Metabolomes of muscle of MIRAS ( A ) patients ; PLS - DA plots ; VIP score plots of top 15 metabolites ; volcano plots of all metabolites . Data information : a Significantly changed metabolites outside the FDR cut - off . b Metabolites not significantly changed between patients and controls . AMP , adenosine monophosphate NAD , nicotinamide adenine dinucleotide TCA , taurocholic

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Metabolomes of muscle of MIRAS (A) patients; PLS-DA plots; VIP score plots of top 15 metabolites; volcano plots of all metabolites. Data information: a Significantly changed metabolites outside the FDR cut-off. b Metabolites not significantly changed between patients and controls. AMP, adenosine monophosphate NAD, nicotinamide adenine dinucleotide TCA, taurocholic acid


Metabolomes of muscle of PEO ( B ) patients ; PLS - DA plots ; VIP score plots of top 15 metabolites ; volcano plots of all metabolites . Data information : a Significantly changed metabolites outside the FDR cut - off . b Metabolites not significantly changed between patients and controls . car . , carnitine NAD , nicotinamide adenine

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Metabolomes of muscle of PEO (B) patients; PLS-DA plots; VIP score plots of top 15 metabolites; volcano plots of all metabolites. Data information: a Significantly changed metabolites outside the FDR cut-off. b Metabolites not significantly changed between patients and controls. car., carnitine NAD, nicotinamide adenine dinucleotide


D Relative values of metabolites in muscle of MIRAS , PEO and MELAS / MIDD patients compared to controls . All data represent mean ± SD . * P = 0 . 031 , * * P = 0 . 008 ( two sample T - test ) . UDP , uridine diphosphate .

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D Relative values of metabolites in muscle of MIRAS, PEO and MELAS/MIDD patients compared to controls. All data represent mean ± SD. *P = 0.031, **P = 0.008 (two sample T-test). UDP, uridine diphosphate.


A Separate specificity and sensitivity ( ROC ) of sorbitol , alanine , myoinositol and cystathionine ( left ) and conventional blood biomarkers lactate and pyruvate , and cytokine FGF21 ( right ) in blood of MIRAS , PEO and MELAS / MIDD patients ( n = 20 ) compared to controls ( n = 30 ) . Data information : ROC analysis : AUC of sorbitol 0 . 81 ( 95 % CI 0 . 68 - 0 . 94 , P = 0 . 0003 ) , alanine 0 . 81 ( 95 % CI 0 . 66 - 0 . 94 , P = 0 . 0003 ) , myoinositol 0 . 79 ( 95 % CI 0 . 66 - 0 . 91 , P = 0 . 0007 ) and cystathionine 0 . 78 ( 95 % CI 0 . 65 - 0 . 91 , P = 0 . 001 ) . AUC of conventional biomarkers : FGF21 0 . 87 ( 95 % CI 0 . 74 - 0 . 99 , P = 0 . 0001 ) , lactate 0 . 86 ( 95 % CI 0 . 76 - 0 . 97 , P = 0 . 0001 ) and pyruvate 0 . 78 ( 95 % CI 0 . 64 - 0 . 93 , P = 0 . 0017 ) .

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A Separate specificity and sensitivity (ROC) of sorbitol, alanine, myoinositol and cystathionine (left) and conventional blood biomarkers lactate and pyruvate, and cytokine FGF21 (right) in blood of MIRAS, PEO and MELAS/MIDD patients (n = 20) compared to controls (n = 30). Data information: ROC analysis: AUC of sorbitol 0.81 (95% CI 0.68 - 0.94, P = 0.0003), alanine 0.81 (95% CI 0.66 - 0.94, P = 0.0003), myoinositol 0.79 (95% CI 0.66 - 0.91, P = 0.0007) and cystathionine 0.78 (95% CI 0.65 - 0.91, P = 0.001). AUC of conventional biomarkers: FGF21 0.87 (95% CI 0.74 - 0.99, P = 0.0001), lactate 0.86 (95% CI 0.76 - 0.97, P = 0.0001) and pyruvate 0.78 (95% CI 0.64 - 0.93, P = 0.0017).


B Combined ' multi - biomarker ' value of sorbitol / alanine / myoinositol / cystathionine ; ROC - analysis ( left ) ; mean centroids MD , IBM and NMD patients , and MIRAS carriers compared to controls ( right ) . Data information : ROC analysis AUC of ' multi - biomarker ' 0 . 94 ( 95 % CI 0 . 89 - 0 . 995 , P = 0 . 0001 ) . Mean centroid data represent mean ± SD . * * P < 0 . 01 , * * * P < 0 . 001 ( 1 - way ANOVA with Dunnett ´ s multiple comparison test ) .

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B Combined 'multi-biomarker' value of sorbitol/alanine/myoinositol/cystathionine; ROC-analysis (left); mean centroids MD, IBM and NMD patients, and MIRAS carriers compared to controls (right). Data information: ROC analysis AUC of 'multi-biomarker' 0.94 (95% CI 0.89 - 0.995, P = 0.0001). Mean centroid data represent mean ± SD. **P < 0.01, ***P < 0.001 (1-way ANOVA with Dunnett´s multiple comparison test).


A Tissue distribution of RIPK2 determined by anti - FLAG immunoprecipitation . Organ homogenates from WT ( wild - type ) and KI ( homozygous FLAG - RIPK2 ) mice were subjected to anti - FLAG immunoprecipitation and immunoblotting .

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A Tissue distribution of RIPK2 determined by anti-FLAG immunoprecipitation. Organ homogenates from WT (wild-type) and KI (homozygous FLAG-RIPK2) mice were subjected to anti-FLAG immunoprecipitation and immunoblotting.


B Inflammatory signaling in wild - type ( WT / WT ) , RIPK2 CRISPR KO ( KO / KO ) and FLAG - RIPK2 heterozygous ( WT / KI ) and homozygous ( KI / KI ) BMDMs . BMDMs were primed with IFNy , stimulated with MDP for indicated times and analysed by immunoblotting .

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B Inflammatory signaling in wild-type (WT/WT), RIPK2 CRISPR KO (KO/KO) and FLAG-RIPK2 heterozygous (WT/KI) and homozygous (KI/KI) BMDMs. BMDMs were primed with IFNy, stimulated with MDP for indicated times and analysed by immunoblotting.


C Cytokine production of RIPK2 CRISPR KO ( KO ) , wild - type ( WT ) and FLAG - RIPK2 heterozygous ( WT / KI ) and homozygous ( KI / KI ) BMDMs in response to MDP . BMDMs were left untreated or treated with IFNy or IFNy and MDP over - night and cytokines were measured by ELISA . N = 5 - 8 mice . Shown is average ± SEM . ns = P > 0 . 05 ; * * = P ≤ 0 . 01 ; * * * * = P ≤ 0 . 0001 ; two - way ANOVA .

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C Cytokine production of RIPK2 CRISPR KO (KO), wild-type (WT) and FLAG-RIPK2 heterozygous (WT/KI) and homozygous (KI/KI) BMDMs in response to MDP. BMDMs were left untreated or treated with IFNy or IFNy and MDP over-night and cytokines were measured by ELISA. N = 5-8 mice. Shown is average ± SEM. ns = P > 0.05; ** = P ≤ 0.01; **** = P ≤ 0.0001; two-way ANOVA.


D Serum cytokines in RIPK2 CRISPR KO ( KO ) , wild - type ( WT ) and FLAG - RIPK2 heterozygous ( WT / KI ) or homozygous ( KI / KI ) mice after i . p . MDP administration . Mice were injected i . p . with PBS or MDP , sacrificed after 4h and serum cytokines were measured by ELISA . N = 3 - 6 mice . Shown is average ± SEM . ns = P > 0 . 05 ; * * = P ≤ 0 . 01 ; two - way ANOVA .

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D Serum cytokines in RIPK2 CRISPR KO (KO), wild-type (WT) and FLAG-RIPK2 heterozygous (WT/KI) or homozygous (KI/KI) mice after i.p. MDP administration. Mice were injected i.p. with PBS or MDP, sacrificed after 4h and serum cytokines were measured by ELISA. N = 3-6 mice. Shown is average ± SEM. ns = P > 0.05; ** = P ≤ 0.01; two-way ANOVA.


A Two step enrichment to isolate ubiquitinated RIPK2 from BMDMs . Protein lysates from FLAG - RIPK2 BMDMs ( A ) were sequentially subjected to ubiquitin enrichment ( UBA , B ) and FLAG pulldown ( C ) prior to protein elution and subsequent mass spectrometry analysis .

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A Two step enrichment to isolate ubiquitinated RIPK2 from BMDMs. Protein lysates from FLAG-RIPK2 BMDMs (A) were sequentially subjected to ubiquitin enrichment (UBA, B) and FLAG pulldown (C) prior to protein elution and subsequent mass spectrometry analysis.


A Activation of the NF - κB pathway by RIPK2 Lysine - and phosphosite mutants . RIPK2 - deficient THP - 1 cells reconstituted with wild - type RIPK2 or RIPK2 mutants were stimulated with L18 - MDP , harvested at indicated time points and activation of the NF - κB pathway was analyzed by immunoblotting .

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A Activation of the NF-κB pathway by RIPK2 Lysine- and phosphosite mutants. RIPK2-deficient THP-1 cells reconstituted with wild-type RIPK2 or RIPK2 mutants were stimulated with L18-MDP, harvested at indicated time points and activation of the NF-κB pathway was analyzed by immunoblotting.


B IL - 8 production of wild - type THP - 1 and RIPK2 - deficient THP - 1 cells reconstituted with wild - type RIPK2 or RIPK2 mutants and stimulated with L18 - MDP was assessed by ELISA . N = 4 - 8 experiments . Shown is average ± SEM . * = P ≤ 0 . 05 ; two - way ANOVA .

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B IL-8 production of wild-type THP-1 and RIPK2-deficient THP-1 cells reconstituted with wild-type RIPK2 or RIPK2 mutants and stimulated with L18-MDP was assessed by ELISA. N = 4-8 experiments. Shown is average ± SEM. * = P ≤ 0.05; two-way ANOVA.


C ) RIPK2 ubiquitination determined by UBA pulldown in RIPK2 - deficient cells reconstituted with wild - type or mutant RIPK2 after stimulation with L18 - MDP .

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C) RIPK2 ubiquitination determined by UBA pulldown in RIPK2-deficient cells reconstituted with wild-type or mutant RIPK2 after stimulation with L18-MDP.


D ) Detection of K63 - and M1 - linked ubiquitin chains by UBA pulldown or pulldown with ubiquitin chain type - specific antibodies in RIPK2 - deficient cells reconstituted with RIPK2 K209R after stimulation with L18 - MDP .

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D) Detection of K63- and M1-linked ubiquitin chains by UBA pulldown or pulldown with ubiquitin chain type-specific antibodies in RIPK2-deficient cells reconstituted with RIPK2 K209R after stimulation with L18-MDP.


