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

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


D 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 O O O O O O 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 O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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

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

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

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 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

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

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 B-CONTROLLED_VAR 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 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

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

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


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 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

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


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 O O O O O O O O O O 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 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

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 O O O O O O O O O O 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

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

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


J Bar graph with 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


K Bar graphs show qPCR 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


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

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

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O 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.


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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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.


RNA - seq of liver 10h post Sham or CLP . Mice ( n = 3 / group ) underwent a sham or CLP operation and were injected with GW7647 ( 10 µg / g ) 6h post - surgery , after 4h ( total of 10h ) , livers were isolated and RNA was prepared . Scatter plot showing log fold change ( LFC ) of all GW7647 - upregulated genes ( LFC > 0 . 8 and P < 0 . 05 ) in sham versus their LFC 10h after CLP . The red line represents the diagonal , the black line represents the real slope ( 0 . 3115 ) of the data .

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

RNA-seq of liver 10h post Sham or CLP. Mice (n = 3/group) underwent a sham or CLP operation and were injected with GW7647 (10 µg/g) 6h post-surgery, after 4h (total of 10h), livers were isolated and RNA was prepared. Scatter plot showing log fold change (LFC) of all GW7647-upregulated genes (LFC > 0.8 and P < 0.05) in sham versus their LFC 10h after CLP. The red line represents the diagonal, the black line represents the real slope (0.3115) of the data.


RNA - seq of liver 10h post Sham or CLP . Mice ( n = 3 / group ) underwent a sham or CLP operation and were injected with GW7647 ( 10 µg / g ) 6h post - surgery , after 4h ( total of 10h ) , livers were isolated and RNA was prepared . Heatmap of differentially expressed genes in sham mice after GW7647 treatment , involved in β - oxidation of fatty acids ( unit scale bar = log2 of the normalized counts ) .

O O O O O 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 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

RNA-seq of liver 10h post Sham or CLP. Mice (n = 3/group) underwent a sham or CLP operation and were injected with GW7647 (10 µg/g) 6h post-surgery, after 4h (total of 10h), livers were isolated and RNA was prepared. Heatmap of differentially expressed genes in sham mice after GW7647 treatment, involved in β-oxidation of fatty acids (unit scale bar = log2 of the normalized counts).


Confirmation of RNA - seq data via qPCR on pure hepatocytes isolated via flow cytometry - based sorting ( n = 3 / group ) . ( E ) Ppara and ( F ) Hmgcs2 mRNA expression is shown as relative expression , normalized to housekeeping genes Hprt and Rpl . P - values were calculated using 2 - way ANOVA analysis .

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

Confirmation of RNA-seq data via qPCR on pure hepatocytes isolated via flow cytometry-based sorting (n = 3/group). (E) Ppara and (F) Hmgcs2 mRNA expression is shown as relative expression, normalized to housekeeping genes Hprt and Rpl. P-values were calculated using 2-way ANOVA analysis.


Mice ( n = 4 / group , data are representative of 2 experiments ) underwent a sham ( with or without starvation ) or CLP operation and liver was isolated on several timepoints post - surgery for RNA preparation and qPCR . Ppara mRNA expression is shown as relative expression , normalized to housekeeping genes Hprt and Rpl . P - values were calculated using 1 - way ANOVA analysis .

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

Mice (n = 4/group, data are representative of 2 experiments) underwent a sham (with or without starvation) or CLP operation and liver was isolated on several timepoints post-surgery for RNA preparation and qPCR. Ppara mRNA expression is shown as relative expression, normalized to housekeeping genes Hprt and Rpl. P-values were calculated using 1-way ANOVA analysis.


PPARA protein levels were analyzed in livers 24h after CLP by Western Blot using actin as a loading control .

PPARA protein levels were analyzed in livers 24h after CLP by Western Blot using actin as a loading control.


Quantification of PPARA western blot . P - value was calculated via 2 - way Student ' s t - test ( n = 4 / group ) .

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

Quantification of PPARA western blot. P-value was calculated via 2-way Student's t-test (n = 4/group).


Pearson correlation between log fold change ( LFC ) Ppara expression levels and body temperature 24h post - sepsis ( n = 38 , r = 0 . 6875 , combined data of 4 independent experiments ) .

Pearson correlation between log fold change (LFC) Ppara expression levels and body temperature 24h post-sepsis (n= 38, r = 0.6875, combined data of 4 independent experiments).


Liver Hmgcs2 mRNA expression at different timepoints post - sepsis , expression is shown as relative expression , normalized to housekeeping genes Hprt and Rpl . P - values were calculated using 1 - way ANOVA analysis ( n = 4 / group , data are representative of 2 experiments ) .

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

Liver Hmgcs2 mRNA expression at different timepoints post-sepsis, expression is shown as relative expression, normalized to housekeeping genes Hprt and Rpl. P-values were calculated using 1-way ANOVA analysis (n = 4/group, data are representative of 2 experiments).


