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 .

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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 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 .

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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 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 .

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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 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

O O O O O 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 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 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

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 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 .

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

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 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 .

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 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 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 ) 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 .

O O O O O O O B-CONTROLLED_VAR 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

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 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 .

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

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 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 .

O O 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 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 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

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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 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

O O O O O O O O O O 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

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 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 .

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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 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 .

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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 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 .

O O O O O 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

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 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 .

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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 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 .

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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 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 ) .

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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 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 .

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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 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 .

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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 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 .

O O O O O O O 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 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

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 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

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 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 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 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 ) .

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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 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 ) .

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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 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 ) .

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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 and FADD - D mice ( Scale bars = 50 μm ) . Shown are typical results from four different fields and 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 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 ) .

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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 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 .

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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 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 .

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 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 .

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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 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 .

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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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 , 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 ) .

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

O O O O O O O O O O O O O O O O O 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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.


O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 ) .

O O O O O O O O O O O O O O O O O O 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 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 .

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

O O O O O O O O O O O O O O O O O O O O O O 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 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 .

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 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 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 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 .

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 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

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 ) 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 ) .

O O O B-MEASURED_VAR I-MEASURED_VAR 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 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 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

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

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

O O O O 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 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 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O 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 - 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 ) .

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 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 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 ) .

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

O O O O O O O O O O O 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 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O 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 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 ) .

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 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 ) .

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 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 ) .

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 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 ) .

O O O O O O O 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 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 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 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 ) .

O O O O O O O O B-CONTROLLED_VAR O O O O O O B-MEASURED_VAR O O O O B-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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 ) .

O O O 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 O O 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 ) .

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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 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 ) .

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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 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 .

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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 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 .

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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 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 .

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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 ) 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 ) .

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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 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 ) .

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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 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 ) .

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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 male control and 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 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 ) .

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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 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 .

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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 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 .

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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 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 ) .

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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 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 ) .

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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 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 ) .

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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 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 ) .

O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 ) .

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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 , and skeletal muscle of control and ad - FADD mice . The 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 ± SEM from three independent experiments . * P = 0 . 0044 ( Student ' s t - test ) .

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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 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 ) .

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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 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 ) .

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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 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 ) .

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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 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 .

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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 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 ) .

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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 and three different experiments . Data are expressed as mean ± SEM . * P = 0 . 0003 ( Student ' s t - test ) .

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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 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 ) .

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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 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 .

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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 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 ) .

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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 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 ) .

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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 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 .

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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 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 ) .

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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 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 ) .

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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 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 ) .

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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 - 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 ) .

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 O O O O O O O O O O O O O O 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 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 ) .

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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 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 .

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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 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 , 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 .

O O O O O O O O B-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 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 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 .

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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 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

O O O O O O O O O O O O O O O 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) 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


( B ) Expression ( RPKM ) of Sox3 and Sox9 in NPC and GPC cultures from RNA - seq experiments . Results are represented as mean ± SEM from 3 - 4 experiments ( n = 3 for NPC and n = 4 for GPC , * * * equals p = 4x10 - 5 , t . test )

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(B) Expression (RPKM) of Sox3 and Sox9 in NPC and GPC cultures from RNA-seq experiments. Results are represented as mean ± SEM from 3-4 experiments (n=3 for NPC and n=4 for GPC, *** equals p=4x10-5, t.test)


( C ) Immunohistochemical analysis of SOX3 and SOX9 expression in ESC derived NPC and GPC cultures . Analysis of TUJ1 , GFAP and SOX10 protein expression in differentiated cultures revealed that NPC give rise to neurons and GPCs to astrocytes and oligodendrocytes . Scale bar represents

O O O O O O O O O O O O O O O O O O O O O O O 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) Immunohistochemical analysis of SOX3 and SOX9 expression in ESC derived NPC and GPC cultures. Analysis of TUJ1, GFAP and SOX10 protein expression in differentiated cultures revealed that NPC give rise to neurons and GPCs to astrocytes and oligodendrocytes. Scale bar represents 40µm


1 ] . ( F ) H3K27Ac read density in NPCs or GPCs around DNA - regions bound by SOX3 in NPCs . SOX3 DNA - regions with different H3K27Ac read density patterns in NPCs and GPCs were categorized as group I and II loci . Graphs show the mean read density of H3K27Ac in NPCs and GPCs at group I and II

