FLMycER cells , primed or not with OHT ( 48h ) , were treated with 135 nm IACS - 010759 ( IACS ) for 48 hours ( B ) H2O2 quantification , expressed as fold - increase of the 405 / 488 nm fluorescence ratio in treated vs . untreated FLMycER cells , expressing either the cytoplasmic ( left ) or mitochondrial ( right ) roGFP2 - ORP1 biosensor . Data information : * p ≤ 0 . 05 ( one - way ANOVA ) . Each point in the graphs in B from an independent biological replicate , each representing the average of thousands of events ( single cells ) in a distinct cell population , normalized to the untreated condition .

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

FLMycER cells, primed or not with OHT (48h), were treated with 135 nm IACS-010759 (IACS) for 48 hours (B) H2O2 quantification, expressed as fold-increase of the 405/488 nm fluorescence ratio in treated vs. untreated FLMycER cells, expressing either the cytoplasmic (left) or mitochondrial (right) roGFP2-ORP1 biosensor. Data information: * p ≤ 0.05 (one-way ANOVA). Each point in the graphs in B from an independent biological replicate, each representing the average of thousands of events (single cells) in a distinct cell population, normalized to the untreated condition.


FLMycER cells , primed or not with OHT ( 48h ) , were treated with 135 nm IACS - 010759 ( IACS ) for 48 hours ( C ) Superoxide anion O2 • - production in treated vs . untreated FLMycER cells , based on dihydroethidium staining . Data information : * p ≤ 0 . 05 ( one - way ANOVA ) . Each point in the graphs in B and C is from an independent biological replicate , each representing the average of thousands of events ( single cells ) in a distinct cell population , normalized to the untreated condition .

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

FLMycER cells, primed or not with OHT (48h), were treated with 135 nm IACS-010759 (IACS) for 48 hours (C) Superoxide anion O2•- production in treated vs. untreated FLMycER cells, based on dihydroethidium staining. Data information: * p ≤ 0.05 (one-way ANOVA). Each point in the graphs in B and C is from an independent biological replicate, each representing the average of thousands of events (single cells) in a distinct cell population, normalized to the untreated condition.


FLMycER cells were primed with100 nM OHT ( 48h ) and / or treated with 135nM IACS - 010759 , as indicated . ( B ) glutathione quantification in FLMycER cells , measured after 40 hours of IACS - 010759 treatment . Data information : n = 3 biological replicates ; error bars : SD . * p ≤ 0 . 05 ; * * p ≤ 0 . 01 ; * * * p ≤ 0 . 001 ; * * * * p ≤ 0 . 0001 ( one - way ANOVA ) .

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

FLMycER cells were primed with100 nM OHT (48h) and/or treated with 135nM IACS-010759, as indicated. (B) glutathione quantification in FLMycER cells, measured after 40 hours of IACS-010759 treatment. Data information: n=3 biological replicates; error bars: SD. * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001 (one-way ANOVA).


( C ) Cell viability , as determined by PI staining after 48 hours of IACS - 010759 treatment , with or without addition of 10 mM N - acetyl - cysteine ( NAC ) . ( D ) Same as ( C ) , with 50 µM buthionine - sulfoximine ( BSO ) . Data information : n = 3 biological replicates ; error bars : SD . * p ≤ 0 . 05 ; * * p ≤ 0 . 01 ; * * * p ≤ 0 . 001 ; * * * * p ≤ 0 . 0001 ( one - way ANOVA ) .

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

(C) Cell viability, as determined by PI staining after 48 hours of IACS-010759 treatment, with or without addition of 10 mM N-acetyl-cysteine (NAC). (D) Same as (C), with 50 µM buthionine-sulfoximine (BSO). Data information: n=3 biological replicates; error bars: SD. * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001 (one-way ANOVA).


( F ) Immunoblot on lysates from FLMycER cells treated as indicated , with the addition of 10 mM NAC concomitantly with IACS - 010759 .

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

(F) Immunoblot on lysates from FLMycER cells treated as indicated, with the addition of 10 mM NAC concomitantly with IACS-010759.


FLMycER cells were primed with 100 nM OHT and treated with 135 nM IACS - 010759 , as indicated . ( B ) NADPH / NADP redox state in FLMycER cells , assayed after 40 hours of IACS - 010759 treatment . n = 3 biological replicates . * * p ≤ 0 . 01 ; * * * * p ≤ 0 . 0001 ( one - way ANOVA ) .

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

FLMycER cells were primed with 100 nM OHT and treated with 135 nM IACS-010759, as indicated. (B) NADPH/NADP redox state in FLMycER cells, assayed after 40 hours of IACS-010759 treatment. n=3 biological replicates. ** p ≤ 0.01; **** p ≤ 0.0001 (one-way ANOVA).


