Figure 4 ( a ) Upper panel : Single - cell UMAP of sequenced PBMCs from 2 NAFLD patients and 4 healthy subjects ; n = 10 , 786 cells . We identified 15 distinct cell types by DICE annotation . Lower panel : Expression patterns of CXCR3 , CXCR4 , ACKR3 , CX3CR1 , CCR6 across different cell types in NAFLD and healthy PBMC samples . ( c ) Graphic shows the percentage composition of identified cell types in NAFLD and healthy PBMCs single - cell analysis . ( d ) Upper panel : Representative flow cytometry dot plots , characterizing the isolated CD8 + T cells and CD8 - immune cell fraction ( mainly CD4 + T cells , NK cells and monocytes ) from PBMCs of healthy and NAFLD subjects . Lower panel : Flow cytometry quantification of % of positively expressing CXCR4 + and CX3CR1 + cells within each immune subset . Sample sizes are n = 14 NAFLD ; n = 6 healthy . ( e ) Heatmap of gene expressions of endothelial antigens relevant to interaction with T lymphocytes in NAFLD ( n = 3 ) and healthy ( n = 3 ) BOECs , with 3 biological replicates per donor . ( f ) Upper panel : Representative flow cytometry histograms of HLA - A / B / C , HLA - DM and IFN - γR1 expressions in NAFLD ( red ) and healthy ( blue ) BOECs . Grey , isotype control . Lower panel : Box - whisker plots reflect mean fluorescence intensity ( a . u . , arbituary unit ) , normalized to isotype control , as a readout for epitope density . Box - whisker plots indicate median ( middle line ) , 25th , 75th percentile ( box ) and the lowest / highest data points ( whiskers ) . ns , non - significant ( t - test ) . n = 4 donors ; * p < 0 . 05 ; ns , not significant ( Mann - Whitney test for HLA - A / B / C and IFN - γR1 ; t - test for HLA - DM ) .

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Figure 4(a) Upper panel: Single-cell UMAP of sequenced PBMCs from 2 NAFLD patients and 4 healthy subjects; n = 10,786 cells. We identified 15 distinct cell types by DICE annotation. Lower panel: Expression patterns of CXCR3, CXCR4, ACKR3, CX3CR1, CCR6 across different cell types in NAFLD and healthy PBMC samples.(c) Graphic shows the percentage composition of identified cell types in NAFLD and healthy PBMCs single-cell analysis.(d) Upper panel: Representative flow cytometry dot plots, characterizing the isolated CD8+ T cells and CD8- immune cell fraction (mainly CD4+ T cells, NK cells and monocytes) from PBMCs of healthy and NAFLD subjects. Lower panel: Flow cytometry quantification of % of positively expressing CXCR4+ and CX3CR1+ cells within each immune subset. Sample sizes are n = 14 NAFLD; n = 6 healthy.(e) Heatmap of gene expressions of endothelial antigens relevant to interaction with T lymphocytes in NAFLD (n = 3) and healthy (n = 3) BOECs, with 3 biological replicates per donor.(f) Upper panel: Representative flow cytometry histograms of HLA-A/B/C, HLA-DM and IFN-γR1 expressions in NAFLD (red) and healthy (blue) BOECs. Grey, isotype control. Lower panel: Box-whisker plots reflect mean fluorescence intensity (a.u., arbituary unit), normalized to isotype control, as a readout for epitope density. Box-whisker plots indicate median (middle line), 25th, 75th percentile (box) and the lowest/ highest data points (whiskers). ns, non-significant (t-test). n = 4 donors; *p<0.05; ns, not significant (Mann-Whitney test for HLA-A/B/C and IFN-γR1; t-test for HLA-DM).


Figure 5 ( a ) Left : Workflow schematic of adhesion assay . Middle : Representative images of labeled THP - 1 and Jurkat cells attached to BOEC monolayers ( Scale bars , 100 µm ) . Right : Box - whisker plots of the numbers of adhered leukocytes were determined fluorometrically . Sample sizes are n = 9 NAFLD and n = 7 healthy , with 3 biological replicates per donor . ( b ) Left : Schematic of chemotaxis assay . Right : Box - whisker plots of the numbers of recruited THP - 1 and Jurkat cells by the BOECs . Box - whisker plots indicate median ( middle line ) , 25th , 75th percentile ( box ) and the lowest / highest data points ( whiskers ) . ( c ) CD8 + T cells and CD8 - cell fractions were isolated from NAFLD and healthy subjects . Box - whisker plots show the number of recruited immune cells to their respective BOECs matched by health condition in coculture chemotaxis assays . Box - whisker plots indicate median ( middle line ) , 25th , 75th percentile ( box ) and the lowest / highest data points ( whiskers ) . ns , non - significant ( t - test ) . Sample sizes are n = 11 NAFLD and n = 6 healthy , with 3 - 4 technical replicates per donor . ( d ) Box - whisker plots show percentages of inhibition on immune cells chemotaxis by treatment with AMD3100 ( CXCR4 small molecule inhibitor ) . Box - whisker plots indicate median ( middle line ) , 25th , 75th percentile ( box ) and the lowest / highest data points ( whiskers ) . ns , non - significant ( t - test ) . Sample sizes are n = 6 NAFLD and n = 4 healthy , with 3 - 4 technical replicates per donor . ( e ) Box - whisker plots show percentages of inhibition on immune cells chemotaxis by treatment with anti - CXCL12 IgG ( CXCL12 neutralizing antibody ) . Box - whisker plots indicate median ( middle line ) , 25th , 75th percentile ( box ) and the lowest / highest data points ( whiskers ) . ns , non - significant ( t - test ) . Sample sizes are n = 5 NAFLD and n = 3 healthy , with 3 - 4 technical replicates per donor . ( f ) Left : Schematic of transendothelial migration assay with patient - derived BOECs and immune cells . Right : Transendothelial electric resistance ( TEER ) measurements of BOEC barrier tightness in the presence and absence of PBMCs and / or AMD3100 . Box - whisker plots indicate median ( middle line ) , 25th , 75th percentile ( box ) and the lowest / highest data points ( whiskers ) . ns , non - significant ( t - test ) . Sample size is n = 5 patient samples , with 3 technical TEER readings recorded per sample . ( g ) Box - whisker plots of the numbers of circulating endothelial cells per million PBMCs in healthy ( n = 6 ) and NAFLD subjects ( n = 11 ) . Box - whisker plots indicate median ( middle line ) , 25th , 75th percentile ( box ) and the lowest / highest data points ( whiskers ) . ns , non - significant ( t - test ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(a) Left: Workflow schematic of adhesion assay. Middle: Representative images of labeled THP-1 and Jurkat cells attached to BOEC monolayers (Scale bars, 100 µm). Right: Box-whisker plots of the numbers of adhered leukocytes were determined fluorometrically. Sample sizes are n = 9 NAFLD and n = 7 healthy, with 3 biological replicates per donor.(b) Left: Schematic of chemotaxis assay. Right: Box-whisker plots of the numbers of recruited THP-1 and Jurkat cells by the BOECs. Box-whisker plots indicate median (middle line), 25th, 75th percentile (box) and the lowest/ highest data points (whiskers).(c) CD8+ T cells and CD8- cell fractions were isolated from NAFLD and healthy subjects. Box-whisker plots show the number of recruited immune cells to their respective BOECs matched by health condition in coculture chemotaxis assays. Box-whisker plots indicate median (middle line), 25th, 75th percentile (box) and the lowest/ highest data points (whiskers). ns, non-significant (t-test). Sample sizes are n = 11 NAFLD and n = 6 healthy, with 3-4 technical replicates per donor.(d) Box-whisker plots show percentages of inhibition on immune cells chemotaxis by treatment with AMD3100 (CXCR4 small molecule inhibitor). Box-whisker plots indicate median (middle line), 25th, 75th percentile (box) and the lowest/ highest data points (whiskers). ns, non-significant (t-test). Sample sizes are n = 6 NAFLD and n = 4 healthy, with 3-4 technical replicates per donor.(e) Box-whisker plots show percentages of inhibition on immune cells chemotaxis by treatment with anti-CXCL12 IgG (CXCL12 neutralizing antibody). Box-whisker plots indicate median (middle line), 25th, 75th percentile (box) and the lowest/ highest data points (whiskers). ns, non-significant (t-test). Sample sizes are n = 5 NAFLD and n = 3 healthy, with 3-4 technical replicates per donor.(f) Left: Schematic of transendothelial migration assay with patient-derived BOECs and immune cells. Right: Transendothelial electric resistance (TEER) measurements of BOEC barrier tightness in the presence and absence of PBMCs and/or AMD3100. Box-whisker plots indicate median (middle line), 25th, 75th percentile (box) and the lowest/ highest data points (whiskers). ns, non-significant (t-test). Sample size is n = 5 patient samples, with 3 technical TEER readings recorded per sample.(g) Box-whisker plots of the numbers of circulating endothelial cells per million PBMCs in healthy (n = 6) and NAFLD subjects (n = 11). Box-whisker plots indicate median (middle line), 25th, 75th percentile (box) and the lowest/ highest data points (whiskers). ns, non-significant (t-test).


