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 B-TISSUE B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD O O O O O B-TISSUE I-TISSUE O O O O O B-TISSUE B-EXP_ASSAY O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-SUBCELLULAR O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O 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-TISSUE O B-TISSUE O B-TISSUE O B-TISSUE 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 B-TISSUE B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-TISSUE I-TISSUE I-TISSUE I-TISSUE O O O O O O B-TISSUE B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-SUBCELLULAR O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O 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-TISSUE O B-TISSUE I-TISSUE O B-TISSUE O B-TISSUE I-TISSUE 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 B-TISSUE B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O B-TISSUE I-TISSUE O O O O O B-TISSUE B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-SUBCELLULAR O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-TISSUE B-EXP_ASSAY O B-GENEPROD I-GENEPROD O O O O O O O O O O O B-TISSUE O O O O O B-TISSUE B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-SUBCELLULAR O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-TISSUE B-EXP_ASSAY O B-GENEPROD O B-SUBCELLULAR B-GENEPROD O O O O O B-TISSUE B-EXP_ASSAY O O O B-TISSUE O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-SUBCELLULAR O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 .

O O O B-TISSUE B-EXP_ASSAY O O O B-TISSUE B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-TISSUE O O O B-ORGANISM O O O O O O O O O O B-TISSUE B-EXP_ASSAY O O B-TISSUE O

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 B-TISSUE B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O B-TISSUE B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O B-TISSUE O O O B-ORGANISM O O O O O O B-TISSUE B-EXP_ASSAY O O B-TISSUE O O O B-TISSUE 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 B-TISSUE B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O B-TISSUE B-EXP_ASSAY O O B-TISSUE 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 B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O B-TISSUE B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O B-TISSUE B-EXP_ASSAY O O B-TISSUE O O O B-TISSUE 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 B-EXP_ASSAY I-EXP_ASSAY O O O O O B-TISSUE B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O B-TISSUE B-EXP_ASSAY O O B-TISSUE O O O B-TISSUE 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 B-EXP_ASSAY O O O O O O O O O B-EXP_ASSAY O O O B-ORGANISM B-TISSUE O O O O B-EXP_ASSAY O O O O B-EXP_ASSAY O O O O B-EXP_ASSAY O O O O O O O O O O O B-EXP_ASSAY 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 B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O O O B-ORGANISM B-TISSUE O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD O O O B-TISSUE O O O O O O O O B-TISSUE I-TISSUE B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-ORGANISM O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O B-SMALL_MOLECULE 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 B-ORGANISM B-TISSUE B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O B-TISSUE O O O O O O B-EXP_ASSAY O B-TISSUE I-TISSUE B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-ORGANISM O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O B-SMALL_MOLECULE 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 B-ORGANISM B-TISSUE O B-GENEPROD I-GENEPROD B-EXP_ASSAY O O O O O O O O O O B-TISSUE O O O O O O O O B-TISSUE I-TISSUE B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-ORGANISM O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O B-SMALL_MOLECULE 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 B-ORGANISM B-TISSUE O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O B-TISSUE I-TISSUE B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-ORGANISM O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O B-SMALL_MOLECULE 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 .

O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O O O B-ORGANISM B-TISSUE O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD O O O B-TISSUE O O O O O O O O B-TISSUE I-TISSUE B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-ORGANISM O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O B-SMALL_MOLECULE O

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 B-EXP_ASSAY O B-GENEPROD O B-GENEPROD I-GENEPROD B-EXP_ASSAY O O O B-GENEPROD O O O B-ORGANISM B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-ORGANISM 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-SMALL_MOLECULE B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O B-TISSUE I-TISSUE O O B-ORGANISM O O O O O O O O B-TISSUE B-EXP_ASSAY O B-TISSUE I-TISSUE O O B-TISSUE O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O B-TISSUE I-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-SUBCELLULAR O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-TISSUE I-TISSUE 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 .

