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 .

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

C Young (10-weeks-old) and aged pituitary gland immunolabelled with ESM-1, Collagen IV and Emcn. Insets with higher magnifications of pars distalis and pars nervosa regions. Combo plot shows quantification of vessel density. Data information: The white and dashed lines in each panel represent the outlines of each organ. (n=5), P-values and two-tailed unpaired t-tests. ns: not significant; **: P < 0.01; ***: P < 0.001. Nuclei: TO-PRO-3. For the Combo plots, the boxes represent mean ± s.d., line in the box is the median, and the lower and upper lines show the minimum and the maximum of the values. The line on the right side of these combined plots represents the sample distribution. Pd: Pars Distalis; Pn: Pars Nervosa. Scale bars for tile scan 3D images 200 µm, for insets 50 µm.


D Young 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 .

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

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

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

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 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 .

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

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


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

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

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


C 3D images of whole cleared young and aged testes stained with α - SMA and Emcn . Asterisks indicate higher magnifications of indicated regions . Combo plots show quantifications of vessel density and numbers of arteries .

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

C 3D images of whole cleared young and aged testes stained with α-SMA and Emcn. Asterisks indicate higher magnifications of indicated regions. Combo plots show quantifications of vessel density and numbers of arteries.


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

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

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


E 3D images acquired on a light sheet microscope of whole cleared young and aged thyroid glands stained with α - SMA and Emcn . Asterisks indicate higher magnifications of regions . Combo plots show quantifications of vessel density and numbers of arteries in young and aged thyroid glands .

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

E 3D images acquired on a light sheet microscope of whole cleared young and aged thyroid glands stained with α-SMA and Emcn. Asterisks indicate higher magnifications of regions. Combo plots show quantifications of vessel density and numbers of arteries in young and aged thyroid glands.


F Heatmap of 3D spatial proteomic data showing qualitative analysis of differences in cell type markers and matrix expressions between young and aged murine endocrine glands . The colour code based on immunolabelling intensities indicates very high expression in red , high expression in yellow , medium in light green , low in dark green and no expression in grey .

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

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 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 .

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

A 3D images (left) with α-SMA and CD31 immunostaining in young and aged human adrenal gland. Middle panel images surface rendered for α-SMA, Vimentin and CD31. Arrowheads indicate arteries. Combo plots with quantifications of the number of arteries and vessel density. Data information: (n=4), P-values, and two-tailed unpaired t-tests for all the above panels. ns: not significant; *: P < 0.05; **: P < 0.01; ****: P < 0.0001. Human samples from donors at ages below 20 years and over 70 years were chosen for young and aged group sets, respectively. Nuclei: DAPI.


B 3D 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 .

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

B 3D images of young and aged human ovary immunolabelled with α-SMA, Vimentin and CD31. Images in the middle panel with a surface rendered for all the channels. Arrowheads indicate arteries. Combo plots with quantifications of the number of arteries and vessel density. Data information: (n=4), P-values, and two-tailed unpaired t-tests for all the above panels. ns: not significant; *: P < 0.05; **: P < 0.01; ****: P < 0.0001. Human samples from donors at ages below 20 years and over 70 years were chosen for young and aged group sets, respectively. Nuclei: DAPI.


C Representative 3D 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 .

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

C Representative 3D images (left) of young and aged human testis with SM22α and Endoglin immunostaining. Middle panel images surface rendered for all the channels. Arrowheads indicate arteries. Combo plots show quantifications of the number of arteries and vessel density. Data information: (n=4), P-values, and two-tailed unpaired t-tests for all the above panels. ns: not significant; *: P < 0.05; **: P < 0.01; ****: P < 0.0001. Human samples from donors at ages below 20 years and over 70 years were chosen for young and aged group sets, respectively. Nuclei: DAPI.


D 3D 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 .

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

D 3D images from young and aged human pancreas staining with α-SMA, Vimentin and Endoglin. 3D images (middle) with a surface rendering for all the channels. Arrowheads indicate arteries. Combined box and whiskers, and scatter plots with the quantifications of the number of arteries and vessel density. Data information: (n=4), P-values, and two-tailed unpaired t-tests for all the above panels. ns: not significant; *: P < 0.05; **: P < 0.01; ****: P < 0.0001. Human samples from donors at ages below 20 years and over 70 years were chosen for young and aged group sets, respectively. Nuclei: DAPI.


E Representative 3D 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 .