Figure 2 ) Animals harboring CRISPR - generated deletion alleles in y48g1c . 1 ( gg682 or gg683 ) were treated with dpy - 11 RNAi and progeny , grown in the absence of dpy - 11 RNAi , were scored for Dpy . Percentage of Dpy animals is shown . N = 3 , error bar is standard deviation , s . d . E ) Fluorescence micrograph of GFP : : ZNFX - 1 and PGL - 1 : : TagRFP fluorescence in animals harboring a CRISPR - generated deletion allele ( gg682 ) in y48g1c . 1 . Scale bar , 5μm .

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Figure 2) Animals harboring CRISPR-generated deletion alleles in y48g1c.1 (gg682 or gg683) were treated with dpy-11 RNAi and progeny, grown in the absence of dpy-11 RNAi, were scored for Dpy. Percentage of Dpy animals is shown. N=3, error bar is standard deviation, s.d.E) Fluorescence micrograph of GFP::ZNFX-1 and PGL-1::TagRFP fluorescence in animals harboring a CRISPR-generated deletion allele (gg682) in y48g1c.1. Scale bar, 5μm.


Figure 3Fluorescence micrograph of gfp : : zsp - 1 at the indicated stages using a Nikon Eclipse Ti microscope equipped with a W1 Yokogawa Spinning disc under a 63X / 1 . 4 Plan Apo oil objective . Scale bar , embryos : 5μm , germline : 10μm . Dashed lines indicate the outline of the adult germline .

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Figure 3Fluorescence micrograph of gfp::zsp-1 at the indicated stages using a Nikon Eclipse Ti microscope equipped with a W1 Yokogawa Spinning disc under a 63X/1.4 Plan Apo oil objective. Scale bar, embryos: 5μm, germline: 10μm. Dashed lines indicate the outline of the adult germline.


Figure 4E ) Germlines dissected from animals expressing the indicated proteins were fixed and stained with α - GFP and α - HA or α - FLAG antibodies , followed by secondary antibodies conjugated with Alexa 488 and CF430 , respectively . Pachytene germ - cell nuclei were imaged with a Leica TCS SP8 STED 3X microscope using 592nm to selectively deactivate fluorophores . Bottom panel . GFP : : ZNFX - 1 and HA : : TagRFP : : ZNFX - 1 expressing animals were crossed and germlines of trans - heterozygous progeny from this cross were fixed and stained and imaged by STED as described above . Scale bar , 0 . 5μm .

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Figure 4E) Germlines dissected from animals expressing the indicated proteins were fixed and stained with α-GFP and α-HA or α-FLAG antibodies, followed by secondary antibodies conjugated with Alexa 488 and CF430, respectively. Pachytene germ-cell nuclei were imaged with a Leica TCS SP8 STED 3X microscope using 592nm to selectively deactivate fluorophores. Bottom panel. GFP::ZNFX-1 and HA::TagRFP::ZNFX-1 expressing animals were crossed and germlines of trans-heterozygous progeny from this cross were fixed and stained and imaged by STED as described above. Scale bar, 0.5μm.


Figure 5Z - stack confocal images from pachytene germ cells from animals of the indicated genotypes were analysed as described in Fig . EV4B and Materials and Methods to determine ; the size of P granules ( panel B ) Granules from 9 total nuclei from 3 different animals ( 3 cells per animal ) were quantified for these analyses . Black lines indicate mean of all data points in each group . P value , student ' s t test , two tailed . Z - stack confocal images from pachytene germ cells from animals of the indicated genotypes were analysed as described to determine ; the size of P or Z granules ( panel C ) ; or the number of P or Z granules surrounding a typical nucleus in wild - type and zsp - 1 ( gg682 ) animals ( panel D ) . Granules from 9 total nuclei from 3 different animals ( 3 cells per animal ) were quantified for these analyses . Black lines indicate mean of all data points in each group . P value , student ' s t test , two tailed . G , H ) GFP : : ZNFX - 1 ( panel G ) fluorescence or PGL - 1 : : TagRFP fluorescence ( panel H ) in the anterior ( A ) or posterior ( P ) section of a one cell embryo . Anterior and posterior regions were determined based on corresponding DIC images . N = 4 embryos for G , N = 3 embryos for H . Error bars are + / - s . d .

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Figure 5Z-stack confocal images from pachytene germ cells from animals of the indicated genotypes were analysed as described in Fig. EV4B and Materials and Methods to determine; the size of P granules (panel B) Granules from 9 total nuclei from 3 different animals (3 cells per animal) were quantified for these analyses. Black lines indicate mean of all data points in each group. P value, student's t test, two tailed.Z-stack confocal images from pachytene germ cells from animals of the indicated genotypes were analysed as described to determine; the size of P or Z granules (panel C); or the number of P or Z granules surrounding a typical nucleus in wild-type and zsp-1(gg682) animals (panel D). Granules from 9 total nuclei from 3 different animals (3 cells per animal) were quantified for these analyses. Black lines indicate mean of all data points in each group. P value, student's t test, two tailed.G,H) GFP::ZNFX-1 (panel G) fluorescence or PGL-1::TagRFP fluorescence (panel H) in the anterior (A) or posterior (P) section of a one cell embryo. Anterior and posterior regions were determined based on corresponding DIC images. N=4 embryos for G, N=3 embryos for H. Error bars are +/- s.d.


Figure 6A ) Heat map showing recovery of ZNFX - 1 fluorescence after photobleaching in Z granules from pachytene stage adult germ cells from wild - type or zsp - 1 ( gg682 ) mutant animals . Scale bar , 0 . 5 μm . B ) Normalized recovery of fluorescence after photobleaching of GFP : : ZNFX - 1 in wild - type or zsp - 1 ( - ) mutant animals over indicated time points . Bleaching event occurred at 0s . Data are represented as ± s . e . m . of n = 13 , and n = 16 individual granules from wild - type and zsp - 1 ( gg682 ) animals , respectively .

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Figure 6A) Heat map showing recovery of ZNFX-1 fluorescence after photobleaching in Z granules from pachytene stage adult germ cells from wild-type or zsp-1(gg682) mutant animals. Scale bar, 0.5 μm. B) Normalized recovery of fluorescence after photobleaching of GFP::ZNFX-1 in wild-type or zsp-1(-) mutant animals over indicated time points. Bleaching event occurred at 0s. Data are represented as ± s.e.m. of n=13, and n=16 individual granules from wild-type and zsp-1(gg682) animals, respectively.


Figure 7A ) Lineages of animals of the indicated genotypes were grown at 25 ° C or 20 ° C for the indicated generations . Each generation six animals were isolated in order to maintain the lineage and to determine numbers of progeny . Data are represented as + / - s . e . m . N = 3 for generation 8 or 9 , N = 6 for all other generations . F ) oma - 1 ( zu405 ) is a temperature sensitive ( ts ) lethal ( embryonic arrest at temperatures > 20 ° C ) allele of oma - 1 ( R . Lin 2003 ) . oma - 1 RNAi suppresses oma - 1 ( zu405ts ) lethality . Animals were exposed to oma - 1 RNAi and the number of embryos that hatched was scored at 20 ° C . N = 3 . Error bars are + / - s . d . Animals harboring mutations in genes specifically required for the inheritance of RNAi would respond normally to direct exposure to oma - 1 dsRNA but fail to inherit this silencing ( Wan et al . 2018 ) . zsp - 1 mutants fail to respond immediately to direct exposure to oma - 1 dsRNA , indicating that ZSP - 1 is not specific to inheritance and , therefore , is generally required for RNAi ( see also Fig . EV5B - D ) .

