H αSMA mRNA expression in TGFβ - treated HFL1 overexpressing two RNase dead mutants ( K599A K907A ) of IRE1α . * P K599A = 0 . 013 , * P K907A = 0 . 0008 compared to WT .

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H αSMA mRNA expression in TGFβ-treated HFL1 cells overexpressing two RNase dead mutants (K599A and K907A) of IRE1α. *P K599A = 0.013, *P K907A = 0.0008 compared to WT.


I αSMA mRNA expression in TGFβ - treated IRE1α - / - or WT MEF . * P = 0 . 025 .

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I αSMA mRNA expression in TGFβ-treated IRE1α -/- or WT MEF cells. *P = 0.025.


A miR - 150 in TGFβ - treated HFL1 fibroblasts effect of IRE1α inhibitor 4μ8C . * P = 0 . 046 compared to control .

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A miR-150 levels in TGFβ-treated HFL1 fibroblasts and the effect of the IRE1α inhibitor 4μ8C. * P = 0.046 compared to control.


B miR - 150 in HFL1 fibroblasts overexpressing two RNase dead mutants ( K599A K907A ) of IRE1α . * P = 0 . 0043 ( K599A ) , P = 0 . 043 ( K907A ) , compared to control .

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B miR-150 levels in HFL1 fibroblasts overexpressing two RNase dead mutants (K599A and K907A) of IRE1α. * P = 0.0043 (K599A), P = 0.043 (K907A), compared to control.


C miR - 150 in TGFβ - treated WT ( IRE1α + / + ) IRE1α - / - MEF . * P = 0 . 018 . Results are expressed as fold change compared to Ctrl .

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C miR-150 levels in TGFβ-treated WT (IRE1α +/+) and IRE1α -/- MEF cells. *P = 0.018. Results are expressed as fold change compared to the Ctrl.


D miR - 150 in IRE1α - / - MEF that were either untransfected ( UT ) or transfected with miRNA inhibitor control ( siCtrl ) or with a miR - 150 - 5p miRNA inhibitor . * P = 0 . 034 compared to UT .

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D miR-150 levels in IRE1α -/- MEF cells that were either untransfected (UT) or transfected with miRNA inhibitor control (siCtrl) or with a miR-150-5p miRNA inhibitor. * P = 0.034 compared to UT.


E αSMA mRNA expression in TGFβ - treated IRE1α - / - MEF that were either untransfected ( UT ) or transfected with miRNA inhibitor control ( siCtrl ) or with a miR150 - 5p miRNA inhibitor . * P = 0 . 043 compared to UT .

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E αSMA mRNA expression in TGFβ-treated IRE1α -/- MEF cells that were either untransfected (UT) or transfected with miRNA inhibitor control (siCtrl) or with a miR150-5p miRNA inhibitor. *P = 0.043 compared to UT.


F miR - 150 in HFL1 fibroblasts transfected with a control plasmid ( Ctrl ) or a miR - 150 expression plasmid ( miR150 ) . * P = 0 . 038 .

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F miR-150 levels in HFL1 fibroblasts transfected with a control plasmid (Ctrl) or a miR-150 expression plasmid (miR150). * P = 0.038.


G Western blot of c - Myb in TGFβ - treated HFL1 fibroblasts that were either untransfected ( UT ) , transfected with a control plasmid ( pCtrl ) or a miR150 expression plasmid ( miR150 ) . * P = 0 . 0206 in UT Ctrl vs . UT TGFβ P = 0 . 0126 in pCtrl Ctrl vs . pCtrl TGFβ .

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G Western blot of c-Myb in TGFβ-treated HFL1 fibroblasts that were either untransfected (UT), transfected with a control plasmid (pCtrl) or a miR150 expression plasmid (miR150). *P = 0.0206 in UT Ctrl vs. UT TGFβ and P = 0.0126 in pCtrl Ctrl vs. pCtrl TGFβ .


H Western blot of αSMA in same experimental setup as in G . * P = 0 . 0405 in UT Ctrl vs . UT TGFβ , P = 0 . 0284 in pCtrl Ctrl vs . pCtrl TGFβ .

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H Western blot of αSMA in the same experimental setup as in G. * P = 0.0405 in UT Ctrl vs. UT TGFβ, and P = 0.0284 in pCtrl Ctrl vs. pCtrl TGFβ.


I c - Myb mRNA in untransfected HFL1 ( UT ) in transfected with a control siRNA ( siCtrl ) or an siRNA targeting c - Myb ( sicMyb ) . * P = 0 . 00001 compared to UT .

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I c-Myb mRNA levels in untransfected HFL1 cells (UT) and in cells transfected with a control siRNA (siCtrl) or an siRNA targeting c-Myb (sicMyb). * P = 0.00001 compared to UT.


J αSMA mRNA expression in untransfected HFL1 ( UT ) in transfected with a control siRNA ( siCtrl ) or with a siRNA targeting c - Myb ( sicMyb ) . * P = 0 . 0042 in UT Ctrl vs . UT TGFβ .

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J αSMA mRNA expression in untransfected HFL1 cells (UT) and in cells transfected with a control siRNA (siCtrl) or with a siRNA targeting c-Myb (sicMyb). * P = 0.0042 in UT Ctrl vs. UT TGFβ.


L In vitro cleavage with recombinant IRE1α miR - 150 ( left ) or XBP - 1 ( right ) .

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L In vitro cleavage assay with recombinant IRE1α and miR-150 (left) or XBP-1 (right).


M miR - 150 expression in HFL - 1 that were pre - treated with TGFβ in absence of presence of 4μ8C then treated with Actinomycin D ( ActD ) for 4 24 hours . * P = 0 . 027 compared to TGFβ .

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M miR-150 expression in HFL-1 cells that were pre-treated with TGFβ in the absence of presence of 4μ8C and then treated with Actinomycin D (ActD) for 4 and 24 hours. * P = 0.027 compared to TGFβ.


A of ER in HFL1 fibroblasts treated with TGFβ in absence ( Ctrl ) or presence of IRE1α inhibitor 4μ8C , or in HFL1 transfected with a siRNA control siRNA ( siCtrl ) or with siRNA targeting XBP - 1 ( siXBP - 1 ) . ER was visualized with ER tracker Red nuclei stained with Hoechst ( left , scale bars = 20 μm ) or by electron microscopy ( right , scale bars = 200 nm ) where red lines mark ER . B of ER relative to nucleus from experimental setup in A . * P = 0 . 0001 in Ctrl , * P = 0 . 0223 in siCtrl .

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A Analysis of ER size in HFL1 fibroblasts treated with TGFβ in the absence (Ctrl) or presence of the IRE1α inhibitor 4μ8C, or in HFL1 cells transfected with a siRNA control siRNA (siCtrl) or with siRNA targeting XBP-1 (siXBP-1). The ER was visualized with ER tracker Red and nuclei stained with Hoechst (left, scale bars = 20 μm) or by electron microscopy (right, scale bars = 200 nm) where red lines mark the ER.B Quantification of ER size relative to the nucleus from the experimental setup in A. * P = 0.0001 in Ctrl, and * P = 0.0223 in siCtrl.


C XBP - 1 mRNA expression in untransfected HFL1 ( UT ) in transfected with control siRNA ( siCtrl ) or siRNA targeting XBP - 1 ( siXBP - 1 ) . * P = 0 , 0001 .

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C XBP-1 mRNA expression in untransfected HFL1 cells (UT) and in cells transfected with control siRNA (siCtrl) or siRNA targeting XBP-1 (siXBP-1). * P = 0,0001.


D Collagen in TGFβ treated HFL1 fibroblasts transfected as described in C . * P = 0 , 005 .

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D Collagen protein levels in TGFβ treated HFL1 fibroblasts transfected as described in C. * P = 0,005.


A effect of IRE1α inhibitor 4μ8C on CCl4 induced liver fibrosis in C57BL / 6mice as measured by histological metavir score . Ctrl ( n = 8 ) ; Ctrl + 4μ8C ( n = 6 ) ; CCl4 ( n = 6 ) ; CCl4 + 4μ8C ( n = 8 ) . * P = 0 , 0077 . B Representative liver tissue sections stained with sirius red for collagen ( top ) αSMA ( bottom ) . Scale bars = 50 μm . C of αSMA positive in pericentral areas in livers from mice in experimental setup in A . * P = 0 , 036 .

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A The effect of the IRE1α inhibitor 4μ8C on CCl4 induced liver fibrosis in C57BL/6mice as measured by the histological metavir score. Ctrl (n = 8) ; Ctrl + 4μ8C (n = 6) ; CCl4 (n = 6); CCl4 + 4μ8C (n = 8). * P = 0,0077.B Representative liver tissue sections stained with sirius red for collagen (top) and αSMA (bottom). Scale bars= 50 μm.C Percentage of αSMA positive cells in pericentral areas in livers from mice in the experimental setup in A. * P = 0,036.


D Liver to from experimental setup in A . * P = 0 , 0031 .

D Liver to body weight ratio from the experimental setup in A. * P = 0,0031.


E BiP mRNA expression in liver tissue from mice presented in A . * P = 0 , 020 .

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E BiP mRNA expression in liver tissue from mice presented in A. * P = 0,020.


F Representative gel showing of spliced unspliced XBP - 1 in liver tissue from control CCl4 - induced cirrhotic mice effect of 4μ8C treatment .

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F Representative gel showing levels of spliced and unspliced XBP-1 in liver tissue from control and CCl4-induced cirrhotic mice and the effect of 4μ8C treatment.


G miR150 expression in liver tissue from mice in A . * P = 0 , 032 compared to untreated controls .

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G miR150 expression in liver tissue from mice in A. * P = 0,032 compared to untreated controls.


H αSMA mRNA in stellate isolated from mouse liver then treated or not with TGFβ ± 4μ8C . * P = 0 , 031 , n = 3 .

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H αSMA mRNA levels in primary stellate cells isolated from mouse liver and then treated or not with TGFβ ± 4μ8C. * P = 0,031, n = 3.


I BiP mRNA in stellate isolated from mouse liver then treated with or without TGFβ ± 4μ8C . * P = 0 , 008 , n = 3 .

I BiP mRNA levels in primary stellate cells isolated from mouse liver and then treated with or without TGFβ ± 4μ8C. * P = 0,008, n = 3.


J Microscopic of mouse hepatic stellate stained for BiP ( top ) or αSMA ( bottom ) . Scale bars = 20 μm .

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J Microscopic images of primary mouse hepatic stellate cells stained for BiP (top) or αSMA (bottom). Scale bars = 20 μm.


K Western blot showing BiP expression in mouse hepatic stellate .

K Western blot showing BiP protein expression in primary mouse hepatic stellate cells.