Oxygen consumption rates ( OCR ) of liver tissue explants 24h post sham or CLP . Liver tissue was isolated 24h post - surgery and OCR was measured via Seahorse with BSA or palmitic acid ( PA ) as a substrate for 42 min . n = 4 / group .

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

Oxygen consumption rates (OCR) of liver tissue explants 24h post sham or CLP. Liver tissue was isolated 24h post-surgery and OCR was measured via Seahorse with BSA or palmitic acid (PA) as a substrate for 42 min. n = 4/group.


Visualization of T42 ( min ) OCR , p - values were calculated using 2 - way ANOVA analysis .

Visualization of T42(min) OCR, p-values were calculated using 2-way ANOVA analysis.


Percentage of body weight that is taken up by the inguinal fat pad ( iWAT , A ) , the mesenteric fat pad ( mWAT , B ) and the perirenal fat pad ( pWAT , C ) 24h post - surgery in sham ( with or without starvation ) and CLP mice . P - values were calculated with a 1 - way ANOVA test . Combined data of 2 independent experiments , n = 6 / group .

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

Percentage of body weight that is taken up by the inguinal fat pad (iWAT, A), the mesenteric fat pad (mWAT, B) and the perirenal fat pad (pWAT, C) 24h post-surgery in sham (with or without starvation) and CLP mice. P-values were calculated with a 1-way ANOVA test. Combined data of 2 independent experiments, n = 6/group.


Visual representation of ( D ) iWAT and ( E ) pWAT sizes in sham , sham starved and CLP mice 24h post - surgery . Data are representative of 3 independent experiments .

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

Visual representation of (D) iWAT and (E) pWAT sizes in sham, sham starved and CLP mice 24h post-surgery. Data are representative of 3 independent experiments.


Mice ( n = 6 - 10 / group ) underwent a sham ( with or without starvation ) or CLP operation and blood was collected 6h and 24h post - surgery . Plasma was isolated and ( F ) FFA concentration and ( G ) glycerol concentration was determined as described in the method section . P - values were calculated with 2 - way ANOVA tests . Combined data of 2 independent experiments .

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

Mice (n=6-10/group) underwent a sham (with or without starvation) or CLP operation and blood was collected 6h and 24h post-surgery. Plasma was isolated and (F) FFA concentration and (G) glycerol concentration was determined as described in the method section. P-values were calculated with 2-way ANOVA tests. Combined data of 2 independent experiments.


Mice ( n = 6 - 10 / group ) underwent a sham ( with or without starvation ) or CLP operation and blood was collected 6h and 24h post - surgery . Normalized abundances of ( H ) palmitic acid and ( I ) palmitoyl - carnitine determined via liquid chromotography - mass spectrometric lipidomics . Values were normalized to IQ values , P - values were calculated with 2 - way ANOVA tests . n = 8 / group .

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

Mice (n=6-10/group) underwent a sham (with or without starvation) or CLP operation and blood was collected 6h and 24h post-surgery. Normalized abundances of (H) palmitic acid and (I) palmitoyl-carnitine determined via liquid chromotography-mass spectrometric lipidomics. Values were normalized to IQ values , P-values were calculated with 2-way ANOVA tests. n = 8/group.


Immunofluorescent images of liver and kidney 24h after sham ( with or without starvation ) or CLP ( n = 6 - 7 / group , combined data of 2 independent experiments ) . White scale bar = 20µm . Cryosections were stained with Acti - stain ( green ) , Hoechst ( blue ) , and LipidTOX ( red ) . Z - stacks were generated in 5 - 10 areas scattered across the entire tissue section .

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

Immunofluorescent images of liver and kidney 24h after sham (with or without starvation) or CLP (n= 6-7/group, combined data of 2 independent experiments). White scale bar = 20µm. Cryosections were stained with Acti-stain (green), Hoechst (blue), and LipidTOX (red). Z-stacks were generated in 5-10 areas scattered across the entire tissue section.


Quantification of lipid peroxidation by determination of ( C ) MDA and ( D ) 4 - HNE concentrations in liver homogenates 24h post - surgery in sham and CLP mice as described in methods ( n = 6 - 7 / group , combined data of 2 independent experiments ) . P - values were calculated with 1 - way ANOVA tests .

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

Quantification of lipid peroxidation by determination of (C) MDA and (D) 4-HNE concentrations in liver homogenates 24h post-surgery in sham and CLP mice as described in methods (n = 6-7/group, combined data of 2 independent experiments). P-values were calculated with 1-way ANOVA tests.


Apoptosis in liver paraffin - fixated sections 24h after sepsis , measured by TUNEL staining , and presented as % PI positive cells / µm² tissue area . ( n = 6 / 7 mice / group , combined data of 2 independent experiments ) . P - values were calculated with 1 - way ANOVA tests .

