A . Normalized expression from promoters ybjC recA in response to NIT stress , measured at population level using GFP reporters 4 μg / mL NIT was added at time zero . oxidative stress promoter ybjC clearly precedes SOS response promoter recA , when measured at population level Error bars show standard deviation of three replicate experiments measured on different days . B . Normalized ybjC recA expression over time in response to addition of 4 μg / mL NIT at time zero in single from one microcolony . Dashed : threshold used to determine response times Lower panels , upper row : Dual - color of oxidative stress reporter ybjC ( green ) SOS reporter recA ( red ) in single at 4 different time points after NIT addition in one microcolony . Note that ybjC signal ( green ) is stronger at t = 2h , whereas recA signal ( red ) is only apparent at t = 4h 6h . Lower row : Constitutively expressed mCherry used for segmentation . C . Response times for ybjC recA from with dual reporters ( Figure 3 ) , combined from two microcolonies . response times for recA are higher than for ybjC response times for both reporters are strongly correlated ( r = 0 . 74 ± 0 . 04 , p = 3 . 8 · 10 - 6 ) . error of correlation coefficient is from subsampling of descendants of single that were present at time of NIT

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A. Normalized expression from the promoters ybjC and recA in response to NIT stress, measured at the population level using GFP reporters 4 μg/mL NIT was added at time zero. The oxidative stress promoter ybjC clearly precedes the SOS response promoter recA, when measured at the population level Error bars show standard deviation of three replicate experiments measured on different days. B. Normalized ybjC and recA expression over time in response to the addition of 4 μg/mL NIT at time zero in single cells from one microcolony. Dashed line: threshold used to determine response times Lower panels, upper row: Dual-color images of the oxidative stress reporter ybjC (green) and the SOS reporter recA (red) in single cells at 4 different time points after NIT addition in one microcolony. Note that the ybjC signal (green) is stronger at t = 2h, whereas the recA signal (red) is only apparent at t = 4h and 6h. Lower row: Constitutively expressed mCherry used for segmentation. C. Response times for ybjC and recA from individual cells with dual reporters (Figure 3), combined from two microcolonies. All response times for recA are higher than for ybjC and response times for both reporters are strongly correlated (r = 0.74 ± 0.04, p = 3.8 · 10-6). The error of the correlation coefficient is from subsampling of descendants of single cells that were present at the time of NIT addition


A . Expression from gadB recA promoters , measured at population level using GFP reporters over time ; 0 . 5 μg / mL TMP was added at time zero . acid stress response ( gadB ) clearly precedes SOS response ( recA ) . Error bars show standard deviation of three replicate experiments measured on different days . B . Normalized expression of gadB recA expression over time in responding to addition of 0 . 5 μg / mL TMP at time zero shown for one microcolony . Dashed : threshold used to determine response times ( Methods ) . Lower panels , upper row : Dual - color of gadB ( blue ) recA ( red ) in single at 4 different time points . Most are predominantly either red or blue , i . e . acid stress SOS response are typically not both strongly expressed in same . Lower row : Constitutively expressed mCherry used for segmentation . C . Scatter plot of response times for gadB recA in single ; there is no clear temporal order no significant correlation ( r = 0 . 08 ± 0 . 08 , p = 0 . 68 ) ; data is combined from three microcolonies . Error is from subsampling of descendants of single present at time point of TMP

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

A. Expression from the gadB and recA promoters, measured at the population level using GFP reporters over time; 0.5 μg/mL TMP was added at time zero. The acid stress response (gadB) clearly precedes the SOS response (recA). Error bars show standard deviation of three replicate experiments measured on different days. B. Normalized expression of gadB and recA expression over time in individual cells responding to the addition of 0.5 μg/mL TMP at time zero shown for one microcolony. Dashed line: threshold used to determine response times (Methods). Lower panels, upper row: Dual-color images of gadB (blue) and recA (red) in single cells at 4 different time points. Most cells are predominantly either red or blue, i.e. acid stress and SOS response are typically not both strongly expressed in the same cells. Lower row: Constitutively expressed mCherry used for segmentation. C. Scatter plot of response times for gadB and recA in single cells; there is no clear temporal order and no significant correlation (r = 0.08 ± 0.08, p = 0.68); data is combined from three microcolonies. Error is from subsampling of descendants of single cells present at the time point of TMP addition


D . Expression of gadB recA in inosine - supplemented medium , averaged over 13 single in a microcolony . Insets : Dual color image of gadB recA segmentation image at t = 6h .

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D. Expression of gadB and recA in inosine-supplemented medium, averaged over 13 single cells in a microcolony. Insets: Dual color image of gadB and recA and segmentation image at t = 6h.


Figure 1 : Endothelial Ang - 2 upregulation correlates with WHO grading in human gliomas . ELISA displaying human Ang - 2 ( A ) Ang - 1 ( B ) level in serum of healthy ( Ang - 2 n = 4 ; Ang - 1 n = 21 ) or with low grade glioma ( WHOII ) ( n = 5 ) , anaplastic astrocytoma ( WHOIII ) ( n = 7 ) or glioblastoma ( WHOIV ) ( n = 39 ) are shown .

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Figure 1: Endothelial Ang-2 upregulation correlates with WHO grading in human gliomas. ELISA displaying human Ang-2 (A) and Ang-1 (B) level in serum of healthy patients (Ang-2 n=4; Ang-1 n=21) or patients with low grade glioma (WHOII) (n=5), anaplastic astrocytoma (WHOIII) (n=7) or glioblastoma (WHOIV) (n=39) are shown.


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Figure 1 : Endothelial Ang - 2 upregulation correlates with WHO grading in human gliomas . ELISA displaying human Ang - 2 ( A ) Ang - 1 ( B ) level in serum of healthy ( Ang - 2 n = 4 ; Ang - 1 n = 21 ) or with low grade glioma ( WHOII ) ( n = 5 ) , anaplastic astrocytoma ( WHOIII ) ( n = 7 ) or glioblastoma ( WHOIV ) ( n = 39 ) are shown . TCGA database was accessed to obtain gene expression level for Ang - 1 , Ang - 2 , Tie2 Tie1 in GBM in comparison to normal brain ( C ) .

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Figure 1: Endothelial Ang-2 upregulation correlates with WHO grading in human gliomas. ELISA displaying human Ang-2 (A) and Ang-1 (B) level in serum of healthy patients (Ang-2 n=4; Ang-1 n=21) or patients with low grade glioma (WHOII) (n=5), anaplastic astrocytoma (WHOIII) (n=7) or glioblastoma (WHOIV) (n=39) are shown. The TCGA database was accessed to obtain gene expression level for Ang-1, Ang-2, Tie2 and Tie1 in GBM in comparison to normal brain (C).


Ang - 2 expression quantitative of Ang - 2 in healthy human brain ( n = 3 ) , low grade diffuse glioma ( n = 14 ) , anaplastic astrocytoma ( n = 12 ) or glioblastoma ( n = 11 ) is shown ( D ) .

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Ang-2 expression and quantitative analysis of Ang-2 in healthy human brain (n=3), low grade diffuse glioma (n=14), anaplastic astrocytoma (n=12) or glioblastoma (n=11) is shown (D).


Co - of Ang - 2 vWF ( endothelial ) , αSMA ( mural ) IBA1 ( microglia ) in different glioblastoma specimen . Normal brain tissue was used to assess Ang - 2 specificity ( E ) .

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Co-staining of Ang-2 and vWF (endothelial cells), αSMA (mural cells) and IBA1 (microglia) in different glioblastoma specimen. Normal brain tissue was used to assess Ang-2 staining specificity (E).


Ang - 2 predicts survival of glioblastoma ( F ) . If not indicated differently , Kruskall - Wallis ( Dunn ´ s post ) was applied , * * p < 0 . 05 , * * * p < 0 . 005 ; data are mean ±

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Ang-2 predicts survival of glioblastoma patients (F). If not indicated differently, Kruskall-Wallis (Dunn´s post test) was applied, ** p < 0.05, *** p < 0.005; data are mean ± SEM


Figure 2 : Endothelial Ang - 2 expression reduced pericyte coverage increased macrophage infiltration in experimental glioblastoma . GL261 were intracerebrally transplanted in wild type or Ang - 2 gain - of - function mice ( Ang - 2 DT ) . Brain sections were stained with antibodies against CD31 desmin ( A ) , microvessel densities ( MVD ) pericyte coverage were determined ( WT n = 14 ; Ang - 2 DT n = 6 ) ( B ) .

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Figure 2: Endothelial Ang-2 expression reduced pericyte coverage and increased macrophage infiltration in experimental glioblastoma. GL261 cells were intracerebrally transplanted in wild type or Ang-2 gain-of-function mice (Ang-2 DT). Brain tumor sections were stained with antibodies against CD31 and desmin (A), and microvessel densities (MVD) and pericyte coverage were determined (WT n=14; Ang-2 DT n=6) (B).


FACS of infiltrating macrophages in Ang - 2 DT normal brain ( w / o ) compared to WT is shown ( C , dot plot ; D , ) ( WT n = 5 ; Ang - 2 DT n = 3 ) .

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FACS analysis of infiltrating macrophages in Ang-2 DT normal brain (w/o tumor) compared to WT is shown (C, dot plot; D, quantification) (WT n=5; Ang-2 DT n=3).