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Figure 7A) Lineages of animals of the indicated genotypes were grown at 25°C or 20°C for the indicated generations. Each generation six animals were isolated in order to maintain the lineage and to determine numbers of progeny. Data are represented as +/- s.e.m. N=3 for generation 8 or 9, N=6 for all other generations.F) oma-1(zu405) is a temperature sensitive (ts) lethal (embryonic arrest at temperatures > 20°C) allele of oma-1 (R. Lin 2003). oma-1 RNAi suppresses oma-1(zu405ts) lethality. Animals were exposed to oma-1 RNAi and the number of embryos that hatched was scored at 20°C. N=3. Error bars are +/- s.d. Animals harboring mutations in genes specifically required for the inheritance of RNAi would respond normally to direct exposure to oma-1 dsRNA but fail to inherit this silencing (Wan et al. 2018). zsp-1 mutants fail to respond immediately to direct exposure to oma-1 dsRNA, indicating that ZSP-1 is not specific to inheritance and, therefore, is generally required for RNAi (see also Fig. EV5B-D).


Figure 1Figure 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 . 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 . 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 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 - 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 patients ( F ) . If not indicated differently , Kruskall - Wallis ( Dunn ´ s post test ) was applied , * * p < 0 . 05 , * * * p < 0 . 005 ; data are mean ±

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1Figure 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.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.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 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-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 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 2Figure 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 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 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 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 ±

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Figure 2Figure 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 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 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 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 3Figure 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 ±

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Figure 3Figure 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 4Figure 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 ) . . 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 ) . 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 ) . Analysis of Glut1 immunoreactivity in GL261 glioblastoma sections ( F ; n = 3 ) . 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 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 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 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 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 )

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4Figure 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).. 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).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).Analysis of Glut1 immunoreactivity in GL261 glioblastoma sections (F; n=3).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 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 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 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 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 5Figure 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 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 cells per blood vessel ( H ) 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 + 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 ±

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Figure 5Figure 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 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 cells per blood vessel (H)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+ 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 6Figure 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 and Ang - 2 + vessels are shown in ( C ) and ( D ) . The ratio of Ang - 2 + vs . CD31 + vessels was determined in ( E ) . Western Blot and corresponding quantification of CD31 and Ang - 2 in control and aflibercept treated mice ( F ) . 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 ±

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6Figure 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 and Ang-2+ vessels are shown in (C) and (D). The ratio of Ang-2+ vs. CD31+ vessels was determined in (E).Western Blot and corresponding quantification of CD31 and Ang-2 in control and aflibercept treated mice (F).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


Figure 1 . SARS - CoV - 2 . For weigh loss record , hACE2 mice ( n = 7 ) and WT mice ( n = 3 ) were experimentally challenged intranasally with SARS - CoV - 2 , and the ACE2 - Mock mice ( n = 3 ) were used as control . And then the weight loss was recorded over 14 days ( a , ANOVA , * * * p < 0 . 001 ) . To screen virus replication , 12 mice were infected in each group , and 3 mice per group were sacrificed and their major organs harvested for viral load and virus titer at 1 dpi , 3 dpi , 5 dpi and 7 dpi respectively . The distribution of SARS - CoV - 2 in the primary organs of ACE2 - HB - 01 mice was detected by qRT - PCR ( b ) . Virus titers of lungs were determined on Vero E6 cells ( c , unpaired t - test , * * * p < 0 . 001 ) the virus isolated from lungs of ACE2 - HB - 01 mice at 3 dpi was observed by electron microscope ( d ) . The specific IgG against SARS - CoV - 2 was detected at 21 dpi by ELISA ( e , unpaired t - test , * * * p < 0 . 001 ) .

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Figure 1.SARS-CoV-2.For weigh loss record, hACE2 mice (n=7) and WT mice (n=3) were experimentally challenged intranasally with SARS-CoV-2, and the ACE2-Mock mice (n=3) were used as control. And then the weight loss was recorded over 14 days (a, ANOVA, ***p<0.001).To screen virus replication, 12 mice were infected in each group, and 3 mice per group were sacrificed and their major organs harvested for viral load and virus titer at 1 dpi, 3 dpi, 5 dpi and 7 dpi respectively. The distribution of SARS-CoV-2 in the primary organs of ACE2-HB-01 mice was detected by qRT-PCR (b).Virus titers of lungs were determined on Vero E6 cells (c, unpaired t-test, ***p<0.001)the virus isolated from lungs of ACE2-HB-01 mice at 3 dpi was observed by electron microscope (d).The specific IgG against SARS-CoV-2 was detected at 21 dpi by ELISA (e, unpaired t-test, ***p<0.001).


Figure 2 . mice . a . Gross pathology and histopathology of lungs from WT - HB - 01 mice ( 3 dpi ) , ACE2 - Mock mice ( 3 dpi ) and ACE2 - HB - 01 mice ( 3 dpi and 5 dpi ) . Postmortem examinations showed focal dark red lesions ( red arrow ) throughout the dorsal of the right middle lobe of the lung at 3dpi . The lesions progressed into multifocally scattered - dark reddish purple areas and palpable nodules ( red arrow ) throughout the right lobe of the lung at 5 dpi . Histopathological observation indicated that moderate interstitial pneumonia with thickened alveolar septa ( black arrows ) and infiltration of lymphocytes ( red arrows ) . The swollen and degenerative alveolar macrophages ( green arrows ) are scattered within the alveolar cavities at 3dpi and 5 dpi . b . Immunohistochemical examination of lungs of each group . The sequential sections were stained by HE and IHC , respectively . The viral antigens were observed in the alveolar macrophage ( green arrows ) , the alveolar epithelia ( blue arrows ) , and the degenerative and being desquamated bronchial epithelial cells ( black arrows ) . Black bar = 100 µm , red bar = 50 µm .

O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2.mice.a. Gross pathology and histopathology of lungs from WT-HB-01 mice (3 dpi), ACE2-Mock mice (3 dpi) and ACE2-HB-01 mice (3 dpi and 5 dpi). Postmortem examinations showed focal dark red lesions (red arrow) throughout the dorsal of the right middle lobe of the lung at 3dpi. The lesions progressed into multifocally scattered-dark reddish purple areas and palpable nodules (red arrow) throughout the right lobe of the lung at 5 dpi. Histopathological observation indicated that moderate interstitial pneumonia with thickened alveolar septa (black arrows) and infiltration of lymphocytes (red arrows). The swollen and degenerative alveolar macrophages (green arrows) are scattered within the alveolar cavities at 3dpi and 5 dpi.b. Immunohistochemical examination of lungs of each group. The sequential sections were stained by HE and IHC, respectively. The viral antigens were observed in the alveolar macrophage (green arrows), the alveolar epithelia (blue arrows), and the degenerative and being desquamated bronchial epithelial cells (black arrows). Black bar = 100 µm, red bar = 50 µm.


Figure 3 . Immunofluorescence analysis of viral antigens in lungs of SARS - CoV - 2 - infected hACE2 mice . Co - localization of SARS - CoV - 2 S protein and hACE2 receptor in hACE2 mouse lungs , the sections were incubated with anti - SARS - CoV - 2 S protein antibody , anti - human ACE2 antibody , and DAPI . The lung sections of ACE2 - Mock mice ( a - d ) . The lung sections of ACE2 - HB - 01 mice ( e - h ) . The white arrows showed the viral S protein ( f ) and hACE2 ( g ) , respectively , the yellow arrow showed the merge of viral S protein and hACE2 ( h ) . White bar = 25 µm .

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Figure 3. Immunofluorescence analysis of viral antigens in lungs of SARS-CoV-2-infected hACE2 mice.Co-localization of SARS-CoV-2 S protein and hACE2 receptor in hACE2 mouse lungs, the sections were incubated with anti-SARS-CoV-2 S protein antibody, anti-human ACE2 antibody, and DAPI. The lung sections of ACE2-Mock mice (a-d). The lung sections of ACE2-HB-01 mice (e-h). The white arrows showed the viral S protein (f) and hACE2 (g), respectively, the yellow arrow showed the merge of viral S protein and hACE2 (h). White bar=25 µm.