C Activation of the NF - κB pathway by RIPK2 pocket mutants . RIPK2 - deficient THP - 1 cells were reconstituted with wild - type RIPK2 or RIPK2 mutants , stimulated with L18 - MDP , harvested at indicated time points and analyzed by immunoblotting .

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C Activation of the NF-κB pathway by RIPK2 pocket mutants. RIPK2-deficient THP-1 cells were reconstituted with wild-type RIPK2 or RIPK2 mutants, stimulated with L18-MDP, harvested at indicated time points and analyzed by immunoblotting.


D IL - 8 production of RIPK2 - deficient THP - 1cells reconstituted with wild - type RIPK2 or RIPK2 mutants and stimulated with L18 - MDP was assessed by ELISA . N = 3 - 8 experiments . Shown is average ± SEM . ns = P > 0 . 05 ; * = P ≤ 0 . 05 ; * * * = P ≤ 0 . 001 ; * * * * = P ≤ 0 . 0001 ; two - way ANOVA .

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D IL-8 production of RIPK2-deficient THP-1cells reconstituted with wild-type RIPK2 or RIPK2 mutants and stimulated with L18-MDP was assessed by ELISA. N = 3-8 experiments. Shown is average ± SEM. ns = P > 0.05; * = P ≤ 0.05; *** = P ≤ 0.001; **** = P ≤ 0.0001; two-way ANOVA.


E Ubiquitination of RIPK2 pocket mutants . RIPK2 - deficient THP - 1 cells were reconstituted with wild - type or mutant RIPK2 , left unstimulated or stimulated with L18 - MDP and subjected to UBA pulldown and immunoblotting .

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E Ubiquitination of RIPK2 pocket mutants. RIPK2-deficient THP-1 cells were reconstituted with wild-type or mutant RIPK2, left unstimulated or stimulated with L18-MDP and subjected to UBA pulldown and immunoblotting.


A : Thermal stability assay using selected RIPK2 mutants . THP - 1 cells expressing RIPK2 were subjected to heat treatment and the non - denatured fraction was analysed by immunoblotting .

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A: Thermal stability assay using selected RIPK2 mutants. THP-1 cells expressing RIPK2 were subjected to heat treatment and the non-denatured fraction was analysed by immunoblotting.


B : Binding of RIPK2 to XIAP - BIR2 . Lysates from RIPK2 - deficient THP - 1 cells reconstituted with WT or mutant RIPK2 were subjected to pulldown experiments with recombinantly expressed XIAP - BIR2 and analysed by immunoblotting .

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B: Binding of RIPK2 to XIAP-BIR2. Lysates from RIPK2-deficient THP-1 cells reconstituted with WT or mutant RIPK2 were subjected to pulldown experiments with recombinantly expressed XIAP-BIR2 and analysed by immunoblotting.


( B ) Gal4 DNA - binding domain fusions of MUPs were tested with Gal4 activation domain fusions of ubiquilins ( murine ) in Y2H .

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(B) Gal4 DNA-binding domain fusions of MUPs were tested with Gal4 activation domain fusions of ubiquilins (murine) in Y2H.


( C ) HEK293 cells transfected with indicated MUP - V5 , myc - UBQLN1 ( human ) , or empty vector were immunoprecipitated ( IP ) with anti - myc or anti - UBQLN1 antibodies . Western blot ( WB ) of IP and lysate were probed as indicated .

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(C) HEK293 cells transfected with indicated MUP-V5, myc-UBQLN1 (human), or empty vector were immunoprecipitated (IP) with anti-myc or anti-UBQLN1 antibodies. Western blot (WB) of IP and lysate were probed as indicated.


( D - E ) WB of input lysate and IP from HEK293 cells expressing myc - UBQLN1 and Rv1926 - V5 ( D ) or Rv1566 - V5 ( E ) . IP was performed using antibodies directed against myc , V5 , or isotype control ( IgG ) .

(D-E) WB of input lysate and IP from HEK293 cells expressing myc-UBQLN1 and Rv1926-V5 (D) or Rv1566-V5 (E). IP was performed using antibodies directed against myc, V5, or isotype control (IgG).


( B ) Mtb were incubated with lysate from HEK293 cells transfected with vector control , myc - UBQLN1 , GFP , or V5 - parkin . WB analysis of the input ( I ) , 5th wash ( W5 ) , and elution ( E ) , probed with indicated antibodies . Actin is a loading control for the cell lysate and Ag85b for the bacterial pellet .

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(B) Mtb were incubated with lysate from HEK293 cells transfected with vector control, myc-UBQLN1, GFP, or V5-parkin. WB analysis of the input (I), 5th wash (W5), and elution (E), probed with indicated antibodies. Actin is a loading control for the cell lysate and Ag85b for the bacterial pellet.


( C ) Mtb were incubated with lysate from HEK293 cells transfected with myc - UBQLN1 - ΔUBA , myc - UBQLN1 - ΔUBL , and myc - UBQLN1 - UBA . WB containing the input , 5th wash , and elution were incubated with anti - myc antibody . The predicted sizes of myc - UBQLN1 - ΔUBL , myc - UBQLN1 - ΔUBA , and myc - UBQLN1 - UBA are 55 kD , 50 kD , and 6 kD , respectively .

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(C) Mtb were incubated with lysate from HEK293 cells transfected with myc-UBQLN1-ΔUBA, myc-UBQLN1-ΔUBL, and myc-UBQLN1-UBA. WB containing the input, 5th wash, and elution were incubated with anti-myc antibody. The predicted sizes of myc-UBQLN1-ΔUBL, myc-UBQLN1-ΔUBA, and myc-UBQLN1-UBA are 55 kD, 50 kD, and 6 kD, respectively.


( D ) Mtb were incubated with recombinant GST - UBQLN1 , GST - UBQLN1 - ΔUBL , or GST - UBQLN1 - ΔUBA . WB containing the input and elution was probed with an anti - GST antibody .

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(D) Mtb were incubated with recombinant GST-UBQLN1, GST-UBQLN1-ΔUBL, or GST-UBQLN1-ΔUBA. WB containing the input and elution was probed with an anti-GST antibody.


( A ) BMDMs were infected with GFP - expressing wt Mtb or ΔesxA for 24h and immunostained with UBQLN1 antibody . IFN - γ was added 1d prior to infection as indicated . ( B ) Quantification of results from A from 3 independent experiments with at least 100 bacteria analyzed per experiment ; * * * P < 0 . 001 , Fisher ' s exact test , comparing the proportion of UBQLN1 positive versus negative Mtb in indicated samples .

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(A) BMDMs were infected with GFP-expressing wt Mtb or ΔesxA for 24h and immunostained with UBQLN1 antibody. IFN-γ was added 1d prior to infection as indicated. (B) Quantification of results from A from 3 independent experiments with at least 100 bacteria analyzed per experiment; ***P< 0.001, Fisher's exact test, comparing the proportion of UBQLN1 positive versus negative Mtb in indicated samples.


( C ) Cellular lysate from BMDMs that were treated with IFN - γ as indicated were analyzed by WB with an antibody that recognizes UBQLN1 and UBQLN2 .

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(C) Cellular lysate from BMDMs that were treated with IFN-γ as indicated were analyzed by WB with an antibody that recognizes UBQLN1 and UBQLN2.


( D ) BMDMs were transfected with siRNAs targeting UBQLN1 or a non - targeting control ( siCON ) and lysates were analyzed by WB using an antibody that recognizes UBQLN1 and UBQLN2 . ( C - D ) Actin served as a loading control .

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(D) BMDMs were transfected with siRNAs targeting UBQLN1 or a non-targeting control (siCON) and lysates were analyzed by WB using an antibody that recognizes UBQLN1 and UBQLN2. (C-D) Actin served as a loading control.


( E ) BMDMs were infected with Mtb live / dead strain and treated with AnTc 24h prior to fixation .

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(E) BMDMs were infected with Mtb live/dead strain and treated with AnTc 24h prior to fixation.


( F ) BMDMs were transfected with siRNAs targeting UBQLN1 or siCON and infected with live / dead Mtb strain . IFN - γ was added 1d prior to infection if indicated . % Viable bacteria were quantified 24 and 48 hpi . Data are from 2 independent experiments . * P < 0 . 05 ; * * * P < 0 . 001 , Fisher ' s exact test , comparing the proportion of viable and non - viable bacteria in indicated samples .

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(F) BMDMs were transfected with siRNAs targeting UBQLN1 or siCON and infected with live/dead Mtb strain. IFN-γ was added 1d prior to infection if indicated. %Viable bacteria were quantified 24 and 48 hpi. Data are from 2 independent experiments. *P<0.05; ***P<0.001, Fisher's exact test, comparing the proportion of viable and non-viable bacteria in indicated samples.


( G ) BMDMs were treated with the indicated siRNAs three days prior to infection and then activated with IFN - γ one day prior to infection with H37Rv or ΔesxA . CFU were enumerated 4 and 96 hpi . * P < 0 . 05 , unpaired Student ' s t - test . Data are representative of three independent experiments .

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(G) BMDMs were treated with the indicated siRNAs three days prior to infection and then activated with IFN-γ one day prior to infection with H37Rv or ΔesxA. CFU were enumerated 4 and 96 hpi. *P< 0.05, unpaired Student's t-test. Data are representative of three independent experiments.


( H ) BMDMs transfected with siRNA targeting UBQLN1 or siCON were infected with wt Mtb ( H37Rv ) and IFN - γ was measured by

(H) BMDMs transfected with siRNA targeting UBQLN1 or siCON were infected with wt Mtb (H37Rv) and IFN-γ was measured by ELISA


( I ) UBQLN1 - silenced BMDMs and controls were infected with H37Rv or ΔesxA . ( H - I ) 24 hpi infected BMDMs were co - cultured with P25TCR - Tg CD4 + T cells , and IFN - γ was measured by ELISA . Data is representative of 3 independent experiments . * P < 0 . 05 , * * P < 0 . 005 , unpaired Student ' s t - test . ( B , F , Γ , H ) Results are mean + / - SEM ; ns , not significant .

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(I) UBQLN1-silenced BMDMs and controls were infected with H37Rv or ΔesxA. (H-I) 24 hpi infected BMDMs were co-cultured with P25TCR-Tg CD4+ T cells, and IFN-γ was measured by ELISA. Data is representative of 3 independent experiments. *P<0.05, **P<0.005, unpaired Student's t-test. (B, F, Γ, H) Results are mean +/- SEM; ns, not significant.


( A ) BMDMs treated with IFN - γ and infected for 24h with GFP - Mtb were immunostained for UBQLN1 and either ubiquitin ( using the FK2 antibody ) or p62 . GFP - LC3 BMDMs infected with DsRed - Mtb were immunostained for UBQLN1 . ( B ) Quantification of those Mtb that co - localize with at least one of the cellular markers shown in A . More than 375 total bacteria were counted in two independent experiments , and the number co - localizing with at least one marker is indicated as " total " under each bar .