L of mRNA expression of CTGF collagen in human hepatic stellate stimulated with TGFβ ± 4μ8C .

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L Quantification of mRNA expression levels of CTGF and collagen in primary human hepatic stellate cells stimulated with TGFβ ±4μ8C.


A effect of IRE1α inhibitor 4μ8C on TGFβ - induced skin fibrosis in C57BL / 6 mice as measured by of sirius red areas in hypodermis . Ctrl ( n = 6 ) ; Ctrl + 4μ8C ( n = 5 ) ; TGFβ ( n = 8 ) ; TGFβ + 4μ8C ( n = 8 ) . * P = 0 , 046 . B Representative skin tissue sections stained with sirius red for collagen ( top ) αSMA ( bottom ) . Scale bars = 50 μm . C αSMA - positive in hypodermis of mice from experimental setup in A . * P = 0 , 024 .

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A The effect of the IRE1α inhibitor 4μ8C on TGFβ-induced skin fibrosis in C57BL/6 mice as measured by the percentage of sirius red areas in the hypodermis. Ctrl (n = 6); Ctrl + 4μ8C (n = 5); TGFβ (n = 8); TGFβ + 4μ8C (n = 8). * P = 0,046.B Representative skin tissue sections stained with sirius red for collagen (top) and αSMA (bottom). Scale bars = 50 μm.C Percentage αSMA-positive cells in the hypodermis of mice from the experimental setup in A. * P = 0,024.


D Representative image of selected for capture microdissection ( scale bars = 100 μm ) . E Expression of αSMA mRNA in samples of hypodermis obtained by capture microdissection . Ctrl ( n = 5 ) , Ctrl + 4μ8C ( n = 5 ) , TGFβ ( n = 5 ) , TGFβ + 4μ8C ( n = 5 ) . * P = 0 , 044 . F miR - 150 expression in same samples as shown in E .

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D Representative image of the area selected for laser capture microdissection (scale bars = 100 μm).E Expression levels of αSMA mRNA in samples of hypodermis obtained by laser capture microdissection. Ctrl (n = 5), Ctrl + 4μ8C (n = 5), TGFβ (n = 5), TGFβ + 4μ8C (n = 5). * P = 0,044.F miR-150 expression levels in the same samples as shown in E.


G Immunohistochemistry of αSMA in skinfibroblasts isolated from healthy mice after treatment with TGFβ ± 4μ8C . Scale bars = 20 μm .

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G Immunohistochemistry staining of αSMA in primary skinfibroblasts isolated from healthy mice after treatment with TGFβ ± 4μ8C. Scale bars = 20 μm.


H αSMA mRNA expression in mouse skin fibroblasts after treatment with TGFβ ± 4μ8C . * P = 0 , 049 , n = 4 .

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H αSMA mRNA expression in primary mouse skin fibroblasts after treatment with TGFβ ± 4μ8C. * P = 0,049, n = 4.


I Agarose gel showing of spliced unspliced XBP - 1 in mouse skin fibroblasts treated as indicated with TGFβ 4μ8C .

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I Agarose gel showing levels of spliced and unspliced XBP-1 in primary mouse skin fibroblasts treated as indicated with TGFβ and 4μ8C.


J BiP mRNA expression in mouse skin fibroblasts after treatment with TGFβ in absence presence of 4μ8C . * P = 0 , 00007 for TGFβ vs . Ctrl P = 0 , 00002 for TGFβ vs . TGFβ + 4μ8C . n = 4 .

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J BiP mRNA expression in primary mouse skin fibroblasts after treatment with TGFβ in the absence and presence of 4μ8C. * P = 0,00007 for TGFβ vs. Ctrl and P = 0,00002 for TGFβ vs. TGFβ + 4μ8C. n=4.


K Western blot showing CHOP expression in mouse skin fibroblasts treated as indicated with TGFβ 4μ8C .

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K Western blot showing CHOP protein expression in primary mouse skin fibroblasts treated as indicated with TGFβ and 4μ8C.


L miR - 150 mRNA in mouse skin fibroblasts treated as indicated with TGFβ 4μ8C . * P = 0 , 00031 compared to untreated control , n = 3 .

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L miR-150 mRNA levels in primary mouse skin fibroblasts treated as indicated with TGFβ and 4μ8C. * P = 0,00031 compared to untreated control, n = 3.


A CTGF mRNA expression in skin ( dSSc ) lung ( LSSc ) fibroblasts isolated from scleroderma treated with IRE1α inhibitor 4μ8C . * P = 0 , 018 for dSSc ( n = 6 ) P = 0 , 003 for LSSc ( n = 3 ) .

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A CTGF mRNA expression levels in skin (dSSc) and lung (LSSc) fibroblasts isolated from scleroderma patients and treated with the IRE1α inhibitor 4μ8C. * P = 0,018 for dSSc (n = 6) and P = 0,003 for LSSc (n = 3).


B Collagen1α2 mRNA expression in same samples as in A . * P = 0 , 0030 for dSSC P = 0 , 0036 for LSSc .

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B Collagen1α2 mRNA expression levels in the same samples as in A. * P = 0,0030 for dSSC and P = 0,0036 for LSSc.


C of collagen in same samples as in A . * P = 0 , 010 .

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C Quantification of collagen protein levels in the same samples as in A. * P = 0,010.


D Immunohistochemistry to detect BiP in fibroblasts isolated from scleroderma treated or not with 4μ8C .

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D Immunohistochemistry staining to detect BiP protein in fibroblasts isolated from scleroderma patients treated or not with 4μ8C.


E mRNA of αSMA in lung fibroblasts isolated from with either cystic fibrosis ( CF ) , chronic obstructive pulmonary disease ( COPD ) or asthma . * P = 0 , 00001 for asthma vs . healthy controls , P = 0 , 00003 for ctrl vs . 4μ8C in asthma .

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E mRNA levels of αSMA in primary lung fibroblasts isolated from patients with either cystic fibrosis (CF), chronic obstructive pulmonary disease (COPD) or asthma. * P = 0,00001 for asthma vs. healthy controls, and P = 0,00003 for ctrl vs. 4μ8C in asthma patients.


F of mRNA for BiP in same samples as in E . * P = 0 , 0023 between asthma healthy controls P = 0 , 0031 between 4μ8C - treated control treatment in fibroblasts from asthma .

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F Quantification of mRNA levels for BiP in same samples as in E. * P = 0,0023 between asthma and healthy controls and P = 0,0031 between 4μ8C-treated and control treatment in fibroblasts from asthma patients.


A , B titers in blood from mice ( n = 5 ) fed an NSD ( 0 . 45 % NaCl ) or HSD ( 4 % NaCl ) for 30 days ( A ) or 7 days ( B ) then intraperitoneally infected with VSV ( 1x108 PFU per gram , 48 hrs ) were analyzed by TCID50 . Data information : Data represent mean of five biological replicates For statistical testing , p values were calculated using two - unpaired Student ' s t - . NS , not significant ( p > 0 . 05 ) . * p < 0 . 05 , * * p < 0 . 01 * * * p < 0 . 001 .

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A, B Virus titers in the blood from mice (n=5) fed an NSD (0.45% NaCl) or HSD (4% NaCl) for 30 days (A) or 7 days (B) and then intraperitoneally infected with VSV (1x108 PFU per gram body, 48 hrs) were analyzed by the TCID50 assay. Data information: Data represent mean and SEM of five biological replicates For all statistical testing, p values were calculated using two-tailed unpaired Student's t-test. NS, not significant (p > 0.05). *p < 0.05, **p < 0.01 and ***p < 0.001.


H RT - qPCR of viral in U937 , THP1 , HT1080 , 2fTGH A549 infected with VSV ( MOI = 1 . 0 , 24 hrs ) immediately after addition of NaCl ( + 34 mM ) . I RAW264 . 7 were infected with VSV / H1N1 / SeV ( MOI = 1 . 0 , 24 hrs ) immediately after addition of NaCl ( + 34 mM ) , or were transfected with HBV - replicon or HCV - replicon then treated with additional NaCl ( + 34 mM ) for 12 hrs . Viral were analyzed by RT - qPCR . Data information : Data show mean SD of four or three biological replicates . For statistical testing , p values were calculated using two - unpaired Student ' s t - . NS , not significant ( p > 0 . 05 ) . * p < 0 . 05 , * * p < 0 . 01 * * * p < 0 . 001 .

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H RT-qPCR analysis of viral RNA levels in U937, THP1, HT1080, 2fTGH and A549 cells infected with VSV (MOI=1.0, 24 hrs) immediately after addition of NaCl (+34 mM). I RAW264.7 cells were infected with VSV/H1N1/SeV (MOI=1.0, 24 hrs) immediately after addition of NaCl (+34 mM), or were transfected with HBV-replicon or HCV-replicon and then treated with additional NaCl (+34 mM) for 12 hrs. Viral RNA levels were analyzed by RT-qPCR. Data information: Data show the mean and SD of four or three biological replicates. For all statistical testing, p values were calculated using two-tailed unpaired Student's t-test. NS, not significant (p > 0.05). *p < 0.05, **p < 0.01 and ***p < 0.001.


A Western blot of VSV - G in Ifnar1 + / + or Ifnar1 - / - MEF infected with VSV ( MOI = 1 . 0 , 24 hrs ) immediately after addition of NaCl ( + 34 mM ) . Data information : are representative of at least two biological

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A Western blot analysis of VSV-G protein levels in Ifnar1+/+ or Ifnar1-/- MEF cells infected with VSV (MOI=1.0, 24 hrs) immediately after addition of NaCl (+34 mM). Data information: are representative of at least two biological replicates


C RAW264 . 7 were treated with mIFNβ ( 50 IU / ml , 20 hrs ) immediately after addition of NaCl ( + 17 , 34 51 mM ) . After washing , were infected with H1N1 ( MOI = 1 . 0 , 24 hrs ) viral were analyzed by RT - qPCR . Data information : Data represent mean SD of three biological replicates , p values were calculated using two - unpaired Student ' s t - For statistical testing : NS , not signiﬁcant ( p > 0 . 05 ) , * * p < 0 . 01 * * * p < 0 . 001 .

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C RAW264.7 cells were treated with mIFNβ (50 IU/ml, 20 hrs) immediately after addition of NaCl (+17, 34 and 51 mM). After washing, cells were infected with H1N1 (MOI=1.0, 24 hrs) and viral RNA levels were analyzed by RT-qPCR. Data information: Data represent mean and SD of three biological replicates, and p values were calculated using two-tailed unpaired Student's t-test For all statistical testing: NS, not signiﬁcant (p > 0.05), **p < 0.01 and ***p < 0.001.


