Figure 4 ( A ) Western blots of lysates of primary neurons transduced with control lentiviruses ( NT ) or lentiviruses to knockdown ( KD ) or overexpress ( OE ) PRR7 . PRR7 knockdown reduced ( 75 . 6 ± 6 . 9 % ) while PRR7 overexpression ( OE ) increased ( 157 . 6 ± 22 . 8 % ) c - Jun levels compared to controls . No change was detected in c - Fos levels . One - way ANOVA test was used to analyze results with Dunett ' s post hoc test ( n = 16 gels for c - Jun and 4 gels for c - Fos ) . ( B ) Control or PRR7 transfected HEK293 cells were treated with cycloheximide for the indicated time points and c - Jun protein levels were measured by Western blotting . PRR7 expression significantly stabilized c - Jun levels at 120 min ( 110 . 0 ± 7 . 5 % vs control 68 . 7 ± 7 . 9 % ) and 240 minutes ( 83 . 6 ± 5 . 2 % vs control 43 . 7 ± 3 . 4 % ) . Values for c - Jun were normalized to their respective 0 min time point ( n = 4 gels for each group performed blind ) . T - tests between similar time points were used to analyze results . ( C ) PRR7 , FBW7 and / or a control protein ( PSD95 ) were transfected into HEK293 cells and c - Jun ubiquitination was measured by immunoprecipitating c - Jun and blotting for ubiquitin . Ubiquitination of c - Jun in the presence of FBW7 ( 178 . 3 ± 26 . 9 % ) was significantly reduced in the presence of PRR7 ( 103 . 7 ± 20 . 6 % ) but not a control protein ( PSD95 ) ( n = 5 gels for all groups ) . ( D ) Western blots of c - Junubiquitination in hippocampal primary neuronal cultures . PRR7 knockdown ( KD ) elevated ( 149 ± 10 . 62 % , n = 6 gels ) c - Junubiquitination level in comparison to control neurons ( NT ) . Statistical significance was determined by t - test . ( E ) Western blots showing knockdown ( KD ) or overexpression ( OE ) of PRR7 in primary hippocampal neurons do not alter levels of other FBW7 targets mTOR or Myc .

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Figure 4(A) Western blots of lysates of primary neurons transduced with control lentiviruses (NT) or lentiviruses to knockdown (KD) or overexpress (OE) PRR7. PRR7 knockdown reduced (75.6 ± 6.9%) while PRR7 overexpression (OE) increased (157.6 ± 22.8%) c-Jun levels compared to controls. No change was detected in c-Fos levels. One-way ANOVA test was used to analyze results with Dunett's post hoc test (n=16 gels for c-Jun and 4 gels for c-Fos).(B) Control or PRR7 transfected HEK293 cells were treated with cycloheximide for the indicated time points and c-Jun protein levels were measured by Western blotting. PRR7 expression significantly stabilized c-Jun levels at 120 min (110.0 ± 7.5% vs control 68.7 ± 7.9%) and 240 minutes (83.6 ± 5.2% vs control 43.7 ± 3.4%). Values for c-Jun were normalized to their respective 0 min time point (n=4 gels for each group performed blind). T-tests between similar time points were used to analyze results.(C) PRR7, FBW7 and/or a control protein (PSD95) were transfected into HEK293 cells and c-Jun ubiquitination was measured by immunoprecipitating c-Jun and blotting for ubiquitin. Ubiquitination of c-Jun in the presence of FBW7 (178.3 ± 26.9%) was significantly reduced in the presence of PRR7 (103.7 ± 20.6%) but not a control protein (PSD95) (n=5 gels for all groups).(D) Western blots of c-Junubiquitination in hippocampal primary neuronal cultures. PRR7 knockdown (KD) elevated (149±10.62%, n=6 gels) c-Junubiquitination level in comparison to control neurons (NT). Statistical significance was determined by t-test.(E) Western blots showing knockdown (KD) or overexpression (OE) of PRR7 in primary hippocampal neurons do not alter levels of other FBW7 targets mTOR or Myc.


Figure 5 ( A ) Co - IPs of PRR7 with c - Jun from lysates of HEK293 cells overexpressing PRR7 . ( B ) Pulldown assays in vitro using purified proteins showed a direct interaction between PRR7 and c - Jun . ( C ) Co - IPs of PRR7 with FBW7 from lysates of HEK293 cells overexpressing PRR7 and FBW7 . ( D ) Pulldown assays in vitro using purified PRR7 and FBW7 . ( E ) Co - IPs of endogenous PRR7 and FBW7 from lysates of ratbrain . ( F ) Co - IPs of PRR7 with c - Jun and FBW7 from lysates of HEK293 cells overexpressing PRR7 and FBW7 . FBW7 expression increased the amount of c - Jun pulled down with PRR7 ( n = 4 gels for each panel , representative blots ) . Lower bar graphs - quantitation for Western blots show c - Jun / FBW7 binding is 41 . 4 ± 13 . 9 % greater in the presence of PRR7 ( normalized for FBW7 expression ) . Similarly , c - Jun / PRR7 binding is 97 . 4 ± 38 . 1 % greater in the presence of FBW7 ( normalized for PRR7 expression ) . Statistical significance was calculated using unpaired t - tests .

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Figure 5(A) Co-IPs of PRR7 with c-Jun from lysates of HEK293 cells overexpressing PRR7.(B) Pulldown assays in vitro using purified proteins showed a direct interaction between PRR7 and c-Jun.(C) Co-IPs of PRR7 with FBW7 from lysates of HEK293 cells overexpressing PRR7 and FBW7.(D) Pulldown assays in vitro using purified PRR7 and FBW7.(E) Co-IPs of endogenous PRR7 and FBW7 from lysates of ratbrain.(F) Co-IPs of PRR7 with c-Jun and FBW7 from lysates of HEK293 cells overexpressing PRR7 and FBW7. FBW7 expression increased the amount of c-Jun pulled down with PRR7 (n=4 gels for each panel, representative blots). Lower bar graphs- quantitation for Western blots show c-Jun/FBW7 binding is 41.4±13.9% greater in the presence of PRR7 (normalized for FBW7 expression). Similarly, c-Jun/PRR7 binding is 97.4±38.1% greater in the presence of FBW7 (normalized for PRR7 expression). Statistical significance was calculated using unpaired t-tests.


Figure 6 ( A ) Western blots of phospho - c - Jun in primary neuronal cultures . Quantification of phospho - c - Jun represents a ratio of phospho - c - Jun to total c - Jun and all ratios were normalized to NT . PRR7 knockdown ( KD ) decreased , while PRR7 overexpression ( OE ) increased phospho - c - Jun levels ( black bars ) . Glutamate stimulation ( gray bars ) increased phospho - c - Jun / c - Jun ratio in NT but not in KD or OE neurons ( n = 7 gels for all groups ) . Significance was determined using two - way ANOVA with Sidak post - hoc test . ( B ) AP - 1 driven luciferase luminescence assays . PRR7 expression in HEK293 cells increased AP - 1 activity ( 190 . 6 ± 13 . 9 % , n = 12 ; RLU = relative light units ) . Statistical significance was determined using t - tests . ( D ) Quantitation of cell death using 30 minutes of PI in - situ staining following 48h of WT - PRR7 - GFP or mNLS - PRR7 - GFP overexpression in primary hippocampal neuronal cultures ( n = 24 cells per group ) . Images represent a max projection of five z - stack sections across the cell body . ( E ) NMDA treatment ( 25 min , 100 µM followed by 14h chase ) caused 83 . 6 ± 3 . 5 % cell death in control neurons compared to 60 . 3 ± 4 . 6 % cell death in neurons with PRR7 expression knocked down . A c - Jun inhibitory peptide reduced NMDA - mediated cell death to 66 . 2 ± 6 . 0 % in control neurons and to 55 . 6 ± 5 . 3 % in neurons knocked down for PRR7 , but this difference was not statistically significant ( n = 5 independent experiments ) . Two - way ANOVA was used in conjugation with Tukey post - hoc test . ( Left ) Representative images showing transduced neurons ( green - GFP ) and PI staining ( red ) . ( F ) Quantification of cell death in response to NMDA stimulation in the presence or absence of PRR7 and either WT c - Jun or a dominant negative c - Jun4A ( 4A ) . Overexpression of WT c - Jun caused extensive neuronal cell death ( 84 . 8 ± 2 . 4 % ) that was reduced in neurons knocked down for PRR7 ( 43 . 8 ± 9 . 2 ) . Overexpressing 4A conferred neuroprotection to NMDA excitotoxicity ( 50 . 8 ± 7 . 9 % ) but was not additive with PRR7 knockdown ( 42 . 3 ± 8 . 1 % ) ( n = 3 independent experiments ) . Significance was determined using two - way ANOVA in conjugation with Tukey post - hoc test . Images are representative 10X widefield micrographs showing transduced neurons ( green - GFP ) and PI staining ( red ) . Data is presented as mean ± sem . All % values represent comparisons to control .

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Figure 6(A) Western blots of phospho-c-Jun in primary neuronal cultures. Quantification of phospho-c-Jun represents a ratio of phospho-c-Jun to total c-Jun and all ratios were normalized to NT. PRR7 knockdown (KD) decreased, while PRR7 overexpression (OE) increased phospho-c-Jun levels (black bars). Glutamate stimulation (gray bars) increased phospho-c-Jun/c-Jun ratio in NT but not in KD or OE neurons (n=7 gels for all groups). Significance was determined using two-way ANOVA with Sidak post-hoc test.(B) AP-1 driven luciferase luminescence assays. PRR7 expression in HEK293 cells increased AP-1 activity (190.6 ± 13.9%, n=12; RLU= relative light units). Statistical significance was determined using t-tests.(D) Quantitation of cell death using 30 minutes of PI in-situ staining following 48h of WT-PRR7-GFP or mNLS-PRR7-GFP overexpression in primary hippocampal neuronal cultures (n=24 cells per group). Images represent a max projection of five z-stack sections across the cell body.(E) NMDA treatment (25 min, 100 µM followed by 14h chase) caused 83.6±3.5% cell death in control neurons compared to 60.3±4.6% cell death in neurons with PRR7 expression knocked down. A c-Jun inhibitory peptide reduced NMDA-mediated cell death to 66.2±6.0% in control neurons and to 55.6±5.3% in neurons knocked down for PRR7, but this difference was not statistically significant (n=5 independent experiments). Two-way ANOVA was used in conjugation with Tukey post-hoc test. (Left) Representative images showing transduced neurons (green- GFP) and PI staining (red).(F) Quantification of cell death in response to NMDA stimulation in the presence or absence of PRR7 and either WT c-Jun or a dominant negative c-Jun4A (4A). Overexpression of WT c-Jun caused extensive neuronal cell death (84.8±2.4%) that was reduced in neurons knocked down for PRR7 (43.8±9.2). Overexpressing 4A conferred neuroprotection to NMDA excitotoxicity (50.8±7.9%) but was not additive with PRR7 knockdown (42.3±8.1%) (n=3 independent experiments). Significance was determined using two-way ANOVA in conjugation with Tukey post-hoc test. Images are representative 10X widefield micrographs showing transduced neurons (green- GFP) and PI staining (red). Data is presented as mean±sem. All % values represent comparisons to control.