Figure 1Cryo - TEM images demonstrate the lipid bilayer EV purified from Molm - 14 and U - 937 cells . Scale bars are 100nm . Peripheral blood ( PB ) plasma of control animals contain no mGFP + foci ( top ) however Molm - 14 - mGFP ( middle ) and U - 937 - mGFP ( bottom ) xenografts contain numerous mGFP - labeled EV ( green ) detectible without vesicle concentration . Scale bars are 5μm . Live - cell microscopy of KSL cells ( left panel ) LT - HSC cells ( right panel ) . Cells were sorted from control mice ( top ) , Molm - 14 - mGFP ( middle ) and U - 937 - mGFP ( bottom ) , stained with Cell Mask ( red ) and nuclear stain Hoechst ( blue ) ; imaged using the GE / API Deltavision ( DV ) widefield microscope ( 60X objective ) to show the uptake of the GFP + EV ( green ) . Scale bars are 5μm .

O B-PANEL_START 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1Cryo-TEM images demonstrate the lipid bilayer EV purified from Molm-14 and U-937 cells. Scale bars are 100nm.Peripheral blood (PB) plasma of control animals contain no mGFP+ foci (top) however Molm-14-mGFP (middle) and U-937-mGFP (bottom) xenografts contain numerous mGFP-labeled EV (green) detectible without vesicle concentration. Scale bars are 5μm.Live-cell microscopy of KSL cells (left panel) LT-HSC cells (right panel). Cells were sorted from control mice (top), Molm-14-mGFP (middle) and U-937-mGFP (bottom), stained with Cell Mask (red) and nuclear stain Hoechst (blue); imaged using the GE/API Deltavision (DV) widefield microscope (60X objective) to show the uptake of the GFP+ EV (green). Scale bars are 5μm.


Figure 2Flow cytometric analysis showing the frequency of LK and KSL cells in lineage - negative cells ( left panel ) and LT - HSC in KSL ( right panel ) in : Molm - 14 - xenograft ( red , n = 10 ) vs . control ( black , n = 10 ) . Data were obtained from at least two independent experiments . Flow cytometric analysis showing the frequency of LK and KSL cells in lineage - negative cells ( left panel ) and LT - HSC in KSL ( right panel ) in : IF injection of EV from Molm - 14 cells ( red , n = 8 ) , AML plasma EV ( Orange , n = 6 ) and human CD34 EV ( Blue , n = 4 ) relative to the vehicle - injected contralateral femurs . Data were obtained from at least two independent experiments . Cell - cycle histograms of KSL ( upper panel ) and LT - HSC ( lower panel ) from Molm - 14 - xenografts or control using Hoechst - 33342 staining . Flow cytometric analysis of the Ki67 - ve percentage of LT - HSC representing the G0 phase of cell - cycle in : ( left panel ) Molm - 14 - xenografts ( red , n = 7 ) vs . control ( black , n = 9 ) ; ( right panel ) IF injection of Molm - 14 - EV , U - 937 EV , HL - 60 EV ( red , n = 5 , 4 , 4 ) vs . human CD34 EV ( blue , n = 3 ) vs . controls ( black ) . Flow cytometric analysis of intracellular P53 levels shown in histograms ( F ) of LT - HSC in : ( left panel ) Molm - 14 - xenograft , n = 6 red vs . non - engrafted control , n = 4 black , or ( right panel ) IF injection of EV from Molm - 14 , U - 937 , HL - 60 ( red , n = 5 , 5 , 3 ) or human CD34 cells ( blue , n = 6 ) normalized to vehicle - injected contralateral femurs . Flow cytometric analysis of intracellular P53 levels shown in histograms of LT - HSC in : ( left panel ) Molm - 14 - xenograft , n = 6 red vs . non - engrafted control , n = 4 black , or ( right panel ) IF injection of EV from Molm - 14 , U - 937 , HL - 60 ( red , n = 5 , 5 , 3 ) or human CD34 cells ( blue , n = 6 ) normalized to vehicle - injected contralateral femurs .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2Flow cytometric analysis showing the frequency of LK and KSL cells in lineage-negative cells (left panel) and LT-HSC in KSL (right panel) in: Molm-14-xenograft (red, n=10) vs. control (black, n=10). Data were obtained from at least two independent experiments.Flow cytometric analysis showing the frequency of LK and KSL cells in lineage-negative cells (left panel) and LT-HSC in KSL (right panel) in: IF injection of EV from Molm-14 cells (red, n=8), AML plasma EV (Orange, n=6) and human CD34 EV (Blue, n=4) relative to the vehicle-injected contralateral femurs. Data were obtained from at least two independent experiments.Cell-cycle histograms of KSL (upper panel) and LT-HSC (lower panel) from Molm-14-xenografts or control using Hoechst-33342 staining.Flow cytometric analysis of the Ki67-ve percentage of LT-HSC representing the G0 phase of cell-cycle in: (left panel) Molm-14-xenografts (red, n=7) vs. control (black, n=9); (right panel) IF injection of Molm-14-EV, U-937 EV, HL-60 EV (red, n=5,4,4) vs. human CD34 EV (blue, n=3) vs. controls (black).Flow cytometric analysis of intracellular P53 levels shown in histograms (F) of LT-HSC in: (left panel) Molm-14-xenograft, n=6 red vs. non-engrafted control, n=4 black, or (right panel) IF injection of EV from Molm-14, U-937, HL-60 (red, n=5,5,3) or human CD34 cells (blue, n=6) normalized to vehicle-injected contralateral femurs.Flow cytometric analysis of intracellular P53 levels shown in histograms of LT-HSC in: (left panel) Molm-14-xenograft, n=6 red vs. non-engrafted control, n=4 black, or (right panel) IF injection of EV from Molm-14, U-937, HL-60 (red, n=5,5,3) or human CD34 cells (blue, n=6) normalized to vehicle-injected contralateral femurs.


Figure 3A heatmap showing the highly - deregulated ribosomal proteins in AML - EV exposed cells . Flow cytometric analysis showing the histograms and MFI of O - propagyl - puromysin ( OPP ) incorporation in LT - HSC in : Molm - 14 - xenografts , red n = 6 vs . non - engrafted controls , black n = 6 , Flow cytometric analysis showing the histograms and MFI of O - propagyl - puromysin ( OPP ) incorporation in LT - HSC in : IF injection of EV from Molm - 14 , U - 937 , HL - 60 ( red , n = 5 , 43 ) , AML patients plasma ( Orange , n = 6 ) , or human CD34 + cells ( blue , n = 3 ) normalized to vehicle - injected contralateral femurs after subtracting the background fluorescence .

O B-PANEL_START O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3A heatmap showing the highly-deregulated ribosomal proteins in AML-EV exposed cells.Flow cytometric analysis showing the histograms and MFI of O-propagyl-puromysin (OPP) incorporation in LT-HSC in: Molm-14-xenografts, red n=6 vs. non-engrafted controls, black n=6,Flow cytometric analysis showing the histograms and MFI of O-propagyl-puromysin (OPP) incorporation in LT-HSC in: IF injection of EV from Molm-14, U-937, HL-60 (red, n=5,43), AML patients plasma (Orange, n=6), or human CD34+ cells (blue, n=3) normalized to vehicle-injected contralateral femurs after subtracting the background fluorescence.