O O O O O B-TISSUE I-TISSUE B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O 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-TISSUE I-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-SUBCELLULAR O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-TISSUE I-TISSUE O O O O O O O

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 B-EXP_ASSAY O B-SMALL_MOLECULE O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O O O O O B-ORGANISM B-TISSUE O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-TISSUE I-TISSUE O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-EXP_ASSAY O O O O B-TISSUE I-TISSUE I-TISSUE O O O O O O B-TISSUE I-TISSUE O O O O O O O O O O O O O O O O O O O O O B-TISSUE I-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-SUBCELLULAR O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-TISSUE I-TISSUE 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 B-GENEPROD I-GENEPROD B-EXP_ASSAY O O B-TISSUE O O O O O O O O O O B-TISSUE O B-TISSUE I-TISSUE O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O B-TISSUE I-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-SUBCELLULAR O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-TISSUE I-TISSUE 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 .

O O B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O B-GENEPROD I-GENEPROD O B-GENEPROD B-CELL_TYPE O O O O B-TISSUE O O O O O O B-GENEPROD O B-GENEPROD B-CELL_TYPE O O O B-TISSUE O

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-SMALL_MOLECULE O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O O O B-TISSUE O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O B-TISSUE I-TISSUE O O O O O O O O O O O O O B-GENEPROD I-GENEPROD O O O B-GENEPROD O B-CELL_TYPE I-CELL_TYPE O O O O O O B-GENEPROD O B-CELL_TYPE I-CELL_TYPE O O O B-EXP_ASSAY O B-CELL_TYPE I-CELL_TYPE O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O B-TISSUE I-TISSUE O O O O O O O O O O O O O O O O O O O O O B-TISSUE I-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-SUBCELLULAR O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-TISSUE I-TISSUE 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-SMALL_MOLECULE O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O O O B-TISSUE O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O B-TISSUE I-TISSUE O O O O O O O O O O O O O B-GENEPROD I-GENEPROD O O O B-GENEPROD O B-CELL_TYPE I-CELL_TYPE 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-SMALL_MOLECULE O B-GENEPROD I-GENEPROD B-EXP_ASSAY O O O B-TISSUE O O O O B-GENEPROD O B-GENEPROD B-TISSUE B-EXP_ASSAY O O O O O B-TISSUE I-TISSUE O O O O O O O O O O O O O O O O O O O O O B-TISSUE I-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-SUBCELLULAR O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-TISSUE I-TISSUE 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 B-TISSUE O O O O O O O B-SMALL_MOLECULE B-GENEPROD B-EXP_ASSAY O O O O O O O B-GENEPROD O O O B-EXP_ASSAY O O B-TISSUE O O O O O O O O O O O B-ORGANISM O O O O O O O O B-GENEPROD O B-TISSUE O O 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-TISSUE I-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-SUBCELLULAR O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-TISSUE I-TISSUE 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 B-TISSUE O O O O O O O O B-SMALL_MOLECULE O O O B-ORGANISM O B-SMALL_MOLECULE O B-GENEPROD I-GENEPROD B-EXP_ASSAY O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD B-TISSUE B-EXP_ASSAY O B-SMALL_MOLECULE O O O B-TISSUE I-TISSUE O O O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O B-SUBCELLULAR O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-TISSUE I-TISSUE 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 B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-SMALL_MOLECULE O O O B-TISSUE I-TISSUE O O O O O O B-SMALL_MOLECULE O O O B-TISSUE I-TISSUE B-EXP_ASSAY O B-SMALL_MOLECULE B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O B-GENEPROD O B-CELL_TYPE I-CELL_TYPE O B-TISSUE O B-SMALL_MOLECULE O O O B-TISSUE I-TISSUE O O O O O O O O B-GENEPROD O B-CELL_TYPE I-CELL_TYPE O O O B-EXP_ASSAY O B-CELL_TYPE I-CELL_TYPE B-EXP_ASSAY O O O O O O O O B-SMALL_MOLECULE O