E ) Primary microglia cultures were obtained using CD11b + magnetic beads from old male mice with or without microglia renewal ( n = 2 per group ) . Cells were exposed to red fluorescent pHrodo E . coli bioparticles and were stained with green fluorescent Bodipy and studied by confocal microscopy . Black and white images illustrate raw intensity data of Bodipy staining . The lower images are corresponding merged channels showing Bodipy in green , phagocytosed bioparticles in red , and the DAPI + nuclei in blue . Repopulated microglia show less lipid droplets and the cytoplasm is replenished with phagocytosed bioparticles . The square regions of the images indicate areas magnified at the side of each image . Scale bar = 20 µm .

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E) Primary microglia cultures were obtained using CD11b+ magnetic beads from old male mice with or without microglia renewal (n=2 per group). Cells were exposed to red fluorescent pHrodo E. coli bioparticles and were stained with green fluorescent Bodipy and studied by confocal microscopy. Black and white images illustrate raw intensity data of Bodipy staining. The lower images are corresponding merged channels showing Bodipy in green, phagocytosed bioparticles in red, and the DAPI+ nuclei in blue. Repopulated microglia show less lipid droplets and the cytoplasm is replenished with phagocytosed bioparticles. The square regions of the images indicate areas magnified at the side of each image. Scale bar = 20 µm.


F ) Electron microscopy images representative of microglia of old male mice with original microglia ( Old ) and with renewed microglia ( Old - Renewed ) ( n = 3 per group ) . Images labelled ( a - d ) are higher magnifications of the areas marked with yellow squares in the images on the left . Lipid droplets are markes with arrows . Large lipid droplets are seen in microglia of old mice , whereas renewed microglia typically show no or small lipid droplets .

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F) Electron microscopy images representative of microglia of old male mice with original microglia (Old) and with renewed microglia (Old-Renewed) (n=3 per group). Images labelled (a-d) are higher magnifications of the areas marked with yellow squares in the images on the left. Lipid droplets are markes with arrows. Large lipid droplets are seen in microglia of old mice, whereas renewed microglia typically show no or small lipid droplets.


C ) . The MRI lesion volume , as illustrated in the corresponding false color images below , showed no differences in infarct volume in mice with ( n = 16 ) or without ( n = 15 ) microglia renewal . Results correspond to day four post - ischemia . Representative MRI images obtained on day four post - ischemia using T2w MRI and day 14 using a Modified Driven Equilibrium Fourier Transform ( MDEFT ) MRI sequence ( 7 . 0 T BioSpec 70 / 30 , Bruker BioSpin ) . Values show data for individual mice and / or the mean ± SD .

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C). The MRI lesion volume, as illustrated in the corresponding false color images below, showed no differences in infarct volume in mice with (n=16) or without (n=15) microglia renewal. Results correspond to day four post-ischemia. Representative MRI images obtained on day four post-ischemia using T2w MRI and day 14 using a Modified Driven Equilibrium Fourier Transform (MDEFT) MRI sequence (7.0 T BioSpec 70/30, Bruker BioSpin). Values show data for individual mice and/or the mean±SD.


3D - brain model , horizontal brain section illustrating transgenic human P301Ltau expression in the entorhinal cortex ( green , EC ) of the ECrTgTaumouse lines , and the propagation of transgenic tau to the dentate gyrus ( DG ) . Tau composition in ECrTgTau and control mouse lines investigated .

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3D-brain model, horizontal brain section illustrating transgenic human P301Ltau expression in the entorhinal cortex (green, EC) of the ECrTgTaumouse lines, and the propagation of transgenic tau to the dentate gyrus (DG). Tau composition in ECrTgTau and control mouse lines investigated.


Immunostained horizontal sections show the expression of human P301Ltau in ECneurons in absence of endogenous mousetau ( ECrTgTau - Mapt0 / 0 ) . Fluorescence in situ hybridization of humantau mRNA combined with immunofluorescent labeling ( Immuno - FISH ) of humantau protein ( huTau ) verifies P301Ltau transgene expression in the EC . Scale bars 50 μm .

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Immunostained horizontal sections show the expression of human P301Ltau in ECneurons in absence of endogenous mousetau (ECrTgTau-Mapt0/0). Fluorescence in situ hybridization of humantau mRNA combined with immunofluorescent labeling (Immuno-FISH) of humantau protein (huTau) verifies P301Ltau transgene expression in the EC. Scale bars 50 μm.


Propagation of humantau protein to neurons in the DG ( white arrows ) in ECrTgTau - Mapt0 / 0 mice . Close - ups show DG neurons from three ECrTgTau - Mapt0 / 0 mice ( DG I - III ) . Immuno - FISH proofs the absence of humantau expression in DG neurons , which have huTau protein but no humantau mRNA . Scale bars 50 μm .

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Propagation of humantau protein to neurons in the DG (white arrows) in ECrTgTau-Mapt0/0 mice. Close-ups show DG neurons from three ECrTgTau-Mapt0/0 mice (DG I-III). Immuno-FISH proofs the absence of humantau expression in DG neurons, which have huTau protein but no humantau mRNA. Scale bars 50 μm.


Immunostained horizontal sections of ECrTgTaumice show the expression of human P301Ltau in ECneurons in presence of endogenous mousetau . Close - ups show DGneurons from three ECrTgTaumice ( DG I - III ) and Immuno - FISH proofs the absence of humantau expression in these DGneurons . Scale bars 50 μm .

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Immunostained horizontal sections of ECrTgTaumice show the expression of human P301Ltau in ECneurons in presence of endogenous mousetau. Close-ups show DGneurons from three ECrTgTaumice (DG I-III) and Immuno-FISH proofs the absence of humantau expression in these DGneurons. Scale bars 50 μm.


Human P301Ltau propagation to DG neurons ( white arrows ) in presence of endogenous mouse tau in ECrTgTau mice . Scale bars 50 µm .

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Human P301Ltau propagation to DG neurons (white arrows) in presence of endogenous mouse tau in ECrTgTau mice. Scale bars 50 µm.


Human ( huTau , antibody Tau13 ) and total tau ( hu + moTau , DAKO ) levels in entorhinal cortex ( EC ) extracts from 18 - month - oldmice show equal human P301Ltau expression in ECrTgTau and ECrTgTau - Mapt0 / 0 mice ( p = 0 . 201 , n = 3 mice / group , one - way ANOVA with Bonferroni correction )

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Human (huTau, antibody Tau13) and total tau (hu+moTau, DAKO) levels in entorhinal cortex (EC) extracts from 18-month-oldmice show equal human P301Ltau expression in ECrTgTau and ECrTgTau-Mapt0/0 mice (p=0.201, n=3 mice/group, one-way ANOVA with Bonferroni correction)


The number of human tau - positive cell bodies in the DG ( p = 0 . 58 , n = 4 sections and 3 mice / group ) and human tau in hippocampal ( HPC ) extracts ( p = 0 . 14 , n = 3 mice / group ) were similar in ECrTgTau - Mapt0 / 0 and ECrTgTau mice ( two - tailed Student ' s T - test ) .

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The number of human tau-positive cell bodies in the DG (p=0.58, n=4 sections and 3 mice/group) and human tau in hippocampal (HPC) extracts (p=0.14, n=3 mice/group) were similar in ECrTgTau-Mapt0/0 and ECrTgTau mice (two-tailed Student's T-test).


Primary cortical neuron cultures that were transduced with AAVeGFP - 2a - P301Ltau at 7 DIV , and fixed and immunostained for GFP and humantau ( Tau13 antibody ) at 14 DIV , show tau donor ( GFP + , huTau + ; ~ 10 % neurons ) and a small number of tau recipient neurons ( GFP - , huTau + ; ~ 1 % neurons ) . Western blot of whole cell lysates verified efficient cleavage ( ~ 95 % ) of eGFP and P301Ltau by the 2a peptide ( n = 3 ) .

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Primary cortical neuron cultures that were transduced with AAVeGFP-2a-P301Ltau at 7 DIV, and fixed and immunostained for GFP and humantau (Tau13 antibody) at 14 DIV, show tau donor (GFP+, huTau+; ~10% neurons) and a small number of tau recipient neurons (GFP-, huTau+; ~1% neurons). Western blot of whole cell lysates verified efficient cleavage (~95%) of eGFP and P301Ltau by the 2a peptide (n=3).


Eight weeks after AAV injection into right EC of aged Mapt0 / 0 mice ( = 3 ) , immunostained brain sections showed that huTauP301L ( red ) propagated to a few DG " recipient neurons " ( white arrows ) . Scale bar 50 µm .

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Eight weeks after AAV injection into right EC of aged Mapt0/0 mice (=3), immunostained brain sections showed that huTauP301L (red) propagated to a few DG "recipient neurons" (white arrows). Scale bar 50 µm.


Unilateral AAV - mediated human P301L tau expression in the EC and DG of age - matched WT mice ( n = 3 ) . Representative images of brain sections show donor neurons in the injected EC and DG , and a few tau recipient neurons ( white arrows ) adjacent to the AAV injection site . Scale bar 50 µm . In the contralateral hemisphere of the same brain section , some tau recipient neurons ( white arrows ) were also present in the ( non - injected ) axonal projection areas in the contralateral EC ( GFP - filled terminal ends ) . Scale bar 100 µm .

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Unilateral AAV-mediated human P301L tau expression in the EC and DG of age-matched WT mice (n=3). Representative images of brain sections show donor neurons in the injected EC and DG, and a few tau recipient neurons (white arrows) adjacent to the AAV injection site. Scale bar 50 µm.In the contralateral hemisphere of the same brain section, some tau recipient neurons (white arrows) were also present in the (non-injected) axonal projection areas in the contralateral EC (GFP-filled terminal ends). Scale bar 100 µm.


A . Brain sections from 18 - month - old ECrTgTau - Mapt0 / 0 and ECrTgTau mice were co - immuolabeled for human tau and misfolded tau ( Alz50 ) . Misfolded tau was only found in EC and DG neurons ( white arrows ) of ECrTgTau but not ECrTgTau - Mapt0 / 0 animals ( n = 4 sections / mouse , 3 mice / group ) . Scale bars 50 μm .

A. Brain sections from 18-month-old ECrTgTau-Mapt0/0 and ECrTgTau mice were co-immuolabeled for human tau and misfolded tau (Alz50). Misfolded tau was only found in EC and DG neurons (white arrows) of ECrTgTau but not ECrTgTau-Mapt0/0 animals (n=4 sections/mouse, 3 mice/group). Scale bars 50 μm.


B + C . Immunofluorescence labeling and stereological counting of microglia in entorhinal cortex ( B ) and astrocytes in hippocampus ( C ) indicated early signs of neurodegeneration in ECrTgTaumice . The significantly increased number of Iba1 - positive microglia in the EC layer II / III of ECrTgtau mice ( compared to WT ) was partially rescued in ECrTgTau - Mapt0 / 0 mice ( non - significant ) . The number of GFAP - positive astrocytes was similar across all genotypes ( non - significant ) . Mean ± SEM , n = 4 sections per mouse , 3 mice / group , one - way ANOVA with Bonferroni correction . Scale bars 100 μm .