Figure 4qRT - PCR gene expression analysis showing the fold change of Pi3K / mTOR associated gene panel in KSL cells sorted from : ( A ) Molm - 14 - xenografted miceqRT - PCR gene expression analysis showing the fold change of Pi3K / mTOR associated gene panel in KSL cells sorted from : IF injected mice with Molm - 14 - EV relative to their controls and normalized to Gapdh endogenous control . Flow cytometric analysis showing the histograms ( C ) Flow cytometric analysis showing the MFI of intracellular pS6KRP in LT - HSC in : ( D ) Molm - 14 - xenograft ( red , n = 9 ) vs . non - engrafted control ( black , n = 7 ) , Flow cytometric analysis showing the MFI of intracellular pS6KRP in LT - HSC in : IF injection of EV from Molm - 14 , U - 937 , HL - 60 ( red , n = 7 , 3 , 3 ) or human CD34 cells ( blue , n = 4 ) normalized to the control contralateral femur with subtraction of background fluorescence . Flow cytometric assessment of protein synthesis showing the MFI of : ( F ) OPP , or ( G ) pS6RP in NIH - 3T3 cells 72 hours after transfection with the indicated miRNA mimics . The results were calculated relative to control ( miR - scramble ) with the background fluorescence subtracted and performed with at least three independent experiments , in technical replicates . Flow cytometric analysis showing the MFI of Raptor in NIH - 3T3 cells 72 hours after transfection with the indicated miRNA mimics . The data were calculated relative to miR - scramble and presented as means bars ± SEM and the background fluorescence was subtracted . Performed with at least three independent experiments , in technical replicates . Dual Luciferase reporter assay . NIH - 3T3 was transfected with the miRNA mimics . Three hours later the cells were transfected with the Raptor 3 ′ UTR cloned into the psiCheck - 2 vector for a total of 48 hours . Data are presented as % RLU ( relative luciferase units ) of the miR - scramble control as means ± SEM from at least three independent experiments , performed in technical replicates . Cell - cycle flow cytometric analysis using Ki67 / Hoechst - 33342 staining of the percentage of LT - HSC in the G0 phase after nucleofection of cKit + cells using the AmaxaTM P3 Primary Cell 4D - Nucleoeftcor kit , ( J ) Cells were nucleofected with miR - Scramble ( CTRL ) or miR - 1246 mimic ( n = 3 ) for 72 hours , Cell - cycle flow cytometric analysis using Ki67 / Hoechst - 33342 staining of the percentage of LT - HSC in the G0 phase after nucleofection of cKit + cells using the AmaxaTM P3 Primary Cell 4D - Nucleoeftcor kit , Cells were nucleofected with Anti - miR - Scramble ( CTRL ) or Anti - miR - 1246 ( n = 5 ) and 1 - hour later co - treatment with Molm - 14 EVs for 72 hours .

O B-PANEL_START O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4qRT-PCR gene expression analysis showing the fold change of Pi3K/mTOR associated gene panel in KSL cells sorted from: (A) Molm-14-xenografted miceqRT-PCR gene expression analysis showing the fold change of Pi3K/mTOR associated gene panel in KSL cells sorted from: IF injected mice with Molm-14-EV relative to their controls and normalized to Gapdh endogenous control.Flow cytometric analysis showing the histograms (C)Flow cytometric analysis showing the MFI of intracellular pS6KRP in LT-HSC in: (D) Molm-14-xenograft (red, n=9) vs. non-engrafted control (black, n=7),Flow cytometric analysis showing the MFI of intracellular pS6KRP in LT-HSC in: IF injection of EV from Molm-14, U-937, HL-60 (red, n=7,3,3) or human CD34 cells (blue, n=4) normalized to the control contralateral femur with subtraction of background fluorescence.Flow cytometric assessment of protein synthesis showing the MFI of: (F) OPP, or (G) pS6RP in NIH-3T3 cells 72 hours after transfection with the indicated miRNA mimics. The results were calculated relative to control (miR-scramble) with the background fluorescence subtracted and performed with at least three independent experiments, in technical replicates.Flow cytometric analysis showing the MFI of Raptor in NIH-3T3 cells 72 hours after transfection with the indicated miRNA mimics. The data were calculated relative to miR-scramble and presented as means bars ± SEM and the background fluorescence was subtracted. Performed with at least three independent experiments, in technical replicates.Dual Luciferase reporter assay. NIH-3T3 was transfected with the miRNA mimics. Three hours later the cells were transfected with the Raptor 3′UTR cloned into the psiCheck-2 vector for a total of 48 hours. Data are presented as %RLU (relative luciferase units) of the miR-scramble control as means ± SEM from at least three independent experiments, performed in technical replicates.Cell-cycle flow cytometric analysis using Ki67/Hoechst-33342 staining of the percentage of LT-HSC in the G0 phase after nucleofection of cKit+ cells using the AmaxaTM P3 Primary Cell 4D-Nucleoeftcor kit, (J) Cells were nucleofected with miR-Scramble (CTRL) or miR-1246 mimic (n=3) for 72 hours,Cell-cycle flow cytometric analysis using Ki67/Hoechst-33342 staining of the percentage of LT-HSC in the G0 phase after nucleofection of cKit+ cells using the AmaxaTM P3 Primary Cell 4D-Nucleoeftcor kit, Cells were nucleofected with Anti-miR-Scramble (CTRL) or Anti-miR-1246 (n=5) and 1-hour later co-treatment with Molm-14 EVs for 72 hours.


Figure 5CFU - C assay showing the relative number of colonies ( left panel ) and the colony subtypes ( right panel ) from KSL cells derived from Molm - 14 - xenografted mice ( red , n = 6 ) vs . non - engrafted control mice ( black , n = 5 ) and in vitro cultured for 7 days in methylcellulose media . Experiments were performed in technical triplicates . " G " Granulocyte , " M " Monocyte , " E " Erythrocytes . LT - HSC assessment after repopulation , recipient female mice ( n = 4 per group ) were sacrificed after 21 days and their LT - HSC were compared using P53 , pS6RP and OPP flow cytometric assays . ( D - E ) Evaluation of the in vivo long - term repopulation capacity . Evaluation of the in vivo long - term repopulation capacity . For secondary transplantation ( n = 6 per group ) , PB samples were collected twice weekly for 12 weeks ( WK ) and the donor chimerism was measured by qPCR of Sex - determining Region Y ( SRY ) normalized to Gapdh gatekeeper as shown in the left panel . Sixteen weeks later , mice were sacrificed and BM male chimerism percentage was assessed as shown in the right panel . Tertiary transplantation , unfractionated BM cells from the secondary recipients ( n = 6 per group , 106 cells per mouse ) were injected into tertiary 150cGy sub - lethally irradiated female recipients and blood SRY levels were assessed for 20 weeks .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5CFU-C assay showing the relative number of colonies (left panel) and the colony subtypes (right panel) from KSL cells derived from Molm-14-xenografted mice (red, n=6) vs. non-engrafted control mice (black, n=5) and in vitro cultured for 7 days in methylcellulose media. Experiments were performed in technical triplicates. "G" Granulocyte, "M" Monocyte, "E" Erythrocytes.LT-HSC assessment after repopulation, recipient female mice (n=4 per group) were sacrificed after 21 days and their LT-HSC were compared using P53, pS6RP and OPP flow cytometric assays. (D-E) Evaluation of the in vivo long-term repopulation capacity.Evaluation of the in vivo long-term repopulation capacity. For secondary transplantation (n=6 per group), PB samples were collected twice weekly for 12 weeks (WK) and the donor chimerism was measured by qPCR of Sex-determining Region Y (SRY) normalized to Gapdh gatekeeper as shown in the left panel. Sixteen weeks later, mice were sacrificed and BM male chimerism percentage was assessed as shown in the right panel. Tertiary transplantation, unfractionated BM cells from the secondary recipients (n=6 per group, 106 cells per mouse) were injected into tertiary 150cGy sub-lethally irradiated female recipients and blood SRY levels were assessed for 20 weeks.