) . Immunohistochemistry for monocytes / macrophages ( F4 / 80 ) neutrophils ( Ly6G ; arrowheads ) in brain tumors derived from WT or Ang - 2 overexpressing mice ( E ) , Quantitative of C ( F ) ( WT n = 14 ; Ang - 2 DT n = 10 )

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). Immunohistochemistry staining for monocytes/macrophages (F4/80) and neutrophils (Ly6G; arrowheads) in brain tumors derived from WT or Ang-2 overexpressing mice (E), Quantitative analysis of C (F) (WT n=14; Ang-2 DT n=10)


Kaplan - Meier survival curve of GL261 - bearing WT Ang - 2 DT mice ( WT n = 17 ; Ang - 2 DT n = 14 ) ( E ) . Statistical analyses were performed using an unpaired Student ´ s t - ( B , F ) , one - way Anova ( Tukey post ) ( D ) , Log - rank Wilcoxon ( G ) * p < 0 . 05 , * * p < 0 . 01 ; data are mean ±

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Kaplan-Meier survival curve of GL261 tumor-bearing WT and Ang-2 DT mice (WT n=17; Ang-2 DT n=14) (E). Statistical analyses were performed using an unpaired Student´s t-test (B, F), one-way Anova (Tukey post test) (D), Log-rank and Wilcoxon (G) * p < 0.05, ** p < 0.01; data are mean ± SEM


Figure 3 : Infiltrating leukocyte subsets in human glioma . Paraffin sections of healthy human brain ( n = 3 ) , low grade glioma ( n = 12 ) , anaplastic astrocytoma ( n = 7 ) glioblastoma ( n = 9 ) were stained for IBA1 ( macrophages / microglia ) , CD15 ( granulocytes ) CD3 ( T - ) ( A ) . of leukocyte subsets in human glioma specimens ( B ) . For statistical Kruskall - Wallis ( Dunn ´ s post ) was applied . * p < 0 . 05 , * * p < 0 . 01 ; data are mean ±

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Figure 3: Infiltrating leukocyte subsets in human glioma. Paraffin sections of healthy human brain (n=3), low grade glioma (n=12), anaplastic astrocytoma (n=7) and glioblastoma (n=9) were stained for IBA1 (macrophages/microglia), CD15 (granulocytes) and CD3 (T-cells) (A). Quantification of leukocyte subsets in human glioma specimens (B). For statistical analysis Kruskall-Wallis (Dunn´s post test) was applied.* p < 0.05, ** p < 0.01; data are mean ± SEM


Figure 4 : Dual anti - Ang - 2 anti - VEGF therapy acts synergistic on vascular normalization macrophage infiltration in experimental GBM . Immunofluorescence of CD31 desmin in GL261 glioblastoma after single dual treatment with anti - Ang - 2 ( AMG386 ) anti - VEGF ( aflibercept ) ( A ) . Corresponding quantitative of microvessel densities pericytes numbers ( B ; control n = 24 ; AMG386 n = 10 ; aflibercept n = 12 ; AMG386 + aflibercept n = 13 ) .

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Figure 4: Dual anti-Ang-2 and anti-VEGF therapy acts synergistic on vascular normalization and macrophage infiltration in experimental GBM. Immunofluorescence staining of CD31 and desmin in GL261 glioblastoma after single and dual treatment with anti-Ang-2 (AMG386) and anti-VEGF (aflibercept) (A). Corresponding quantitative analysis of microvessel densities and pericytes numbers (B; control n=24; AMG386 n=10; aflibercept n=12; AMG386 + aflibercept n=13).


. for mouse IgG ( red ) as a surrogate parameter for vascular leakage CD31 ( green ) is shown in GL261 glioblastoma sections following different anti - angiogenic treatments ( C ) with in ( D ; n = 3 ) .

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. Staining for mouse IgG (red) as a surrogate parameter for vascular leakage and CD31 (green) is shown in GL261 glioblastoma sections following different anti-angiogenic treatments (C) with quantification in (D; n=3).


of necrotic areas in GL261 glioblastoma sections after anti - angiogenic treatment is shown as of whole ( E ; control , aflibercept , AMG386 + aflibercept n = 4 ; AMG386 n = 3 ) .

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Quantification of necrotic areas in GL261 glioblastoma sections after anti-angiogenic treatment is shown as percent of whole tumor area (E; control, aflibercept, AMG386 + aflibercept n=4; AMG386 n=3).


of Glut1 immunoreactivity in GL261 glioblastoma sections ( F ; n = 3 ) .

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Analysis of Glut1 immunoreactivity in GL261 glioblastoma sections (F; n=3).


Double - immunofluorescence stainings of macrophages ( F4 / 80 ) vessels ( vWF ) in mouse GBM after treatment with AMG386 , aflibercept or combination of both are shown in ( G )

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Double-immunofluorescence stainings of macrophages (F4/80) and tumor vessels (vWF) in mouse GBM after treatment with AMG386, aflibercept or the combination of both are shown in (G)


Quantitatve of infiltrating leukocytes ( F4 / 80 + , CD3 + , Ly - 6G + ) following anti - angiogenic treatment is displayed in ( H ; control n = 28 ; AMG386 n = 12 ; aflibercept n = 11 ; AMG386 + aflibercept n = 12 ) .

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Quantitatve analysis of tumor infiltrating leukocytes (F4/80+, CD3+, Ly-6G+) following anti-angiogenic treatment is displayed in (H; control n=28; AMG386 n=12; aflibercept n=11; AMG386 + aflibercept n=12).


Kaplan - Meier survival of GL261 tumors grown in C57BL / 6 mice following anti - angiogenic treatment ( I ; control n = 38 ; AMG386 n = 13 ; aflibercept n = 12 ; AMG386 + aflibercept n = 13 ) .

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Kaplan-Meier survival analysis of GL261 tumors grown in C57BL/6 mice following anti-angiogenic treatment (I; control n=38; AMG386 n=13; aflibercept n=12; AMG386 + aflibercept n=13).


Double - immunofluorescence stainings with anti - F4 / 80 anti - CD206 in brain sections of mice treated with anti - Ang - 2 ( AMG386 ) , anti - VEGF ( aflibercept ) or combination of both are shown in ( J ) . Corresponding quantitative analyses of infiltrating F4 / 80 + CD206 + , of CD206 + vs . F4 / 80 + upon anti - angiogenic therapy are displayed in ( K ; control n = 21 ; AMG386 n = 13 ; aflibercept n = 9 ; AMG386 + aflibercept n = 4 ) .

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Double-immunofluorescence stainings with anti-F4/80 and anti-CD206 in brain tumor sections of mice treated with anti-Ang-2 (AMG386), anti-VEGF (aflibercept) or the combination of both are shown in (J). Corresponding quantitative analyses of tumor infiltrating F4/80+ cells and CD206+ cells, and the ratio of CD206+ vs. F4/80+ upon anti-angiogenic therapy are displayed in (K; control n=21; AMG386 n=13; aflibercept n=9; AMG386 + aflibercept n=4).


Flow cytometry of infiltrating macrophages following enzymatic dissociation of mouse GL261 brain tumors plus / minus anti - angiogenic therapy . of CD206 + ( L ) MHC class IIHI ( M ) among CD45 + CD11b + GR1 - F4 / 80 + are displayed ( L , M ; control n = 4 ; AMG386 n = 4 ; aflibercept n = 5 ; AMG386 + aflibercept n = 5 ) . Statistical analyses were performed using one - way ANOVA Tukey ' s multiple comparison except for Log - rank Wilcoxon ( I ) Kruskall Wallis ( B ) . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 005 , # p < 0 . 005 of aflibercept + AMG386 vs aflibercept ; data are mean ± ( A - K )

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Flow cytometry of tumor infiltrating macrophages following enzymatic dissociation of mouse GL261 brain tumors plus/minus anti-angiogenic therapy. Percent of CD206+ (L) and MHC class IIHI cells (M) among CD45+CD11b+GR1-F4/80+ cells are displayed (L,M; control n=4; AMG386 n=4; aflibercept n=5; AMG386 + aflibercept n=5). Statistical analyses were performed using one-way ANOVA and Tukey's multiple comparison except for Log-rank and Wilcoxon (I) and Kruskall Wallis (B). * p < 0.05, ** p < 0.01, *** p < 0.005, # p < 0.005 of aflibercept + AMG386 vs aflibercept; data are mean ± SEM (A-K)


Figure 5 : Anti - VEGF therapy led to decreased infiltration of CD68 + macrophages in human GBM . Anti - CD68 ( A ) , anti - CSFR1 ( B ) anti - CD206 ( C ) immunohistochemistry of samples derived from treatment - naive GBM ( n = 24 ) , post - radiochemotherapy ( S / RTx / CTx ) ( n = 7 ) post - radiochemotherapy + bevacizumab ( S / RTx / CTx / Bev ) ( n = 29 ) therapy is shown .

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Figure 5: Anti-VEGF therapy led to decreased infiltration of CD68+ macrophages in human GBM. Anti-CD68 (A), anti-CSFR1 (B) and anti-CD206 (C) immunohistochemistry of patient samples derived from treatment-naive GBM (n=24), post-radiochemotherapy (S/RTx/CTx) (n=7) and post-radiochemotherapy + bevacizumab (S/RTx/CTx/Bev) (n=29) therapy is shown.


Corresponding of infiltrating is displayed ( anti - CD68 , D ) , ( anti - CSF1R , E ) ( anti - CD206 , F ) . of CD206 + vs . CD68 + ( G ) .

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Corresponding quantification of tumor infiltrating cells is displayed (anti-CD68, D), (anti-CSF1R, E) and (anti-CD206, F). Ratio of CD206+ vs. CD68+ cells (G).


Amount of CD206 + perivascular per blood vessel ( H )

Amount of CD206+ perivascular cells per blood vessel (H)


Double of Ang - 2 ( brown ) CD206 ( red ) of a S / RTx / CTx / Bev , arrowheads indicating perivascular CD206 + around Ang - 2 + vessels ( I ) . Arrows pointing on parenchymal CD206 + ( I ) .

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Double staining of Ang-2 (brown) and CD206 (red) of a S / RTx / CTx / Bev patient, arrowheads indicating perivascular CD206+ cells around Ang-2+ vessels (I). Arrows pointing on parenchymal CD206+ cells (I).


) . CD206 + in relation to of hypoxic ( CAIX positive ) ( J ) . Statistical : Kruskall - Wallis ( Dunn ´ s post ) . * p < 0 . 05 , * * p < 0 . 01 ; data are mean ±

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). CD206+ cells in relation to the percentage of hypoxic (CAIX positive) tumor area (J). Statistical analysis: Kruskall-Wallis (Dunn´s post test). * p < 0.05, ** p < 0.01; data are mean ± SEM


Figure 6 : Bevacizumab therapy leads to reduced vessel density increased Ang - 2 expression . Immunohistochemistry stainings with antibodies directed against CD31 ( A ) Ang - 2 ( B ) in treatment - naive GBM , post - radiochemotherapy ( S / RTx / CTx ) post - radiochemotherapy + bevacizumab ( S / RTx / CTx / Bev ) therapy are displayed .

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Figure 6: Bevacizumab therapy leads to reduced vessel density and increased Ang-2 expression. Immunohistochemistry stainings with antibodies directed against CD31 (A) and Ang-2 (B) in treatment-naive GBM, post-radiochemotherapy (S/RTx/CTx) and post-radiochemotherapy + bevacizumab (S/RTx/CTx/Bev) therapy are displayed.