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

Apoptosis in liver paraffin-fixated sections 24h after sepsis, measured by TUNEL staining, and presented as % PI positive cells/µm² tissue area. (n= 6/7 mice/group, combined data of 2 independent experiments). P-values were calculated with 1-way ANOVA tests.


Mice were pre - treated with Pemafibrate ( 1mg / kg ) or vehicle ( 0 , 9 % NaCl ) for 1 week before being subjected to CLP . Survival was monitored during 9 days , after which no further deaths occurred . Survival curve was analyzed via a Log rank test . Combined data of 3 experiments , n = 20 / group .

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

Mice were pre-treated with Pemafibrate (1mg/kg) or vehicle (0,9% NaCl) for 1 week before being subjected to CLP. Survival was monitored during 9 days, after which no further deaths occurred. Survival curve was analyzed via a Log rank test. Combined data of 3 experiments, n = 20/group.


Mice were pre - treated with Pemafibrate ( 1mg / kg ) or vehicle ( 0 , 9 % NaCl ) for 1 week before being subjected to CLP . Liver samples were isolated 24h after CLP ( n = 5 - 7 / group , combined data of 2 independent experiments ) , mRNA was prepared and gene expression levels of Ppara was analyzed via qPCR . Gene expression values are shown as relative expression , normalized to housekeeping genes Hprt and Rpl . P - values were calculated via 2 - way ANOVA test .

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

Mice were pre-treated with Pemafibrate (1mg/kg) or vehicle (0,9% NaCl) for 1 week before being subjected to CLP. Liver samples were isolated 24h after CLP (n= 5-7/group, combined data of 2 independent experiments), mRNA was prepared and gene expression levels of Ppara was analyzed via qPCR. Gene expression values are shown as relative expression, normalized to housekeeping genes Hprt and Rpl. P-values were calculated via 2-way ANOVA test.


Mice were pre - treated with Pemafibrate ( 1mg / kg ) or vehicle ( 0 , 9 % NaCl ) for 1 week before being subjected to CLP . Plasma was isolated 24h after sepsis and FFA concentration and ( D ) glycerol concentration were determined as described in the method section . P - values were calculated with 2 - way ANOVA tests . n = 5 - 7 / group , combined data of 2 independent experiments . Arrows represent the % of decrease caused by pemafibrate treatment during sepsis .

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

Mice were pre-treated with Pemafibrate (1mg/kg) or vehicle (0,9% NaCl) for 1 week before being subjected to CLP. Plasma was isolated 24h after sepsis and FFA concentration and (D) glycerol concentration were determined as described in the method section. P-values were calculated with 2-way ANOVA tests. n = 5-7/group, combined data of 2 independent experiments. Arrows represent the % of decrease caused by pemafibrate treatment during sepsis.


Mice were pre - treated with Pemafibrate ( 1mg / kg ) or vehicle ( 0 , 9 % NaCl ) for 1 week before being subjected to CLP . Immunofluorescent images of cryosections of liver 24h post - surgery that were stained with Acti - stain ( green ) , hoechst ( blue ) and LipidTOX ( red ) . Z - stacks were generated in 5 - 10 areas scattered across the entire tissue section . White scale bar = 20µm .

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

Mice were pre-treated with Pemafibrate (1mg/kg) or vehicle (0,9% NaCl) for 1 week before being subjected to CLP. Immunofluorescent images of cryosections of liver 24h post-surgery that were stained with Acti-stain (green), hoechst (blue) and LipidTOX (red). Z-stacks were generated in 5-10 areas scattered across the entire tissue section. White scale bar = 20µm.


Mice were pre - treated with Pemafibrate ( 1mg / kg ) or vehicle ( 0 , 9 % NaCl ) for 1 week before being subjected to CLP . Quantification of lipid peroxidation by determination of MDA concentration in liver homogenates 24h post - surgery in CLP mice ( n = 6 - 7 / group , combined data of 2 independent experiments ) , as described in methods . P - values were calculated with 2 - way ANOVA tests . Arrows represent the % of decrease caused by pemafibrate treatment during sepsis .

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 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 B-CONTROLLED_VAR O O O O

Mice were pre-treated with Pemafibrate (1mg/kg) or vehicle (0,9% NaCl) for 1 week before being subjected to CLP. Quantification of lipid peroxidation by determination of MDA concentration in liver homogenates 24h post-surgery in CLP mice (n= 6-7/group, combined data of 2 independent experiments ), as described in methods. P-values were calculated with 2-way ANOVA tests. Arrows represent the % of decrease caused by pemafibrate treatment during sepsis.


Mice were pre - treated with Pemafibrate ( 1mg / kg ) or vehicle ( 0 , 9 % NaCl ) for 1 week before being subjected to CLP . Quantification of lipid peroxidation by determination of MDA concentration in kidney homogenates 24h post - surgery in CLP mice ( n = 6 - 7 / group , combined data of 2 independent experiments ) , as described in methods . P - values were calculated with 2 - way ANOVA tests . Arrows represent the % of decrease caused by pemafibrate treatment during sepsis .