1]. (F) H3K27Ac read density in NPCs or GPCs around DNA-regions bound by SOX3 in NPCs. SOX3 DNA-regions with different H3K27Ac read density patterns in NPCs and GPCs were categorized as group I and II loci. Graphs show the mean read density of H3K27Ac in NPCs and GPCs at group I and II loci


( G ) Expression pattern of genes associated with group I and II loci ( from Fig . 2E ) within differentially expressed gene sets . Significance calculated by prop . test R , ( * * * ) P < 0 . 001 . ( H ) Venn diagram shows overlap between SOX3 binding in NPCs and GPCs . Bar graph shows expression pattern of genes continuously bound by SOX3 NPCs and GPCs . ( I ) Venn diagram shows overlap between SOX3 and SOX9 binding in GPCs . Bar graph shows expression pattern of genes co - bound by SOX3 and SOX9 in GPCs . ( J ) ChIP - seq peak graphics around the astrocyte gene Fgfbp3 . ChIP - seq peaks are derived from three different experiments ; SOX3 ChIPs in NPCs , SOX3 ChIPs in GPCs , SOX9 ChIPs in GPCs . Both ChIP - seq reads and called peak regions ( underlying black lines ) are shown for all data sets . Bar graphs shows the distribution of differentially expressed genes that are bound by all three factors . P - values ( phyper , R ) were calculated from the total number of protein coding genes in mm10 assembly ( 23 ´ 389 )

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(G) Expression pattern of genes associated with group I and II loci (from Fig. 2E) within differentially expressed gene sets. Significance calculated by prop.test R, (***) P<0.001. (H) Venn diagram shows overlap between SOX3 binding in NPCs and GPCs. Bar graph shows expression pattern of genes continuously bound by SOX3 NPCs and GPCs. (I) Venn diagram shows overlap between SOX3 and SOX9 binding in GPCs. Bar graph shows expression pattern of genes co-bound by SOX3 and SOX9 in GPCs. (J) ChIP-seq peak graphics around the astrocyte gene Fgfbp3. ChIP-seq peaks are derived from three different experiments; SOX3 ChIPs in NPCs, SOX3 ChIPs in GPCs, SOX9 ChIPs in GPCs. Both ChIP-seq reads and called peak regions (underlying black lines) are shown for all data sets. Bar graphs shows the distribution of differentially expressed genes that are bound by all three factors. P-values (phyper, R) were calculated from the total number of protein coding genes in mm10 assembly (23´389)


( A ) Graphical view showing SOX9 and SOX10 expression among spinal cord cells analyzed with scRNA -

(A) Graphical view showing SOX9 and SOX10 expression among spinal cord cells analyzed with scRNA-seq


( B ) Bar graph shows enrichment of genes bound by SOX10 in rat oligodendrocytes within the differentially expressed gene sets . Yellow bar represents enrichment within NPC / GPC genes , green bar within neuronal genes , blue bar within astrocytic genes and red bar within oligodendrocytic genes . SOX10 ChIP - seq data from [

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(B) Bar graph shows enrichment of genes bound by SOX10 in rat oligodendrocytes within the differentially expressed gene sets. Yellow bar represents enrichment within NPC/GPC genes, green bar within neuronal genes, blue bar within astrocytic genes and red bar within oligodendrocytic genes. SOX10 ChIP-seq data from [37


( F ) Expression of Nfia , Ehf and Zbtb3 among spinal cord cells analyzed with scRNA - seq . Red color indicates cells with RPKM values above 55 for Nfia , 5 for Zbtb3 and 5 for Ehf . n = 3 for SOX9 and 2 for SOX3 . P - values ( phyper , R ) were calculated from the total number of genes for mm10 ( 23 ´ 389 )

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(F) Expression of Nfia, Ehf and Zbtb3 among spinal cord cells analyzed with scRNA-seq. Red color indicates cells with RPKM values above 55 for Nfia, 5 for Zbtb3 and 5 for Ehf. n=3 for SOX9 and 2 for SOX3. P-values (phyper, R) were calculated from the total number of genes for mm10 (23´389)