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

E Representative 3D images (left) show α-SMA and CD31 immunostaining in young and aged human thyroid gland. Middle panel images surface rendered for α-SMA, Vimentin and CD31. Arrowheads indicate arteries. Combo plots with quantifications of the number of arteries and vessel density. Data information: (n=4), P-values, and two-tailed unpaired t-tests for all the above panels. ns: not significant; *: P < 0.05; **: P < 0.01; ****: P < 0.0001. Human samples from donors at ages below 20 years and over 70 years were chosen for young and aged group sets, respectively. Nuclei: DAPI.


F qPCR analysis of Efnb2 , Cdh5 and Kdr expression ( normalized to Actb ) in aged human endocrine glands compared to young glands . Data information : ( n = 4 ) , P - values , and two - tailed unpaired t - tests for all the above panels . ns : not significant ; * : P < 0 . 05 ; * * : P < 0 . 01 ; * * * * : P < 0 . 0001 . Human samples from donors at ages below 20 years and over 70 years were chosen for young and aged group sets , respectively .

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

F qPCR analysis of Efnb2, Cdh5 and Kdr expression (normalized to Actb) in aged human endocrine glands compared to young glands. Data information: (n=4), P-values, and two-tailed unpaired t-tests for all the above panels. ns: not significant; *: P < 0.05; **: P < 0.01; ****: P < 0.0001. Human samples from donors at ages below 20 years and over 70 years were chosen for young and aged group sets, respectively.


A Representative 3D 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 .

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

A Representative 3D images with Insulin and Caveolin-1 immunostaining in pancreatic islets from young mice. Combined box and whiskers, and scatter plots show the vessel density analysis in the pancreatic islets and non-islet areas from young and aged mice. Data information: The white and dashed lines in panel A, B, D, F, H and I represent the outlines of pancreatic islets. (n=5), P-values, and two-tailed unpaired t-tests in panel (A, E and I) and one-way ANOVA tests with Tukey's multiple comparisons tests in panel (G and H). *: P < 0.05; **: P < 0.01; ***: P < 0.001; ****: P < 0.0001. Nuclei: TO-PRO-3. Is: pancreatic islet. f.u: fluorescence unit.


B Representative 3D 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 .

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

B Representative 3D images with adult pancreatic islets immunolabeled with Ki67 and VE-Cadherin. Inset (*) shows single-cell resolution and surface rendering for all the channels. Data information: The white and dashed lines in panel A, B, D, F, H and I represent the outlines of pancreatic islets. (n=5), P-values, and two-tailed unpaired t-tests in panel (A, E and I) and one-way ANOVA tests with Tukey's multiple comparisons tests in panel (G and H). *: P < 0.05; **: P < 0.01; ***: P < 0.001; ****: P < 0.0001. Nuclei: TO-PRO-3. Is: pancreatic islet. f.u: fluorescence unit.


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

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

C Representative tile scan 3D image with Insulin, ESM-1 and Emcn immunostaining in 19-weeks-old mouse pancreas. Higher magnification at the top of the right panel shows the ESM-1+ pancreatic islets. Inset (*) shows single-cell resolution and surface rendering for ESM-1 and Emcn. The white and dashed line in the tile scan image represents the outline of one lobe of the pancreas, and the dashed lines in the inset represents the pancreatic islet. Data information: The white and dashed lines in panel A, B, D, F, H and I represent the outlines of pancreatic islets. (n=5), P-values, and two-tailed unpaired t-tests in panel (A, E and I) and one-way ANOVA tests with Tukey's multiple comparisons tests in panel (G and H). *: P < 0.05; **: P < 0.01; ***: P < 0.001; ****: P < 0.0001. Nuclei: TO-PRO-3. Is: pancreatic islet. f.u: fluorescence unit.


D Representative 3D 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 .

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

D Representative 3D images show CD31 and Emcn expressions in young pancreas. Insets (* and #) show higher magnifications of capillaries in the pancreatic islets and non-islet areas. Data information: The white and dashed lines in panel A, B, D, F, H and I represent the outlines of pancreatic islets. (n=5), P-values, and two-tailed unpaired t-tests in panel (A, E and I) and one-way ANOVA tests with Tukey's multiple comparisons tests in panel (G and H). *: P < 0.05; **: P < 0.01; ***: P < 0.001; ****: P < 0.0001. Nuclei: TO-PRO-3. Is: pancreatic islet. f.u: fluorescence unit.