Mice were pre-treated with Pemafibrate (1mg/kg) or vehicle (0,9% NaCl) for 1 week before being subjected to CLP. Quantification of lipid peroxidation by determination of MDA concentration in kidney homogenates 24h post-surgery in CLP mice (n= 6-7/group, combined data of 2 independent experiments ), as described in methods. P-values were calculated with 2-way ANOVA tests. Arrows represent the % of decrease caused by pemafibrate treatment during sepsis.


Mice were pre - treated with Pemafibrate ( 1mg / kg ) or vehicle ( 0 , 9 % NaCl ) for 1 week before being subjected to CLP . Oxygen consumption rates ( OCR ) of liver tissue explants 24h post CLP ( vehicle or pemafibrate treated ) after supplementation of palmitic acid ( PA ) , measured via Seahorse . Visualization of T42 ( min ) OCR . P - values were calculates using 2 - way ANOVA analysis . One experiment , n = 3 / group .

O 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-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

Mice were pre-treated with Pemafibrate (1mg/kg) or vehicle (0,9% NaCl) for 1 week before being subjected to CLP. Oxygen consumption rates (OCR) of liver tissue explants 24h post CLP (vehicle or pemafibrate treated) after supplementation of palmitic acid (PA), measured via Seahorse. Visualization of T42(min) OCR. P-values were calculates using 2-way ANOVA analysis. One experiment, n = 3/group.


Mice were pre - treated with pemafibrate ( 1mg / kg ) or vehicle ( 0 , 9 % NaCl ) for 1 week before being subjected to CLP . Plasma was collected 24h post - surgery and ( A ) Alanine - amino transferase ( ALT ) , were measured as described in methods . P - values were calculated with 2 - way ANOVA tests . n = 6 - 7 / group , combined data of 2 independent experiments .

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

Mice were pre-treated with pemafibrate (1mg/kg) or vehicle (0,9% NaCl) for 1 week before being subjected to CLP. Plasma was collected 24h post-surgery and (A) Alanine-amino transferase (ALT), were measured as described in methods. P-values were calculated with 2-way ANOVA tests. n = 6-7/group, combined data of 2 independent experiments.


Mice were pre - treated with pemafibrate ( 1mg / kg ) or vehicle ( 0 , 9 % NaCl ) for 1 week before being subjected to CLP . Plasma was collected 24h post - surgery and creatinine concentrations , were measured as described in methods . P - values were calculated with 2 - way ANOVA tests . n = 6 - 7 / group , combined data of 2 independent experiments .

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 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

Mice were pre-treated with pemafibrate (1mg/kg) or vehicle (0,9% NaCl) for 1 week before being subjected to CLP. Plasma was collected 24h post-surgery and creatinine concentrations, were measured as described in methods. P-values were calculated with 2-way ANOVA tests. n = 6-7/group, combined data of 2 independent experiments.


Mice were pre - treated with pemafibrate ( 1mg / kg ) or vehicle ( 0 , 9 % NaCl ) for 1 week before being subjected to CLP . Plasma was collected 24h post - surgery and IL - 6 levels were measured as described in methods . P - values were calculated with 2 - way ANOVA tests . n = 6 - 7 / group , combined data of 2 independent experiments .

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

Mice were pre-treated with pemafibrate (1mg/kg) or vehicle (0,9% NaCl) for 1 week before being subjected to CLP. Plasma was collected 24h post-surgery and IL-6 levels were measured as described in methods. P-values were calculated with 2-way ANOVA tests. n = 6-7/group, combined data of 2 independent experiments.


Mice were pre - treated with pemafibrate ( 1mg / kg ) or vehicle ( 0 , 9 % NaCl ) for 1 week before being subjected to CLP . Apoptosis in liver paraffin - fixated sections 24h after sepsis , measured with TUNEL staining and depicted as % of PI positive cells / µm² tissue area . P - values were calculated with 2 - way ANOVA tests . n = 6 - 7 / group , combined data of 2 independent experiments .

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

Mice were pre-treated with pemafibrate (1mg/kg) or vehicle (0,9% NaCl) for 1 week before being subjected to CLP. Apoptosis in liver paraffin-fixated sections 24h after sepsis, measured with TUNEL staining and depicted as % of PI positive cells/µm² tissue area. P-values were calculated with 2-way ANOVA tests. n = 6-7/group, combined data of 2 independent experiments.


Mice were pre - treated with pemafibrate ( 1mg / kg ) or vehicle ( 0 , 9 % NaCl ) for 1 week before being subjected to CLP . Apoptosis in kidney paraffin - fixated sections 24h after sepsis , measured with TUNEL staining and depicted as % of PI positive cells / µm² tissue area . P - values were calculated with 2 - way ANOVA tests . n = 6 - 7 / group , combined data of 2 independent experiments .