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(A) BMDMs treated with IFN-γ and infected for 24h with GFP-Mtb were immunostained for UBQLN1 and either ubiquitin (using the FK2 antibody) or p62. GFP-LC3 BMDMs infected with DsRed-Mtb were immunostained for UBQLN1. (B) Quantification of those Mtb that co-localize with at least one of the cellular markers shown in A. More than 375 total bacteria were counted in two independent experiments, and the number co-localizing with at least one marker is indicated as "total" under each bar.


( C ) Wt macrophages ( Atg16L1flox / flox ; Cre - ) and autophagy - deficient macrophages ( Atg16L1flox / flox Lyz - cre ; Cre + ) were activated with IFN - γ or not , followed by infection with the live / dead Mtb reporter strain . % viable was quantified after addition of AnTc . * * P < 0 . 005 , * * * P < 0 . 0001 , Fisher ' s exact test ( in which the proportions of viable to non - viable Mtb was compared ) .

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(C) Wt macrophages (Atg16L1flox/flox; Cre-) and autophagy-deficient macrophages (Atg16L1flox/flox Lyz-cre; Cre+) were activated with IFN-γ or not, followed by infection with the live/dead Mtb reporter strain. % viable was quantified after addition of AnTc. **P<0.005, ***P<0.0001, Fisher's exact test (in which the proportions of viable to non-viable Mtb was compared).


( D ) Atg16L1flox / flox Cre + and Cre - BMDMs treated with IFN - γ and infected for 24h with GFP - Mtb were immunostained for UBQLN1 and p62 . The number of Mtb co - localizating with the cellular markers is shown . ( C - D ) At least 100 bacteria were analyzed in three independent experiments .

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(D) Atg16L1flox/flox Cre+ and Cre- BMDMs treated with IFN-γ and infected for 24h with GFP-Mtb were immunostained for UBQLN1 and p62. The number of Mtb co-localizating with the cellular markers is shown. (C-D) At least 100 bacteria were analyzed in three independent experiments.


( E ) Images from IFN - γ treated BMDMs of indicated genotype infected with GFP - Mtb and immunostained with UBQLN1 and p62 . Arrows indicates p62 + UBQLN1 + Mtb .

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(E) Images from IFN-γ treated BMDMs of indicated genotype infected with GFP-Mtb and immunostained with UBQLN1 and p62. Arrows indicates p62+ UBQLN1+ Mtb.


( A - C ) BMDMs transfected with siRNA targeting UBQLN1 or control siRNA . Images show IFN - γ treated macrophages infected with GFP - Mtb ( A - B ) or DsRed - Mtb ( C ) for 24h . Cells were immunostained for ubiquitin ( Ub ) with the FK2 antibody ( A ) or p62 ( B ) , which are shown in red . ( C ) GFP - LC3 BMDMs were infected with DsRed - Mtb . LC3 is shown in green , whereas Mtb are red . ( A - C ) Quantification shows the percentage of Mtb that colocalized with the indicated cellular marker in macrophages that were treated with IFN - γ as indicated . Results are mean + / - SEM from two independent experiments , with at least 100 bacteria analyzed per experiment . * * * P < 0 . 001 , Fisher ' s exact test ( in which the proportion of Mtb associated with the cellular marker in a given condition was compared to the proportion found in the sample treated with siCON and IFN - γ ) .

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

(A-C) BMDMs transfected with siRNA targeting UBQLN1 or control siRNA. Images show IFN-γ treated macrophages infected with GFP-Mtb (A-B) or DsRed-Mtb (C) for 24h. Cells were immunostained for ubiquitin (Ub) with the FK2 antibody (A) or p62 (B), which are shown in red. (C) GFP-LC3 BMDMs were infected with DsRed-Mtb. LC3 is shown in green, whereas Mtb are red. (A-C) Quantification shows the percentage of Mtb that colocalized with the indicated cellular marker in macrophages that were treated with IFN-γ as indicated. Results are mean +/- SEM from two independent experiments, with at least 100 bacteria analyzed per experiment. ***P<0.001, Fisher's exact test (in which the proportion of Mtb associated with the cellular marker in a given condition was compared to the proportion found in the sample treated with siCON and IFN-γ).


( A ) WT and Park2 - / - BMDMs were pretreated with IFN - γ , infected with GFP - Mtb for 24h , and immunostained with the FK2 antibody . Bacterial colocalization with FK2 immunoreactivity was quantified from 2 independent experiments .

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(A) WT and Park2-/- BMDMs were pretreated with IFN-γ, infected with GFP-Mtb for 24h, and immunostained with the FK2 antibody. Bacterial colocalization with FK2 immunoreactivity was quantified from 2 independent experiments.


( B ) Park2 - / - BMDMs pretreated or not treated with IFN - γ were infected with GFP - Mtb for 24h and immunostained with UBQLN1 specific antibody ; Arrows indicate Mtb co - localized with UBQLN1 . ns - not significant .

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(B) Park2-/- BMDMs pretreated or not treated with IFN-γ were infected with GFP-Mtb for 24h and immunostained with UBQLN1 specific antibody; Arrows indicate Mtb co-localized with UBQLN1. ns- not significant.


( C ) Park2 - / - BMDMs were transfected with siRNA targeting UBQLN1 or control siRNA 3d prior to infection , treated with IFN - γ , and infected with GFP - Mtb for 24h prior to immunostaining for ubiquitin ( FK2 antibody ) . Ubiquitin ( FK2 + ) bacteria were quantified from two independent experiments . ( A , C ) * * P < 0 . 01 , Fisher ' s exact test ( comparing the proportion of FK2 positive bacteria in indicated samples ) . Results are mean + / - SEM . A minimum of 100 Mtb were counted in each experiment .

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(C) Park2-/- BMDMs were transfected with siRNA targeting UBQLN1 or control siRNA 3d prior to infection, treated with IFN-γ, and infected with GFP-Mtb for 24h prior to immunostaining for ubiquitin (FK2 antibody). Ubiquitin (FK2+) bacteria were quantified from two independent experiments. (A, C) **P<0.01, Fisher's exact test (comparing the proportion of FK2 positive bacteria in indicated samples). Results are mean +/- SEM. A minimum of 100 Mtb were counted in each experiment.


C . Confocal images of U2OS cells transiently expressing lifeAct - GFP wild type and mutants of lifeAct residues interacting with F - actin , cells were additionally stained with SiR - actin to confirm the actin filaments . The line scan as indicated with yellow line on the cells shows the extent of lifeAct ( green ) and SiR - actin ( magenta ) co - staining of actin structures . Scale bar = 5µm .

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C. Confocal images of U2OS cells transiently expressing lifeAct-GFP wild type and mutants of lifeAct residues interacting with F-actin, cells were additionally stained with SiR-actin to confirm the actin filaments. The line scan as indicated with yellow line on the cells shows the extent of lifeAct (green) and SiR-actin (magenta) co-staining of actin structures. Scale bar = 5µm.


D . Binding affinities calculated from titration data of co - sedimentation assays of lifeAct - 14 ( 1 . 2µM ) and lifeAct - 17 ( 2 . 2µM ) as indicated . Data points for each concentration were averaged from 3 independent experiments , error bars represent S . D between independent experiments .

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D. Binding affinities calculated from titration data of co-sedimentation assays of lifeAct-14 (1.2µM) and lifeAct-17 (2.2µM) as indicated. Data points for each concentration were averaged from 3 independent experiments, error bars represent S.D between independent experiments.


C . Representative TIRF images of lifeAct binding experiments as indicated . The remaining lifeAct concentration images are shown in Supplement Figure 4 . Scale bar = 5µm .

C. Representative TIRF images of lifeAct binding experiments as indicated. The remaining lifeAct concentration images are shown in Supplement Figure 4. Scale bar = 5µm.


D . Mean ratio of lifeAct fluorescent intensity bound to phalloidin and jasplakinolide F - actin ( mean and s . e . m ; n = 2 or 3 independent experiments with > 50 actin filament for each set ) .

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D. Mean ratio of lifeAct fluorescent intensity bound to phalloidin and jasplakinolide F-actin (mean and s.e.m; n = 2 or 3 independent experiments with >50 actin filament for each set).


A . Representative Coomassie stained SDS - PAGE gels of UTRN - ABD and UTRN - V87D co - sedimentation with F - actin . Pellet ( top ) and supernatant ( bottom ) fractions of individual co - sedimentation reactions of increasing utrophin concentrations , uncropped gel images of all the co - sedimentation reactions are presented in Supplement Figure 6 .

A. Representative Coomassie stained SDS-PAGE gels of UTRN-ABD and UTRN-V87D co-sedimentation with F-actin. Pellet (top) and supernatant (bottom) fractions of individual co-sedimentation reactions of increasing utrophin concentrations, uncropped gel images of all the co-sedimentation reactions are presented in Supplement Figure 6.


B . Apparent Kd indicated was calculated from the titration data of co - sedimentation assays of utrophin wildtype ( 1 . 8µM ) and mutants ; H29A ( 2 . 8µM ) , I22D ( 38µM ) , V87D ( > 100µM ) and N109A ( 55µM ) as indicated . Data points for each concentration were averaged from 3 independent experiments , error bars represent S . D between independent experiments .

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B. Apparent Kd indicated was calculated from the titration data of co-sedimentation assays of utrophin wildtype (1.8µM) and mutants; H29A (2.8µM), I22D (38µM), V87D (>100µM) and N109A (55µM) as indicated. Data points for each concentration were averaged from 3 independent experiments, error bars represent S.D between independent experiments.


C . Confocal images of U20S cells transfected with GFP tagged UTRN - mini and mCherry UTRN - ABD , stained with SIR - Actin shows F - actin structures , mainly stress fibers . Scale bar = 5µm .

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C. Confocal images of U20S cells transfected with GFP tagged UTRN-mini and mCherry UTRN-ABD, stained with SIR-Actin shows F-actin structures, mainly stress fibers. Scale bar = 5µm.


A A neutral red - stained mouse pancreas is excised and digested with collagenase . Islets are then separated by hand picking and further digested into single islet cells . Scale bars : 10 mm ( left image ) ; 200 µm ( middle image ) ; 60 µm ( right image ) .

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A A neutral red-stained mouse pancreas is excised and digested with collagenase. Islets are then separated by hand picking and further digested into single islet cells. Scale bars: 10 mm (left image); 200 µm (middle image); 60 µm (right image).


B Epidydimal fat pads are isolated and processed mechanically and enzymatically . The obtained mixture , containing MVF and ECM , is filtrated for the purification of MVF . Scale bars : 20 mm ( left image ) ; 25 µm ( right image ) .

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B Epidydimal fat pads are isolated and processed mechanically and enzymatically. The obtained mixture, containing MVF and ECM, is filtrated for the purification of MVF. Scale bars: 20 mm (left image); 25 µm (right image).


C Western blots of CD31 , α - SMA and GAPDH in whole cell extracts of eEND , HDMEC , HUVEC and MVF .

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C Western blots of CD31, α-SMA and GAPDH in whole cell extracts of eEND, HDMEC, HUVEC and MVF.