FLMycER cells were primed with 100 nM OHT and treated with 135 nM IACS - 010759 , as indicated . ( E - G ) Cell viability after 40 hours of IACS - 010759 treatment in the presence of ( E ) 50 µM dehydroepiandrosterone ( DHEA ) , ( F ) 10 µM 6 - aminonicotinamide ( 6AN ) or ( G ) 1 mM 2 - deoxyglucose ( 2DG ) . See Supplemental Figure3F for detailed statistical analysis . Data information : In E , G ) n = 6 ; in ( F ) n = 9 ; all biological replicates ; error bars : SD .

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

FLMycER cells were primed with 100 nM OHT and treated with 135 nM IACS-010759, as indicated. (E-G) Cell viability after 40 hours of IACS-010759 treatment in the presence of (E) 50 µM dehydroepiandrosterone (DHEA), (F) 10 µM 6- aminonicotinamide (6AN) or (G) 1 mM 2-deoxyglucose (2DG). See Supplemental Figure3F for detailed statistical analysis. Data information: In E,G) n=6; in (F) n=9; all biological replicates; error bars: SD.


( A ) Viability of FLMycER and BaFMycER cells primed or not with 100 nM OHT and treated with 135 nM IACS - 010759 for 48h and / or with ascorbate at the indicated concentration for 6h . n = 3 biological replicates ; error bars : SD .

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

(A) Viability of FLMycER and BaFMycER cells primed or not with 100 nM OHT and treated with 135 nM IACS-010759 for 48h and/or with ascorbate at the indicated concentration for 6h. n=3 biological replicates; error bars: SD.


( C ) Cell viability of FL5 . 12cells at the end of treatment ( 48h IACS - 010759 , 6h Asc ) , in the presence or absence of 50 µM deferoxamine ( DFX , added 1 hour before Asc ) . n = 3 biological replicates ; error bars : SD .

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

(C) Cell viability of FL5.12cells at the end of treatment (48h IACS-010759,6h Asc), in the presence or absence of 50 µM deferoxamine (DFX, added 1 hour before Asc). n=3 biological replicates; error bars: SD.


( D ) Quantification of lipid peroxides in FL5 . 12 cells treated with 135 nM IACS - 010759 for 24h and / or 400 µM Asc for 3 hours . * * p ≤ 0 . 01 ; * p ≤ 0 . 05 ( one - way ANOVA ) .

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

(D) Quantification of lipid peroxides in FL5.12 cells treated with 135 nM IACS-010759 for 24h and/ or 400 µM Asc for 3 hours. ** p ≤ 0.01; * p ≤ 0.05 (one-way ANOVA).


The DLBCL cell lines DoHH2 , SU - DHL - 6 , Karpas 422 and HBL - 1 were treated with the indicated concentrations of IACS - 010759 and ascorbate , and cell viability determined after 24 hours . ( A ) Live cell counts . n = 3 biological replicates ; error bars : SD .

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

The DLBCL cell lines DoHH2, SU-DHL-6, Karpas 422 and HBL-1 were treated with the indicated concentrations of IACS-010759 and ascorbate, and cell viability determined after 24 hours. (A) Live cell counts. n=3 biological replicates; error bars: SD.


The indicated lymphoma cell lines and PDX samples were xenografted in recipient animals and tumors allowed to develop to detectable sizes prior to randomization and treatment with IACS - 010759 ( IACS ) by oral gavage and / or ascorbate ( Asc ) by intraperitoneal injection , as indicated ( see Materials and methods ) . Daily doses and the number of animals per group ( n ) are indicated in parenthesis . ( A , B ) Tumor progression ( mm3 ) in CD1 nude mice bearing subcutaneous Ramos ( A ) or DoHH2 tumors ( B ) . Data information : In each panel , the plot on the right shows the data for individual groups on the indicated day . Error bars : SD ; n = animals per group . Right : one - way between - group ANOVA . * p ≤ 0 . 05 ; * * p ≤ 0 . 01 ; * * * 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 B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

The indicated lymphoma cell lines and PDX samples were xenografted in recipient animals and tumors allowed to develop to detectable sizes prior to randomization and treatment with IACS-010759 (IACS) by oral gavage and/or ascorbate (Asc) by intraperitoneal injection, as indicated (see Materials and methods). Daily doses and the number of animals per group (n) are indicated in parenthesis. (A, B) Tumor progression (mm3) in CD1 nude mice bearing subcutaneous Ramos (A) or DoHH2 tumors (B). Data information: In each panel, the plot on the right shows the data for individual groups on the indicated day. Error bars: SD; n = animals per group. Right: one-way between-group ANOVA. * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001.