F RT - qPCR of Ifit1 , Isg54 Isg15 mRNA in HEK293T pretreated with additional NaCl ( + 34 mM ) for 12 hrs then treated with IFNα ( 1 , 000 IU / ml ) as indicated . Data information : Data represent mean SD of three biological replicates , p values were calculated using two - unpaired Student ' s t - For statistical testing : NS , not signiﬁcant ( p > 0 . 05 ) , * * p < 0 . 01 * * * p < 0 . 001 .

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F RT-qPCR analysis of Ifit1, Isg54 and Isg15 mRNA levels in HEK293T pretreated with additional NaCl (+34 mM) for 12 hrs and then treated with IFNα (1,000 IU/ml) as indicated. Data information: Data represent mean and SD of three biological replicates, and p values were calculated using two-tailed unpaired Student's t-test For all statistical testing: NS, not signiﬁcant (p > 0.05), **p < 0.01 and ***p < 0.001.


G RAW264 . 7 were treated with mIFNβ ( 500 IU / ml ) for 12 hrs immediately after addition of NaCl ( + 34 mM ) or control ddH2O ( Ctrl ) . Whole lysates were subjected to a quantitative proteomic with a TMT2 - plex method to identify differentially expressed . upregulated downregulated were analyzed in volcano plot . most dramatically downregulated ISG , Viperin ( Rsad2 ) , was marked in dotted frame .

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G RAW264.7 cells were treated with mIFNβ (500 IU/ml) for 12 hrs immediately after addition of NaCl (+34 mM) or control ddH2O (Ctrl). Whole cell lysates were subjected to a quantitative proteomic analysis with a TMT2-plex method to identify the differentially expressed proteins. The upregulated and downregulated proteins were analyzed in the volcano plot. The most dramatically downregulated ISG protein, Viperin (Rsad2), was marked in the dotted frame.


H Western blot of Viperin in RAW264 . 7 treated with mIFNβ ( 100 500 IU / ml , 12 hrs ) immediately after addition of NaCl ( + 34 mM ) . I Western blot of Viperin in HT1080 treated with IFNα ( 1 , 000 IU / ml ) NaCl ( + 34 mM ) as ( H ) . J Western blot of Viperin in MEF infected with VSV ( MOI = 0 . 5 1 . 0 , 24 hrs ) immediately after addition of NaCl ( + 34 mM ) . K Western blot of Viperin in THP1 infected with H1N1 ( MOI = 1 . 0 ) immediately after addition of NaCl ( + 34 mM ) . Data information : Data are representative of at least two biological

O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

H Western blot analysis of Viperin protein in RAW264.7 treated with mIFNβ (100 and 500 IU/ml, 12 hrs) immediately after addition of NaCl (+34 mM). I Western blot analysis of Viperin protein in HT1080 treated with IFNα (1,000 IU/ml) and NaCl (+34 mM) as (H). J Western blot analysis of Viperin protein in MEF cells infected with VSV (MOI=0.5 and 1.0, 24 hrs) immediately after addition of NaCl (+34 mM). K Western blot analysis of Viperin protein in THP1 infected with H1N1 (MOI= 1.0) immediately after addition of NaCl (+34 mM). Data information: Data are representative of at least two biological replicates


N Survival curves of 8 - week - old Rsad2 + / + or Rsad2 - / - mice administrated with an NSD or HSD for 7 days then intraperitoneally infected with VSV ( 1 × 108 PFU per gram mouse , n = 10 ) . Data information : Data ( N ) show mean of ten biological replicates , p value was calculated using logrank ( Mantel - Cox ) . For statistical testing : NS , not signiﬁcant ( p > 0 . 05 ) , * * p < 0 . 01 * * * p < 0 . 001 .

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N Survival curves of 8-week-old Rsad2+/+ or Rsad2-/- mice administrated with an NSD or HSD for 7 days and then intraperitoneally infected with VSV (1×108 PFU per gram body mouse, n=10). Data information: Data (N) show the mean and SEM of ten biological replicates, and p value was calculated using logrank (Mantel-Cox) tests. For all statistical testing: NS, not signiﬁcant (p > 0.05), **p < 0.01 and ***p < 0.001.


B Western blot of FH - Viperin in stable FH - Viperin - expressing HeLa pretreated with additional NaCl ( + 34 mM ) then treated with CHX ( 50 μM ) for 6 12 hrs . Data information : Data are representative of at least two biological replicates .

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B Western blot analysis of FH-Viperin protein in stable FH-Viperin-expressing HeLa cells pretreated with additional NaCl (+34 mM) and then treated with CHX (50 μM) for 6 and 12 hrs. Data information: Data are representative of at least two biological replicates.


C RAW264 . 7 were treated with MG132 ( 10 μM ) additional NaCl ( + 34 mM ) , followed by mIFNβ ( 300 IU / ml ) treatment for 12 hrs . Viperin were analyzed by immunoblotting . Data information : Data are representative of at least two biological replicates .

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C RAW264.7 were treated with MG132 (10 μM) and additional NaCl (+34 mM), followed by mIFNβ (300 IU/ml) treatment for 12 hrs. Viperin protein levels were analyzed by immunoblotting. Data information: Data are representative of at least two biological replicates.


J Western blot of Viperin in 2fTGH transfected with control shRNAs ( shCtrl ) or shRNAs against USP33 ( shUSP33 ) then treated with IFNα ( 1 , 000 IU / ml ) for different times . Data information : Data are representative of at least two biological replicates .

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J Western blot analysis of Viperin in 2fTGH cells transfected with control shRNAs (shCtrl) or shRNAs against USP33 (shUSP33) and then treated with IFNα (1,000 IU/ml) for different times. Data information: Data are representative of at least two biological replicates.


M Western blot of Viperin in Usp33 - / - MEF transfected with FH - USP33 ( wild type , WT ; C194S H673Q , DM ) then stimulated with mIFNβ ( 500 IU / ml ) for 12 hrs . Data information : Data are representative of at least two biological replicates .

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M Western blot analysis of Viperin protein in Usp33-/- MEF cells transfected with FH-USP33 (wild type, WT; C194S and H673Q, DM) and then stimulated with mIFNβ (500 IU/ml) for 12 hrs. Data information: Data are representative of at least two biological replicates.


O Western blot of Flag - Viperin in stable Flag - Viperin - expressing HEK293T transfected with shCtrl or shUSP33 then treated with CHX ( 50 μM ) as indicated . Data information : Data are representative of at least two biological replicates .

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O Western blot analysis of Flag-Viperin in stable Flag-Viperin-expressing HEK293T cells transfected with shCtrl or shUSP33 and then treated with CHX (50 μM) as indicated. Data information: Data are representative of at least two biological replicates.


D Immunoprecipitation of Viperin ubiquitination in HEK293T co - transfected with Flag - Viperin , HA - Ub shUSP33 , then treated with additional NaCl ( + 34 mM ) for 12 hrs . Data information : Data are representative of at least two biological

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D Immunoprecipitation analysis of Viperin ubiquitination in HEK293T cells co-transfected with Flag-Viperin, HA-Ub and shUSP33, then treated with additional NaCl (+34 mM) for 12 hrs. Data information: Data are representative of at least two biological replicates


E Western blot of Viperin in 2fTGH transfected with shCtrl or shUSP33 then infected with VSV ( MOI = 1 . 0 , 12 hrs ) immediately after addition of NaCl ( + 34 mM ) . Data information : Data are representative of at least two biological

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E Western blot analysis of Viperin levels in 2fTGH cells transfected with shCtrl or shUSP33 and then infected with VSV (MOI=1.0, 12 hrs) immediately after addition of NaCl (+34 mM). Data information: Data are representative of at least two biological replicates


G RT - qPCR of VSV in 2fTGH transfected with shCtrl or shUSP33 then infected with VSV ( MOI = 1 . 0 , 24 hrs ) immediately after addition of NaCl ( + 34 mM ) . Data information : Data ( G ) represent mean SD of three biological replicates , p values were calculated using two - unpaired Student ' s t - For statistical testing : NS , not signiﬁcant ( p > 0 . 05 ) . * * * p < 0 . 001 .

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G RT-qPCR analysis of VSV RNA levels in 2fTGH cells transfected with shCtrl or shUSP33 and then infected with VSV (MOI=1.0, 24 hrs) immediately after addition of NaCl (+34 mM). Data information: Data (G) represent mean and SD of three biological replicates, and p values were calculated using two-tailed unpaired Student's t-test For all statistical testing: NS, not signiﬁcant (p > 0.05). ***p < 0.001.


I Survival curves of 8 - week - old Usp33 - / - mice fed an NSD ( 0 . 5 % NaCl ) or HSD ( 4 % NaCl ) for 7 days then infected with VSV ( 1x108 PFU per gram mouse , n = 10 ) . Data information : ; Data ( I ) show mean of ten biological replicates , p value was calculated using logrank ( Mantel - Cox ) . For statistical testing : NS , not signiﬁcant ( p > 0 . 05 ) . * * * p < 0 . 001 .

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I Survival curves of 8-week-old Usp33-/- mice fed an NSD (0.5% NaCl) or HSD (4% NaCl) for 7 days and then infected with VSV (1x108 PFU per gram body mouse, n=10). Data information: ; Data (I) show mean and SEM of ten biological replicates, and p value was calculated using logrank (Mantel-Cox) tests. For all statistical testing: NS, not signiﬁcant (p > 0.05). ***p < 0.001.


G Immunoprecipitation of ubiquitination of Flag - USP33 in p97 - WT or p97 - KO 2fTGH co - transfected with Flag - USP33 HA - Ub then treated with NaCl ( + 34 mM ) for 12 hrs . Data information : Data are representative of at least two biological replicates .

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G Immunoprecipitation analysis of ubiquitination of Flag-USP33 in p97-WT or p97-KO 2fTGH cells co-transfected with Flag-USP33 and HA-Ub and then treated with NaCl (+34 mM) for 12 hrs. Data information: Data are representative of at least two biological replicates.


L Immunoprecipitation of Viperin ubiquitination in HT1080 transfected with shCtrl or shp97 ( 1 # or 2 # ) then treated with IFNα ( 1 , 000 IU / ml , 12 hrs ) immediately after addition of NaCl ( + 34 mM ) . Data information : Data are representative of at least two biological replicates .

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L Immunoprecipitation analysis of Viperin ubiquitination in HT1080 cells transfected with shCtrl or shp97 (1# or 2#) and then treated with IFNα (1,000 IU/ml, 12 hrs) immediately after addition of NaCl (+34 mM). Data information: Data are representative of at least two biological replicates.


N Western blot of Myc - Viperin in HEK293T co - transfected with Myc - Viperin shCtrl or shp97 , then treated with additional NaCl ( + 34 mM ) for 12 hrs . Data information : Data are representative of at least two biological replicates .