D Immunofluorescence stainings of CD31 / α - SMA and CD31 / PDGFR - β in MVF . Cell nuclei are stained with Hoechst 33342 ( blue ) . Scale bar : 20 µm .

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D Immunofluorescence stainings of CD31 / α-SMA and CD31 / PDGFR-β in MVF. Cell nuclei are stained with Hoechst 33342 (blue). Scale bar: 20 µm.


F PI and PI + MVF on days - 5 , - 3 and 0 . Scale bar : 200 µm .

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F PI and PI+MVF on days -5, -3 and 0. Scale bar: 200 µm.


G Surface morphology of FI , CI , PI and PI + MVF on day 0 as visualized by scanning electron microscopy . Scale bar : 50 µm .

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G Surface morphology of FI, CI, PI and PI+MVF on day 0 as visualized by scanning electron microscopy. Scale bar: 50 µm.


A Neutral red and trypan blue stainings of FI , CI , PI and PI + MVF . CI incubated for 24 h with 0 . 2 % H2O2 served as control . Scale bar : 100 µm .

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A Neutral red and trypan blue stainings of FI, CI, PI and PI+MVF. CI incubated for 24 h with 0.2% H2O2 served as control. Scale bar: 100 µm.


B Representative flow cytometric scatterplots of propidium iodide / annexin V - stained cells from PI + MVF and CI + H2O2 ( positive control ) . C Quantitative analysis of propidium iodide / annexin V - stained cells from FI , CI , PI , PI + MVF and CI + H2O2 subdivided in necrotic , necroptotic , apoptotic and vital cells in % of total cell number ( n = 3 each ) .

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B Representative flow cytometric scatterplots of propidium iodide / annexin V-stained cells from PI+MVF and CI+H2O2 (positive control). C Quantitative analysis of propidium iodide / annexin V-stained cells from FI, CI, PI, PI+MVF and CI+H2O2 subdivided in necrotic, necroptotic, apoptotic and vital cells in % of total cell number (n = 3 each).


D Schematic illustration of the mitochondrial ROS - sensor mito - roGFP2 - Orp1 . The fluorescence maximum of this sensor shifts from 488 nm to 405 nm with increasing mitochondrial H2O2 levels , as shown by representative fluorescence images of FI . Scale bar : 70 µm .

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D Schematic illustration of the mitochondrial ROS-sensor mito-roGFP2-Orp1. The fluorescence maximum of this sensor shifts from 488 nm to 405 nm with increasing mitochondrial H2O2 levels, as shown by representative fluorescence images of FI. Scale bar: 70 µm.


E Quantitative analysis of the ROS sensor ratio ( 405 nm / 488 nm ) of FI , CI , PI and PI + MVF ( n = 25 each ) . Mean ± SD . One - way ANOVA and Tukey ' s multiple comparisons post hoc test were used for statistical analysis . * P < 0 . 05 vs . FI ; # P < 0 . 05 vs . CI ; + P < 0 . 05 vs . PI .

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E Quantitative analysis of the ROS sensor ratio (405 nm / 488 nm) of FI, CI, PI and PI+MVF (n = 25 each). Mean ± SD. One-way ANOVA and Tukey's multiple comparisons post hoc test were used for statistical analysis. *P < 0.05 vs. FI; #P < 0.05 vs. CI; +P < 0.05 vs. PI.


F Representative Western blot analysis of β - actin and HO - 1 in whole cell extracts from FI , CI , PI and PI + MVF . G Quantitative analysis of HO - 1 expression in FI , CI , PI and PI + MVF ( n = 3 each ) . Mean ± SD . One - way ANOVA and Tukey ' s multiple comparisons post hoc test were used for statistical analysis . * P < 0 . 05 vs . FI ; # P < 0 . 05 vs . CI ; + P < 0 . 05 vs . PI .

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F Representative Western blot analysis of β-actin and HO-1 in whole cell extracts from FI, CI, PI and PI+MVF. G Quantitative analysis of HO-1 expression in FI, CI, PI and PI+MVF (n = 3 each). Mean ± SD. One-way ANOVA and Tukey's multiple comparisons post hoc test were used for statistical analysis. *P < 0.05 vs. FI; #P < 0.05 vs. CI; +P < 0.05 vs. PI.


H Representative immunofluorescence stainings of HO - 1 / insulin and HO - 1 / CD31 in PI + MVF . Cell nuclei are stained with Hoechst 33342 ( blue ) . Scale bar : 50 µm .

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H Representative immunofluorescence stainings of HO-1 / insulin and HO-1 / CD31 in PI+MVF. Cell nuclei are stained with Hoechst 33342 (blue). Scale bar: 50 µm.


I Representative Western blot analysis of β - actin and HO - 1 in whole cell extracts from PI and MVF .

I Representative Western blot analysis of β-actin and HO-1 in whole cell extracts from PI and MVF.


A Representative immunofluorescence stainings of insulin / glucagon and insulin / somatostatin in FI , CI , PI and PI + MVF . Cell nuclei are stained with Hoechst 33342 ( blue ) . Scale bar : 60 µm . B - D Quantitative analysis of insulin - ( B ) , glucagon - ( C ) and somatostatin - positive cells ( D ) in FI , CI , PI and PI + MVF in % of all islet or organoid cells ( n = 20 each ) . Mean ± SD . One - way ANOVA and Tukey ' s multiple comparisons post hoc test were used for statistical analysis . * P < 0 . 05 vs . FI ; # P < 0 . 05 vs . CI ; + P < 0 . 05 vs . PI .

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A Representative immunofluorescence stainings of insulin / glucagon and insulin / somatostatin in FI, CI, PI and PI+MVF. Cell nuclei are stained with Hoechst 33342 (blue). Scale bar: 60 µm. B-D Quantitative analysis of insulin- (B), glucagon- (C) and somatostatin-positive cells (D) in FI, CI, PI and PI+MVF in % of all islet or organoid cells (n = 20 each). Mean ± SD. One-way ANOVA and Tukey's multiple comparisons post hoc test were used for statistical analysis. *P < 0.05 vs. FI; #P < 0.05 vs. CI; +P < 0.05 vs. PI.


E Western blot analysis of β - actin and insulin in whole cell extracts from FI , CI , PI and PI + MVF .

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E Western blot analysis of β-actin and insulin in whole cell extracts from FI, CI, PI and PI+MVF.


Insulin secretion ( µU / mL ) ( normalized to DNA content ) of FI , CI , PI and PI + MVF exposed to 1 . 1 mM and 16 . 5 mM glucose ( n = 3 each ) . Mean ± SD . One - way ANOVA and Tukey ' s multiple comparisons post hoc test as well as an unpaired Student ' s t - test were used for statistical analysis . * P < 0 . 05 vs . insulin secretion at 1 . 1 mM glucose within each individual group ; + P < 0 . 05 vs . insulin secretion of PI at 16 . 5 mM glucose .

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Insulin secretion (µU/mL) (normalized to DNA content) of FI, CI, PI and PI+MVF exposed to 1.1 mM and 16.5 mM glucose (n = 3 each). Mean ± SD. One-way ANOVA and Tukey's multiple comparisons post hoc test as well as an unpaired Student's t-test were used for statistical analysis. *P < 0.05 vs. insulin secretion at 1.1 mM glucose within each individual group; +P < 0.05 vs. insulin secretion of PI at 16.5 mM glucose.


A Representative immunofluorescence stainings of insulin and CD31 in CI , FI , PI and PI + MVF . Cell nuclei are stained with Hoechst 33342 ( blue ) . Scale bar : 100 µm . B Quantitative analysis of CD31 - positive cells in FI , CI , PI and PI + MVF in % of all islet or islet organoid cells ( n = 20 each ) . Mean ± SD . One - way ANOVA and Tukey ' s multiple comparisons post hoc test were used for statistical analysis . * P < 0 . 05 vs . FI ; # P < 0 . 05 vs . CI ; + P < 0 . 05 vs . PI .

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A Representative immunofluorescence stainings of insulin and CD31 in CI, FI, PI and PI+MVF. Cell nuclei are stained with Hoechst 33342 (blue). Scale bar: 100 µm. B Quantitative analysis of CD31-positive cells in FI, CI, PI and PI+MVF in % of all islet or islet organoid cells (n = 20 each). Mean ± SD. One-way ANOVA and Tukey's multiple comparisons post hoc test were used for statistical analysis. *P < 0.05 vs. FI; #P < 0.05 vs. CI; +P < 0.05 vs. PI.


C Representative Western blot analysis of CD31 and β - actin in whole cell extracts from CI , FI , PI and PI + MVF .

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C Representative Western blot analysis of CD31 and β-actin in whole cell extracts from CI, FI, PI and PI+MVF.


D Quantitative analysis of CD31 expression in FI , CI , PI and PI + MVF in % of FI ( n = 3 ) . Mean ± SD . One - way ANOVA and Tukey ' s multiple comparisons post hoc test were used for statistical analysis . * P < 0 . 05 vs . FI ; # P < 0 . 05 vs . CI ; + P < 0 . 05 vs . PI .

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D Quantitative analysis of CD31 expression in FI, CI, PI and PI+MVF in % of FI (n = 3). Mean ± SD. One-way ANOVA and Tukey's multiple comparisons post hoc test were used for statistical analysis. *P < 0.05 vs. FI; #P < 0.05 vs. CI; +P < 0.05 vs. PI.


E Confocal microscopic images of CD31 and cell nuclei ( blue ) in PI + MVF , demonstrating endothelial cells ( arrowheads ) . Scale bar : 100 µm .

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E Confocal microscopic images of CD31 and cell nuclei (blue) in PI+MVF, demonstrating endothelial cells (arrowheads). Scale bar: 100 µm.


F Representative immunofluorescence staining of Ki67 and CD31 in PI + MVF . Cell nuclei are stained with Hoechst 33342 ( blue ) . Arrows mark endothelial cells expressing Ki67 . Scale bar : 100 µm .

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F Representative immunofluorescence staining of Ki67 and CD31 in PI+MVF. Cell nuclei are stained with Hoechst 33342 (blue). Arrows mark endothelial cells expressing Ki67. Scale bar: 100 µm.


G Quantitative analysis of CD31 / Ki67 - positive cells in FI , CI , PI and PI + MVF in % of all endothelial cells ( n = 10 each ) . Mean ± SD . One - way ANOVA and Tukey ' s multiple comparisons post hoc test were used for statistical analysis . * P < 0 . 05 vs . FI ; # P < 0 . 05 vs . CI ; + P < 0 . 05 vs . PI .

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G Quantitative analysis of CD31 / Ki67-positive cells in FI, CI, PI and PI+MVF in % of all endothelial cells (n = 10 each). Mean ± SD. One-way ANOVA and Tukey's multiple comparisons post hoc test were used for statistical analysis. *P < 0.05 vs. FI; #P < 0.05 vs. CI; +P < 0.05 vs. PI.


