M . [ left ] CD105 normalized MFI of the EC cells at P30 in Male Ctrl ( 100 . 00 ± 7 . 39 ) , Male Poly I : C ( 99 . 58 ± 6 . 23 ) , Female Ctrl ( 100 . 30 ± 1 . 61 ) , Female Poly I : C ( 96 . 67 ± 3 . 72 ) . [ right ] Representative histograms indicating mean fluorescence intensity ( MFI ) of CD105 on EC in Male Ctrl ( blue hollow ) , Male Poly I : C ( solid light blue ) , Female Ctrl ( purple hollow ) , Female Poly I : C ( solid purple ) samples . N . [ left ] Quantitative data of the normalized MFI of CD105 in PCs at P30 in Male Ctrl ( 100 . 00 ± 3 . 43 ) , Male Poly I : C ( 98 . 01 ± 5 . 14 ) , Female Ctrl ( 98 . 57 ± 4 . 16 ) , Female Poly I : C ( 87 . 99 ± 4 . 21 ) . Mann - Whitney U test , * p < . 05 . [ right ] Representative histograms for CD105 expression on PCs in Male Ctrl ( blue hollow ) , Male Poly I : C ( solid light blue ) , Female Ctrl ( purple hollow ) , Female Poly I : C ( solid purple ) samples . Data information : Embryos prenatally exposed to vehicle ( Ctrl ) or Poly I : C on GD9 were dissected , and cortex single - cell suspension was analyzed by multi - color flow cytometry . Ctrl males = white bars with blu border . Poly I : C males = blu bars with blu border . Ctrl females = white bars with purple border . Poly I : C females = purple bars with purple border . Bars represent mean ± SEM , numbers in bars indicate the number of animals .

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

M. [left] CD105 normalized MFI of the EC cells at P30 in Male Ctrl (100.00 ± 7.39), Male Poly I:C (99.58 ± 6.23), Female Ctrl (100.30 ± 1.61), Female Poly I:C (96.67 ± 3.72). [right] Representative histograms indicating mean fluorescence intensity (MFI) of CD105 on EC in Male Ctrl (blue hollow), Male Poly I:C (solid light blue), Female Ctrl (purple hollow), Female Poly I:C (solid purple) samples. N. [left] Quantitative data of the normalized MFI of CD105 in PCs at P30 in Male Ctrl (100.00 ± 3.43), Male Poly I:C (98.01 ± 5.14), Female Ctrl (98.57 ± 4.16), Female Poly I:C (87.99 ± 4.21). Mann-Whitney U test, *p < .05. [right] Representative histograms for CD105 expression on PCs in Male Ctrl (blue hollow), Male Poly I:C (solid light blue), Female Ctrl (purple hollow), Female Poly I:C (solid purple) samples. Data information: Embryos prenatally exposed to vehicle (Ctrl) or Poly I:C on GD9 were dissected, and cortex single-cell suspension was analyzed by multi-color flow cytometry. Ctrl males = white bars with blu border. Poly I:C males = blu bars with blu border. Ctrl females = white bars with purple border. Poly I:C females = purple bars with purple border. Bars represent mean ± SEM, numbers in bars indicate the number of animals.


O . [ top ] Representative images of CD13 ( red ) positive pericytes covering Lectin - positive vessels ( green ) in sections of P90 untreated male brains . [ bottom ] 3D rendering , Scale bar = 20 µm , enlargement scale bar = 10 µm . P . Percentage of CD13 - positive pericyte coverage at P90 in Male Ctrl ( 53 . 42 ± 9 . 22 ) and Poly I : C ( 27 . 73 ± 5 . 29 ) . Student ' s t test , * p < . 05 . Data information : Ctrl males = white bars with blu border . Poly I : C males = blu bars with blu border . Bars represent mean ± SEM , numbers in bars indicate the number of animals .