Figure 6Serial replating CFU - C analysis presented as mean ± SEM showing : ( B ) the number of colonies per 1000 plated cells , and ( C ) the number of cells per colony , for each week time - point . DNA - damage assessment in KSL from primary recipients and after two turns of replating . ( D , F , H ) Number of γH2AX foci in control - derived and Molm - 14 - xenograft - derived : ( D ) Primary recipient KSL , n = 211 and 321 , All cells were stained with phosphoH2axser139 and the nuclear stain Hoechst ( blue ) , imaged using the GE / API DV widefield microscope ( 60X objective ) to show the γH2AX foci ( green ) and counted using Imaris software . qRT - PCR gene expression analysis showing the fold - change of select DNA - damage response - genes panel in Molm - 14 - xenograft - derived : ( E ) Primary recipient KSL , n = 6 Data indicated by bars of fold - change calculated relative to matching controls , normalized to the endogenous control Gapdh and presented as means ± SEM . Number of γH2AX foci in control - derived and Molm - 14 - xenograft - derived : cells from W1 plating , n = 218 and 369 All cells were stained with phosphoH2axser139 and the nuclear stain Hoechst ( blue ) , imaged using the GE / API DV widefield microscope ( 60X objective ) to show the γH2AX foci ( green ) and counted using Imaris software . Scale bars are 2μm . qRT - PCR gene expression analysis showing the fold - change of select DNA - damage response - genes panel in Molm - 14 - xenograft - derived : Cells from W1 plating , n = 4 , Data indicated by bars of fold - change calculated relative to matching controls , normalized to the endogenous control Gapdh and presented as means ± SEM . Number of γH2AX foci in control - derived and Molm - 14 - xenograft - derived : cells from W2 plating , n = 770 and 853 . All cells were stained with phosphoH2axser139 and the nuclear stain Hoechst ( blue ) , imaged using the GE / API DV widefield microscope ( 60X objective ) to show the γH2AX foci ( green ) and counted using Imaris software . qRT - PCR gene expression analysis showing the fold - change of select DNA - damage response - genes panel in Molm - 14 - xenograft - derived : Cells from W2 plating n = 4 . Data indicated by bars of fold - change calculated relative to matching controls , normalized to the endogenous control Gapdh and presented as means ± SEM . umber of γH2AX foci , respectively , in control - and Molm - 14 - xenograft - derived : ( K ) KSL , n = 101 / 204 , and ( L ) LT - HSC , n = 33 / 53 . Images were processed similar to D - F . Scale bars are 3μm . Statistics : Student ' s t - test . ( * * P < 0 . 01 ) . qRT - PCR analysis of the DNA - damage - genes panel in Molm - 14 - xenograft - derived : ( M ) KSL , and ( N ) LT - HSC , relative to matched controls . Data depicted as fold - change relative to matching controls , normalized to the endogenous control Gapdh and presented as mean ± SEM .

O B-PANEL_START O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6Serial replating CFU-C analysis presented as mean ± SEM showing: (B) the number of colonies per 1000 plated cells, and (C) the number of cells per colony, for each week time-point.DNA-damage assessment in KSL from primary recipients and after two turns of replating. (D, F, H) Number of γH2AX foci in control-derived and Molm-14-xenograft-derived: (D) Primary recipient KSL, n=211 and 321, All cells were stained with phosphoH2axser139 and the nuclear stain Hoechst (blue), imaged using the GE/API DV widefield microscope (60X objective) to show the γH2AX foci (green) and counted using Imaris software.qRT-PCR gene expression analysis showing the fold-change of select DNA-damage response-genes panel in Molm-14-xenograft-derived: (E) Primary recipient KSL, n=6 Data indicated by bars of fold-change calculated relative to matching controls, normalized to the endogenous control Gapdh and presented as means ± SEM.Number of γH2AX foci in control-derived and Molm-14-xenograft-derived: cells from W1 plating, n=218 and 369 All cells were stained with phosphoH2axser139 and the nuclear stain Hoechst (blue), imaged using the GE/API DV widefield microscope (60X objective) to show the γH2AX foci (green) and counted using Imaris software. Scale bars are 2μm.qRT-PCR gene expression analysis showing the fold-change of select DNA-damage response-genes panel in Molm-14-xenograft-derived: Cells from W1 plating, n=4, Data indicated by bars of fold-change calculated relative to matching controls, normalized to the endogenous control Gapdh and presented as means ± SEM.Number of γH2AX foci in control-derived and Molm-14-xenograft-derived: cells from W2 plating, n=770 and 853. All cells were stained with phosphoH2axser139 and the nuclear stain Hoechst (blue), imaged using the GE/API DV widefield microscope (60X objective) to show the γH2AX foci (green) and counted using Imaris software.qRT-PCR gene expression analysis showing the fold-change of select DNA-damage response-genes panel in Molm-14-xenograft-derived: Cells from W2 plating n=4. Data indicated by bars of fold-change calculated relative to matching controls, normalized to the endogenous control Gapdh and presented as means ± SEM.umber of γH2AX foci, respectively, in control- and Molm-14-xenograft-derived: (K) KSL, n=101 / 204, and (L) LT-HSC, n=33 / 53. Images were processed similar to D-F. Scale bars are 3μm. Statistics: Student's t-test. (** P< 0.01).qRT-PCR analysis of the DNA-damage-genes panel in Molm-14-xenograft-derived: (M) KSL, and (N) LT-HSC, relative to matched controls. Data depicted as fold-change relative to matching controls, normalized to the endogenous control Gapdh and presented as mean ± SEM.


Figure 2A . Left : Snapshots of actin comet tails assembled in Assembly conditions : 4 . 5 µm polystyrene beads coated with 400 nM Strep - SNAP - WA ; 3 µM actin , 6 µM profilin , 90 nM Arp2 / 3 complex , 15 nM capping protein . Right : Tracking of the comet shown in the snapshots . Time is encoded in color . B . Left : Snapshots of actin comet tails assembled in Disassembly conditions : same as assembly conditions with 200 nM ADF / cofilin added . Right : Tracking of the comet shown in the snapshots . Time is encoded in color . C . Left : Snapshots of actin comet tails assembled in Recycling conditions : Disassembly conditions with 400 nM Cyclase Associated Protein ( CAP ) added . Right : Tracking of the comet shown in the snapshots . Time is encoded in color . E . Estimation of half - life of bead motility ( from an exponential fit to the bead velocity curve ) for each independent dataset in the different dynamic conditions ( 1 point per independent dataset ) . Data information : Mean and standard deviation are represented . Assembly : n = 20 comet tails . Disassembly : n = 13 comet tails . Recycling : n = 16 comet tails .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2A. Left: Snapshots of actin comet tails assembled in Assembly conditions: 4.5 µm polystyrene beads coated with 400 nM Strep-SNAP-WA; 3 µM actin, 6 µM profilin, 90 nM Arp2/3 complex, 15 nM capping protein. Right: Tracking of the comet shown in the snapshots. Time is encoded in color. B. Left: Snapshots of actin comet tails assembled in Disassembly conditions: same as assembly conditions with 200 nM ADF/cofilin added. Right: Tracking of the comet shown in the snapshots. Time is encoded in color. C. Left: Snapshots of actin comet tails assembled in Recycling conditions: Disassembly conditions with 400 nM Cyclase Associated Protein (CAP) added. Right: Tracking of the comet shown in the snapshots. Time is encoded in color.E. Estimation of half-life of bead motility (from an exponential fit to the bead velocity curve) for each independent dataset in the different dynamic conditions (1 point per independent dataset). Data information: Mean and standard deviation are represented. Assembly: n = 20 comet tails. Disassembly: n = 13 comet tails. Recycling: n = 16 comet tails.


Figure 3B . Example of bead velocity as a function of time in recycling conditions . The integral of this curve ( or area under curve ) represents the length of actin polymerized in the comets during the time of the experiment . C . Example of cumulative length of actin polymerized as a function of time .

O B-PANEL_START O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O

Figure 3B. Example of bead velocity as a function of time in recycling conditions. The integral of this curve (or area under curve) represents the length of actin polymerized in the comets during the time of the experiment.C. Example of cumulative length of actin polymerized as a function of time.


Figure 4A . Left : Snapshots of actin comet tails in recycling conditions with the indicated ATP concentrations . Right : Tracking of the comet shown in the snapshots . Time is encoded in color .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4A. Left: Snapshots of actin comet tails in recycling conditions with the indicated ATP concentrations. Right: Tracking of the comet shown in the snapshots. Time is encoded in color.