The indicated lymphoma cell lines and PDX samples were xenografted in recipient animals and tumors allowed to develop to detectable sizes prior to randomization and treatment with IACS - 010759 ( IACS ) by oral gavage and / or ascorbate ( Asc ) by intraperitoneal injection , as indicated ( see Materials and methods ) . Daily doses and the number of animals per group ( n ) are indicated in parenthesis . ( C , D ) Tumor progression in NSG mice injected with the luciferase - positive DHL - derived PDX lines DFBL - 20954 ( C ) or DFBL - 69487 ( D ) , as determined by in vivo imaging and quantification of bilateral femur radiance efficiency . Examples from the control and double - treated groups are shown at the bottom . Data information : In each panel , the plot on the right shows the data for individual groups on the indicated day . Error bars : SD ; n = animals per group . Right : one - way between - group ANOVA . * p ≤ 0 . 05 ; * * p ≤ 0 . 01 ; * * * 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 B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

The indicated lymphoma cell lines and PDX samples were xenografted in recipient animals and tumors allowed to develop to detectable sizes prior to randomization and treatment with IACS-010759 (IACS) by oral gavage and/or ascorbate (Asc) by intraperitoneal injection, as indicated (see Materials and methods). Daily doses and the number of animals per group (n) are indicated in parenthesis. (C, D) Tumor progression in NSG mice injected with the luciferase-positive DHL-derived PDX lines DFBL-20954 (C) or DFBL-69487 (D), as determined by in vivo imaging and quantification of bilateral femur radiance efficiency. Examples from the control and double-treated groups are shown at the bottom. Data information: In each panel, the plot on the right shows the data for individual groups on the indicated day. Error bars: SD; n = animals per group. Right: one-way between-group ANOVA. * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001.


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 ) .

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

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

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

C Quantitative real time PCR for 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 .

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

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

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

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

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 .

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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 ) .

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

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 ) .

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

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 .

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

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


E Basal 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 .

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 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

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 .

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 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

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 ) .

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

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


A Typical growth curves of control 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 ) .

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

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

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 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 ) .

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

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

O O O O 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 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

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 ) .

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

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

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


F Mice were initially maintained at room 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 ) .

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

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

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

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 ) .

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

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

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


I Basal 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 ) .

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

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 ) .

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

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 .

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

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 ) .

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

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

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

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

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

E-H Serum levels of 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 ) .

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

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


A Top , representative 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 .

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

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

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

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 ) .

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

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 ) .

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

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


G Quantitative real time PCR for UCP3 , PGC - 1α , PPARd 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 .

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

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


H Quantitative real time PCR for UCP1 , PPARa , CPT1 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 ) .

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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 .

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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.


Confocal microscopy of IBA - 1 ( green staining ) immunohistochemistry of RPE flatmounts ( RPE autofluorescence visible as orange due to its autofluorescence in the red and green channel ) from a healthy donor ( A ) , a geographic atrophy lesion ( B ) , and large drusen ( C and D ) . ( A , B , D ) : orthogonal Z - stack projection ; ( C ) : oblique Z - stack projection and dissecting microscope appearance of postmortem large drusen after removal of the overlaying retina ( inset ) .

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Confocal microscopy of IBA-1 (green staining) immunohistochemistry of RPE flatmounts (RPE autofluorescence visible as orange due to its autofluorescence in the red and green channel) from a healthy donor (A), a geographic atrophy lesion (B), and large drusen (C and D). (A, B, D): orthogonal Z-stack projection; (C): oblique Z-stack projection and dissecting microscope appearance of postmortem large drusen after removal of the overlaying retina (inset).


Double - labeling on the subretinal side of the retina ( to avoid masking by RPE autofluorescence ) of IBA - 1 + ( E , green fluorescence ) and CD18 ( F , red fluorescence ; G , merge ) .

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Double-labeling on the subretinal side of the retina (to avoid masking by RPE autofluorescence) of IBA-1+ (E, green fluorescence) and CD18 (F, red fluorescence; G, merge).


Orthogonal and lateral Z - stack of a subretinal IBA - 1 + ( green fluorescence ) MPs adjacent to the RPE ( orange autofluorescence ) in the vicinity of a large drusen .

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Orthogonal and lateral Z-stack of a subretinal IBA-1+ (green fluorescence) MPs adjacent to the RPE (orange autofluorescence) in the vicinity of a large drusen.


APOE ( A ) and IBA - 1 ( B , merge in C ) double - labeling on paraffin sections of surgical specimens of human tonsils .

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APOE (A) and IBA-1 (B, merge in C) double-labeling on paraffin sections of surgical specimens of human tonsils.


APOE ( D ) and IBA - 1 ( E , merge in F ) double - labeling on the subretinal aspect of a large drusen overlaying retina ( to avoid specific signal masking by the RPE orange autofluorescence ) .

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APOE (D) and IBA-1 (E, merge in F) double-labeling on the subretinal aspect of a large drusen overlaying retina (to avoid specific signal masking by the RPE orange autofluorescence).


APOE ( red staining ) immunohistochemistry of control donor tissue ( G ) and adjacent to GA lesion ( H ) .

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APOE (red staining) immunohistochemistry of control donor tissue (G) and adjacent to GA lesion (H).


IBA - 1 ( green staining , RPE far - red autofluorescence ) double - labeling of the GA lesion shown in ( H ) . Representative images from five donor eyes with large drusen , five donor eyes with GA , and three healthy donors between 70 and 89 years old .

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IBA-1 (green staining, RPE far-red autofluorescence) double-labeling of the GA lesion shown in (H). Representative images from five donor eyes with large drusen, five donor eyes with GA, and three healthy donors between 70 and 89 years old.