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N Western blot analysis of Myc-Viperin in HEK293T cells co-transfected with Myc-Viperin and shCtrl or shp97, and then treated with additional NaCl (+34 mM) for 12 hrs. Data information: Data are representative of at least two biological replicates.


E , F Mass spectrometry of acetylation of Flag - p97 ( E ) in RAW264 . 7 transfected with Flag - p97 then treated with additional NaCl ( + 34 mM ) or control ddH2O ( Ctrl ) for 12 hrs . of p97 - K663 site was shown in ( F ) .

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E, F Mass spectrometry analysis of acetylation of Flag-p97 (E) in RAW264.7 transfected with Flag-p97 and then treated with additional NaCl (+34 mM) or control ddH2O (Ctrl) for 12 hrs. The intensity of p97-K663 site was shown in (F).


H Immunoprecipitation of interaction between USP33 Myc - p97 in HEK293T transfected with Myc - p97 ( WT or K663R ) . Data information : Data are representative of at least two biological replicates .

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H Immunoprecipitation analysis of the interaction between USP33 and Myc-p97 in HEK293T cells transfected with Myc-p97 (WT or K663R). Data information: Data are representative of at least two biological replicates.


L Western blot of FH - USP33 in p97 - KO HEK293T co - transfected with Myc - p97 ( WT or K663R ) Flag - USP33 , then treated with additional NaCl ( + 34 mM ) for 12 hrs . Data information : Data are representative of at least two biological replicates .

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L Western blot analysis of FH-USP33 levels in p97-KO HEK293T cells co-transfected with Myc-p97 (WT or K663R) and Flag-USP33, and then treated with additional NaCl (+34 mM) for 12 hrs. Data information: Data are representative of at least two biological replicates.


M Immunoprecipitation of Viperin ubiquitination in p97 - KO 2fTGH co - transfected with Myc - p97 ( WT or K663R ) , Flag - Viperin HA - Ub as indicated , then treated with additional NaCl ( + 34 mM ) for 12 hrs . Data information : Data are representative of at least two biological replicates .

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M Immunoprecipitation analysis of Viperin ubiquitination in p97-KO 2fTGH cells co-transfected with Myc-p97 (WT or K663R), Flag-Viperin and HA-Ub as indicated, and then treated with additional NaCl (+34 mM) for 12 hrs. Data information: Data are representative of at least two biological replicates.


B Western blot of USP33 in RAW264 . 7 cultured in media containing reduced of NaCl ( - 17 - 34 mM ) for 12 hrs . C Western blot of Viperin in HT1080 treated with IFNα ( 1 , 000 IU / ml , 12 hrs ) in media containing reduced of NaCl ( - 17 , - 34 - 51 mM ) . Data information : Data are representative of at least two biological

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B Western blot analysis of USP33 in RAW264.7 cells cultured in media containing reduced concentration of NaCl (-17 and -34 mM) for 12 hrs. C Western blot analysis of Viperin in HT1080 cells treated with IFNα (1,000 IU/ml, 12 hrs) in media containing reduced concentration of NaCl (-17, -34 and -51 mM). Data information: Data are representative of at least two biological replicates


D RAW264 . 7 were infected with VSV ( MOI = 1 . 0 , 12 hrs ) in media containing reduced of NaCl ( - 17 - 34 mM ) . Viral were analyzed by RT - qPCR . Data information : Data ( D ) represent mean SD of four biological replicates For statistical testing : p values were calculated using two - unpaired Student ' s t - . N . S , not significant ( p > 0 . 05 ) * * p < 0 . 01 , * * * p < 0 . 001 ( two - unpaired Student ' s t - ) .

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D RAW264.7 cells were infected with VSV (MOI=1.0, 12 hrs) in media containing reduced concentration of NaCl (-17 and -34 mM). Viral RNA levels were analyzed by RT-qPCR. Data information: Data (D) represent mean and SD of four biological replicates For all statistical testing: p values were calculated using two-tailed unpaired Student's t-test. N.S, not significant (p > 0.05) and **p < 0.01, ***p < 0.001 (two-tailed unpaired Student's t-test).


H Mice ( n = 5 ) were intraperitoneally infected with VSV ( 1x108 PFU per gram ) . 12 hrs post infection , mice were fed an NSD ( 0 . 45 % NaCl ) or LSD ( 0 . 15 % NaCl ) for 3 days . Then VSV in mouse spleen tissues were detected by RT - qPCR . I titers in mouse blood ( n = 5 ) from ( H ) were analyzed by TCID50 . Data information : Data show mean of five biological replicates . For statistical testing : p values were calculated using two - unpaired Student ' s t - . N . S , not significant ( p > 0 . 05 ) * * p < 0 . 01 , * * * p < 0 . 001 ( two - unpaired Student ' s t - ) .

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H Mice (n=5) were intraperitoneally infected with VSV (1x108 PFU per gram body). 12 hrs post infection, mice were fed an NSD (0.45% NaCl) or LSD (0.15% NaCl) for 3 days. Then VSV RNA levels in mouse spleen tissues were detected by RT-qPCR. I Virus titers in the mouse blood (n=5) from (H) were analyzed by the TCID50 assay. Data information: Data show mean and SEM of five biological replicates. For all statistical testing: p values were calculated using two-tailed unpaired Student's t-test. N.S, not significant (p > 0.05) and **p < 0.01, ***p < 0.001 (two-tailed unpaired Student's t-test).


A Immunoblot of PPAR - α Tubulin ( control ) ( top ) in WAT from WT FADD - D mice with relative ( bottom ) . Data are expressed as mean ± from three independent experiments . * P = 0 . 0035 ( Student ' s t - ) .

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A Immunoblot of PPAR-α and Tubulin (control) (top) in WAT from WT and FADD-D mice with relative quantification (bottom). Data are expressed as mean ± SEM from three independent experiments. *P = 0.0035 (Student's t-test).


B Immunostaining of PPAR - α expression in WAT from WT FADD - D mice ( Scale bars = 50 μm ) . Shown are typical results from four different fields three different experiments .

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B Immunostaining of PPAR-α expression in WAT from WT and FADD-D mice (Scale bars = 50 μm). Shown are typical results from four different fields and three different experiments.


C Quantitative real time PCR for PPAR - α PPAR - α target genes , using from epididymalfat from 10 - week - old WT FADD - D mice fed a SD . Data are expressed as mean ± from two independent experiments ( n = 6 total for each genotype ) . * P = 0 . 0022 , * * P = 0 . 0083 , * * * P = 0 . 0107 , # P = 0 . 0011 , # # P = 0 . 0037 ( Student ' s t - ) .

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C Quantitative real time PCR for PPAR-α and PPAR-α target genes, using RNA from epididymalfat from 10-week-old male WT and FADD-D mice fed a SD. Data are expressed as mean ± SEM from two independent experiments (n = 6 total for each genotype). *P = 0.0022, **P = 0.0083, ***P = 0.0107, #P = 0.0011, ##P = 0.0037 (Student's t-test).


D ChIP of CPT1b promoter in adipocytes isolated from WT , FADD - D ad - FADDmice . Soluble from adipocytes was immunoprecipitated with control mouse IgG or antibodies against PPAR - α . experiments were performed in triplicate .

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D ChIP assay of CPT1b promoter in adipocytes isolated from WT, FADD-D and ad-FADDmice. Soluble chromatin from adipocytes was immunoprecipitated with control mouse IgG or antibodies against PPAR-α. The experiments were performed in triplicate.


E ChIP of CPT1b promoter in adipocytes isolated from WT FADD - D mice . Soluble from adipocytes was immunoprecipitated with control mouse IgG or antibodies against FADD . experiments were performed in triplicate .

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E ChIP assay of CPT1b promoter in adipocytes isolated from WT and FADD-D mice. Soluble chromatin from adipocytes was immunoprecipitated with control mouse IgG or antibodies against FADD. The experiments were performed in triplicate.


F PPAR - α RIP140coimmunoprecipitate in WT but not FADD knockout or FADD - D MEFs . PPAR - α was immunoprecipitated ( IP ) from lysates of WT or FADD knockout MEFs . Data shown are representative of three independent experiments having similar results .

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F PPAR-α and RIP140coimmunoprecipitate in WT but not FADD knockout or FADD-D MEFs. PPAR-α was immunoprecipitated (IP) from the cell lysates of WT or FADD knockout MEFs. Data shown are representative of three independent experiments having similar results.


G PPAR - α RIP140coimmunoprecipitate in WT but not FADD knockout MEFs upon WY - 14 , 643 stimulation . Ligand - dependent interactions were examined in presence of 100 μM WY - 14 , 643 where indicated . Data shown are representative of three independent experiments having similar results .

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G PPAR-α and RIP140coimmunoprecipitate in WT but not FADD knockout MEFs upon WY-14,643 stimulation. Ligand-dependent interactions were examined in the presence of 100 μM WY-14,643 where indicated. Data shown are representative of three independent experiments having similar results.


H Coimmunoprecipitation of FADD RIP140 in 293T . As indicated , lysates from transfected with plasmids encoding Flag - RIP140 , HA - FADD - A or HA - FADD - D were subjected to immunoprecipitation with an anti - Flag antibody . FADD - A FADD - D were detected using an anti - HA antibody . Data shown are representative of three independent experiments having similar results .

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H Coimmunoprecipitation analysis of FADD and RIP140 in 293T cells. As indicated, lysates from the cells transfected with plasmids encoding Flag-RIP140, HA-FADD-A or HA-FADD-D were subjected to immunoprecipitation with an anti-Flag antibody. FADD-A and FADD-D were detected using an anti-HA antibody. Data shown are representative of three independent experiments having similar results.


I Coimmunoprecipitation of FADD PPAR - α in 293T . As indicated , lysates from transfected with plasmids encoding Flag - FADD - A , Flag - FADD - D or HA - PPAR - α were subjected to immunoprecipitation with an anti - Flag antibody . PPAR - α was visualized by anti - HA . Data shown are representative of three independent experiments having similar results .

O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O

I Coimmunoprecipitation analysis of FADD and PPAR-α in 293T cells. As indicated, lysates from the cells transfected with plasmids encoding Flag-FADD-A, Flag-FADD-D or HA-PPAR-α were subjected to immunoprecipitation with an anti-Flag antibody. PPAR-α was visualized by anti-HA. Data shown are representative of three independent experiments having similar results.


A Left , weights of WT FADD - D mice on either a SD ( n = 8 for each genotype ) or a HFD ( n = 6 for each genotype ) . Right , Food intake per mouse fed a HFD measured over 20 days normalized by ( n = 10 for each genotype ) . Results are means ± . * P = 0 . 0007 ( Student ' s t - ) .