( A ) Luc - reporter assays in P19 cells . Luc - reporters containing SOX3 and SOX9 bound CRMs around the astrocyte specific genes Fgfbp3 and Ttyh1 were strongly activated by SOX9 in synergy with NFIA ; an activation that could be efficiently blocked by SOX3 . No activation of these reporters could be detected with SOX10 or SOX11 . ( B ) Luc - reporters containing SOX9 and SOX10 bound DNA - regions around the astrocyte genes Aldh1l1 and Atp1a2 , which are not expressed in SOX10 + oligodendrocytes , could not be activated by SOX9 or SOX10

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(A) Luc-reporter assays in P19 cells. Luc-reporters containing SOX3 and SOX9 bound CRMs around the astrocyte specific genes Fgfbp3 and Ttyh1 were strongly activated by SOX9 in synergy with NFIA; an activation that could be efficiently blocked by SOX3. No activation of these reporters could be detected with SOX10 or SOX11. (B) Luc-reporters containing SOX9 and SOX10 bound DNA-regions around the astrocyte genes Aldh1l1 and Atp1a2, which are not expressed in SOX10+ oligodendrocytes, could not be activated by SOX9 or SOX10


( C ) Luc - reporters containing SOX9 and SOX10 bound CRMs around the oligodendrocyte specific genes Plekhb1 and Mid2 were activated in an additive manner by SOX9 and SOX10 . No activation could be detected with SOX11 . ( D ) Luc - reporters containing SOX3 and SOX11 bound CRMs of the neuronal genes Tubb3 and Lhx2 , which are sequentially bound by SOX3 and SOX11 , could be activated by SOX11 and efficiently repressed by SOX3 . No activation could be detected with

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Luc-reporters containing SOX9 and SOX10 bound CRMs around the oligodendrocyte specific genes Plekhb1 and Mid2 were activated in an additive manner by SOX9 and SOX10. No activation could be detected with SOX11. (D) Luc-reporters containing SOX3 and SOX11 bound CRMs of the neuronal genes Tubb3 and Lhx2, which are sequentially bound by SOX3 and SOX11, could be activated by SOX11 and efficiently repressed by SOX3. No activation could be detected with SOX10


( E ) Forced expression of SOX3 for 96 hrs in differentiating GPCs blocked the formation of astrocytes but not oligodendrocytes . Scale bar , 25µm . ( F ) Statistical view of Fig . 5E . Bars represents mean values ( of 3 experiments ) of cells expressing GFAP or SOX10 in GFP or Sox3 - transduced cells . Error bars in figure 5 indicate standard deviation of 3 - 7 individual samples and three - star significance > 0 . 001 ( t - test ) . ( * ) 0 , 05 < P < 0 , 01 ; ( * * ) 0 , 01 < P < 0 , 001 ; ( * * * ) P < 0 ,

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(E) Forced expression of SOX3 for 96 hrs in differentiating GPCs blocked the formation of astrocytes but not oligodendrocytes. Scale bar, 25µm. (F) Statistical view of Fig. 5E. Bars represents mean values (of 3 experiments) of cells expressing GFAP or SOX10 in GFP or Sox3-transduced cells. Error bars in figure 5 indicate standard deviation of 3-7 individual samples and three-star significance > 0.001 (t-test). (*) 0,05<P<0,01; (**) 0,01<P<0,001; (***) P<0,001


( A ) SOX3 over - expression experiments in chicken spinal cord at E4 . 48 hrs post electroporation , transfected side showed decreased expression of FGFR2 , NFIA and Fgfr3 . Bar : 50µm . ( B ) 24 hrs after miss - expression of a dominant negative version of SOXB1 ( dnSOXB1 ) , premature expression of FGFR2 , NFIA and Fgfr3 were found in the transfected side . Bar : 40µm . ( C ) 24 hrs after SOX9 over - expression in chicken spinal cord ( at E4 ) , increased expression of FGFR2 , NFIA and Fgfr3 was shown compared to the un - transfected control side . Bar : 40µm . ( D ) Sox3 and Sox9 co - electroporation in chicken spinal cord at E4 could rescue the effect of SOX9 overexpression and FGFR2 , NFIA and Fgfr3 levels were comparable to the un - transfected control side . Embryos were analyzed 24 hrs post electroporation . Bar : 40µm . ( E