Immunohistochemistry of APOE ( red ) and IBA - 1 ( green ) on a section ( A , blue Hoechst ) and the subretinal side of a retinal flatmount ( B and C ) from a 12 - month - old Cx3cr1GFP / GFP mouse ( representative of 3 independent experiments , experiments omitting the primary antibody immunostaining served as negative controls ) . ONL : outer nuclear layer ; RPE : retinal pigment epithelium .

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Immunohistochemistry of APOE (red) and IBA-1 (green) on a section (A, blue Hoechst) and the subretinal side of a retinal flatmount (B and C) from a 12-month-old Cx3cr1GFP/GFP mouse (representative of 3 independent experiments, experiments omitting the primary antibody immunostaining served as negative controls). ONL: outer nuclear layer; RPE: retinal pigment epithelium.


Quantitative RT - PCR of ApoEmRNA normalized with S26mRNA of C57BL / 6J and Cx3cr1GFP / GFP monocytes cultured for 24 h in contact with POS of an overlaying retinal explant ( n = 4 / group per experiment ; Mann - Whitney U - test , * P = 0 . 0286 , the experiment was repeated twice with similar results ) . Quantitative RT - PCR of ApoEmRNA normalized with S26mRNA of C57BL / 6J and Cx3cr1GFP / GFP FACS - sorted microglial cells , freshly extracted from adult brain ( n = 4 - 5 / group ; Mann - Whitney U - test , C57BL / 6J versus Cx3cr1GFP / GFP * P = 0 . 0159 ) . Quantitative RT - PCR of ApoEmRNA normalized with S26mRNA of C57BL / 6J and Cx3cr1GFP / GFP peritoneal macrophages cultured for 24 h with CX3CL1 . ( n = 4 / group per experiment ; Mann - Whitney U - test , * P = 0 . 0286 . The experiment was repeated twice with 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Quantitative RT-PCR of ApoEmRNA normalized with S26mRNA of C57BL/6J and Cx3cr1GFP/GFP monocytes cultured for 24 h in contact with POS of an overlaying retinal explant (n = 4/group per experiment; Mann-Whitney U-test, *P = 0.0286, the experiment was repeated twice with similar results).Quantitative RT-PCR of ApoEmRNA normalized with S26mRNA of C57BL/6J and Cx3cr1GFP/GFP FACS-sorted microglial cells, freshly extracted from adult brain (n = 4-5/group; Mann-Whitney U-test, C57BL/6J versus Cx3cr1GFP/GFP *P = 0.0159).Quantitative RT-PCR of ApoEmRNA normalized with S26mRNA of C57BL/6J and Cx3cr1GFP/GFP peritoneal macrophages cultured for 24 h with CX3CL1. (n = 4/group per experiment; Mann-Whitney U-test, *P = 0.0286. The experiment was repeated twice with similar results).


APOE Western blot analysis of equivalent amounts of supernatant protein from CX3CL1 - exposed C57BL / 6J and Cx3cr1GFP / GFP peritoneal macrophages at 24 h . Soluble Mer receptor tyrosine kinase that is released constitutively from cultured macrophages served as a loading control . The experiment was repeated twice with similar results .

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APOE Western blot analysis of equivalent amounts of supernatant protein from CX3CL1-exposed C57BL/6J and Cx3cr1GFP/GFP peritoneal macrophages at 24 h. Soluble Mer receptor tyrosine kinase that is released constitutively from cultured macrophages served as a loading control. The experiment was repeated twice with similar results.


12 - month - old IBA - 1 stained RPE flatmounts of Cx3cr1GFP / GFP ( H ) and Cx3cr1GFP / GFPApoE − / − ( I ) mice . Quantification of subretinal IBA - 1 + mononuclear phagocytes in 2 - month - old ( left ) and 12 - month - old ( right ) mice of the indicated strains ( n = 9 - 20 / group ANOVA / Dunnett test at 12 months : Cx3cr1GFP / GFP versus any other group * P < 0 . 0001 ; Mann - Whitney U - test at 12 months of Cx3cr1GFP / GFP versus Cx3cr1GFP / GFPApoE − / − * P < 0 . 0001 ) .

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12-month-old IBA-1 stained RPE flatmounts of Cx3cr1GFP/GFP (H) and Cx3cr1GFP/GFPApoE−/− (I) mice.Quantification of subretinal IBA-1+ mononuclear phagocytes in 2-month-old (left) and 12-month-old (right) mice of the indicated strains (n = 9-20/group ANOVA/Dunnett test at 12 months: Cx3cr1GFP/GFP versus any other group *P < 0.0001; Mann-Whitney U-test at 12 months of Cx3cr1GFP/GFP versus Cx3cr1GFP/GFPApoE−/− *P < 0.0001).