E Representative flow cytometry dot plots showing CD31 + Emcnhi and CD31 + Emcnlo EC populations in young and aged pancreas . Combo plot shows the population of CD31 + Emcnhi ECs in young and aged pancreas .

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

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

F Representative 3D images (left) with Insulin, Ki67 and VE-Cadherin immunostaining on young and aged pancreas. 3D images (middle) show CXCR4, Ki67 and VE-Cadherin in young and aged pancreatic islet. Insets (yellow asterisks) show single-cell resolution and surface rendering for Ki67 and CXCR4. Arrowheads represent Ki67+ β-cell. G Combo plot shows the quantification of Ki67+ β-cells normalized to the total number of β-cells per islet in juvenile (3-weeks-old), adult (12-weeks-old) and aged (55-weeks-old) pancreatic islets. Data information: The white and dashed lines in panel A, B, D, F, H and I represent the outlines of pancreatic islets. (n=5), P-values, and two-tailed unpaired t-tests in panel (A, E and I) and one-way ANOVA tests with Tukey's multiple comparisons tests in panel (G and H). *: P < 0.05; **: P < 0.01; ***: P < 0.001; ****: P < 0.0001. Nuclei: TO-PRO-3. Is: pancreatic islet. f.u: fluorescence unit.


F Representative 3D 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 .

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

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


H Representative 3D 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 .

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

H Representative 3D images with Insulin, CD31 and Emcn immunostaining in young and aged pancreas. Combo plot shows the CD31+ Emcnhi vessel density in juvenile, adult and aged pancreatic islets. Data information: The white and dashed lines in panel A, B, D, F, H and I represent the outlines of pancreatic islets. (n=5), P-values, and two-tailed unpaired t-tests in panel (A, E and I) and one-way ANOVA tests with Tukey's multiple comparisons tests in panel (G and H). *: P < 0.05; **: P < 0.01; ***: P < 0.001; ****: P < 0.0001. Nuclei: TO-PRO-3. Is: pancreatic islet. f.u: fluorescence unit.


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

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

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 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 .

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

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


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

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

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 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 .

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

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

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

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

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


G Representative 3D 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 .

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

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 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 .

O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR 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 analysis of Pdgfa , Pdgfb , Igf1 , Igf2 , Cxcl12 , Hgf and Kitl expression ( normalized to Actb ) in purified CD31 + Emcnhi ECs compared to CD31 + Emcnlo ECs .

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

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


J Bar graph with the quantification of Ki67 + β - cell numbers per islet ( f . u ) and Ki67 + EC numbers per islet ( f . u ) . 45 pancreatic islets were analysed and quantified from 9 young mice .

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

J Bar graph with the quantification of Ki67+ β-cell numbers per islet (f.u) and Ki67+ EC numbers per islet (f.u). 45 pancreatic islets were analysed and quantified from 9 young mice.


K Bar graphs show qPCR analysis of Dll4 , Emcn , Kdr , Flt4 , Sele , Mmp9 , Plxnd1 , Robo4 , Unc5b , Cdh5 , Cldn5 , Gja1 , Gja4 , Icam1 , Icam2 , Jam3 , Ntn1 , Ntn2 , Ocln , Pecam1 , Tjp1 , Tjp2 , Tjp3 and Vcam1 expression ( normalized to Actb ) in purified CD31 + Emcnhi ECs compared to CD31 + Emcnlo ECs from murine pancreas .

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

K Bar graphs show qPCR analysis of Dll4, Emcn, Kdr, Flt4, Sele, Mmp9, Plxnd1, Robo4, Unc5b, Cdh5, Cldn5, Gja1, Gja4, Icam1, Icam2, Jam3, Ntn1, Ntn2, Ocln, Pecam1, Tjp1, Tjp2, Tjp3 and Vcam1 expression (normalized to Actb) in purified CD31+ Emcnhi ECs compared to CD31+ Emcnlo ECs from murine pancreas.


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

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

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


A Representative tile scan 3D 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 .

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

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

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 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 .