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

O. [top] Representative images of CD13 (red) positive pericytes covering Lectin-positive vessels (green) in sections of P90 untreated male brains. [bottom] 3D rendering, Scale bar = 20 µm, enlargement scale bar = 10 µm. P. Percentage of CD13-positive pericyte coverage at P90 in Male Ctrl (53.42 ± 9.22) and Poly I:C (27.73 ± 5.29). Student's t test, *p < .05. Data information: Ctrl males = white bars with blu border. Poly I:C males = blu bars with blu border. Bars represent mean ± SEM, numbers in bars indicate the number of animals.


Q . Percentage of PDGFrB - positive pericyte coverage at P90 in Male Ctrl ( 66 . 15 ± 3 . 09 ) and Poly I : C ( 55 . 08 ± 3 . 10 ) . Student ' s t test , * p < . 05 . Data information : Ctrl males = white bars with blu border . Poly I : C males = blu bars with blu border . Bars represent mean ± SEM , numbers in bars indicate the number of animals .

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

Q. Percentage of PDGFrB-positive pericyte coverage at P90 in Male Ctrl (66.15 ± 3.09) and Poly I:C (55.08 ± 3.10). Student's t test, *p < .05. Data information: Ctrl males = white bars with blu border. Poly I:C males = blu bars with blu border. Bars represent mean ± SEM, numbers in bars indicate the number of animals.


R . Representative images of PDGFrB ( red ) and NG2 ( white ) positive pericytes covering Lectin - positive vessels ( blu ) in sections of P90 untreated male brains . Scale bar = 10 µm , enlargement scale bar = 5 µm .

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

R. Representative images of PDGFrB (red) and NG2 (white) positive pericytes covering Lectin-positive vessels (blu) in sections of P90 untreated male brains. Scale bar = 10 µm, enlargement scale bar = 5 µm.


S . Percentage of CD13 - positive pericyte coverage at P90 in Female Ctrl ( 71 . 57 ± 8 . 37 ) and Poly I : C ( 69 . 13 ± 6 . 63 ) . T . Percentage of PDGFrB - positive pericyte coverage at P90 in Female Ctrl ( 66 . 77 ± 11 . 76 ) and Poly I : C ( 70 . 56 ± 8 . 32 ) . Data information : Ctrl females = white bars with purple border . Poly I : C females = purple bars with purple border . Bars represent mean ± SEM , numbers in bars indicate the number of animals .

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

S. Percentage of CD13-positive pericyte coverage at P90 in Female Ctrl (71.57 ± 8.37) and Poly I:C (69.13 ± 6.63). T. Percentage of PDGFrB-positive pericyte coverage at P90 in Female Ctrl (66.77 ± 11.76) and Poly I:C (70.56 ± 8.32). Data information: Ctrl females = white bars with purple border. Poly I:C females = purple bars with purple border. Bars represent mean ± SEM, numbers in bars indicate the number of animals.


A . Representative images of brain acute bleedings detected through hemoglobin IHC in P30 Ctrl and Poly I : C MIA male offspring . Scale bar = 500 µm , enlargement scale bar = 50 µm . B . Representative images of brain acute bleedings detected through hemoglobin IHC in P90 Ctrl and Poly I : C MIA male offspring . Scale bar = 500 µm , enlargement scale bar = 50 µm . C . Quantification of hemoglobin ( Hb ) positive signal in the brain of P30 and P90 male MIA offspring , expressed as percentage of the area . P30 Ctrl = 0 . 00 ± 0 . 00 , P30 Poly I : C = 0 . 01 ± 0 . 00 , P90 Ctrl = 0 . 00 ± 0 . 00 , P90 Poly I : C = 0 . 01 ± 0 . 00 . Mann - Whitney U test , * * * p < . 001 . Data information : Ctrl males = white bars with blu border . Poly I : C males = blu bars with blu border . Numbers in bars indicate the number of animals ( N ) and slices ( n ) . Bars represent mean ± SEM .