Quantification of subretinal IBA - 1 + mononuclear phagocytes after 4 days of light challenge of 2 - month - old mice of the indicated strains ( n = 10 - 25 / group ANOVA / Dunnett test : Cx3cr1GFP / GFP versus any other group * P < 0 . 0001 ; Mann - Whitney U - test of Cx3cr1GFP / GFP versus Cx3cr1GFP / GFPApoE − / − * P < 0 . 0001 ) .

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Quantification of subretinal IBA-1+ mononuclear phagocytes after 4 days of light challenge of 2-month-old mice of the indicated strains (n = 10-25/group ANOVA/Dunnett test: Cx3cr1GFP/GFP versus any other group *P < 0.0001; Mann-Whitney U-test of Cx3cr1GFP/GFP versus Cx3cr1GFP/GFPApoE−/− *P < 0.0001).


IBA - 1 - ( green ) and CD102 - ( red ) stained RPE flatmounts 7 days after laser injury of 2 - month - old Cx3cr1GFP / GFP ( E ) and Cx3cr1GFP / GFPApoE − / − ( F ) mice . Quantification of subretinal IBA - 1 + mononuclear phagocytes on the RPE counted on the RPE at a distance of 0 - 500 μm to CD102 + CNV 7 days after the laser injury of 2 - month - old mice of the indicated strains ( n = 8 - 10 eyes / group ANOVA / Dunnett test : Cx3cr1GFP / GFP versus any other group * P < 0 . 0001 ; Mann - Whitney U - test of Cx3cr1GFP / GFP versus Cx3cr1GFP / GFPApoE − / − * P < 0 . 0001 ) .

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IBA-1- (green) and CD102- (red) stained RPE flatmounts 7 days after laser injury of 2-month-old Cx3cr1GFP/GFP (E) and Cx3cr1GFP/GFPApoE−/− (F) mice.Quantification of subretinal IBA-1+mononuclear phagocytes on the RPE counted on the RPE at a distance of 0-500 μm to CD102+CNV 7 days after the laser injury of 2-month-old mice of the indicated strains (n = 8-10 eyes/group ANOVA/Dunnett test: Cx3cr1GFP/GFP versus any other group *P < 0.0001; Mann-Whitney U-test of Cx3cr1GFP/GFP versus Cx3cr1GFP/GFPApoE−/− *P < 0.0001).


Representative image of a RPE flatmount 12 h after the subretinal injection ( red marking ) of 4 μl PBS with 12 , 000 CFSE - stained thioglycollate - elicited peritoneal cells that contain 70 % macrophages ( inset close - up view ) . Quantifications of CFSE + F4 / 80 + macrophages at different time points after subretinal injections of C57BL / 6J and Cx3cr1GFP / GFP CFSE + macrophages ( n = 5 / per group ( 12 h ) and n = 6 / per group thereafter ; Mann - Whitney U - test , C57BL / 6J versus Cx3cr1GFP / GFP : 1 day n = 20 / group * P < 0 . 0001 ; 2 day n = 6 / group * P = 0 . 0317 ) .

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Representative image of a RPE flatmount 12 h after the subretinal injection (red marking) of 4 μl PBS with 12,000 CFSE-stained thioglycollate-elicited peritoneal cells that contain 70% macrophages (inset close-up view).Quantifications of CFSE+F4/80+ macrophages at different time points after subretinal injections of C57BL/6J and Cx3cr1GFP/GFP CFSE+ macrophages (n = 5/per group (12 h) and n = 6/per group thereafter; Mann-Whitney U-test, C57BL/6J versus Cx3cr1GFP/GFP: 1 day n = 20/group *P < 0.0001; 2 day n = 6/group *P = 0.0317).


Representative image of TUNEL / Hoechst double - staining 12 h after subretinal injection of C57BL / 6J CFSE + macrophages ( experiment repeated three times , inset close - up view ) .

Representative image of TUNEL/Hoechst double-staining 12 h after subretinal injection of C57BL/6J CFSE+ macrophages (experiment repeated three times, inset close-up view).


Representative cytometry images of SSC - A / CFSE and CD11b / F4 / 80 gated analysis of eye cell suspensions prepared 24 h after the injection of Cx3cr1GFP / GFP CFSE + macrophages and cytometric quantification of eye cell suspensions at 24 h after the injection of C57BL / 6J and Cx3cr1GFP / GFP macrophages into C57BL / 6J ( n = 16 - 20 / group ; Mann - Whitney U - test , * P = 0 . 0024 ) .

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Representative cytometry images of SSC-A/CFSE and CD11b/F4/80 gated analysis of eye cell suspensions prepared 24 h after the injection of Cx3cr1GFP/GFP CFSE+ macrophages and cytometric quantification of eye cell suspensions at 24 h after the injection of C57BL/6J and Cx3cr1GFP/GFP macrophages into C57BL/6J (n = 16-20/group; Mann-Whitney U-test, *P = 0.0024).