Quantitative analyses of microvessel densities Ang - 2 + vessels are shown in ( C ) ( D ) . of Ang - 2 + vs . CD31 + vessels was determined in ( E ) .

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Quantitative analyses of microvessel densities and Ang-2+ vessels are shown in (C) and (D). The ratio of Ang-2+ vs. CD31+ vessels was determined in (E).


Western Blot corresponding of CD31 Ang - 2 in control aflibercept treated mice ( F ) .

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Western Blot and corresponding quantification of CD31 and Ang-2 in control and aflibercept treated mice (F).


CAIX of treatment - naive GBM S / RTx / CTx / Bev ( G ) . Analyses of hypoxic ( CAIX + ) with regard to whole in human GBM ( H ) . - infiltrating lymphocytes ( CD8 / CD3 ) in human ( I ) . Kruskall - Wallis ( Dunn ´ s post ) . * p < 0 . 05 , * * * p < 0 . 005 ; treatment - naive GBM n = 26 , S / RTx / CTx n = 7 , S / RTx / CTx / Bev n = 29 ; data are mean ±

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CAIX staining of treatment-naive GBM and S / RTx / CTx / Bev patients (G). Analyses of hypoxic (CAIX+) area with regard to the whole tumor in human GBM (H). Tumor-infiltrating lymphocytes (CD8/CD3) in human patients (I). Kruskall-Wallis (Dunn´s post test). * p < 0.05, *** p < 0.005; treatment-naive GBM n=26, S/RTx/CTx n=7, S/RTx/CTx/Bev n=29; data are mean ± SEM


Whole - mount view of E14 . 5 - 15 . 5 hearts from Npr3 - sCreER ; R26 - tdTomato Npr3 - CreER ; R26 - tdTomato mice . Immunostaining for tdTomato , ESR CDH5 on heart sections . C . M . , compact myocardium ; T . M . , trabecular myocardium . Arrowheads , tdTomato + ESR - endocardial .

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Whole-mount fluorescence view of E14.5-15.5 hearts from Npr3-sCreER;R26-tdTomato and Npr3-CreER;R26-tdTomato mice. Immunostaining for tdTomato, ESR and CDH5 on heart sections. C.M., compact myocardium; T.M., trabecular myocardium. Arrowheads, tdTomato+ESR- endocardial cells.


of of tdTomato + or ESR + endothelial . Data are mean ± s . e . m . ; n = 5 .

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Quantification of the percentage of tdTomato+ or ESR+ endothelial cells. Data are mean ± s.e.m.; n = 5.


Isolation of tdTomato - or tdTomato + from E14 . 5 Npr3 - sCreER ; R26 - tdTomato Npr3 - CreER ; R26 - tdTomato mice by FACS .

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Isolation of tdTomato- or tdTomato+ cells from E14.5 Npr3-sCreER;R26-tdTomato and Npr3-CreER;R26-tdTomato mice by FACS.


qRT - PCR of Cre , ESR , Npr3 Cdh5 from tdTomato + . Data are mean ± s . e . m . ; n = 5 ; * P < 0 . 05 .

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qRT-PCR of Cre, ESR, Npr3 and Cdh5 from tdTomato+ cells. Data are mean ± s.e.m.; n = 5; *P < 0.05.


Western blotting of ESR GAPDH in tdTomato - tdTomato + . Data are mean ± s . e . m . ; n = 5 ; * P < 0 . 05 ; n . s . , non - significant . Scale bars , 500 µm . Each figure is representative of 5 biological samples .

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Western blotting of ESR and GAPDH in tdTomato- and tdTomato+ cells. Data are mean ± s.e.m.; n = 5; *P < 0.05; n.s., non-significant. Scale bars, 500 µm. Each figure is representative of 5 individual biological samples.


Whole - mount of E14 . 5 Npr3 - CreER ; R26 - Confetti Npr3 - sCreER ; R26 - Confetti mouse hearts . No Tam is used as control for leakiness of Npr3 - sCreER .

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Whole-mount fluorescence images of E14.5 Npr3-CreER;R26-Confetti and Npr3-sCreER;R26-Confetti mouse hearts. No Tam is used as control for leakiness of Npr3-sCreER.


Immunostaining for CDH5 on heart sections shows a significantly increase of endothelial labeling in Npr3 - sCreER ; R26 - Confetti heart compared with Npr3 - CreER ; R26 - Confetti heart ( Tam ) . * P < 0 . 05 ; Data are mean ± s . e . m . ; n = 5 . Scale bars , 500 µm in c ; 100 µm in d . Each image is representative of 5 biological samples .

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Immunostaining for CDH5 on heart sections shows a significantly increase of endothelial cell labeling in Npr3-sCreER;R26-Confetti heart compared with Npr3-CreER;R26-Confetti heart (Tam). *P < 0.05; Data are mean ± s.e.m.; n = 5. Scale bars, 500 µm in c; 100 µm in d. Each image is representative of 5 individual biological samples.


Whole mount of E14 . 5 or E15 . 5 Npr3 - sCreER ; R26 - LZLT Npr3 - CreER ; R26 - LZLT mouse hearts . No tam is as control for detecting leakiness of sCreER or CreER . Representative Immunostaining for tdTomato , ESR CDH5 on heart sections .

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Whole mount fluorescent images of E14.5 or E15.5 Npr3-sCreER;R26-LZLT and Npr3-CreER;R26-LZLT mouse hearts. No tam is as control for detecting leakiness of sCreER or CreER. Representative Immunostaining images for tdTomato, ESR and CDH5 on heart sections.


of of endocardial expressing tdTomato in Npr3 - sCreER ; R26 - LZLT ( sCreER ) or Npr3 - CreER ; R26 - LZLT ( CreER ) hearts . Data are mean ± s . e . m . ; n = 5 ; * P < 0 . 05 . Scale bars , 500 µm . Each figure is a representative of 5 biological samples .

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Quantification of the percentage of endocardial cells expressing tdTomato in Npr3-sCreER;R26-LZLT (sCreER) or Npr3-CreER;R26-LZLT (CreER) hearts. Data are mean ± s.e.m.; n = 5; *P < 0.05. Scale bars, 500 µm. Each figure is a representative of 5 individual biological samples.


Immunostaining for VEGFR2 on Npr3 - sCreER ; Kdrflox / flox heart section from mice without tamoxifen treatment ( No Tam ) .

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Immunostaining for VEGFR2 on Npr3-sCreER;Kdrflox/flox heart section from mice without tamoxifen treatment (No Tam).


Immunostaining for VEGFR2 on mouse heart sections after tamoxifen treatment ( Tam ) . A , atrium ; V , ventricle ; TM , trabecular myocardium .

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Immunostaining for VEGFR2 on mouse heart sections after tamoxifen treatment (Tam). A, atrium; V, ventricle; TM, trabecular myocardium.


Immunostaining for tdTomato , VEGFR2 CDH5 on heart sections from mice of four different genotypes . data showed of VEGFR2 + endocardial ( ECs ) in tdTomato + ( tdT + ) in TM regions . Cartoon ( bottom ) indicate tdTomato + ( red ) VEGFR2 + ( green ) in TM or compact myocardium ( CM ) . Data are mean ± s . e . m . ; n = 5 . Scale bars , 100 µm . Each image is a representative of 5 biological samples .

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Immunostaining for tdTomato, VEGFR2 and CDH5 on heart sections from mice of four different genotypes. Quantification data showed the percentage of VEGFR2+ endocardial cells (ECs) in tdTomato+ (tdT+) cells in the TM regions. Cartoon images (bottom) indicate tdTomato+ cells (red) and VEGFR2+ cells (green) in the TM or compact myocardium (CM). Data are mean ± s.e.m.; n = 5. Scale bars, 100 µm. Each image is a representative of 5 individual biological samples.


Whole - mount of Col1a2 - sCreER ; R26 - tdTomato or Col1a2 - CreER ; R26 - tdTomato hearts . Inserts indicate bright - field .

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Whole-mount fluorescence images of Col1a2-sCreER;R26-tdTomato or Col1a2-CreER;R26-tdTomato hearts. Inserts indicate bright-field images.


Immunostaining for tdTomato PDGFRa on heart sections shows similar patterning of tdTomato PDGFRa signals .

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Immunostaining for tdTomato and PDGFRa on heart sections shows similar patterning of tdTomato and PDGFRa signals.


Immunostaining for tdTomato ESR on heart sections . Arrowheads indicate valve mesenchyme . of of PDGFRa + expressing tdTomato in Col1a2 - sCreER ; R26 - tdTomato ( sCreER ) or Col1a2 - CreER ; R26 - tdTomato ( CreER ) hearts . n . s . , non - significant ; Data are mean ± s . e . m . ; n = 6 ; n . s . , non - significant . of of tdTomato + expressing ESR . * P < 0 . 05 ; Data are mean ± s . e . m . ; n = 6 ; * P < 0 . 05 .

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Immunostaining for tdTomato and ESR on heart sections. Arrowheads indicate valve mesenchyme. Quantification of the percentage of PDGFRa+ cells expressing tdTomato in Col1a2-sCreER;R26-tdTomato (sCreER) or Col1a2-CreER;R26-tdTomato (CreER) hearts. n.s., non-significant; Data are mean ± s.e.m.; n = 6; n.s., non-significant. Quantification of the percentage of tdTomato+ cells expressing ESR. *P < 0.05; Data are mean ± s.e.m.; n = 6; *P < 0.05.


Immunostaining for PDGFRa in heart sections shows a significant increase of fibroblast labeling in valves of Col1a2 - sCreER ; R26 - Confetti heart compared with Col1a2 - CreER ; R26 - Confetti heart ( Tam ) . of of fibroblasts expressing reporters in cardiac valves . Data are mean ± s . e . m . ; n = 5 ; * P < 0 . 05 . Scale bars , 500 µm in c - e ; 100 µm in h . Each figure is a representative of 5 - 6 biological samples .

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Immunostaining for PDGFRa in heart sections shows a significant increase of fibroblast labeling in valves of Col1a2-sCreER;R26-Confetti heart compared with Col1a2-CreER;R26-Confetti heart (Tam). Quantification of the percentage of fibroblasts expressing fluorescent reporters in cardiac valves. Data are mean ± s.e.m.; n = 5; *P < 0.05. Scale bars, 500 µm in c-e; 100 µm in h. Each figure is a representative of 5-6 individual biological samples.