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

A. Representative images of brain acute bleedings detected through hemoglobin IHC in P30 Ctrl and Poly I:C MIA male offspring. Scale bar = 500 µm, enlargement scale bar = 50 µm. B. Representative images of brain acute bleedings detected through hemoglobin IHC in P90 Ctrl and Poly I:C MIA male offspring. Scale bar = 500 µm, enlargement scale bar = 50 µm. C. Quantification of hemoglobin (Hb) positive signal in the brain of P30 and P90 male MIA offspring, expressed as percentage of the area. P30 Ctrl = 0.00 ± 0.00, P30 Poly I:C = 0.01 ± 0.00, P90 Ctrl = 0.00 ± 0.00, P90 Poly I:C = 0.01 ± 0.00. Mann-Whitney U test, ***p < .001. Data information: Ctrl males = white bars with blu border. Poly I:C males = blu bars with blu border. Numbers in bars indicate the number of animals (N) and slices (n). Bars represent mean ± SEM.


D . Representative 3D rendering of iron deposits detected by DAB - enhanced Perls staining in P30 Ctrl and Poly I : C MIA male offspring brains . Brain volume is rendered gold - transparent with positive signal for the staining color - coded by surface dimension . Scale bar = 100 µm .

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D. Representative 3D rendering of iron deposits detected by DAB-enhanced Perls staining in P30 Ctrl and Poly I:C MIA male offspring brains. Brain volume is rendered gold-transparent with positive signal for the staining color-coded by surface dimension. Scale bar = 100 µm.


E . Quantification of DAB - enhanced histochemical iron stains in the brain of P30 Ctrl ( 0 . 00 ± 0 . 00 ) and Poly I : C ( 0 . 01 ± 0 . 00 ) male MIA offspring , expressed as Perls - positive percentage of the volume . Mann - Whitney U test , * p < . 05 . Data information : Ctrl males = white bars with blu border . Poly I : C males = blu bars with blu border . Numbers in bars indicate the number of animals ( N ) and slices ( n ) . Bars represent mean ± SEM .

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

E. Quantification of DAB-enhanced histochemical iron stains in the brain of P30 Ctrl (0.00 ± 0.00) and Poly I:C (0.01 ± 0.00) male MIA offspring, expressed as Perls-positive percentage of the volume. Mann-Whitney U test, *p < .05. Data information: Ctrl males = white bars with blu border. Poly I:C males = blu bars with blu border. Numbers in bars indicate the number of animals (N) and slices (n). Bars represent mean ± SEM.


F . Poisson distribution R value of Perls / DAB positive staining of P30 male MIA offspring , respectively in the Coronal ( Ctrl = 1 . 19 ± 0 . 04 , Poly I : C = 0 . 94 ± 0 . 01 ; Mann - Whitney U test , * * p < . 01 ) , Axial ( Ctrl = 1 . 16 ± 0 . 08 , Poly I : C = 1 . 03 ± 0 . 03 ) , and Sagittal ( Ctrl = 1 . 09 ± 0 . 09 , Poly I : C = 1 . 01 ± 0 . 05 ) planes . Data information : Ctrl males = white bars with blu border . Poly I : C males = blu bars with blu border . Numbers in bars indicate the number of animals ( N ) and slices ( n ) . Bars represent mean ± SEM .

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F. Poisson distribution R value of Perls/DAB positive staining of P30 male MIA offspring, respectively in the Coronal (Ctrl = 1.19 ± 0.04, Poly I:C = 0.94 ± 0.01; Mann-Whitney U test, **p < .01), Axial (Ctrl = 1.16 ± 0.08, Poly I:C = 1.03 ± 0.03), and Sagittal (Ctrl = 1.09 ± 0.09, Poly I:C = 1.01 ± 0.05) planes. Data information: Ctrl males = white bars with blu border. Poly I:C males = blu bars with blu border. Numbers in bars indicate the number of animals (N) and slices (n). Bars represent mean ± SEM.