Quantification of subretinal CFSE + cells on RPE and retinal flatmounts 24 h after subretinal injections of CFSE + magnetic - bead - sorted bone marrow - derived monocytes ( Mo ) from C57BL / 6J and Cx3cr1GFP / GFPmice into C57BL / 6Jmice ( n = 8 - 12 / group ; Mann - Whitney U - test , * P = 0 . 0006 ) . Quantification of subretinal CFSE + cells on RPE and retinal flatmounts 24 h after subretinal injections of CFSE + CD11bFACS - sorted brainmicroglial cells from C57BL / 6J and Cx3cr1GFP / GFPmice into C57BL / 6Jmice ( n = 9 - 12 / group ; Mann - Whitney U - test , * P = 0 . 0087 ) .

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Quantification of subretinal CFSE+ cells on RPE and retinal flatmounts 24 h after subretinal injections of CFSE+ magnetic-bead-sorted bone marrow-derived monocytes (Mo) from C57BL/6J and Cx3cr1GFP/GFPmice into C57BL/6Jmice (n = 8-12/group; Mann-Whitney U-test, *P = 0.0006).Quantification of subretinal CFSE+ cells on RPE and retinal flatmounts 24 h after subretinal injections of CFSE+CD11bFACS-sorted brainmicroglial cells from C57BL/6J and Cx3cr1GFP/GFPmice into C57BL/6Jmice (n = 9-12/group; Mann-Whitney U-test, *P = 0.0087).


Quantification of subretinal CFSE + F4 / 80 + macrophages on RPE and retinal flatmounts 24 h after subretinal injections of CFSE + macrophages from C57BL / 6J , Cx3cr1GFP / GFP , Cx3cr1GFP / GFPApoE − / − , and ApoE − / − mice into C57BL / 6Jmice ( n = 8 - 12 / group ; one - way ANOVA / Dunnett test of Cx3cr1GFP / GFP versus any other group * P ≤ 0 . 0001 ; Mann - Whitney U - test , Cx3cr1GFP / GFP versus Cx3cr1GFP / GFPApoE − / − * P = 0 . 0006 ) .

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Quantification of subretinal CFSE+F4/80+macrophages on RPE and retinal flatmounts 24 h after subretinal injections of CFSE+macrophages from C57BL/6J, Cx3cr1GFP/GFP, Cx3cr1GFP/GFPApoE−/−, and ApoE−/−mice into C57BL/6Jmice (n = 8-12/group; one-way ANOVA/Dunnett test of Cx3cr1GFP/GFP versus any other group *P ≤ 0.0001; Mann-Whitney U-test, Cx3cr1GFP/GFP versus Cx3cr1GFP/GFPApoE−/− *P = 0.0006).


Quantification of subretinal CFSE + F4 / 80 + macrophages on RPE and retinal flatmounts 24 h after subretinal injections of C57BL / 6J CFSE + macrophages into C57BL / 6J and with exogenously added APOE3 at 1 , 10 , or 100 μg / ml calculated intraocular concentrations ( n = 6 - 7 / group ; one - way ANOVA / Dunnett test : C57BL / 6J versus 10 μg * P = 0 . 0488 ; C57BL / 6J versus 100 μg ‡ P = 0 . 006 . Mann - Whitney U - test : C57BL / 6J versus 10 μg * P = 0 . 0012 ; C57BL / 6J versus 100 μg ‡ P = 0 . 0013 ) .

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Quantification of subretinal CFSE+F4/80+ macrophages on RPE and retinal flatmounts 24 h after subretinal injections of C57BL/6J CFSE+ macrophages into C57BL/6J and with exogenously added APOE3 at 1, 10, or 100 μg/ml calculated intraocular concentrations (n = 6-7/group; one-way ANOVA/Dunnett test: C57BL/6J versus 10 μg *P = 0.0488; C57BL/6J versus 100 μg ‡P = 0.006. Mann-Whitney U-test: C57BL/6J versus 10 μg *P = 0.0012; C57BL/6J versus 100 μg ‡P = 0.0013).


Quantitative RT - PCR of FasLmRNA normalized with β - actinmRNA of 2 - month - , 12 - month - , and 2 - month - old mice after 4 days of light challenge of C57BL / 6 and Cx3cr1GFP / GFP mouse RPE / choroid plexus ( aging : n = 5 - 6 / group ; Mann - Whitney U - test : 12 months * P = 0 . 0129 ; 4 days light challenge : n = 7 - 8 / group ; * P = 0 . 029 ) .

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Quantitative RT-PCR of FasLmRNA normalized with β-actinmRNA of 2-month-, 12-month-, and 2-month-old mice after 4 days of light challenge of C57BL/6 and Cx3cr1GFP/GFP mouse RPE/choroid plexus (aging: n = 5-6/group; Mann-Whitney U-test: 12 months *P = 0.0129; 4 days light challenge: n = 7-8/group; *P = 0.029).


Immunohistochemistry of FASL ( red ) and IBA - 1 ( green ) of a RPE flatmounts and sections ( insets , Hoechst staining blue ) from a 12 - month - old WT ( B ) and Cx3cr1GFP / GFP ( C ) mouse ( representative of three independent experiments , immunostainings omitting the primary antibody served as negative controls ) .