Mice were pre-treated with pemafibrate (1mg/kg) or vehicle (0,9% NaCl) for 1 week before being subjected to CLP. Apoptosis in kidney paraffin-fixated sections 24h after sepsis, measured with TUNEL staining and depicted as % of PI positive cells/µm² tissue area. P-values were calculated with 2-way ANOVA tests. n = 6-7/group, combined data of 2 independent experiments.


Mice were pre - treated with pemafibrate ( 1mg / kg ) or vehicle ( 0 , 9 % NaCl ) for 1 week before being subjected to CLP . Systemic bacterial load ( CFU / ml blood ) 24h post - sepsis in vehicle or pemafibrate treated mice . P - values were calculated with 2 - way Student ' s t - test .

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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O

Mice were pre-treated with pemafibrate (1mg/kg) or vehicle (0,9% NaCl) for 1 week before being subjected to CLP. Systemic bacterial load (CFU/ml blood) 24h post-sepsis in vehicle or pemafibrate treated mice. P-values were calculated with 2-way Student's t-test.


Pemafibrate ( 1 mg / kg ) or vehicle ( 0 , 9 % NaCl ) was administered at different timepoints before and after the induction of sepsis and survival was monitored during 9 days , after which no further deaths occurred . Survival curve was analyzed via Log rank

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

Pemafibrate (1 mg/kg) or vehicle (0,9% NaCl) was administered at different timepoints before and after the induction of sepsis and survival was monitored during 9 days, after which no further deaths occurred. Survival curve was analyzed via Log rank tests


Mice were injected with the PPARα antagonist GW6471 ( 10µg / g ) or vehicle ( DMSO ) 3h pre - and 3h post - CLP . Survival was monitored during 9 days , after which no further deaths occurred . Survival curve was analyzed via a Log rank test . Combined data of 2 independent experiments , n = 16 / group .

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

Mice were injected with the PPARα antagonist GW6471 (10µg/g) or vehicle (DMSO) 3h pre- and 3h post-CLP. Survival was monitored during 9 days, after which no further deaths occurred. Survival curve was analyzed via a Log rank test. Combined data of 2 independent experiments, n = 16/group.


Blood samples were collected from healthy volunteers and septic patients ( n = 13 septic patients , = 15 healthy controls ) . Plasma was prepared and ( A ) FFA and ( B ) glycerol concentrations were determined as described in the methods . P - values were calculated with two - way Student t - tests .

O O O O O O 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 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

Blood samples were collected from healthy volunteers and septic patients (n= 13 septic patients, = 15 healthy controls). Plasma was prepared and (A) FFA and (B) glycerol concentrations were determined as described in the methods. P-values were calculated with two-way Student t-tests.


( C ) Confocal images of formaldehyde fixed GFP + S . flexneri following 20 min incubation with 2 mM GTP and 10 µM Alexa - Fluor647 - labeled protein . Bacteria associated with hGBP1 mutants after 20 min were quantified . Mean frequencies ± SEM of combined data from two independent experiments are shown . Significance was determined by one - way ANOVA with Tukey ' s multiple comparison test . * * * , P ≤ 0 . 001 . Scale bars equal 5 µm . Flow diagram depicts effects of K51A , R48A , and H74A hGBP1 mutations on nucleotide binding and hydrolysis .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Confocal images of formaldehyde fixed GFP+ S. flexneri following 20 min incubation with 2 mM GTP and 10 µM Alexa-Fluor647-labeled protein. Bacteria associated with hGBP1 mutants after 20 min were quantified. Mean frequencies ± SEM of combined data from two independent experiments are shown. Significance was determined by one-way ANOVA with Tukey's multiple comparison test. ***, P ≤ 0.001. Scale bars equal 5 µm. Flow diagram depicts effects of K51A, R48A, and H74A hGBP1 mutations on nucleotide binding and hydrolysis.


( A ) Translocation of ectopically expressed mCherry - hGBP1 to cytosolic GFP + S . flexneri ΔipaH9 . 8 in HeLa hGBP1 - KO cells was monitored by time - lapse microscopy . Individual time frames of Movie EV1 starting at 55 min post infection ( mpi ) are shown .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Translocation of ectopically expressed mCherry-hGBP1 to cytosolic GFP+ S. flexneri ΔipaH9.8 in HeLa hGBP1-KO cells was monitored by time-lapse microscopy. Individual time frames of Movie EV1 starting at 55 min post infection (mpi) are shown.


( B ) Confocal time - lapse microscopy was used to image 10 µM Alexa - Fluor647 - hGBP1F supplemented with 2 mM GTP in the presence or absence of formaldehyde fixed GFP + S . flexneri . Individual time frames of Movie EV2 depict hGBP1F fluorescence intensity . Merged images of hGBP1F and S . flexneri fluorescence are shown for the 60 min time points .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Confocal time-lapse microscopy was used to image 10 µM Alexa-Fluor647-hGBP1F supplemented with 2 mM GTP in the presence or absence of formaldehyde fixed GFP+ S. flexneri. Individual time frames of Movie EV2 depict hGBP1F fluorescence intensity. Merged images of hGBP1F and S. flexneri fluorescence are shown for the 60 min time points.