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B+C. Immunofluorescence labeling and stereological counting of microglia in entorhinal cortex (B) and astrocytes in hippocampus (C) indicated early signs of neurodegeneration in ECrTgTaumice. The significantly increased number of Iba1-positive microglia in the EC layer II/III of ECrTgtau mice (compared to WT) was partially rescued in ECrTgTau-Mapt0/0 mice (non-significant). The number of GFAP-positive astrocytes was similar across all genotypes (non-significant). Mean±SEM, n=4 sections per mouse, 3 mice/group, one-way ANOVA with Bonferroni correction. Scale bars 100 μm.


Human , mouse and total tau protein levels in cortical TBS - extracts of rTg4510 , rTg4510 - Mapt0 / 0 , and control mice : The amount of human tau ( Tau13 antibody ) was comparable in rTg4510 and rTg4510 - Mapt0 / 0 , moTau ( Tau / 5 ) was comparable in WT and rTg4510 , and total tau levels ( hu + moTau , DAKO antibody ) were ( expected ) highest in rTg4510 mice . Mean ± SEM , n = 3 mice / group , non - significant .

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Human, mouse and total tau protein levels in cortical TBS-extracts of rTg4510, rTg4510-Mapt0/0, and control mice: The amount of human tau (Tau13 antibody) was comparable in rTg4510 and rTg4510-Mapt0/0, moTau (Tau/5) was comparable in WT and rTg4510, and total tau levels (hu+moTau, DAKO antibody) were (expected) highest in rTg4510 mice. Mean±SEM, n=3 mice/group, non-significant.


Whole brain weights of 9 - month - old animals revealed pronounced brain matter loss in rTg4510 compared to WT mice ( weight loss > 16 % ) , which was rescued in rTg4510 - Mapt0 / 0 mice to > 96 % . Mean ± SEM , n = 5 mice / group .

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Whole brain weights of 9-month-old animals revealed pronounced brain matter loss in rTg4510 compared to WT mice (weight loss >16%), which was rescued in rTg4510-Mapt0/0 mice to >96%. Mean±SEM, n=5 mice/group.


Cortical thickness measured adjacent to HPC , from CTX surface to corpus callosum , was decreased in rTg4510 mice by ≈ 25 % compared to WT mice . rTg4510 - Mapt0 / 0 showed no CTX thinning compared to Mapt0 / 0 or WT mcie . Mean ± SEM , n = 3 mice / group .

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Cortical thickness measured adjacent to HPC, from CTX surface to corpus callosum, was decreased in rTg4510 mice by ≈25% compared to WT mice. rTg4510-Mapt0/0 showed no CTX thinning compared to Mapt0/0 or WT mcie. Mean±SEM, n=3 mice/group.


The number of neurons ( NeuN + cells ) in the cortex of rTg4510 mice was significantly reduced to ≈ 67 % compared to both WT and rTg4510 - Mapt0 / 0 . Mean ± SEM , n = 3 mice / group .

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The number of neurons (NeuN+ cells) in the cortex of rTg4510 mice was significantly reduced to ≈67% compared to both WT and rTg4510-Mapt0/0. Mean±SEM, n=3 mice/group.


The volume of hippocampal region CA1 , with CT the most effected regions in rTg4510 mice , was significantly reduced in rTg4510 by ~ 70 % volume ; rTg4510 - Mapt0 / 0 had significantly larger CA1 volume left ( reduced by only ~ 40 % ) . Mean ± SEM , n = 3 mice / group .

The volume of hippocampal region CA1, with CT the most effected regions in rTg4510 mice, was significantly reduced in rTg4510 by ~70% volume; rTg4510-Mapt0/0 had significantly larger CA1 volume left (reduced by only ~40%). Mean±SEM, n=3 mice/group.


rTg4510 showed strong signs of neuroinflammation with extremely high numbers of activated astroglia ( GFAP + , red ) and microglia ( Iba1 + , white ) in the CTX compared to WT mice . Both astro - and microgliosis was reduced by ~ 50 % in rTg4510 - Mapt0 / 0 mice . Mean ± SEM , n = 3 sections / mouse and 5 mice per / group . For all panels : two - tailed Student ' s T - test and one - way ANOVA with Bonferroni for multiple comparison , ns not significant , scale bars 100 µm .

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rTg4510 showed strong signs of neuroinflammation with extremely high numbers of activated astroglia (GFAP+, red) and microglia (Iba1+, white) in the CTX compared to WT mice. Both astro- and microgliosis was reduced by ~50% in rTg4510-Mapt0/0 mice. Mean±SEM, n=3 sections/mouse and 5 mice per/group. For all panels: two-tailed Student's T-test and one-way ANOVA with Bonferroni for multiple comparison, ns not significant, scale bars 100 µm.


Cortical extracts from rTg4510 - Mapt0 / 0 brains had significantly less phospho - tau ( CP13 , PHF1 , 12E8 ) than rTg4510 extracts . Compared to WT mice , both transgenic tau lines had high levels of phospho - tau . Mean ± SEM , n = 3 mice / group .

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Cortical extracts from rTg4510-Mapt0/0 brains had significantly less phospho-tau (CP13, PHF1, 12E8) than rTg4510 extracts. Compared to WT mice, both transgenic tau lines had high levels of phospho-tau. Mean±SEM, n=3 mice/group.


Representative images of gallyas silver stained aggregated tau in cortices from 12 - month - old mice unravel stunning differences in the degree of tau pathology in rTg4510 compared to rTg4510 - Mapt0 / 0 mice . Mean ± SEM , n = 3 mice / group .

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Representative images of gallyas silver stained aggregated tau in cortices from 12-month-old mice unravel stunning differences in the degree of tau pathology in rTg4510 compared to rTg4510-Mapt0/0 mice. Mean±SEM, n=3 mice/group.


Higher magnification images of silver ( 12 month - old ) and Thioflavine - S ( 9month - old ) stained cortices show mature tangles ( white arrows in Thio - S stain ) in rTg4510 and rTg4510 - Mapt0 / 0 mice ; enhanced pathological changes such as neuritic tau accumulation and neuropil vacuolation around NFTs are found only in rTg4510 mice . Stereological counting revealed similar numbers of cortical NFTs between in rTg4510 and rTg4510 - Mapt0 / 0 mice at 9 and 12 month of age . Because of the pronounced neuronal death only in rTg4510 mice , the percentage of tangle - bearing neurons was ≈ 1 . 6 to 1 . 8 - fold higher higher in 9 and 12 - month old rTg4510 mice . Mean ± SEM , n = 3 sections / mouse , 3 mice / group .

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Higher magnification images of silver (12 month-old) and Thioflavine-S (9month-old) stained cortices show mature tangles (white arrows in Thio-S stain) in rTg4510 and rTg4510-Mapt0/0 mice; enhanced pathological changes such as neuritic tau accumulation and neuropil vacuolation around NFTs are found only in rTg4510 mice. Stereological counting revealed similar numbers of cortical NFTs between in rTg4510 and rTg4510-Mapt0/0 mice at 9 and 12 month of age. Because of the pronounced neuronal death only in rTg4510 mice, the percentage of tangle-bearing neurons was ≈1.6 to 1.8-fold higher higher in 9 and 12-month old rTg4510 mice. Mean±SEM, n=3 sections/mouse, 3 mice/group.


Immuno - FISH for huTau mRNA ( green ) and phospho - tau ( PHF1 , red ) shows obvious differences in the distribution of neurons in cortex layer II / III : 9 - month - old rTg4510 mice had more neurons filled with NFT - like phosphorylated tau ( PHF1 + , red ) , and rTg4510 - Mapt0 / 0 mice had significantly more huTau mRNA positive neurons ( FISH + ) . rTg4510 - Mapt0 / 0 mice had also more neurons still expressing both PHF1 and huTau mRNA ( PHF1 + FISH + ) , suggesting a reduced neurotoxicity of P301Ltau expression in rTg4510 - Mapt0 / 0 mice . Mean ± SEM , n = 3 sections per mouse , 3 mice / group . For all panels : two - tailed Student ' s T - test and one - way ANOVA with Bonferroni for multiple comparison , ns not significant , scale bars 100 μm .

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Immuno-FISH for huTau mRNA (green) and phospho-tau (PHF1, red) shows obvious differences in the distribution of neurons in cortex layer II/III: 9-month-old rTg4510 mice had more neurons filled with NFT-like phosphorylated tau (PHF1+, red), and rTg4510-Mapt0/0 mice had significantly more huTau mRNA positive neurons (FISH+). rTg4510-Mapt0/0 mice had also more neurons still expressing both PHF1 and huTau mRNA (PHF1+ FISH+), suggesting a reduced neurotoxicity of P301Ltau expression in rTg4510-Mapt0/0 mice. Mean±SEM, n=3 sections per mouse, 3 mice/group. For all panels: two-tailed Student's T-test and one-way ANOVA with Bonferroni for multiple comparison, ns not significant, scale bars 100 μm.


Immuno - FISH showing EC neurons having both misfolded somatic tau ( Alz50 , red ) and human tau mRNA ( green ; white arrows ) in rTg4510 - Mapt0 / 0 but rarely in rTg4510 mice . n = 3 sections , 2 mice / group . Scale bars 50 μm .

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Immuno-FISH showing EC neurons having both misfolded somatic tau (Alz50, red) and human tau mRNA (green; white arrows) in rTg4510-Mapt0/0 but rarely in rTg4510 mice. n=3 sections, 2 mice/group. Scale bars 50 μm.


Extraction of cortices revealed similar human tau ( Tau13 ) in TBS - extracts ( not significant ) but significantly more human tau in TritonX - 100 ( TX - 100 ) extracts of rTg4510 - Mapt0 / 0 compared to rTg4510 mice . Mean ± SEM , n = 3 mice / group , two - tailed Student ' s T test , ns non significant .

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Extraction of cortices revealed similar human tau (Tau13) in TBS-extracts (not significant) but significantly more human tau in TritonX-100 (TX-100) extracts of rTg4510-Mapt0/0 compared to rTg4510 mice. Mean±SEM, n =3 mice/group, two-tailed Student's T test, ns non significant.


Native gel electrophoresis of cortical TBS - extracts showed small differences in HMW ( oligomeric ) human tau between rTg4510 and rTg4510 - Mapt0 / 0 brains . Western blot lanes were averaged across ~ 2 / 3 of the width ( black rectangular and arrow in Tau13 blot ) . The mean ± SEM ( n = 3 mice / group ) of these averages was plotted as longitudinal lane profiles . Differences in HMW tau are indicated by red and black arrows .

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Native gel electrophoresis of cortical TBS-extracts showed small differences in HMW (oligomeric) human tau between rTg4510 and rTg4510-Mapt0/0 brains. Western blot lanes were averaged across ~2/3 of the width (black rectangular and arrow in Tau13 blot). The mean±SEM (n=3 mice/group) of these averages was plotted as longitudinal lane profiles. Differences in HMW tau are indicated by red and black arrows.