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Immunohistochemistry of FASL (red) and IBA-1 (green) of a RPE flatmounts and sections (insets, Hoechst staining blue) from a 12-month-old WT (B) and Cx3cr1GFP/GFP(C) mouse (representative of three independent experiments, immunostainings omitting the primary antibody served as negative controls).


Quantitative RT - PCR of FasLmRNA normalized with β - actinmRNA of 12 - month - old C57BL / 6 RPE / choroid plexus extracts 3 h after subretinal injection of C57BL / 6J macrophages or Cx3cr1GFP / GFP macrophages ( n = 6 per group ) , Mann - Whitney U - test , * P = 0 . 0087 .

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Quantitative RT-PCR of FasLmRNA normalized with β-actinmRNA of 12-month-old C57BL/6 RPE/choroid plexus extracts 3 h after subretinal injection of C57BL/6J macrophages or Cx3cr1GFP/GFP macrophages (n = 6 per group), Mann-Whitney U-test, *P = 0.0087.


IBA - 1 - stained RPE flatmounts of 2 - month - old C57BL / 6J , Faslgld / gld , and Faslpr / lpr mice after 4 days of light challenge . Quantification of subretinal IBA - 1 + mononuclear phagocytes in control ( left ) and 4 - day light - challenged ( right ) 2 - month - old mice of the indicated strains ( n = 6 - 10 / group ANOVA / Dunnett test at 4 - day light challenge : C57BL / 6J versus FasLgld / gld and C57BL / 6J versus Faslpr / lpr both * P < 0 . 0001 ; Mann - Whitney U - test at 4 - day light challenge : C57BL / 6J versus FasLgld / gld * P < 0 . 0001 ; C57BL / 6J versus Faslpr / lpr * P < 0 . 0001 ) .

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IBA-1-stained RPE flatmounts of 2-month-old C57BL/6J, Faslgld/gld, and Faslpr/lpr mice after 4 days of light challenge.Quantification of subretinal IBA-1+ mononuclear phagocytes in control (left) and 4-day light-challenged (right) 2-month-old mice of the indicated strains (n = 6-10/group ANOVA/Dunnett test at 4-day light challenge: C57BL/6J versus FasLgld/gld and C57BL/6J versus Faslpr/lpr both *P < 0.0001; Mann-Whitney U-test at 4-day light challenge: C57BL/6J versus FasLgld/gld *P < 0.0001; C57BL/6J versus Faslpr/lpr *P < 0.0001).


Quantification of subretinal CFSE + F4 / 80 + macrophages on RPE and retinal flatmounts 24 h after subretinal injection of C57BL / 6JCFSE + macrophages into C57BL / 6J and FasLgld / gldmice and Faslpr / lprmacrophages into C57BL / 6Jmice ( n = 10 - 15 / group , one - way ANOVA / Dunnett test : C57BL / 6Jmacrophages inj . into C57BL / 6Jmice versus C57BL / 6Jmacrophages inj . into Fasgld / gldmice * P = 0 . 0002 ; C57BL / 6Jmacrophages inj . into C57BL / 6Jmice versus Faslpr / lprmacrophages inj . into C57BL / 6Jmice * P < 0 . 0001 . Mann - Whitney U - test : C57BL / 6Jmacrophages inj . into C57BL / 6Jmice versus C57BL / 6Jmacrophages inj . into FasLgld / gldmice * P < 0 . 0001 ; C57BL / 6Jmacrophages inj . into C57BL / 6Jmice versus Faslpr / lprmacrophages inj . into C57BL / 6Jmice * P < 0 . 0001 ) .

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

Quantification of subretinal CFSE+F4/80+macrophages on RPE and retinal flatmounts 24 h after subretinal injection of C57BL/6JCFSE+macrophages into C57BL/6J and FasLgld/gldmice and Faslpr/lprmacrophages into C57BL/6Jmice (n = 10-15/group, one-way ANOVA/Dunnett test: C57BL/6Jmacrophages inj. into C57BL/6Jmice versus C57BL/6Jmacrophages inj. into Fasgld/gldmice *P = 0.0002; C57BL/6Jmacrophages inj. into C57BL/6Jmice versus Faslpr/lprmacrophages inj. into C57BL/6Jmice *P < 0.0001. Mann-Whitney U-test: C57BL/6Jmacrophages inj. into C57BL/6Jmice versus C57BL/6Jmacrophages inj. into FasLgld/gldmice *P < 0.0001; C57BL/6Jmacrophages inj. into C57BL/6Jmice versus Faslpr/lprmacrophages inj. into C57BL/6Jmice *P < 0.0001).


Quantification of subretinal CFSE + F4 / 80 + macrophages on RPE and retinal flatmounts 24 h after subretinal injection of Cx3cr1GFP / GFP CFSE + macrophages into C57BL / 6J with or without the Fas agonist MegaFasL ( calculated intraocular concentrations 1 ng / ml ; n = 7 - 8 ; Mann - Whitney U - test : * P = 0 . 014 ) .