( A ) Comparative expression during murine pre - implantation development of Dppa2 ( red ) , Dppa4 ( blue ) , Dux ( black ) , Zscan4c ( ZGA marker , green ) . Each dot represents average value of single - -

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(A) Comparative expression during murine pre-implantation embryo development of Dppa2 (red), Dppa4 (blue), Dux (black), and Zscan4c (ZGA marker, green). Each dot represents the average value of single-cell RNA-seq


( C ) Sashimi plot representing coverage on Dppa4 gene of an sequencing of murine zygotes , early 2C , mid 2C late 2C Arcs depict splicing events , numbers their relative frequency ( i . e . reads across junctions ) .

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(C) Sashimi plot representing coverage on the Dppa4 gene of an RNA sequencing analysis of murine zygotes, early 2C, mid 2C and late 2C Arcs depict splicing events, and numbers their relative frequency (i.e. reads across junctions).


( A ) Average expression of Dppa2 Dppa4 in a single - - seq of mESCs sorted for expression of both Tomato GFP reporters driven by MERVL Zscan4 promoters , respectively , double - negative population .

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(A) Average expression of Dppa2 and Dppa4 in a single-cell RNA-seq analysis of mESCs sorted for expression of both Tomato and GFP reporters driven by MERVL and Zscan4 promoters, respectively, and the double-negative population.


( B ) Fraction of GFP + in mESCs carrying a MERVL - GFP reporter depleted of Dppa2 or Dppa4 using shRNAs specific to transcripts or a control . Bars represent average , error bars SD ; n = 3 . * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 , unpaired t - .

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(B) Fraction of GFP+ cells in mESCs carrying a MERVL-GFP reporter and depleted of Dppa2 or Dppa4 using shRNAs specific to the transcripts or a control. Bars represent the average, error bars the SD; n = 3. ** p ≤ 0.01, *** p ≤ 0.001, unpaired t-test.


( C ) Dppa2 , Dppa4 , Dux Zscan4 gene expression in mESCs carrying a MERVL - GFP reporter depleted of Dppa2 or Dppa4 using shRNAs specific to transcripts or a control . Expression was normalized to Actb . Bars represent average , error bars SD ; n = 3 . * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 , unpaired t - .

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(C) Dppa2, Dppa4, Dux and Zscan4 gene expression in mESCs carrying a MERVL-GFP reporter and depleted of Dppa2 or Dppa4 using shRNAs specific to the transcripts or a control. Expression was normalized to Actb. Bars represent the average, error bars the SD; n = 3. ** p ≤ 0.01, *** p ≤ 0.001, unpaired t-test.


- seq of WT Dppa2 ( D ) KO mESC clones . dot plot displays average gene expression of three independent clones from each type ; n = 3 .

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RNA-seq analysis of WT and Dppa2 (D) KO mESC clones. The dot plot displays the average gene expression of three independent clones from each cell type; n = 3.


- seq of WT Dppa4 ( E ) KO mESC clones . dot plot displays average gene expression of three independent clones from each type ; n = 3 .

RNA-seq analysis of WT and Dppa4 (E) KO mESC clones. The dot plot displays the average gene expression of three independent clones from each cell type; n = 3.


( F ) Comparative expression by qPCR of Dux , two downstream targets of DUX ( Zscan4 Tdpoz4 ) , two DUX - independent targets of DPPA2 DPPA4 ( Mael Tdrd1 ) in mESCs depleted of endogenous DPPA2 DPPA4 overexpressing ectopically DUX or GFP as a control . Expression was normalized to Actb . Bars represent average , error bars SD ; n =

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(F) Comparative expression by qPCR of Dux, two downstream targets of DUX (Zscan4 and Tdpoz4), and two DUX-independent targets of DPPA2 and DPPA4 (Mael and Tdrd1) in mESCs depleted of endogenous DPPA2 and DPPA4 and overexpressing ectopically DUX or GFP as a control. Expression was normalized to Actb. Bars represent the average, error bars the SD; n = 3


( H ) Comparative expression of DUX - dependent , DPPA2 DPPA4 - dependent , of genes in mESCs sorted for expression of both Tomato GFP reporters driven by MERVL Zscan4 promoters , respectively , double - negative population ( unpaired t - ) .

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(H) Comparative expression of DUX-dependent, DPPA2 and DPPA4-dependent, and the rest of the genes in mESCs sorted for expression of both Tomato and GFP reporters driven by MERVL and Zscan4 promoters, respectively, and the double-negative population (unpaired t-test).


( A ) Comparative expression by qPCR of Dux , two downstream targets of DUX ( Zscan4 Tdpoz4 ) , three targets of DPPA2 DPPA4 independent of DUX ( Mael , Tdrd1 , Prex2 ) in mESCs depleted of endogenous DPPA2 DPPA4 overexpressing ectopically DPPA2 , DPPA4 or GFP as a control Expression was normalized to Actb . Bars represent average , error bars SD ; n = 3 . * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 , unpaired t - .

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(A) Comparative expression by qPCR of Dux, two downstream targets of DUX (Zscan4 and Tdpoz4), and three targets of DPPA2 and DPPA4 independent of DUX (Mael, Tdrd1, Prex2) in mESCs depleted of endogenous DPPA2 and DPPA4 and overexpressing ectopically DPPA2, DPPA4 or GFP as a control Expression was normalized to Actb. Bars represent the average, error bars the SD; n = 3. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, unpaired t-test.


( B ) Average coverage normalized for sequencing depth of ChIP - seq signal of DPPA2 , DPPA4 or DPPA4 truncated of SAP domain overexpressed in mESCs in a window of 500bp around Dux gene ( n = 2 ) . Total input ( TI ) is shown in gray . Peaks over Dux gene were called in DPPA2 , DPPA4 DPPA4ΔSAP ChIP - seq .

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(B) Average coverage normalized for sequencing depth of the ChIP-seq signal of DPPA2, DPPA4 or DPPA4 truncated of the SAP domain overexpressed in mESCs in a window of 500bp around the Dux gene (n=2). Total input (TI) is shown in gray. Peaks over the Dux gene were called in DPPA2, DPPA4 and DPPA4ΔSAP ChIP-seq.


( C ) showing distribution of DUX , DPPA2 , DPPA4 DPPA4ΔSAP coverage in a ± 5kb window around TSS of DUX - dependent , DPPA2 DPPA4 - dependent random genes in mESCs .

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(C) Heatmap showing the distribution of DUX, DPPA2, DPPA4 and DPPA4ΔSAP coverage in a ± 5kb window around the TSS of DUX-dependent, DPPA2 and DPPA4-dependent and random genes in mESCs.


( D ) Boxplot / Jitterplot representing of GC nucleotide contents in peaks from DPPA2 , DPPA4 DUX ChIP , in a random shuffle of peaks .

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(D) Boxplot/Jitterplot representing the percentage of GC nucleotide contents in the peaks from DPPA2, DPPA4 and DUX ChIP, and in a random shuffle of peaks.


Total expression of transposable element subfamilies in ( A ) Dppa2 KO compared to WT mESC clones .

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Total expression of transposable element subfamilies in (A) Dppa2 KO compared to WT mESC clones.


Total expression of transposable element subfamilies in ( B ) Dppa4 KO compared to WT mESC clones .

Total expression of transposable element subfamilies in (B) Dppa4 KO compared to WT mESC clones.


( C ) - seq of MERVL - int , L1Md _ A L1Md _ T in mESCs sorted for expression of both Tomato GFP reporters driven by MERVL Zscan4 promoters , respectively , double - negative population .

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(C) RNA-seq analysis of MERVL-int, L1Md_A and L1Md_T in mESCs sorted for expression of both Tomato and GFP reporters driven by MERVL and Zscan4 promoters, respectively, and the double-negative population.


( D ) displaying enrichment of DUX , DPPA2 , DPPA4 DPPA4ΔSAP binding at different transposable element families . Every subfamily enriched ( pval < 0 . 05 ) in at least one DPPA2 , DPPA4 or DPPA4ΔSAP replicate are shown .

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(D) Heatmap displaying the enrichment of DUX, DPPA2, DPPA4 and DPPA4ΔSAP binding at different transposable element families. Every subfamily enriched (pval < 0.05) in at least one DPPA2, DPPA4 or DPPA4ΔSAP replicate are shown.


( E ) MSA plot of DPPA2 , DPPA4 DPPA4ΔSAP ChIP - seq in mESCs showing enrichment over L1Md _ A , L1Md _ T MERVL - int sequences . Gaps are in grey sequences in with overlap of color - coded ChIP - seq signals . Upper plot shows average coverage of signal over aligned TEs .

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(E) MSA plot of DPPA2, DPPA4 and DPPA4ΔSAP ChIP-seq in mESCs showing enrichment over L1Md_A, L1Md_T and MERVL-int sequences. Gaps are in grey and sequences in white with overlap of color-coded ChIP-seq signals. Upper plot shows average coverage of the signal over the aligned TEs.


Proliferation curves of parental AGO2KO HeLaS3 are plotted ( n = 6 experimental replicates ) .

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Proliferation curves of parental and AGO2KO HeLaS3 cells are plotted (n=6 experimental replicates).


Colony - forming activity of HeLaS3 AGO2KO was determined by colony - formation ( n = 3 experimental replicates ) .

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Colony-forming activity of HeLaS3 and AGO2KO cells was determined by colony-formation assay (n=3 experimental replicates).


Tumour growth curves of xenografts derived from HeLaS3 or AGO2KO are shown as tumour diameter at different time points ( n = 5 mice for each group ) .

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Tumour growth curves of xenografts derived from HeLaS3 or AGO2KO cells are shown as tumour diameter at different time points (n=5 mice for each group).


Xenograft tumour weights were measured 30 days post - injection ( n = 5 mice for each group ) .

Xenograft tumour weights were measured 30 days post- injection (n=5 mice for each group).


HeLaS3 were transduced with a lentiviral vector coding for human TERC ( HeLaS3 _ TERC ) or for GFP , as a control ( HeLaS3 _ GFP ) . relative expression of TERC was measured by RT - qPCR . HPRT1 was used as internal control ( n = 3 experimental replicates ) .