TBS - brain extracts were applied to a HEK293 cell tau aggregation seeding assay ( Holmes et al , 2014 , Sanders et al , 2014 ) , in which TauRDP301S - CFP and TauRDP301S - YFP are co - expressed intracellularly . The formation of intracellular fluorescent TauRDP301S aggregates leads to Foerster resonance energy transfer ( FRET ) activity between co - aggregated CFP and YFP - tags and correlates with the tau aggregation seeding activity of the applied brain extracts . After 24 hours , cells treated with extract ( 0 . 5 µg and 1 . 0 µg total protein per 96 well ) from 9 - month - old rTg4510 had significantly more intracellular YFP - positive ( white arrows ) aggregates compared to rTg4510 - Mapt0 / 0 extracts ; FRET activity of TauRDP301S aggregates appeared similar for both rTg4510 and rTg4510 - Mapt0 / 0 . WT and Mapt0 / 0 extracts never showed seeding activity . Addition of lipofectamine corrected for differences in cellular uptake of tau and led to similar differences in seeding activity between rTg4510 and rTg4510 - Mapt0 / 0 mice . Two - tailed Student ' s T - test , mean ± SEM ns , not significant , Insets 100 µm , Scale bars 50 µm .

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

TBS-brain extracts were applied to a HEK293 cell tau aggregation seeding assay(Holmes et al, 2014, Sanders et al, 2014), in which TauRDP301S-CFP and TauRDP301S-YFP are co-expressed intracellularly. The formation of intracellular fluorescent TauRDP301S aggregates leads to Foerster resonance energy transfer (FRET) activity between co-aggregated CFP and YFP-tags and correlates with the tau aggregation seeding activity of the applied brain extracts. After 24 hours, cells treated with extract (0.5 µg and 1.0 µg total protein per 96 well) from 9-month-old rTg4510 had significantly more intracellular YFP-positive (white arrows) aggregates compared to rTg4510-Mapt0/0 extracts; FRET activity of TauRDP301S aggregates appeared similar for both rTg4510 and rTg4510-Mapt0/0. WT and Mapt0/0 extracts never showed seeding activity. Addition of lipofectamine corrected for differences in cellular uptake of tau and led to similar differences in seeding activity between rTg4510 and rTg4510-Mapt0/0 mice. Two-tailed Student's T-test, mean±SEM ns, not significant, Insets 100 µm, Scale bars 50 µm.


( A ) C57BL / 6 mice were inoculated with 6x105 B16Bl6 parental or dnTNF - R1 cells on day 0 and treated daily with 7µg mTNF p . l . from day 10 . Tumor growth is shown as mean tumor size index ( TSI ) + SEM ( n = 6 ) . The line under the graph represents the treatment period .

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(A) C57BL/6 mice were inoculated with 6x105 B16Bl6 parental or dnTNF-R1 cells on day 0 and treated daily with 7µg mTNF p.l. from day 10. Tumor growth is shown as mean tumor size index (TSI) + SEM (n = 6). The line under the graph represents the treatment period.


( B ) B16Bl6 tumor growth after daily p . l . treatment with 7µg mTNF in C57BL / 6J wild - type ( WT ) , TNF - R1 - / - , conditional TNF - R1 reactivation knockout ( p55cneo / cneo ) , or p55cneo / cneo mice with Cre expression in endothelium ( Flk1Cre ) or all cells ( DelCre ) . Tumor growth is shown as mean TSI + SEM ( n = 7 for WT and Flk1Cre , 8 for DelCre , 9 for TNF - R1 - / - and 10 for p55cneo / cneo ) . The line under the graph represents the treatment period .

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(B) B16Bl6 tumor growth after daily p.l. treatment with 7µg mTNF in C57BL/6J wild-type (WT), TNF-R1-/-, conditional TNF-R1 reactivation knockout (p55cneo/cneo), or p55cneo/cneo mice with Cre expression in endothelium (Flk1Cre) or all cells (DelCre). Tumor growth is shown as mean TSI + SEM (n = 7 for WT and Flk1Cre, 8 for DelCre, 9 for TNF-R1-/- and 10 for p55cneo/cneo). The line under the graph represents the treatment period.


( C ) PCR analysis for the detection of wild - type , conditional knockout ( cneo ) or reactivated ( lox ) TNF - R1 allele in whole tissues , lung endothelial cells ( LECs ) and lung single - cell suspension depleted of CD31 + cells .

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(C) PCR analysis for the detection of wild-type, conditional knockout (cneo) or reactivated (lox) TNF-R1 allele in whole tissues, lung endothelial cells (LECs) and lung single-cell suspension depleted of CD31+ cells.


( D ) Toxicity of a single i . v . bolus injection of the indicated dose of mTNF . Mean rectal body temperature + SEM and cumulative survival rates are shown ( n = 4 for Flk1Cre 10µg ; 6 for DelCre 10µg , Flk1Cre 10µg and p55cneo / cneo 15µg ; 7 for WT 10µg ; 9 for WT 8µg , 10 for DelCre 20µg ; and 5 for all other groups ) . For continuity of the temperature graphs , dead mice were included with a temperature of 20 ° C .

(D) Toxicity of a single i.v. bolus injection of the indicated dose of mTNF. Mean rectal body temperature + SEM and cumulative survival rates are shown (n = 4 for Flk1Cre 10µg; 6 for DelCre 10µg, Flk1Cre 10µg and p55cneo/cneo 15µg; 7 for WT 10µg; 9 for WT 8µg, 10 for DelCre 20µg; and 5 for all other groups). For continuity of the temperature graphs, dead mice were included with a temperature of 20°C.


( B , C ) Viability of L929 parental or mCD20 - expressing cells after 72h stimulation with mTNF or sc mTNF mutant Y86F fused to a BcII10 or mCD20 VHH ( B ) and viability of MCF7 parental or hCD20 - expressing cells after 72h stimulation with hTNF or sc hTNF mutant Y87F fused to a BcII10 or hCD20 VHH ( C ) . Cell viability was measured via an ATP luminescence assay . Each point is the mean of three replicates and error bars are SEM .

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(B,C) Viability of L929 parental or mCD20-expressing cells after 72h stimulation with mTNF or sc mTNF mutant Y86F fused to a BcII10 or mCD20 VHH (B) and viability of MCF7 parental or hCD20-expressing cells after 72h stimulation with hTNF or sc hTNF mutant Y87F fused to a BcII10 or hCD20 VHH (C). Cell viability was measured via an ATP luminescence assay. Each point is the mean of three replicates and error bars are SEM.


( D ) IL - 8 concentration in HUVEC supernatant after 24h stimulation with the indicated concentration of stimulus , measured via ELISA .

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(D) IL-8 concentration in HUVEC supernatant after 24h stimulation with the indicated concentration of stimulus, measured via ELISA.


( A ) Toxicity of wt sc mTNF and mutant Y86F after injection ( ↑ ) in naïve C56BL / 6 mice . First injection was i . v . , other injections were i . p . Mean rectal body temperature ± SEM and cumulative survival rates are shown ( n = 4 ) . For continuity of the temperature graphs , dead mice were included with a temperature of 20 ° C .

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(A) Toxicity of wt sc mTNF and mutant Y86F after injection (↑) in naïve C56BL/6 mice. First injection was i.v., other injections were i.p. Mean rectal body temperature ± SEM and cumulative survival rates are shown (n = 4). For continuity of the temperature graphs, dead mice were included with a temperature of 20°C.


( B ) Plasma IL - 6 concentrations measured via ELISA . Blood samples were taken 6 hours after i . v . injection of wt sc mTNF or mutant Y86F .

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(B) Plasma IL-6 concentrations measured via ELISA. Blood samples were taken 6 hours after i.v. injection of wt sc mTNF or mutant Y86F.


( C ) Immunohistochemical staining for PECAM - 1 ( red ) , ICAM - 1 ( green ) and DNA ( blue ) of B16Bl6 tumors after p . l . treatment with 7µg wt mTNF or 50µg of mCD13 - AFR . Scale bar is 50µm .

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(C) Immunohistochemical staining for PECAM-1 (red), ICAM-1 (green) and DNA (blue) of B16Bl6 tumors after p.l. treatment with 7µg wt mTNF or 50µg of mCD13-AFR. Scale bar is 50µm.


( D ) Expression of ICAM - 1 , E - selectin and VEGF - R2 in B16Bl6 tumor 6h after treatment , by qPCR analysis of whole tumor RNA . Error bars are SEM . ns , non - significant ; * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 by one - way ANOVA with Bonferroni ' s multiple comparison test .

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(D) Expression of ICAM-1, E-selectin and VEGF-R2 in B16Bl6 tumor 6h after treatment, by qPCR analysis of whole tumor RNA. Error bars are SEM. ns, non-significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001 by one-way ANOVA with Bonferroni's multiple comparison test.


( E ) Tumor growth and body weight change after daily p . l . injection of indicated ( equimolar ) doses of mCD13 - AFR , non - targeted AFR ( BcII10 - AFR ) or mCD13 VHH control ( CD13 - hIFNα2 ) in the B16Bl6 melanoma model . The line under the graph represents the treatment period . Tumor growth is shown as mean TSI , error bars are SEM ( n = 5 ) . * * * , p < 0 . 001 by two - way ANOVA with Bonferroni ' s multiple comparison test .

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(E) Tumor growth and body weight change after daily p.l. injection of indicated (equimolar) doses of mCD13-AFR, non-targeted AFR (BcII10-AFR) or mCD13 VHH control (CD13-hIFNα2) in the B16Bl6 melanoma model. The line under the graph represents the treatment period. Tumor growth is shown as mean TSI, error bars are SEM (n = 5). ***, p < 0.001 by two-way ANOVA with Bonferroni's multiple comparison test.


( F ) B16Bl6 tumor growth after daily p . l . treatment with indicated doses of mCD13 - AFR , Wortmannin ( Wm ) , Birinapant ( Bir ) or combinations thereof . Wortmannin and Birinapant were given 1 hour before CD13 - AFR . Tumor growth is shown as mean TSI + SEM ( n = 4 for Wm and Bir , 5 for other groups ) . The line under the graph represents the treatment period . TSI of individual tumors of the indicated treatment groups at day 17 after tumor inoculation .

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(F) B16Bl6 tumor growth after daily p.l. treatment with indicated doses of mCD13-AFR, Wortmannin (Wm), Birinapant (Bir) or combinations thereof. Wortmannin and Birinapant were given 1 hour before CD13-AFR. Tumor growth is shown as mean TSI + SEM (n = 4 for Wm and Bir, 5 for other groups). The line under the graph represents the treatment period. TSI of individual tumors of the indicated treatment groups at day 17 after tumor inoculation.


( A ) Schematic representation of the CAR construct used , consisting of N - terminal anti - hCD70 VHH , hCD8α hinge and transmembrane ( TM ) domain , CD137 co - stimulatory domain and C - terminal CD3ζ activation domain . ( B , C ) SKOV3 tumor growth in NSG mice after i . v . injection of 6 x 106 hCD70 CAR T - cells on day 12 and p . l . treatment with 50µg mCD13 - AFR ( ↑ ) . Tumor growth is shown as mean TSI , error bars are SEM ( n = 5 ) ( B ) or as TSI of individual mice ( C ) .