O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A Left, Body weights of male WT and FADD-D mice on either a SD (n = 8 for each genotype) or a HFD (n = 6 for each genotype). Right, Food intake per mouse fed a HFD measured over 20 days normalized by body weight (n = 10 for each genotype). Results are means ± SEM. *P = 0.0007 (Student's t-test).


B Representative of fat pads organs of 10 - week - old FADD - D WT mice .

O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O

B Representative photographs of fat pads and organs of 10-week-old male FADD-D and WT mice.


C Left , fat pad weights as a of ( BW ) in absolute amounts ( inset ) from FADD - D WT mice . Mice were fed a SD or a HFD until 15 of ( n = 6 for each genotype ) . Results are means ± . * P = 0 . 0012 , * * P = 0 . 0002 , * * * P = 0 . 0027 , * * * * P = 0 . 0014 , ^ P = 0 . 0038 , ^ ^ P = 0 . 0019 , # P = 0 . 0045 , # # P = 0 . 0007 , * ^ P = 0 . 0252 , * ^ ^ P = 0 . 0082 , # # # P = 0 . 0275 , # # # # P = 0 . 0071 ( one - way ANOVA ) . Middle , fat pad weights of FADD - D WT mice on a HFD at 30 of ( n = 6 for each genotype ) . Inset , triacylglycerol ( TAG ) content in epididymal WAT . Data are expressed as means ± . * P = 0 . 0003 , * * P = 0 . 0005 , * * * P = 0 . 0019 , * * * * P = 0 . 0011 ( Student ' s t - ) . Right , of liver normalized by for FADD - D WT at of 15 ( n = 6 for each genotype ) . Data are expressed as means ± . NS , not statistically significant ( Student ' s t - ) . Epi , epididymal fat ; Ren , renal fat ; Ing , inguinal fat .

O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C Left, fat pad weights as a percentage of body weight (BW) and in absolute amounts (inset) from male FADD-D and WT mice. Mice were fed a SD or a HFD until 15 weeks of age (n = 6 for each genotype). Results are means ± SEM. *P = 0.0012, **P = 0.0002, ***P = 0.0027, ****P = 0.0014, ^P = 0.0038, ^^P = 0.0019, #P = 0.0045, ##P = 0.0007, *^P = 0.0252, *^^P = 0.0082, ###P = 0.0275, ####P = 0.0071 (one-way ANOVA). Middle, fat pad weights of male FADD-D and WT mice on a HFD at 30 weeks of age (n = 6 for each genotype). Inset, triacylglycerol (TAG) content in epididymal WAT. Data are expressed as means ± SEM. *P = 0.0003, **P = 0.0005, ***P = 0.0019, ****P = 0.0011 (Student's t-test). Right, weight of liver normalized by body weight for male FADD-D and WT littermates at the age of 15 weeks (n = 6 for each genotype). Data are expressed as means ± SEM. NS, not statistically significant (Student's t-test). Epi, epididymal fat; Ren, renal fat; Ing, inguinal fat.


D of brown adipose tissue normalized by ( n = 5 for each genotype ) . Data are expressed as means ± . * P = 0 . 0008 ( Student ' s t - ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D Weight of brown adipose tissue normalized by body weight (n = 5 for each genotype). Data are expressed as means ± SEM. *P = 0.0008 (Student's t-test).


E MRI of fat content : standard non - fat - suppressed ( left ) , fat - suppressed ( middle ) fat - only ( right ) . Representative of three independent experiments ( n = 6 total for each genotype ) showing similar results are shown .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E MRI of body fat content: standard non-fat-suppressed (left), fat-suppressed (middle) and fat-only (right). Representative images of three independent experiments (n = 6 total for each genotype) showing similar results are shown.


F Paraffin - embedded sections of WAT BAT from WT FADD - D mice were stained with H & amp ; amp ; E . Scale bar = 50 μm . Shown are typical results from four different fields three different experiments .

O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F Paraffin-embedded sections of WAT and BAT from WT and FADD-D mice were stained with H&amp;E. Scale bar = 50 μm. Shown are typical results from four different fields and three different experiments.


G Left , relative amounts of liver muscle triacylglycerol contents in 10 - month - old WT FADD - D mice ( n = 6 for each genotype ) . Right , relative amounts of cholesterol in liver muscle of same group of WT FADD - D mice ( n = 6 for each genotype ) . Data are expressed as means ± . * P = 0 . 0015 , * * P = 0 . 0027 , * * * P = 0 . 0318 ( Student ' s t - ) . NS , not statistically significant .

O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

G Left, relative amounts of liver and muscle triacylglycerol contents in 10-month-old WT and FADD-D mice (n = 6 for each genotype). Right, relative amounts of cholesterol in the liver and muscle of the same group of WT and FADD-D mice (n = 6 for each genotype). Data are expressed as means ± SEM. *P = 0.0015, **P = 0.0027, ***P = 0.0318 (Student's t-test). NS, not statistically significant.


H Left , plasma triacylglycerol in 10 - month - old WT FADD - D mice following fasting for 20 h ( n = 5 for each genotype ) . Right , plasma cholesterol in same group of WT FADD - D mice ( n = 5 for each genotype ) . Data are expressed as means ± . * P = 0 . 0124 ( Student ' s t - ) . NS , not statistically significant .

O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

H Left, plasma triacylglycerol levels in 10-month-old WT and FADD-D mice following fasting for 20 h (n = 5 for each genotype). Right, plasma cholesterol levels in the same group of WT and FADD-D mice (n = 5 for each genotype). Data are expressed as means ± SEM. *P = 0.0124 (Student's t-test). NS, not statistically significant.


A Serum free fatty acid ( FFA ) triacylglycerol in WT FADD - D mice fed a HFD for 20 days ( n = 5 for each genotype ) . Data are expressed as means ± . * P = 0 . 0059 , * * P = 0 . 0012 ( Student ' s t - ) .

O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A Serum free fatty acid (FFA) levels and triacylglycerol levels in WT and FADD-D mice fed a HFD for 20 days (n = 5 for each genotype). Data are expressed as means ± SEM. *P = 0.0059, **P = 0.0012 (Student's t-test).


B Serum of leptin in WT FADD - D mice ( n = 4 for each genotype ) fed a SD or a HFD . Data are expressed as means ± . * P = 0 . 0032 , * * P = 0 . 0003 , * * * P = 0 . 0038 ( one - way ANOVA ) .

O O O B-MEASURED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B Serum levels of leptin in WT and FADD-D mice (n = 4 for each genotype) fed a SD or a HFD. Data are expressed as means ± SEM. *P = 0.0032, **P = 0.0003, ***P = 0.0038 (one-way ANOVA).


C of inguinal WAT , BAT , liver from WT FADD - D mice fed a HFD for 20 days . WAT BAT were stained with hematoxylin eosin . Liver tissues were stained with Oil red O to demonstrate lipid accumulation counterstained with hematoxylin . Scale bars = 50 μm . Shown are typical results from four different fields three different experiments .

O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C Morphology of inguinal WAT, BAT, and liver from WT and FADD-D mice fed a HFD for 20 days. WAT and BAT were stained with hematoxylin and eosin. Liver tissues were stained with Oil red O to demonstrate lipid accumulation and counterstained with hematoxylin. Scale bars = 50 μm. Shown are typical results from four different fields and three different experiments.


D Comparison of in WAT of WT FADD - D mice maintained on SD HFD ( n = 5 for each genotype ) .

O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O

D Comparison of cell size in WAT of WT and FADD-D mice maintained on SD and HFD (n = 5 for each genotype).


E FADD - D mice lost more as compared with WT on 20 - h fasting ( n = 4 for each genotype ) . Data are expressed as means ± . * P = 0 . 0085 ( Student ' s t - ) .

O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E FADD-D mice lost more weight as compared with WT on 20-h fasting (n = 4 for each genotype). Data are expressed as means ± SEM. *P = 0.0085 (Student's t-test).


F Whole - oxygen consumption rate ( VO2 ) during 24 hr in WT FADD - D mice fed a SD ( n = 4 for each genotype ) . Data are expressed as means ± . * P = 0 . 0055 , * * P = 0 . 0081 ( Student ' s t - ) .

O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F Whole-body oxygen consumption rate (VO2) during 24 hr in WT and FADD-D mice fed a SD (n = 4 for each genotype). Data are expressed as means ± SEM. *P = 0.0055, **P = 0.0081 (Student's t-test).


G Average respiratory exchange ( VCO2 / VO2 ) ( RER ) for period in WT FADD - D mice ( n = 5 for each genotype ) . Data are expressed as means ± . * P = 0 . 0042 , * * P = 0 . 0079 ( Student ' s t - ) .

O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

G Average respiratory exchange ratio (VCO2/VO2) (RER) for the dark and light period in WT and FADD-D mice (n = 5 for each genotype). Data are expressed as means ± SEM. *P = 0.0042, **P = 0.0079 (Student's t-test).


H Rectal ( n = 5 for each genotype ) . Data are expressed as means ± . * P = 0 . 0217 ( Student ' s t - ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

H Rectal temperature (n = 5 for each genotype). Data are expressed as means ± SEM. *P = 0.0217 (Student's t-test).


I Average values of energy expenditure ( EE ) for 24 hr period in WT FADD - D mice ( n = 5 for each genotype ) . Data are expressed as means ± . * P = 0 . 0054 ( Student ' s t - ) .

O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

I Average values of energy expenditure (EE) for the 24 hr period in WT and FADD-D mice (n = 5 for each genotype). Data are expressed as means ± SEM. *P = 0.0054 (Student's t-test).


J Physical activity for period in WT FADD - D mice ( n = 5 for each genotype ) . Data are expressed as means ± . NS , not statistically significant ( Student ' s t - ) .

O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

J Physical activity for the dark and light period in WT and FADD-D mice (n = 5 for each genotype). Data are expressed as means ± SEM. NS, not statistically significant (Student's t-test).


A Left , transmission electron microscopy of adipocytes from WAT BAT of WT FADD - D mice . Arrowheads indicate mitochondria lipid droplets ( L ) ( × 5 , 000 magnification ) . Right , of mitochondria in WAT BAT of WT FADD - D mice , respectively . Data are expressed as mean ± from three independent experiments ( n = 6 total for each genotype ) . * P = 0 . 0007 , * * P = 0 . 0083 ( Student ' s t - ) .

O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A Left, transmission electron microscopy analysis of adipocytes from WAT and BAT of WT and FADD-D mice. Arrowheads indicate mitochondria and lipid droplets (L) (× 5,000 magnification). Right, quantification of mitochondria in WAT and BAT of WT and FADD-D mice, respectively. Data are expressed as mean ± SEM from three independent experiments (n = 6 total for each genotype). *P = 0.0007, **P = 0.0083 (Student's t-test).