Figure 5B , C . Test of reaction mix aging without beads . Reaction mix was prepared without beads and left on the bench at room temperature . Then at different time intervals , fresh beads were added to the mix . After 24 hours ( right part of the graph ) , All ( fresh ) proteins in the assay were added one by one with fresh the beads . Velocity of fresh beads was estimated for those different times . Composition of the reaction mix : 3 µM actin , 6 µM profilin , 90 nM Arp2 / 3 , 15 nM Capping protein , 200 nM cofilin , 400 nM Cyclase Associated Protein ( CAP ) . t = 0 : N = 4 , n = 494 comets . t = 6 hours : N = 1 , n = 65 comets . t = 24 hours : N = 2 , n = 46 comets . t = 24 hours + fresh mix : N = 1 , n = 32 comets . t = 24 hours + actin : N = 2 , n = 108 comets . t = 24 hours + profilin : N = 1 , n = 10 comets . t = 24 hours + capping protein : N = 1 , n = 12 comets . t = 24 hours + Arp2 / 3 complex : N = 1 , n = 11 comets . t = 24 hours + ADF / cofilin : N = 1 , n = 17 comets . t = 24 hours + CAP : N = 1 , n = 7 comets . Individual points for each comet are represented with mean and standard deviation superimposed . D . Test of actin monomer aging . Actin monomers were prepared in salt - free motility buffer ( Ca - ATP monomers , see Methods ) or in salt - free motility buffer with EGTA and MgCl2 ( Mg - ATP monomers , see Methods ) and left on the bench at room temperature overnight . The day after , aged actin monomers were added to a fresh mixture ( without actin monomers ) in presence of fresh beads . Velocity of beads was measured in the different conditions . Composition of the reaction mix : 3 µM actin , 6 µM profilin , 90 nM Arp2 / 3 , 15 nM Capping protein , 200 nM cofilin , 400 nM Cyclase Associated Protein ( CAP ) . Ca - ATP actin monomers fresh : N = 1 , n = 50 comets . Ca - ATP actin monomers ( old ) N = 1 , n = 46 comets . Mg - ATP actin monomers ( fresh ) : N = 2 , n = 121 comets . Mg - ATP actin monomers ( old ) : N = 2 , n = 134 comets . Individual points for each comet are represented with mean and standard deviation superimposed .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5B, C. Test of reaction mix aging without beads. Reaction mix was prepared without beads and left on the bench at room temperature. Then at different time intervals, fresh beads were added to the mix. After 24 hours (right part of the graph), All (fresh) proteins in the assay were added one by one with fresh the beads. Velocity of fresh beads was estimated for those different times. Composition of the reaction mix: 3 µM actin, 6 µM profilin, 90 nM Arp2/3, 15 nM Capping protein, 200 nM cofilin, 400 nM Cyclase Associated Protein (CAP). t = 0: N = 4, n = 494 comets. t = 6 hours: N = 1, n = 65 comets. t = 24 hours: N = 2, n = 46 comets. t = 24 hours + fresh mix: N = 1, n = 32 comets. t = 24 hours + actin: N = 2, n = 108 comets. t = 24 hours + profilin: N = 1, n = 10 comets. t = 24 hours + capping protein: N = 1, n = 12 comets. t = 24 hours + Arp2/3 complex: N = 1, n = 11 comets. t = 24 hours + ADF/cofilin: N = 1, n = 17 comets. t = 24 hours + CAP: N = 1, n = 7 comets. Individual points for each comet are represented with mean and standard deviation superimposed.D. Test of actin monomer aging. Actin monomers were prepared in salt-free motility buffer (Ca-ATP monomers, see Methods) or in salt-free motility buffer with EGTA and MgCl2 (Mg-ATP monomers, see Methods) and left on the bench at room temperature overnight. The day after, aged actin monomers were added to a fresh mixture (without actin monomers) in presence of fresh beads . Velocity of beads was measured in the different conditions. Composition of the reaction mix: 3 µM actin, 6 µM profilin, 90 nM Arp2/3, 15 nM Capping protein, 200 nM cofilin, 400 nM Cyclase Associated Protein (CAP). Ca-ATP actin monomers fresh: N = 1, n = 50 comets. Ca-ATP actin monomers (old) N = 1, n = 46 comets. Mg-ATP actin monomers (fresh): N = 2, n = 121 comets. Mg-ATP actin monomers (old): N = 2, n = 134 comets. Individual points for each comet are represented with mean and standard deviation superimposed.


Figure 1C , Olaparib and ETC - 159 synergize to prevent the growth of HPAF - II xenografts in mice . NSG mice with established HPAF - II subcutaneous xenografts were randomized into four groups . Mice were gavaged daily with ETC - 159 ( 10 mg / kg ) , Olaparib ( 50 mg / kg ) or a combination of ETC - 159 ( 10 mg / kg ) and Olaparib ( 50 mg / kg ) . Treatment was initiated after HPAF - II tumors were established . ( C ) Tumor volumes were measured starting from day 0 and during the course of treatment as shown . Data points represent the mean ± SD . n = 7 - 8 tumors / group . p - values indicate significant difference compared to the vehicle group .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1C, Olaparib and ETC-159 synergize to prevent the growth of HPAF-II xenografts in mice. NSG mice with established HPAF-II subcutaneous xenografts were randomized into four groups. Mice were gavaged daily with ETC-159 (10 mg/kg), Olaparib (50 mg/kg) or a combination of ETC-159 (10 mg/kg) and Olaparib (50 mg/kg). Treatment was initiated after HPAF-II tumors were established. (C) Tumor volumes were measured starting from day 0 and during the course of treatment as shown. Data points represent the mean ± SD. n = 7-8 tumors/group. p-values indicate significant difference compared to the vehicle group.


Figure 2DNA repair genes involved in HR and FA pathways were differentially expressed over time in response to PORCN inhibition in HPAF - II orthotopic xenografts . Comparison of log2 fold change ( FDR < 10 % ) in the expression of Wnt - activated genes over time across multiple DNA repair pathways shows that genes regulating HR and FA pathways have higher fold changes compared to genes regulating BER , NER or other pathways . BER : base excision repair ; HR : homologous recombination ; FA : Fanconi anemia ; MMR : mismatch repair ; NER : nucleotide excision repair ; NHEJ : non - homologous end joining ; TLS : translesion DNA synthesis . The horizontal line represents median of 4 - 6 replicates with the upper and the lower edges of the box representing the 75th and 25th percentile of the data respectively and the whiskers representing 1 . 5 x interquartile range . H , I . Wnt inhibition reduces the expression of HR and FA pathway genes in a RNF43 - mutant pancreatic cancer patient - derived xenograft and AsPC - 1 cells . ( H ) Pancreatic canc er PDX with G371fs RNF43 mutation treated with vehicle or ETC - 159 ( 30 mg / kg ) for 21 days were analysed for changes in the expression of the indicated genes measured by qRT - PCR . Each data point represents an individual tumor . The horizontal lines represent mean of 3 tumors / group . ( I ) AsPC - 1 cells were seeded in low adherence plates , treated with DMSO or ETC - 159 ( 100 nM ) for 72 hours and the expression of indicated genes was measured by qRT - PCR .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2DNA repair genes involved in HR and FA pathways were differentially expressed over time in response to PORCN inhibition in HPAF-II orthotopic xenografts. Comparison of log2 fold change (FDR < 10%) in the expression of Wnt-activated genes over time across multiple DNA repair pathways shows that genes regulating HR and FA pathways have higher fold changes compared to genes regulating BER, NER or other pathways. BER: base excision repair; HR: homologous recombination; FA: Fanconi anemia; MMR: mismatch repair; NER: nucleotide excision repair; NHEJ: non-homologous end joining; TLS: translesion DNA synthesis. The horizontal line represents median of 4-6 replicates with the upper and the lower edges of the box representing the 75th and 25th percentile of the data respectively and the whiskers representing 1.5 x interquartile range.H, I. Wnt inhibition reduces the expression of HR and FA pathway genes in a RNF43-mutant pancreatic cancer patient-derived xenograft and AsPC-1 cells. (H) Pancreatic cancer PDX with G371fs RNF43 mutation treated with vehicle or ETC-159 (30 mg/kg) for 21 days were analysed for changes in the expression of the indicated genes measured by qRT-PCR. Each data point represents an individual tumor. The horizontal lines represent mean of 3 tumors/group. (I) AsPC-1 cells were seeded in low adherence plates, treated with DMSO or ETC-159 (100 nM) for 72 hours and the expression of indicated genes was measured by qRT-PCR.