B-CELL_TYPE I-CELL_TYPE O B-TISSUE O B-SUBCELLULAR O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-TISSUE I-TISSUE 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 B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-SMALL_MOLECULE O O O B-TISSUE I-TISSUE O O O O O O B-SMALL_MOLECULE O O O B-TISSUE I-TISSUE B-EXP_ASSAY O B-SMALL_MOLECULE B-GENEPROD I-GENEPROD I-GENEPROD 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 B-TISSUE O O B-GENEPROD O O O O B-ORGANISM O B-SMALL_MOLECULE O B-GENEPROD I-GENEPROD B-EXP_ASSAY O O O O O O O O O B-GENEPROD O B-GENEPROD B-TISSUE B-EXP_ASSAY O B-GENEPROD O O O B-TISSUE I-TISSUE O O O O O B-SMALL_MOLECULE O O B-TISSUE O B-EXP_ASSAY O B-SUBCELLULAR O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-TISSUE I-TISSUE 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 B-TISSUE O O B-GENEPROD O O O O B-ORGANISM O B-SMALL_MOLECULE O B-GENEPROD I-GENEPROD B-EXP_ASSAY 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 B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-TISSUE O B-GENEPROD O O O O B-ORGANISM O O O O O O O B-GENEPROD O O O O B-TISSUE B-EXP_ASSAY O B-SMALL_MOLECULE B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O B-GENEPROD O B-CELL_TYPE I-CELL_TYPE O B-TISSUE O B-GENEPROD O O O O B-TISSUE I-TISSUE O O O O O O O O B-GENEPROD O B-CELL_TYPE I-CELL_TYPE O O B-EXP_ASSAY O B-CELL_TYPE I-CELL_TYPE O O O O O O O O B-SMALL_MOLECULE O B-CELL_TYPE I-CELL_TYPE O B-TISSUE O B-SUBCELLULAR O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-TISSUE I-TISSUE 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 B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-TISSUE O B-GENEPROD O O O O B-ORGANISM O O O O O O O B-GENEPROD O O O O B-TISSUE B-EXP_ASSAY O B-SMALL_MOLECULE B-GENEPROD I-GENEPROD I-GENEPROD 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 B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD B-EXP_ASSAY O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD B-CELL_TYPE O O B-GENEPROD O B-GENEPROD B-CELL_TYPE 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 B-GENEPROD O B-CELL_TYPE I-CELL_TYPE B-EXP_ASSAY O B-TISSUE O O O O O B-GENEPROD O B-CELL_TYPE B-EXP_ASSAY O B-TISSUE O O O O O O O B-TISSUE I-TISSUE O O O O O O B-ORGANISM 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 B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD B-EXP_ASSAY O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD B-CELL_TYPE O O B-GENEPROD O B-GENEPROD B-CELL_TYPE O B-ORGANISM B-TISSUE 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 B-EXP_ASSAY O B-GENEPROD B-EXP_ASSAY O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD B-CELL_TYPE O O O O O B-ORGANISM B-TISSUE 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 B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD O B-TISSUE O O O O O B-GENEPROD O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD B-TISSUE B-EXP_ASSAY O B-GENEPROD O O O O B-TISSUE I-TISSUE O O 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-GENEPROD I-GENEPROD O B-CELL_TYPE B-EXP_ASSAY O B-TISSUE O O O O O O O O B-ORGANISM B-TISSUE O O O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD B-EXP_ASSAY O B-GENEPROD O O O O B-TISSUE O O O O O O O O O O O O O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O B-TISSUE I-TISSUE O B-GENEPROD O O O B-ORGANISM O O O O O O O O O O O O O B-TISSUE O O B-TISSUE O O O O O O O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O B-SMALL_MOLECULE B-GENEPROD I-GENEPROD I-GENEPROD O B-TISSUE I-TISSUE O B-GENEPROD O O O B-ORGANISM O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O B-TISSUE O B-EXP_ASSAY O O O O O O O O B-SMALL_MOLECULE O B-CELL_TYPE I-CELL_TYPE O B-TISSUE O B-GENEPROD O O O O B-ORGANISM O O O O 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-TISSUE O O O O O O O O O O O O O O O O B-TISSUE I-TISSUE O O O O O O O O O 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-SUBCELLULAR O B-SMALL_MOLECULE O O B-TISSUE I-TISSUE O O O O O O O O O O O O O O O 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 B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD O B-TISSUE O O O O O B-GENEPROD O O O O B-ORGANISM 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.