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(A) Schematic representation of the CAR construct used, consisting of N-terminal anti-hCD70 VHH, hCD8α hinge and transmembrane (TM) domain, CD137 co-stimulatory domain and C-terminal CD3ζ activation domain. (B,C) SKOV3 tumor growth in NSG mice after i.v. injection of 6 x 106 hCD70 CAR T-cells on day 12 and p.l. treatment with 50µg mCD13-AFR (↑). Tumor growth is shown as mean TSI, error bars are SEM (n = 5) (B) or as TSI of individual mice (C).


( D ) Flow cytometric analysis of hCD45 + hCD3 + eGFP + CAR T - cells in SKOV3 tumors on day 18 after tumor inoculation . Mice were injected with 6 x 106 CAR T - cells on day 12 and treated with PBS or CD13 -

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(D) Flow cytometric analysis of hCD45+hCD3+eGFP+ CAR T-cells in SKOV3 tumors on day 18 after tumor inoculation. Mice were injected with 6 x 106 CAR T-cells on day 12 and treated with PBS or CD13-AFR


( E ) B16Bl6 tumor growth and body weight change after daily p . l . injection of 30µg mCD8 - AFN , 50µg mCD13 - AFR or a combination thereof . Tumor growth is shown as mean TSI + SEM ( n = 6 ) . The line under the graph represents the treatment period . ( F ) TSI of individual tumors of the indicated treatment groups at day 19 after tumor inoculation .

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(E) B16Bl6 tumor growth and body weight change after daily p.l. injection of 30µg mCD8-AFN, 50µg mCD13-AFR or a combination thereof. Tumor growth is shown as mean TSI + SEM (n = 6). The line under the graph represents the treatment period. (F) TSI of individual tumors of the indicated treatment groups at day 19 after tumor inoculation.


( A , B ) B16Bl6 tumor growth after daily p . l . treatment with 7µg mTNF , 30µg hTNF alone or in combination with 10000 IU mIFN - γ in wt C57BL / 6 mice ( A ) or homozygous Flk1 dnIFN - γR1 transgenic mice ( B ) . The line under the graph represents the treatment period . Tumor growth is depicted as mean TSI , error bars are SEM ( n = 5 ) .

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(A,B) B16Bl6 tumor growth after daily p.l. treatment with 7µg mTNF, 30µg hTNF alone or in combination with 10000 IU mIFN-γ in wt C57BL/6 mice (A) or homozygous Flk1 dnIFN-γR1 transgenic mice (B). The line under the graph represents the treatment period. Tumor growth is depicted as mean TSI, error bars are SEM (n = 5).


( C ) IL - 8 concentration in HUVEC supernatant after 24h stimulation with the indicated concentration of sc hTNF mutant Y87F ( Y87F ) , hCD13 - AFR or wt hTNF , alone or in combination with 200ng / ml hIFN - γ , measured via ELISA .

(C) IL-8 concentration in HUVEC supernatant after 24h stimulation with the indicated concentration of sc hTNF mutant Y87F (Y87F), hCD13-AFR or wt hTNF, alone or in combination with 200ng/ml hIFN-γ, measured via ELISA.


( E ) Viability of B16Bl6 parental or mCD20 - expressing cells after 72h stimulation with wt mIFN - γ or del8 mutant fused to a BcII10 or mCD20 VHH , in the presence of 60ng / ml mTNF . Cell viability was measured via an ATP luminescence assay .

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(E) Viability of B16Bl6 parental or mCD20-expressing cells after 72h stimulation with wt mIFN-γ or del8 mutant fused to a BcII10 or mCD20 VHH, in the presence of 60ng/ml mTNF. Cell viability was measured via an ATP luminescence assay.


( A , B ) Tumor growth , body weight change and rectal body temperature after daily p . l . treatment with mCD13 - AFR , mCD13 - AFN - II or combination thereof , in the B16Bl6 model in C57BL / 6 mice ( A ) or RL model in NSG mice ( B ) . Tumor growth is shown as mean TSI , error bars are SEM ( n = 5 ) ( upper panel ) , or as TSI of individual mice ( lower panel ) .

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(A,B) Tumor growth, body weight change and rectal body temperature after daily p.l. treatment with mCD13-AFR, mCD13-AFN-II or combination thereof, in the B16Bl6 model in C57BL/6 mice (A) or RL model in NSG mice (B). Tumor growth is shown as mean TSI, error bars are SEM (n = 5) (upper panel), or as TSI of individual mice (lower panel).


( C ) Immunohistochemical staining for PECAM - 1 ( red ) , cleaved Caspase - 3 ( green ) and DNA ( blue ) of B16Bl6 tumors after p . l . treatment with 50µg mCD13 - AFR and 23 . 8µg mCD13 - AFN - II . Scale bar is 100µm .

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(C) Immunohistochemical staining for PECAM-1 (red), cleaved Caspase-3 (green) and DNA (blue) of B16Bl6 tumors after p.l. treatment with 50µg mCD13-AFR and 23.8µg mCD13-AFN-II. Scale bar is 100µm.


( A - C ) . Mice body weight gain ( A ) , the lean mass and fat mass ( B ) and gWAT index ( C ) . At 8 weeks of age , male C57BL / 6 mice were switched to HFD . After 12 weeks of HFD feeding , mice were divided into three groups receiving non - exercise , endurance exercise or resistance exercise for 14 days . ( n = 8 per group ) .

(A-C). Mice body weight gain (A), the lean mass and fat mass (B) and gWAT index (C). At 8 weeks of age, male C57BL/6 mice were switched to HFD. After 12 weeks of HFD feeding, mice were divided into three groups receiving non-exercise, endurance exercise or resistance exercise for 14 days. (n = 8 per group).


( D ) . Relative changes in metabolites in response to resistance exercise . Heat maps show changes of metabolites in the serums from mice receiving resistance exercise or non - exercise . Male C57BL / 6 mice ( 10 weeks ) fed with normal chow were divided into two groups receiving either non - exercise or resistance exercise for 40 min ( n = 6 per group ) . Shades of yellow and blue represent fold increase and fold decrease of a metabolite , respectively ( see color scale ) .

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(D). Relative changes in metabolites in response to resistance exercise. Heat maps show changes of metabolites in the serums from mice receiving resistance exercise or non-exercise. Male C57BL/6 mice (10 weeks) fed with normal chow were divided into two groups receiving either non-exercise or resistance exercise for 40 min (n = 6 per group). Shades of yellow and blue represent fold increase and fold decrease of a metabolite, respectively (see color scale).


( G ) . Two tailed Pearson ' s correlation coefficient analysis of plasma AKG level and body mass index ( BMI ) in Chinese adults ( 10 males and 35 females ) .

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

(G). Two tailed Pearson's correlation coefficient analysis of plasma AKG level and body mass index (BMI) in Chinese adults (10 males and 35 females).


( H ) . Serum AKG concentration - time profile obtained before and after 40 - min resistant exercise . At 8 weeks of age , male C57BL / 6 mice were switched to HFD and continuously fed with HFD for 12 weeks . At 20 weeks of age , mice received resistance exercise for 40 min . Another group of chow fed male C57BL / 6 mice ( 10 weeks ) received resistance exercise for 40 min . The serum AKG level were tested at 0 , 1 , 2 , 4 and 6 hrs after exercise ( n = 8 - 10 per group ) .

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(H). Serum AKG concentration-time profile obtained before and after 40-min resistant exercise. At 8 weeks of age, male C57BL/6 mice were switched to HFD and continuously fed with HFD for 12 weeks. At 20 weeks of age, mice received resistance exercise for 40 min. Another group of chow fed male C57BL/6 mice (10 weeks) received resistance exercise for 40 min. The serum AKG level were tested at 0, 1, 2, 4 and 6 hrs after exercise (n = 8-10 per group).


( I ) . Serum AKG levels after exercise . Male C57BL / 6 mice ( 10 weeks ) fed with normal chow were divided into three groups receiving non - exercise , running wheel free access for 1 day or resistance exercise for 40 min . ( n = 8 - 10 per group ) .

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(I). Serum AKG levels after exercise. Male C57BL/6 mice (10 weeks) fed with normal chow were divided into three groups receiving non-exercise, running wheel free access for 1 day or resistance exercise for 40 min.(n = 8-10 per group).


( J ) . Muscles AKG levels after exercise . Male C57BL / 6 mice ( 10 weeks ) fed with normal chow were divided into two groups receiving either non - exercise or resistance exercise for 40 min ( n = 6 per group ) .

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(J). Muscles AKG levels after exercise. Male C57BL/6 mice (10 weeks) fed with normal chow were divided into two groups receiving either non-exercise or resistance exercise for 40 min (n = 6 per group).


Muscle ICDHm ( K ) , enzyme activity after exercise . Male C57BL / 6 mice ( 10 weeks ) fed with normal chow were divided into three groups receiving non - exercise , running wheel free access for 1day or resistance exercise for 40 min ( n = 8 - 9 per group ) .

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Muscle ICDHm (K), enzyme activity after exercise. Male C57BL/6 mice (10 weeks) fed with normal chow were divided into three groups receiving non-exercise, running wheel free access for 1day or resistance exercise for 40 min (n = 8-9 per group).


GDH ( L ) , enzyme activity after exercise . Male C57BL / 6 mice ( 10 weeks ) fed with normal chow were divided into three groups receiving non - exercise , running wheel free access for 1day or resistance exercise for 40 min ( n = 8 - 9 per group ) .

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GDH (L), enzyme activity after exercise. Male C57BL/6 mice (10 weeks) fed with normal chow were divided into three groups receiving non-exercise, running wheel free access for 1day or resistance exercise for 40 min (n = 8-9 per group).


α - KGDH ( M ) enzyme activity after exercise . Male C57BL / 6 mice ( 10 weeks ) fed with normal chow were divided into three groups receiving non - exercise , running wheel free access for 1day or resistance exercise for 40 min ( n = 8 - 9 per group ) .

α-KGDH (M) enzyme activity after exercise. Male C57BL/6 mice (10 weeks) fed with normal chow were divided into three groups receiving non-exercise, running wheel free access for 1day or resistance exercise for 40 min (n = 8-9 per group).


( A ) . Serum AKG concentration - time profile obtained from male C57BL / 6 mice ( 10 weeks ) fed with normal chow before or after AKG gavage ( 10 mg / kg ) . The serum AKG level were tested at 0 , 1 , 2 , 4 and 6 hrs after gavage ( n = 8 per group ) .

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(A). Serum AKG concentration-time profile obtained from male C57BL/6 mice (10 weeks) fed with normal chow before or after AKG gavage (10 mg/kg). The serum AKG level were tested at 0, 1, 2, 4 and 6 hrs after gavage (n = 8 per group).


( B - D ) . Water intake ( B ) , cumulative food intake ( C ) and body weight gain ( D ) of male C57BL / 6 mice . At 12 weeks of age , mice were switched to HFD and received tap water or water supplemented with 2 % AKG for 11 weeks ( n = 9 per group ) .

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(B-D). Water intake (B), cumulative food intake (C) and body weight gain (D) of male C57BL/6 mice. At 12 weeks of age, mice were switched to HFD and received tap water or water supplemented with 2% AKG for 11 weeks (n = 9 per group).