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

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


H 3D images with CXCR4 and Emcn immunostaining in Gja1i ∆ EC and control pancreas . The scale bars are 50 µm . I Representative 3D images show CXCR4 , Ki67 and VE - Cadherin immunostaining in Gja1i ∆ EC and control pancreas . The scale bars are 50 µm . J Combo plots show the percentage of CXCR4 + β - cells per islet and Ki67 + β - cells normalized to the total number of β - cells per islet in Gja1i ∆ EC mutant versus the littermate control mice . ( n = 5 ) , P - values , and two - tailed unpaired t - tests . K Box whiskers , and scatter plot shows the quantification of CD31 + Emcnhi vessel density in aged Gja1i ∆ EC mutant and control pancreatic islets . Data represent mean ± s . d . ( n = 4 ) , P - value , and two - tailed unpaired t - test . Combo plot ( below ) shows the quantification of β - cells on thick murine pancreatic sections . Data represent mean ± s . d . ( n = 4 ) , P - value , and two - tailed unpaired t - test . Data information : The dashed lines in panel C , E , G , H and I represent the outlines of pancreatic islets . Two - tailed Student ' s t - test were performed for the statistical analysis . * * : P < 0 . 01 ; * * * * : P < 0 . 0001 . Nuclei : TO - PRO -

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

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

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

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


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

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

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


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

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

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


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 ) .

O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O 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).


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

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

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


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

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

(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 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 .

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

(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 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 .

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

(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 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 .

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

(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 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 .

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

(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 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 .

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

(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 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 .

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

(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 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 .

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

(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 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 .

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

(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 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 .

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

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

(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.


( a ) Stable 293T cells in the presence of pro - caspase - 1 , pro - IL - 1β and ASC overexpression were transfected with control vector or BRLF1 with control vector , IFI16 , NLRP3 , RIG - I or AIM2 for 36 h . After the cells were infected with HSV - 1 ( MOI = 1 ) for 12 h , the cells were collected , and the cell lysates were analyzed by western blotting to determine the levels of pro - caspase - 1 , cleaved caspase - 1 , pro - IL - 1β and mature IL - 1β as indicated .

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

(a) Stable 293T cells in the presence of pro-caspase-1, pro-IL-1β and ASC overexpression were transfected with control vector or BRLF1 with control vector, IFI16, NLRP3, RIG-I or AIM2 for 36 h. After the cells were infected with HSV-1 (MOI=1) for 12 h, the cells were collected, and the cell lysates were analyzed by western blotting to determine the levels of pro-caspase-1, cleaved caspase-1, pro-IL-1β and mature IL-1β as indicated.


( b ) Scramble ( sc ) or shRNAs against RIG - I and empty control vector ( ctr ) , BRLF1 - or BRLF1 L578A - expressing plasmids were co - transfected into EBV - ΔBRLF1 - harboring HNE1 cells . Thirty - six hours after the transfection , cell pellets were collected , after which the cleavage of caspase - 1 and IL - 1β was measured as indicated .

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

(b) Scramble (sc) or shRNAs against RIG-I and empty control vector (ctr), BRLF1- or BRLF1 L578A-expressing plasmids were co-transfected into EBV-ΔBRLF1-harboring HNE1 cells. Thirty-six hours after the transfection, cell pellets were collected, after which the cleavage of caspase-1 and IL-1β was measured as indicated.


( c ) Analysis of ASC oligomerization . ASC - expressing plasmid was transfected into 293T cells or HNE1 cells with empty vector or GFP - BRLF1 in the presence of AIM2 - or RIG - I - expressing plasmid . Twelve hours after HSV - 1 infection ( MOI = 1 ) in 293T cells , or 48 h after EBV - ΔBRLF1 infection ( MOI = 1000 ) in HNE1 cells , the cell pellets were collected , and cell lysates were treated with DSS to induce chemical cross - linking and analyzed by western blotting for ASC oligomerization .

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

(c) Analysis of ASC oligomerization. ASC-expressing plasmid was transfected into 293T cells or HNE1 cells with empty vector or GFP-BRLF1 in the presence of AIM2- or RIG-I-expressing plasmid. Twelve hours after HSV-1 infection (MOI=1) in 293T cells, or 48 h after EBV-ΔBRLF1 infection (MOI=1000) in HNE1 cells, the cell pellets were collected, and cell lysates were treated with DSS to induce chemical cross-linking and analyzed by western blotting for ASC oligomerization.


( d ) 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 .

(d) 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.


( e ) 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 .

(e) 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.


Jurkat cells ( a ) were infected with EBV - WT or EBV - ΔBRLF1 virus ( MOI = 500 ) for 36 h . Then , the cells were collected , and the total RNAs were extracted , reverse - transcribed and analyzed by real - time PCR . Color gradient depicts the mean mRNA expression normalized to the housekeeping gene GAPDH and compared to the mock group .