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HeLaS3 cells were transduced with a lentiviral vector coding for human TERC (HeLaS3_TERC) or for GFP, as a control (HeLaS3_GFP). The relative expression of TERC was measured by RT-qPCR. HPRT1 was used as internal control (n = 3 experimental replicates).


relative expression of terc - sRNA was measured in HeLaS3 _ TERC HeLaS3 _ GFP by RT - qPCR . RNU44 was used as internal control ( n = 3 experimental replicates ) .

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The relative expression of terc-sRNA was measured in HeLaS3_TERC and HeLaS3_GFP by RT-qPCR. RNU44 was used as internal control (n = 3 experimental replicates).


Average telomere was measured from genomic of parental AGO2KO HeLaS3 , by qPCR amplification of telomere repeats ( T ) multicopy gene Alu ( R ) , as a reference . relative telomere ( T / R ) was plotted ( n = 3 experimental replicates )

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Average telomere length was measured from genomic DNA of parental and AGO2KO HeLaS3, by qPCR amplification of telomere repeats (T) and multicopy gene Alu (R), as a reference. The relative telomere length (T/R) was plotted (n=3 experimental replicates)


Representative of metaphase spreads obtained from HeLaS3 AGO2KO stained for telomeric sequences centromere 2 alphoid .

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Representative images of metaphase spreads obtained from HeLaS3 and AGO2KO cells stained for telomeric sequences and centromere 2 alphoid DNA.


Representative telomere distributions in parental AGO2KO HeLaS3 . Average telomere standard deviation , as well as total number of telomeres analyzed , are indicated . Notice marked shift towards short telomeres in AGO2KO compared with parental HeLa S3 .

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Representative telomere length distributions in parental and AGO2KO HeLaS3. Average telomere length and the standard deviation, as well as the total number of telomeres analyzed, are indicated. Notice the marked shift towards short telomeres in AGO2KO cells compared with the parental HeLa S3 cell line.


Telomerase activity was detected by TRAP in parental HeLaS3 AGO2KO . 293T were used as a positive control . As negative controls , telomerase was heat - inactivated in extracts , in last lane no were added in lysis buffer . of telomerase products ( 6 bp - ladder ) was determined by ImageLab Software ( Biorad ) normalized with of Internal Control ( IC ) ( n = 3 experimental replicates ) .

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Telomerase activity was detected by TRAP in parental HeLaS3 and AGO2KO cells. 293T cells were used as a positive control. As negative controls, telomerase was heat- inactivated in cell extracts, in the last lane no cells were added in lysis buffer. Intensity of the telomerase products (6 bp-ladder) was determined by ImageLab Software (Biorad) and normalized with the intensity of the Internal Control (IC) (n=3 experimental replicates).


AGO2KO were transduced with a lentiviral vector coding for FLAG - HA - tagged AGO2 ( AGO2KO _ FH AGO2 ) or for GFP as a control ( AGO2KO _ GFP ) . of telomerase activity in AGO2KO _ FH AGO2 AGO2KO _ GFP , as assessed by TRAP , was plotted ( n = 3 experimental replicates ) .

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AGO2KO cells were transduced with a lentiviral vector coding for FLAG-HA-tagged AGO2 (AGO2KO_FH AGO2) or for GFP as a control (AGO2KO_GFP). Quantification of telomerase activity in AGO2KO_FH AGO2 and AGO2KO_GFP, as assessed by TRAP, was plotted (n=3 experimental replicates).


HeLaS3 were transduced with a lentiviral vector coding for FLAG - HA - tagged AGO2 ( HeLaS3 _ FH AGO2 ) or for GFP as a control ( HeLaS3 _ GFP ) . of telomerase activity in HeLaS3 _ FH AGO2 HeLaS3 _ GFP , as assessed by TRAP , was plotted ( n = 3 experimental replicates ) .

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HeLaS3 cells were transduced with a lentiviral vector coding for FLAG-HA-tagged AGO2 (HeLaS3_FH AGO2) or for GFP as a control (HeLaS3_GFP). Quantification of telomerase activity in HeLaS3_FH AGO2 and HeLaS3_GFP, as assessed by TRAP, was plotted (n=3 experimental replicates).


relative expression of TERC was measured by RT - qPCR from total of HeLaS3 AGO2KO . HPRT1 was used as internal control ( n = 3 experimental replicates ) . qPCR of 3 ' - extended TERC transcript in cDNA from HeLaS3 AGO2KO generated using random hexamers . HPRT1 was used for normalization ( n = 3 experimental replicates ) . qPCR of TERC transcript in cDNA from HeLaS3 AGO2KO generated using oligo ( dT ) 10 priming . HPRT1 was used for normalization ( n = 3 experimental replicates ) .

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The relative expression of TERC was measured by RT-qPCR from total RNA of HeLaS3 and AGO2KO cells. HPRT1 was used as internal control (n = 3 experimental replicates). qPCR of 3'-extended TERC transcript in cDNA from HeLaS3 and AGO2KO cells generated using random hexamers. HPRT1 was used for normalization (n=3 experimental replicates). qPCR of TERC transcript in cDNA from HeLaS3 and AGO2KO cells generated using oligo(dT)10 priming. HPRT1 was used for normalization (n=3 experimental replicates).


Whole extract of HeLaS3 AGO2KO was analyzed by western blot for presence of TERT . GAPDH was used as loading control . Data are representative of 4 independent experiments . TERT level was quantified by ImageLab Software ( Biorad ) . GAPDH was use as internal control ( n = 4 experimental replicates )

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Whole cell extract of HeLaS3 and AGO2KO was analyzed by western blot for the presence of TERT protein. GAPDH was used as loading control. Data are representative of 4 independent experiments. TERT protein level was quantified by ImageLab Software (Biorad). GAPDH was use as internal control (n=4 experimental replicates)


HeLaS3 AGO2KO extracts ( n = 3 ) , were immunoprecipitated using an anti - TERT antibody or IgG as mock IP . TERC HOTAIR ( as a negative control ) abundance was assessed by RT - qPCR . HPRT1 was used for normalization enrichment in TERT - RIP as compared to IgG RIP was plotted .

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HeLaS3 and AGO2KO cell extracts (n=3), were immunoprecipitated using an anti-TERT antibody or IgG as mock IP. TERC and HOTAIR (as a negative control) abundance was assessed by RT-qPCR. HPRT1 was used for normalization and enrichment in TERT-RIP as compared to IgG RIP was plotted.


AGO2KO _ FH AGO2 AGO2KO _ GFP extracts ( n = 3 experimental replicates ) were immunoprecipitated using an anti - TERT antibody or IgG as mock IP . TERC HOTAIR ( as a negative control ) abundance was assessed by RT - qPCR . HPRT1 was used for normalization enrichment in TERT - RIP as compared to IgG RIP was plotted .

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AGO2KO_FH AGO2 and AGO2KO_GFP cell extracts (n=3 experimental replicates) were immunoprecipitated using an anti-TERT antibody or IgG as mock IP. TERC and HOTAIR (as a negative control) abundance was assessed by RT-qPCR. HPRT1 was used for normalization and enrichment in TERT-RIP as compared to IgG RIP was plotted.


HeLaS3 whole - extract was immunoprecipitated using anti - AGO2 antibody or IgG , as mock IP . Whole lysates ( input ) immunoprecipitates were analyzed by western blot with anti - TERT antibody . As a positive control , presence in immunoprecipitates of GW182 , a known AGO2 - interacting , was assessed . Data are representative of three independent experiments .

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HeLaS3 whole-cell extract was immunoprecipitated using anti- AGO2 antibody or IgG, as mock IP. Whole cell lysates (input) and immunoprecipitates were analyzed by western blot with anti-TERT antibody. As a positive control, the presence in immunoprecipitates of GW182, a known AGO2-interacting protein, was assessed. Data are representative of three independent experiments.


HeLaS3 whole - extract was immunoprecipitated using anti - AGO2 antibody or IgG , as mock IP . TERC enrichment in AGO2 RIP as compared to IgG RIP was assessed by RT - qPCR . 7SK was used for normalization ( n = 4 experimental replicates ) .

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HeLaS3 whole-cell extract was immunoprecipitated using anti- AGO2 antibody or IgG, as mock IP. TERC RNA enrichment in AGO2 RIP as compared to IgG RIP was assessed by RT-qPCR . 7SK RNA was used for normalization (n=4 experimental replicates).


RIP was performed from HeLaS3 _ FH AGO2 HeLaS3 _ GFP extract using anti - HA antibody or IgG , as negative control , followed by TERC detection by RT - qPCR . 7SK was used for normalization ( n = 3 experimental replicates ) .

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RIP assay was performed from HeLaS3_FH AGO2 and HeLaS3_GFP cell extract using anti-HA antibody or IgG, as negative control, followed by TERC detection by RT-qPCR. 7SK RNA was used for normalization (n=3 experimental replicates).


HeLaS3 were transduced with a lentiviral vector coding for wild - type TERC ( HeLaS3 _ TERCwt ) , TERC mutated in position 313 - 340 ( HeLaS3 _ TERC ( 313 - 340mut ) ) or TERC delated in position 12 - 31 ( HeLaS3 _ TERC ( Δ 12 - 31 ) ) . Whole - lysates from HeLaS3 _ TERCwt , HeLaS3 _ TERC ( 313 - 340mut ) HeLaS3 _ TERC ( Δ 12 - 31 ) was immunoprecipitated using an anti - AGO2 antibody in order to assessed impact of TERC mutations on AGO2 binding . For each experiment enrichment of ectoTERC as compared to 7SK in RIP samples was normalized on amount of ectoTERC 7SK in input samples . Association of AGO2 with TERC ( 313 - 340mut TERC ( Δ 12 - 31 ) was compared to association with TERCwt ( n = 3 experimental replicates ) .

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HeLaS3 cells were transduced with a lentiviral vector coding for wild-type TERC (HeLaS3_TERCwt), TERC mutated in position 313-340 (HeLaS3_TERC (313-340mut)) or TERC delated in position 12-31 (HeLaS3_TERC (Δ 12-31)). Whole-cell lysates from HeLaS3_TERCwt, HeLaS3_TERC(313-340mut) and HeLaS3_TERC (Δ 12-31) was immunoprecipitated using an anti-AGO2 antibody in order to assessed the impact of TERC mutations on AGO2 binding. For each experiment the enrichment of ectoTERC as compared to 7SK RNA in RIP samples was normalized on the amount of ectoTERC and 7SK RNA in input samples. Association of AGO2 with TERC(313-340mut and TERC (Δ 12-31) was compared to the association with TERCwt (n=3 experimental replicates).