Fat and lean mass ( E ) of body composition from male mice after 11 weeks of AKG supplementation ( n = 9 per group ) .

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Fat and lean mass (E) of body composition from male mice after 11 weeks of AKG supplementation (n = 9 per group).


representative images ( F ) of body composition from male mice after 11 weeks of AKG supplementation ( n = 9 per group ) .

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

representative images (F) of body composition from male mice after 11 weeks of AKG supplementation (n = 9 per group).


( G - I ) . Weight index of BAT ( G ) , gWAT ( H ) and iWAT ( I ) from male mice after 11 weeks of AKG supplementation ( n = 9 per group ) .

O O O O O O 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

(G-I). Weight index of BAT (G), gWAT (H) and iWAT (I) from male mice after 11 weeks of AKG supplementation (n = 9 per group).


( J ) . Serum AKG level of male mice after 11 weeks of AKG supplementation ( n = 9 per group ) .

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

(J). Serum AKG level of male mice after 11 weeks of AKG supplementation (n = 9 per group).


( K - L ) . Representative images ( K ) and quantification ( L ) of gWAT and iWAT HE staining from male mice after 11 weeks of AKG supplementation ( n = 9 per group ) .

O O O O O 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

(K-L). Representative images (K) and quantification (L) of gWAT and iWAT HE staining from male mice after 11 weeks of AKG supplementation (n = 9 per group).


( M - O ) . Water intake ( M ) , cumulative food intake ( N ) and body weight gain ( O ) of female C57BL / 6 mice . At 12 weeks of age , mice were switched to HFD and received tap water or water supplemented with 2 % AKG for 11 weeks ( n = 9 per group ) .

(M-O). Water intake (M), cumulative food intake (N) and body weight gain (O) of female C57BL/6 mice. At 12 weeks of age, mice were switched to HFD and received tap water or water supplemented with 2% AKG for 11 weeks (n = 9 per group).


( P - Q ) . Fat and lean mass index ( P ) and representative image ( Q ) of body composition from female mice after 11 weeks of AKG supplementation ( n = 9 per group ) .

(P-Q). Fat and lean mass index (P) and representative image (Q) of body composition from female mice after 11 weeks of AKG supplementation (n = 9 per group).


( R - T ) . Weight index of BAT ( R ) , gWAT ( S ) and iWAT ( T ) from female mice after 11 weeks of AKG supplementation ( n = 9 per group ) .

(R-T). Weight index of BAT (R), gWAT (S) and iWAT (T) from female mice after 11 weeks of AKG supplementation (n = 9 per group).


( U - V ) . Representative images ( U ) and quantification ( V ) of gWAT and iWAT HE staining from female mice after 11 weeks of AKG supplementation ( n = 9 per group ) .

(U-V). Representative images (U) and quantification (V) of gWAT and iWAT HE staining from female mice after 11 weeks of AKG supplementation (n = 9 per group).


( A - B ) . Oxygen consumption in male C57BL / 6 mice after 11 weeks of AKG supplementation ( n = 8 per group ) .

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

(A-B). Oxygen consumption in male C57BL/6 mice after 11 weeks of AKG supplementation (n = 8 per group).


( C ) . Body temperature of male mice after 11 weeks of AKG supplementation ( n = 9 per group ) .

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

(C). Body temperature of male mice after 11 weeks of AKG supplementation (n = 9 per group).


( D - E ) . Representative images ( D ) and quantification ( E ) of BAT thermogenesis induced by 6 hr cold exposure at 4 ℃ in male mice supplemented with AKG for 11 weeks ( n = 9 per group ) .

O O O O O O O O O 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

(D-E). Representative images (D) and quantification (E) of BAT thermogenesis induced by 6 hr cold exposure at 4℃ in male mice supplemented with AKG for 11 weeks (n = 9 per group).


The mRNA expression of thermogenic genes ( F ) in BAT of male mice after 11 weeks of AKG supplementation ( n = 3 - 6 per group ) .

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

The mRNA expression of thermogenic genes (F) in BAT of male mice after 11 weeks of AKG supplementation (n = 3-6 per group).


Immunoblots and quantification ( G ) of UCP1 protein in BAT of male mice after 11 weeks of AKG supplementation ( n = 3 - 6 per group ) .

Immunoblots and quantification (G) of UCP1 protein in BAT of male mice after 11 weeks of AKG supplementation (n = 3-6 per group).


( H - I ) . DAB staining ( H ) and quantification ( I ) of UCP1 in BAT of male mice supplemented with AKG for 11 weeks ( n = 9 per group ) .

O 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

(H-I). DAB staining (H) and quantification (I) of UCP1 in BAT of male mice supplemented with AKG for 11 weeks (n = 9 per group).


( J ) . Serum levels of NEFA in male mice supplemented with AKG for 11 weeks ( n = 9 per group ) .

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

(J). Serum levels of NEFA in male mice supplemented with AKG for 11 weeks (n = 9 per group).


( K ) . The mRNA expression of CD137 , CD40 , TBX1 , TMEM26 , CITED1 and slc27a1 in iWAT of male mice supplemented with AKG for 11 weeks ( n = 8 per group ) .

O O 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

(K). The mRNA expression of CD137, CD40, TBX1, TMEM26, CITED1 and slc27a1 in iWAT of male mice supplemented with AKG for 11 weeks (n = 8 per group).


( L - M ) . Respiratory exchange ratio ( RER ) in male C57BL / 6 mice after 11 weeks of AKG supplementation ( n = 8 per group ) .

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

(L-M). Respiratory exchange ratio (RER) in male C57BL/6 mice after 11 weeks of AKG supplementation (n = 8 per group).


( N - O ) . Immunoblots ( N ) and quantification ( O ) of p - HSL and ATGL protein in gWAT of male mice after 11 weeks of AKG supplementation ( n = 3 per group ) .

O O O O O O O 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

(N-O). Immunoblots (N) and quantification (O) of p-HSL and ATGL protein in gWAT of male mice after 11 weeks of AKG supplementation (n = 3 per group).


( P - Q ) . Representative images ( P ) and quantification ( Q ) of p - HSL DAB staining in gWAT and iWAT of male mice after 11 weeks of AKG supplementation ( n = 9 per group ) .

O O O O O O O O O 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

(P-Q). Representative images (P) and quantification (Q) of p-HSL DAB staining in gWAT and iWAT of male mice after 11 weeks of AKG supplementation (n = 9 per group).


( R ) . The mRNA expression of PPARγ , FASN and ACC in the gWAT and iWAT from male mice supplemented with AKG for 11 weeks ( n = 6 per group ) .

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

(R). The mRNA expression of PPARγ, FASN and ACC in the gWAT and iWAT from male mice supplemented with AKG for 11 weeks (n = 6 per group).


( S - V ) . Serum levels of E ( S ) , NE ( T ) , T3 ( U ) , and T4 ( V ) in male mice supplemented with AKG for 11 weeks ( n = 8 - 9 per group ) .

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

(S-V). Serum levels of E (S), NE (T), T3 (U), and T4 (V) in male mice supplemented with AKG for 11 weeks (n = 8-9 per group).


( A ) . Serum AKG concentration - time profile obtained from male C57BL / 6 mice ( 10 weeks ) fed with normal chow before or after i . p AKG ( 10 mg / kg body weight ) . The serum AKG level were tested at 0 , 1 , 2 , 4 and 6 hrs after injection ( n = 8 per group ) .

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 B-CONTROLLED_VAR 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

(A). Serum AKG concentration-time profile obtained from male C57BL/6 mice (10 weeks) fed with normal chow before or after i.p AKG (10 mg/kg body weight). The serum AKG level were tested at 0, 1, 2, 4 and 6 hrs after injection (n = 8 per group).


( B - C ) . Representative images ( B ) and quantification ( C ) of BAT thermogenesis after 6 hrs cold exposure at 4 ℃ . Male C57BL / 6 mice ( 10 weeks ) were i . p . injected with 10 mg / kg AKG or saline and immediately exposed to cold stress at 4 ℃ ( n = 8 per group ) .

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

(B-C). Representative images (B) and quantification (C) of BAT thermogenesis after 6 hrs cold exposure at 4℃. Male C57BL/6 mice (10 weeks) were i.p. injected with 10 mg/kg AKG or saline and immediately exposed to cold stress at 4℃ (n = 8 per group).


( D ) . Immunoblots and quantification of p - HSL and ATGL in the gWAT of male C57BL / 6 mice ( 10 weeks ) 3 hrs after i . p . injection of 10 mg / kg AKG or saline ( n = 3 per group ) .

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

(D). Immunoblots and quantification of p-HSL and ATGL in the gWAT of male C57BL/6 mice (10 weeks) 3 hrs after i.p. injection of 10 mg/kg AKG or saline (n = 3 per group).


( E ) . Serum E level in AKG treated male mice 3 hrs after i . p . injection ( n = 8 per group ) .

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

(E). Serum E level in AKG treated male mice 3 hrs after i.p. injection (n = 8 per group).


Oxygen consumption ( F - G ) in male C57BL / 6 mice ( 10 weeks ) i . p . injected with saline , 10 mg / kg AKG , 1 mg / kg SR59230A ( ADRB3 inhibitor ) or AKG + SR59230A ( n = 8 per group ) . All injections were performed at 7 : 00 am of second day . Data was summarized in bar graph ( G and I ) by light or dark cycle of second day .

O O O O O 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 B-CONTROLLED_VAR 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

Oxygen consumption (F-G) in male C57BL/6 mice (10 weeks) i.p. injected with saline, 10 mg/kg AKG, 1 mg/kg SR59230A (ADRB3 inhibitor) or AKG + SR59230A (n = 8 per group). All injections were performed at 7:00 am of second day. Data was summarized in bar graph (G and I) by light or dark cycle of second day.


RER ( H - I ) in male C57BL / 6 mice ( 10 weeks ) i . p . injected with saline , 10 mg / kg AKG , 1 mg / kg SR59230A ( ADRB3 inhibitor ) or AKG + SR59230A ( n = 8 per group ) . All injections were performed at 7 : 00 am of second day . Data was summarized in bar graph ( G and I ) by light or dark cycle of second day .

O O O O 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 B-CONTROLLED_VAR 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

RER (H-I) in male C57BL/6 mice (10 weeks) i.p. injected with saline, 10 mg/kg AKG, 1 mg/kg SR59230A (ADRB3 inhibitor) or AKG + SR59230A (n = 8 per group). All injections were performed at 7:00 am of second day. Data was summarized in bar graph (G and I) by light or dark cycle of second day.


( J - N ) . Body weight gain ( J ) , cumulative food intake ( K ) , body composition ( L ) , fat weight ( M ) of sham or adrenalectomized male C57BL / 6 mice . Male mice were adrenalectomized at 8 weeks of age . Two weeks after surgeries , male mice were switched to HFD and given free access to tap water or 2 % AKG for 9 weeks ( n = 8 per group ) .