Figure 1Cell count of Ronin fl / del and Ronin del / del ESC . Data represent mean ± SD , n = three independent experiments . Annexin V cell death assay of Ronin fl / del and Ronin del / del cultured for 6 days . Two - tailed unpaired Student ' s t test , data represent mean ± SD , n = three independent experiments . Ronin fl / del and Ronin del / del ESC stained for Ronin ( left panel ) or Oct4 ( right panel ) . The nuclei are counterstained with DAPI .

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Figure 1Cell count of Ronin fl/del and Ronin del/del ESC. Data represent mean ± SD, n = three independent experiments.Annexin V cell death assay of Ronin fl/del and Ronin del/del cultured for 6 days. Two-tailed unpaired Student's t test, data represent mean ± SD, n = three independent experiments.Ronin fl/del and Ronin del/del ESC stained for Ronin (left panel) or Oct4 (right panel). The nuclei are counterstained with DAPI.


Figure 2Haematoxylin / eosin staining of paraffin sections of Ronin fl / del and ecto - Ronin teratomas . In vitro differentiation of Ronin fl / del , ecto - Ronin ( grown in the presence of Dox ) and Ronin del / del ESC .

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Figure 2Haematoxylin/eosin staining of paraffin sections of Ronin fl/del and ecto-Ronin teratomas.In vitro differentiation of Ronin fl/del, ecto-Ronin (grown in the presence of Dox) and Ronin del/del ESC.


Figure 3Volcano plot of gene expression in Ronin del / del day 4 vs . Ronin fl / del ( left panel ) and Ronin del / del day 8 vs . Ronin fl / del ESC ( right panel ) with annotated naïve and primed pluripotency markers .

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Figure 3Volcano plot of gene expression in Ronin del/del day 4 vs. Ronin fl/del (left panel) and Ronin del/del day 8 vs. Ronin fl/del ESC (right panel) with annotated naïve and primed pluripotency markers.


Figure 4Volcano plot of gene expression in Ronin del / del day 8 vs . Ronin del / del day 4 ESC . Analysis of nascent protein synthesis using OP - puro assay . Ronin fl / del cells treated with protein synthesis inhibitor ( cycloheximide ) are used as negative control . The nuclei are counterstained with DAPI . Quantification of the OP - puro signal , normalised to DAPI . Ordinary one - way Anova data represent mean ± SD ; n = three independent experiments for each genotype .

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Figure 4Volcano plot of gene expression in Ronin del/del day 8 vs. Ronin del/del day 4 ESC.Analysis of nascent protein synthesis using OP-puro assay. Ronin fl/del cells treated with protein synthesis inhibitor (cycloheximide) are used as negative control. The nuclei are counterstained with DAPI.   Quantification of the OP-puro signal, normalised to DAPI. Ordinary one-way Anova data represent mean ± SD; n = three independent experiments for each genotype.


Figure 5Transmission electron microscopy analysis of mitochondrial structure in Ronin fl / del and Ronin del / del ESC . Mitochondria analysed , n > 150 for each condition . Quantification of the OCR analysis . Kruskal - Wallis test , data represent mean ± SD , n = 16 technical replicates per genotype in three independent experiments .

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Figure 5Transmission electron microscopy analysis of mitochondrial structure in Ronin fl/del and Ronin del/del ESC. Mitochondria analysed, n > 150 for each condition.Quantification of the OCR analysis. Kruskal-Wallis test, data represent mean ± SD, n = 16 technical replicates per genotype in three independent experiments.


Figure 6Blastocyst ( E4 . 5 ) and diapause embryos stained for Ronin , Sox2 and Sox17 . Egg cylinder stage ( E5 . 5 ) embryo stained for Ronin and Sox17 . Nuclei are counterstained with DAPI . Transmission electron microscopy analysis of mitochondrial morphology in the epiblast of E4 . 5 , diapause and E5 . 5 embryo . Mitochondria analysed , E4 . 5 ( n mitochondria = 55 ) , diapause ( n mitochondria = 62 ) , E5 . 5 ( n mitochondria = 65 ) .

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Figure 6Blastocyst (E4.5) and diapause embryos stained for Ronin, Sox2 and Sox17. Egg cylinder stage (E5.5) embryo stained for Ronin and Sox17. Nuclei are counterstained with DAPI.Transmission electron microscopy analysis of mitochondrial morphology in the epiblast of E4.5, diapause and E5.5 embryo. Mitochondria analysed, E4.5 (n mitochondria = 55), diapause (n mitochondria = 62), E5.5 (n mitochondria = 65).


Figure 7E4 . 5 chimeric embryos stained for tdTomato and DAPI . Quantification of donor cells ' contribution in E4 . 5 blastocysts , Mann - Whitney test , data represent mean ± SD ; Ronin fl / del n embryos = 26 , Ronin del / del n embryos = 27 . Diapause embryos stained for tdTomato and DAPI . Quantification of donor cells ' contribution in diapause embryos , unpaired Student ' s t test , data represent mean ± SD , Ronin fl / del n embryos = 4 , Ronin del / del n embryos = 12 . E5 . 5 chimeric embryos stained for tdTomato and DAPI . Quantification of donor cells ' contribution in E5 . 5 embryos , unpaired Student ' s t test , data represent mean ± SD , Ronin fl / del n embryos = 21 , Ronin del / del n embryos = 15 . Reactivated E5 . 5 chimeric embryos stained for tdTomato and DAPI . Quantification of donor cells ' contribution in reactivated E5 . 5 embryos , Mann - Whitney test , data represent mean ± SD , Ronin fl / del n embryos = 17 , Ronin del / del n embryos = 12 .

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Figure 7E4.5 chimeric embryos stained for tdTomato and DAPI.   Quantification of donor cells' contribution in E4.5 blastocysts, Mann-Whitney test, data represent mean ± SD; Ronin fl/del n embryos = 26, Ronin del/del n embryos = 27.   Diapause embryos stained for tdTomato and DAPI.   Quantification of donor cells' contribution in diapause embryos, unpaired Student's t test, data represent mean ± SD, Ronin fl/del n embryos = 4, Ronin del/del n embryos = 12.   E5.5 chimeric embryos stained for tdTomato and DAPI.   Quantification of donor cells' contribution in E5.5 embryos, unpaired Student's t test, data represent mean ± SD, Ronin fl/del n embryos = 21, Ronin del/del n embryos = 15.   Reactivated E5.5 chimeric embryos stained for tdTomato and DAPI.   Quantification of donor cells' contribution in reactivated E5.5 embryos, Mann-Whitney test, data represent mean ± SD, Ronin fl/del n embryos = 17, Ronin del/del n embryos = 12.


Figure 1 . Testing an assay for SARS - CoV - 2 using synthetic RNA targets . We show data from both fluorescent ( a ) and lateral flow detection ( b ) . Target concentrations ( in cp / μl ) are indicated . RPA NTC : water input into RPA ; Det NTC : water input into Cas13 detection reaction . In ( a ) , error bars indicate one standard deviation based on n = 3 technical replicates .

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Figure 1.Testing an assay for SARS-CoV-2 using synthetic RNA targets. We show data from both fluorescent (a) and lateral flow detection (b). Target concentrations (in cp/μl) are indicated. RPA NTC: water input into RPA; Det NTC: water input into Cas13 detection reaction. In (a), error bars indicate one standard deviation based on n=3 technical replicates.


Figure 1A Representative 3D tile scan images of young adrenal gland , ovary , pituitary gland , testis , pancreas and thyroid gland stained with the antibodies indicated in the panel . Scale bars are 200 µm . The white and dashed lines represent the outlines of each organ . B Representative 3D images at single - cell resolution level of adrenal gland , ovary , pituitary gland , testis , pancreas and thyroid gland . Insets ( * ) indicate higher magnification images of regions with single - cell resolution and surface rendering for all markers . Scale bars for tile scan 3D images are 200 µm , for single - cell resolution insets are 5 µm . The dashed lines represent the outlines of each organ . C Exemplar image illustrates the cell - cell interactome analysis with the distance estimates of CD68 + macrophages to blood vessels ( Endoglin ) in cortex , medulla and the transition zone ( TZ ) of an aged adrenal gland . The colour code indicates the distance which is very close in violet and very far in red . The scale bar is 50 µm . The white and dashed lines represent the outline of the transition zone .

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Figure 1A Representative 3D tile scan images of young adrenal gland, ovary, pituitary gland, testis, pancreas and thyroid gland stained with the antibodies indicated in the panel. Scale bars are 200 µm. The white and dashed lines represent the outlines of each organ.B Representative 3D images at single-cell resolution level of adrenal gland, ovary, pituitary gland, testis, pancreas and thyroid gland. Insets (*) indicate higher magnification images of regions with single-cell resolution and surface rendering for all markers. Scale bars for tile scan 3D images are 200 µm, for single-cell resolution insets are 5 µm. The dashed lines represent the outlines of each organ.C Exemplar image illustrates the cell-cell interactome analysis with the distance estimates of CD68+ macrophages to blood vessels (Endoglin) in cortex, medulla and the transition zone (TZ) of an aged adrenal gland. The colour code indicates the distance which is very close in violet and very far in red. The scale bar is 50 µm. The white and dashed lines represent the outline of the transition zone.