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

Jurkat cells (a) were infected with EBV-WT or EBV-ΔBRLF1 virus (MOI=500) for 36 h. Then, the cells were collected, and the total RNAs were extracted, reverse-transcribed and analyzed by real-time PCR. Color gradient depicts the mean mRNA expression normalized to the housekeeping gene GAPDH and compared to the mock group.


NK - 92MI cells ( b ) were infected with EBV - WT or EBV - ΔBRLF1 virus ( MOI = 500 ) for 36 h . Then , the cells were collected , and the total RNAs were extracted , reverse - transcribed and analyzed by real - time PCR . Color gradient depicts the mean mRNA expression normalized to the housekeeping gene GAPDH and compared to the mock group .

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

NK-92MI cells (b) were infected with EBV-WT or EBV-ΔBRLF1 virus (MOI=500) for 36 h. Then, the cells were collected, and the total RNAs were extracted, reverse-transcribed and analyzed by real-time PCR. Color gradient depicts the mean mRNA expression normalized to the housekeeping gene GAPDH and compared to the mock group.


Jurkat and NK - 92MI cells were cultured with conditional medium from EBV - WT or EBV - ΔBRLF1 - infected Ramos cells ( paracrine ) , or Jurkat and NK - 92MI cells were directly infected with EBV - WT or EBV - ΔBRLF1 viruses ( autocrine ) . ( c ) The cell pellets were collected and the whole cell extracts were analyzed by western blotting to detect TAK1 , IKKα / β and IκB phosphorylation .

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

Jurkat and NK-92MI cells were cultured with conditional medium from EBV-WT or EBV-ΔBRLF1-infected Ramos cells (paracrine), or Jurkat and NK-92MI cells were directly infected with EBV-WT or EBV-ΔBRLF1 viruses (autocrine). (c) The cell pellets were collected and the whole cell extracts were analyzed by western blotting to detect TAK1, IKKα/β and IκB phosphorylation.


The cell killing activity of Jurkat cells ( d ) were measured by the detection of the LDH release from the K562 target cells .

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

The cell killing activity of Jurkat cells (d) were measured by the detection of the LDH release from the K562 target cells.


The cell killing activity of NK - 92MI cells ( e ) were measured by the detection of the LDH release from the K562 target cells .

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

The cell killing activity of NK-92MI cells (e) were measured by the detection of the LDH release from the K562 target cells.


Anti - IL - 1β and / or anti - IL - 18 neutralizing antibodies ( NA ) , TAT - Flag or TAT - N572 peptide were added into EBV - WT - or EBV - ΔBRLF1 - infected Jurkat cells or NK - 92MI cells for 24 h . ( f ) TAK1 , IKKα and IκB phosphorylation were detected as indicated .

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

Anti-IL-1β and/or anti-IL-18 neutralizing antibodies (NA), TAT-Flag or TAT-N572 peptide were added into EBV-WT- or EBV-ΔBRLF1-infected Jurkat cells or NK-92MI cells for 24 h. (f) TAK1, IKKα and IκB phosphorylation were detected as indicated.


cell killing activity of Jurkat cells ( g ) were measured .

cell killing activity of Jurkat cells (g) were measured.


cell killing activity of NK - 92MI cells ( h ) were measured .

cell killing activity of NK-92MI cells (h) were measured.


( a - d ) PBMCs were isolated and infected with EBV - WT or EBV - ΔBRLF1 virus ( MOI = 500 ) . Thirty - six hours after infection , the cells were harvested and the total RNAs were extracted , reverse - transcribed and subjected to real - time PCR analysis . The differential expression of receptors and cytokines ( a ) , cytotoxic genes ( b ) , T cell - specific genes ( c ) and NK cell - specific genes ( d ) are shown . The values are shown as the mean ± standard deviations of five analyses of PBMCs from five different health donors .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-d) PBMCs were isolated and infected with EBV-WT or EBV-ΔBRLF1 virus (MOI=500). Thirty-six hours after infection, the cells were harvested and the total RNAs were extracted, reverse-transcribed and subjected to real-time PCR analysis. The differential expression of receptors and cytokines (a), cytotoxic genes (b), T cell-specific genes (c) and NK cell-specific genes (d) are shown. The values are shown as the mean ± standard deviations of five analyses of PBMCs from five different health donors.