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

(J-N). Body weight gain (J), cumulative food intake (K), body composition (L), fat weight (M) of sham or adrenalectomized male C57BL/6 mice. Male mice were adrenalectomized at 8 weeks of age. Two weeks after surgeries, male mice were switched to HFD and given free access to tap water or 2% AKG for 9 weeks (n = 8 per group).


serum NEFA ( N ) of sham or adrenalectomized male C57BL / 6 mice . Male mice were adrenalectomized at 8 weeks of age . Two weeks after surgeries , male mice were switched to HFD and given free access to tap water or 2 % AKG for 9 weeks ( n = 8 per group ) .

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

serum NEFA (N) of sham or adrenalectomized male C57BL/6 mice. Male mice were adrenalectomized at 8 weeks of age. Two weeks after surgeries, male mice were switched to HFD and given free access to tap water or 2% AKG for 9 weeks (n = 8 per group).


( O - P ) . Representative images ( O ) and quantification ( P ) of BAT thermogenesis after 6 hrs cold exposure at 4 ℃ in sham or adrenalectomized male mice treated with AKG for 9 weeks ( n = 8 per group ) .

O O O O O O O O O O O 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-P). Representative images (O) and quantification (P) of BAT thermogenesis after 6 hrs cold exposure at 4℃ in sham or adrenalectomized male mice treated with AKG for 9 weeks (n = 8 per group).


( Q ) . The mRNA expression of themogenic genes in the BAT of sham or adrenalectomized male mice treated with AKG for 9 weeks ( n = 6 per group ) .

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

(Q). The mRNA expression of themogenic genes in the BAT of sham or adrenalectomized male mice treated with AKG for 9 weeks (n = 6 per group).


( R - S ) . Immunoblots ( R ) and quantification ( S ) of p - HSL and ATGL protein in the gWAT of sham or adrenalectomized male mice treated with AKG for 9 weeks ( n = 4 per group ) .

O O O O O O O O O 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

(R-S). Immunoblots (R) and quantification (S) of p-HSL and ATGL protein in the gWAT of sham or adrenalectomized male mice treated with AKG for 9 weeks (n = 4 per group).


( T - U ) . Immunoblots ( T ) and quantification ( U ) of UCP1 protein in the BAT of sham or adrenalectomized male mice treated with AKG for 9 weeks ( n = 4 per group ) .

O O O O O 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

(T-U). Immunoblots (T) and quantification (U) of UCP1 protein in the BAT of sham or adrenalectomized male mice treated with AKG for 9 weeks (n = 4 per group).


( A ) . The mRNA expression of AKG - sensing genes in the adrenal gland tissue of 12 - weeks male C57BL / 6 mice ( n = 4 per group ) .

O O O O O O O O O O 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). The mRNA expression of AKG-sensing genes in the adrenal gland tissue of 12-weeks male C57BL/6 mice (n = 4 per group).


( B ) . The mRNA expression of OXGR1 in different tissues of 12 - weeks male C57BL / 6 mice ( n = 3 per group ) .

O O O O O 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). The mRNA expression of OXGR1 in different tissues of 12-weeks male C57BL/6 mice (n = 3 per group).


( C ) . OXGR1 localization in adrenal gland medulla indicated by fluorescent staining of OXGR1 ( green ) .

(C). OXGR1 localization in adrenal gland medulla indicated by fluorescent staining of OXGR1 (green).


( D ) . The mRNA expression of OXGR1 in the adrenal gland of male mice 3 hrs after i . p . injection of saline or 10 mg / kg AKG , or immediately after 40 - mins resistance exercise ( n = 8 per group ) .

O O O O O O O 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

(D). The mRNA expression of OXGR1 in the adrenal gland of male mice 3 hrs after i.p. injection of saline or 10 mg/kg AKG, or immediately after 40-mins resistance exercise (n=8 per group).


( E ) . Immunoblots and quantification of OXGR1 protein expression in adrenal chromaffin cells treated with negative control ( NC ) siRNA or siOXGR1 ( n = 3 per group ) .

O O O O O O O O O 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). Immunoblots and quantification of OXGR1 protein expression in adrenal chromaffin cells treated with negative control (NC) siRNA or siOXGR1 (n = 3 per group).


( F ) . E level in the medium from adrenal chromaffin cell cultured with vehicle + NC , vehicle + siOXGR1 , AKG ( 100 μM ) + NC or AKG + siOXGR1 for 30 mins ( n = 8 per group ) .

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

(F). E level in the medium from adrenal chromaffin cell cultured with vehicle + NC, vehicle + siOXGR1, AKG (100 μM) + NC or AKG + siOXGR1 for 30 mins (n = 8 per group).


( G ) . Intracellular calcium ion [ Ca2 + ] changes in adrenal medulla cell cultured with vehicle + NC , vehicle + siOXGR1 , AKG ( 100 μM ) + NC or AKG + siOXGR1 ( n = 30 per group ) .

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

(G). Intracellular calcium ion [Ca2+] changes in adrenal medulla cell cultured with vehicle + NC, vehicle + siOXGR1, AKG (100 μM) + NC or AKG + siOXGR1 (n = 30 per group).


( H - I ) . Body weight gain ( H ) and cumulative food intake ( I ) of male WT control ( littermates ) or OXGR1 global knock out ( OXGR1KO ) mice . At 12 weeks of age , both control and KO mice were switched to HFD and further divided into two groups , receiving tap water or water supplemented with 2 % AKG for 13 weeks ( n = 8 per group ) .

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

(H-I). Body weight gain (H) and cumulative food intake (I) of male WT control (littermates) or OXGR1 global knock out (OXGR1KO) mice. At 12 weeks of age, both control and KO mice were switched to HFD and further divided into two groups, receiving tap water or water supplemented with 2% AKG for 13 weeks (n = 8 per group).


( J - K ) . Representative images of body composition ( J ) and fat and lean mass index ( K ) of male WT or OXGR1KO mice treated with AKG for 13 weeks ( n = 8 per group ) .

O O O O O O O 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

(J-K). Representative images of body composition (J) and fat and lean mass index (K) of male WT or OXGR1KO mice treated with AKG for 13 weeks (n = 8 per group).


( L - M ) . Weight index of iWAT ( L ) and gWAT ( M ) in male WT or OXGR1KO mice treated with AKG for 13 weeks ( n = 8 per group ) .

O O 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

(L-M). Weight index of iWAT (L) and gWAT (M) in male WT or OXGR1KO mice treated with AKG for 13 weeks (n = 8 per group).


( N - O ) . Immunoblots ( N ) and quantification ( O ) of p - HSL and ATGL protein in gWAT of male WT or OXGR1KO mice treated with AKG for 13 weeks ( n = 4 per group ) .

O O O O O O O O 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

(N-O). Immunoblots (N) and quantification (O) of p-HSL and ATGL protein in gWAT of male WT or OXGR1KO mice treated with AKG for 13 weeks (n = 4 per group).


Oxygen consumption ( P - Q ) of male WT or OXGR1KO mice treated with AKG for 13 weeks ( n = 8 per group ) .

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

Oxygen consumption (P-Q) of male WT or OXGR1KO mice treated with AKG for 13 weeks (n = 8 per group).


RER ( R - S ) of male WT or OXGR1KO mice treated with AKG for 13 weeks ( n = 8 per group ) .

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

RER (R-S) of male WT or OXGR1KO mice treated with AKG for 13 weeks (n = 8 per group).


Body weight gain and cumulative food intake of male OXGR1 adrenal - specific reexpression mice ( OXGR1REAG ) . Male OXGR1KO mice ( 8 weeks ) were adrenal - specifically injected with control HBAAV2 / 9 - GFP ( OXGR1KO control ) or HBAAV2 / 9 - OXGR1 ( OXGR1REAG ) . Two weeks after injections , mice were switched to HFD and further divided into two groups , receiving tap water or water supplemented with 2 % AKG for 12 weeks ( n = 8 per group ) .

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

Body weight gain and cumulative food intake of male OXGR1 adrenal-specific reexpression mice (OXGR1REAG). Male OXGR1KO mice (8 weeks) were adrenal-specifically injected with control HBAAV2/9-GFP (OXGR1KO control) or HBAAV2/9-OXGR1 (OXGR1REAG). Two weeks after injections, mice were switched to HFD and further divided into two groups, receiving tap water or water supplemented with 2% AKG for 12 weeks (n = 8 per group).


Representative image of body composition and fat and lean mass index of male OXGR1REAG mice treated with AKG for 12 weeks ( n = 8 per group ) .

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

Representative image of body composition and fat and lean mass index of male OXGR1REAG mice treated with AKG for 12 weeks (n = 8 per group).


Weight index of gWAT and iWAT in male OXGR1REAG mice treated with AKG for 12 weeks ( n = 8 per group ) .

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

Weight index of gWAT and iWAT in male OXGR1REAG mice treated with AKG for 12 weeks (n = 8 per group).


Immunoblots and quantification of p - HSL and ATGL protein in gWAT of male OXGR1REAG mice treated with AKG for 12 weeks ( n = 8 per group ) .

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

Immunoblots and quantification of p-HSL and ATGL protein in gWAT of male OXGR1REAG mice treated with AKG for 12 weeks (n = 8 per group).


Oxygen consumption of male OXGR1REAG mice treated with AKG for 12 weeks ( n = 8 per group ) .

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

Oxygen consumption of male OXGR1REAG mice treated with AKG for 12 weeks (n = 8 per group).


RER of male OXGR1REAG mice treated with AKG for 12 weeks ( n = 8 per group ) .

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

RER of male OXGR1REAG mice treated with AKG for 12 weeks (n = 8 per group).


Body weight gain in male WT littermates and OXGR1KO mice . At 8 weeks of age , male C57BL / 6 WT control or OXGR1KO mice were switched to HFD . After 12 weeks of HFD feeding , mice were further divided into two groups , receiving non - exercise or resistance exercise for 14 days . ( n = 8 per group ) .

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

Body weight gain in male WT littermates and OXGR1KO mice. At 8 weeks of age, male C57BL/6 WT control or OXGR1KO mice were switched to HFD. After 12 weeks of HFD feeding, mice were further divided into two groups, receiving non-exercise or resistance exercise for 14 days. (n = 8 per group).


Exercise - induced body weight loss in male WT littermates and OXGR1KO mice . Body weights from exercise mice were subtracted by the average body weight of non - exercise control group for each genotype ( n = 8 per group ) .

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

Exercise-induced body weight loss in male WT littermates and OXGR1KO mice. Body weights from exercise mice were subtracted by the average body weight of non-exercise control group for each genotype (n = 8 per group).


Exercise - induced fat mass loss in male WT littermates and OXGR1KO mice . Fat mass from exercise mice were subtracted by the average fat mass of non - exercise control group for each genotype ( n = 8 per group ) .

Exercise-induced fat mass loss in male WT littermates and OXGR1KO mice. Fat mass from exercise mice were subtracted by the average fat mass of non-exercise control group for each genotype (n = 8 per group).


Cumulative food intake of male WT littermates and OXGR1KO mice after 14 - day resistance exercise ( n = 8 per group ) .

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

Cumulative food intake of male WT littermates and OXGR1KO mice after 14-day resistance exercise (n = 8 per group).


Weight index of gWAT ( E ) and iWAT ( F ) of male OXGR1KO mice after 14 - days resistance exercise ( n = 8 per group ) .