G Representative 3D of ESM - 1 Caveolin - 1 in pancreatic islets from Gja1i ∆ EC control mice . Insets ( * # ) show higher magnifications with surface rendering for ESM - 1 Caveolin - 1 immunostaining . Combo plot with of ESM - 1 + numbers per islet ( f . u ) . ( n = 5 ) , P - value , two - unpaired t - . scale bars for 3D are 50 µm , 5 µm for higher magnifications with single - resolution . 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 : TO - PRO - 3 as indicated . Is : pancreatic islet . f . u : unit .

O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-TISSUE I-TISSUE O B-GENEPROD O O O B-ORGANISM O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-EXP_ASSAY O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-TISSUE I-TISSUE O O O O O O O O O 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-SUBCELLULAR O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-TISSUE I-TISSUE O O O O O O O

G Representative 3D images of ESM-1 and Caveolin-1 in pancreatic islets from Gja1i∆EC and control mice. Insets (* and #) show higher magnifications with surface rendering for ESM-1 and Caveolin-1 immunostaining. Combo plot with the quantification of ESM-1+ cell numbers per islet (f.u). (n=5), P-value, and two-tailed unpaired t-test. The scale bars for the 3D images are 50 µm, and 5 µm for the higher magnifications with single-cell resolution images. The dashed lines in panel C, E, G, H and I represent the outlines of pancreatic islets. Two-tailed Student's t-test were performed for the statistical analysis. **: P < 0.01; ****: P < 0.0001. Nuclei: TO-PRO-3 as indicated. Is: pancreatic islet. f.u: fluorescence unit.


H 3D with CXCR4 Emcn immunostaining in Gja1i ∆ EC control pancreas . scale bars are 50 µm . I Representative 3D show CXCR4 , Ki67 VE - Cadherin immunostaining in Gja1i ∆ EC control pancreas . scale bars are 50 µm . J Combo plots show of CXCR4 + β - per islet Ki67 + β - normalized to total number of β - per islet in Gja1i ∆ EC mutant versus littermate control mice . ( n = 5 ) , P - values , two - unpaired t - . K Box whiskers , scatter plot shows of CD31 + Emcnhi vessel density in aged Gja1i ∆ EC mutant control pancreatic islets . Data represent mean ± s . d . ( n = 4 ) , P - value , two - unpaired t - . Combo plot ( below ) shows of β - on thick murine pancreatic sections . Data represent mean ± s . d . ( n = 4 ) , P - value , two - unpaired t - . Data information : 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 : TO - PRO -

O O O B-GENEPROD I-GENEPROD B-EXP_ASSAY O B-GENEPROD O O O B-TISSUE O O O O O O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O B-GENEPROD O O O B-TISSUE O O O O O O O O O O O O B-GENEPROD O B-CELL_TYPE I-CELL_TYPE O B-TISSUE B-GENEPROD O B-CELL_TYPE I-CELL_TYPE O O O B-EXP_ASSAY O B-CELL_TYPE I-CELL_TYPE O B-TISSUE O B-GENEPROD O O O O O O B-ORGANISM O 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-GENEPROD O B-GENEPROD B-TISSUE B-EXP_ASSAY O O B-GENEPROD O O O O B-TISSUE I-TISSUE O O O O O O O O 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-CELL_TYPE I-CELL_TYPE O O B-ORGANISM B-TISSUE O O O O O O O O O O O O O O O O O O O O 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-TISSUE I-TISSUE O O O O O O O O O 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-SUBCELLULAR O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE

H 3D images with CXCR4 and Emcn immunostaining in Gja1i∆EC and control pancreas. The scale bars are 50 µm. I Representative 3D images show CXCR4, Ki67 and VE-Cadherin immunostaining in Gja1i∆EC and control pancreas. The scale bars are 50 µm. J Combo plots show the percentage of CXCR4+ β-cells per islet and Ki67+ β-cells normalized to the total number of β-cells per islet in Gja1i∆EC mutant versus the littermate control mice. (n=5), P-values, and two-tailed unpaired t-tests. K Box whiskers, and scatter plot shows the quantification of CD31+ Emcnhi vessel density in aged Gja1i∆EC mutant and control pancreatic islets. Data represent mean±s.d. (n=4), P-value, and two-tailed unpaired t-test. Combo plot (below) shows the quantification of β-cells on thick murine pancreatic sections. Data represent mean±s.d. (n=4), P-value, and two-tailed unpaired t-test. Data information: The dashed lines in panel C, E, G, H and I represent the outlines of pancreatic islets. Two-tailed Student's t-test were performed for the statistical analysis. **: P < 0.01; ****: P < 0.0001. Nuclei: TO-PRO-3


H 3D with CXCR4 Emcn immunostaining in Gja1i ∆ EC control pancreas . scale bars are 50 µm .

O O O B-GENEPROD I-GENEPROD B-EXP_ASSAY O B-GENEPROD O O O B-TISSUE O O O O O O O

H 3D images with CXCR4 and Emcn immunostaining in Gja1i∆EC and control pancreas. The scale bars are 50 µm.


( a ) THP - 1 were transiently infected with BRLF1 - expressing or empty control lentivirus . Twenty - four hours later , control or BRLF1 - expressing THP - 1 were primed with 40 ng / ml TPA overnight then were either uninfected or infected with HSV - 1 ( MOI = 1 ) in serum - free medium for 12 h . supernatants whole lysates were collected analyzed by western blotting to detect pro - caspase - 1 ( p45 ) , caspase - 1 ( p20 ) , pro - IL - 1β , mature IL - 1β , pro - IL - 18 mature IL - 18 as indicated .

O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O B-GENEPROD O O O O O B-ORGANISM O O O O O O O O O B-GENEPROD O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O B-TISSUE O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O

(a) THP-1 cells were transiently infected with BRLF1-expressing or empty control lentivirus. Twenty-four hours later, the control or BRLF1-expressing THP-1 cells were primed with 40 ng/ml TPA overnight and then were either uninfected or infected with HSV-1 (MOI=1) in serum-free medium for 12 h. The supernatants and whole cell lysates were collected and analyzed by western blotting to detect pro-caspase-1 (p45), cleaved caspase-1 (p20), pro-IL-1β, mature IL-1β, pro-IL-18 and mature IL-18 as indicated.


release of mature IL - 1β ( b ) from THP - 1 were measured by enzyme - linked immunosorbent ( ELISA ) following HSV - 1 infection .

O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O B-ORGANISM I-ORGANISM I-ORGANISM O O

The release of mature IL-1β (b) from THP-1 cells were measured by enzyme-linked immunosorbent assay (ELISA) following HSV-1 infection.


release of IL - 18 ( c ) from THP - 1 were measured by enzyme - linked immunosorbent ( ELISA ) following HSV - 1 infection .

O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O B-ORGANISM I-ORGANISM I-ORGANISM O O

The release of IL-18 (c) from THP-1 cells were measured by enzyme-linked immunosorbent assay (ELISA) following HSV-1 infection.


Ramos were infected with wild type EBV - WT or BRLF1 - deficient EBV - ΔBRLF1 at a high titer ( MOI = 100 ) . After being transduced with empty control or BZLF1 - expressing lentivirus , supernatants pellets of EBV - WT - or EBV - ΔBRLF1 - harboring Ramos were collected after 12 h in serum - free culture , then of EBNA1 , BRLF1 , BZLF1 , pro - caspase - 1 ( p45 ) , caspase - 1 ( p20 ) , pro - IL - 1β , mature IL - 1β , pro - IL - 18 , mature IL - 18 , GSDMD N - GSDMD in supernatants whole - lysates were analyzed as indicated ( d ) .