Figure 2A Young and aged adrenal gland immunolabelled with HSPG2 , NG2 and Podocalyxin . Higher magnification insets show cortex ( Co ) and medulla ( Me ) . Combined box and whiskers , and scatter plot with quantification of vessel density in young and aged adrenal glands . Data information : The white and dashed lines in each panel represent the outlines of each organ . ( n = 5 ) , P - values and two - tailed unpaired t - tests . ns : not significant ; * * : P < 0 . 01 ; * * * : P < 0 . 001 . Nuclei : TO - PRO - 3 . For the Combo plots , the boxes represent mean ± s . d . , line in the box is the median , and the lower and upper lines show the minimum and the maximum of the values . The line on the right side of these combined plots represents the sample distribution . Co : Cortex ; Me : Medulla Scale bars for tile scan 3D images 200 µm , for insets 50 µm . B Young and aged ovary immunolabelled with Sca - 1 , PDGFRβ and Emcn . Higher magnification insets show cortex and medulla . Combo plot with quantification of vessel density in young and aged ovary . Data information : The white and dashed lines in each panel represent the outlines of each organ . ( n = 5 ) , P - values and two - tailed unpaired t - tests . ns : not significant ; * * : P < 0 . 01 ; * * * : P < 0 . 001 . Nuclei : TO - PRO - 3 . For the Combo plots , the boxes represent mean ± s . d . , line in the box is the median , and the lower and upper lines show the minimum and the maximum of the values . The line on the right side of these combined plots represents the sample distribution . Co : Cortex ; Me : Medulla Scale bars for tile scan 3D images 200 µm , for insets 50 µm . C Young ( 10 - weeks - old ) and aged pituitary gland immunolabelled with ESM - 1 , Collagen IV and Emcn . Insets with higher magnifications of pars distalis and pars nervosa regions . Combo plot shows quantification of vessel density . Data information : The white and dashed lines in each panel represent the outlines of each organ . ( n = 5 ) , P - values and two - tailed unpaired t - tests . ns : not significant ; * * : P < 0 . 01 ; * * * : P < 0 . 001 . Nuclei : TO - PRO - 3 . For the Combo plots , the boxes represent mean ± s . d . , line in the box is the median , and the lower and upper lines show the minimum and the maximum of the values . The line on the right side of these combined plots represents the sample distribution . Pd : Pars Distalis ; Pn : Pars Nervosa . Scale bars for tile scan 3D images 200 µm , for insets 50 µm . D Young and aged testis immunolabelled with Tie - 2 and HSPG2 . Higher magnifications of cortex and medulla . Combo plot shows quantification of vessel density . Data information : The white and dashed lines in each panel represent the outlines of each organ . ( n = 5 ) , P - values and two - tailed unpaired t - tests . ns : not significant ; * * : P < 0 . 01 ; * * * : P < 0 . 001 . Nuclei : TO - PRO - 3 . For the Combo plots , the boxes represent mean ± s . d . , line in the box is the median , and the lower and upper lines show the minimum and the maximum of the values . The line on the right side of these combined plots represents the sample distribution . Scale bars for tile scan 3D images 200 µm , for insets 50 µm . E Young and aged pancreas immunolabelled with SM22α and Endoglin . Insets representative of higher magnifications of regions in young and aged pancreas . Combo plot shows quantification of vessel density . Data information : The white and dashed lines in each panel represent the outlines of each organ . ( n = 5 ) , P - values and two - tailed unpaired t - tests . ns : not significant ; * * : P < 0 . 01 ; * * * : P < 0 . 001 . Nuclei : TO - PRO - 3 . For the Combo plots , the boxes represent mean ± s . d . , line in the box is the median , and the lower and upper lines show the minimum and the maximum of the values . The line on the right side of these combined plots represents the sample distribution . Scale bars for tile scan 3D images 200 µm , for insets 50 µm . F Young and aged thyroid gland immunolabelled with CXCR4 , Laminin and Emcn . Combo plot shows quantification of vessel density in young and aged thyroid glands . Insets : periphery and centre of the thyroid gland . The white and dashed lines in each panel represent the outlines of each organ . ( n = 5 ) , P - values and two - tailed unpaired t - tests . ns : not significant ; * * : P < 0 . 01 ; * * * : P < 0 . 001 . Nuclei : TO - PRO - 3 . For the Combo plots , the boxes represent mean ± s . d . , line in the box is the median , and the lower and upper lines show the minimum and the maximum of the values . The line on the right side of these combined plots represents the sample distribution . ; Pe : Periphery Ct : Center Scale bars for tile scan 3D images 200 µm , for insets 50 µm .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2A Young and aged adrenal gland immunolabelled with HSPG2, NG2 and Podocalyxin. Higher magnification insets show cortex (Co) and medulla (Me). Combined box and whiskers, and scatter plot with quantification of vessel density in young and aged adrenal glands. Data information: The white and dashed lines in each panel represent the outlines of each organ. (n=5), P-values and two-tailed unpaired t-tests. ns: not significant; **: P < 0.01; ***: P < 0.001. Nuclei: TO-PRO-3. For the Combo plots, the boxes represent mean ± s.d., line in the box is the median, and the lower and upper lines show the minimum and the maximum of the values. The line on the right side of these combined plots represents the sample distribution. Co: Cortex; Me: Medulla Scale bars for tile scan 3D images 200 µm, for insets 50 µm. B Young and aged ovary immunolabelled with Sca-1, PDGFRβ and Emcn. Higher magnification insets show cortex and medulla. Combo plot with quantification of vessel density in young and aged ovary. Data information: The white and dashed lines in each panel represent the outlines of each organ. (n=5), P-values and two-tailed unpaired t-tests. ns: not significant; **: P < 0.01; ***: P < 0.001. Nuclei: TO-PRO-3. For the Combo plots, the boxes represent mean ± s.d., line in the box is the median, and the lower and upper lines show the minimum and the maximum of the values. The line on the right side of these combined plots represents the sample distribution. Co: Cortex; Me: Medulla Scale bars for tile scan 3D images 200 µm, for insets 50 µm.  C Young (10-weeks-old) and aged pituitary gland immunolabelled with ESM-1, Collagen IV and Emcn. Insets with higher magnifications of pars distalis and pars nervosa regions. Combo plot shows quantification of vessel density. Data information: The white and dashed lines in each panel represent the outlines of each organ. (n=5), P-values and two-tailed unpaired t-tests. ns: not significant; **: P < 0.01; ***: P < 0.001. Nuclei: TO-PRO-3. For the Combo plots, the boxes represent mean ± s.d., line in the box is the median, and the lower and upper lines show the minimum and the maximum of the values. The line on the right side of these combined plots represents the sample distribution. Pd: Pars Distalis; Pn: Pars Nervosa. Scale bars for tile scan 3D images 200 µm, for insets 50 µm.  D Young and aged testis immunolabelled with Tie-2 and HSPG2. Higher magnifications of cortex and medulla. Combo plot shows quantification of vessel density. Data information: The white and dashed lines in each panel represent the outlines of each organ. (n=5), P-values and two-tailed unpaired t-tests. ns: not significant; **: P < 0.01; ***: P < 0.001. Nuclei: TO-PRO-3. For the Combo plots, the boxes represent mean ± s.d., line in the box is the median, and the lower and upper lines show the minimum and the maximum of the values. The line on the right side of these combined plots represents the sample distribution. Scale bars for tile scan 3D images 200 µm, for insets 50 µm.  E Young and aged pancreas immunolabelled with SM22α and Endoglin. Insets representative of higher magnifications of regions in young and aged pancreas. Combo plot shows quantification of vessel density. Data information: The white and dashed lines in each panel represent the outlines of each organ. (n=5), P-values and two-tailed unpaired t-tests. ns: not significant; **: P < 0.01; ***: P < 0.001. Nuclei: TO-PRO-3. For the Combo plots, the boxes represent mean ± s.d., line in the box is the median, and the lower and upper lines show the minimum and the maximum of the values. The line on the right side of these combined plots represents the sample distribution. Scale bars for tile scan 3D images 200 µm, for insets 50 µm.  F Young and aged thyroid gland immunolabelled with CXCR4, Laminin and Emcn. Combo plot shows quantification of vessel density in young and aged thyroid glands. Insets: periphery and centre of the thyroid gland. The white and dashed lines in each panel represent the outlines of each organ. (n=5), P-values and two-tailed unpaired t-tests. ns: not significant; **: P < 0.01; ***: P < 0.001. Nuclei: TO-PRO-3. For the Combo plots, the boxes represent mean ± s.d., line in the box is the median, and the lower and upper lines show the minimum and the maximum of the values. The line on the right side of these combined plots represents the sample distribution. ; Pe: Periphery Ct: Center Scale bars for tile scan 3D images 200 µm, for insets 50 µm.


Figure 3 A Whole - endocrine gland imaging of young and aged adrenal glands immunolabelled with α - SMA and Emcn acquired on a light - sheet microscope . Asterisks indicate higher magnifications of regions in adrenal glands from young and aged mice . Combined box and whiskers , and scatter plots with quantifications of vessel density and numbers of arteries . B Whole - endocrine gland light - sheet imaging - based 3D images of young and aged ovaries stained with α - SMA and Emcn . Asterisks represent higher magnifications of regions in ovaries from young and aged mice . Combo plots show quantifications of vessel density and numbers of arteries in young and aged ovary . C 3D images of whole cleared young and aged testes stained with α - SMA and Emcn . Asterisks indicate higher magnifications of indicated regions . Combo plots show quantifications of vessel density and numbers of arteries . D Light - sheet imaging of whole cleared young and aged pancreases immunolabelled with α - SMA and Emcn . Asterisks indicate higher magnifications of regions . Combo plots with quantifications of vessel density and numbers of arteries in young and aged pancreas . E 3D images acquired on a light sheet microscope of whole cleared young and aged thyroid glands stained with α - SMA and Emcn . Asterisks indicate higher magnifications of regions . Combo plots show quantifications of vessel density and numbers of arteries in young and aged thyroid glands . F Heatmap of 3D spatial proteomic data showing qualitative analysis of differences in cell type markers and matrix expressions between young and aged murine endocrine glands . The colour code based on immunolabelling intensities indicates very high expression in red , high expression in yellow , medium in light green , low in dark green and no expression in grey .