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) SOX3 over-expression experiments in chicken spinal cord at E4. 48 hrs post electroporation, transfected side showed decreased expression of FGFR2, NFIA and Fgfr3. Bar: 50µm. (B) 24 hrs after miss-expression of a dominant negative version of SOXB1 (dnSOXB1), premature expression of FGFR2, NFIA and Fgfr3 were found in the transfected side. Bar: 40µm. (C) 24 hrs after SOX9 over-expression in chicken spinal cord (at E4), increased expression of FGFR2, NFIA and Fgfr3 was shown compared to the un-transfected control side. Bar: 40µm. (D) Sox3 and Sox9 co-electroporation in chicken spinal cord at E4 could rescue the effect of SOX9 overexpression and FGFR2, NFIA and Fgfr3 levels were comparable to the un-transfected control side. Embryos were analyzed 24 hrs post electroporation. Bar: 40µm. (E


( B ) Representative images of cells released from mitosis for 1 , 2 or 3h in EdU - containing medium . Top , DNA staining with DAPI ; Bottom , EdU immunofluorescence . L , M and H indicate cells with low , medium or high levels of EdU incorporation . The barplot shows the quantification of the proportion of replicating cells at each time point .

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 O O O O O O O O O

(B) Representative images of cells released from mitosis for 1, 2 or 3h in EdU-containing medium. Top, DNA staining with DAPI; Bottom, EdU immunofluorescence. L, M and H indicate cells with low, medium or high levels of EdU incorporation. The barplot shows the quantification of the proportion of replicating cells at each time point.


Y - axis , z - score ; X - axis , timepoints . A indicates asynchronous cells , M mitotic cells and the numbers represent the minutes after release from the mitotic block ) . Dots show individual replicates and lines averages ( n = 3 for A and M ; n = 2 for each post - mitotic sample ) . Pre - mRNA levels are shown for pluripotency genes ( Oct4 , Klf4 , Esrrb ) and cell cycle regulators ( E2f1 , Cdk2 , Rb ) ; mRNA levels for individual histone genes ( only averages 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Y-axis, z-score; X-axis, timepoints. A indicates asynchronous cells, M mitotic cells and the numbers represent the minutes after release from the mitotic block). Dots show individual replicates and lines averages (n=3 for A and M; n=2 for each post-mitotic sample). Pre-mRNA levels are shown for pluripotency genes (Oct4, Klf4, Esrrb) and cell cycle regulators (E2f1, Cdk2, Rb); mRNA levels for individual histone genes (only averages are shown).


( C ) Percentage of genes presenting at least 1 TF binding site as detected by ChIP - seq within 25kb centred on the TSS , calculated for each gene category shown in ( A ) .

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(C) Percentage of genes presenting at least 1 TF binding site as detected by ChIP-seq within 25kb centred on the TSS, calculated for each gene category shown in (A).


( B ) Heatmaps presented for untreated ( - IAA ; left ) or CTCF - depleted ( + IAA ; right ) CTCF - AID cells .

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(B) Heatmaps presented for untreated (-IAA; left) or CTCF-depleted (+IAA; right) CTCF-AID cells.


( A ) Gene expression changes ( z - score ) in asynchronous cells and during post - mitotic release of CTCF - AID cells treated or not with IAA to deplete CTCF ( blue , presence of CTCF ; red , absence ) . Samples are indicated on the X - axis ( A : asynchronous cells ; M : mitotic cells ; numbers : minutes since release from mitotic block ) . Six gene groups are shown , based on the time of their response to CTCF depletion ( asy , late , early ) and the direction of the transcriptional change ( up , down ) . The number of genes of each group is indicated . The yellow shadow areas indicate times showing expression changes . The boxplots show the median ( bar ) , 25 - 75 % percentiles ( box ) and 1 . 5 - fold inter - quartile range ( whiskers ) .