( e - h ) FACS analysis of T and NK cell activation in PBMCs in the presence of EBV - WT or EBV - ΔBRLF1 primary infection . The PBMCs were left uninfected or infected with EBV - WT or EBV - ΔBRLF1 virus for 36 h , and the EBV - ΔBRLF1 - infected lymphocytes were incubated with additional TAT - Flag or TAT - N572 peptide for 12 h . Immunofluorescent PE - CD3 and APC - CD25 antibodies were used to detect T cell activation ( e , g ) , and PE - CD56 and APC - CD69 were used to detect NK cell activation ( f , h ) . Representative images of the FACS analysis of T cell and NK cell activation are shown ( e , f ) , and the percentages of activated T cells and NK cells were calculated and are shown as the mean ± standard deviation ( g , h ) for lymphocytes from 5 different health donors ;

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

(e-h) FACS analysis of T and NK cell activation in PBMCs in the presence of EBV-WT or EBV-ΔBRLF1 primary infection. The PBMCs were left uninfected or infected with EBV-WT or EBV-ΔBRLF1 virus for 36 h, and the EBV-ΔBRLF1-infected lymphocytes were incubated with additional TAT-Flag or TAT-N572 peptide for 12 h. Immunofluorescent PE-CD3 and APC-CD25 antibodies were used to detect T cell activation (e, g), and PE-CD56 and APC-CD69 were used to detect NK cell activation (f, h). Representative images of the FACS analysis of T cell and NK cell activation are shown (e, f), and the percentages of activated T cells and NK cells were calculated and are shown as the mean ± standard deviation (g, h) for lymphocytes from 5 different health donors;


B Representative images showing co - expression of the stage - specific markers Nestin , Tbr2 , Doublecortin ( DCX ) , and Calbindin ( all in green ) , with the β - Galactosidase ( β - Gal , red ) reporter in 8 - week old BATGAL and Axin2LacZ / + mice . Nuclei are counterstained with DAPI ( in blue ) . Scale bar = 10µm . Insets show a 1 . 5x magnification of selected cells ( position indicated by dashed box ) . Scale bar = 5µm .

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

B Representative images showing co-expression of the stage-specific markers Nestin, Tbr2, Doublecortin (DCX), and Calbindin (all in green), with the β-Galactosidase (β-Gal, red) reporter in 8-week old BATGAL and Axin2LacZ/+ mice. Nuclei are counterstained with DAPI (in blue). Scale bar= 10µm. Insets show a 1.5x magnification of selected cells (position indicated by dashed box). Scale bar= 5µm.


C Fraction of Nestin - , Tbr2 - , DCX - and Calbindin - positive cells expressing β - Gal in BATGAL and Axin2LacZ / + animals show stage specific canonical Wnt signaling during adult hippocampal neurogenesis [ BATGAL : Tbr2 vs . DCX p = 0 . 0056 , DCX vs . Calbindin p < 0 . 0001 ; Axin2LacZ : Nestin vs . Tbr2 p = 0 . 0003 , Tbr2 vs . DCX p = 0 . 0115 , DCX vs . Calbindin p < 0 . 0001 ] ( n = 3 animals per mouse model and marker ) .

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

C Fraction of Nestin-, Tbr2-, DCX- and Calbindin-positive cells expressing β-Gal in BATGAL and Axin2LacZ/+ animals show stage specific canonical Wnt signaling during adult hippocampal neurogenesis [BATGAL: Tbr2 vs. DCX p=0.0056, DCX vs. Calbindin p<0.0001; Axin2LacZ: Nestin vs. Tbr2 p=0.0003, Tbr2 vs. DCX p=0.0115, DCX vs. Calbindin p<0.0001] (n= 3 animals per mouse model and marker).


D Detection of the corrected total cell fluorescence ( CTCF ) of the β - Gal reporter in Nestin - , Tbr2 - , DCX - and Calbindin - positive cells in BATGAL mice corroborate stage specific activity pattern of canonical Wnt signaling during lineage progression [ Nestin vs . Tbr2 p < 0 . 0001 , Tbr2 vs . DCX p = 0 . 0029 , DCX vs . Calbindin p < 0 . 0001 ] ( Nestin : n = 236 cells , Tbr2 : n = 134 cells , DCX : n = 200 cells , Calbindin : n = 300 cells ) .

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

D Detection of the corrected total cell fluorescence (CTCF) of the β-Gal reporter in Nestin-, Tbr2-, DCX- and Calbindin-positive cells in BATGAL mice corroborate stage specific activity pattern of canonical Wnt signaling during lineage progression [Nestin vs. Tbr2 p <0.0001, Tbr2 vs. DCX p=0.0029, DCX vs. Calbindin p<0.0001] (Nestin: n= 236 cells, Tbr2: n= 134 cells, DCX: n= 200 cells, Calbindin: n= 300 cells).


F Quantification of BrdU + cells expressing β - Gal reveals biphasic activity of canonical Wnt signaling during adult hippocampal neurogenesis [ 0 vs . 7dpi p = 0 . 0036 , 7 vs . 28dpi p = 0 . 0115 , 7 vs . 42dpi p = 0 . 0007 ] ( 0dpi time - point ; n = 4 animals ) , 3dpi ( n = 3 animals ) , 7dpi ( n = 3 animals ) , 14dpi ( n = 4 animals ) , 28dpi ( n = 6 animals ) and 42dpi ( n = 6 animals ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Quantification of BrdU+ cells expressing β-Gal reveals biphasic activity of canonical Wnt signaling during adult hippocampal neurogenesis [0 vs. 7dpi p=0.0036, 7 vs. 28dpi p=0.0115, 7 vs. 42dpi p=0.0007] (0dpi time-point; n= 4 animals), 3dpi (n= 3 animals), 7dpi (n= 3 animals), 14dpi (n= 4 animals), 28dpi (n= 6 animals) and 42dpi (n= 6 animals).


G Total cell fluorescence of β - Gal in BrdU + cells indicate a biphasic pattern in canonical Wnt signaling strength during lineage progression [ 0 vs . 3dpi p < 0 . 0001 , 7 vs . 14dpi p < 0 . 0001 , 14 vs . 28dpi p < 0 . 0001 , 28 vs . 42dpi p = 0 . 0333 ] ( 0dpi n = 194 cells , 3dpi n = 245 cells , 7dpi n = 224 cells , 14dpi n = 72 cells , 28dpi n = 103 cells and 42dpi n = 114 cells )

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

G Total cell fluorescence of β-Gal in BrdU+ cells indicate a biphasic pattern in canonical Wnt signaling strength during lineage progression [0 vs. 3dpi p<0.0001, 7 vs. 14dpi p<0.0001, 14 vs. 28dpi p<0.0001, 28 vs. 42dpi p=0.0333] (0dpi n= 194 cells, 3dpi n= 245 cells, 7dpi n= 224 cells, 14dpi n= 72 cells, 28dpi n= 103 cells and 42dpi n= 114 cells)


B Representative images of transduced adult - born neurons at 17dpi . CAG - dnLEF - IRES - GFP ( dnLEF , green ) and CAG - RFP ( control , red ) double transduced cells ( arrows ) and CAG - RFP single transduced cells ( arrowheads ) . Scale bar = 20

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

B Representative images of transduced adult-born neurons at 17dpi. CAG-dnLEF-IRES-GFP (dnLEF, green) and CAG-RFP (control, red) double transduced cells (arrows) and CAG-RFP single transduced cells (arrowheads). Scale bar= 20 µm


C Representative reconstructions of control and dnLEF neurons . Scale bar = 20

C Representative reconstructions of control and dnLEF neurons. Scale bar= 20 µm


D Analysis of morphology showed a reduction in dendritic length in dnLEF transduced neurons [ p < 0 . 0001 ] , while the number of branch points remained comparable [ p = 0 . 7914 ] . Sholl analysis displayed a reduction in dendritic complexity in dnLEF transduced neurons and indicated decreased growth of terminal dendritic branches [ p < 0 . 0001 ] ( control : n = 20 cells from 3 animals , dnLEF : n = 38 cells from 6 animals ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Analysis of morphology showed a reduction in dendritic length in dnLEF transduced neurons [p<0.0001], while the number of branch points remained comparable [p=0.7914]. Sholl analysis displayed a reduction in dendritic complexity in dnLEF transduced neurons and indicated decreased growth of terminal dendritic branches [p<0.0001] (control: n= 20 cells from 3 animals, dnLEF: n= 38 cells from 6 animals).


E DnLEF neurons displayed basal dendrites [ p < 0 . 0001 ] ( control : n = 20 cells from 3 animals , dnLEF : n = 38 cells from 6 animals ) .

O O O O O O O O O O O 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 DnLEF neurons displayed basal dendrites [p<0.0001] (control: n= 20 cells from 3 animals, dnLEF: n= 38 cells from 6 animals).


G Representative images depicting CAG - GFP - IRES - Cre transduced adult - born neurons in control and β - catex3 mice at 17dpi . Scale bar =

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

G Representative images depicting CAG-GFP-IRES-Cre transduced adult-born neurons in control and β-catex3 mice at 17dpi. Scale bar= 20µm


H Representative reconstructions of control and β - catex3 neurons . Scale bar =

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

H Representative reconstructions of control and β-catex3 neurons. Scale bar= 20µm


I Quantification showed decreased dendritic length of β - catex3 neurons [ p < 0 . 0001 ] ; no difference was apparent in number of branch points [ p = 0 . 3256 ] . Sholl analysis displayed a less complex dendritic tree of β - catex3 neurons [ p < 0 . 0001 ] ( control : n = 20 cells from 5 animals , β - catex3 : n = 20 cells from 5 animals ) .

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

I Quantification showed decreased dendritic length of β-catex3 neurons [p<0.0001]; no difference was apparent in number of branch points [p=0.3256]. Sholl analysis displayed a less complex dendritic tree of β-catex3 neurons [p<0.0001] (control: n= 20 cells from 5 animals, β-catex3: n= 20 cells from 5 animals).


J The number of basal dendrites was increased in β - catex3 neurons [ p = 0 . 0003 ] ( control : n = 20 cells from 5 animals , β - catex3 : n = 20 cells from 5 animals ) .

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

J The number of basal dendrites was increased in β-catex3 neurons [p=0.0003] (control: n= 20 cells from 5 animals, β-catex3: n= 20 cells from 5 animals).


B Representative reconstructions of control and β - catex3 iDCX neurons at 3dpT . Scale bar = 20µm .

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

B Representative reconstructions of control and β-catex3 iDCX neurons at 3dpT. Scale bar= 20µm.


C Analysis of morphology showed increased dendritic length in β - catex3 iDCX neurons at 3dpT [ p = 0 . 0320 ] . The number of branch points was unaltered [ p = 0 . 0651 ] . Sholl analysis indicated increased growth of terminal dendrite branches in neurons of β - catex3 iDCX mice [ p < 0 . 0001 ] ( control : n = 25 cells from 9 animals ; β - catex3 iDCX : n = 25 cells from 8 animals ) . D Analysis of morphology showed no difference in dendritic length [ p = 0 . 1029 ] and branch point number [ p = 0 . 3726 ] between experimental groups at 13dpT . Sholl analysis indicated a decrease in length of terminal dendrites in neurons of β - catex3 iDCX mice [ p < 0 . 01 ] ( control : n = 23 cells from 9 animals , β - catex3 iDCX : n = 26 cells from 8 animals ) .

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

C Analysis of morphology showed increased dendritic length in β-catex3 iDCX neurons at 3dpT [p=0.0320]. The number of branch points was unaltered [p=0.0651]. Sholl analysis indicated increased growth of terminal dendrite branches in neurons of β-catex3 iDCX mice [p<0.0001] (control: n = 25 cells from 9 animals; β-catex3 iDCX: n = 25 cells from 8 animals). D Analysis of morphology showed no difference in dendritic length [p=0.1029] and branch point number [p=0.3726] between experimental groups at 13dpT. Sholl analysis indicated a decrease in length of terminal dendrites in neurons of β-catex3 iDCX mice [p<0.01] (control: n= 23 cells from 9 animals, β-catex3 iDCX: n= 26 cells from 8 animals).


E Spine densities were comparable between experimental groups at 3dpT [ iML p = 0 . 1512 , oML p = 0 . 3841 ] . 13dpT β - catex3 iDCX neurons showed significantly increased spine density in the inner molecular layer [ iML p < 0 . 0001 , oML p = 0 . 0402 ] ( 3dpT : control : n = 20 cells from 3 animals , β - catex3 iDCX : n = 20 cells from 4 animals ; 13dpT control : n = 20 cells from 5 animals , β - catex3 iDCX : n = 20 cells from 7 animals ) . Representative images showing dendritic segments in the inner molecular layer of the DG at 13dpT . Scale bars = 1µm .

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

E Spine densities were comparable between experimental groups at 3dpT [iML p=0.1512, oML p=0.3841]. 13dpT β-catex3 iDCX neurons showed significantly increased spine density in the inner molecular layer [iML p<0.0001 , oML p=0.0402] (3dpT: control: n= 20 cells from 3 animals, β-catex3 iDCX: n= 20 cells from 4 animals; 13dpT control: n= 20 cells from 5 animals, β-catex3 iDCX: n= 20 cells from 7 animals). Representative images showing dendritic segments in the inner molecular layer of the DG at 13dpT. Scale bars= 1µm.