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

Figure 3 A Whole-endocrine gland imaging of young and aged adrenal glands immunolabelled with α-SMA and Emcn acquired on a light-sheet microscope. Asterisks indicate higher magnifications of regions in adrenal glands from young and aged mice. Combined box and whiskers, and scatter plots with quantifications of vessel density and numbers of arteries.  B Whole-endocrine gland light-sheet imaging-based 3D images of young and aged ovaries stained with α-SMA and Emcn. Asterisks represent higher magnifications of regions in ovaries from young and aged mice. Combo plots show quantifications of vessel density and numbers of arteries in young and aged ovary.  C 3D images of whole cleared young and aged testes stained with α-SMA and Emcn. Asterisks indicate higher magnifications of indicated regions. Combo plots show quantifications of vessel density and numbers of arteries.  D Light-sheet imaging of whole cleared young and aged pancreases immunolabelled with α-SMA and Emcn. Asterisks indicate higher magnifications of regions. Combo plots with quantifications of vessel density and numbers of arteries in young and aged pancreas.  E 3D images acquired on a light sheet microscope of whole cleared young and aged thyroid glands stained with α-SMA and Emcn. Asterisks indicate higher magnifications of regions. Combo plots show quantifications of vessel density and numbers of arteries in young and aged thyroid glands.  F Heatmap of 3D spatial proteomic data showing qualitative analysis of differences in cell type markers and matrix expressions between young and aged murine endocrine glands. The colour code based on immunolabelling intensities indicates very high expression in red, high expression in yellow, medium in light green, low in dark green and no expression in grey.


Figure 4 A 3D images ( left ) with α - SMA and CD31 immunostaining in young and aged human adrenal gland . Middle panel images surface rendered for α - SMA , Vimentin and CD31 . Arrowheads indicate arteries . Combo plots with quantifications of the number of arteries and vessel density . Data information : ( n = 4 ) , P - values , and two - tailed unpaired t - tests for all the above panels . ns : not significant ; * : P < 0 . 05 ; * * : P < 0 . 01 ; * * * * : P < 0 . 0001 . Human samples from donors at ages below 20 years and over 70 years were chosen for young and aged group sets , respectively . Nuclei : DAPI . B 3D images of young and aged human ovary immunolabelled with α - SMA , Vimentin and CD31 . Images in the middle panel with a surface rendered for all the channels . Arrowheads indicate arteries . Combo plots with quantifications of the number of arteries and vessel density . Data information : ( n = 4 ) , P - values , and two - tailed unpaired t - tests for all the above panels . ns : not significant ; * : P < 0 . 05 ; * * : P < 0 . 01 ; * * * * : P < 0 . 0001 . Human samples from donors at ages below 20 years and over 70 years were chosen for young and aged group sets , respectively . Nuclei : DAPI . C Representative 3D images ( left ) of young and aged human testis with SM22α and Endoglin immunostaining . Middle panel images surface rendered for all the channels . Arrowheads indicate arteries . Combo plots show quantifications of the number of arteries and vessel density . Data information : ( n = 4 ) , P - values , and two - tailed unpaired t - tests for all the above panels . ns : not significant ; * : P < 0 . 05 ; * * : P < 0 . 01 ; * * * * : P < 0 . 0001 . Human samples from donors at ages below 20 years and over 70 years were chosen for young and aged group sets , respectively . Nuclei : DAPI . D 3D images from young and aged human pancreas staining with α - SMA , Vimentin and Endoglin . 3D images ( middle ) with a surface rendering for all the channels . Arrowheads indicate arteries . Combined box and whiskers , and scatter plots with the quantifications of the number of arteries and vessel density . Data information : ( n = 4 ) , P - values , and two - tailed unpaired t - tests for all the above panels . ns : not significant ; * : P < 0 . 05 ; * * : P < 0 . 01 ; * * * * : P < 0 . 0001 . Human samples from donors at ages below 20 years and over 70 years were chosen for young and aged group sets , respectively . Nuclei : DAPI . E Representative 3D images ( left ) show α - SMA and CD31 immunostaining in young and aged human thyroid gland . Middle panel images surface rendered for α - SMA , Vimentin and CD31 . Arrowheads indicate arteries . Combo plots with quantifications of the number of arteries and vessel density . Data information : ( n = 4 ) , P - values , and two - tailed unpaired t - tests for all the above panels . ns : not significant ; * : P < 0 . 05 ; * * : P < 0 . 01 ; * * * * : P < 0 . 0001 . Human samples from donors at ages below 20 years and over 70 years were chosen for young and aged group sets , respectively . Nuclei : DAPI . F qPCR analysis of Efnb2 , Cdh5 and Kdr expression ( normalized to Actb ) in aged human endocrine glands compared to young glands . Data information : ( n = 4 ) , P - values , and two - tailed unpaired t - tests for all the above panels . ns : not significant ; * : P < 0 . 05 ; * * : P < 0 . 01 ; * * * * : P < 0 . 0001 . Human samples from donors at ages below 20 years and over 70 years were chosen for young and aged group sets , respectively .

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

Figure 4 A 3D images (left) with α-SMA and CD31 immunostaining in young and aged human adrenal gland. Middle panel images surface rendered for α-SMA, Vimentin and CD31. Arrowheads indicate arteries. Combo plots with quantifications of the number of arteries and vessel density. Data information: (n=4), P-values, and two-tailed unpaired t-tests for all the above panels. ns: not significant; *: P < 0.05; **: P < 0.01; ****: P < 0.0001. Human samples from donors at ages below 20 years and over 70 years were chosen for young and aged group sets, respectively. Nuclei: DAPI.  B 3D images of young and aged human ovary immunolabelled with α-SMA, Vimentin and CD31. Images in the middle panel with a surface rendered for all the channels. Arrowheads indicate arteries. Combo plots with quantifications of the number of arteries and vessel density. Data information: (n=4), P-values, and two-tailed unpaired t-tests for all the above panels. ns: not significant; *: P < 0.05; **: P < 0.01; ****: P < 0.0001. Human samples from donors at ages below 20 years and over 70 years were chosen for young and aged group sets, respectively. Nuclei: DAPI.  C Representative 3D images (left) of young and aged human testis with SM22α and Endoglin immunostaining. Middle panel images surface rendered for all the channels. Arrowheads indicate arteries. Combo plots show quantifications of the number of arteries and vessel density. Data information: (n=4), P-values, and two-tailed unpaired t-tests for all the above panels. ns: not significant; *: P < 0.05; **: P < 0.01; ****: P < 0.0001. Human samples from donors at ages below 20 years and over 70 years were chosen for young and aged group sets, respectively. Nuclei: DAPI.  D 3D images from young and aged human pancreas staining with α-SMA, Vimentin and Endoglin. 3D images (middle) with a surface rendering for all the channels. Arrowheads indicate arteries. Combined box and whiskers, and scatter plots with the quantifications of the number of arteries and vessel density. Data information: (n=4), P-values, and two-tailed unpaired t-tests for all the above panels. ns: not significant; *: P < 0.05; **: P < 0.01; ****: P < 0.0001. Human samples from donors at ages below 20 years and over 70 years were chosen for young and aged group sets, respectively. Nuclei: DAPI.  E Representative 3D images (left) show α-SMA and CD31 immunostaining in young and aged human thyroid gland. Middle panel images surface rendered for α-SMA, Vimentin and CD31. Arrowheads indicate arteries. Combo plots with quantifications of the number of arteries and vessel density. Data information: (n=4), P-values, and two-tailed unpaired t-tests for all the above panels. ns: not significant; *: P < 0.05; **: P < 0.01; ****: P < 0.0001. Human samples from donors at ages below 20 years and over 70 years were chosen for young and aged group sets, respectively. Nuclei: DAPI.  F qPCR analysis of Efnb2, Cdh5 and Kdr expression (normalized to Actb) in aged human endocrine glands compared to young glands. Data information: (n=4), P-values, and two-tailed unpaired t-tests for all the above panels. ns: not significant; *: P < 0.05; **: P < 0.01; ****: P < 0.0001. Human samples from donors at ages below 20 years and over 70 years were chosen for young and aged group sets, respectively.