O O O O O 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

(A) Gene expression changes (z-score) in asynchronous cells and during post-mitotic release of CTCF-AID cells treated or not with IAA to deplete CTCF (blue, presence of CTCF; red, absence). Samples are indicated on the X-axis (A: asynchronous cells; M: mitotic cells; numbers: minutes since release from mitotic block). Six gene groups are shown, based on the time of their response to CTCF depletion (asy, late, early) and the direction of the transcriptional change (up, down). The number of genes of each group is indicated. The yellow shadow areas indicate times showing expression changes. The boxplots show the median (bar), 25-75% percentiles (box) and 1.5-fold inter-quartile range (whiskers).


( C ) Average binding profile ( X - axis , reads per million ) of CTCF ( top ) and the Cohesin subunit SMC1 ( bottom ) , across 8kb centred on the TSS of genes responding to CTCF depletion exclusively in asynchronous cells ( left ) or either late or early upon mitotic release ( middle and right , respectively ) . Interphase is shown in blue and mitosis in red .

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(C) Average binding profile (X-axis, reads per million) of CTCF (top) and the Cohesin subunit SMC1 (bottom), across 8kb centred on the TSS of genes responding to CTCF depletion exclusively in asynchronous cells (left) or either late or early upon mitotic release (middle and right, respectively). Interphase is shown in blue and mitosis in red.


B . Western blot of PTEN variants in a panel of human cancer cell lines using a rabbit monoclonal antibody against the C - terminal region of PTEN The PTEN null prostate cancer cell line PC3 was used as a negative control .

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B. Western blot of PTEN variants in a panel of human cancer cell lines using a rabbit monoclonal antibody against the C-terminal region of PTEN The PTEN null prostate cancer cell line PC3 was used as a negative control.


D . Verification of expression of FLAG - tagged PTENα , PTENβ , and PTENε in PtenFLAG knock - in mice . Various tissues from PtenFLAG knock - in mice or control wild - type mice were lysed for immunoprecipitation with anti - FLAG M2 magnetic beads before being immunoblotted with the anti - PTENα

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D. Verification of expression of FLAG-tagged PTENα, PTENβ, and PTENε in PtenFLAG knock-in mice. Various tissues from PtenFLAG knock-in mice or control wild-type mice were lysed for immunoprecipitation with anti-FLAG M2 magnetic beads before being immunoblotted with the anti-PTENα antibody


B . Reduction of PTENε in response to eIF2A knockdown . Hela cells were infected with lentivirus expressing eIF2A shRNA , eIF5 shRNA , or scramble shRNA separately . The knockdown of eIF2A or eIF5 was validated by western blot ( left panel ) . Lysates of Hela cells were collected and subjected to western blot analysis with antibodies against PTEN , and GAPDH ( right panel ) .

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B. Reduction of PTENε in response to eIF2A knockdown. Hela cells were infected with lentivirus expressing eIF2A shRNA, eIF5 shRNA, or scramble shRNA separately. The knockdown of eIF2A or eIF5 was validated by western blot (left panel). Lysates of Hela cells were collected and subjected to western blot analysis with antibodies against PTEN, and GAPDH (right panel).


C . Overexpression of eIF2A induces the relative expression of PTENε . FLAG - tagged eIF2A or eIF5 were overexpressed in DU145 cells before western blot analysis of PTENε expression with an antibody against PTEN . GAPDH was used as a loading control .

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C. Overexpression of eIF2A induces the relative expression of PTENε. FLAG-tagged eIF2A or eIF5 were overexpressed in DU145 cells before western blot analysis of PTENε expression with an antibody against PTEN. GAPDH was used as a loading control.


C . The transfected Hela PTEN - / - cells were stained with an anti - β - catenin antibody and DAPI before being imaged with confocal microscopy .

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C. The transfected Hela PTEN-/- cells were stained with an anti-β-catenin antibody and DAPI before being imaged with confocal microscopy.


To determine the sequences that are critical for the membrane localization of PTENε . Hela were transfected with constructs Thirty - six hours after transfection , transfected Hela cells were stained with anti - β - catenin antibody and DAPI , followed by imaging with confocal microscopy . The scale bars represent 5 μm ( F ) .

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To determine the sequences that are critical for the membrane localization of PTENε. Hela were transfected with constructs Thirty-six hours after transfection, transfected Hela cells were stained with anti-β-catenin antibody and DAPI, followed by imaging with confocal microscopy. The scale bars represent 5 μm (F).