( C ) Images were taken at 45 min after addition of 10 µM Alexa - Fluor647 - hGBP1F to formaldehyde fixed GFP + S . flexneri in the presence of indicated nucleotides ( GTP , natural substrate ; GppNHp , non - hydrolysable GTP analog ; GTPγS , slowly hydrolysable GTP analog ; GDP · AlFX , GTP transition state analog ) . hGBP1 - associated bacteria after 45 min were quantified . Combined data from two independent experiments are shown as mean ± SEM . Significance was determined by one - way ANOVA with Tukey ' s multiple comparison test . * * * * , P ≤ 0 . 0001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Images were taken at 45 min after addition of 10 µM Alexa-Fluor647-hGBP1F to formaldehyde fixed GFP+ S. flexneri in the presence of indicated nucleotides (GTP, natural substrate; GppNHp, non-hydrolysable GTP analog; GTPγS, slowly hydrolysable GTP analog; GDP·AlFX, GTP transition state analog). hGBP1-associated bacteria after 45 min were quantified. Combined data from two independent experiments are shown as mean ± SEM. Significance was determined by one-way ANOVA with Tukey's multiple comparison test. ****, P ≤ 0.0001.


( A ) Polymerization of 10 µM hGBP1F and hGBP1FR584 - 586A in the presence of 2 mM GTP was monitored over time by absorption spectroscopy at 350 nm . Absorbance signals were superimposed with nucleotide composition of the same solution analyzed at defined time points , revealing the characteristic first phase of slow polymer nucleation and the second phase of fast polymer growth and cooperative hydrolysis .

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

(A) Polymerization of 10 µM hGBP1F and hGBP1FR584-586A in the presence of 2 mM GTP was monitored over time by absorption spectroscopy at 350 nm. Absorbance signals were superimposed with nucleotide composition of the same solution analyzed at defined time points, revealing the characteristic first phase of slow polymer nucleation and the second phase of fast polymer growth and cooperative hydrolysis.


( B ) Scanning - electron micrographs of live S . flexneri incubated with no protein or with 5 µM of either hGBP1F , hGBP1FR584 - 586A , or hGBP1FR48A ( non - polymerizing mutant ) in the presence of 2 mM GTP for 4 min . Arrowheads point to unattached hGBP1 polymers , arrows point to hGBP1 polymers attached to bacteria , and asterisks mark polymeric structures that appear to fuse with bacterial surfaces .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Scanning-electron micrographs of live S. flexneri incubated with no protein or with 5 µM of either hGBP1F, hGBP1FR584-586A, or hGBP1FR48A (non-polymerizing mutant) in the presence of 2 mM GTP for 4 min. Arrowheads point to unattached hGBP1 polymers, arrows point to hGBP1 polymers attached to bacteria, and asterisks mark polymeric structures that appear to fuse with bacterial surfaces.


( C ) Confocal time - lapse microscopy frames of formaldehyde fixed GFP + S . flexneri following admixture of 10 µM Alexa - Fluor647 - labeled hGBP1F or hGBP1FR584 - 586A and 2 mM GTP . Binding of hGBP1F polymers to bacteria at 5 min and enclosure of bacteria with hGBP1F protein coats after 60 min were quantified . Mean frequencies ± SEM of combined data from at least three independent experiments are shown . Significance was determined by by unpaired t - tests , two - tailed . * * * , P ≤ 0 . 001 ; * * * * , P ≤ 0 . 0001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Confocal time-lapse microscopy frames of formaldehyde fixed GFP+ S. flexneri following admixture of 10 µM Alexa-Fluor647-labeled hGBP1F or hGBP1FR584-586A and 2 mM GTP. Binding of hGBP1F polymers to bacteria at 5 min and enclosure of bacteria with hGBP1F protein coats after 60 min were quantified. Mean frequencies ± SEM of combined data from at least three independent experiments are shown. Significance was determined by by unpaired t-tests, two-tailed. ***, P ≤ 0.001; ****, P ≤ 0.0001.


( A ) Representative confocal images taken at 60 min after admixture of 2 mM GTP and 10 µM Alexa - Fluor647 - or Alexa - Fluor488 - hGBP1F to formaldehyde fixed gram - negative and gram - positive bacteria expressing GFP ( S . enterica Typhimurium [ ST ] , L . monocytogenes [ Lm ] ) , or RFP ( S . flexneri [ Sf ] , uropathogenic E . coli [ UPEC ] ) , or dsRed ( L . pneumophila [ Lp ] , S . aureus [ Sa ] ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 confocal images taken at 60 min after admixture of 2 mM GTP and 10 µM Alexa-Fluor647- or Alexa-Fluor488-hGBP1F to formaldehyde fixed gram-negative and gram-positive bacteria expressing GFP (S. enterica Typhimurium [ST], L. monocytogenes [Lm]), or RFP (S. flexneri [Sf], uropathogenic E. coli [UPEC]), or dsRed (L. pneumophila [Lp], S. aureus [Sa]).