Figure 5 A Representative 3D images with Insulin and Caveolin - 1 immunostaining in pancreatic islets from young mice . Combined box and whiskers , and scatter plots show the vessel density analysis in the pancreatic islets and non - islet areas from young and aged mice . Data information : The white and dashed lines in panel A , B , D , F , H and I represent the outlines of pancreatic islets . ( n = 5 ) , P - values , and two - tailed unpaired t - tests in panel ( A , E and I ) and one - way ANOVA tests with Tukey ' s multiple comparisons tests in panel ( G and H ) . * : P < 0 . 05 ; * * : P < 0 . 01 ; * * * : P < 0 . 001 ; * * * * : P < 0 . 0001 . Nuclei : TO - PRO - 3 . Is : pancreatic islet . f . u : fluorescence unit . B Representative 3D images with adult pancreatic islets immunolabeled with Ki67 and VE - Cadherin . Inset ( * ) shows single - cell resolution and surface rendering for all the channels . Data information : The white and dashed lines in panel A , B , D , F , H and I represent the outlines of pancreatic islets . ( n = 5 ) , P - values , and two - tailed unpaired t - tests in panel ( A , E and I ) and one - way ANOVA tests with Tukey ' s multiple comparisons tests in panel ( G and H ) . * : P < 0 . 05 ; * * : P < 0 . 01 ; * * * : P < 0 . 001 ; * * * * : P < 0 . 0001 . Nuclei : TO - PRO - 3 . Is : pancreatic islet . f . u : fluorescence unit . C Representative tile scan 3D image with Insulin , ESM - 1 and Emcn immunostaining in 19 - weeks - old mouse pancreas . Higher magnification at the top of the right panel shows the ESM - 1 + pancreatic islets . Inset ( * ) shows single - cell resolution and surface rendering for ESM - 1 and Emcn . The white and dashed line in the tile scan image represents the outline of one lobe of the pancreas , and the dashed lines in the inset represents the pancreatic islet . Data information : The white and dashed lines in panel A , B , D , F , H and I represent the outlines of pancreatic islets . ( n = 5 ) , P - values , and two - tailed unpaired t - tests in panel ( A , E and I ) and one - way ANOVA tests with Tukey ' s multiple comparisons tests in panel ( G and H ) . * : P < 0 . 05 ; * * : P < 0 . 01 ; * * * : P < 0 . 001 ; * * * * : P < 0 . 0001 . Nuclei : TO - PRO - 3 . Is : pancreatic islet . f . u : fluorescence unit . D Representative 3D images show CD31 and Emcn expressions in young pancreas . Insets ( * and # ) show higher magnifications of capillaries in the pancreatic islets and non - islet areas . Data information : The white and dashed lines in panel A , B , D , F , H and I represent the outlines of pancreatic islets . ( n = 5 ) , P - values , and two - tailed unpaired t - tests in panel ( A , E and I ) and one - way ANOVA tests with Tukey ' s multiple comparisons tests in panel ( G and H ) . * : P < 0 . 05 ; * * : P < 0 . 01 ; * * * : P < 0 . 001 ; * * * * : P < 0 . 0001 . Nuclei : TO - PRO - 3 . Is : pancreatic islet . f . u : fluorescence unit . E Representative flow cytometry dot plots showing CD31 + Emcnhi and CD31 + Emcnlo EC populations in young and aged pancreas . Combo plot shows the population of CD31 + Emcnhi ECs in young and aged pancreas . F Representative 3D images ( left ) with Insulin , Ki67 and VE - Cadherin immunostaining on young and aged pancreas . 3D images ( middle ) show CXCR4 , Ki67 and VE - Cadherin in young and aged pancreatic islet . Insets ( yellow asterisks ) show single - cell resolution and surface rendering for Ki67 and CXCR4 . Arrowheads represent Ki67 + β - cell . G Combo plot shows the quantification of Ki67 + β - cells normalized to the total number of β - cells per islet in juvenile ( 3 - weeks - old ) , adult ( 12 - weeks - old ) and aged ( 55 - weeks - old ) pancreatic islets . Data information : The white and dashed lines in panel A , B , D , F , H and I represent the outlines of pancreatic islets . ( n = 5 ) , P - values , and two - tailed unpaired t - tests in panel ( A , E and I ) and one - way ANOVA tests with Tukey ' s multiple comparisons tests in panel ( G and H ) . * : P < 0 . 05 ; * * : P < 0 . 01 ; * * * : P < 0 . 001 ; * * * * : P < 0 . 0001 . Nuclei : TO - PRO - 3 . Is : pancreatic islet . f . u : fluorescence unit . H Representative 3D images with Insulin , CD31 and Emcn immunostaining in young and aged pancreas . Combo plot shows the CD31 + Emcnhi vessel density in juvenile , adult and aged pancreatic islets . Data information : The white and dashed lines in panel A , B , D , F , H and I represent the outlines of pancreatic islets . ( n = 5 ) , P - values , and two - tailed unpaired t - tests in panel ( A , E and I ) and one - way ANOVA tests with Tukey ' s multiple comparisons tests in panel ( G and H ) . * : P < 0 . 05 ; * * : P < 0 . 01 ; * * * : P < 0 . 001 ; * * * * : P < 0 . 0001 . Nuclei : TO - PRO - 3 . Is : pancreatic islet . f . u : fluorescence unit . I Representative 3D images of adult ( 10 - weeks - old ) and aged pancreas ( 55 - weeks - old ) with Insulin and VEGFA immunostaining . Combo plot ( middle ) shows the VEGFA as determined by ELISA assay in isolated islets from 10 - weeks - old adult and 55 - weeks - old aged mice . Combo plot ( right ) shows the percentage of VEGFA per islet ( f . u ) . Data information : The white and dashed lines in panel A , B , D , F , H and I represent the outlines of pancreatic islets . ( n = 5 ) , P - values , and two - tailed unpaired t - tests in panel ( A , E and I ) and one - way ANOVA tests with Tukey ' s multiple comparisons tests in panel ( G and H ) . * : P < 0 . 05 ; * * : P < 0 . 01 ; * * * : P < 0 . 001 ; * * * * : P < 0 . 0001 . Nuclei : TO - PRO - 3 . Is : pancreatic islet . f . u : fluorescence unit .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5 A Representative 3D images with Insulin and Caveolin-1 immunostaining in pancreatic islets from young mice. Combined box and whiskers, and scatter plots show the vessel density analysis in the pancreatic islets and non-islet areas from young and aged mice. Data information: The white and dashed lines in panel A, B, D, F, H and I represent the outlines of pancreatic islets. (n=5), P-values, and two-tailed unpaired t-tests in panel (A, E and I) and one-way ANOVA tests with Tukey's multiple comparisons tests in panel (G and H). *: P < 0.05; **: P < 0.01; ***: P < 0.001; ****: P < 0.0001. Nuclei: TO-PRO-3. Is: pancreatic islet. f.u: fluorescence unit.  B Representative 3D images with adult pancreatic islets immunolabeled with Ki67 and VE-Cadherin. Inset (*) shows single-cell resolution and surface rendering for all the channels. Data information: The white and dashed lines in panel A, B, D, F, H and I represent the outlines of pancreatic islets. (n=5), P-values, and two-tailed unpaired t-tests in panel (A, E and I) and one-way ANOVA tests with Tukey's multiple comparisons tests in panel (G and H). *: P < 0.05; **: P < 0.01; ***: P < 0.001; ****: P < 0.0001. Nuclei: TO-PRO-3. Is: pancreatic islet. f.u: fluorescence unit.  C Representative tile scan 3D image with Insulin, ESM-1 and Emcn immunostaining in 19-weeks-old mouse pancreas. Higher magnification at the top of the right panel shows the ESM-1+ pancreatic islets. Inset (*) shows single-cell resolution and surface rendering for ESM-1 and Emcn. The white and dashed line in the tile scan image represents the outline of one lobe of the pancreas, and the dashed lines in the inset represents the pancreatic islet. Data information: The white and dashed lines in panel A, B, D, F, H and I represent the outlines of pancreatic islets. (n=5), P-values, and two-tailed unpaired t-tests in panel (A, E and I) and one-way ANOVA tests with Tukey's multiple comparisons tests in panel (G and H). *: P < 0.05; **: P < 0.01; ***: P < 0.001; ****: P < 0.0001. Nuclei: TO-PRO-3. Is: pancreatic islet. f.u: fluorescence unit. D Representative 3D images show CD31 and Emcn expressions in young pancreas. Insets (* and #) show higher magnifications of capillaries in the pancreatic islets and non-islet areas. Data information: The white and dashed lines in panel A, B, D, F, H and I represent the outlines of pancreatic islets. (n=5), P-values, and two-tailed unpaired t-tests in panel (A, E and I) and one-way ANOVA tests with Tukey's multiple comparisons tests in panel (G and H). *: P < 0.05; **: P < 0.01; ***: P < 0.001; ****: P < 0.0001. Nuclei: TO-PRO-3. Is: pancreatic islet. f.u: fluorescence unit. E Representative flow cytometry dot plots showing CD31+ Emcnhi and CD31+ Emcnlo EC populations in young and aged pancreas. Combo plot shows the population of CD31+ Emcnhi ECs in young and aged pancreas.  F Representative 3D images (left) with Insulin, Ki67 and VE-Cadherin immunostaining on young and aged pancreas. 3D images (middle) show CXCR4, Ki67 and VE-Cadherin in young and aged pancreatic islet. Insets (yellow asterisks) show single-cell resolution and surface rendering for Ki67 and CXCR4. Arrowheads represent Ki67+ β-cell. G Combo plot shows the quantification of Ki67+ β-cells normalized to the total number of β-cells per islet in juvenile (3-weeks-old), adult (12-weeks-old) and aged (55-weeks-old) pancreatic islets. Data information: The white and dashed lines in panel A, B, D, F, H and I represent the outlines of pancreatic islets. (n=5), P-values, and two-tailed unpaired t-tests in panel (A, E and I) and one-way ANOVA tests with Tukey's multiple comparisons tests in panel (G and H). *: P < 0.05; **: P < 0.01; ***: P < 0.001; ****: P < 0.0001. Nuclei: TO-PRO-3. Is: pancreatic islet. f.u: fluorescence unit. H Representative 3D images with Insulin, CD31 and Emcn immunostaining in young and aged pancreas. Combo plot shows the CD31+ Emcnhi vessel density in juvenile, adult and aged pancreatic islets. Data information: The white and dashed lines in panel A, B, D, F, H and I represent the outlines of pancreatic islets. (n=5), P-values, and two-tailed unpaired t-tests in panel (A, E and I) and one-way ANOVA tests with Tukey's multiple comparisons tests in panel (G and H). *: P < 0.05; **: P < 0.01; ***: P < 0.001; ****: P < 0.0001. Nuclei: TO-PRO-3. Is: pancreatic islet. f.u: fluorescence unit.  I Representative 3D images of adult (10-weeks-old) and aged pancreas (55-weeks-old) with Insulin and VEGFA immunostaining. Combo plot (middle) shows the VEGFA as determined by ELISA assay in isolated islets from 10-weeks-old adult and 55-weeks-old aged mice. Combo plot (right) shows the percentage of VEGFA per islet (f.u). Data information: The white and dashed lines in panel A, B, D, F, H and I represent the outlines of pancreatic islets. (n=5), P-values, and two-tailed unpaired t-tests in panel (A, E and I) and one-way ANOVA tests with Tukey's multiple comparisons tests in panel (G and H). *: P < 0.05; **: P < 0.01; ***: P < 0.001; ****: P < 0.0001. Nuclei: TO-PRO-3. Is: pancreatic islet. f.u: fluorescence unit.