B Quantitative real time PCR for UCP1 , Dio2 , PPARd , UCP3 , CPT1 PGC1 - α , using from epididymalfat from 10 - week - old WT FADD - D mice on a SD . Data are expressed as mean ± from three independent experiments ( n = 6 total for each genotype ) . * P = 0 . 0014 , * * P = 0 . 0025 , * * * P = 0 . 0006 , * * * * P = 0 . 0007 , # P = 0 . 0003 , # # P = 0 . 0008 ( Student ' s t - ) .

O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B Quantitative real time PCR for UCP1, Dio2, PPARd, UCP3, CPT1 and PGC1-α, using RNA from epididymalfat from 10-week-old male WT and FADD-D mice on a SD. Data are expressed as mean ± SEM from three independent experiments (n = 6 total for each genotype). *P = 0.0014, **P = 0.0025, ***P = 0.0006, ****P = 0.0007, #P = 0.0003, ##P = 0.0008 (Student's t-test).


C Quantitative real time PCR for UCP3 , CPT1 , PPARd PGC1 - α , using from skeletal muscle from 10 - week - old WT FADD - D mice on a SD . Data are expressed as mean ± from three independent experiments ( n = 6 total for each genotype ) . * P = 0 . 0027 , * * P = 0 . 0329 , * * * P = 0 . 0092 , * * * * P = 0 . 0085 ( Student ' s t - ) .

O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C Quantitative real time PCR for UCP3, CPT1, PPARd and PGC1-α, using RNA from skeletal muscle from 10-week-old male WT and FADD-D mice on a SD. Data are expressed as mean ± SEM from three independent experiments (n = 6 total for each genotype). *P = 0.0027, **P = 0.0329, ***P = 0.0092, ****P = 0.0085 (Student's t-test).


D Quantitative real time PCR for UCP1 , using from BAT from 10 - week - old WT FADD - D mice on a SD . Data are expressed as mean ± from three independent experiments ( n = 6 total for each genotype ) . * P = 0 . 0018 ( Student ' s t - ) .

O O O O O O B-MEASURED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D Quantitative real time PCR for UCP1, using RNA from BAT from 10-week-old male WT and FADD-D mice on a SD. Data are expressed as mean ± SEM from three independent experiments (n = 6 total for each genotype). *P = 0.0018 (Student's t-test).


E Immunoblotting of UCP1 Tubulin ( control ) ( top ) with relative ( bottom ) , using total lysates of BAT from 10 - week - old WT FADD - D mice on a SD . Data are expressed as mean ± from three independent experiments . * P = 0 . 0217 ( Student ' s t - ) .

O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E Immunoblotting of UCP1 and Tubulin (control) (top) with relative quantification (bottom), using total cell lysates of BAT from 10-week-old male WT and FADD-D mice on a SD. Data are expressed as mean ± SEM from three independent experiments. *P = 0.0217 (Student's t-test).


F Oxygen consumption in isolated BAT from 10 - week - old WT FADD - D mice on a SD ( n = 4 for each genotype ) . Data are expressed as mean ± . * P = 0 . 0025 , * * P = 0 . 0006 ( Student ' s t - ) .

O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F Oxygen consumption in isolated BAT from 10-week-old male WT and FADD-D mice on a SD (n = 4 for each genotype). Data are expressed as mean ± SEM. *P = 0.0025, **P = 0.0006 (Student's t-test).


G Quantitative real time PCR for PGC1α , C / EBPα PPARγ , using from BAT from 10 - week - old WT FADD - D mice on a SD . Data are expressed as mean ± from two independent experiments ( n = 4 total for each genotype ) . * P = 0 . 0113 ( Student ' s t - ) . NS , not statistically significant .

O O O O O O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

G Quantitative real time PCR for PGC1α, C/EBPα and PPARγ, using RNA from BAT from 10-week-old male WT and FADD-D mice on a SD. Data are expressed as mean ± SEM from two independent experiments (n = 4 total for each genotype). *P = 0.0113 (Student's t-test). NS, not statistically significant.


H β - oxidation of adipocytes isolated from WT ( n = 5 ) FADD - D mice ( n = 4 ) . 1 - 14C labeled oleic acid was added to medium containing isolated adipocytes ( 5 × 105 ) , 14CO2 trapped by filter paper soaked with hyamine hydroxide was measured after a 3 h - incubation by a scintillation counter . Data are expressed as mean ± . * P = 0 . 0022 ( Student ' s t - ) .

O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

H β-oxidation analysis of white adipocytes isolated from WT (n = 5) and FADD-D mice (n = 4). 1-14C labeled oleic acid was added to the medium containing isolated adipocytes (5×105), and 14CO2 trapped by the filter paper soaked with hyamine hydroxide was measured after a 3 h-incubation by a scintillation counter. Data are expressed as mean ± SEM. *P = 0.0022 (Student's t-test).


I Basal stimulated ( + 100 nM isoproterenol ) lipolysis , as measured by glycerol ( left ) fatty ( right ) released from explants of epididymal WAT from overnight fasted 10 - week - old WT FADD - D mice fed a HFD ( n = 4 for each genotype ) . Data are expressed as mean ± . * P = 0 . 0078 , * * P = 0 . 0065 , * * * P = 0 . 0008 , ^ P = 0 . 0133 , ^ ^ P = 0 . 0053 , ^ ^ ^ P = 0 . 0006 , ^ ^ ^ ^ P = 0 . 0001 , # P = 0 . 0082 , # # P = 0 . 0051 , # # # P = 0 . 0009 , * ^ P = 0 . 0151 , * ^ ^ P = 0 . 0019 , * * ^ ^ P = 0 . 0005 , * * * ^ P = 0 . 0002 ( Student ' s t - ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

I Basal and stimulated (+ 100 nM isoproterenol) lipolysis, as measured by glycerol (left) and fatty acids (right) released from explants of epididymal WAT from overnight fasted 10-week-old male WT and FADD-D mice fed a HFD (n = 4 for each genotype). Data are expressed as mean ± SEM. *P = 0.0078, **P = 0.0065, ***P = 0.0008, ^P = 0.0133, ^^P = 0.0053, ^^^P = 0.0006, ^^^^P = 0.0001, #P = 0.0082, ##P = 0.0051, ###P = 0.0009, *^P = 0.0151, *^^P = 0.0019, **^^P = 0.0005, ***^P = 0.0002 (Student's t-test).


J Molar of FFA to glycerol release from WAT explants ( n = 4 for each genotype ) . Data are expressed as mean ± . * P = 0 . 0412 , * * P = 0 . 0033 ( Student ' s t - ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

J Molar ratio of FFA to glycerol release from WAT explants (n = 4 for each genotype). Data are expressed as mean ± SEM. *P = 0.0412, **P = 0.0033 (Student's t-test).


A Quantitative real time PCR for enzymes related to lipid metabolism , using from epididymalfat from 10 - week - old WT FADD - D mice on a SD . Data are expressed as mean ± from two independent experiments ( n = 5 total for each genotype ) . * P = 0 . 0162 , * * P = 0 . 0105 , * * * P = 0 . 0063 ( Student ' s t - ) . NS , not statistically significant .

O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A Quantitative real time PCR for enzymes related to lipid metabolism, using RNA from epididymalfat from 10-week-old male WT and FADD-D mice on a SD. Data are expressed as mean ± SEM from two independent experiments (n = 5 total for each genotype). *P = 0.0162, **P = 0.0105, ***P = 0.0063 (Student's t-test). NS, not statistically significant.


B cAMP abundance in epididymalWAT from 10 - week - old WT FADD - D mice on a HFD ( n = 4 for each genotype ) . Data are expressed as mean ± . * P = 0 . 0033 ( Student ' s t - ) .

O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B cAMP abundance in epididymalWAT from 10-week-old male WT and FADD-D mice on a HFD (n = 4 for each genotype). Data are expressed as mean ± SEM. *P = 0.0033 (Student's t-test).


C Basal forskolin - stimulated adenylyl cyclase activity in epididymalWAT from 10 - week - old WT FADD - D mice on a HFD ( n = 4 for each genotype ) . Data are expressed as mean ± . * P = 0 . 0177 , * P = 0 . 0091 ( Student ' s t - ) .

O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C Basal and forskolin-stimulated adenylyl cyclase activity in epididymalWAT from 10-week-old male WT and FADD-D mice on a HFD (n = 4 for each genotype). Data are expressed as mean ± SEM. *P = 0.0177, *P = 0.0091 (Student's t-test).


D Immunoblotting of phosphorylated HSL ( p - HSL ) , HSL , p - PKA substrate , desnutrin Tubulin ( control ) ( left ) with relative ( right ) . Data are expressed as mean ± from three independent experiments . * P = 0 . 0032 , * * P = 0 . 0011 ( Student ' s t - ) . NS , not statistically significant .

O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O B-MEASURED_VAR O O O B-MEASURED_VAR O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D Immunoblotting of phosphorylated HSL (p-HSL), HSL, p-PKA substrate, desnutrin and Tubulin (control) (left) with relative quantification (right). Data are expressed as mean ± SEM from three independent experiments. *P = 0.0032, **P = 0.0011 (Student's t-test). NS, not statistically significant.


E Basal stimulated ( + 200 nM isoproterenol ) lipolysis in epididymalWAT of 10 - week - old WT FADD - D mice ( n = 4 for each genotype ) treated with or without 10 μM BAY . Data are expressed as mean ± . * P = 0 . 0019 , * * P = 00038 , ^ P = 0 . 0035 , ^ ^ P = 0 . 0053 ( one - way ANOVA ) . NS , not statistically significant .

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E Basal and stimulated (+200 nM isoproterenol) lipolysis in epididymalWAT of 10-week-old male WT and FADD-D mice (n = 4 for each genotype) treated with or without 10 μM BAY. Data are expressed as mean ± SEM. *P = 0.0019, **P = 00038, ^P = 0.0035, ^^P = 0.0053 (one-way ANOVA). NS, not statistically significant.


F Basal stimulated ( + 200 nM isoproterenol ) lipolysis in isolated adipocytes from 10 - week - old WT FADD - D mice ( n = 4 for each genotype ) treated with or without 10 μM BAY . Data are expressed as mean ± . * P = 0 . 0033 , * * P = 0 . 0051 , ^ P = 0 . 004 , ^ ^ P = 0 . 0057 ( one - way ANOVA ) . NS , not statistically significant .

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F Basal and stimulated (+200 nM isoproterenol) lipolysis in isolated adipocytes from 10-week-old WT and FADD-D mice (n = 4 for each genotype) treated with or without 10 μM BAY. Data are expressed as mean ± SEM. *P = 0.0033, **P = 0.0051, ^P = 0.004, ^^P = 0.0057 (one-way ANOVA). NS, not statistically significant.