( B ) ( Left panel ) Confocal time - lapse microscopy frames of Alexa - Fluor488 - LPS - O55 : B5 after addition of 5 µM Alexa - Fluor647 - hGBP1F supplemented with 2 mM GTP . ( Right panel ) Confocal images taken of 5 µM Alexa - Fluor488 - LPS - O55 : B5 20 min after addition of 5 µM Alexa - Fluor647 - hGBP1F or - hGBP1FR48A supplemented with 2 mM GTP . Graphs depict average aggregate area of Alexa - Fluor488 - LPS - O55 : B5 supplemented with 2 mM GTP and 5 µM of the indicated hGBP1 variant . Mean area ± SEM of combined data from three independent experiments . Significance was determined by one - way ANOVA with Tukey ' s multiple comparison test . . n . s . , not significant ; * * * , P ≤ 0 . 001 ; * * * * , P ≤ 0 . 0001 .

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 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 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

(B) (Left panel) Confocal time-lapse microscopy frames of Alexa-Fluor488-LPS-O55:B5 after addition of 5 µM Alexa-Fluor647-hGBP1F supplemented with 2 mM GTP. (Right panel) Confocal images taken of 5 µM Alexa-Fluor488-LPS-O55:B5 20 min after addition of 5 µM Alexa-Fluor647-hGBP1F or -hGBP1FR48A supplemented with 2 mM GTP. Graphs depict average aggregate area of Alexa-Fluor488-LPS-O55:B5 supplemented with 2 mM GTP and 5 µM of the indicated hGBP1 variant. Mean area ± SEM of combined data from three independent experiments. Significance was determined by one-way ANOVA with Tukey's multiple comparison test.. n.s., not significant; ***, P ≤ 0.001; ****, P ≤ 0.0001.


( C ) Polymerization of 5 µM hGBP1F induced by addition of 2 mM GTP in the presence and absence of 5 µM LPS - O55 : B5 was monitored by absorption spectroscopy at 350 nm . The absorbance signal was superimposed with nucleotide composition of the same solution analyzed at defined time points . Maximal hydrolysis rates ( turnover numbers ) were determined for hGBP1F variants in the presence and absence of 5 µM LPS - O55 : B5 . Graphs show mean turnover numbers ± SEM of combined data from at least three independent experiments . Significance was determined by two - way ANOVA with Tukey ' s multiple comparison test . n . s . , not significant ; * * * , P ≤ 0 . 001 ; * * * * , P ≤ 0 . 0001 .

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 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 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

(C) Polymerization of 5 µM hGBP1F induced by addition of 2 mM GTP in the presence and absence of 5 µM LPS-O55:B5 was monitored by absorption spectroscopy at 350 nm. The absorbance signal was superimposed with nucleotide composition of the same solution analyzed at defined time points. Maximal hydrolysis rates (turnover numbers) were determined for hGBP1F variants in the presence and absence of 5 µM LPS-O55:B5. Graphs show mean turnover numbers ± SEM of combined data from at least three independent experiments. Significance was determined by two-way ANOVA with Tukey's multiple comparison test. n.s., not significant; ***, P ≤ 0.001; ****, P ≤ 0.0001.


( D ) Formaldehyde fixed GFP + S . flexneri supplemented with varying LPS - O55 : B5 concentrations were mixed with 5 µM hGBP1F and 2 mM GTP . After 60 min hGBP1F - enclosed S . flexneri were quantified . Combined data from three independent experiments are shown as mean ± SEM . Significance was determined by one - way ANOVA with Tukey ' s multiple comparison test . * * * * , P ≤ 0 . 0001 .

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

(D) Formaldehyde fixed GFP+ S. flexneri supplemented with varying LPS-O55:B5 concentrations were mixed with 5 µM hGBP1F and 2 mM GTP. After 60 min hGBP1F-enclosed S. flexneri were quantified. Combined data from three independent experiments are shown as mean ± SEM. Significance was determined by one-way ANOVA with Tukey's multiple comparison test. ****, P ≤ 0.0001.


( E ) Confocal images of formaldehyde fixed GFP - , RFP - , or dsRed - expressing gram - negative bacteria 60 min after addition of 5 µM Alexa - Fluor647 - or Alexa - Fluor488 - hGBP1F and 2 mM GTP in the presence and absence of 100 µM LPS - O55 : B5 . After 60 min hGBP1F - enclosed gram - negative bacteria were quantified . Combined data from three independent experiments are shown as mean ± SEM . Significance was determined by two - way ANOVA with Tukey ' s multiple comparison test . * * * * , P ≤ 0 . 0001 .