Figure 6 A Representative 3D images of pancreas from adult ( 18 - weeks - old ) sunitinib - treated and control mice with Insulin , CD31 and Emcn immunostaining . B Combined box and whiskers , and scatter plot ( left ) shows the quantification of CD31 + Emcnhi vessel density in sunitinib - treated and control pancreatic islets . Combo plot ( right ) shows ELISA based analysis of Insulin . Nuclei : DAPI or TO - PRO - 3 as indicated . Is : pancreatic islet . f . u : fluorescence unit . C Representative 3D images ( upper panel ) show CXCR4 , Ki67 and VE - Cadherin in sunitinib - treated and control pancreatic islet . 3D images ( bottom ) show sunitinib - treated and control pancreatic islets immunolabeled with Insulin and VE - Cadherin . D Combo plot ( top ) shows the percentage of CXCR4 + β - cells per islet in sunitinib - treated and control pancreatic islet . Combo plot ( middle ) shows the quantification of Ki67 + β - cells normalized to the total number of β - cell numbers . Combo plot ( bottom ) shows the percentage of Insulin + β - cells per islet . Nuclei : DAPI or TO - PRO - 3 as indicated . Is : pancreatic islet . f . u : fluorescence unit . E Representative 3D images of pancreas from adult Vegfr2i ∆ EC mutant and control mice with Insulin , CD31 and Emcn immunostainings . F Combo plot ( left ) with quantification of CD31 + Emcnhi vessel density in Vegfr2i ∆ EC and control pancreatic islet . Right combo plot with insulin estimation in pancreas by ELISA assay . Nuclei : DAPI or TO - PRO - 3 as indicated . Is : pancreatic islet . f . u : fluorescence unit . G Representative 3D images in the upper panel show CXCR4 , Ki67 and VE - Cadherin in islets from Vegfr2i ∆ EC mutant and control mice . 3D images ( lower panel ) show Vegfr2i ∆ EC mutant and control islets immunolabeled with Insulin and VE - Cadherin . H Combo plot ( top ) shows the percentage of CXCR4 + β - cells per islet in Vegfr2i ∆ EC mutant and control pancreatic islet . Combo plot ( middle ) shows the quantification of Ki67 + β - cells normalized to the number of β - cells . Combo plot ( bottom ) shows the percentage of Insulin + β - cells per islet . Nuclei : DAPI or TO - PRO - 3 as indicated . Is : pancreatic islet . f . u : fluorescence unit . I Graph shows qPCR analysis of Pdgfa , Pdgfb , Igf1 , Igf2 , Cxcl12 , Hgf and Kitl expression ( normalized to Actb ) in purified CD31 + Emcnhi ECs compared to CD31 + Emcnlo ECs . J Bar graph with the quantification of Ki67 + β - cell numbers per islet ( f . u ) and Ki67 + EC numbers per islet ( f . u ) . 45 pancreatic islets were analysed and quantified from 9 young mice . K Bar graphs show qPCR analysis of Dll4 , Emcn , Kdr , Flt4 , Sele , Mmp9 , Plxnd1 , Robo4 , Unc5b , Cdh5 , Cldn5 , Gja1 , Gja4 , Icam1 , Icam2 , Jam3 , Ntn1 , Ntn2 , Ocln , Pecam1 , Tjp1 , Tjp2 , Tjp3 and Vcam1 expression ( normalized to Actb ) in purified CD31 + Emcnhi ECs compared to CD31 + Emcnlo ECs from murine pancreas . L Graph shows qPCR analysis of Gja1 expression ( normalized to Actb ) in purified CD31 + Emcnhi ECs from juvenile , adult and aged murine pancreas .

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

Figure 6 A Representative 3D images of pancreas from adult (18-weeks-old) sunitinib-treated and control mice with Insulin, CD31 and Emcn immunostaining. B Combined box and whiskers, and scatter plot (left) shows the quantification of CD31+ Emcnhi vessel density in sunitinib-treated and control pancreatic islets. Combo plot (right) shows ELISA based analysis of Insulin. Nuclei: DAPI or TO-PRO-3 as indicated. Is: pancreatic islet. f.u: fluorescence unit.  C Representative 3D images (upper panel) show CXCR4, Ki67 and VE-Cadherin in sunitinib-treated and control pancreatic islet. 3D images (bottom) show sunitinib-treated and control pancreatic islets immunolabeled with Insulin and VE-Cadherin. D Combo plot (top) shows the percentage of CXCR4+ β-cells per islet in sunitinib-treated and control pancreatic islet. Combo plot (middle) shows the quantification of Ki67+ β-cells normalized to the total number of β-cell numbers. Combo plot (bottom) shows the percentage of Insulin+ β-cells per islet. Nuclei: DAPI or TO-PRO-3 as indicated. Is: pancreatic islet. f.u: fluorescence unit. E Representative 3D images of pancreas from adult Vegfr2i∆EC mutant and control mice with Insulin, CD31 and Emcn immunostainings. F Combo plot (left) with quantification of CD31+ Emcnhi vessel density in Vegfr2i∆EC and control pancreatic islet. Right combo plot with insulin estimation in pancreas by ELISA assay. Nuclei: DAPI or TO-PRO-3 as indicated. Is: pancreatic islet. f.u: fluorescence unit. G Representative 3D images in the upper panel show CXCR4, Ki67 and VE-Cadherin in islets from Vegfr2i∆EC mutant and control mice. 3D images (lower panel) show Vegfr2i∆EC mutant and control islets immunolabeled with Insulin and VE-Cadherin. H Combo plot (top) shows the percentage of CXCR4+ β-cells per islet in Vegfr2i∆EC mutant and control pancreatic islet. Combo plot (middle) shows the quantification of Ki67+ β-cells normalized to the number of β-cells. Combo plot (bottom) shows the percentage of Insulin+ β-cells per islet. Nuclei: DAPI or TO-PRO-3 as indicated. Is: pancreatic islet. f.u: fluorescence unit. I Graph shows qPCR analysis of Pdgfa, Pdgfb, Igf1, Igf2, Cxcl12, Hgf and Kitl expression (normalized to Actb) in purified CD31+ Emcnhi ECs compared to CD31+ Emcnlo ECs.  J Bar graph with the quantification of Ki67+ β-cell numbers per islet (f.u) and Ki67+ EC numbers per islet (f.u). 45 pancreatic islets were analysed and quantified from 9 young mice.  K Bar graphs show qPCR analysis of Dll4, Emcn, Kdr, Flt4, Sele, Mmp9, Plxnd1, Robo4, Unc5b, Cdh5, Cldn5, Gja1, Gja4, Icam1, Icam2, Jam3, Ntn1, Ntn2, Ocln, Pecam1, Tjp1, Tjp2, Tjp3 and Vcam1 expression (normalized to Actb) in purified CD31+ Emcnhi ECs compared to CD31+ Emcnlo ECs from murine pancreas.  L Graph shows qPCR analysis of Gja1 expression (normalized to Actb) in purified CD31+ Emcnhi ECs from juvenile, adult and aged murine pancreas.


Figure 7 A Representative tile scan 3D images with α - SMA , Isolectin , FABP4 and Emcn in the pancreas from 17 - weeks - old Gja1i ∆ EC mutant and control mice . Insets show higher magnifications of regions . B Box whiskers , and scatter plot ( left ) shows the quantification of CD31 + Emcnhi vessel density in Gja1i ∆ EC mutant and control pancreatic islets . ( n = 7 ) , P - value , and two - tailed unpaired t - test . Combo plot ( right ) shows the quantification of P - Histone H3 + EC numbers per islet ( f . u ) counted on thick murine pancreatic sections . ( n = 5 ) , P - value , and two - tailed unpaired t - test . C 3D images show CD31 and Emcn immunostaining in the Gja1i ∆ EC mutant and control pancreas . The scale bars are 50 µm . D Combined violin , scatter and sample distribution plot shows the quantification of β - cell mass ( % ) in pancreatic islets from Gja1i ∆ EC and control mice . Data represent mean ± s . d . ( n = 140 islets from 7 pancreas ) , P - value , and two - tailed unpaired t - test . E Representative 3D images with immunostaining of α - SMA , Insulin and VE - Cadherin in the pancreatic islets from Gja1i ∆ EC and control mice . The scale bars are 50 µm . F Combo plot ( left ) shows insulin measurement in lysate of pancreas by ELISA . Combo plot ( right ) shows the percentage of Insulin + β - cells per islet in Gja1i ∆ EC mutant and control mice . ( n = 5 ) , P - values , and two - tailed unpaired t - tests . Data information : The dashed lines in panel A represent the outlines of pancreas . The dashed lines in panel C , E , G , H and I represent the outlines of pancreatic islets . Two - tailed Student ' s t - test were performed for the statistical analysis . * * : P < 0 . 01 ; * * * * : P < 0 . 0001 . Nuclei : DAPI Is : pancreatic islet . f . u : fluorescence unit . The line on the right side of the combo plots represent the sample distribution . Scale bars are 200 µm for tile scan images of longitudinal thick sections across the entire glands , and 50 µm for the higher magnification insets images . G Representative 3D images of ESM - 1 and Caveolin - 1 in pancreatic islets from Gja1i ∆ EC and control mice . Insets ( * and # ) show higher magnifications with surface rendering for ESM - 1 and Caveolin - 1 immunostaining . Combo plot with the quantification of ESM - 1 + cell numbers per islet ( f . u ) . ( n = 5 ) , P - value , and two - tailed unpaired t - test . The scale bars for the 3D images are 50 µm , and 5 µm for the higher magnifications with single - cell resolution images . The dashed lines in panel C , E , G , H and I represent the outlines of pancreatic islets . Two - tailed Student ' s t - test were performed for the statistical analysis . * * : P < 0 . 01 ; * * * * : P < 0 . 0001 . Nuclei : TO - PRO - 3 as indicated . Is : pancreatic islet . f . u : fluorescence unit . H 3D images with CXCR4 and Emcn immunostaining in Gja1i ∆ EC and control pancreas . The scale bars are 50 µm . I Representative 3D images show CXCR4 , Ki67 and VE - Cadherin immunostaining in Gja1i ∆ EC and control pancreas . The scale bars are 50 µm . J Combo plots show the percentage of CXCR4 + β - cells per islet and Ki67 + β - cells normalized to the total number of β - cells per islet in Gja1i ∆ EC mutant versus the littermate control mice . ( n = 5 ) , P - values , and two - tailed unpaired t - tests . K Box whiskers , and scatter plot shows the quantification of CD31 + Emcnhi vessel density in aged Gja1i ∆ EC mutant and control pancreatic islets . Data represent mean ± s . d . ( n = 4 ) , P - value , and two - tailed unpaired t - test . Combo plot ( below ) shows the quantification of β - cells on thick murine pancreatic sections . Data represent mean ± s . d . ( n = 4 ) , P - value , and two - tailed unpaired t - test . Data information : The dashed lines in panel C , E , G , H and I represent the outlines of pancreatic islets . Two - tailed Student ' s t - test were performed for the statistical analysis . * * : P < 0 . 01 ; * * * * : P < 0 . 0001 . Nuclei : TO - PRO -