B-CELL_LINE O O O O O B-ORGANISM O O O B-GENEPROD O O B-ORGANISM O O O O O O O O O O O O O O O O O O O B-GENEPROD O O B-ORGANISM O O O O B-ORGANISM O O O O B-ORGANISM O O O O B-CELL_LINE O O O O O O B-TISSUE O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O

Ramos cells were infected with wild type EBV-WT or BRLF1-deficient EBV-ΔBRLF1 virus at a high titer (MOI=100). After being transduced with empty control or BZLF1-expressing lentivirus, the supernatants and cell pellets of EBV-WT- or EBV-ΔBRLF1-harboring Ramos cells were collected after 12 h in serum-free culture, and then the levels of EBNA1, BRLF1, BZLF1, pro-caspase-1 (p45), cleaved caspase-1 (p20), pro-IL-1β, mature IL-1β, pro-IL-18, mature IL-18, GSDMD and cleaved N-GSDMD in the supernatants and whole-cell lysates were analyzed as indicated (d).


release of mature IL - 1β ( e ) from Ramos were measured by enzyme - linked immunosorbent ( ELISA ) .

O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-CELL_LINE O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O

The release of mature IL-1β (e) from Ramos cells were measured by enzyme-linked immunosorbent assay (ELISA).


release of mature IL - 18 ( f ) from Ramos were measured by enzyme - linked immunosorbent ( ELISA ) .

The release of mature IL-18 (f) from Ramos cells were measured by enzyme-linked immunosorbent assay (ELISA).


( g ) After being transduced with control or BZLF1 - expressing lentivirus for 48 h , EBV - WT - EBV - ΔBRLF1 infected Ramos were stained with PI measured using flow cytometry . Representative of pyroptotic are shown percentages of pyroptotic were calculated in duplicate .

O O O O O O O O O B-GENEPROD O O B-ORGANISM O O O O B-ORGANISM O O O B-ORGANISM O O O B-CELL_LINE O B-EXP_ASSAY O B-SMALL_MOLECULE O O B-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY O O O O B-EXP_ASSAY O O O O O

(g) After being transduced with control or BZLF1-expressing lentivirus for 48 h, EBV-WT- and EBV-ΔBRLF1 infected Ramos cells were stained with PI and measured using fluorescent flow cytometry. Representative images of pyroptotic cells are shown and the percentages of pyroptotic cells were calculated in duplicate.


( a ) GST - tagged BRLF1 - expressing plasmid or control vector was transfected into A549 . After HSV - 1 infection ( MOI = 1 ) for 12 h , were collected , whole extracts were subjected to immunoprecipitation with GST affinity beads . immunoprecipitated complexes were then separated by SDS - PAGE , image obtained from silver is shown .

O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O B-CELL_LINE O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O O O B-EXP_ASSAY O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY O O O

(a) GST-tagged BRLF1-expressing plasmid or control vector was transfected into A549 cells. After HSV-1 infection (MOI=1) for 12 h, the cells were collected, and the whole cell extracts were subjected to immunoprecipitation with GST affinity beads. The immunoprecipitated complexes were then separated by SDS-PAGE, and the image obtained from silver staining is shown.


( b ) Scramble ( sc ) or shRNAs against POLR3F POLR3G with empty control vector ( ctr ) or BRLF1 - expressing plasmid were transfected into EBV - ΔBRLF1 - harboring HNE1 A549 , followed by HSV - 1 infection . Thirty - six hours after transfection of HNE1 , 12 h after HSV - 1 infection ( MOI = 1 ) of A549 , pellets were collected then cleavage of caspase - 1 IL - 1β were measured as indicated . release of mature IL - 1β IL - 18 from HNE - 1 A549 were measured by enzyme - linked immunosorbent ( ELISA ) after ultrafiltration with Amicon ® Ultra - 0 . 5 centrifugal filter devices .

O O O O O O O O O O B-GENEPROD I-GENEPROD O O O O O O O O B-GENEPROD O O O O O O B-ORGANISM O O O O B-CELL_LINE I-CELL_LINE O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O B-CELL_LINE O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O B-CELL_LINE O O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O O O O O O O O

(b) Scramble (sc) or shRNAs against POLR3F and POLR3G with empty control vector (ctr) or BRLF1-expressing plasmid were transfected into EBV-ΔBRLF1-harboring HNE1 cells and A549 cells, followed by HSV-1 infection. Thirty-six hours after the transfection of the HNE1 cells, and 12 h after HSV-1 infection (MOI=1) of the A549 cells, the cell pellets were collected and then the cleavage of caspase-1 and IL-1β were measured as indicated. The release of mature IL-1β and IL-18 from HNE-1 and A549 cells were measured by enzyme-linked immunosorbent assay (ELISA) after ultrafiltration and concentration with Amicon® Ultra-0.5 centrifugal filter devices.