Figure 3Olaparib and ETC - 159 combination is more effective in preventing the growth of PaTu8988T xenografts in mice . NSG mice with established PaTu8988T orthotopic xenografts were randomized into four groups . Mice were gavaged daily with ETC - 159 ( 30 mg / kg ) , olaparib ( 50 mg / kg ) or a combination of ETC - 159 and olaparib . Treatment was initiated after the tumors were established . Tumor weights in the respective groups at the end of the 18 days treatment are shown . Each dot represents an individual tumor and the horizontal lines represent the mean . p - values were calculated with Mann - Whitney U test . Wnt inhibition through ETC - 159 does not change the expression of HR and FA pathway genes in Panc 08 . 13 cells . Panc 08 . 13 cells were cultured in low adherence plates and treated with ETC - 159 for 72 hours . Total RNA was then isolated and expression of indicated genes was measured by qRT - PCR . The horizontal lines represent mean of replicates .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3Olaparib and ETC-159 combination is more effective in preventing the growth of PaTu8988T xenografts in mice. NSG mice with established PaTu8988T orthotopic xenografts were randomized into four groups. Mice were gavaged daily with ETC-159 (30 mg/kg), olaparib (50 mg/kg) or a combination of ETC-159 and olaparib. Treatment was initiated after the tumors were established. Tumor weights in the respective groups at the end of the 18 days treatment are shown. Each dot represents an individual tumor and the horizontal lines represent the mean. p-values were calculated with Mann-Whitney U test.Wnt inhibition through ETC-159 does not change the expression of HR and FA pathway genes in Panc 08.13 cells. Panc 08.13 cells were cultured in low adherence plates and treated with ETC-159 for 72 hours. Total RNA was then isolated and expression of indicated genes was measured by qRT-PCR. The horizontal lines represent mean of replicates.


Figure 4 Stabilized β - catenin prevents the downregulation of HR and FA pathway genes upon Wnt inhibition . Mice bearing HPAF - II xenografts without or with stabilized β - catenin were treated with ETC - 159 ( 37 . 5mg / kg bid ) for 56 hours before tumors were harvested and the expression of indicated genes was measured by qRT - PCR . ETC - 159 induced reduction in the expression of HR and FA pathway genes was blocked in xenografts with stabilized β - catenin . The horizontal lines represent mean of replicates E , F . G007 - LK and olaparib synergistically inhibit the growth of COLO320HSR colorectal cancer cells with APC mutation . COLO320HSR cells were plated at a low density and treated with G007 - LK , olaparib or the combination of the two inhibitors at an equivalent dose as described in Figure 1A . ( E ) Representative image from two independent experiments is shown . ( F ) The Combination Index ( CI ) values of olaparib and G007 - LK calculated from two independent experiments using the Chou - Talalay CompuSyn software

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

Figure 4 Stabilized β-catenin prevents the downregulation of HR and FA pathway genes upon Wnt inhibition. Mice bearing HPAF-II xenografts without or with stabilized β-catenin were treated with ETC-159 (37.5mg/kg bid) for 56 hours before tumors were harvested and the expression of indicated genes was measured by qRT-PCR. ETC-159 induced reduction in the expression of HR and FA pathway genes was blocked in xenografts with stabilized β-catenin. The horizontal lines represent mean of replicates E, F. G007-LK and olaparib synergistically inhibit the growth of COLO320HSR colorectal cancer cells with APC mutation. COLO320HSR cells were plated at a low density and treated with G007-LK, olaparib or the combination of the two inhibitors at an equivalent dose as described in Figure 1A. (E) Representative image from two independent experiments is shown. (F) The Combination Index (CI) values of olaparib and G007-LK calculated from two independent experiments using the Chou-Talalay CompuSyn software


Figure 5β - catenin regulates the expression of MYBL2 in HPAF - II cells . HPAF - II cells were either treated with ETC - 159 ( 100 nM ) or transfected with siRNA against β - catenin alone or in the presence of ETC - 159 for 48 hours . The relative expression of MYBL2 as measured by qRT - PCR is shown . The horizontal lines represent mean of replicates . Expression of DNA repair genes in Wnt - addicted cells is regulated by MYBL2 . HPAF - II cells were transfected with two independent siRNAs against MYBL2 or treated with ETC - 159 ( 100 nM ) for 48 hours . Total RNA was isolated and expression of MYBL2 and DNA repair genes was measured by qRT - PCR . Data are representative of three independent experiments . The horizontal lines represent mean of replicates . H - K . Expression of MYBL2 is downregulated upon ETC - 159 or G007 - LK treatment in multiple Wnt - high tumors . Expression of MYBL2 in AsPC - 1 xenograft tumors and colorectal cancer PDX ( as measured by RNA - seq ) or in pancreatic cancer PDX with RNF43 mutation ( measured by qRT - PCR ) is reduced with ETC - 159 treatment . G007 - LK reduces MYBL2 expression in COL320HSR cells ( measured by qRT - PCR ) . p - values were calculated with Mann - Whitney U test . Each data point represents an individual tumor or replicate . n = 4 - 6 samples / group . The horizontal lines represent median of 4 - 6 replicates with the upper and the lower edges of the boxes representing the 75th and the 25th percentile of the data respectively and the whiskers representing 1 . 5x interquartile range .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5β-catenin regulates the expression of MYBL2 in HPAF-II cells. HPAF-II cells were either treated with ETC-159 (100 nM) or transfected with siRNA against β-catenin alone or in the presence of ETC-159 for 48 hours. The relative expression of MYBL2 as measured by qRT-PCR is shown. The horizontal lines represent mean of replicates.Expression of DNA repair genes in Wnt-addicted cells is regulated by MYBL2. HPAF-II cells were transfected with two independent siRNAs against MYBL2 or treated with ETC-159 (100 nM) for 48 hours. Total RNA was isolated and expression of MYBL2 and DNA repair genes was measured by qRT-PCR. Data are representative of three independent experiments. The horizontal lines represent mean of replicates.H-K. Expression of MYBL2 is downregulated upon ETC-159 or G007-LK treatment in multiple Wnt-high tumors. Expression of MYBL2 in AsPC-1 xenograft tumors and colorectal cancer PDX (as measured by RNA-seq) or in pancreatic cancer PDX with RNF43 mutation (measured by qRT-PCR) is reduced with ETC-159 treatment. G007-LK reduces MYBL2 expression in COL320HSR cells (measured by qRT-PCR). p-values were calculated with Mann-Whitney U test. Each data point represents an individual tumor or replicate. n = 4-6 samples/group. The horizontal lines represent median of 4-6 replicates with the upper and the lower edges of the boxes representing the 75th and the 25th percentile of the data respectively and the whiskers representing 1.5x interquartile range.


Figure 6F , Wnt inhibition induces senescence , which is further enhanced by co - treatment with olaparib . ( F ) Representative images of tumor sections from the four treatment groups ( treated for 21 days ) stained for SA - β - galactosidase , a senescence marker , and counterstained with nuclear fast red . Blue color indicates positive staining for senescent cells . The horizontal lines represent mean of replicates . p - values were calculated by Mann - Whitney U test . Wnt inhibition potentiates DNA double strand breaks induced by olaparib treatment . ( H ) HPAF - II cells were treated with DMSO , ETC - 159 ( 50 nM ) , olaparib ( 20 µM ) or both for 7 days . 53BP1 foci per cell were then assessed by immunofluorescence . The horizontal lines represent mean of replicates . p - values were calculated by Mann - Whitney U test . I . Wnt inhibition potentiates DNA double strand breaks induced by olaparib treatment . ( I ) Representative immunofluorescence images of 53BP1 foci ( red ) and nuclei counterstained with DAPI ( blue ) are shown for each group .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6F, Wnt inhibition induces senescence, which is further enhanced by co-treatment with olaparib. (F) Representative images of tumor sections from the four treatment groups (treated for 21 days) stained for SA-β-galactosidase, a senescence marker, and counterstained with nuclear fast red. Blue color indicates positive staining for senescent cells. The horizontal lines represent mean of replicates. p-values were calculated by Mann-Whitney U test.Wnt inhibition potentiates DNA double strand breaks induced by olaparib treatment. (H) HPAF-II cells were treated with DMSO, ETC-159 (50 nM), olaparib (20 µM) or both for 7 days. 53BP1 foci per cell were then assessed by immunofluorescence. The horizontal lines represent mean of replicates. p-values were calculated by Mann-Whitney U test.I. Wnt inhibition potentiates DNA double strand breaks induced by olaparib treatment. (I) Representative immunofluorescence images of 53BP1 foci (red) and nuclei counterstained with DAPI (blue) are shown for each group.