HeLaS3 AGO2KO were transfected with a synthetic terc - sRNA or an siRNA with no target in humans ( siCtl ) , as a control . Four days after transfection , telomerase activity in 100 - 50 - lysate was assessed by TRAP . of telomerase products ( 6 bp - ladder ) was determined by ImageLab Software ( Biorad ) normalized with of Internal Control ( IC ) , ( n = 4 experimental replicates )

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HeLaS3 and AGO2KO cells were transfected with a synthetic terc-sRNA or an siRNA with no target in humans (siCtl), as a control. Four days after transfection, telomerase activity in 100- and 50 cell-lysate was assessed by TRAP. Intensity of the telomerase products (6 bp-ladder) was determined by ImageLab Software (Biorad) and normalized with the intensity of the Internal Control (IC), (n=4 experimental replicates)


Comparison of colony forming unit ( CFU ) in feces from vehicle - ABX - treated mice ( n = 5 mice / group ) . n . d . , non - detectable .

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Comparison of bacterial colony forming unit (CFU) in feces from vehicle- and ABX-treated mice (n = 5 mice/group). n.d., non-detectable.


Representative image measurements of lengths for CD31 + / LYVE - 1 + lacteal , CD31 + / LYVE - 1 - capillary plexus DAPI + villus .

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Representative image and measurements of lengths for CD31+/LYVE-1+ lacteal, CD31+/LYVE-1- capillary plexus and DAPI+ villus.


comparisons of villi lengths absolute relative lacteal lengths in duodenum ( DD ) , jejunum ( JJ ) ileum ( IL ) of vehicle - or ABX - treated mice . Each dot indicates mean value of 5 - 10 villi in a mouse ( n = 6 mice / group ) .

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Images and comparisons of villi lengths and absolute and relative lacteal lengths in duodenum (DD), jejunum (JJ) and ileum (IL) of vehicle- or ABX-treated mice. Each dot indicates mean value of 5-10 villi in a mouse (n = 6 mice/group).


comparisons of number of Prox1 + LECs within initial 100 μm portion ( F ) presented as relative ( FI ) ) in CD31 + / LYVE - 1 + lacteals of jejunum from vehicle - or ABX - treated mice . Dotted lines outline villi . Each dot indicates mean value of 5 - 10 villi in a mouse ( n = 6 mice / group ) . AU , arbitrary unit .

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Images and comparisons of the number of Prox1+ LECs within initial 100 μm portion (F) presented as relative fluorescence intensity (FI)) in CD31+/LYVE-1+ lacteals of jejunum from vehicle- or ABX-treated mice. Dotted lines outline villi. Each dot indicates mean value of 5-10 villi in a mouse (n = 6 mice/group). AU, arbitrary unit.


comparisons of VEGFR3 expression ( G ) , presented as relative ( FI ) ) in CD31 + / LYVE - 1 + lacteals of jejunum from vehicle - or ABX - treated mice . Dotted lines outline villi . Each dot indicates mean value of 5 - 10 villi in a mouse ( n = 6 mice / group ) . AU , arbitrary unit .

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Images and comparisons of the VEGFR3 expression (G), presented as relative fluorescence intensity (FI)) in CD31+/LYVE-1+ lacteals of jejunum from vehicle- or ABX-treated mice. Dotted lines outline villi. Each dot indicates mean value of 5-10 villi in a mouse (n = 6 mice/group). AU, arbitrary unit.


comparison of VE - cadherin LEC junctions in lacteals of jejunum between vehicle - ABX - treated mice . Each boxed portion is highly magnified for visualization of detailed distributions of VE - cadherin to distinguish button - like ( red arrowheads ) , zipper - like ( open arrowheads ) mixed LEC junctions in lacteals ( n = 6 mice / group , 5 - 10 villi / mouse ) . Scale bars , 50 μm .

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Images and comparison of VE-cadherin LEC junctions in lacteals of jejunum between vehicle- and ABX-treated mice. Each boxed portion is highly magnified for visualization of detailed distributions of VE-cadherin to distinguish button-like (red arrowheads), zipper-like (open arrowheads) and mixed LEC junctions in lacteals (n = 6 mice/group, 5-10 villi/mouse). Scale bars, 50 μm.


Representative intravital imaging of lipid clearance from lamina propria via lacteals . Asterisks indicate site in lamina propria where ( FI ) was measured . Relative FI divided by mean FI of controls at 16 min ( left ) normalized FI divided by FI at 16 min in each villus ( right ) after initial loading of BODIPY - FA were quantified . For normalization of FI , villi that showed peak earlier or later than 16 min were excluded ( n = 5 - 7 mice / group , 5 - 10 villi / mouse ) . Scale bars , 100 μm .

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Representative intravital imaging and quantification of lipid clearance from lamina propria via lacteals. Asterisks indicate the site in lamina propria where fluorescence intensity (FI) was measured. Relative FI divided by mean FI of controls at 16 min (left) and normalized FI divided by FI at 16 min in each villus (right) after initial loading of BODIPY-FA were quantified. For normalization of FI, villi that showed peak intensity earlier or later than 16 min were excluded (n = 5-7 mice/group, 5-10 villi/mouse). Scale bars, 100 μm.


Representative from transmission electron microscopic examination of lacteals of frequency of open junctions ( n = 4 mice / group , 4 - 6 / mouse ) . Jejunum of vehicle - ABX - treated mice were harvested 2 hours after oral lipid loading . Note that junction between LECs ( red colored bidirectional arrows ) is open in vehicle - treated mice , but not in ABX - treated mice . CM , chylomicron ; IS , interstitium of lamina propria ; LC , large caveola ; Lu , lacteal lumen ; Ve , vesicle containing lipoproteins . Scale bars , 1 μm .

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Representative images from transmission electron microscopic examination of lacteals and quantification of frequency of open junctions (n = 4 mice/group, 4-6 images/mouse). Jejunum of vehicle- and ABX-treated mice were harvested 2 hours after oral lipid loading. Note that the junction between LECs (red colored bidirectional arrows) is open in vehicle-treated mice, but not in ABX-treated mice. CM, chylomicron; IS, interstitium of lamina propria; LC, large caveola; Lu, lacteal lumen; Ve, vesicle containing lipoproteins. Scale bars, 1 μm.


of frequency of open junctions ( n = 4 mice / group , 4 - 6 / mouse ) . Note that junction between LECs ( red colored bidirectional arrows ) is open in vehicle - treated mice , but not in ABX - treated mice .

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quantification of frequency of open junctions (n = 4 mice/group, 4-6 images/mouse). Note that the junction between LECs (red colored bidirectional arrows) is open in vehicle-treated mice, but not in ABX-treated mice.


Comparisons of serum triglyceride ( TG ) free fatty ( FFA ) at indicated time points in vehicle - ABX - treated mice ( n = 5 - 7 mice / group ) .

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Comparisons of serum triglyceride (TG) and free fatty acids (FFA) at indicated time points in vehicle- and ABX- treated mice (n = 5-7 mice/group).


comparisons of absolute relative lacteal lengths in jejunum between vehicle - or ABX - treated mice at P7 , P14 , P28 . Each dot indicates mean value of 5 - 10 villi in a mouse ( n = 6 mice / group ) . Scale bars , 100 μm .

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Images and comparisons of absolute and relative lacteal lengths in jejunum between vehicle- or ABX-treated mice at P7, P14, and P28. Each dot indicates mean value of 5-10 villi in a mouse (n = 6 mice/group). Scale bars, 100 μm.


comparisons of number of Prox1 + LECs within initial 100 μm portion in CD31 + / LYVE - 1 + lacteals of jejunum from vehicle - or ABX - treated mice . Dotted lines outline villi . Each dot indicates mean value of 5 - 10 villi in a mouse ( n = 6 mice / group ) . Scale bars , 100 μm .

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

Images and comparisons of number of Prox1+ LECs within initial 100 μm portion in CD31+/LYVE-1+ lacteals of jejunum from vehicle- or ABX-treated mice. Dotted lines outline villi. Each dot indicates mean value of 5-10 villi in a mouse (n = 6 mice/group). Scale bars, 100 μm.


comparisons of proportions of each VE - cadherin LEC junctions in CD31 + / LYVE - 1 + lacteals in jejunum between vehicle - or ABX - treated mice at P7 , P14 , P28 . Button - like ( red arrowheads ) zipper - like ( open arrowheads ) junctions are indicated in each magnified box in right ( n = 6 mice / group , 5 - 10 villi / mouse ) . Scale bars , 25 μm .

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

Images and comparisons of proportions of each VE-cadherin LEC junctions in CD31+/LYVE-1+ lacteals in jejunum between vehicle- or ABX-treated mice at P7, P14, and P28. Button-like (red arrowheads) and zipper-like (open arrowheads) junctions are indicated in each magnified box in right (n = 6 mice/group, 5-10 villi/mouse). Scale bars, 25 μm.


comparisons of absolute relative lacteal lengths in duodenum ( DD ) , jejunum ( JJ ) ileum ( IL ) of 8 - week old SPF , GF or CONV mice . Each dot indicates mean value of 5 - 10 villi in a mouse ( n = 6 mice / group ) . Scale bars , 100 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

Images and comparisons of absolute and relative lacteal lengths in duodenum (DD), jejunum (JJ) and ileum (IL) of 8-week old SPF, GF or CONV mice. Each dot indicates mean value of 5-10 villi in a mouse (n = 6 mice/group). Scale bars, 100 μm.


comparisons of number of Prox1 + LECs within initial 100 μm portion ( C ) VEGFR3 expression ( D , presented as relative ( FI ) ) in CD31 + / LYVE - 1 + lacteals of jejunum from SPF , GF CONV mice . Each dot indicates mean value of 5 - 10 villi in a mouse ( n = 6 mice / group ) . AU , arbitrary unit . Scale bars , 100 μm .

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

Images and comparisons of the number of Prox1+ LECs within initial 100 μm portion (C) and VEGFR3 expression (D, presented as relative fluorescent intensity (FI)) in CD31+/LYVE-1+ lacteals of jejunum from SPF, GF and CONV mice. Each dot indicates mean value of 5-10 villi in a mouse (n = 6 mice/group). AU, arbitrary unit. Scale bars, 100 μm.


comparison of proportions of each VE - cadherin LEC junctions in CD31 + / LYVE - 1 + lacteals in jejunum among SPF , GF CONV mice . Button - like ( red arrowheads ) zipper - like ( open arrowheads ) junctions are indicated in each magnified box in below ( n = 6 mice / group , 5 - 10 villi / mouse ) . Scale bars , 25 μm .