( c ) GFP - tagged BRLF1 wild type , BRLF1 L578A or empty vector was transfected into 293T with GST - tagged POLR3F or POLR3G expressing plasmids for 48 h . were collected immunoprecipitation with GST - affinity beads was performed , then whole lysates immunoprecipitated complexes were analyzed as indicated .

O O O B-GENEPROD O O B-GENEPROD O O O B-GENEPROD O O O O O O O B-CELL_LINE O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O O O O O B-EXP_ASSAY O B-GENEPROD O O O O O O O O O B-EXP_ASSAY O O O O O O

(c) GFP-tagged BRLF1 wild type, BRLF1 L578A or empty vector was transfected into 293T cells with GST-tagged POLR3F or POLR3G expressing plasmids for 48 h. Cells were collected and immunoprecipitation with GST-affinity beads was performed, and then whole cell lysates and immunoprecipitated complexes were analyzed as indicated.


( d ) GFP - tagged empty vector or GFP - BRLF1 - or GFP - BRLF1 L578A - expressing plasmids were transfected into A549 , after which were then infected with HSV - 1 , or transfected into HNE - 1 - EBV - ΔBRLF1 . were then collected lysed . Immunoprecipitation with GST - affinity beads was subsequently performed , then whole - lysates immunoprecipitated complexes were analyzed as indicated .

O O O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O O O O B-CELL_LINE O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O B-ORGANISM O O O O O O O O B-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O B-EXP_ASSAY O O O O O O

(d) GFP-tagged empty vector or GFP-BRLF1- or GFP-BRLF1 L578A-expressing plasmids were transfected into A549 cells, after which the cells were then infected with HSV-1, or transfected into HNE-1-EBV-ΔBRLF1 cells. The cells were then collected and lysed. Immunoprecipitation with GST-affinity beads was subsequently performed, and then whole-cell lysates and immunoprecipitated complexes were analyzed as indicated.


( e ) P3HR - 1 were induced by TPA plus NaB into lytic infection , were then collected . Immunoprecipitation with anti - mouse IgG or BRLF1 antibody was performed , after which whole - lysates immunoprecipitated complexes were analyzed as indicated .

O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O B-EXP_ASSAY O O O O O O B-GENEPROD O O O O O O O O O B-EXP_ASSAY O O O O O O

(e) P3HR-1 cells were induced by TPA plus NaB into lytic infection, and the cells were then collected. Immunoprecipitation with anti-mouse IgG or BRLF1 antibody was performed, after which whole-cell lysates and immunoprecipitated complexes were analyzed as indicated.


( a ) After infected with wild type EBV - WT or BRLF1 - deficient EBV - ΔBRLF1 at a high titer ( MOI = 100 ) , POLR3 - dependent transcripts in Ramos were detected by - seq . relative of small transcripts are shown as fold changes in in mock ( mock group vs . mock group ) , EBV - WT ( EBV - WT group vs . mock group ) EBV - ΔBRLF1 ( EBV - ΔBRLF1 group vs . mock group ) comparisons .

O O O O O O O O B-ORGANISM O O O B-GENEPROD O O B-ORGANISM O O O O O O O O O O O O B-GENEPROD O O O O B-CELL_LINE O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O B-ORGANISM O O O O O O O O B-ORGANISM O O O B-ORGANISM O O O O O O O O O O

(a) After infected with wild type EBV-WT or BRLF1-deficient EBV-ΔBRLF1 virus at a high titer (MOI=100), POLR3-dependent RNA transcripts in Ramos cells were detected by RNA-seq analysis. The relative levels of cellular small RNA transcripts are shown as RNA fold changes in the heatmap in the mock (mock group vs. mock group), EBV-WT (EBV-WT group vs. mock group) and EBV-ΔBRLF1 (EBV-ΔBRLF1 group vs. mock group) comparisons.