G . Representative images of iron deposits detected through Perls staining in P90 Ctrl and Poly I : C MIA male offspring brains . Scale bar = 500 µm , enlargement scale bar = 100 µm . H . Quantification of Perls - positive staining in the brain of P90 Ctrl ( 0 . 25 ± 0 . 05 ) and Poly I : C ( 0 . 59 ± 0 . 10 ) male MIA offspring , expressed as percentage of the area . Mann - Whitney U test , * * p < . 01 . Data information : Ctrl males = white bars with blu border . Poly I : C males = blu bars with blu border . Numbers in bars indicate the number of animals ( N ) and slices ( n ) . Bars represent mean ± SEM .

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

G. Representative images of iron deposits detected through Perls staining in P90 Ctrl and Poly I:C MIA male offspring brains. Scale bar = 500 µm, enlargement scale bar = 100 µm. H. Quantification of Perls-positive staining in the brain of P90 Ctrl (0.25 ± 0.05) and Poly I:C (0.59 ± 0.10) male MIA offspring, expressed as percentage of the area. Mann-Whitney U test, **p < .01. Data information: Ctrl males = white bars with blu border. Poly I:C males = blu bars with blu border. Numbers in bars indicate the number of animals (N) and slices (n). Bars represent mean ± SEM.


I . Representative image of marble burying test at 0 and 30 minutes . 15 glass marbles were evenly spaced on a 5 x 3 grid . Marbles buried by two - thirds at least are indicated by a red circle or by a cross within a red circle when fully covered by the sawdust . J . Quantification of buried marble ( n ° ) by P90 Ctrl Male ( 3 . 33 ± 1 . 67 ) , Poly I : C Male ( 5 . 72 ± 2 . 10 ) , Ctrl Female ( 3 . 83 ± 1 . 72 ) and Poly I : C Female ( 3 . 00 ± 2 . 00 ) mice . Student ' s t test , * * p < . 01 . Data information : Ctrl males = white bars with blu border . Poly I : C males = blu bars with blu border . Ctrl females = white bars with purple border . Poly I : C females = purple bars with purple border . Numbers in bars indicate the number of animals ( N ) and slices ( n ) . Bars represent mean ± SEM .

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

I. Representative image of marble burying test at 0 and 30 minutes. 15 glass marbles were evenly spaced on a 5 x 3 grid. Marbles buried by two-thirds at least are indicated by a red circle or by a cross within a red circle when fully covered by the sawdust. J. Quantification of buried marble (n°) by P90 Ctrl Male (3.33 ± 1.67), Poly I:C Male (5.72 ± 2.10), Ctrl Female (3.83 ± 1.72) and Poly I:C Female (3.00 ± 2.00) mice. Student's t test, **p < .01. Data information: Ctrl males = white bars with blu border. Poly I:C males = blu bars with blu border. Ctrl females = white bars with purple border. Poly I:C females = purple bars with purple border. Numbers in bars indicate the number of animals (N) and slices (n). Bars represent mean ± SEM.


K . Quantification of self - grooming time ( sec ) by P90 Ctrl Males ( 19 . 34 ± 13 . 00 ) , Poly I : C Males ( 43 . 38 ± 35 . 21 ) , Ctrl Females ( 19 . 86 ± 18 . 06 ) and Poly I : C Females ( 16 . 93 ± 11 . 19 ) mice . Student ' s t test , * p < . 05 . Data information : Ctrl males = white bars with blu border . Poly I : C males = blu bars with blu border . Ctrl females = white bars with purple border . Poly I : C females = purple bars with purple border . Numbers in bars indicate the number of animals ( N ) and slices ( n ) . Bars represent mean ± SEM .

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

K. Quantification of self-grooming time (sec) by P90 Ctrl Males (19.34 ± 13.00), Poly I:C Males (43.38 ± 35.21), Ctrl Females (19.86 ± 18.06) and Poly I:C Females (16.93 ± 11.19) mice. Student's t test, *p < .05. Data information: Ctrl males = white bars with blu border. Poly I:C males = blu bars with blu border. Ctrl females = white bars with purple border. Poly I:C females = purple bars with purple border. Numbers in bars indicate the number of animals (N) and slices (n). Bars represent mean ± SEM.