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

Images and comparison of proportions of each VE-cadherin LEC junctions in CD31+/LYVE-1+ lacteals in jejunum among SPF, GF and CONV mice. Button-like (red arrowheads) and zipper-like (open arrowheads) junctions are indicated in each magnified box in below (n = 6 mice/group, 5-10 villi/mouse). Scale bars, 25 μm.


comparison of VEGFR3 expression ( presented as relative ( FI ) ) in CD31 + / LYVE - 1 + lacteals of jejunum between WT VR3iΔLEC mice . Each dot indicates mean value of 5 - 10 villi in a mouse ( n = 6 mice / group ) . AU , arbitrary unit . Scale bars , 100 μm .

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

Images and comparison of VEGFR3 expression (presented as relative fluorescent intensity (FI)) in CD31+/LYVE-1+ lacteals of jejunum between WT and VR3iΔLEC mice. Each dot indicates mean value of 5-10 villi in a mouse (n = 6 mice/group). AU, arbitrary unit. Scale bars, 100 μm.


comparisons of absolute relative lacteal lengths in duodenum ( DD ) , jejunum ( JJ ) ileum ( IL ) of WT VR3iΔLEC mice . Note that , in addition to alterations in lacteal lengths in VR3iΔLEC mice , diameters of lacteal also shrank by VEGFR3 ablation . Each dot indicates mean value of 5 - 10 villi in a mouse ( n = 6 mice / group ) . Scale bars , 100 μm .

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

Images and comparisons of absolute and relative lacteal lengths in duodenum (DD), jejunum (JJ) and ileum (IL) of WT and VR3iΔLEC mice. Note that, in addition to the alterations in the lacteal lengths in VR3iΔLEC mice, diameters of the lacteal also shrank by VEGFR3 ablation. Each dot indicates mean value of 5-10 villi in a mouse (n = 6 mice/group). Scale bars, 100 μm.


comparisons of number of Prox1 + LECs within initial 100 μm portion in CD31 + / LYVE - 1 + lacteals of jejunum from WT VR3iΔLEC mice . Dotted lines outline villi . Each dot indicates mean value of 5 - 10 villi in a mouse ( n = 6 mice / group ) . Scale bars , 100 μm .

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

Images and comparisons of number of Prox1+ LECs within initial 100 μm portion in CD31+/LYVE-1+ lacteals of jejunum from WT and VR3iΔLEC mice. Dotted lines outline villi. Each dot indicates mean value of 5-10 villi in a mouse (n = 6 mice/group). Scale bars, 100 μm.


comparison of proportions of each VE - cadherin LEC junctions in CD31 + / LYVE - 1 + lacteals in jejunum from WT VR3iΔLEC mice . Button - like ( red arrowheads ) zipper - like ( open arrowheads ) junctions are indicated in each magnified box in below ( n = 6 mice / group , 5 - 10 villi / mouse ) . Scale bars , 25 μm .

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

Images and comparison of proportions of each VE-cadherin LEC junctions in CD31+/LYVE-1+ lacteals in jejunum from WT and VR3iΔLEC mice. Button-like (red arrowheads) and zipper-like (open arrowheads) junctions are indicated in each magnified box in below (n = 6 mice/group, 5-10 villi/mouse). Scale bars, 25 μm.


Comparisons of serum triglyceride ( TG ) at indicated time points in WT VR3iΔLEC mice ( n = 6 mice / group ) .

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

Comparisons of serum triglyceride (TG) at indicated time points in WT and VR3iΔLEC mice (n = 6 mice/group).


Comparison of of VEGF - C in whole tissue of jejunum ileum from SPF , GF , CONV mice ( n = 3 - 5 mice / group ) .

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

Comparison of protein levels of VEGF-C in the whole tissue of jejunum and ileum from SPF, GF, and CONV mice (n = 3-5 mice/group).


Comparison of mRNA of VEGF - C in whole tissue of jejunum ileum from SPF , GF , CONV mice ( n = 3 - 5 mice / group ) .

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

Comparison of mRNA levels of VEGF-C in the whole tissue of jejunum and ileum from SPF, GF, and CONV mice (n = 3-5 mice/group).


Comparison of mRNA of VEGF - C in sorted CD45 - stromal CD45 + MHCII + F4 / 80 + macrophages from jejunum ileum of vehicle - treated mice . mean of transcription of VEGF - C in CD45 - stromal was normalized to 1 , relative in macrophages were presented as fold change ( n = 4 mice / group ) .

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 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

Comparison of mRNA levels of VEGF-C in sorted CD45- stromal cells and CD45+ MHCII+ F4/80+ macrophages from jejunum and ileum of vehicle-treated mice. The mean of transcription levels of VEGF-C in CD45- stromal cells was normalized to 1, and the relative levels in macrophages were presented as fold change (n = 4 mice/group).


Comparisons of VEGF - C transcription level in CD45 - stromal ( left ) CD45 + MHCII + F4 / 80 + macrophages ( right ) in jejunum ileum of vehicle - ABX - treated mice . mean of transcription of VEGF - C in vehicle - treated mice was normalized to 1 , relative in ABX - treated mice were presented as fold change ( n = 5 - 6 mice / group ) .

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 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

Comparisons of VEGF-C transcription level in CD45- stromal cells (left) and CD45+ MHCII+ F4/80+ macrophages (right) in jejunum and ileum of vehicle- and ABX-treated mice. The mean of transcription levels of VEGF-C in vehicle-treated mice was normalized to 1, and the relative levels in ABX-treated mice were presented as fold change (n = 5 -6 mice/group).


comparison of F4 / 80 + macrophages in villi of duodenum ( DD ) , jejunum ( JJ ) , ileum ( IL ) from vehicle - or ABX - treated mice . Each dot indicates mean value of 5 sites in a mouse ( n = 6 mice / group ) . AU , arbitrary unit . Scale bars , 100 μm .

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

Images and comparison of F4/80+ macrophages in the villi of duodenum (DD), jejunum (JJ), and ileum (IL) from vehicle- or ABX-treated mice. Each dot indicates mean value of 5 sites in a mouse (n = 6 mice/group). AU, arbitrary unit. Scale bars, 100 μm.


Representative flow cytometric comparison of MHCII + F4 / 80 + macrophage gated on DAPI - CD45 + ( E ) macrophage gated on DAPI - CD45 + from jejunum ileum of vehicle - ABX - treated mice ( n = 5 mice / group ) .

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

Representative flow cytometric analysis and comparison of MHCII+ F4/80+ macrophage gated on DAPI- CD45+ cells (E) macrophage gated on DAPI- CD45+ cells from jejunum and ileum of vehicle- and ABX-treated mice (n = 5 mice/group).


Representative flow cytometric comparison of CX3CR1 + F4 / 80 + macrophage gated on DAPI - CD45 + MHCII + ( F ) macrophage gated on DAPI - CD45 + from jejunum ileum of vehicle - ABX - treated mice ( n = 5 mice / group ) .

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

Representative flow cytometric analysis and comparison of CX3CR1+ F4/80+ macrophage gated on DAPI- CD45+ MHCII+ cells (F) macrophage gated on DAPI- CD45+ cells from jejunum and ileum of vehicle- and ABX-treated mice (n = 5 mice/group).


Representative flow cytometric comparison of MHCII + F4 / 80 + macrophage gated on DAPI - CD45 + from jejunum ileum of WT DTR mice ( n = 5 mice / group ) .

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

Representative flow cytometric analysis and comparison of MHCII+ F4/80+ macrophage gated on DAPI- CD45+ cells from jejunum and ileum of WT and DTR mice (n = 5 mice/group).


Comparison of mRNA of VEGF - C in whole tissue of jejunum ileum from WT DTR mice ( n = 5 mice / group ) .

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

Comparison of mRNA levels of VEGF-C in the whole tissue of jejunum and ileum from WT and DTR mice (n = 5 mice/group).


comparisons of absolute relative lacteal lengths in duodenum ( DD ) , jejunum ( JJ ) ileum ( IL ) of WT DTR mice . Each dot indicates mean value of 5 - 10 villi in a mouse ( n = 6 mice / group ) . Scale bars , 100 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

Images and comparisons of absolute and relative lacteal lengths in duodenum (DD), jejunum (JJ) and ileum (IL) of WT and DTR mice. Each dot indicates mean value of 5-10 villi in a mouse (n = 6 mice/group). Scale bars, 100 μm.


comparisons of number of Prox1 + LECs within initial 100 μm portion ( upper ) VEGFR3 expression ( lower , presented as relative ( FI ) ) in CD31 + / LYVE - 1 + lacteals of jejunum from WT DTR mice . Each dot indicates mean value of 5 - 10 villi in a mouse ( n = 6 mice / group ) . AU , arbitrary unit . Scale bars , 100 μm .

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

Images and comparisons of number of Prox1+ LECs within initial 100 μm portion (upper) and VEGFR3 expression (lower, presented as relative fluorescent intensity (FI)) in CD31+/LYVE-1+ lacteals of jejunum from WT and DTR mice. Each dot indicates mean value of 5-10 villi in a mouse (n = 6 mice/group). AU, arbitrary unit. Scale bars, 100 μm.


comparison of proportions of each VE - cadherin LEC junctions in CD31 + / LYVE - 1 + lacteals in jejunum between WT DTR mice . Button - like ( red arrowheads ) zipper - like ( open arrowheads ) junctions are indicated in each magnified box in below ( n = 6 mice / group , 5 - 10 villi / mouse ) . Scale bars , 25 μm .

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

Images and comparison of proportions of each VE-cadherin LEC junctions in CD31+/LYVE-1+ lacteals in jejunum between WT and DTR mice. Button-like (red arrowheads) and zipper-like (open arrowheads) junctions are indicated in each magnified box in below (n = 6 mice/group, 5-10 villi/mouse). Scale bars, 25 μm.


Representative flow cytometric comparison of MHCII + F4 / 80 + macrophage gated on DAPI - CD45 + from jejunum ileum of WT MyD88ΔMP mice ( n = 5 mice / group ) .