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

Weight index of gWAT (E) and iWAT (F) of male OXGR1KO mice after 14-days resistance exercise (n = 8 per group).


Body composition of male OXGR1KO mice after 14 - days resistance exercise ( n = 8 per group ) .

Body composition of male OXGR1KO mice after 14-days resistance exercise (n = 8 per group).


Serum AKG levels of male WT and OXGR1KO mice after resistance exercise . Male WT and OXGR1KO mice ( 10 weeks ) fed with normal chow were receiving resistance exercise for 40 min ( n = 8 per group ) . The serum AKG levels were tested before and immediately after exercise .

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

Serum AKG levels of male WT and OXGR1KO mice after resistance exercise. Male WT and OXGR1KO mice (10 weeks) fed with normal chow were receiving resistance exercise for 40 min (n = 8 per group). The serum AKG levels were tested before and immediately after exercise.


Serum E level in male OXGR1KO mice after 14 - day resistance exercise ( n = 8 per group ) .

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

Serum E level in male OXGR1KO mice after 14-day resistance exercise (n = 8 per group).


The mRNA expression in the BAT of male OXGR1KO mice after 14 - day resistance exercise ( n = 4 per group ) .

The mRNA expression in the BAT of male OXGR1KO mice after 14-day resistance exercise (n = 4 per group).


protein expression of UCP1 in the BAT of male OXGR1KO mice after 14 - day resistance exercise ( n = 4 per group ) .

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

protein expression of UCP1 in the BAT of male OXGR1KO mice after 14-day resistance exercise (n = 4 per group).


the mRNA expression of HSL and ATGL n the gWAT of male OXGR1KO mice after 14 - day resistance exercise ( n = 4 per group ) .

O O O O 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 mRNA expression of HSL and ATGL n the gWAT of male OXGR1KO mice after 14-day resistance exercise (n = 4 per group).


Oxygen consumption in male OXGR1KO mice after 14 - day resistance exercise ( n = 8 per group ) .

Oxygen consumption in male OXGR1KO mice after 14-day resistance exercise (n = 8 per group).


RER in male OXGR1KO mice after 14 - day resistance exercise ( n = 8 per group ) .

RER in male OXGR1KO mice after 14-day resistance exercise (n = 8 per group).


Ingenuity Pathway Analysis ( IPA ) of AKG - induced transcriptome signature in adrenal chromaffin cell treated with AKG . The mRAN was extracted from adrenal chromaffin cells after 3 - hrs incubation of vehicle or AKG ( 100 μM ) . ( n = 3 per group ) .

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

Ingenuity Pathway Analysis (IPA) of AKG-induced transcriptome signature in adrenal chromaffin cell treated with AKG. The mRAN was extracted from adrenal chromaffin cells after 3-hrs incubation of vehicle or AKG (100 μM). (n = 3 per group).


Immunoblots and quantification of p - IKK / IKK , p65 , p - IκB / IκB and OXGR1 protein in adrenal chromaffin cells cultured with vehicle + NC , vehicle + siOXGR1 , AKG ( 100 μM ) + NC or AKG + siOXGR1 for 3 hrs ( n = 3 per group ) .

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

Immunoblots and quantification of p-IKK/IKK, p65, p-IκB/IκB and OXGR1 protein in adrenal chromaffin cells cultured with vehicle + NC, vehicle + siOXGR1, AKG (100 μM) + NC or AKG + siOXGR1 for 3 hrs (n = 3 per group).


p65 translocation in adrenal chromaffin cells cultured with vehicle + NC , vehicle + siOXGR1 , AKG ( 100 μM ) + NC or AKG + siOXGR1 for 3 hrs ( n = 3 per group ) . Scale bars , 100 μm .

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

p65 translocation in adrenal chromaffin cells cultured with vehicle + NC, vehicle + siOXGR1, AKG (100 μM) + NC or AKG + siOXGR1 for 3 hrs (n = 3 per group). Scale bars, 100 μm.


Immunoblots and quantification of p - IKK / IKK , p65 and p - IκB / IκB protein in adrenal chromaffin cells cultured with vehicle , AKG ( 100 μM ) , IKK inhibitor IKK16 or AKG + IKK16 for 3 hrs .

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

Immunoblots and quantification of p-IKK/IKK, p65 and p-IκB/IκB protein in adrenal chromaffin cells cultured with vehicle, AKG (100 μM), IKK inhibitor IKK16 or AKG + IKK16 for 3 hrs.


E level in the medium from adrenal chromaffin cell cultured with vehicle , AKG ( 100 μM ) , IKK16 or AKG + IKK16 for 3 hrs ( n = 10 per group ) .

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

E level in the medium from adrenal chromaffin cell cultured with vehicle, AKG (100 μM), IKK16 or AKG + IKK16 for 3 hrs (n = 10 per group).


B . ITC binding assays of the UVR8 and HY5 VP - peptides versus the COP1 WD40 domain . The top panel represents the heats detected during each injection . The bottom panel represents the integrated heats of each injection , fitted to a one - site binding model ( solid line ) . The following concentrations were typically used ( titrant into cell ) : UVR8 - COP1 ( 2500 μM in 175 μM ) ; HY5 - COP1 ( 1500 μM in 175 μM ) . The insets show the dissociation constants ( Kd ) and stoichiometries of binding ( N ) ( ± standard deviation ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. ITC binding assays of the UVR8 and HY5 VP-peptides versus the COP1 WD40 domain. The top panel represents the heats detected during each injection. The bottom panel represents the integrated heats of each injection, fitted to a one-site binding model (solid line). The following concentrations were typically used (titrant into cell): UVR8 - COP1 (2500 μM in 175 μM); HY5 - COP1 (1500 μM in 175 μM). The insets show the dissociation constants (Kd) and stoichiometries of binding (N) (± standard deviation).


E . ITC assays of the HY5 and UVR8 VP - peptides versus the different COP1 WD40 domain mutants The following concentrations were typically used ( titrant into cell ) : UVR8 - COP1Lys422Ala ( 1000 μM in 100 μM ) ; UVR8 - COP1Tyr441Ala ( 2500 μM in 90 μM ) ; UVR8 - COP1Trp467Ala ( 2500 μM in 90 μM ) ; HY5 - COP1Lys422Ala ( 1500 μM in 175 μM ) ; HY5 - COP1Tyr441Ala ( 1600 μM in 138 μM ) ; HY5 - COP1Trp467Ala ( 1600 μM in 112 μM ) . The insets show the dissociation constants ( Kd ) . The stoichiometries of binding ( N ) for UVR8 - COP1Lys422Ala = 0 . 87 ± 0 . 2 ; HY5 - COP1Lys422Ala = 0 . 83 ± 0 . 2 ; HY5 - COP1Tyr441Ala = 0 . 92 ± 0 . 3 ( All measurements ± standard deviation ; n . d . : no detectable binding ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. ITC assays of the HY5 and UVR8 VP-peptides versus the different COP1 WD40 domain mutants The following concentrations were typically used (titrant into cell): UVR8 - COP1Lys422Ala (1000 μM in 100 μM); UVR8 - COP1Tyr441Ala (2500 μM in 90 μM); UVR8 - COP1Trp467Ala (2500 μM in 90 μM); HY5 - COP1Lys422Ala (1500 μM in 175 μM); HY5 - COP1Tyr441Ala (1600 μM in 138 μM); HY5 - COP1Trp467Ala (1600 μM in 112 μM). The insets show the dissociation constants (Kd). The stoichiometries of binding (N) for UVR8 - COP1Lys422Ala = 0.87 ± 0.2; HY5 - COP1Lys422Ala = 0.83 ± 0.2; HY5 - COP1Tyr441Ala = 0.92 ± 0.3 (All measurements ± standard deviation; n.d.: no detectable binding).


Images of representative individuals ( F ) of 4 - day - old seedlings grown with or without supplemental UV - B . The scale bar for all lines represent 5 mm except for cop1 - 5 where the scale bars represent 1 mm . Data Information , lines used : wild - type ( Ws ) , uvr8 - 7 , cop1 - 4 , cop1 - 5 / Pro35S : YFP - COP1 ( WT ) , cop1 - 5 / Pro35S : YFP - COP1Lys422Ala , cop1 - 5 / Pro35S : YFP - COP1Tyr441Ala , cop1 - 5 / Pro35S : YFP - COP1Trp467Ala and cop1 - 5 . # 1 and # 2 : independent transgenic lines .

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

Images of representative individuals (F) of 4-day-old seedlings grown with or without supplemental UV-B. The scale bar for all lines represent 5 mm except for cop1-5 where the scale bars represent 1 mm. Data Information , lines used: wild-type (Ws), uvr8-7, cop1-4, cop1-5/Pro35S:YFP-COP1 (WT), cop1-5/Pro35S:YFP-COP1Lys422Ala, cop1-5/Pro35S:YFP-COP1Tyr441Ala, cop1-5/Pro35S:YFP-COP1Trp467Ala and cop1-5. #1 and #2: independent transgenic lines.


quantification of hypocotyl lengths ( G ) of 4 - day - old seedlings grown with or without supplemental UV - B . The scale bar for all lines represent 5 mm except for cop1 - 5 where the scale bars represent 1 mm . Violin and box plots are shown for n > 60 seedlings ; upper and lower hinges correspond to the first and third quartiles , the horizontal line in the interior of the box indicates the median . Data Information : lines used : wild - type ( Ws ) , uvr8 - 7 , cop1 - 4 , cop1 - 5 / Pro35S : YFP - COP1 ( WT ) , cop1 - 5 / Pro35S : YFP - COP1Lys422Ala , cop1 - 5 / Pro35S : YFP - COP1Tyr441Ala , cop1 - 5 / Pro35S : YFP - COP1Trp467Ala and cop1 - 5 . # 1 and # 2 : independent transgenic lines .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 hypocotyl lengths (G) of 4-day-old seedlings grown with or without supplemental UV-B. The scale bar for all lines represent 5 mm except for cop1-5 where the scale bars represent 1 mm. Violin and box plots are shown for n > 60 seedlings; upper and lower hinges correspond to the first and third quartiles, the horizontal line in the interior of the box indicates the median. Data Information: lines used: wild-type (Ws), uvr8-7, cop1-4, cop1-5/Pro35S:YFP-COP1 (WT), cop1-5/Pro35S:YFP-COP1Lys422Ala, cop1-5/Pro35S:YFP-COP1Tyr441Ala, cop1-5/Pro35S:YFP-COP1Trp467Ala and cop1-5. #1 and #2: independent transgenic lines.


Quantitative real - time PCR analysis of ( H ) HY5 expression . Four - day - old seedlings grown in white light were exposed to narrowband UV - B for 2 hours ( + UV - B ) , or not ( - UV - B ) . Error bars represent SEM of 3 biological replicates . Data Information : lines used : wild - type ( Ws ) , uvr8 - 7 , cop1 - 4 , cop1 - 5 / Pro35S : YFP - COP1 ( WT ) , cop1 - 5 / Pro35S : YFP - COP1Lys422Ala , cop1 - 5 / Pro35S : YFP - COP1Tyr441Ala , cop1 - 5 / Pro35S : YFP - COP1Trp467Ala and cop1 - 5 . # 1 and # 2 : independent transgenic lines .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 real-time PCR analysis of (H) HY5 expression. Four-day-old seedlings grown in white light were exposed to narrowband UV-B for 2 hours (+UV-B), or not (-UV-B). Error bars represent SEM of 3 biological replicates. Data Information: lines used: wild-type (Ws), uvr8-7, cop1-4, cop1-5/Pro35S:YFP-COP1 (WT), cop1-5/Pro35S:YFP-COP1Lys422Ala, cop1-5/Pro35S:YFP-COP1Tyr441Ala, cop1-5/Pro35S:YFP-COP1Trp467Ala and cop1-5. #1 and #2: independent transgenic lines.


Quantitative real - time PCR analysis of ( I ) RUP2 expression . Four - day - old seedlings grown in white light were exposed to narrowband UV - B for 2 hours ( + UV - B ) , or not ( - UV - B ) . Error bars represent SEM of 3 biological replicates . Data Information lines used : wild - type ( Ws ) , uvr8 - 7 , cop1 - 4 , cop1 - 5 / Pro35S : YFP - COP1 ( WT ) , cop1 - 5 / Pro35S : YFP - COP1Lys422Ala , cop1 - 5 / Pro35S : YFP - COP1Tyr441Ala , cop1 - 5 / Pro35S : YFP - COP1Trp467Ala and cop1 - 5 . # 1 and # 2 : independent transgenic lines .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 real-time PCR analysis of (I) RUP2 expression. Four-day-old seedlings grown in white light were exposed to narrowband UV-B for 2 hours (+UV-B), or not (-UV-B). Error bars represent SEM of 3 biological replicates. Data Information lines used: wild-type (Ws), uvr8-7, cop1-4, cop1-5/Pro35S:YFP-COP1 (WT), cop1-5/Pro35S:YFP-COP1Lys422Ala, cop1-5/Pro35S:YFP-COP1Tyr441Ala, cop1-5/Pro35S:YFP-COP1Trp467Ala and cop1-5. #1 and #2: independent transgenic lines.


A . ITC assay between the COP1 WD40 domain and full - length UVR8 pre - monomerized by UV - B light . Integrated heats are shown in solid , purple squares . For comparison , an ITC experiment between UVR8 VP peptide and the COP1 WD40 domain ( from Fig 1B ) is also shown in open , purple squares . The following concentrations were typically used ( titrant into cell ) : COP1 - UVR8 + UV - B ( 130 μM in 20 μM ) . The inset shows the dissociation constant ( Kd ) , stoichiometry of binding ( N ) ( ± standard deviation ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. ITC assay between the COP1 WD40 domain and full-length UVR8 pre-monomerized by UV-B light. Integrated heats are shown in solid, purple squares. For comparison, an ITC experiment between UVR8 VP peptide and the COP1 WD40 domain (from Fig 1B) is also shown in open, purple squares. The following concentrations were typically used (titrant into cell): COP1 - UVR8 +UV-B (130 μM in 20 μM). The inset shows the dissociation constant (Kd), stoichiometry of binding (N) (± standard deviation).


B . Binding kinetics of UVR8 pre - monomerized by UV - B versus the COP1 WD40 domain obtained by grating - coupled interferometry ( GCI ) . Sensorgrams of UVR8 injected are shown in red , with their respective 1 : 1 binding model fits in black . The following amounts were typically used : ligand - COP1 ( 2000 pg / mm2 ) ; analyte - UVR8 + UV - B ( highest concentration 2 μM ) . ka = association rate constant , kd = dissociation rate constant , Kd = dissociation constant .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Binding kinetics of UVR8 pre-monomerized by UV-B versus the COP1 WD40 domain obtained by grating-coupled interferometry (GCI). Sensorgrams of UVR8 injected are shown in red, with their respective 1:1 binding model fits in black. The following amounts were typically used: ligand - COP1 (2000 pg/mm2); analyte - UVR8 +UV-B (highest concentration 2 μM). ka = association rate constant, kd = dissociation rate constant, Kd = dissociation constant.


D . Binding kinetics of UVR8ValPro / AlaAla pre - monomerized by UV - B versus the COP1 WD40 domain obtained by GCI experiments . Sensorgrams of UVR8 injected are shown in red , with their respective 1 : 1 binding model fits in black . The following amounts were typically used : ligand - COP1 ( 2000 pg / mm2 ) ; analyte - UVR8ValPro / AlaAla + UV - B ( highest concentration 2 μM ) . ka = association rate constant , kd = disassociation rate constant , Kd = dissociation constant .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Binding kinetics of UVR8ValPro/AlaAla pre-monomerized by UV-B versus the COP1 WD40 domain obtained by GCI experiments. Sensorgrams of UVR8 injected are shown in red, with their respective 1:1 binding model fits in black. The following amounts were typically used: ligand - COP1 (2000 pg/mm2); analyte - UVR8ValPro/AlaAla +UV-B (highest concentration 2 μM). ka = association rate constant, kd = disassociation rate constant, Kd = dissociation constant.


E . Binding kinetics of HY5 versus the COP1 WD40 domain obtained by GCI experiments . Sensorgrams of HY5 injected are shown in red , with their respective 1 : 1 binding model fits in black . The following amounts were typically used : ligand - COP1 ( 2000 pg / mm2 ) ; analyte - HY5 ( highest concentration 2 μM ) . ka = association rate constant , kd = dissociation rate constant , Kd = dissociation constant .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Binding kinetics of HY5 versus the COP1 WD40 domain obtained by GCI experiments. Sensorgrams of HY5 injected are shown in red, with their respective 1:1 binding model fits in black. The following amounts were typically used: ligand - COP1 (2000 pg/mm2); analyte - HY5 (highest concentration 2 μM). ka = association rate constant, kd = dissociation rate constant, Kd = dissociation constant.


F . Yeast 3 - hybrid analysis of the COP1 - HY5 interaction in the presence of UVR8 . ( Top ) Normalized Miller Units were calculated as a ratio of β - galactosidase activity in yeast grown under UV - B ( + UV - B ) versus yeast grown without UV - B ( - UV - B ) . Additionally , normalized Miller Units are reported separately here for yeast grown on media without or with 1 mM methionine , corresponding to induction ( - Met ) or repression ( + Met ) of Met25 promoter - driven UVR8 expression , respectively . Means and SEM for 3 biological repetitions are shown . ( Bottom ) representative filter lift assays . AD , activation domain ; BD , DNA binding domain ; Met , methionine .

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

F. Yeast 3-hybrid analysis of the COP1 - HY5 interaction in the presence of UVR8. (Top) Normalized Miller Units were calculated as a ratio of β-galactosidase activity in yeast grown under UV-B (+ UV-B) versus yeast grown without UV-B (- UV-B). Additionally, normalized Miller Units are reported separately here for yeast grown on media without or with 1 mM methionine, corresponding to induction (- Met) or repression (+ Met) of Met25 promoter-driven UVR8 expression, respectively. Means and SEM for 3 biological repetitions are shown. (Bottom) representative filter lift assays. AD, activation domain; BD, DNA binding domain; Met, methionine.


B . Representative image showing the phenotype of wild - type ( Ws ) , uvr8 - 7 and uvr8 - 7 / Pro35S : UVR8HY5C44 , uvr8 - 7 / Pro35S : UVR8HY5VP and uvr8 - 7 / Pro35S : UVR8TRIB1 seedlings grown for 4 days under white light ( - UV - B ) or white light supplemented with UV - B ( + UV - B ) . The scale bar represents 5 mm .

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

B. Representative image showing the phenotype of wild-type (Ws), uvr8-7 and uvr8-7/Pro35S:UVR8HY5C44, uvr8-7/Pro35S:UVR8HY5VP and uvr8-7/Pro35S:UVR8TRIB1 seedlings grown for 4 days under white light (- UV-B) or white light supplemented with UV-B (+ UV-B). The scale bar represents 5 mm.


C . Quantification of hypocotyl length data Violin and box plots are shown for n > 60 seedlings ; upper and lower hinges correspond to the first and third quartiles , the horizontal line in the interior of the box indicates the median .

O O O O O O O O O O O O O O O O O O O O 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. Quantification of hypocotyl length data Violin and box plots are shown for n > 60 seedlings; upper and lower hinges correspond to the first and third quartiles, the horizontal line in the interior of the box indicates the median.


D . Anthocyanin accumulation in seedlings Average and SEM are shown ( n = 3 ) .

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

D. Anthocyanin accumulation in seedlings Average and SEM are shown (n = 3).


E . Quantitative real - time PCR analysis of RUP2 , CHS , HY5 and ELIP2 expression in wild - type ( Ws ) , uvr8 - 7 and uvr8 - 7 / Pro35S : UVR8HY5C44 , uvr8 - 7 / Pro35S : UVR8HY5VP and uvr8 - 7 / Pro35S : UVR8TRIB1 4 - day old seedlings grown under white light in response to 2h of UV - B ( + UV - B vs . - UV - B ) . Error bars represent SEM of 3 biological replicates . Note : the primers used to detect the HY5 transcript abundance also bind to an identical region present in UVR8HY5C44 chimera .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Quantitative real-time PCR analysis of RUP2, CHS, HY5 and ELIP2 expression in wild-type (Ws), uvr8-7 and uvr8-7/Pro35S:UVR8HY5C44, uvr8-7/Pro35S:UVR8HY5VP and uvr8-7/Pro35S:UVR8TRIB1 4-day old seedlings grown under white light in response to 2h of UV-B (+UV-B vs. -UV-B). Error bars represent SEM of 3 biological replicates. Note: the primers used to detect the HY5 transcript abundance also bind to an identical region present in UVR8HY5C44 chimera.


F . Yeast 3 - hybrid analysis of the COP1 - HY5 interaction in the presence of the UVR8HY5C44 chimera . ( Left pane ) Normalized Miller Units were calculated as a ratio of β - galactosidase activity in yeast grown under UV - B ( + UV - B ) versus yeast grown without UV - B ( - UV - B ) . Additionally , normalized Miller Units are reported separately here for yeast grown on media without or with 1 mM methionine , corresponding to induction ( - Met ) or repression ( + Met ) of Met25 promoter - driven UVR8 expression , respectively . Means and SEM for 3 biological repetitions are shown . ( Right panel ) Representative filter lift assays of the yeast analyzed in left panel . AD , activation domain ; BD , DNA binding domain ; Met , methionine .

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

F. Yeast 3-hybrid analysis of the COP1-HY5 interaction in the presence of the UVR8HY5C44 chimera. (Left pane) Normalized Miller Units were calculated as a ratio of β-galactosidase activity in yeast grown under UV-B (+ UV-B) versus yeast grown without UV-B (- UV-B). Additionally, normalized Miller Units are reported separately here for yeast grown on media without or with 1 mM methionine, corresponding to induction (- Met) or repression (+ Met) of Met25 promoter-driven UVR8 expression, respectively. Means and SEM for 3 biological repetitions are shown. (Right panel) Representative filter lift assays of the yeast analyzed in left panel. AD, activation domain; BD, DNA binding domain; Met, methionine.