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

Figure 7 A Representative tile scan 3D images with α-SMA, Isolectin, FABP4 and Emcn in the pancreas from 17-weeks-old Gja1i∆EC mutant and control mice. Insets show higher magnifications of regions. B Box whiskers, and scatter plot (left) shows the quantification of CD31+ Emcnhi vessel density in Gja1i∆EC mutant and control pancreatic islets. (n=7), P-value, and two-tailed unpaired t-test. Combo plot (right) shows the quantification of P-Histone H3+ EC numbers per islet (f.u) counted on thick murine pancreatic sections. (n=5), P-value, and two-tailed unpaired t-test. C 3D images show CD31 and Emcn immunostaining in the Gja1i∆EC mutant and control pancreas. The scale bars are 50 µm. D Combined violin, scatter and sample distribution plot shows the quantification of β-cell mass (%) in pancreatic islets from Gja1i∆EC and control mice. Data represent mean ± s.d. (n=140 islets from 7 pancreas), P-value, and two-tailed unpaired t-test. E Representative 3D images with immunostaining of α-SMA, Insulin and VE-Cadherin in the pancreatic islets from Gja1i∆EC and control mice. The scale bars are 50 µm. F Combo plot (left) shows insulin measurement in lysate of pancreas by ELISA. Combo plot (right) shows the percentage of Insulin+ β-cells per islet in Gja1i∆EC mutant and control mice. (n=5), P-values, and two-tailed unpaired t-tests. Data information: The dashed lines in panel A represent the outlines of pancreas. The dashed lines in panel C, E, G, H and I represent the outlines of pancreatic islets. Two-tailed Student's t-test were performed for the statistical analysis. **: P < 0.01; ****: P < 0.0001. Nuclei: DAPI Is: pancreatic islet. f.u: fluorescence unit. The line on the right side of the combo plots represent the sample distribution. Scale bars are 200 µm for tile scan images of longitudinal thick sections across the entire glands, and 50 µm for the higher magnification insets images. G Representative 3D images of ESM-1 and Caveolin-1 in pancreatic islets from Gja1i∆EC and control mice. Insets (* and #) show higher magnifications with surface rendering for ESM-1 and Caveolin-1 immunostaining. Combo plot with the quantification of ESM-1+ cell numbers per islet (f.u). (n=5), P-value, and two-tailed unpaired t-test. The scale bars for the 3D images are 50 µm, and 5 µm for the higher magnifications with single-cell resolution images. The dashed lines in panel C, E, G, H and I represent the outlines of pancreatic islets. Two-tailed Student's t-test were performed for the statistical analysis. **: P < 0.01; ****: P < 0.0001. Nuclei: TO-PRO-3 as indicated. Is: pancreatic islet. f.u: fluorescence unit.  H 3D images with CXCR4 and Emcn immunostaining in Gja1i∆EC and control pancreas. The scale bars are 50 µm. I Representative 3D images show CXCR4, Ki67 and VE-Cadherin immunostaining in Gja1i∆EC and control pancreas. The scale bars are 50 µm. J Combo plots show the percentage of CXCR4+ β-cells per islet and Ki67+ β-cells normalized to the total number of β-cells per islet in Gja1i∆EC mutant versus the littermate control mice. (n=5), P-values, and two-tailed unpaired t-tests. K Box whiskers, and scatter plot shows the quantification of CD31+ Emcnhi vessel density in aged Gja1i∆EC mutant and control pancreatic islets. Data represent mean±s.d. (n=4), P-value, and two-tailed unpaired t-test. Combo plot (below) shows the quantification of β-cells on thick murine pancreatic sections. Data represent mean±s.d. (n=4), P-value, and two-tailed unpaired t-test. Data information: The dashed lines in panel C, E, G, H and I represent the outlines of pancreatic islets. Two-tailed Student's t-test were performed for the statistical analysis. **: P < 0.01; ****: P < 0.0001. Nuclei: TO-PRO-3


Figure 1RNA - seq of liver 10h post Sham or CLP . Mice ( n = 3 / group ) underwent a sham or CLP operation and were injected with GW7647 ( 10 µg / g ) 6h post - surgery , after 4h ( total of 10h ) , livers were isolated and RNA was prepared . Scatter plot showing log fold change ( LFC ) of all GW7647 - upregulated genes ( LFC > 0 . 8 and P < 0 . 05 ) in sham versus their LFC 10h after CLP . The red line represents the diagonal , the black line represents the real slope ( 0 . 3115 ) of the data . RNA - seq of liver 10h post Sham or CLP . Mice ( n = 3 / group ) underwent a sham or CLP operation and were injected with GW7647 ( 10 µg / g ) 6h post - surgery , after 4h ( total of 10h ) , livers were isolated and RNA was prepared . Heatmap of differentially expressed genes in sham mice after GW7647 treatment , involved in β - oxidation of fatty acids ( unit scale bar = log2 of the normalized counts ) . Confirmation of RNA - seq data via qPCR on pure hepatocytes isolated via flow cytometry - based sorting ( n = 3 / group ) . ( E ) Ppara and ( F ) Hmgcs2 mRNA expression is shown as relative expression , normalized to housekeeping genes Hprt and Rpl . P - values were calculated using 2 - way ANOVA analysis .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1RNA-seq of liver 10h post Sham or CLP. Mice (n = 3/group) underwent a sham or CLP operation and were injected with GW7647 (10 µg/g) 6h post-surgery, after 4h (total of 10h), livers were isolated and RNA was prepared. Scatter plot showing log fold change (LFC) of all GW7647-upregulated genes (LFC > 0.8 and P < 0.05) in sham versus their LFC 10h after CLP. The red line represents the diagonal, the black line represents the real slope (0.3115) of the data.RNA-seq of liver 10h post Sham or CLP. Mice (n = 3/group) underwent a sham or CLP operation and were injected with GW7647 (10 µg/g) 6h post-surgery, after 4h (total of 10h), livers were isolated and RNA was prepared. Heatmap of differentially expressed genes in sham mice after GW7647 treatment, involved in β-oxidation of fatty acids (unit scale bar = log2 of the normalized counts).Confirmation of RNA-seq data via qPCR on pure hepatocytes isolated via flow cytometry-based sorting (n = 3/group). (E) Ppara and (F) Hmgcs2 mRNA expression is shown as relative expression, normalized to housekeeping genes Hprt and Rpl. P-values were calculated using 2-way ANOVA analysis.


Figure 2Mice ( n = 4 / group , data are representative of 2 experiments ) underwent a sham ( with or without starvation ) or CLP operation and liver was isolated on several timepoints post - surgery for RNA preparation and qPCR . Ppara mRNA expression is shown as relative expression , normalized to housekeeping genes Hprt and Rpl . P - values were calculated using 1 - way ANOVA analysis . PPARA protein levels were analyzed in livers 24h after CLP by Western Blot using actin as a loading control . Quantification of PPARA western blot . P - value was calculated via 2 - way Student ' s t - test ( n = 4 / group ) . Pearson correlation between log fold change ( LFC ) Ppara expression levels and body temperature 24h post - sepsis ( n = 38 , r = 0 . 6875 , combined data of 4 independent experiments ) . Liver Hmgcs2 mRNA expression at different timepoints post - sepsis , expression is shown as relative expression , normalized to housekeeping genes Hprt and Rpl . P - values were calculated using 1 - way ANOVA analysis ( n = 4 / group , data are representative of 2 experiments ) . Oxygen consumption rates ( OCR ) of liver tissue explants 24h post sham or CLP . Liver tissue was isolated 24h post - surgery and OCR was measured via Seahorse with BSA or palmitic acid ( PA ) as a substrate for 42 min . n = 4 / group . Visualization of T42 ( min ) OCR , p - values were calculated using 2 - way ANOVA analysis .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O 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-PANEL_START O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O

Figure 2Mice (n = 4/group, data are representative of 2 experiments) underwent a sham (with or without starvation) or CLP operation and liver was isolated on several timepoints post-surgery for RNA preparation and qPCR. Ppara mRNA expression is shown as relative expression, normalized to housekeeping genes Hprt and Rpl. P-values were calculated using 1-way ANOVA analysis.PPARA protein levels were analyzed in livers 24h after CLP by Western Blot using actin as a loading control.Quantification of PPARA western blot. P-value was calculated via 2-way Student's t-test (n = 4/group).Pearson correlation between log fold change (LFC) Ppara expression levels and body temperature 24h post-sepsis (n= 38, r = 0.6875, combined data of 4 independent experiments).Liver Hmgcs2 mRNA expression at different timepoints post-sepsis, expression is shown as relative expression, normalized to housekeeping genes Hprt and Rpl. P-values were calculated using 1-way ANOVA analysis (n = 4/group, data are representative of 2 experiments).Oxygen consumption rates (OCR) of liver tissue explants 24h post sham or CLP. Liver tissue was isolated 24h post-surgery and OCR was measured via Seahorse with BSA or palmitic acid (PA) as a substrate for 42 min. n = 4/group.Visualization of T42(min) OCR, p-values were calculated using 2-way ANOVA analysis.


Figure 3Percentage of body weight that is taken up by the inguinal fat pad ( iWAT , A ) , the mesenteric fat pad ( mWAT , B ) and the perirenal fat pad ( pWAT , C ) 24h post - surgery in sham ( with or without starvation ) and CLP mice . P - values were calculated with a 1 - way ANOVA test . Combined data of 2 independent experiments , n = 6 / group . Visual representation of ( D ) iWAT and ( E ) pWAT sizes in sham , sham starved and CLP mice 24h post - surgery . Data are representative of 3 independent experiments . Mice ( n = 6 - 10 / group ) underwent a sham ( with or without starvation ) or CLP operation and blood was collected 6h and 24h post - surgery . Plasma was isolated and ( F ) FFA concentration and ( G ) glycerol concentration was determined as described in the method section . P - values were calculated with 2 - way ANOVA tests . Combined data of 2 independent experiments . Mice ( n = 6 - 10 / group ) underwent a sham ( with or without starvation ) or CLP operation and blood was collected 6h and 24h post - surgery . Normalized abundances of ( H ) palmitic acid and ( I ) palmitoyl - carnitine determined via liquid chromotography - mass spectrometric lipidomics . Values were normalized to IQ values , P - values were calculated with 2 - way ANOVA tests . n = 8 / group .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3Percentage of body weight that is taken up by the inguinal fat pad (iWAT, A), the mesenteric fat pad (mWAT, B) and the perirenal fat pad (pWAT, C) 24h post-surgery in sham (with or without starvation) and CLP mice. P-values were calculated with a 1-way ANOVA test. Combined data of 2 independent experiments, n = 6/group.Visual representation of (D) iWAT and (E) pWAT sizes in sham, sham starved and CLP mice 24h post-surgery. Data are representative of 3 independent experiments.Mice (n=6-10/group) underwent a sham (with or without starvation) or CLP operation and blood was collected 6h and 24h post-surgery. Plasma was isolated and (F) FFA concentration and (G) glycerol concentration was determined as described in the method section. P-values were calculated with 2-way ANOVA tests. Combined data of 2 independent experiments.Mice (n=6-10/group) underwent a sham (with or without starvation) or CLP operation and blood was collected 6h and 24h post-surgery. Normalized abundances of (H) palmitic acid and (I) palmitoyl-carnitine determined via liquid chromotography-mass spectrometric lipidomics. Values were normalized to IQ values , P-values were calculated with 2-way ANOVA tests. n = 8/group.


Figure 4Immunofluorescent images of liver and kidney 24h after sham ( with or without starvation ) or CLP ( n = 6 - 7 / group , combined data of 2 independent experiments ) . White scale bar = 20µm . Cryosections were stained with Acti - stain ( green ) , Hoechst ( blue ) , and LipidTOX ( red ) . Z - stacks were generated in 5 - 10 areas scattered across the entire tissue section . Quantification of lipid peroxidation by determination of ( C ) MDA and ( D ) 4 - HNE concentrations in liver homogenates 24h post - surgery in sham and CLP mice as described in methods ( n = 6 - 7 / group , combined data of 2 independent experiments ) . P - values were calculated with 1 - way ANOVA tests . Apoptosis in liver paraffin - fixated sections 24h after sepsis , measured by TUNEL staining , and presented as % PI positive cells / µm² tissue area . ( n = 6 / 7 mice / group , combined data of 2 independent experiments ) . P - values were calculated with 1 - way ANOVA tests .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4Immunofluorescent images of liver and kidney 24h after sham (with or without starvation) or CLP (n= 6-7/group, combined data of 2 independent experiments). White scale bar = 20µm. Cryosections were stained with Acti-stain (green), Hoechst (blue), and LipidTOX (red). Z-stacks were generated in 5-10 areas scattered across the entire tissue section.Quantification of lipid peroxidation by determination of (C) MDA and (D) 4-HNE concentrations in liver homogenates 24h post-surgery in sham and CLP mice as described in methods (n = 6-7/group, combined data of 2 independent experiments). P-values were calculated with 1-way ANOVA tests.Apoptosis in liver paraffin-fixated sections 24h after sepsis, measured by TUNEL staining, and presented as % PI positive cells/µm² tissue area. (n= 6/7 mice/group, combined data of 2 independent experiments). P-values were calculated with 1-way ANOVA tests.


Figure 5Mice were pre - treated with Pemafibrate ( 1mg / kg ) or vehicle ( 0 , 9 % NaCl ) for 1 week before being subjected to CLP . Survival was monitored during 9 days , after which no further deaths occurred . Survival curve was analyzed via a Log rank test . Combined data of 3 experiments , n = 20 / group . Mice were pre - treated with Pemafibrate ( 1mg / kg ) or vehicle ( 0 , 9 % NaCl ) for 1 week before being subjected to CLP . Liver samples were isolated 24h after CLP ( n = 5 - 7 / group , combined data of 2 independent experiments ) , mRNA was prepared and gene expression levels of Ppara was analyzed via qPCR . Gene expression values are shown as relative expression , normalized to housekeeping genes Hprt and Rpl . P - values were calculated via 2 - way ANOVA test . Mice were pre - treated with Pemafibrate ( 1mg / kg ) or vehicle ( 0 , 9 % NaCl ) for 1 week before being subjected to CLP . Plasma was isolated 24h after sepsis and FFA concentration and ( D ) glycerol concentration were determined as described in the method section . P - values were calculated with 2 - way ANOVA tests . n = 5 - 7 / group , combined data of 2 independent experiments . Arrows represent the % of decrease caused by pemafibrate treatment during sepsis . Mice were pre - treated with Pemafibrate ( 1mg / kg ) or vehicle ( 0 , 9 % NaCl ) for 1 week before being subjected to CLP . Immunofluorescent images of cryosections of liver 24h post - surgery that were stained with Acti - stain ( green ) , hoechst ( blue ) and LipidTOX ( red ) . Z - stacks were generated in 5 - 10 areas scattered across the entire tissue section . White scale bar = 20µm . Mice were pre - treated with Pemafibrate ( 1mg / kg ) or vehicle ( 0 , 9 % NaCl ) for 1 week before being subjected to CLP . Quantification of lipid peroxidation by determination of MDA concentration in liver homogenates 24h post - surgery in CLP mice ( n = 6 - 7 / group , combined data of 2 independent experiments ) , as described in methods . P - values were calculated with 2 - way ANOVA tests . Arrows represent the % of decrease caused by pemafibrate treatment during sepsis . Mice were pre - treated with Pemafibrate ( 1mg / kg ) or vehicle ( 0 , 9 % NaCl ) for 1 week before being subjected to CLP . Quantification of lipid peroxidation by determination of MDA concentration in kidney homogenates 24h post - surgery in CLP mice ( n = 6 - 7 / group , combined data of 2 independent experiments ) , as described in methods . P - values were calculated with 2 - way ANOVA tests . Arrows represent the % of decrease caused by pemafibrate treatment during sepsis . Mice were pre - treated with Pemafibrate ( 1mg / kg ) or vehicle ( 0 , 9 % NaCl ) for 1 week before being subjected to CLP . Oxygen consumption rates ( OCR ) of liver tissue explants 24h post CLP ( vehicle or pemafibrate treated ) after supplementation of palmitic acid ( PA ) , measured via Seahorse . Visualization of T42 ( min ) OCR . P - values were calculates using 2 - way ANOVA analysis . One experiment , n = 3 / group .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5Mice were pre-treated with Pemafibrate (1mg/kg) or vehicle (0,9% NaCl) for 1 week before being subjected to CLP. Survival was monitored during 9 days, after which no further deaths occurred. Survival curve was analyzed via a Log rank test. Combined data of 3 experiments, n = 20/group.Mice were pre-treated with Pemafibrate (1mg/kg) or vehicle (0,9% NaCl) for 1 week before being subjected to CLP. Liver samples were isolated 24h after CLP (n= 5-7/group, combined data of 2 independent experiments), mRNA was prepared and gene expression levels of Ppara was analyzed via qPCR. Gene expression values are shown as relative expression, normalized to housekeeping genes Hprt and Rpl. P-values were calculated via 2-way ANOVA test.Mice were pre-treated with Pemafibrate (1mg/kg) or vehicle (0,9% NaCl) for 1 week before being subjected to CLP. Plasma was isolated 24h after sepsis and FFA concentration and (D) glycerol concentration were determined as described in the method section. P-values were calculated with 2-way ANOVA tests. n = 5-7/group, combined data of 2 independent experiments. Arrows represent the % of decrease caused by pemafibrate treatment during sepsis.Mice were pre-treated with Pemafibrate (1mg/kg) or vehicle (0,9% NaCl) for 1 week before being subjected to CLP. Immunofluorescent images of cryosections of liver 24h post-surgery that were stained with Acti-stain (green), hoechst (blue) and LipidTOX (red). Z-stacks were generated in 5-10 areas scattered across the entire tissue section. White scale bar = 20µm.Mice were pre-treated with Pemafibrate (1mg/kg) or vehicle (0,9% NaCl) for 1 week before being subjected to CLP. Quantification of lipid peroxidation by determination of MDA concentration in liver homogenates 24h post-surgery in CLP mice (n= 6-7/group, combined data of 2 independent experiments ), as described in methods. P-values were calculated with 2-way ANOVA tests. Arrows represent the % of decrease caused by pemafibrate treatment during sepsis.Mice were pre-treated with Pemafibrate (1mg/kg) or vehicle (0,9% NaCl) for 1 week before being subjected to CLP. Quantification of lipid peroxidation by determination of MDA concentration in kidney homogenates 24h post-surgery in CLP mice (n= 6-7/group, combined data of 2 independent experiments ), as described in methods. P-values were calculated with 2-way ANOVA tests. Arrows represent the % of decrease caused by pemafibrate treatment during sepsis.Mice were pre-treated with Pemafibrate (1mg/kg) or vehicle (0,9% NaCl) for 1 week before being subjected to CLP. Oxygen consumption rates (OCR) of liver tissue explants 24h post CLP (vehicle or pemafibrate treated) after supplementation of palmitic acid (PA), measured via Seahorse. Visualization of T42(min) OCR. P-values were calculates using 2-way ANOVA analysis. One experiment, n = 3/group.