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

Representative flow cytometric analysis and comparison of MHCII+ F4/80+ macrophage gated on DAPI- CD45+ cells from jejunum and ileum of WT and MyD88ΔMP mice (n = 5 mice/group).


Comparisons of mRNA of VEGF - C in sorted MHCII + F4 / 80 + macrophages from jejunum ileum of WT MyD88ΔMP mice . mean of transcription of VEGF - C in WT mice was normalized to 1 , relative level in MyD88ΔMP mice were presented as fold change ( n = 5 mice / group ) .

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

Comparisons of mRNA levels of VEGF-C in sorted MHCII+ F4/80+ macrophages from jejunum and ileum of WT and MyD88ΔMP mice. The mean of transcription levels of VEGF-C in WT mice was normalized to 1, and the relative level in MyD88ΔMP mice were presented as fold change (n = 5 mice/group).


comparisons of absolute relative lacteal lengths in duodenum ( DD ) , jejunum ( JJ ) ileum ( IL ) of WT MyD88ΔMP mice . Each dot indicates mean value of 5 - 10 villi in a mouse ( n = 6 mice / group ) . Scale bars , 100 μm .

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

Images and comparisons of absolute and relative lacteal lengths in duodenum (DD), jejunum (JJ) and ileum (IL) of WT and MyD88ΔMP mice. Each dot indicates mean value of 5-10 villi in a mouse (n = 6 mice/group). Scale bars, 100 μm.


comparisons of number of Prox1 + LECs within initial 100 μm portion ( upper ) VEGFR3 expression ( lower , presented as relative ( FI ) ) in CD31 + / LYVE - 1 + lacteals of jejunum from WT MyD88ΔMP mice . Each dot indicates mean value of 5 - 10 villi . Each dot indicates mean value of 5 - 10 villi in a mouse ( n = 6 mice / group ) . AU , arbitrary unit . Scale bars , 100 μm .

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

Images and comparisons of number of Prox1+ LECs within initial 100 μm portion (upper) and VEGFR3 expression (lower, presented as relative fluorescent intensity (FI)) in CD31+/LYVE-1+ lacteals of jejunum from WT and MyD88ΔMP mice. Each dot indicates mean value of 5-10 villi. Each dot indicates mean value of 5-10 villi in a mouse (n = 6 mice/group). AU, arbitrary unit. Scale bars, 100 μm.


comparison of proportions of each VE - cadherin LEC junctions in CD31 + / LYVE - 1 + lacteals in jejunum between WT MyD88ΔMP mice . Button - like ( red arrowheads ) zipper - like ( open arrowheads ) junctions are indicated in each magnified box in below ( n = 6 mice / group , 5 - 10 villi / mouse ) . Scale bars , 25 μm .

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

Images and comparison of proportions of each VE-cadherin LEC junctions in CD31+/LYVE-1+ lacteals in jejunum between WT and MyD88ΔMP mice. Button-like (red arrowheads) and zipper-like (open arrowheads) junctions are indicated in each magnified box in below (n = 6 mice/group, 5-10 villi/mouse). Scale bars, 25 μm.


Representative confocal of - type - specific promoter : : GFP : AGO1 translational fusions .

Representative confocal images of cell-type-specific promoter::GFP:AGO1 translational fusions.


Immunoblot of FLAG : AGO1 IPed in each root - tip layer . Scale bars : 50μM , NT : Non - transformed . CB : Coomassie blue .

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

Immunoblot analysis of FLAG:AGO1 IPed in each root-tip layer. Scale bars: 50μM, NT: Non-transformed. CB: Coomassie blue.


Left : input IPed AGO1 following post - lysis addition of a radiolabeled siRNA duplex ( siGFP ) . Right : northern of AGO1 - bound sRNAs .

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 B-MEASURED_VAR O O O O

Left: input and IPed AGO1 following post-lysis addition of a radiolabeled siRNA duplex (siGFP). Right: northern analysis of AGO1-bound sRNAs.


Layer - specific distribution of AGO1 - loaded miRNAs .

Layer-specific distribution of all AGO1-loaded miRNAs.


Northern of layer - specific AGO1 - loaded miRNAs using sRNAs IPed independently from that used for deep - sequencing . Bar - plots underneath each panel display deep - sequencing results as normalized reads .

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

Northern analysis of layer-specific AGO1-loaded miRNAs using sRNAs IPed independently from that used for deep-sequencing. Bar-plots underneath each panel display the deep-sequencing results as normalized reads counts.


Representative confocal of - type - specific promoter : : GFP transcriptional fusions in hyl1 vs WT roots .

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

Representative confocal images of cell-type-specific promoter::GFP transcriptional fusions in hyl1 vs WT roots.


Hierarchical clustering of validated miRNA targets between different layers in either WT or hyl1 roots .

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

Hierarchical clustering of validated miRNA targets between different cell layers in either WT or hyl1 roots.


qRT - PCR validation of three distinct classes of direct miRNA target transcripts from total root - , total root polysome - or epidermis - specific polysome - associated . AT2G37050 kinase is positively regulated by LAC17 . Scale bars : 50μM . qPCR values were normalized to internal control ACTIN2 .

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

qRT-PCR validation of three distinct classes of direct miRNA target transcripts from total root-, total root polysome- or epidermis-specific polysome-associated RNA. The AT2G37050 kinase is positively regulated by LAC17. Scale bars: 50μM. All qPCR values were normalized to the internal control ACTIN2.


miRoot snapshots of whole - root ( left ) layer - specific ( right ) LAC17 mRNA accumulation in WT versus hyl1 , as assessed by polysome profiling .

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

miRoot snapshots of whole-root (left) and layer-specific (right) LAC17 mRNA accumulation in WT versus hyl1, as assessed by polysome profiling.


miRoot snapshot of - type specific miRNAs showing , in this case , a sum group of miR156 family . right panel shows gradient of expression revealed upon removal of stele ( blue box ) .

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

miRoot snapshot of cell-type specific miRNAs showing, in this case, a sum group of the miR156 family. The right panel shows the gradient of expression revealed upon removal of the stele (blue box).


Snapshot of SPL6 miR156 translatome in WT hyl1 backgrounds . Removal of stele ( blue box ) reveals subtleties of miR156 - mediated gene regulation in other layers , resulting in a near - mirror image of ( A ) .

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

Snapshot of the SPL6 miR156 translatome in WT and hyl1 backgrounds. Removal of the stele (blue box) reveals subtleties of miR156-mediated gene regulation in the other layers, resulting in a near-mirror image of (A).


Confocal image of pmiR396a : : H2B : GFP . Normalized read from AGO1 - IP - Seq independent northern of AGO1 - loaded miR396 .

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

Confocal image of pmiR396a::H2B:GFP. Normalized read counts from AGO1-IP-Seq and independent northern analysis of AGO1-loaded miR396.


Representative confocal image of pGRF2 : : H2B : GFP translational fusion .

Representative confocal image of the pGRF2::H2B:GFP translational fusion.


Focused miRoot of GRF2 translatome signal in WT versus hyl1 columella . Graph : GRF2 normalized read .

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

Focused miRoot analysis of the GRF2 translatome signal in WT versus hyl1 columella. Graph: GRF2 normalized read counts.


Representative confocal of pGRF2 : : GRF2 : GFP translational fusion in WT or hyl1 backgrounds .

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

Representative confocal images of the pGRF2::GRF2:GFP translational fusion in WT or hyl1 backgrounds.


Focused miRoot of PLT1 translatome signal in WT versus hyl1 columella .

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Focused miRoot analysis of the PLT1 translatome signal in WT versus hyl1 columella.


Layer - specific normalized read of miR169 paralogs ( separated into 4 different groups on identity : a , b - c , d - g , h - n ) from AGO1 - IP - deep - Seq independent northern , as in Fig . 1F .

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Layer-specific normalized read counts of miR169 paralogs (separated into 4 different groups based on sequence identity: a, b-c, d-g, h-n) from AGO1-IP-deep-Seq and independent northern analysis, as in Fig.1F.


- type specific AGO1 - IP signals for each miR169 - group with ( upper ) or without ( lower panel ) miRoot scale normalization option .

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Cell-type specific AGO1-IP signals for each miR169 sequence-group with (upper) or without (lower panel) the miRoot scale normalization option.


Predicted base - pairing of indicated miR169 - group to four NF - YA target transcripts . Pairing with expectation value greater than 3 was not considered . color gradient ( pale : low , : strong ) qualitatively approximates pairing contribution of each miR169 - group according to upper panel of ( B ) . miRoot snapshots of layer - specific translatome signals of four out - of - seven miR169 NF - YA targets in WT versus hyl1 backgrounds .

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Predicted base-pairing of indicated miR169 sequence-group to four NF-YA target transcripts. Pairing with expectation value greater than 3 was not considered. The color gradient (pale: low, dark: strong) qualitatively approximates the pairing contribution of each miR169 sequence-group according to the upper panel of (B). miRoot snapshots of layer-specific translatome signals of four out-of-seven miR169 NF-YA targets in WT versus hyl1 backgrounds.


Browser representation independent northern AGO1 - loaded miR165 / 166 .

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Browser representation and independent northern analysis and AGO1-loaded miR165/166.


miRoot snapshot of PHB translatome signals in WT versus hyl1 backgrounds .

miRoot snapshot of PHB translatome signals in WT versus hyl1 backgrounds.


Representative confocal of pmiR395 ( a / c / e ) : : H2B : GFP transcriptional fusions under SO4 - - deficient or SO4 - - proficient growth conditions . Northern of AGO1 - loaded miR395 under conditions in ( C ) .

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Representative confocal images of pmiR395(a/c/e)::H2B:GFP transcriptional fusions under SO4--deficient or SO4--proficient growth conditions. Northern analysis of AGO1-loaded miR395 under the conditions in (C).


Representative confocal of pAPS4 : : APS4 : GFP translational fusion under conditions in ( C ) .

Representative confocal images of the pAPS4::APS4:GFP translational fusion under the conditions in (C).


Representative confocal image of pSHR : : GFP : P19 translational fusion ( B ) principle of P19 - miRNA duplex sequestration .

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Representative confocal image of the pSHR::GFP:P19 translational fusion and (B) principle of P19-based miRNA duplex sequestration.


Binding of miR395 by SHR : : HA : P19 under SO4 - - sufficient ( + ) - deficient ( - ) growth conditions .

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Binding of miR395 by SHR::HA:P19 under SO4--sufficient (+) and -deficient (-) growth conditions.