H Flow cytometric analysis of BrdU - positive cells from PI + MVF treated with DMSO ( vehicle ) or linsitinib during the entire fusion process ( n = 5 each ) . Mean ± SD . Unpaired Student ' s t - test was used for statistical analysis . * P < 0 . 05 vs . vehicle .

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H Flow cytometric analysis of BrdU-positive cells from PI+MVF treated with DMSO (vehicle) or linsitinib during the entire fusion process (n = 5 each). Mean ± SD. Unpaired Student's t-test was used for statistical analysis. *P < 0.05 vs. vehicle.


I Representative Western blot analysis of pAkt , Akt , pERK1 / 2 and ERK1 / 2 from MVF cell extracts . MVF were treated with vehicle or insulin for 3 h . Quantitative analysis of pERK1 / 2 / ERK1 / 2 ( J ) ratio in PI + MVF treated with vehicle or insulin for 3 h . Data are expressed as relative intensities ( n = 3 each ) . Mean ± SD . Unpaired Student ' s t - test was used for statistical analysis . * P < 0 . 05 vs . vehicle .

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

I Representative Western blot analysis of pAkt, Akt, pERK1/2 and ERK1/2 from MVF cell extracts. MVF were treated with vehicle or insulin for 3 h. Quantitative analysis of pERK1/2 / ERK1/2 (J) ratio in PI+MVF treated with vehicle or insulin for 3 h. Data are expressed as relative intensities (n = 3 each). Mean ± SD. Unpaired Student's t-test was used for statistical analysis. *P < 0.05 vs. vehicle.


Quantitative analysis of pAkt / Akt ( K ) ratio in PI + MVF treated with vehicle or insulin for 3 h . Data are expressed as relative intensities ( n = 3 each ) . Mean ± SD . Unpaired Student ' s t - test was used for statistical analysis . * P < 0 . 05 vs . vehicle .

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

Quantitative analysis of pAkt / Akt (K) ratio in PI+MVF treated with vehicle or insulin for 3 h. Data are expressed as relative intensities (n = 3 each). Mean ± SD. Unpaired Student's t-test was used for statistical analysis. *P < 0.05 vs. vehicle.


L Representative bright field image of angiogenic sprouting from PI + MVF on 3 day of the sprouting assay . Scale bar : 150 µm . Insert : Immunofluorescence staining of insulin ( red ) and CD31 ( green ) of sprouted PI + MVF . Cell nuclei are stained with Hoechst 33342 ( blue ) . Scale bar : 30 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 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

L Representative bright field image of angiogenic sprouting from PI+MVF on 3 day of the sprouting assay. Scale bar: 150 µm. Insert: Immunofluorescence staining of insulin (red) and CD31 (green) of sprouted PI+MVF. Cell nuclei are stained with Hoechst 33342 (blue). Scale bar: 30 µm.


M Quantitative analysis of sprouting area of FI , CI , PI and PI + MVF in % of the initial size ( day 0 ) ( n = 10 each ) . Mean ± SD . One - way ANOVA and Tukey ' s multiple comparisons post hoc test were used for statistical analysis . * P < 0 . 05 vs . FI ; # P < 0 . 05 vs . CI ; + P < 0 . 05 vs . PI .

M Quantitative analysis of sprouting area of FI, CI, PI and PI+MVF in % of the initial size (day 0) (n = 10 each). Mean ± SD. One-way ANOVA and Tukey's multiple comparisons post hoc test were used for statistical analysis. *P < 0.05 vs. FI; #P < 0.05 vs. CI; +P < 0.05 vs. PI.


B Bright field images of hFI , hCI , hPI and hPI + MVF . Scale bar : 100 µm .

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

B Bright field images of hFI, hCI, hPI and hPI+MVF. Scale bar: 100 µm.


C Representative immunohistochemical stainings of insulin and CD31 in hFI , hCI , hPI and hPI + MVF . Cell nuclei are stained with Hoechst 33342 ( blue ) . Scale bar : 100 µm .

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

C Representative immunohistochemical stainings of insulin and CD31 in hFI, hCI, hPI and hPI+MVF. Cell nuclei are stained with Hoechst 33342 (blue). Scale bar: 100 µm.


D - G Quantitative analysis of insulin - ( D ) , glucagon - ( E ) , somatostatin - ( F ) and CD31 - positive cells ( G ) in hFI , hCI , hPI and hPI + MVF in % of all islet or islet organoid cells ( n = 20 each ) . Mean ± SD . One - way ANOVA and Tukey ' s multiple comparisons post hoc test were used for statistical analysis . * P < 0 . 05 vs . hFI ; # P < 0 . 05 vs . hCI ; + P < 0 . 05 vs . hPI .

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

D-G Quantitative analysis of insulin-(D), glucagon-(E), somatostatin-(F) and CD31-positive cells (G) in hFI, hCI, hPI and hPI+MVF in % of all islet or islet organoid cells (n = 20 each). Mean ± SD. One-way ANOVA and Tukey's multiple comparisons post hoc test were used for statistical analysis. *P < 0.05 vs. hFI; #P < 0.05 vs. hCI; +P < 0.05 vs. hPI.


H Representative bright field images of angiogenic sprouting of hFI , hCI , hPI and hPI + MVF on day 3 of the sprouting assay . Scale bar : 150 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

H Representative bright field images of angiogenic sprouting of hFI, hCI, hPI and hPI+MVF on day 3 of the sprouting assay. Scale bar: 150 µm.


I Quantitative analysis of sprouting area of hFI , hCI , hPI and hPI + MVF in % of initial size ( day 0 ) ( n = 10 each ) . Mean ± SD . One - way ANOVA and Tukey ' s multiple comparisons post hoc test were used for statistical analysis . * P < 0 . 05 vs . hFI ; # P < 0 . 05 vs . hCI ; + P < 0 . 05 vs . hPI .

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

I Quantitative analysis of sprouting area of hFI, hCI, hPI and hPI+MVF in % of initial size (day 0) (n = 10 each). Mean ± SD. One-way ANOVA and Tukey's multiple comparisons post hoc test were used for statistical analysis. *P < 0.05 vs. hFI; #P < 0.05 vs. hCI; +P < 0.05 vs. hPI.


J Quantitative analysis of C - peptide secretion ( pg / mL ) ( normalized to DNA concentration ) of hFI and hPI + MVF exposed to 1 . 1 mM and 16 . 5 mM glucose ( n = 3 each ) . Mean ± SD .

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

J Quantitative analysis of C-peptide secretion (pg/mL) (normalized to DNA concentration) of hFI and hPI+MVF exposed to 1.1 mM and 16.5 mM glucose (n = 3 each). Mean ± SD.


B Observation window of a dorsal skinfold chamber with 8 neutral red - stained transplanted FI on day 0 . Scale bar : 5 mm .

B Observation window of a dorsal skinfold chamber with 8 neutral red-stained transplanted FI on day 0. Scale bar: 5 mm.


C Take rate ( % of transplanted islets ) on day 14 after islet transplantation into the dorsal skinfold chamber ( n = 8 each ) . Mean ± SD . One - way ANOVA and Tukey ' s multiple comparisons post hoc test were used for statistical analysis . + P < 0 . 05 vs . PI .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Take rate (% of transplanted islets) on day 14 after islet transplantation into the dorsal skinfold chamber (n = 8 each). Mean ± SD. One-way ANOVA and Tukey's multiple comparisons post hoc test were used for statistical analysis. +P < 0.05 vs. PI.


D Representative intravital fluorescent microscopic images of FI , CI , PI and PI + MVF within the dorsal skinfold chamber on day 0 and 14 . FITC - labeled dextran is used for the visualization of blood - perfused microvessels . Grafts are marked by broken lines . Scale bar : 150 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

D Representative intravital fluorescent microscopic images of FI, CI, PI and PI+MVF within the dorsal skinfold chamber on day 0 and 14. FITC-labeled dextran is used for the visualization of blood-perfused microvessels. Grafts are marked by broken lines. Scale bar: 150 μm.


E , F Quantitative analysis of the functional capillary density ( cm / cm - 2 ) ( E ) and revascularized area ( mm2 ) ( F ) of transplanted FI , CI , PI and PI + MVF on day 0 , 3 , 6 , 10 and 14 ( n = 8 each ) . Mean ± SD . One - way ANOVA and Tukey ' s multiple comparisons post hoc test were used for statistical analysis . * P < 0 . 05 vs . FI ; # P < 0 . 05 vs . CI ; + P < 0 . 05 vs . PI .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, F Quantitative analysis of the functional capillary density (cm/cm-2) (E) and revascularized area (mm2) (F) of transplanted FI, CI, PI and PI+MVF on day 0, 3, 6, 10 and 14 (n = 8 each). Mean ± SD. One-way ANOVA and Tukey's multiple comparisons post hoc test were used for statistical analysis. *P < 0.05 vs. FI; #P < 0.05 vs. CI; +P < 0.05 vs. PI.


G Representative intravital fluorescent microscopic images of FI , CI , PI and PI + MVF within the dorsal skinfold chamber on day 14 . Rhodamine 6G is used to visualize blood perfused vital endocrine cells ( bright signals ) . Scale bar : 150 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

G Representative intravital fluorescent microscopic images of FI, CI, PI and PI+MVF within the dorsal skinfold chamber on day 14. Rhodamine 6G is used to visualize blood perfused vital endocrine cells (bright signals). Scale bar: 150 µm.


H Quantitative analysis of the rhodamine 6G - positive area ( % of islet size ) within transplanted FI , CI , PI and PI + MVF on day 0 , 3 , 6 , 10 and 14 . Mean ± SD . One - way ANOVA and Tukey ' s multiple comparisons post hoc test were used for statistical analysis . * P < 0 . 05 vs . FI ; # P < 0 . 05 vs . CI ; + P < 0 . 05 vs . PI .

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

H Quantitative analysis of the rhodamine 6G-positive area (% of islet size) within transplanted FI, CI, PI and PI+MVF on day 0, 3, 6, 10 and 14. Mean ± SD. One-way ANOVA and Tukey's multiple comparisons post hoc test were used for statistical analysis. *P < 0.05 vs. FI; #P < 0.05 vs. CI; +P < 0.05 vs. PI.


A Representative immunofluorescent stainings of insulin / glucagon , insulin / somatostatin and insulin / CD31 in FI , CI , PI and PI + MVF on day 14 after transplantation . Cell nuclei are stained with Hoechst 33342 ( blue ) . Arrows mark intra - islet endothelial cells . Scale bar : 70 µm . B - E Quantitative analysis of insulin - ( B ) , glucagon - ( C ) , somatostatin - ( D ) and intra - islet CD31 - positive cells ( E ) within FI , CI , PI and PI + MVF on day 14 after transplantation ( n = 10 each ) . Mean ± SD . One - way ANOVA and Tukey ' s multiple comparisons post hoc test were used for statistical analysis . * P < 0 . 05 vs . FI ; # P < 0 . 05 vs . CI ; + P < 0 . 05 vs . PI .