( b ) EBV - ΔBRLF1 - harboring HNE1 were transfected with control vector , BRLF1 or BRLF1 L578A in absence or presence of BZLF1 for 48 h . total RNAs were extracted subjected to real - time PCR .

O O O B-ORGANISM O O O O B-CELL_LINE O O O O O O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O

(b) EBV-ΔBRLF1-harboring HNE1 cells were transfected with control vector, BRLF1 or BRLF1 L578A in the absence or presence of BZLF1 for 48 h. The total RNAs were extracted and subjected to real-time PCR analysis.


( c ) A549 were transfected with control vector , BRLF1 or BRLF1 L578A for 36 h . After were infected with HSV - 1 ( MOI = 1 ) for 12 h , total RNAs were extracted subjected to real - time PCR .

O O O B-CELL_LINE O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O

(c) A549 cells were transfected with control vector, BRLF1 or BRLF1 L578A for 36 h. After the cells were infected with HSV-1(MOI=1) for 12 h, the total RNAs were extracted and subjected to real-time PCR analysis.


( d ) EBER1 5 ' - pppRNA detection using splint - ligation . EBV - WT - or EBV - ΔBRLF1 - harboring HNE1 were cotransfected with empty vector , BRLF1 or BRLF1 L578A , BRLF1 plus BZLF1 , or BRLF1 L578A plus BZLF1 for 48 h . total RNAs were extracted , 5 μg of each was subjected to splint - ligation with FAM - labeled probe to quantify 5 ′ - monophosphorylated EBER1 total EBER1 . ligation products were separated using urea - PAGE , visualized by a scanner analyzed using ImageJ software . of 5 ' - pRNA density to total density relative 5 ' - pppRNA level were calculated for three independent experiments .

O O O B-GENEPROD O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-ORGANISM O O O O B-ORGANISM O O O O B-CELL_LINE O O O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O B-SMALL_MOLECULE O B-EXP_ASSAY O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O

(d) EBER1 5'-pppRNA detection using splint-ligation. EBV-WT- or EBV-ΔBRLF1-harboring HNE1 cells were cotransfected with empty vector, BRLF1 or BRLF1 L578A, BRLF1 plus BZLF1, or BRLF1 L578A plus BZLF1 for 48 h. The total RNAs were extracted, and 5 μg of each RNA was subjected to splint-ligation with FAM-labeled probe to quantify the 5′-monophosphorylated EBER1 and total EBER1 RNA. The ligation products were separated using urea-PAGE, visualized by a fluorescent scanner and analyzed using ImageJ software. The ratio of 5'-pRNA density to total RNA density and the relative 5'-pppRNA level were calculated for three independent experiments.


( e ) EBV - WT - harboring HNE1 were transfected with control vector , BRLF1 or BRLF1 L578A - expressing plasmid in presence of GST - tagged polymerase subunit POLR3F for 48 h . were harvested ChIP was performed with anti - GST affinity beads . POLR3F - binding promoter fragments of BSRF1 , BMLF1 , EBER1 , EBER2 , EBNA1 , 7SK , RN7SL1 GAPDH as negative control in ChIP samples were detected by real - time PCR assays .

O O O B-ORGANISM O O O O B-CELL_LINE O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O O B-SUBCELLULAR O B-GENEPROD O O O O O O B-EXP_ASSAY O O O O O B-GENEPROD O O O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD O O O O B-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O

(e) EBV-WT-harboring HNE1 cells were transfected with control vector, BRLF1 or BRLF1 L578A-expressing plasmid in the presence of GST-tagged RNA polymerase subunit POLR3F for 48 h. The cells were harvested and ChIP assay was performed with anti-GST affinity beads. The POLR3F-binding promoter fragments of BSRF1, BMLF1, EBER1, EBER2, EBNA1, 7SK, RN7SL1 and the GAPDH as negative control in the ChIP samples were detected by real-time PCR assays.