G Immunoblotting of phosphorylated PKC ( p - PKC ) , ERK , phosphorylated ERK ( p - ERK ) β - actin ( control ) ( left ) with relative ( right ) . Data are expressed as mean ± from three independent experiments . * P = 0 . 0119 , * * P = 0 . 0034 ( Student ' s t - ) .

O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O B-MEASURED_VAR O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

G Immunoblotting of phosphorylated PKC (p-PKC), ERK, phosphorylated ERK (p-ERK) and β-actin (control) (left) with relative quantification (right). Data are expressed as mean ± SEM from three independent experiments. *P = 0.0119, **P = 0.0034 (Student's t-test).


A Typical growth curves of control ad - FADDmice maintained on a SD ( left panel ) or a HFD ( right panel ) ( n = 8 for each genotype ) . Data are expressed as mean ± . * P = 0 . 0435 , * * P = 0 . 0371 , ^ P = 0 . 0217 , # P = 0 . 0236 , # # P = 0 . 0158 , # # # P = 0 . 0113 ( Student ' s t - ) .

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A Typical growth curves of control and ad-FADDmice maintained on a SD (left panel) or a HFD (right panel) (n = 8 for each genotype). Data are expressed as mean ± SEM. *P = 0.0435, **P =0.0371, ^P = 0.0217, #P = 0.0236, ##P = 0.0158, ###P = 0.0113 (Student's t-test).


B composition by NMR to show fat mass fat mass in control ad - FADDmice after 10 of HFD ( n = 6 for each genotype ) . Data are expressed as mean ± . * P = 0 . 0027 , * * P = 0 . 0012 ( Student ' s t - ) .

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B Body composition by NMR to show the fat mass and fat mass ratio in control and ad-FADDmice after 10 weeks of HFD (n = 6 for each genotype). Data are expressed as mean ± SEM. *P = 0.0027, **P = 0.0012 (Student's t-test).


C Food intake in 15 - week - old control ad - FADDmice fed a HFD ( n = 8 for each genotype ) .

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C Food intake in 15-week-old male control and ad-FADDmice fed a HFD (n = 8 for each genotype).


D E Representative pictures of hematoxylin eosin - stained sections of epididymalWAT from control ad - FADDmice at 10 ( D ) after 10 of an HFD ( E ) are shown . Scale bar = 50 μm . Shown are typical results from four different fields three different experiments . F average cross - sectional of adipocytes in epididymalWAT is shown in bar graph ( n = 6 for each genotype ) . Data are expressed as mean ± . * P = 0 . 002 ( Student ' s t - ) . NS , not statistically significant . G distribution of adipocyte cross - sectional in epididymalWAT of HFD - fed control or ad - FADDmice is shown in bar graph ( n = 6 for each genotype ) .

O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O

D and E Representative pictures of hematoxylin and eosin-stained sections of epididymalWAT from control and ad-FADDmice at 10 weeks (D) and after 10 weeks of an HFD (E) are shown. Scale bar = 50 μm. Shown are typical results from four different fields and three different experiments.F The average cross-sectional area of adipocytes in epididymalWAT is shown in the bar graph (n = 6 for each genotype). Data are expressed as mean ± SEM. *P = 0.002 (Student's t-test). NS, not statistically significant.G The distribution of adipocyte cross-sectional area in epididymalWAT of HFD-fed control or ad-FADDmice is shown in the bar graph (n = 6 for each genotype).


H Representative pictures of hematoxylin eosin - stained sections of brown adipose tissue from HFD - fed control ad - FADDmice are shown . Scale bar = 50 μm . Shown are typical results from four different fields three different experiments .

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H Representative pictures of hematoxylin and eosin-stained sections of brown adipose tissue from HFD-fed control and ad-FADDmice are shown. Scale bar = 50 μm. Shown are typical results from four different fields and three different experiments.


A - C HFD - fed control ad - FADDmice were housed in a computer - controlled open - circuit indirect calorimeter to determine ( A ) oxygen consumption , ( B ) carbon dioxide production , ( C ) respiratory exchange ( RER ) during ( 8 am - 8 pm ) ( 8 pm - 8 am ) periods ( n = 5 for each genotype ) . Data are expressed as mean ± . * P = 0 . 0379 , * * P = 0 . 0453 , * * * P = 0 . 0445 ( Student ' s t - ) . NS , not statistically significant .

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A-C HFD-fed control and ad-FADDmice were housed in a computer-controlled open-circuit indirect calorimeter to determine (A) oxygen consumption, (B) carbon dioxide production, and (C) respiratory exchange ratio (RER) during the light (8 am - 8 pm) and dark (8 pm - 8 am) periods (n = 5 for each genotype). Data are expressed as mean ± SEM. *P = 0.0379, **P = 0.0453, ***P = 0.0445 (Student's t-test). NS, not statistically significant.


D result of real - time quantitative PCR for UCP1 in brown adipose tissue from HFD - fed control ad - FADDmice is shown in bar graph . Data are expressed as mean ± from two independent experiments ( n = 4 total for each genotype ) . * P = 0 . 0172 ( Student ' s t - ) .

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D The result of real-time quantitative PCR analysis for UCP1 in brown adipose tissue from HFD-fed control and ad-FADDmice is shown in the bar graph. Data are expressed as mean ± SEM from two independent experiments (n = 4 total for each genotype). *P = 0.0172 (Student's t-test).


E Immunoblot of UCP1 Tubulin ( control ) ( top ) in BAT from control ad - FADDmice with relative ( bottom ) . Data are expressed as mean ± from three independent experiments . * P = 0 . 0053 ( Student ' s t - ) .

O O O B-MEASURED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E Immunoblot of UCP1 and Tubulin (control) (top) in BAT from control and ad-FADDmice with relative quantification (bottom). Data are expressed as mean ± SEM from three independent experiments. *P = 0.0053 (Student's t-test).


F Mice were initially maintained at room then transferred to cold incubator ( 5 ° C ) . Rectal was recorded at indicated time points using thermoprobe ( n = 6 for each genotype ) . Results are means ± . * P = 0 . 0466 , * * P = 0 . 0357 , # P = 0 . 0229 , # # P = 0 . 0138 , # # # P = 0 . 0206 ( Student ' s t - ) .

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F Mice were initially maintained at room temperature and then transferred to cold incubator (5°C). Rectal body temperature was recorded at the indicated time points using thermoprobe (n = 6 for each genotype). Results are means ± SEM. *P = 0.0466, **P = 0.0357, #P = 0.0229, ##P = 0.0138, ###P = 0.0206 (Student's t-test).


A B Blood glucose serum insulin in 2 - h GTT 12 after HFD . Inset graphs in ( A ) ( B ) depict respective of under curve ( AUC ) . Data shown are from one experiment ( n = 6 for each genotype ) , representative of a total of two independent experiments . Results are means ± . * P = 0 . 0287 , * * P = 0 . 0173 , * * * P = 0 . 0332 , * * * * P = 0 . 0453 , # P = 0 . 0077 , # # P = 0 . 0344 , # # # P = 0 . 0412 , # # # # P = 0 . 0495 , ^ P = 0 . 0435 , ^ ^ P = 0 . 0304 ( Student ' s t - ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A and B Blood glucose levels and serum insulin levels in 2-h GTT 12 weeks after HFD. Inset graphs in (A) and (B) depict the respective analysis of the area under the curve (AUC). Data shown are from one experiment (n = 6 for each genotype), representative of a total of two independent experiments. Results are means ± SEM. *P = 0.0287, **P = 0.0173, ***P = 0.0332, ****P = 0.0453, #P = 0.0077, ##P = 0.0344, ###P = 0.0412, ####P = 0.0495, ^P = 0.0435, ^^P = 0.0304 (Student's t-test).


C ITT 12 after HFD . Data shown are from one experiment ( n = 6 for each genotype ) , representative of a total of two independent experiments . Results are means ± . * P = 0 . 0127 , * * P = 0 . 0232 , * * * P = 0 . 0373 , * * * * P = 0 . 0385 ( Student ' s t - ) .

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C ITT 12 weeks after HFD. Data shown are from one experiment (n = 6 for each genotype), representative of a total of two independent experiments. Results are means ± SEM. *P = 0.0127, **P = 0.0232, ***P = 0.0373, ****P = 0.0385 (Student's t-test).


D Insulin - stimulated Akt phosphorylation ( Ser473 ) in WAT , liver , skeletal muscle of control ad - FADD mice . experiments were performed in triplicate .

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D Insulin-stimulated Akt phosphorylation (Ser473) in WAT, liver, and skeletal muscle of control and ad-FADD mice. The experiments were performed in triplicate.


E Adiponectin mRNA was measured in isolated epididymal adipocytes by RT - PCR . Data are expressed as mean ± from three independent experiments . * P = 0 . 0044 ( Student ' s t - ) .

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E Adiponectin mRNA was measured in isolated epididymal adipocytes by RT-PCR. Data are expressed as mean ± SEM from three independent experiments. *P = 0.0044 (Student's t-test).


F - H Total adiponectin ( F ) , HMW adiponectin ( G ) , of HMW to total adiponectin ( H ) . n = 5 for each genotype . Data are expressed as mean ± . * P = 0 . 0217 , * * P = 0 . 0116 , * * * P = 0 . 0175 ( Student ' s t - ) .

O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F-H Total adiponectin levels (F), HMW adiponectin (G), and the ratio of HMW to total adiponectin (H). n = 5 for each genotype. Data are expressed as mean ± SEM. *P = 0.0217, **P = 0.0116, ***P = 0.0175 (Student's t-test).


I Basal insulin - stimulated glucose uptake in control FADD deficient isolated adipocytes . Data shown are from one experiment ( n = 4 for each genotype ) , representative of a total of two independent experiments . Results are means ± . * P = 0 . 0073 ( Student ' s t - ) .

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I Basal and insulin-stimulated glucose uptake in control and FADD deficient primary isolated adipocytes. Data shown are from one experiment (n = 4 for each genotype), representative of a total of two independent experiments. Results are means ± SEM. *P = 0.0073 (Student's t-test).


J Basal insulin - stimulated glucose uptake in 3T3L1 differentiated to adipocytes infected with lentivirus containing FADD shRNA or scrambled shRNA control . Data are expressed as mean ± from three independent experiments . * P = 0 . 0092 ( Student ' s t - ) .

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J Basal and insulin-stimulated glucose uptake in 3T3L1 cells differentiated to adipocytes and infected with lentivirus containing FADD shRNA or scrambled shRNA control. Data are expressed as mean ± SEM from three independent experiments. *P = 0.0092 (Student's t-test).