( D ) Western blot of whole zebrafish larvae , Smyhc1 stained with the F59 antibody . smyhc1 + / + larvae express Smyhc1 at 24hpf , but not at 48hpf . smyhc1 - / - larvae do not express Smyhc1 .

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(D) Western blot of whole zebrafish larvae, Smyhc1 stained with the F59 antibody. smyhc1+/+ larvae express Smyhc1 at 24hpf, but not at 48hpf. smyhc1-/- larvae do not express Smyhc1.


( E ) Smyhc1 immunohistochemistry stain of 24 hpf ( hour post fertilization ) zebrafish larvae . Filamentous actin is stained with phalloidin ( red ) . Nuclei are stained with DAPI ( blue ) . Smyhc1 is stained with the F59 antibody ( green ) ( Elworthy et al , 2008 ) . smyhc1 + / + , smyhc1R673H / + , and smyhc1R673H / R673H larvae display Smyhc1 in the muscle fibers at 24hpf . smyhc1 - / - larvae at 24 do not stain positively for Smyhc1 . Scale bar represents 50 µm .

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(E) Smyhc1 immunohistochemistry stain of 24 hpf (hour post fertilization) zebrafish larvae. Filamentous actin is stained with phalloidin (red). Nuclei are stained with DAPI (blue). Smyhc1 is stained with the F59 antibody (green) (Elworthy et al, 2008). smyhc1+/+, smyhc1R673H/+, and smyhc1R673H/R673H larvae display Smyhc1 in the muscle fibers at 24hpf. smyhc1-/- larvae at 24 do not stain positively for Smyhc1. Scale bar represents 50 µm.


( A ) Morphologies of smyhc1 + / + , smyhc1 - / + and smyhc1 - / - embryos are grossly normal at 2 days post fertilization ( dpf ) , despite complete paralysis of smyhc1 - / - embryos . In contrast , the smyhc1R673H / + embryos have notochord kinks in larval stages ( arrow head ) , while smyhc1R673H / R673H embryos have much more severe notochord kinks that compress and distort the body axis ( arrow heads ) . The smyhc1R673H / - embryos completely phenocopy smyhc1R673H / R673H embryos . ( B ) Quantification of smyhc1R673H embryonic phenotypes at 2 dpf .

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(A) Morphologies of smyhc1+/+, smyhc1-/+ and smyhc1-/- embryos are grossly normal at 2 days post fertilization (dpf), despite complete paralysis of smyhc1-/- embryos. In contrast, the smyhc1R673H/+ embryos have notochord kinks in larval stages (arrow head), while smyhc1R673H/R673H embryos have much more severe notochord kinks that compress and distort the body axis (arrow heads). The smyhc1R673H/- embryos completely phenocopy smyhc1R673H/R673H embryos. (B) Quantification of smyhc1R673H embryonic phenotypes at 2 dpf.


( B ) The predicted mendelian ( 1 : 2 : 1 ) and experimental ratio of genotypes of progeny from smyhc1R673H / + in - crosses demonstrates survival at the indicated ages . This ratio quickly skewed toward an overrepresentation of smyhc1 + / + fish with no representation of smyhc1R673H / R673H after 5 dpf . There is also dropout of smyhc1 + / R673H fish into adulthood . Number of fish counted per group is indicated .

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(B) The predicted mendelian (1:2:1) and experimental ratio of genotypes of progeny from smyhc1R673H/+ in-crosses demonstrates survival at the indicated ages. This ratio quickly skewed toward an overrepresentation of smyhc1+/+ fish with no representation of smyhc1R673H/R673H after 5 dpf. There is also dropout of smyhc1+/R673H fish into adulthood. