( G ) High resolution respirometry analyses performed in intact in an Oroboros instrument . ROUTINE : basal oxygen consumption rate ( in pmol O2 / sec ) per million in DMEM medium . LEAK is non - phosphorylating respiration in presence of ATP synthase inhibitor oligomycin . ETS : Maximal respiration rate in presence of uncoupler CCCP . AOX : oxygen consumption in presence of antimycin A , inhibiting cIII2 activity but not that of AOX . ROTENONE : Oxygen consumption in presence of cI inhibitor rotenone . In cases this was equal to background . Results are expressed as mean ± SD ( n = 2 biological replicates ) . 2 - way ANOVA with Tukey ' s multiple comparisons * * * * p < 0 . 0001 .

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

(G) High resolution respirometry analyses performed in intact cells in an Oroboros instrument. ROUTINE: cellular basal oxygen consumption rate (in pmol O2/sec) per million cells in DMEM medium. LEAK is the non-phosphorylating respiration in the presence of the ATP synthase inhibitor oligomycin. ETS: Maximal respiration rate in the presence of the uncoupler CCCP. AOX: oxygen consumption in the presence of antimycin A, inhibiting cIII2 activity but not that of AOX. ROTENONE: Oxygen consumption in the presence of the cI inhibitor rotenone. In all cases this was equal to the background. Results are expressed as mean ± SD (n=2 biological replicates). 2-way ANOVA with Tukey's multiple comparisons test ****p<0.0001.


( H ) Respirometry analyses , in WT EV controls AOXHA - expressing untreated or treated with 2 . 5 µM antimycin A for 7 days ( + AA ) . Results are expressed as mean ± SD ( n = 4 biological replicates ) . 2 - way ANOVA with Tukey ' s multiple comparisons * * * * p < 0 . 0001 .

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(H) Respirometry analyses , in WT EV controls and AOXHA- expressing cells untreated or treated with 2.5 µM antimycin A for 7 days (+AA). Results are expressed as mean ± SD (n=4 biological replicates). 2-way ANOVA with Tukey's multiple comparisons test ****p<0.0001.


( I ) 1D BNGE followed by cI - IGA ( right ) or Western blot immunodetection of cIII2 subunit CYC1 ( left ) in digitonin - solubilized samples from WT EV controls AOXHA - expressing untreated ( - ) or treated ( + ) with 2 . 5 µM antimycin A for 7 days .

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(I) 1D BNGE followed by cI-IGA (right) or Western blot and immunodetection of cIII2 subunit CYC1 (left) in digitonin-solubilized samples from WT EV controls and AOXHA- expressing cells untreated (-) or treated (+) with 2.5 µM antimycin A for 7 days.


( A ) Complexome profiles of two cV structural assembly modules . graphs were generated as in Figure 3 but in this case , peptide values for subunits belonging to each module were averaged to simplify . represented values are mean ± of two reciprocal labeling experiments .

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(A) Complexome profiles of the two cV structural and assembly modules. The graphs were generated as in Figure 3 but in this case, the peptide intensity values for the individual subunits belonging to each module were averaged to simplify the analysis. The represented values are the mean ± SEM of the two reciprocal labeling experiments.


( B ) Complexome profiles of three detected cII subunits . represented values are mean ± of two reciprocal labeling experiments .

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(B) Complexome profiles of the three detected cII subunits. The represented values are the mean ± SEM of the two reciprocal labeling experiments.


( C ) Complexome profiles of different cIV assembly modules of last subunit to be incorporated ( NDUFA4 ) . , peptide values for subunits belonging to each assembly module were averaged to simplify . represented values are mean ± of two reciprocal labeling experiments . bar graph represents of total under curve in profiles corresponding to each cIV module . plotted values are mean ± SD ( n = 2 ) . 2 - way ANOVA with Sidak ' s multiple comparisons * * p = 0 . 0088 ( Early ) ; * * p = 0 . 0063 ( MT - CO2 ) ; * * p = 0 . 0040 ( NDUFA4 ) ; * p = 0 . 0264 .

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(C) Complexome profiles of the different cIV assembly modules and of the last subunit to be incorporated (NDUFA4). , the peptide intensity values for the individual subunits belonging to each assembly module were averaged to simplify the analysis. The represented values are the mean ± SEM of the two reciprocal labeling experiments. The bar graph represents the quantification of the total area under the curve in the profiles corresponding to each cIV module. The plotted values are mean ± SD (n=2). 2-way ANOVA with Sidak's multiple comparisons test **p=0.0088 (Early); **p=0.0063 (MT-CO2); **p=0.0040 (NDUFA4); *p=0.0264.


( D ) 1D BNGE , Western blot immunodetection of two cIV subunits ( MT - CO1 COX7B ) in samples from WT ∆ 4 - CYB solubilized with DDM digitonin ( Dig ) .

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(D) 1D BNGE, Western blot and immunodetection of two cIV subunits (MT-CO1 and COX7B) in samples from WT and ∆4-CYB cells solubilized with DDM and digitonin (Dig).


D ) of zone 1 - 3 uninfected hepatocytes day 5 post plating ( + / - s . d . ) of 4 different donors ( blue dots ) . For each donor , > 500 were characterized for final percentages . Kruskal Wallis ( p = 0 . 0002 ) followed by Dunn ' s multiple comparisons where Z1 / hGK + + / hGS - versus Z2 / hGK + / hGS - has a p value of 0 . 0140 ( other comparisons of Z1 / hGK + + / hGS - vs Z3 / hGK - / hGS + Z2 / hGK + / hGS - vs Z3 / hGK - / hGS + were non - significant ) .

D) Percentage of zone 1-3 uninfected hepatocytes day 5 post plating (+/- s.d.) of 4 different donors (blue dots). For each donor, >500 cells were characterized for the final percentages. Kruskal Wallis test (p = 0.0002) followed by Dunn's multiple comparisons test where Z1/hGK++/hGS- versus Z2/hGK+/hGS- has a p value of 0.0140 (the other comparisons of Z1/hGK++/hGS- vs Z3/hGK-/hGS+ and Z2/hGK+/hGS- vs Z3/hGK-/hGS+ were non-significant).


C : Expression of typical liver stage markers : Circumsporozoite ( CSP ) top left ; Exported 2 ( EXP2 ) top right ; Glyceraldehyde 3 - phosphate dehydrogenase ( GAPDH ) bottom left ; Merozoite surface 1 ( MSP1 ) bottom right . Scale bar is 25 µm .

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C: Expression of typical liver stage protein markers: Circumsporozoite protein (CSP) top left; Exported Protein 2 (EXP2) top right; Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) bottom left; and Merozoite surface protein 1 (MSP1) bottom right. Scale bar is 25 µm.


D ) schizonts ( from total number of schizonts in a well + / - s . d . ) located in different hepatocyte sub - populations by NF54 , NF135 , NF175 on day 3 p . i . . Each dot represents a biological replicate . For each , at least 100 schizonts were characterized from 2x 96 wells for final . values from three biological replicates were used in a RM ANOVA followed by a Tukey ' s multiple comparison . of schizonts in Z1 / hGK + + / hGS - hepatocytes is not different between three strains . of schizonts in Z2 / hGK + / hGS - for NF54 is significantly different from NF135 ( p = 0 . 0015 ) NF175 ( p = 0 . 0015 ) . of schizonts in Z3 / hGK - / hGS + hepatocytes for NF54 is significantly different from NF135 ( p = 0 . 0002 ) NF175 ( p = 0 . 0 . 0002 ) . Data information : * p < 0 . 05 ; * * p < 0 . 005 ; * * * p < 0 . 001 ; * * * * p < 0 .

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

D) Percentage schizonts (from total number of schizonts in a well +/- s.d.) located in the different hepatocyte sub-populations by NF54, NF135, NF175 on day 3 p.i.. Each dot represents a biological replicate. For each parasite line, at least 100 schizonts were characterized from 2x 96 wells for the final percentage. The values from three biological replicates were used in a RM ANOVA test followed by a Tukey's multiple comparison test. Percentage of schizonts in Z1/hGK++/hGS- hepatocytes is not different between the three parasite strains. Percentage of schizonts in Z2/hGK+/hGS- for NF54 is significantly different from NF135 (p = 0.0015) and NF175 (p = 0.0015). Percentage of schizonts in Z3/hGK-/hGS+ hepatocytes for NF54 is significantly different from NF135 (p = 0.0002) and NF175 (p = 0.0.0002). Data information: * p <0.05; ** p < 0.005; *** p <0.001; **** p <0.0001


F ) of schizonts in Z1 / hGK + + / hGS - , Z2 / hGK + / hGS - Z3 / hGK - / hGS + hepatocytes on day 3 post invasion for different Pf strains . For each , at least 100 schizonts were measured from each biological replicate . median values + / - s . d . ( as sizes within population is not normally distributed ) from three biological replicates were used in a RM ANOVA followed by a Tukey ' s multiple comparison . Data information : * p < 0 . 05 ; * * p < 0 . 005 ; * * * p < 0 . 001 ; * * * * p < 0 .

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F) Size of schizonts in Z1/hGK++/hGS-, Z2/hGK+/hGS- and Z3/hGK-/hGS+ hepatocytes on day 3 post invasion for the different Pf strains. For each parasite line, at least 100 schizonts were measured from each biological replicate. The median values +/- s.d. (as the sizes within the population is not normally distributed) from three biological replicates were used in a RM ANOVA test followed by a Tukey's multiple comparison test. Data information: * p <0.05; ** p < 0.005; *** p <0.001; **** p <0.0001


hGS interaction with NF135 ( A ) , NF175 ( B ) schizonts on days 3 ( top ) , 5 ( middle ) 7 ( bottom ) p . i . . hGS ( green ) , PfHSP70 ( red ) , DAPI ( blue ) phalloidin ( grey ) . U panel is composed an uninfected zone 3 hepatocyte on day 3 p . i . . Scale bar is 25 µm .

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hGS interaction with NF135 (A), NF175 (B) schizonts on days 3 (top), 5 (middle) and 7 (bottom) p.i.. hGS (green), PfHSP70 (red), DAPI (blue) and phalloidin (grey). The U panel is composed an uninfected zone 3 hepatocyte on day 3 p.i.. Scale bar is 25 µm.


hGS interaction with NF54 ( C ) schizonts on days 3 ( top ) , 5 ( middle ) 7 ( bottom ) p . i . . hGS ( green ) , PfHSP70 ( red ) , DAPI ( blue ) phalloidin ( grey ) . U panel is composed an uninfected zone 3 hepatocyte on day 3 p . i . . Scale bar is 25 µm .

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hGS interaction with NF54 (C) schizonts on days 3 (top), 5 (middle) and 7 (bottom) p.i.. hGS (green), PfHSP70 (red), DAPI (blue) and phalloidin (grey). The U panel is composed an uninfected zone 3 hepatocyte on day 3 p.i.. Scale bar is 25 µm.


D ) Median sizes ( + / - s . d . ) of NF135 ( green ) , NF175 ( purple ) NF54 ( blue ) schizonts divided on presence ( solid ) or absence ( dashed ) of hGS pattern on high content microscopy from three different liver donors where each donor has at least 100 schizonts were measured for each of three biological replicates . A two - way RM ANOVA was performed on median followed by a Tukey ' s multiple comparisons . significant differences between hGS + hGS - population within a is showed with asterisks ( * p < 0 . 05 ; * * p < 0 . 005 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 ) . See Appendix Fig S1 for raw measurements .

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D) Median sizes (+/- s.d.) of NF135 (green), NF175 (purple) and NF54 (blue) schizonts divided based on the presence (solid line) or absence (dashed line) of hGS staining pattern based on high content fluorescence microscopy from three different liver donors where each donor has at least 100 schizonts were measured for each of three biological replicates. A two-way RM ANOVA was performed on the median followed by a Tukey's multiple comparisons test. The significant differences between hGS+ and hGS- population within a strain is showed with asterisks (* p <0.05; ** p < 0.005; *** p <0.001; **** p <0.0001). See Appendix Fig S1 for the raw measurements.


A ) total hGS signal within intracellular NF135 B ) NF175 schizonts on day 3 5 . Each graph represents a biological replicate where each dot represents an schizont .

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A) The total hGS signal within intracellular NF135 and B) NF175 schizonts on day 3 and 5. Each graph represents a biological replicate where each dot represents an individual schizont.


A ) Normalized + / - s . d . of intracellular schizont of NF54 ( blue ) NF135 ( green ) after treatment of GS inhibitors at different concentrations of AIP . Median of schizonts were normalized to that of H2O - treated NF135 ( which is set at 100 % ) . AIP was added one day post invasion kept on for 48 hours . Each dot represents a biological replicate where ≥ 100 schizonts were measured on day 5 p . i . . A two - way ANOVA was performed followed by a Tukey ' s multiple comparison with * * * * < 0 . 0001 to compare conditions within same . A two - way ANOVA was performed followed by a Sidak ' s multiple comparisons with * * * * < 0 . 0001 to compare conditions between strains ) .

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

A) Normalized percentage +/- s.d. of intracellular schizont size of NF54 (blue) and NF135 (green) after treatment of GS inhibitors at different concentrations of AIP. Median size of the schizonts were normalized to that of H2O-treated NF135 (which is set at 100%). AIP was added one day post invasion and kept on for 48 hours. Each dot represents a biological replicate where ≥100 schizonts were measured on day 5 p.i.. A two-way ANOVA was performed followed by a Tukey's multiple comparison test with the **** < 0.0001 to compare conditions within the same parasite strain. A two-way ANOVA was performed followed by a Sidak's multiple comparisons test with **** <0.0001 to compare conditions between parasite strains).


B ) difference between GS positive ( closed circle ) negative ( open circle ) NF135 schizonts after treatment of AIP . Each dot represents median of a biological replicate where ≥ 100 schizonts were measured . Three biological replicates were performed with median from each plotted + / - s . d . . A two - way ANOVA was performed followed by a Dunnett ' s multiple comparison with * * * = 0 . 0002 * * * * = 0 . 0001 .

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B) Size difference between the GS positive (closed circle) and negative (open circle) NF135 schizonts after treatment of AIP. Each dot represents the median of a biological replicate where ≥100 schizonts were measured. Three biological replicates were performed with the median from each plotted +/- s.d.. A two-way ANOVA was performed followed by a Dunnett's multiple comparison test with *** = 0.0002 and **** = 0.0001.


A . Tunicamycin ( Tn , 2 µg / ml ) increased Nox4 in H9c2 . Tubulin was used as a loading control . n = 4 - 6 / group . * , significant compared to baseline .

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A. Tunicamycin (Tn, 2 µg/ml) increased Nox4 protein levels in H9c2 cells. Tubulin was used as a loading control. n=4-6/group. *, significant compared to baseline.


B . Thapsigargin ( Tp , 1 µg / ml ) increased Nox4 in H9c2 . n = 4 - 6 / group . * , significant compared to baseline .

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B. Thapsigargin (Tp, 1 µg/ml) increased Nox4 protein levels in H9c2 cells. n=4-6/group. *, significant compared to baseline.


C . Effect of Nox4 on unfolded response . Nox4 was depleted in H9c2 by shRNA - mediated knockdown ( Ad . shNox4 ) or were treated with a control adenovirus ( Ad . Ctl ) . In with Nox4 knockdown , tunicamycin treatment resulted in lower increases in of ER chaperones Grp94 , Grp78 calreticulin than in conrol . of ATF4 were substantially lower in Nox4 - depleted than control but of ATF6 ( ATF6c ) were similar . Histone was used as a loading control . relative mRNA of Xbp1 - s ( a readout of IRE1 signaling ) were unaltered after Nox4 knockdown . Mean data are shown in Appendix Fig S1E . Similar results were obtained with an independent siRNA approach ( Appendix Fig S2A ) . D . Effect of adenoviral - mediated overexpression of Nox4 ( Ad . Nox4 ) or a control β - galactosidase ( Ad . β - Gal ) on tunicamycin responses of H9c2 . Nox4 enhanced increase in ER chaperones ATF4 but did not affect tunicamycin - induced changes in ATF6c caused minor reduction in Xbp1s mRNA . Mean data are shown in Appendix Fig S2B .

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C. Effect of Nox4 on the unfolded protein response. Nox4 was depleted in H9c2 cells by shRNA-mediated knockdown (Ad.shNox4) or cells were treated with a control adenovirus (Ad.Ctl). In cells with Nox4 knockdown, tunicamycin treatment resulted in lower increases in protein levels of the ER chaperones Grp94, Grp78 and calreticulin than in conrol cells. Nuclear protein levels of ATF4 were substantially lower in Nox4-depleted cells than control cells but the levels of cleaved ATF6 (ATF6c) were similar. Histone was used as a loading control. The relative mRNA levels of Xbp1-s (a readout of IRE1 signaling) were unaltered after Nox4 knockdown. Mean data are shown in Appendix Fig S1E. Similar results were obtained with an independent siRNA approach (Appendix Fig S2A).D. Effect of adenoviral-mediated overexpression of Nox4 (Ad.Nox4) or a control β-galactosidase protein (Ad.β-Gal) on tunicamycin responses of H9c2 cells. Nox4 enhanced the increase in cellular ER chaperones and nuclear ATF4 levels but did not affect tunicamycin-induced changes in nuclear ATF6c levels and caused minor reduction in Xbp1s mRNA levels. Mean data are shown in Appendix Fig S2B.


E , F . Effect of Nox4 knockdown or overexpression , respectively , on tunicamycin - induced changes in mRNA of ATF4 target genes . n = 4 / group . Psat1 , phosphoserine aminotransferase ; Phgdh , 3 - phosphoglycerate dehydrogenase ; Asns , asparagine synthetase ; Slc6a9 , glycine transporter 1 .

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E,F. Effect of Nox4 knockdown or overexpression, respectively, on the tunicamycin-induced changes in mRNA levels of ATF4 target genes. n=4/group. Psat1, phosphoserine aminotransferase; Phgdh, 3-phosphoglycerate dehydrogenase; Asns, asparagine synthetase; Slc6a9, glycine transporter 1.


G . Effect of ATF4 silencing with two different siRNAs on Nox4 in tunicamycin - treated H9c2 . Scrambled siRNAs were used as a control ( Ctl ) . Representative immunoblots shown to top ( captions at bottom of bar graphs refer also to immunoblots ) ; tubulin was used as a loading control . n = 4 / group . * , significant compared to baseline ; # , significant comparing siATF4 versus corresponding siCtl .

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G. Effect of ATF4 silencing with two different siRNAs on Nox4 protein levels in tunicamycin-treated H9c2 cells. Scrambled siRNAs were used as a control (Ctl). Representative immunoblots shown to the top (captions at bottom of bar graphs refer also to the immunoblots); tubulin was used as a loading control. n=4/group. *, significant compared to baseline; #, significant comparing siATF4 versus corresponding siCtl.


H . Effect of ATF4 overexpression on Nox4 mRNA . n = 3 / group .

H. Effect of ATF4 overexpression on Nox4 mRNA and protein levels. n=3/group.


A . knockdown of endogenous Nox4 resulted in a substantial inhibition of tunicamycin - induced eIF2a phosphorylation in H9c2 , with no change in phospho - Thr980 - PERK ( PERK - P ) . GADD34 were significantly decreased after Nox4 knockdown while there was no change in PP1 . B . Overexpression of Nox4 in H9c2 caused prolongation of tunicamycin - induced eIF2a phosphorylation , with minimal change in phospho - PERK . C , D . Mean of phosphorylated eIF2a relative to total eIF2a in tunicamycin - treated after Nox4 knockdown or overexpression , respectively . n = 3 / group . * , significant compared to baseline ; # , significant comparing Nox4 knockdown ( Ad . shNox4 ) or overexpression ( Ad . Nox4 ) versus corresponding controls ( Ad . Ctl or Ad . β - Gal , respectively ) .

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A. The knockdown of endogenous Nox4 resulted in a substantial inhibition of tunicamycin-induced eIF2a phosphorylation in H9c2 cells, with no change in phospho-Thr980-PERK (PERK-P) levels. GADD34 levels were significantly decreased after Nox4 knockdown while there was no change in PP1 protein levels.B. Overexpression of Nox4 in H9c2 cells caused prolongation of tunicamycin-induced eIF2a phosphorylation, with minimal change in phospho-PERK levels.C,D. Mean levels of phosphorylated eIF2a relative to total eIF2a protein in tunicamycin-treated cells after Nox4 knockdown or overexpression, respectively. n=3/group. *, significant compared to baseline; #, significant comparing Nox4 knockdown (Ad.shNox4) or overexpression (Ad.Nox4) versus corresponding controls (Ad.Ctl or Ad.β-Gal, respectively).


E , F . Effect of Nox4 knockdown or overexpression , respectively , on okadaic acid - resistant Ser / Thr phosphatase activity in fractions of tunicamycin - treated H9c2 . n = 4 / group . * p < 0 . 05 , * * p < 0 . 01 cf . baseline ; # , p < 0 . 05 , # # , p < 0 . 01 comparing Nox4 knockdown ( Ad . shNox4 ) or overexpression ( Ad . Nox4 ) versus corresponding controls ( Ad . Ctl or Ad . β - Gal , respectively ) .

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E,F. Effect of Nox4 knockdown or overexpression, respectively, on okadaic acid-resistant Ser/Thr phosphatase activity in membrane fractions of tunicamycin-treated H9c2 cells. n=4/group. *p<0.05, **p<0.01 cf. baseline; #, p<0.05, ##, p<0.01 comparing Nox4 knockdown (Ad.shNox4) or overexpression (Ad.Nox4) versus corresponding controls (Ad.Ctl or Ad.β-Gal, respectively).


G , H . Nox4 knockdown or overexpression , respectively , had no effect on phosphorylation of glycogen synthase at Ser641 ( GS - P ) or Histone H3 at Ser57 ( H3 - P ) in H9c2 .

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G,H. Nox4 knockdown or overexpression, respectively, had no effect on the phosphorylation of glycogen synthase at Ser641 (GS-P) or Histone H3 at Ser57 (H3-P) in H9c2 cells.


I . Nox4 - / - MEF ( KO ) showed blunted tunicamycin - induced increases in of phospho - eIF2a , ATF4 ER chaperones as compared to wild - type ( WT ) MEFs , a response that was rescued by re - introduction of Nox4 ( KO + Nox4 ) . latter had no effect on GS - P or H3 - P .

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I. Nox4-/- MEF cells (KO) showed blunted tunicamycin-induced increases in levels of phospho-eIF2a, ATF4 and ER chaperones as compared to wild-type (WT) MEFs, a response that was rescued by re-introduction of Nox4 (KO+Nox4). The latter had no effect on GS-P or H3-P levels.


A . Sub - localization of Nox4 . Tunicamycin ( Tn 2 µg / ml , 6h ) increased Nox4 in H9c2 as assessed by spinning disk confocal microscopy ( scale bar , 10 µm ) . 3D SIM ( scale bars , 2 µm ) showed localisation of Nox4 ( green ) to ER , which was labeled with an anti - KDEL antibody ( red ) . At higher magnification ( right ) , yellow dots denote co - localization of Nox4 KDEL signals . nuclei were stained with DAPI ( blue ) . 1 Z slice from 3D stack is shown .

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A. Sub-cellular localization of Nox4. Tunicamycin (Tn 2 µg/ml, 6h) increased Nox4 levels in H9c2 cells as assessed by spinning disk confocal microscopy (scale bar, 10 µm).3D SIM images (scale bars, 2 µm) showed localisation of Nox4 (green) to the ER, which was labeled with an anti-KDEL antibody (red). At higher magnification (right), yellow dots denote co-localization of Nox4 and KDEL signals. Cell nuclei were stained with DAPI (blue). 1 Z slice from 3D stack is shown.


B . Progressive enrichment of GADD34 , PP1 , eIF2α Nox4 in fractions of tunicamycin - treated H9c2 .

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B. Progressive enrichment of GADD34, PP1, eIF2α and Nox4 in membrane fractions of tunicamycin-treated H9c2 cells.


C . After sucrose gradient fractionation of lysates of tunicamycin - treated H9c2 , GADD34 , PP1 Nox4 co - eluted in fractions 12 13 ( F12 , F13 ) .

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C. After sucrose gradient fractionation of lysates of tunicamycin-treated H9c2 cells, GADD34, PP1 and Nox4 co-eluted in fractions 12 and 13 (F12, F13).


D . Immunoprecipitation ( IP ) of pooled fractions 12 / 13 with an anti - GADD34 antibody revealed presence of both PP1 Nox4 .

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D. Immunoprecipitation (IP) of pooled fractions 12/13 with an anti-GADD34 antibody revealed the presence of both PP1 and Nox4.


E . association of Nox4 with GADD34 was validated in HEK293 co - transfected with Nox4 either Flag - tagged or non - tagged GADD34 , followed by IP with an anti - Flag antibody .

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E. The association of Nox4 with GADD34 was validated in HEK293 cells co-transfected with Nox4 and either Flag-tagged or non-tagged GADD34, followed by IP with an anti-Flag antibody.


F . Co - transfection of HEK293 with GADD34 - Flag different myc - tagged Nox4 constructs , followed by IP with an anti - myc antibody . GADD34 binds to full - Nox4 ( FL ) Nox4 transmembrane domain ( TD ) but not C - terminal domain ( CD ) .

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F. Co-transfection of HEK293 cells with GADD34-Flag and different myc-tagged Nox4 constructs, followed by IP with an anti-myc antibody. GADD34 binds to full-length Nox4 (FL) and the Nox4 transmembrane domain (TD) but not the C-terminal domain (CD).


G . Representative pseudocolor of simultaneous ER ROS measurement with HyPer ER HyPerRed Cyto , respectively , in tunicamycin - treated MEF . Redox - insensitive mutant probes were used as negative controls to exclude pH changes . Extracellular H2O2 ( 200 nM ) was added as a positive control . pseudocolor scale is shown along left vertical edge of each image . KO = Nox4 - / - . Scale bars , 2 µm .

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G. Representative pseudocolor images of simultaneous ER and cytosolic ROS measurement with HyPer ER and HyPerRed Cyto, respectively, in tunicamycin-treated MEF cells. Redox-insensitive mutant probes were used as negative controls and to exclude pH changes. Extracellular H2O2 (200 nM) was added as a positive control. The pseudocolor scale is shown along the left vertical edge of each image. KO = Nox4-/-. Scale bars, 2 µm.


H . Transfection of HEK293 with PP1 GADD34 increased PP1 activity ( bar graph ) decreased phospho - eIF2 ( immunoblots ) . Co - transfection of full - Nox4 reduced PP1 activity increased phospho - eIF2 ( captions at bottom refer both to bar graphs immunoblots ) . These effects were abrogated when either Nox4 P437H or Nox4 transmembrane domain ( TD ) were transfected . Nox4 did not affect phosphorylation of glycogen synthase ( GS - P ) or histone H3 ( H3 - P ) . experiments were n = 3 / group . Values below immunoblots are mean ± for phospho - eIF2 / total - eIF2 . * , p < 0 . 05 comparing third fourth lanes ; of significance for comparisons of PP1 activities are shown above bar columns . See also Fig EV1 Appendix Fig S4 .

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H. Transfection of HEK293 cells with PP1 and GADD34 increased PP1 activity (bar graph) and decreased phospho-eIF2 levels (immunoblots). Co-transfection of full-length Nox4 reduced PP1 activity and increased phospho-eIF2 levels (captions at bottom refer both to the bar graphs and immunoblots). These effects were abrogated when either Nox4 P437H or the Nox4 transmembrane domain (TD) were transfected. Nox4 did not affect phosphorylation of glycogen synthase (GS-P) or histone H3 (H3-P). All experiments were n=3/group. Values below the immunoblots are mean ± SEM levels for phospho-eIF2/total-eIF2. *, p<0.05 comparing third and fourth lanes; Levels of significance for comparisons of PP1 activities are shown above the bar columns. See also Fig EV1 and Appendix Fig S4.


A . Recombinant PP1 was inhibited by H2O2 ( 0 . 2 mM ) activity was not restored by glutathione ( GSH ) , cysteine ( Cys ) or dithiothreitol ( DTT ) . A Cys127Ser / Cys273Ser PP1 mutant was inhibited by H2O2 similarly to wild - type PP1 . Values above bars denote level of significance for inhibitory effect of H2O2 .

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A. Recombinant PP1 was inhibited by H2O2 (0.2 mM) and activity was not restored by glutathione (GSH), cysteine (Cys) or dithiothreitol (DTT). A Cys127Ser/Cys273Ser PP1 mutant was inhibited by H2O2 similarly to wild-type PP1. Values above bars denote level of significance for the inhibitory effect of H2O2.


B . Ascorbate ( Asc ) dose - dependently restored PP1 activity . # , significant effect of Asc compared to H2O2 alone .

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B. Ascorbate (Asc) dose-dependently restored PP1 activity. #, significant effect of Asc compared to H2O2 alone.


C . EPR spectra of PP1 incubated with ascorbate ( 1 mM ) alone ( a ) or PP1 exposed to H2O2 followed by catalase treatment , then incubation with ascorbate ( b ) . Panel b shows a typical spectrum for ascorbyl radical ( hyperfine splitting constant , aH = 1 . 8G ) , similar to positive control obtained by exposing ascorbate to H2O2 ( c ) . Panel d shows that no ascorbyl radical is detected if H2O2 is degraded by catalase in absence of PP1 , prior to ascorbate addition .

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C. EPR spectra of PP1 incubated with ascorbate (1 mM) alone (a) or PP1 exposed to H2O2 followed by catalase treatment, then incubation with ascorbate (b). Panel b shows a typical spectrum for the ascorbyl radical (hyperfine splitting constant, aH = 1.8G), similar to the positive control obtained by exposing ascorbate to H2O2 (c). Panel d shows that no ascorbyl radical is detected if H2O2 is degraded by catalase in the absence of PP1, prior to ascorbate addition.


D . Cartoon representation of active site of H2O2 - treated PP1 as in Fig EV2D . 2mFo - DFc electron density map at 2 . 2 Å resolution is shown in yellow at 1 . 1 level . H2O2 - treatment causes an overall shrinkage of PP1 coordination sphere by 0 . 12 Å compared to ascorbate - treated crystals consistent with oxidation of dinuclear center . This increases energy barrier for catalytic steps involving - OH - attack on phosphorous center of bridging phosphate rupture of P - O scissile bond with assistance of H125 ( black arrows ) . Reported coordination distances in Å are averaged over two PP1 molecules in crystallographic asymmetric unit . See also Appendix Table S1 .

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D. Cartoon representation of the active site of H2O2-treated PP1 as in Fig EV2D. 2mFo-DFc electron density map at the 2.2 Å resolution is shown in yellow at the 1.1 level. H2O2-treatment causes an overall shrinkage of the PP1 coordination sphere by 0.12 Å compared to ascorbate-treated crystals consistent with the oxidation of the dinuclear center. This increases the energy barrier for the catalytic steps involving -OH- attack on the phosphorous center of the bridging phosphate and rupture of the P-O scissile bond with the assistance of H125 (black arrows). Reported coordination distances in Å are averaged over the two PP1 molecules in the crystallographic asymmetric unit. See also Appendix Table S1.


E . Effect of Nox4 on GADD34 / PP1 - mediated eIF2 dephosphorylation in transfected HEK293 . Nox4 increased eIF2 phosphorylation in transfected with GADD34 WT PP1 , resulted in even higher phospho - eIF2α in transfected with N124D or D64N PP1 variants .

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E. Effect of Nox4 on GADD34/PP1-mediated eIF2 dephosphorylation in transfected HEK293 cells. Nox4 increased eIF2 phosphorylation in cells transfected with GADD34 and WT PP1, and resulted in even higher phospho-eIF2α levels in cells transfected with N124D or D64N PP1 variants.


F . Effect of ascorbate ( Asc , 0 . 5 mM ) on phosphatase inhibition in tunicamycin - treated H9c2 with overexpression or knockdown of Nox4 ( Ad . Nox4 Ad . shNox4 , respectively ) . In control , ascorbate enhanced tunicamycin - stimulated increases in phosphatase activity . Phosphatase activity was lower in Nox4 - overexpressing than control but was normalized by ascorbate to same level as in control . In Nox4 knockdown , tunicamycin - induced increases in phosphatase activity were enhanced ascorbate had minimal additional effect .

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F. Effect of ascorbate (Asc, 0.5 mM) on phosphatase inhibition in tunicamycin-treated H9c2 cells with overexpression or knockdown of Nox4 (Ad.Nox4 and Ad.shNox4, respectively). In control cells, ascorbate enhanced tunicamycin-stimulated increases in phosphatase activity. Phosphatase activity was lower in Nox4-overexpressing than control cells but was normalized by ascorbate to the same level as in control cells. In Nox4 knockdown cells, tunicamycin-induced increases in phosphatase activity were enhanced and ascorbate had minimal additional effect.


A . H9c2 treated with tunicamycin ( 2 µg / ml , 12h ) showed significantly lower survival when endogenous Nox4 was silenced ( siNox4 ) as compared to treated with a scrambled siRNA ( siCtl ) . survival was restored by treatment with either guanabenz ( Gbz , 5 µM ) or salubrinal ( Sal , 50 µM ) but was unaffected by clonidine ( Cld , 5 µM ) . n = 3 / group .

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A. H9c2 cells treated with tunicamycin (2 µg/ml, 12h) showed significantly lower survival when endogenous Nox4 was silenced (siNox4) as compared to cells treated with a scrambled siRNA (siCtl). Cell survival was restored by treatment with either guanabenz (Gbz, 5 µM) or salubrinal (Sal, 50 µM) but was unaffected by clonidine (Cld, 5 µM). n=3/group.


B . Nox4 - depleted had lower of phospho - eIF2α ATF4 than control but these were restored in presence of guanabenz ( Gbz ) .

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B. Nox4-depleted cells had lower levels of phospho-eIF2α and ATF4 than control cells but these were restored in the presence of guanabenz (Gbz).


D . Hearts from Nox4 knockout ( KO ) mice WT controls were subjected to global ischemia followed by aerobic reperfusion ( I / R ) . Infarct assessed by triphenyltetrazolium chloride ( TTC ) was greater in Nox4 KO hearts compared to WT was significantly reduced by guanabenz ( Gbz ) . In representative heart sections shown at top , denotes infarct red viable . Scale bars , 1 mm . Numbers of hearts are indicated within bars .

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D. Hearts from Nox4 knockout (KO) mice and WT controls were subjected to global ischemia followed by aerobic reperfusion (I/R). Infarct size assessed by triphenyltetrazolium chloride (TTC) staining was greater in Nox4 KO hearts compared to WT and was significantly reduced by guanabenz (Gbz). In the representative heart sections shown at the top, white denotes infarct area and red the viable area. Scale bars, 1 mm. Numbers of hearts are indicated within the bars.


E . Immunoblotting of heart homogenates after I / R showed lower of phospho - eIF2a , ATF4 ER chaperones , higher of caspase - 12 , in Nox4 KO compared to WT . Tubulin was used as a loading control . Treatment with guanabenz ( Gbz ) reversed these changes ( blots shown to right ) .

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E. Immunoblotting of heart homogenates after I/R showed lower levels of phospho-eIF2a, ATF4 and ER chaperones, and higher levels of cleaved caspase-12, in Nox4 KO compared to WT. Tubulin was used as a loading control. Treatment with guanabenz (Gbz) reversed these changes (blots shown to the right).


A . Plasma urea were elevated to a greater extent in tunicamycin - treated Nox4 KO mice than WT . Co - treatment with guanabenz ( Gbz ) reduced urea in both groups . Numbers of are indicated within bars .

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A. Plasma urea levels were elevated to a greater extent in tunicamycin-treated Nox4 KO mice than WT. Co-treatment with guanabenz (Gbz) reduced urea levels in both groups. Numbers of animals are indicated within bars.


B . 48 hours after systemic tunicamycin treatment , kidneys of Nox4 KO mice showed a marked surface pallor ( bottom right ) .

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B. 48 hours after systemic tunicamycin treatment, kidneys of Nox4 KO mice showed a marked surface pallor (bottom right).


C . TUNEL revealed a significantly higher number of apoptotic in tunicamycin - treated Nox4 KO mice . n = 4 / group .

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C. TUNEL staining revealed a significantly higher number of apoptotic cells in tunicamycin-treated Nox4 KO mice. n=4/group.


D . Immunoblotting of kidney homogenates showed significantly elevated caspase - 12 PARP in tunicamycin - treated Nox4 KO mice compared to WT .

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D. Immunoblotting of kidney homogenates showed significantly elevated cleaved caspase-12 and cleaved PARP levels in tunicamycin-treated Nox4 KO mice compared to WT.


E . Survival curves showed that a very high proportion of Nox4 KO mice died after AKI . Guanabenz ( Gbz ) treatment dramatically improved survival in tunicamycin - treated KO mice . Number of as indicated . of significance by Kaplan Meier are reported to right .

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E. Survival curves showed that a very high proportion of Nox4 KO mice died after AKI. Guanabenz (Gbz) treatment dramatically improved survival in tunicamycin-treated KO mice. Number of animals as indicated. Levels of significance by Kaplan Meier analysis are reported to the right.


H - I . Fibulin 7 immunostaining in 2 - month - vs . 2 - year - old tail section ( H ) its per basal epidermal / basement ( normalized to 2 - month - old ) ( I ) . Dotted box areas were enlarged in lower panels . arrows indicate fibulin 7 basement .

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H-I. Fibulin 7 immunostaining in 2-month- vs. 2-year-old tail section (H) and its intensity quantification per basal epidermal stem cell/basement membrane (normalized to 2-month-old) (I). Dotted box areas were enlarged in the lower panels. White arrows indicate fibulin 7 basement membrane staining.


D - E . Ki67 immunostaining in 1 - week - chase mice ( D ) its quantitation ( E ) . arrows show Ki67 tdTomato double positive , which were counted summarized in ( E ) . Scale bar : 20 μm . N = 5 WT / het 3 KO mice .

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D-E. Ki67 immunostaining in 1-week-chase mice (D) and its quantitation (E). White arrows show Ki67 and tdTomato double positive cells, which were counted and summarized in (E). Scale bar: 20 μm. N=5 WT/het and 3 KO mice.


F . Representative pictures from tail wounds of 1 - year - old Fbln7 WT KO mice . G . Measurements of wound over time in Fbln7 WT ( N = 5 ) KO ( N = 7 ) mice .

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F. Representative pictures from tail wounds of 1-year-old Fbln7 WT and KO mice. G. Measurements of wound area over time in Fbln7 WT (N=5) and KO (N=7) mice.


H - J . Hematoxylin eosin from tail sections of wounded Fbln7 WT vs . KO mice ( H ) quantitation of re - epithelialization ( I ) thickness at healing front ( J ) . Scale bar : 200 μm . N = 3 WT 5 KO mice .

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H-J. Hematoxylin and eosin staining from tail sections of wounded Fbln7 WT vs. KO mice (H) and quantitation of the re-epithelialization length (I) and thickness at the healing front (J). Scale bar: 200 μm. N=3 WT and 5 KO mice.


F - G . K31 K14 immunostaining in tail sections of 2 - month - vs . 2 - year - old C57BL / 6J WT mice ( F ) ( G ) . Dotted box areas are enlarged in lower panels . Scale bar : 50 μm . of K14 in suprabasal was normalized to basal per . N = 4 ( 2 - 3 - month - old ) N = 5 ( 2 - year - old ) C57BL / 6J mice ( G ) .

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F-G. K31 and K14 immunostaining in tail sections of 2-month- vs. 2-year-old C57BL/6J WT mice (F) and the quantification (G). Dotted box areas are enlarged in the lower panels. Scale bar: 50 μm. Quantification of K14 intensity in the suprabasal area was normalized to basal area per cell. N=4 (2-3-month-old) and N=5 (2-year-old) C57BL/6J mice (G).


H - J . K31 K14 immunostaining in tail sections of 1 - year - old Fbln7 WT vs . KO compared with 2 - month - old Fbln7 KO mice ( H ) quantitation ( I , J ) . Scale bar : 50 μm . N = 5 for both WT KO of 2 - 3 - month - old mice ( I ) . N = 5 N = 7 for 1 - year - old WT vs . KO , respectively ( J ) . Data information ( F - J )

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H-J. K31 and K14 immunostaining in tail sections of 1-year-old Fbln7 WT vs. KO compared with 2-month-old Fbln7 KO mice (H) and the quantitation (I, J). Scale bar: 50 μm. N=5 for both WT and KO of 2-3-month-old mice (I). N=5 and N=7 for 1-year-old WT vs. KO, respectively (J). Data information (F-J)


A . Collagen IV immunostaining in tail sections of 1 - year - old Fbln7 WT vs . KO . Dotted box regions are enlarged .

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A. Collagen IV immunostaining in tail sections of 1-year-old Fbln7 WT vs. KO. Dotted box regions are enlarged.


B . of Collagen IV basement per in Fbln7 KO mice normalized to WT mice . N = 4 WT N = 6 KO mice . Graphs show mean ± S . D . Mann - Whitney . * * ; P < 0 . 01 , ns ; not significant .

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B. Quantification of Collagen IV basement membrane intensity per cell in Fbln7 KO mice normalized to WT mice. N=4 WT and N=6 KO mice. Graphs show mean ± S.D. Mann-Whitney test. **; P<0.01, ns; not significant.


A Representative 3D tile scan of young adrenal , ovary , pituitary , testis , pancreas thyroid stained with antibodies indicated in panel . Scale bars are 200 µm . dashed lines represent outlines of each organ .

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A 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 at single - resolution level of adrenal , ovary , pituitary , testis , pancreas thyroid . Insets ( * ) indicate higher magnification of regions with single - resolution surface rendering for markers . Scale bars for tile scan 3D are 200 µm , for single - resolution insets are 5 µm . dashed lines represent outlines of each organ .

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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 - interactome with estimates of CD68 + macrophages to blood vessels ( Endoglin ) in cortex , medulla transition zone ( TZ ) of an aged adrenal . colour code indicates which is very close in violet very far in red . scale bar is 50 µm . dashed lines represent outline of transition zone .

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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.


A Young aged adrenal immunolabelled with HSPG2 , NG2 Podocalyxin . Higher magnification insets show cortex ( Co ) medulla ( Me ) . Combined box whiskers , scatter plot with of vessel density in young aged adrenal . Data information : dashed lines in each panel represent outlines of each organ . ( n = 5 ) , P - values two - unpaired t - . ns : not significant ; * * : P < 0 . 01 ; * * * : P < 0 . 001 . Nuclei : TO - PRO - 3 . For Combo plots , boxes represent mean ± s . d . , in box is median , lower upper lines show minimum maximum of values . on right side of these combined plots represents sample distribution . Co : Cortex ; Me : Medulla Scale bars for tile scan 3D 200 µm , for insets 50 µm .

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A 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 aged ovary immunolabelled with Sca - 1 , PDGFRβ Emcn . Higher magnification insets show cortex medulla . Combo plot with of vessel density in young aged ovary . Data information : dashed lines in each panel represent outlines of each organ . ( n = 5 ) , P - values two - unpaired t - . ns : not significant ; * * : P < 0 . 01 ; * * * : P < 0 . 001 . Nuclei : TO - PRO - 3 . For Combo plots , boxes represent mean ± s . d . , in box is median , lower upper lines show minimum maximum of values . on right side of these combined plots represents sample distribution . Co : Cortex ; Me : Medulla Scale bars for tile scan 3D 200 µm , for insets 50 µm .

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

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 - - old ) aged pituitary immunolabelled with ESM - 1 , Collagen IV Emcn . Insets with higher magnifications of pars distalis pars nervosa regions . Combo plot shows of vessel density . Data information : dashed lines in each panel represent outlines of each organ . ( n = 5 ) , P - values two - unpaired t - . ns : not significant ; * * : P < 0 . 01 ; * * * : P < 0 . 001 . Nuclei : TO - PRO - 3 . For Combo plots , boxes represent mean ± s . d . , in box is median , lower upper lines show minimum maximum of values . on right side of these combined plots represents sample distribution . Pd : Pars Distalis ; Pn : Pars Nervosa . Scale bars for tile scan 3D 200 µm , for insets 50 µm .

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

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 aged testis immunolabelled with Tie - 2 HSPG2 . Higher magnifications of cortex medulla . Combo plot shows of vessel density . Data information : dashed lines in each panel represent outlines of each organ . ( n = 5 ) , P - values two - unpaired t - . ns : not significant ; * * : P < 0 . 01 ; * * * : P < 0 . 001 . Nuclei : TO - PRO - 3 . For Combo plots , boxes represent mean ± s . d . , in box is median , lower upper lines show minimum maximum of values . on right side of these combined plots represents sample distribution . Scale bars for tile scan 3D 200 µm , for insets 50 µm .

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

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 aged pancreas immunolabelled with SM22α Endoglin . Insets representative of higher magnifications of regions in young aged pancreas . Combo plot shows of vessel density . Data information : dashed lines in each panel represent outlines of each organ . ( n = 5 ) , P - values two - unpaired t - . ns : not significant ; * * : P < 0 . 01 ; * * * : P < 0 . 001 . Nuclei : TO - PRO - 3 . For Combo plots , boxes represent mean ± s . d . , in box is median , lower upper lines show minimum maximum of values . on right side of these combined plots represents sample distribution . Scale bars for tile scan 3D 200 µm , for insets 50 µm .

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

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 aged thyroid immunolabelled with CXCR4 , Laminin Emcn . Combo plot shows of vessel density in young aged thyroid . Insets : periphery centre of thyroid . dashed lines in each panel represent outlines of each organ . ( n = 5 ) , P - values two - unpaired t - . ns : not significant ; * * : P < 0 . 01 ; * * * : P < 0 . 001 . Nuclei : TO - PRO - 3 . For Combo plots , boxes represent mean ± s . d . , in box is median , lower upper lines show minimum maximum of values . on right side of these combined plots represents sample distribution . ; Pe : Periphery Ct : Center Scale bars for tile scan 3D 200 µm , for insets 50 µm .

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

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.


A Whole - endocrine imaging of young aged adrenal immunolabelled with α - SMA Emcn acquired on a - sheet microscope . Asterisks indicate higher magnifications of regions in adrenal from young aged mice . Combined box whiskers , scatter plots with quantifications of vessel density numbers of arteries .

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 - sheet imaging - 3D of young aged ovaries stained with α - SMA Emcn . Asterisks represent higher magnifications of regions in ovaries from young aged mice . Combo plots show quantifications of vessel density numbers of arteries in young aged ovary .

O O O O O O O O O O O O O O O O O O O O O O O O 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 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 of whole cleared young aged testes stained with α - SMA Emcn . Asterisks indicate higher magnifications of indicated regions . Combo plots show quantifications of vessel density numbers of arteries .

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

C 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 - sheet imaging of whole cleared young aged pancreases immunolabelled with α - SMA Emcn . Asterisks indicate higher magnifications of regions . Combo plots with quantifications of vessel density numbers of arteries in young aged 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

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 acquired on a sheet microscope of whole cleared young aged thyroid stained with α - SMA Emcn . Asterisks indicate higher magnifications of regions . Combo plots show quantifications of vessel density numbers of arteries in young aged thyroid .

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

E 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 of 3D spatial proteomic data showing qualitative of differences in type markers matrix expressions between young aged murine endocrine . colour code on immunolabelling intensities indicates very high expression in red , high expression in yellow , medium in green , low in green 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

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.


A 3D ( left ) with α - SMA CD31 immunostaining in young aged human adrenal . Middle panel surface rendered for α - SMA , Vimentin CD31 . Arrowheads indicate arteries . Combo plots with quantifications of number of arteries vessel density . Data information : ( n = 4 ) , P - values , two - unpaired t - for above panels . ns : not significant ; * : P < 0 . 05 ; * * : P < 0 . 01 ; * * * * : P < 0 . 0001 . Human samples from donors at ages below 20 years over 70 years were chosen for young aged group sets , respectively . Nuclei : DAPI .

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

A 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 of young aged human ovary immunolabelled with α - SMA , Vimentin CD31 . in middle panel with a surface rendered for channels . Arrowheads indicate arteries . Combo plots with quantifications of number of arteries vessel density . Data information : ( n = 4 ) , P - values , two - unpaired t - for above panels . ns : not significant ; * : P < 0 . 05 ; * * : P < 0 . 01 ; * * * * : P < 0 . 0001 . Human samples from donors at ages below 20 years over 70 years were chosen for young aged group sets , respectively . Nuclei : DAPI .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 ( left ) of young aged human testis with SM22α Endoglin immunostaining . Middle panel surface rendered for channels . Arrowheads indicate arteries . Combo plots show quantifications of number of arteries vessel density . Data information : ( n = 4 ) , P - values , two - unpaired t - for above panels . ns : not significant ; * : P < 0 . 05 ; * * : P < 0 . 01 ; * * * * : P < 0 . 0001 . Human samples from donors at ages below 20 years over 70 years were chosen for young aged group sets , respectively . Nuclei : DAPI .

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

C Representative 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 from young aged human pancreas with α - SMA , Vimentin Endoglin . 3D ( middle ) with a surface rendering for channels . Arrowheads indicate arteries . Combined box whiskers , scatter plots with quantifications of number of arteries vessel density . Data information : ( n = 4 ) , P - values , two - unpaired t - for above panels . ns : not significant ; * : P < 0 . 05 ; * * : P < 0 . 01 ; * * * * : P < 0 . 0001 . Human samples from donors at ages below 20 years over 70 years were chosen for young aged group sets , respectively . Nuclei : DAPI .

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

D 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 ( left ) show α - SMA CD31 immunostaining in young aged human thyroid . Middle panel surface rendered for α - SMA , Vimentin CD31 . Arrowheads indicate arteries . Combo plots with quantifications of number of arteries vessel density . Data information : ( n = 4 ) , P - values , two - unpaired t - for above panels . ns : not significant ; * : P < 0 . 05 ; * * : P < 0 . 01 ; * * * * : P < 0 . 0001 . Human samples from donors at ages below 20 years over 70 years were chosen for young 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 of Efnb2 , Cdh5 Kdr expression ( normalized to Actb ) in aged human endocrine compared to young . Data information : ( n = 4 ) , P - values , two - unpaired t - for above panels . ns : not significant ; * : P < 0 . 05 ; * * : P < 0 . 01 ; * * * * : P < 0 . 0001 . Human samples from donors at ages below 20 years over 70 years were chosen for young 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

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.


A Representative 3D with Insulin Caveolin - 1 immunostaining in pancreatic islets from young mice . Combined box whiskers , scatter plots show vessel density in pancreatic islets non - islet areas from young aged mice . Data information : dashed lines in panel A , B , D , F , H I represent outlines of pancreatic islets . ( n = 5 ) , P - values , two - unpaired t - in panel ( A , E I ) one - way ANOVA with Tukey ' s multiple comparisons in panel ( G H ) . * : P < 0 . 05 ; * * : P < 0 . 01 ; * * * : P < 0 . 001 ; * * * * : P < 0 . 0001 . Nuclei : TO - PRO - 3 . Is : pancreatic islet . f . u : unit .

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

A Representative 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 with adult pancreatic islets immunolabeled with Ki67 VE - Cadherin . Inset ( * ) shows single - resolution surface rendering for channels . Data information : dashed lines in panel A , B , D , F , H I represent outlines of pancreatic islets . ( n = 5 ) , P - values , two - unpaired t - in panel ( A , E I ) one - way ANOVA with Tukey ' s multiple comparisons in panel ( G H ) . * : P < 0 . 05 ; * * : P < 0 . 01 ; * * * : P < 0 . 001 ; * * * * : P < 0 . 0001 . Nuclei : TO - PRO - 3 . Is : pancreatic islet . f . u : 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 Emcn immunostaining in 19 - - old mouse pancreas . Higher magnification at top of right panel shows ESM - 1 + pancreatic islets . Inset ( * ) shows single - resolution surface rendering for ESM - 1 Emcn . dashed in tile scan image represents outline of one lobe of pancreas , dashed lines in inset represents pancreatic islet . Data information : dashed lines in panel A , B , D , F , H I represent outlines of pancreatic islets . ( n = 5 ) , P - values , two - unpaired t - in panel ( A , E I ) one - way ANOVA with Tukey ' s multiple comparisons in panel ( G H ) . * : P < 0 . 05 ; * * : P < 0 . 01 ; * * * : P < 0 . 001 ; * * * * : P < 0 . 0001 . Nuclei : TO - PRO - 3 . Is : pancreatic islet . f . u : unit .

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

C Representative 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 show CD31 Emcn expressions in young pancreas . Insets ( * # ) show higher magnifications of capillaries in pancreatic islets non - islet areas . Data information : dashed lines in panel A , B , D , F , H I represent outlines of pancreatic islets . ( n = 5 ) , P - values , two - unpaired t - in panel ( A , E I ) one - way ANOVA with Tukey ' s multiple comparisons in panel ( G H ) . * : P < 0 . 05 ; * * : P < 0 . 01 ; * * * : P < 0 . 001 ; * * * * : P < 0 . 0001 . Nuclei : TO - PRO - 3 . Is : pancreatic islet . f . u : 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

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 CD31 + Emcnlo EC populations in young aged pancreas . Combo plot shows population of CD31 + Emcnhi ECs in young aged pancreas .

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 ( left ) with Insulin , Ki67 VE - Cadherin immunostaining on young aged pancreas . 3D ( middle ) show CXCR4 , Ki67 VE - Cadherin in young aged pancreatic islet . Insets ( yellow asterisks ) show single - resolution surface rendering for Ki67 CXCR4 . Arrowheads represent Ki67 + β - . G Combo plot shows of Ki67 + β - normalized to total number of β - per islet in juvenile ( 3 - - old ) , adult ( 12 - - old ) aged ( 55 - - old ) pancreatic islets . Data information : dashed lines in panel A , B , D , F , H I represent outlines of pancreatic islets . ( n = 5 ) , P - values , two - unpaired t - in panel ( A , E I ) one - way ANOVA with Tukey ' s multiple comparisons in panel ( G H ) . * : P < 0 . 05 ; * * : P < 0 . 01 ; * * * : P < 0 . 001 ; * * * * : P < 0 . 0001 . Nuclei : TO - PRO - 3 . Is : pancreatic islet . f . u : unit .

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

F 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.


F Representative 3D ( left ) with Insulin , Ki67 VE - Cadherin immunostaining on young aged pancreas . 3D ( middle ) show CXCR4 , Ki67 VE - Cadherin in young aged pancreatic islet . Insets ( yellow asterisks ) show single - resolution surface rendering for Ki67 CXCR4 . Arrowheads represent Ki67 + β - .

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

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.


H Representative 3D with Insulin , CD31 Emcn immunostaining in young aged pancreas . Combo plot shows CD31 + Emcnhi vessel density in juvenile , adult aged pancreatic islets . Data information : dashed lines in panel A , B , D , F , H I represent outlines of pancreatic islets . ( n = 5 ) , P - values , two - unpaired t - in panel ( A , E I ) one - way ANOVA with Tukey ' s multiple comparisons in panel ( G H ) . * : P < 0 . 05 ; * * : P < 0 . 01 ; * * * : P < 0 . 001 ; * * * * : P < 0 . 0001 . Nuclei : TO - PRO - 3 . Is : pancreatic islet . f . u : unit .

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

H Representative 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 of adult ( 10 - - old ) aged pancreas ( 55 - - old ) with Insulin VEGFA immunostaining . Combo plot ( middle ) shows VEGFA as determined by ELISA in isolated islets from 10 - - old adult 55 - - old aged mice . Combo plot ( right ) shows of VEGFA per islet ( f . u ) . Data information : dashed lines in panel A , B , D , F , H I represent outlines of pancreatic islets . ( n = 5 ) , P - values , two - unpaired t - in panel ( A , E I ) one - way ANOVA with Tukey ' s multiple comparisons in panel ( G H ) . * : P < 0 . 05 ; * * : P < 0 . 01 ; * * * : P < 0 . 001 ; * * * * : P < 0 . 0001 . Nuclei : TO - PRO - 3 . Is : pancreatic islet . f . u : unit .

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

I Representative 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.


A Representative 3D of pancreas from adult ( 18 - - old ) sunitinib - treated control mice with Insulin , CD31 Emcn immunostaining . B Combined box whiskers , scatter plot ( left ) shows of CD31 + Emcnhi vessel density in sunitinib - treated control pancreatic islets . Combo plot ( right ) shows ELISA of Insulin . Nuclei : DAPI or TO - PRO - 3 as indicated . Is : pancreatic islet . f . u : unit .

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

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 ( upper panel ) show CXCR4 , Ki67 VE - Cadherin in sunitinib - treated control pancreatic islet . 3D ( bottom ) show sunitinib - treated control pancreatic islets immunolabeled with Insulin VE - Cadherin . D Combo plot ( top ) shows of CXCR4 + β - per islet in sunitinib - treated control pancreatic islet . Combo plot ( middle ) shows of Ki67 + β - normalized to total number of β - numbers . Combo plot ( bottom ) shows of Insulin + β - per islet . Nuclei : DAPI or TO - PRO - 3 as indicated . Is : pancreatic islet . f . u : unit .

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

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.


C Representative 3D ( upper panel ) show CXCR4 , Ki67 VE - Cadherin in sunitinib - treated control pancreatic islet . 3D ( bottom ) show sunitinib - treated control pancreatic islets immunolabeled with Insulin VE - Cadherin .

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

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.


E Representative 3D of pancreas from adult Vegfr2i ∆ EC mutant control mice with Insulin , CD31 Emcn immunostainings . F Combo plot ( left ) with of CD31 + Emcnhi vessel density in Vegfr2i ∆ EC control pancreatic islet . Right combo plot with insulin estimation in pancreas by ELISA . Nuclei : DAPI or TO - PRO - 3 as indicated . Is : pancreatic islet . f . u : unit .

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

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.


E Representative 3D of pancreas from adult Vegfr2i ∆ EC mutant control mice with Insulin , CD31 Emcn immunostainings .

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

E Representative 3D images of pancreas from adult Vegfr2i∆EC mutant and control mice with Insulin, CD31 and Emcn immunostainings.


G Representative 3D in upper panel show CXCR4 , Ki67 VE - Cadherin in islets from Vegfr2i ∆ EC mutant control mice . 3D ( lower panel ) show Vegfr2i ∆ EC mutant control islets immunolabeled with Insulin VE - Cadherin . H Combo plot ( top ) shows of CXCR4 + β - per islet in Vegfr2i ∆ EC mutant control pancreatic islet . Combo plot ( middle ) shows of Ki67 + β - normalized to number of β - . Combo plot ( bottom ) shows of Insulin + β - per islet . Nuclei : DAPI or TO - PRO - 3 as indicated . Is : pancreatic islet . f . u : 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 B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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.


G Representative 3D in upper panel show CXCR4 , Ki67 VE - Cadherin in islets from Vegfr2i ∆ EC mutant control mice . 3D ( lower panel ) show Vegfr2i ∆ EC mutant control islets immunolabeled with Insulin VE - Cadherin .

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

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.


I Graph shows qPCR of Pdgfa , Pdgfb , Igf1 , Igf2 , Cxcl12 , Hgf Kitl expression ( normalized to Actb ) in purified CD31 + Emcnhi ECs compared to CD31 + Emcnlo ECs .

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

I 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 of Ki67 + β - numbers per islet ( f . u ) Ki67 + EC numbers per islet ( f . u ) . 45 pancreatic islets were analysed quantified from 9 young mice .

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

J 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 of Dll4 , Emcn , Kdr , Flt4 , Sele , Mmp9 , Plxnd1 , Robo4 , Unc5b , Cdh5 , Cldn5 , Gja1 , Gja4 , Icam1 , Icam2 , Jam3 , Ntn1 , Ntn2 , Ocln , Pecam1 , Tjp1 , Tjp2 , Tjp3 Vcam1 expression ( normalized to Actb ) in purified CD31 + Emcnhi ECs compared to CD31 + Emcnlo ECs from 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

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 of Gja1 expression ( normalized to Actb ) in purified CD31 + Emcnhi ECs from juvenile , adult 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

L Graph shows qPCR analysis of Gja1 expression (normalized to Actb) in purified CD31+ Emcnhi ECs from juvenile, adult and aged murine pancreas.


A Representative tile scan 3D with α - SMA , Isolectin , FABP4 Emcn in pancreas from 17 - - old Gja1i ∆ EC mutant control mice . Insets show higher magnifications of regions . B Box whiskers , scatter plot ( left ) shows of CD31 + Emcnhi vessel density in Gja1i ∆ EC mutant control pancreatic islets . ( n = 7 ) , P - value , two - unpaired t - . Combo plot ( right ) shows of P - Histone H3 + EC numbers per islet ( f . u ) counted on thick murine pancreatic sections . ( n = 5 ) , P - value , two - unpaired t - . C 3D show CD31 Emcn immunostaining in Gja1i ∆ EC mutant control pancreas . scale bars are 50 µm . D Combined violin , scatter sample distribution plot shows of β - mass ( % ) in pancreatic islets from Gja1i ∆ EC control mice . Data represent mean ± s . d . ( n = 140 islets from 7 pancreas ) , P - value , two - unpaired t - . E Representative 3D with immunostaining of α - SMA , Insulin VE - Cadherin in pancreatic islets from Gja1i ∆ EC control mice . scale bars are 50 µm . F Combo plot ( left ) shows insulin measurement in lysate of pancreas by ELISA . Combo plot ( right ) shows of Insulin + β - per islet in Gja1i ∆ EC mutant control mice . ( n = 5 ) , P - values , two - unpaired t - . Data information : dashed lines in panel A represent outlines of pancreas . dashed lines in panel C , E , G , H I represent outlines of pancreatic islets . Two - Student ' s t - were performed for statistical . * * : P < 0 . 01 ; * * * * : P < 0 . 0001 . Nuclei : DAPI Is : pancreatic islet . f . u : unit . on right side of combo plots represent sample distribution . Scale bars are 200 µm for tile scan of longitudinal thick sections across entire , 50 µm for higher magnification insets .

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

A Representative 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.


A Representative tile scan 3D with α - SMA , Isolectin , FABP4 Emcn in pancreas from 17 - - old Gja1i ∆ EC mutant control mice . Insets show higher magnifications of regions .

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

A Representative 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.