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

Quantification of subretinal CFSE+F4/80+ macrophages on RPE and retinal flatmounts 24 h after subretinal injection of Cx3cr1GFP/GFP CFSE+ macrophages into C57BL/6J with or without the Fas agonist MegaFasL (calculated intraocular concentrations 1 ng/ml; n = 7-8; Mann-Whitney U-test: *P = 0.014).


In vitro MegaFasL - induced apoptosis of monocytes and macrophages of the indicated genotypes cultured for 24 h . TUNEL + nucleus quantification expressed as percentage of non - MegaFasL - exposed control .

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

In vitro MegaFasL-induced apoptosis of monocytes and macrophages of the indicated genotypes cultured for 24 h. TUNEL+ nucleus quantification expressed as percentage of non-MegaFasL-exposed control.


Quantitative RT - PCR of FasLmRNA normalized with β - actinmRNA of 12 - month - old C57BL / 6 RPE / choroid plexus 3 h after subretinal injection of PBS ( PBS ; n = 36 ) , IL - 6 ( n = 17 ) , and APOE3 ( n = 21 ) ; calculated intraocular concentrations : 5 ng / ml and 10 μg / ml , respectively ; one - way ANOVA / Dunnett post hoc test PBS versus IL - 6 * P = 0 . 038 ; t - test PBS versus IL - 6 * P = 0 . 043 .

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

Quantitative RT-PCR of FasLmRNA normalized with β-actinmRNA of 12-month-old C57BL/6 RPE/choroid plexus 3 h after subretinal injection of PBS (PBS; n = 36), IL-6 (n = 17), and APOE3 (n = 21); calculated intraocular concentrations: 5 ng/ml and 10 μg/ml, respectively; one-way ANOVA/Dunnett post hoc test PBS versus IL-6 *P = 0.038; t-test PBS versus IL-6 *P = 0.043.


Mouse IL - 6 ELISA of supernatants from C57BL / 6J peritoneal macrophages incubated for 24 h in control medium , lipid - free APOE3 ( 5 μg / ml ) , APOE3 ( 5 μg / ml ) , and polymyxin B ( 25 μg / ml ) , heat - denatured APOE3 ( dAPOE3 , 5 μg / ml ) , APOE3 ( 5 μg / ml ) , and rat IgG1 isotype control ( IgG , 100 μg / ml ) or APOE3 ( 5 μg / ml ) and rat anti - CD14 antibody ( aCD14 Ab , 100 μg / ml ) or APOE3 ( 5 μg / ml ) and rat anti - TLR2 antibody ( aTLR2 Ab , 100 μg / ml ) ( n = 5 - 6 / group ; one - way ANOVA / Bonferroni multi - comparison tests : APOE3 versus CTL * P < 0 . 0001 ; dAPOE3 versus APOE3 # P < 0 . 0001 ; APOE3 IgG versus CTL P < 0 . 0001 ; APOE3 IgG versus APOE3 aCD14 Ab § P < 0 . 0001 ; APOE3 IgG versus APOE3 aTLR2 Ab + + P < 0 . 0001 . Mann - Whitney U - test : APOE3 versus CTL * P = 0 . 0043 ; dAPOE3 versus APOE3 # P = 0 . 0117 ; APOE3 IgG versus CTL P = 0 . 0080 ; APOE3 IgG versus APOE3 aCD14 Ab § P = 0 . 0117 ; APOE3 IgG versus APOE3 aTLR2 Ab + + P = 0 . 0079 . The experiment was repeated twice with 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Mouse IL-6 ELISA of supernatants from C57BL/6J peritoneal macrophages incubated for 24 h in control medium, lipid-free APOE3 (5 μg/ml), APOE3 (5 μg/ml), and polymyxin B (25 μg/ml), heat-denatured APOE3 (dAPOE3, 5 μg/ml), APOE3 (5 μg/ml), and rat IgG1 isotype control (IgG, 100 μg/ml) or APOE3 (5 μg/ml) and rat anti-CD14 antibody (aCD14 Ab, 100 μg/ml) or APOE3 (5 μg/ml) and rat anti-TLR2 antibody (aTLR2 Ab, 100 μg/ml) (n = 5-6/group; one-way ANOVA/Bonferroni multi-comparison tests: APOE3 versus CTL *P < 0.0001; dAPOE3 versus APOE3 #P < 0.0001; APOE3 IgG versus CTL P < 0.0001; APOE3 IgG versus APOE3 aCD14 Ab §P < 0.0001; APOE3 IgG versus APOE3 aTLR2 Ab ++P <0.0001. Mann-Whitney U-test: APOE3 versus CTL *P = 0.0043; dAPOE3 versus APOE3 #P = 0.0117; APOE3 IgG versus CTL P = 0.0080; APOE3 IgG versus APOE3 aCD14 Ab §P = 0.0117; APOE3 IgG versus APOE3 aTLR2 Ab ++P = 0.0079. The experiment was repeated twice with similar results).