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

(E) Confocal images of formaldehyde fixed GFP-, RFP-, or dsRed-expressing gram-negative bacteria 60 min after addition of 5 µM Alexa-Fluor647- or Alexa-Fluor488-hGBP1F and 2 mM GTP in the presence and absence of 100 µM LPS-O55:B5. After 60 min hGBP1F-enclosed gram-negative bacteria were quantified. Combined data from three independent experiments are shown as mean ± SEM. Significance was determined by two-way ANOVA with Tukey's multiple comparison test. ****, P ≤ 0.0001.


( B ) Time - lapse microscopy of fixed GFP + co - isogenic S . flexneri wild type and rfaL mutant after adding 10 µM Alexa - Fluor647 - hGBP1F and 2 mM GTP . Bacteria bound by hGBP1F at 5 min and hGBP1F - enclosed bacteria after 60 min were quantified . Combined data from three independent experiments are shown as mean ± SEM . Significance was determined by unpaired t - tests , two - tailed . n . s . , not significant ; * * * * , P ≤ 0 . 0001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Time-lapse microscopy of fixed GFP+ co-isogenic S. flexneri wild type and rfaL mutant after adding 10 µM Alexa-Fluor647-hGBP1F and 2 mM GTP. Bacteria bound by hGBP1F at 5 min and hGBP1F-enclosed bacteria after 60 min were quantified. Combined data from three independent experiments are shown as mean ± SEM. Significance was determined by unpaired t-tests, two-tailed. n.s., not significant; ****, P ≤ 0.0001.


( C ) Scanning - electron micrographs of live wild type and rfaL mutant strains incubated with no protein or with 5 µM of hGBP1F in the presence of 2 mM GTP for 4 min . Arrowheads point to unattached hGBP1 polymers , arrows point to hGBP1 polymers attached to bacteria , and asterisks mark polymeric structures that appear to fuse with bacterial

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Scanning-electron micrographs of live wild type and rfaL mutant strains incubated with no protein or with 5 µM of hGBP1F in the presence of 2 mM GTP for 4 min. Arrowheads point to unattached hGBP1 polymers, arrows point to hGBP1 polymers attached to bacteria, and asterisks mark polymeric structures that appear to fuse with bacterial surfaces


( E ) Confocal images of formaldehyde fixed , co - isogenic GFP + wild type and mutant S . flexneri strains harboring rfb regions of E . coli serotypes O8 and O25 after 60 min of incubation time in the presence of 10 µM Alexa - Fluor647 - hGBP1F and 2 mM GTP .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Confocal images of formaldehyde fixed, co-isogenic GFP+ wild type and mutant S. flexneri strains harboring rfb regions of E. coli serotypes O8 and O25 after 60 min of incubation time in the presence of 10 µM Alexa-Fluor647-hGBP1F and 2 mM GTP.


( A ) Following incubation for 1 . 5 h with buffer in the absence or presence of 10 µM hGBP1F and 5 mM GTP , live bacteria were treated with antibiotics ( 2 . 5 µg / ml PMB or 5 mg / ml lysozyme ) for 30 min or left untreated . The number of viable bacteria was subsequently determined by colony forming unit ( CFU ) counts . Graphs show mean CFUs ± SEM of combined data from at least three independent experiments . Significance was determined by two - way ANOVA with Tukey ' s multiple comparison test . n . s . , not significant ; * * * * , P ≤ 0 . 0001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_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 O O O O O O 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) Following incubation for 1.5 h with buffer in the absence or presence of 10 µM hGBP1F and 5 mM GTP, live bacteria were treated with antibiotics (2.5 µg/ml PMB or 5 mg/ml lysozyme) for 30 min or left untreated. The number of viable bacteria was subsequently determined by colony forming unit (CFU) counts. Graphs show mean CFUs ± SEM of combined data from at least three independent experiments. Significance was determined by two-way ANOVA with Tukey's multiple comparison test. n.s., not significant; ****, P ≤ 0.0001.


( B ) Formaldehyde fixed RFP + UPEC were mixed with 10 µM Alexa - Fluor488 - hGBP1F or - hGBP1FR584 - 586A supplemented with 2 mM GTP . Following 60 min of incubation hGBP1F - enclosed UPEC were quantified . Graphs depict mean ± SEM of combined data from three independent experiments . Significance was determined by unpaired t - test , two - tailed . * * * * , P ≤ 0 . 0001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Formaldehyde fixed RFP+ UPEC were mixed with 10 µM Alexa-Fluor488-hGBP1F or -hGBP1FR584-586A supplemented with 2 mM GTP. Following 60 min of incubation hGBP1F-enclosed UPEC were quantified. Graphs depict mean ± SEM of combined data from three independent experiments. Significance was determined by unpaired t-test, two-tailed. ****, P ≤ 0.0001.