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

A Representative immunofluorescent stainings of insulin / glucagon, insulin / somatostatin and insulin / CD31 in FI, CI, PI and PI+MVF on day 14 after transplantation. Cell nuclei are stained with Hoechst 33342 (blue). Arrows mark intra-islet endothelial cells. Scale bar: 70 µm. B-E Quantitative analysis of insulin- (B), glucagon- (C), somatostatin- (D) and intra-islet CD31-positive cells (E) within FI, CI, PI and PI+MVF on day 14 after transplantation (n = 10 each). Mean ± SD. One-way ANOVA and Tukey's multiple comparisons post hoc test were used for statistical analysis. *P < 0.05 vs. FI; #P < 0.05 vs. CI; +P < 0.05 vs. PI.


B Quantitative analysis of the body weight ( g ) of nondiabetic and diabetic mice transplanted with FI or PI + MVF from day - 8 to day 28 ( n = 7 each ) . Mean ± SD .

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

B Quantitative analysis of the body weight (g) of nondiabetic and diabetic mice transplanted with FI or PI+MVF from day -8 to day 28 (n = 7 each). Mean ± SD.


C AUC of the body weight of nondiabetic and diabetic mice transplanted with FI or PI + MVF from day 0 to day 28 ( n = 7 each ) . Mean ± SD .

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

C AUC of the body weight of nondiabetic and diabetic mice transplanted with FI or PI+MVF from day 0 to day 28 (n = 7 each). Mean ± SD.


D Quantitative analysis of blood glucose level ( mg / dL ) of diabetic mice transplanted with FI or PI + MVF and nondiabetic controls from day - 8 to day 28 ( n = 7 each ) . Mean ± SD . One - way ANOVA and Tukey ' s multiple comparisons post hoc test were used for statistical analysis . * P < 0 . 05 vs . FI ; # P < 0 . 05 vs . PI + MVF .

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

D Quantitative analysis of blood glucose level (mg/dL) of diabetic mice transplanted with FI or PI+MVF and nondiabetic controls from day -8 to day 28 (n = 7 each). Mean ± SD. One-way ANOVA and Tukey's multiple comparisons post hoc test were used for statistical analysis. *P < 0.05 vs. FI; #P < 0.05 vs. PI+MVF.


E AUC of the blood glucose levels of diabetic mice transplanted with FI or PI + MVF and nondiabetic controls from day 0 to day 28 ( n = 7 each ) . Mean ± SD . One - way ANOVA and Tukey ' s multiple comparisons post hoc test were used for statistical analysis . * P < 0 . 05 vs . FI ; # P < 0 . 05 vs . PI + MVF .

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

E AUC of the blood glucose levels of diabetic mice transplanted with FI or PI+MVF and nondiabetic controls from day 0 to day 28 (n = 7 each). Mean ± SD. One-way ANOVA and Tukey's multiple comparisons post hoc test were used for statistical analysis. *P < 0.05 vs. FI; #P < 0.05 vs. PI+MVF.


F Quantitative analysis of blood glucose level ( mg / dL ) according to the IPGTT of diabetic mice transplanted with FI or PI + MVF and nondiabetic controls to the indicated time points ( n = 5 each ) . Mean ± SD . One - way ANOVA and Tukey ' s multiple comparisons post hoc test were used for statistical analysis . * P < 0 . 05 vs . FI .

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

F Quantitative analysis of blood glucose level (mg/dL) according to the IPGTT of diabetic mice transplanted with FI or PI+MVF and nondiabetic controls to the indicated time points (n = 5 each). Mean ± SD. One-way ANOVA and Tukey's multiple comparisons post hoc test were used for statistical analysis. *P < 0.05 vs. FI.


G AUC of IPGTT of diabetic mice transplanted with FI or PI + MVF and nondiabetic controls ( n = 5 each ) . Mean ± SD . One - way ANOVA and Tukey ' s multiple comparisons post hoc test were used for statistical analysis . * P < 0 . 05 vs . FI ; # P < 0 . 05 vs . PI + MVF .

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

G AUC of IPGTT of diabetic mice transplanted with FI or PI+MVF and nondiabetic controls (n = 5 each). Mean ± SD. One-way ANOVA and Tukey's multiple comparisons post hoc test were used for statistical analysis. *P < 0.05 vs. FI; #P < 0.05 vs. PI+MVF.


H Plasma insulin level ( µU / mL ) of diabetic mice transplanted with FI or PI + MVF and nondiabetic controls at 180 min after IPGTT ( n = 4 each ) . Mean ± SD .

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

H Plasma insulin level (µU/mL) of diabetic mice transplanted with FI or PI+MVF and nondiabetic controls at 180 min after IPGTT (n = 4 each). Mean ± SD.


I Representative immunofluorescent stainings of insulin / CD31 in FI and PI + MVF on day 28 after islet transplantation . Scale bar : 50 µm .

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

I Representative immunofluorescent stainings of insulin / CD31 in FI and PI+MVF on day 28 after islet transplantation. Scale bar: 50 µm.


J Quantitative analysis of insulin - ( β - cells ) , glucagon - ( α - cells ) , somatostatin - ( δ - cells ) and CD31 - positive ( endothelial ) cells within transplanted FI and PI + MVF on day 28 after islet transplantation . Data are expressed in % of total islet cells ( n = 25 ) . Mean ± SD .

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

J Quantitative analysis of insulin- (β-cells), glucagon- (α-cells), somatostatin- (δ-cells) and CD31-positive (endothelial) cells within transplanted FI and PI+MVF on day 28 after islet transplantation. Data are expressed in % of total islet cells (n = 25). Mean ± SD.


K Quantitative analysis of islet cells in transplanted FI and PI + MVF per HPF ( n = 70 each ) . Mean ± SD . Unpaired Student ' s t - test was used for statistical analysis . * P < 0 . 05 vs . FI .

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

K Quantitative analysis of islet cells in transplanted FI and PI+MVF per HPF (n = 70 each). Mean ± SD. Unpaired Student's t-test was used for statistical analysis. *P < 0.05 vs. FI.


A . NS1 dimer with one subunit in grey and the other colored by domain ( green , β - roll ; orange , wing with magenta connector subdomain ; blue , central β - ladder ) . N - linked glycosylation sites are shown in sticks , and the glycans are shown in spheres . B . Topology diagram for the NS1 monomer , colored as the pattern in ( A ) . Glycosylation sites are indicated with red hexagons and disulfide bonds with yellow circles . C . Side views of the NS1 dimer from the wing ( left ) and the end of the β - ladder ( right ) . The β - roll ( green ) , connector subdomain ( orange ) and the intertwined loop ( yellow ) of the wing domain form a discontinuous protrusion on one face of the β - ladder with the spaghetti loop ( cyan ) on the other face . The wing domain is omitted from the left image for clarity .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. NS1 dimer with one subunit in grey and the other colored by domain (green, β-roll; orange, wing with magenta connector subdomain; blue, central β-ladder). N-linked glycosylation sites are shown in sticks, and the glycans are shown in spheres.B. Topology diagram for the NS1 monomer, colored as the pattern in (A). Glycosylation sites are indicated with red hexagons and disulfide bonds with yellow circles.C. Side views of the NS1 dimer from the wing (left) and the end of the β-ladder (right). The β-roll (green), connector subdomain (orange) and the intertwined loop (yellow) of the wing domain form a discontinuous protrusion on one face of the β-ladder with the spaghetti loop (cyan) on the other face. The wing domain is omitted from the left image for clarity.


The ZIKV NS1 hexamer is modeled by using the full - length DENV2 NS1 structure ( PDB code : 4O6B ) ( Akey et al , 2014 ) . Each monomer is colored by domains ( green , β - roll ; orange , wing ; magenta , connector subdomain ; blue , central β - ladder ) . The intertwined loop " spike " of ZIKV NS1 contributing to membrane association is colored in red and indicated by dotted 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

The ZIKV NS1 hexamer is modeled by using the full-length DENV2 NS1 structure (PDB code: 4O6B) (Akey et al, 2014). Each monomer is colored by domains (green, β-roll; orange, wing; magenta, connector subdomain; blue, central β-ladder). The intertwined loop "spike" of ZIKV NS1 contributing to membrane association is colored in red and indicated by dotted lines.


ABC . Electrostatic surface views of NS1 from DENV2 ( PDB : 4O6B , A ) and WNV ( PDB : 4O6C , B ) ( Akey et al , 2014 ) , as well as ZIKV © , showing diverse characteristics on certain regions . Surfaces are colored by electrostatic potential at neutral pH from - 2 kT ( red ) to + 2 kT ( blue ) using PyMOL ( http : / / pymol . org / ) .

ABC. Electrostatic surface views of NS1 from DENV2 (PDB: 4O6B, A) and WNV (PDB: 4O6C, B) (Akey et al, 2014), as well as ZIKV©, showing diverse characteristics on certain regions. Surfaces are colored by electrostatic potential at neutral pH from -2 kT (red) to +2 kT (blue) using PyMOL (http://pymol.org/).


A . The NS1 surface is colored using the ConSurf server ( Ashkenazy et al , 2016 ) according to sequence conservation from the most conserved ( dark magenta ) to the most divergent ( dark cyan ) based on an alignment of NS1 sequences from 73 flaviviruses .

A. The NS1 surface is colored using the ConSurf server (Ashkenazy et al, 2016) according to sequence conservation from the most conserved (dark magenta) to the most divergent (dark cyan) based on an alignment of NS1 sequences from 73 flaviviruses.


B . Models of intertwined loop " spike " interacting with the cellular membrane . The disordered " spike " regions are indicated by dash lines in DENV2 and WNV NS1 structures , which consist of hydrophobic or positively charged amino acids .

O O O O O O O O O O O O O O O O O O O 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. Models of intertwined loop "spike" interacting with the cellular membrane. The disordered "spike" regions are indicated by dash lines in DENV2 and WNV NS1 structures, which consist of hydrophobic or positively charged amino acids.


A . B - factor putty representation of the intertwined loop structure . Shades of blue and a narrower tube indicate regions with lower B - factors , whereas orange to red and a wider tube indicate regions with higher B - factors . The figure was prepared using PyMOL ( http : / / pymol . org / ) . B . The 2Fo - Fc electron density map for the intertwined loop ( yellow ) " spike " region residues contoured at 1 . 0 sigma is represented in blue . C . Detailed interactions of the highly conserved residues W115 and W118 with the neighboring residues to stabilize the conformation of the intertwined loop . W115 forms a hydrogen - bonding interaction with E74 from α2 helix . W118 inserts into the hydrophobic interspace between α1 and α2 helix , formed by P36 , L42 , V71 , L75 and I78 , and W118 also interact with P36 and D37 by hydrogen - bonding interactions .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. B-factor putty representation of the intertwined loop structure. Shades of blue and a narrower tube indicate regions with lower B-factors, whereas orange to red and a wider tube indicate regions with higher B-factors. The figure was prepared using PyMOL (http://pymol.org/).B. The 2Fo-Fc electron density map for the intertwined loop (yellow) "spike" region residues contoured at 1.0 sigma is represented in blue.C. Detailed interactions of the highly conserved residues W115 and W118 with the neighboring residues to stabilize the conformation of the intertwined loop. W115 forms a hydrogen-bonding interaction with E74 from α2 helix. W118 inserts into the hydrophobic interspace between α1 and α2 helix, formed by P36, L42, V71, L75 and I78, and W118 also interact with P36 and D37 by hydrogen-bonding interactions.


Immunofluorescence staining of RANKL during in vitro adipogenesis of BMSCs from AdipoqCre ; ranklfl / fl mice ( Rankl - / - ) and ranklfl / fl mice ( Rankl + / + ) . Scale bar = 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

Immunofluorescence staining of RANKL during in vitro adipogenesis of BMSCs from AdipoqCre; ranklfl/fl mice (Rankl-/-) and ranklfl/fl mice (Rankl+/+). Scale bar=50 μm.


Micro - CT and statistical analyses of BMD , BV / TV , Tb . N and Tb . Sp in trabecular bone from AdipoqCre ; ranklfl / fl ( Rankl - / - ) and ranklfl / fl ( Rankl + / + ) mice at 4 weeks , 8 weeks and 16 weeks ( n = 5 independent biological replicates ) . Scale bar = 1 mm . Data were compared using an unpaired t - test ( * * indicates P < 0 . 01 ) , error bars are standard deviations .

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

Micro-CT and statistical analyses of BMD, BV/TV, Tb.N and Tb.Sp in trabecular bone from AdipoqCre; ranklfl/fl (Rankl-/-) and ranklfl/fl (Rankl+/+) mice at 4 weeks, 8 weeks and 16 weeks (n=5 independent biological replicates). Scale bar=1 mm. Data were compared using an unpaired t-test (** indicates P < 0.01), error bars are standard deviations.


Representative images of TRAP staining of femur from AdipoqCre ; ranklfl / fl ( Rankl - / - ) and ranklfl / fl ( Rankl + / + ) mice at 8 weeks . Scale bar = 200 μm . Panel a1 and a4 indicate trabecular bone , a2 and a5 stand for growth plate , a3 and a6 denote cortical bone .

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

Representative images of TRAP staining of femur from AdipoqCre; ranklfl/fl (Rankl-/-) and ranklfl/fl (Rankl+/+) mice at 8 weeks. Scale bar=200 μm. Panel a1 and a4 indicate trabecular bone, a2 and a5 stand for growth plate, a3 and a6 denote cortical bone.


Immunofluorescence staining of osteocalcin from 8 - week - old mice ( n = 6 mice in Rankl - / - group and 4 mice in Rankl + / + group ) . Scale bar = 50 μm . TB : trabecular bone . Statistical analyses of Ocn + cells ( n = 6 independent microscopic vision fields from random mice samples ) . Data were compared using an unpaired t ‐ test ( * * indicates P < 0 . 01 ) , error bars are standard deviations .

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

Immunofluorescence staining of osteocalcin from 8-week-old mice (n=6 mice in Rankl-/- group and 4 mice in Rankl+/+ group). Scale bar=50 μm. TB: trabecular bone. Statistical analyses of Ocn+ cells (n=6 independent microscopic vision fields from random mice samples). Data were compared using an unpaired t‐test (** indicates P < 0.01), error bars are standard deviations.


Calcein staining of trabecular bone at 8 weeks . Scale bar = 50 μm . TB : trabecular bone . Images are representative of 6 independent biological replicates in Rankl - / - and 5 replicates in Rankl + / + group . Mineral apposition rate ( MAR ) of trabecular bone at 8 weeks . ( n = 6 mice in Rankl - / - group and 5 mice in Rankl + / + group ) . Data were compared using an unpaired t ‐ test ( * * indicates P < 0 . 01 ) , error bars are standard deviations .

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

Calcein staining of trabecular bone at 8 weeks. Scale bar=50 μm. TB: trabecular bone. Images are representative of 6 independent biological replicates in Rankl-/- and 5 replicates in Rankl+/+ group. Mineral apposition rate (MAR) of trabecular bone at 8 weeks.(n=6 mice in Rankl-/- group and 5 mice in Rankl+/+ group). Data were compared using an unpaired t‐test ( ** indicates P < 0.01), error bars are standard deviations.


Serum CTX - 1 , OCN and bone marrow RANKL levels from Rankl - / - and Rankl + / + mice at 4 weeks ( n = 5 and 5 ) , 8 weeks ( n = 5 and 6 ) and 16 weeks ( n = 5 and 5 ) . Data were compared using an unpaired t ‐ test ( * indicates P < 0 . 05 , * * indicates P < 0 . 01 ) , error bars are standard deviations . .

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

Serum CTX-1, OCN and bone marrow RANKL levels from Rankl-/- and Rankl+/+ mice at 4 weeks (n=5 and 5), 8 weeks (n=5 and 6) and 16 weeks (n=5 and 5). Data were compared using an unpaired t‐test (* indicates P < 0.05, ** indicates P < 0.01), error bars are standard deviations..


Micro - CT analyses of trabecular bone from Grounded and Tail suspended model ( n = 5 independent biological replicates ) . Scale bar = 1 mm . Data were compared using an unpaired t ‐ test ( * indicates P < 0 . 05 , * * indicates P < 0 . 01 ) , error bars are standard deviations .

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

Micro-CT analyses of trabecular bone from Grounded and Tail suspended model (n=5 independent biological replicates). Scale bar=1 mm. Data were compared using an unpaired t‐test (* indicates P < 0.05, ** indicates P < 0.01), error bars are standard deviations.


Micro - CT analyses of sham and OVX model of AdipoqCre ; ranklfl / fl ( Rankl - / - ) and ranklfl / fl ( Rankl + / + ) mice ( n = 5 independent biological replicates ) . Scale bar = 1 mm . Data were compared using an unpaired t ‐ test ( * * indicates P < 0 . 01 ) , error bars are standard deviations .

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

Micro-CT analyses of sham and OVX model of AdipoqCre; ranklfl/fl (Rankl-/-) and ranklfl/fl (Rankl+/+) mice (n=5 independent biological replicates). Scale bar=1 mm. Data were compared using an unpaired t‐test (** indicates P < 0.01), error bars are standard deviations.


TRAP staining and statistical analyses of numbers of osteoclasts in Sham and OVX model ( Images are representative of 5 independent biological replicates in ( C ) , and n = 5 independent microscopic vision fields in ( D ) ) . Scale bar = 100 µm . Data were compared using an unpaired t ‐ test ( * * indicates P < 0 . 01 ) , error bars are standard deviations .

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

TRAP staining and statistical analyses of numbers of osteoclasts in Sham and OVX model (Images are representative of 5 independent biological replicates in (C), and n=5 independent microscopic vision fields in (D)). Scale bar=100 µm. Data were compared using an unpaired t‐test (** indicates P < 0.01), error bars are standard deviations.


Immunofluorescence staining of OCN and statistical analyses of the number of osteoblasts ( Images are representative of 5 independent biological replicates in ( E ) , and n = 5 independent microscopic vision fields in ( F ) ) . Scale bar = 50 µm . TB : trabecular bone . Data were compared using an unpaired t ‐ test ( * * indicates P < 0 . 01 ) , error bars are standard deviations .

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

Immunofluorescence staining of OCN and statistical analyses of the number of osteoblasts (Images are representative of 5 independent biological replicates in (E), and n=5 independent microscopic vision fields in (F)). Scale bar=50 µm. TB: trabecular bone. Data were compared using an unpaired t‐test (** indicates P < 0.01), error bars are standard deviations.


Three - dimensional CT analyses of BMD , BV / TV , Tb . N , and Tb . Sp in trabecular bone from Vehicle ( n = 5 independent biological replicates ) and ROS ( n = 6 independent biological replicates ) model of AdipoqCre ; ranklfl / fl ( Rankl - / - ) and ranklfl / fl ( Rankl + / + ) mice . Scale bar = 1 mm . Data were compared using an unpaired t ‐ test ( * * indicates P < 0 . 01 ) , error bars are standard deviations .

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

Three-dimensional CT analyses of BMD, BV/TV, Tb.N, and Tb.Sp in trabecular bone from Vehicle (n=5 independent biological replicates) and ROS (n=6 independent biological replicates) model of AdipoqCre; ranklfl/fl (Rankl-/-) and ranklfl/fl (Rankl+/+) mice. Scale bar=1 mm. Data were compared using an unpaired t‐test (** indicates P < 0.01), error bars are standard deviations.


TRAP staining and statistical analyses of numbers of osteoclasts in the trabecular bone from Vehicle ( n = 6 and 5 independent biological replicates ) and ROS ( n = 6 and 6 independent biological replicates ) treatment . Scale bar = 100 µm . Data were compared using an unpaired t ‐ test ( * * indicates P < 0 . 01 ) , error bars are standard deviations . n = 5 independent microscopic vision fields in ( D ) .

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

TRAP staining and statistical analyses of numbers of osteoclasts in the trabecular bone from Vehicle (n=6 and 5 independent biological replicates) and ROS (n=6 and 6 independent biological replicates) treatment. Scale bar=100 µm. Data were compared using an unpaired t‐test (** indicates P < 0.01), error bars are standard deviations. n=5 independent microscopic vision fields in (D).


OCN staining and statistical analyses of the number of osteoblasts in femurs from Vehicle ( n = 6 and 5 independent biological replicates ) and ROS ( n = 6 and 6 independent biological replicates ) treatment . Scale bar = 50 µm . TB : trabecular bone . Data were compared using an unpaired t ‐ test ( * * indicates P < 0 . 01 ) , error bars are standard deviations . n = 5 independent microscopic vision fields in ( F ) .

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

OCN staining and statistical analyses of the number of osteoblasts in femurs from Vehicle (n=6 and 5 independent biological replicates) and ROS (n=6 and 6 independent biological replicates) treatment. Scale bar=50 µm. TB: trabecular bone. Data were compared using an unpaired t‐test (** indicates P < 0.01), error bars are standard deviations. n=5 independent microscopic vision fields in (F).


A - C Five - day treatment of DIO male mice with vehicle ( white ) , liraglutide ( 10 nmol / kg ) ( gray ) , RM - 493 ( 3 . 6 μmol / kg ) ( black ) , or liraglutide ( 10 nmol / kg ) and RM - 493 ( 3 . 6 μmol / kg ) ( checkered ) . Effects on ( A ) body weight and ( B , C ) body composition . Compounds were administered by daily subcutaneous injections . Data represent means ± SEM ; n = 8 ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 .

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

A-C Five-day treatment of DIO male mice with vehicle (white), liraglutide (10 nmol/kg) (gray), RM-493 (3.6 μmol/kg) (black), or liraglutide (10 nmol/kg) and RM-493 (3.6 μmol/kg) (checkered). Effects on (A) body weight and (B, C) body composition. Compounds were administered by daily subcutaneous injections. Data represent means ± SEM;n = 8; *P < 0.05, **P < 0.01, ***P < 0.001.


A - G Five - day treatment of DIO male mice with vehicle ( white ) , liraglutide ( 10 nmol / kg ) ( gray ) , RM - 493 ( 3 . 6 μmol / kg ) ( black ) , or liraglutide ( 10 nmol / kg ) and RM - 493 ( 3 . 6 μmol / kg ) ( checkered ) . Effects on ( A , B ) energy expenditure . Compounds were administered by daily subcutaneous injections . Data represent means ± SEM ; n = 8 ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 .

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

A-G Five-day treatment of DIO male mice with vehicle (white), liraglutide (10 nmol/kg) (gray), RM-493 (3.6 μmol/kg) (black), or liraglutide (10 nmol/kg) and RM-493 (3.6 μmol/kg) (checkered). Effects on (A, B) energy expenditure. Compounds were administered by daily subcutaneous injections. Data represent means ± SEM;n = 8; *P < 0.05, **P < 0.01, ***P < 0.001.


A - G Five - day treatment of DIO male mice with vehicle ( white ) , liraglutide ( 10 nmol / kg ) ( gray ) , RM - 493 ( 3 . 6 μmol / kg ) ( black ) , or liraglutide ( 10 nmol / kg ) and RM - 493 ( 3 . 6 μmol / kg ) ( checkered ) . Effects on ( C ) respiratory exchange ratio ( RER ) . Compounds were administered by daily subcutaneous injections . Data represent means ± SEM ; n = 8 ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 .

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

A-G Five-day treatment of DIO male mice with vehicle (white), liraglutide (10 nmol/kg) (gray), RM-493 (3.6 μmol/kg) (black), or liraglutide (10 nmol/kg) and RM-493 (3.6 μmol/kg) (checkered). Effects on (C) respiratory exchange ratio (RER). Compounds were administered by daily subcutaneous injections. Data represent means ± SEM;n = 8; *P < 0.05, **P < 0.01, ***P < 0.001.


A - G Five - day treatment of DIO male mice with vehicle ( white ) , liraglutide ( 10 nmol / kg ) ( gray ) , RM - 493 ( 3 . 6 μmol / kg ) ( black ) , or liraglutide ( 10 nmol / kg ) and RM - 493 ( 3 . 6 μmol / kg ) ( checkered ) . Effects on ( D ) locomotor activity . Compounds were administered by daily subcutaneous injections . Data represent means ± SEM ; n = 8 ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 .

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

A-G Five-day treatment of DIO male mice with vehicle (white), liraglutide (10 nmol/kg) (gray), RM-493 (3.6 μmol/kg) (black), or liraglutide (10 nmol/kg) and RM-493 (3.6 μmol/kg) (checkered). Effects on (D) locomotor activity. Compounds were administered by daily subcutaneous injections. Data represent means ± SEM;n = 8; *P < 0.05, **P < 0.01, ***P < 0.001.


A - G Five - day treatment of DIO male mice with vehicle ( white ) , liraglutide ( 10 nmol / kg ) ( gray ) , RM - 493 ( 3 . 6 μmol / kg ) ( black ) , or liraglutide ( 10 nmol / kg ) and RM - 493 ( 3 . 6 μmol / kg ) ( checkered ) . Effects on ( E ) cumulative food intake , ( F ) meal number and ( G ) meal size . Compounds were administered by daily subcutaneous injections . Data represent means ± SEM ; n = 8 ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 .

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

A-G Five-day treatment of DIO male mice with vehicle (white), liraglutide (10 nmol/kg) (gray), RM-493 (3.6 μmol/kg) (black), or liraglutide (10 nmol/kg) and RM-493 (3.6 μmol/kg) (checkered). Effects on (E) cumulative food intake, (F) meal number and (G) meal size. Compounds were administered by daily subcutaneous injections. Data represent means ± SEM;n = 8; *P < 0.05, **P < 0.01, ***P < 0.001.


A , B Treatment - induced changes in hypothalamic gene expression in DIO mice treated for 2 ( A ) or 5 ( B ) days with vehicle ( white ) , liraglutide ( 10 nmol / kg ) ( gray ) , RM - 493 ( 3 . 6 μmol / kg ) ( black ) , or liraglutide ( 10 nmol / kg ) and RM - 493 ( 3 . 6 μmol / kg ) ( checkered ) . Compounds were administered by daily subcutaneous injections , and the last injection was provided 2 h prior to tissues sampling . Data represent means ± SEM ; n = 8 ; * P < 0 . 05 , * * P < 0 . 01 , § P < 0 , 001 , comparison between treatment and vehicle control . # P < 0 . 05 , # # P < 0 . 01 , # # # P < 0 . 001 , comparison to liraglutide . + P < 0 . 05 , comparison to RM - 493 .

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

A, B Treatment-induced changes in hypothalamic gene expression in DIO mice treated for 2 (A) or 5 (B) days with vehicle (white), liraglutide (10 nmol/kg) (gray), RM-493 (3.6 μmol/kg) (black), or liraglutide (10 nmol/kg) and RM-493 (3.6 μmol/kg) (checkered). Compounds were administered by daily subcutaneous injections, and the last injection was provided 2 h prior to tissues sampling. Data represent means ± SEM;n = 8; *P < 0.05, **P < 0.01, §P < 0,001, comparison between treatment and vehicle control. #P < 0.05, ##P < 0.01, ###P < 0.001, comparison to liraglutide. +P < 0.05, comparison to RM-493.


A - I Glucose metabolic parameters were assessed following 5 days of treatment of DIO male mice with vehicle ( white ) , liraglutide ( 10 nmol / kg ) ( gray ) , RM - 493 ( 3 . 6 μmol / kg ) ( black ) , or liraglutide ( 10 nmol / kg ) and RM - 493 ( 3 . 6 μmol / kg ) ( checkered ) . ( A ) Fasted blood glucose levels . Data represent means ± SEM ; n = 8 in ( A - H ) ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 .

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

A-I Glucose metabolic parameters were assessed following 5 days of treatment of DIO male mice with vehicle (white), liraglutide (10 nmol/kg) (gray), RM-493 (3.6 μmol/kg) (black), or liraglutide (10 nmol/kg) and RM-493 (3.6 μmol/kg) (checkered). (A) Fasted blood glucose levels. Data represent means ± SEM;n = 8 in (A-H); *P < 0.05, **P < 0.01, ***P < 0.001.


A - I Glucose metabolic parameters were assessed following 5 days of treatment of DIO male mice with vehicle ( white ) , liraglutide ( 10 nmol / kg ) ( gray ) , RM - 493 ( 3 . 6 μmol / kg ) ( black ) , or liraglutide ( 10 nmol / kg ) and RM - 493 ( 3 . 6 μmol / kg ) ( checkered ) . ( B , C ) glucose tolerance . Data represent means ± SEM ; n = 8 in ( A - H ) ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 .

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

A-I Glucose metabolic parameters were assessed following 5 days of treatment of DIO male mice with vehicle (white), liraglutide (10 nmol/kg) (gray), RM-493 (3.6 μmol/kg) (black), or liraglutide (10 nmol/kg) and RM-493 (3.6 μmol/kg) (checkered).(B, C) glucose tolerance. Data represent means ± SEM;n = 8 in (A-H); *P < 0.05, **P < 0.01, ***P < 0.001.


A - I Glucose metabolic parameters were assessed following 5 days of treatment of DIO male mice with vehicle ( white ) , liraglutide ( 10 nmol / kg ) ( gray ) , RM - 493 ( 3 . 6 μmol / kg ) ( black ) , or liraglutide ( 10 nmol / kg ) and RM - 493 ( 3 . 6 μmol / kg ) ( checkered ) . ( D ) fasted insulin levels , . Data represent means ± SEM ; n = 8 in ( A - H ) ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 .

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

A-I Glucose metabolic parameters were assessed following 5 days of treatment of DIO male mice with vehicle (white), liraglutide (10 nmol/kg) (gray), RM-493 (3.6 μmol/kg) (black), or liraglutide (10 nmol/kg) and RM-493 (3.6 μmol/kg) (checkered). (D) fasted insulin levels,. Data represent means ± SEM;n = 8 in (A-H); *P < 0.05, **P < 0.01, ***P < 0.001.


A - I Glucose metabolic parameters were assessed following 5 days of treatment of DIO male mice with vehicle ( white ) , liraglutide ( 10 nmol / kg ) ( gray ) , RM - 493 ( 3 . 6 μmol / kg ) ( black ) , or liraglutide ( 10 nmol / kg ) and RM - 493 ( 3 . 6 μmol / kg ) ( checkered ) . ( E , F ) glucose - induced insulin secretion . Data represent means ± SEM ; n = 8 in ( A - H ) ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 .

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

A-I Glucose metabolic parameters were assessed following 5 days of treatment of DIO male mice with vehicle (white), liraglutide (10 nmol/kg) (gray), RM-493 (3.6 μmol/kg) (black), or liraglutide (10 nmol/kg) and RM-493 (3.6 μmol/kg) (checkered). (E, F) glucose-induced insulin secretion. Data represent means ± SEM;n = 8 in (A-H); *P < 0.05, **P < 0.01, ***P < 0.001.


A - I Glucose metabolic parameters were assessed following 5 days of treatment of DIO male mice with vehicle ( white ) , liraglutide ( 10 nmol / kg ) ( gray ) , RM - 493 ( 3 . 6 μmol / kg ) ( black ) , or liraglutide ( 10 nmol / kg ) and RM - 493 ( 3 . 6 μmol / kg ) ( checkered ) . ( G ) HOMA - IR . Data represent means ± SEM ; n = 8 in ( A - H ) ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 .

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

A-I Glucose metabolic parameters were assessed following 5 days of treatment of DIO male mice with vehicle (white), liraglutide (10 nmol/kg) (gray), RM-493 (3.6 μmol/kg) (black), or liraglutide (10 nmol/kg) and RM-493 (3.6 μmol/kg) (checkered). (G) HOMA-IR. Data represent means ± SEM;n = 8 in (A-H); *P < 0.05, **P < 0.01, ***P < 0.001.


A - I Glucose metabolic parameters were assessed following 5 days of treatment of DIO male mice with vehicle ( white ) , liraglutide ( 10 nmol / kg ) ( gray ) , RM - 493 ( 3 . 6 μmol / kg ) ( black ) , or liraglutide ( 10 nmol / kg ) and RM - 493 ( 3 . 6 μmol / kg ) ( checkered ) . ( H ) insulin sensitivity . Data represent means ± SEM ; n = 8 in ( A - H ) ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 .

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 B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O 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-I Glucose metabolic parameters were assessed following 5 days of treatment of DIO male mice with vehicle (white), liraglutide (10 nmol/kg) (gray), RM-493 (3.6 μmol/kg) (black), or liraglutide (10 nmol/kg) and RM-493 (3.6 μmol/kg) (checkered). (H) insulin sensitivity. Data represent means ± SEM;n = 8 in (A-H); *P < 0.05, **P < 0.01, ***P < 0.001.