K Basal insulin - stimulated glucose uptake in control FADD deficient isolated adipocytes treated with DMSO or 5 μM MK886 . Data are expressed as mean ± from four independent experiments . * P = 0 . 0135 ( one - way ANOVA ) . NS , not statistically significant .

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K Basal and insulin-stimulated glucose uptake in control and FADD deficient primary isolated adipocytes treated with DMSO or 5 μM MK886. Data are expressed as mean ± SEM from four independent experiments. *P = 0.0135 (one-way ANOVA). NS, not statistically significant.


A Relative mRNA of macrophage marker F4 / 80 in SVCs isolated from epididymal adipose tissue . Data are expressed as mean ± from four independent experiments . * P = 0 . 0279 ( Student ' s t - ) .

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A Relative mRNA levels of the macrophage marker F4/80 in SVCs isolated from epididymal adipose tissue. Data are expressed as mean ± SEM from four independent experiments. *P = 0.0279 (Student's t-test).


B F4 / 80 immunostaining in epi - WAT . Scale bars = 50 μm . Shown are typical results from four different fields three different experiments . Data are expressed as mean ± . * P = 0 . 0003 ( Student ' s t - ) .

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B F4/80 immunostaining in epi-WAT. Scale bars = 50 μm. Shown are typical results from four different fields and three different experiments. Data are expressed as mean ± SEM. *P = 0.0003 (Student's t-test).


C Relative mRNA of proinflammatory M1 - like macrophage marker CD11c in SVCs isolated from epididymal adipose tissue . Data are expressed as mean ± from five independent experiments . * P = 0 . 0221 ( Student ' s t - ) .

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C Relative mRNA levels of the proinflammatory M1-like macrophage marker CD11c in SVCs isolated from epididymal adipose tissue. Data are expressed as mean ± SEM from five independent experiments. *P = 0.0221 (Student's t-test).


D Relative mRNA of inflammatory anti - inflammatory cytokines in Epi - WAT . Data are expressed as mean ± from four independent experiments . * P = 0 . 0201 , * * P = 0 . 0159 , * * * P = 0 . 0263 , ^ P = 0 . 0227 , ^ ^ P = 0 . 0215 , ^ ^ ^ P = 0 . 0312 , # P = 0 . 0231 , # # P = 0 . 0255 , # # # P = 0 . 0094 ( Student ' s t - ) . NS , not statistically significant .

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D Relative mRNA levels of inflammatory and anti-inflammatory cytokines in Epi-WAT. Data are expressed as mean ± SEM from four independent experiments. *P = 0.0201, **P = 0.0159, ***P = 0.0263, ^P = 0.0227, ^^P = 0.0215, ^^^P = 0.0312, #P = 0.0231, ##P = 0.0255, ###P = 0.0094 (Student's t-test). NS, not statistically significant.


E - H Serum of TNF - α ( E ) . n = 5 for each genotype . Data are expressed as mean ± . * P = 0 . 0077 , * * P = 0 . 0052 , * * * P = 0 . 0114 , * * * * P = 0 . 0127 ( Student ' s t - ) .

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E-H Serum levels of TNF-α (E). n = 5 for each genotype. Data are expressed as mean ± SEM. *P = 0.0077, **P = 0.0052, ***P = 0.0114, ****P = 0.0127 (Student's t-test).


E - H Serum of IL - 6 ( F ) , IL - 12p40 ( G ) , IL - 10 ( H ) . n = 5 for each genotype . Data are expressed as mean ± . * P = 0 . 0077 , * * P = 0 . 0052 , * * * P = 0 . 0114 , * * * * P = 0 . 0127 ( Student ' s t - ) .

O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E-H Serum levels of IL-6 (F), IL-12p40 (G), and IL-10 (H). n = 5 for each genotype. Data are expressed as mean ± SEM. *P = 0.0077, **P = 0.0052, ***P = 0.0114, ****P = 0.0127 (Student's t-test).


A Top , representative of 20 - week - old ob / ob FADD - D / ob / ob mice fed a SD . Bottom , representative of their livers epididymalWAT .

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A Top, representative photographs of 20-week-old male ob/ob and FADD-D/ob/ob mice fed a SD. Bottom, representative photographs of their livers and epididymalWAT.


B Comparison of weights of WAT depots from WT , FADD - D , ob / ob FADD - D / ob / ob mice . n = 5 for each genotype . Data are expressed as mean ± . * P = 0 . 0007 , * * P = 0 . 0022 , * * * P = 0 . 0011 , # P = 0 . 0009 , # # P = 0 . 0003 , # # # P = 0 . 0027 ( one - way ANOVA ) .

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B Comparison of weights of WAT depots from WT, FADD-D, ob/ob and FADD-D/ob/ob mice. n = 5 for each genotype. Data are expressed as mean ± SEM. *P = 0.0007, **P = 0.0022, ***P = 0.0011, #P = 0.0009, ##P = 0.0003, ###P = 0.0027 (one-way ANOVA).


C Basal stimulated lipolysis measured by fatty acid release from explants of epididymalWAT in 10 - week - old ob / ob FADD - D / ob / ob mice fed a HFD . n = 4 for each genotype . Data are expressed as mean ± . * P = 0 . 0003 , * * P = 0 . 001 ( one - way ANOVA ) .

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C Basal and stimulated lipolysis measured by fatty acid release from explants of epididymalWAT in 10-week-old male ob/ob and FADD-D/ob/ob mice fed a HFD. n = 4 for each genotype. Data are expressed as mean ± SEM. *P = 0.0003, **P = 0.001 (one-way ANOVA).


D cAMP level in WAT of 10 - week - old WT , FADD - D , ob / ob FADD - D / ob / ob mice fed a HFD . n = 4 for each genotype . Data are expressed as mean ± . * P = 0 . 0017 , * * P = 0 . 001 ( one - way ANOVA ) .

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D cAMP level in WAT of 10-week-old male WT, FADD-D, ob/ob and FADD-D/ob/ob mice fed a HFD. n = 4 for each genotype. Data are expressed as mean ± SEM. *P = 0.0017, **P = 0.001 (one-way ANOVA).


E Whole - oxygen consumption rate ( VO2 ) during 24 hr in WT , FADD - D , ob / ob , FADD - D / ob / ob mice fed a SD . n = 4 for each genotype . Data are expressed as mean ± . * P = 0 . 0053 , * * P = 0 . 0025 , * * * P = 0 . 0062 , * * * * P = 0 . 0011 ( one - way ANOVA ) .

O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E Whole-body oxygen consumption rate (VO2) during 24 hr in WT, FADD-D, ob/ob, and FADD-D/ob/ob mice fed a SD. n = 4 for each genotype. Data are expressed as mean ± SEM. *P = 0.0053, **P = 0.0025, ***P = 0.0062, ****P = 0.0011 (one-way ANOVA).


F Quantitative real time PCR for Dio2 , PPARd UCP1 , using from epididymal fat from 20 - week - old ob / ob FADD - D / ob / ob mice fed a SD . Data are expressed as mean ± from four independent experiments . * P = 0 . 0009 , * * P = 0 . 0015 , * * * P = 0 . 0003 ( Student ' s t - ) .

O O O O O O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F Quantitative real time PCR for Dio2, PPARd and UCP1, using RNA from epididymal fat from 20-week-old male ob/ob and FADD-D/ob/ob mice fed a SD. Data are expressed as mean ± SEM from four independent experiments. *P = 0.0009, **P = 0.0015, ***P = 0.0003 (Student's t-test).


G Quantitative real time PCR for UCP3 , PGC - 1α , PPARd CPT1 , using from skeletal muscle from 20 - week - old ob / ob FADD - D / ob / ob mice fed a SD . Data are expressed as mean ± from four independent experiments . * P = 0 . 0105 , * * P = 0 . 0033 , * * * P = 0 . 0058 ( Student ' s t - ) . NS , not statistically significant .

O O O O O O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

G Quantitative real time PCR for UCP3, PGC-1α, PPARd and CPT1, using RNA from skeletal muscle from 20-week-old male ob/ob and FADD-D/ob/ob mice fed a SD. Data are expressed as mean ± SEM from four independent experiments. *P = 0.0105, **P = 0.0033, ***P = 0.0058 (Student's t-test). NS, not statistically significant.


H Quantitative real time PCR for UCP1 , PPARa , CPT1 PGC - 1α , using from brown adipose fat from 20 - week - old ob / ob FADD - D / ob / ob mice fed a SD . Data are expressed as mean ± from four independent experiments . * P = 0 . 0012 * * P = 0 . 0129 , * * * P = 0 . 0103 , * * * * P = 0 . 0207 ( Student ' s t - ) .

O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

H Quantitative real time PCR for UCP1, PPARa, CPT1 and PGC-1α, using RNA from brown adipose fat from 20-week-old male ob/ob and FADD-D/ob/ob mice fed a SD. Data are expressed as mean ± SEM from four independent experiments. *P = 0.0012 **P = 0.0129, ***P = 0.0103, ****P = 0.0207 (Student's t-test).


I Oxidation of [ 1 - 14C ] oleic acid to 14CO2by adipocytes isolated from WT , FADD - D , ob / ob , FADD - D / ob / ob mice . Data shown are from one experiment ( n = 3 for each genotype ) , representative of a total of two independent experiments . Results are means ± . * P = 0 . 0031 , * * P = 0 . 0075 ( one - way ANOVA ) . NS , not statistically significant .

O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

I Oxidation of [1-14C] oleic acid to 14CO2by adipocytes isolated from WT, FADD-D, ob/ob, and FADD-D/ob/ob mice. Data shown are from one experiment (n = 3 for each genotype), representative of a total of two independent experiments. Results are means ± SEM. *P = 0.0031, **P = 0.0075 (one-way ANOVA). NS, not statistically significant.


J Immunoblot of VLCAD , LCAD , MCAD , PTL Tubulin ( control ) , using extracts from epididymal fat from 15 - week - old WT , FADD - D , ob / ob FADD - D / ob / ob mice fed a SD . Data shown are representative of three independent experiments having similar results .

O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O

J Immunoblot of VLCAD, LCAD, MCAD, PTL and Tubulin (control), using protein extracts from epididymal fat from 15-week-old male WT, FADD-D, ob/ob and FADD-D/ob/ob mice fed a SD. Data shown are representative of three independent experiments having similar results.


( C ) Figures show presence of differentially expressed genes within analyzed single . Black color indicates with RPKM values above 40 for Pax3 , 300 for Stmn2 , 200 for Slc1a3 100 for

O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O

(C) Figures show the presence of differentially expressed genes within the analyzed single cells. Black color indicates cells with RPKM values above 40 for Pax3, 300 for Stmn2, 200 for Slc1a3 and 100 for Mbp