interaction between IRX3 and the Ucp1 promoter using a commercial kit (Millipore, 17–371) in accordance with the manufacturer's instructions.

    Techniques: Expressing, In Vitro, Isolation, Mouse Assay, Pyrolysis Gas Chromatography

    The association of IRX3 and human obesity. (A) Relative mRNA levels of IRX3 in oWAT or sWAT from normal weight ( n = 9) and obese subjects ( n = 21) measured by qPCR. Gene expression was normalized to βACTIN . (B) Comparison of the frequency of the IRX3 rare variants in normal weight subjects and obese patients. (C) Sequence conservation analysis of the IRX3 orthologs related to variants identified in obese subjects and controls. Mutant sites are shown with a red box. Dark gray represents residues that are completely conserved, mild gray partially conserved, and light gray shows similar residues. (D) Protein expression levels of WT and mutant IRX3 in HEK293T cells. GFP antibody was used to indicate IRX3, and actin antibody served as a loading control. (E) Ucp1 promoter-luciferase reporter activity was measured in HEK293T cells transfected with pEGFP-C1 vector, WT or mutant IRX3 for 48 h ( n = 3). Normalized luciferase activities are shown as fold change and compared to WT IRX3 ( n = 4). oWAT, omental white adipose tissue. sWAT, subcutaneous white adipose tissue. Data were presented as mean ± s.e.m. * P

    Journal: EBioMedicine

    Article Title: IRX3 Promotes the Browning of White Adipocytes and Its Rare Variants are Associated with Human Obesity Risk

    doi: 10.1016/j.ebiom.2017.09.010

    Figure Lengend Snippet: The association of IRX3 and human obesity. (A) Relative mRNA levels of IRX3 in oWAT or sWAT from normal weight ( n = 9) and obese subjects ( n = 21) measured by qPCR. Gene expression was normalized to βACTIN . (B) Comparison of the frequency of the IRX3 rare variants in normal weight subjects and obese patients. (C) Sequence conservation analysis of the IRX3 orthologs related to variants identified in obese subjects and controls. Mutant sites are shown with a red box. Dark gray represents residues that are completely conserved, mild gray partially conserved, and light gray shows similar residues. (D) Protein expression levels of WT and mutant IRX3 in HEK293T cells. GFP antibody was used to indicate IRX3, and actin antibody served as a loading control. (E) Ucp1 promoter-luciferase reporter activity was measured in HEK293T cells transfected with pEGFP-C1 vector, WT or mutant IRX3 for 48 h ( n = 3). Normalized luciferase activities are shown as fold change and compared to WT IRX3 ( n = 4). oWAT, omental white adipose tissue. sWAT, subcutaneous white adipose tissue. Data were presented as mean ± s.e.m. * P

    Article Snippet: 2.14 Chromatin Immunoprecipitation Assay Chromatin immunoprecipitation (ChIP) assay was carried out to investigate the interaction between IRX3 and the Ucp1 promoter using a commercial kit (Millipore, 17–371) in accordance with the manufacturer's instructions.

    Techniques: Real-time Polymerase Chain Reaction, Expressing, Sequencing, Mutagenesis, Luciferase, Activity Assay, Transfection, Plasmid Preparation

    IRX3/Irx3 expression is induced in browning adipose tissues. (A) Relative mRNA levels of Ucp1 and Irx3 in IWAT from mice under cold stress (4 °C) for one week ( n = 6) and at 25 °C ( n = 8). (B–C) Relative mRNA levels of Ucp1 and Irx3 in IWAT (B) and BAT (C) from mice subjected to PBS or CL-316,243 for 10 days. For A–C, gene expression was normalized to 36b4 . (D–E) Protein levels of Ucp1 and Irx3 (left) and the quantification value of Irx3 relative to Hsp90 (right) in IWAT (D) and BAT (E) in the mice subjected to PBS or CL-316,243 treatment. (F) Representative images of immunohistochemical staining in IWAT from mice subjected to PBS or CL-316,243 treatment (scale bar, 50 μm for the upper and middle panels, and 100 μm for the bottom panel). (G) PET-CT, gross image, HE staining (scale bar, 20 μm), and electron microscopy (scale bar, 5 μm) of the browning WAT from pheochromocytoma patients. (H) Representative images of immunohistochemical staining in oWAT from pheochromocytoma patients (scale bar, 100 μm for the upper panel, and 50 μm for the bottom panel). Data were presented as mean ± s.e.m. * P

    Journal: EBioMedicine

    Article Title: IRX3 Promotes the Browning of White Adipocytes and Its Rare Variants are Associated with Human Obesity Risk

    doi: 10.1016/j.ebiom.2017.09.010

    Figure Lengend Snippet: IRX3/Irx3 expression is induced in browning adipose tissues. (A) Relative mRNA levels of Ucp1 and Irx3 in IWAT from mice under cold stress (4 °C) for one week ( n = 6) and at 25 °C ( n = 8). (B–C) Relative mRNA levels of Ucp1 and Irx3 in IWAT (B) and BAT (C) from mice subjected to PBS or CL-316,243 for 10 days. For A–C, gene expression was normalized to 36b4 . (D–E) Protein levels of Ucp1 and Irx3 (left) and the quantification value of Irx3 relative to Hsp90 (right) in IWAT (D) and BAT (E) in the mice subjected to PBS or CL-316,243 treatment. (F) Representative images of immunohistochemical staining in IWAT from mice subjected to PBS or CL-316,243 treatment (scale bar, 50 μm for the upper and middle panels, and 100 μm for the bottom panel). (G) PET-CT, gross image, HE staining (scale bar, 20 μm), and electron microscopy (scale bar, 5 μm) of the browning WAT from pheochromocytoma patients. (H) Representative images of immunohistochemical staining in oWAT from pheochromocytoma patients (scale bar, 100 μm for the upper panel, and 50 μm for the bottom panel). Data were presented as mean ± s.e.m. * P

    Article Snippet: 2.14 Chromatin Immunoprecipitation Assay Chromatin immunoprecipitation (ChIP) assay was carried out to investigate the interaction between IRX3 and the Ucp1 promoter using a commercial kit (Millipore, 17–371) in accordance with the manufacturer's instructions.

    Techniques: Expressing, Mouse Assay, Immunohistochemistry, Staining, Positron Emission Tomography, Electron Microscopy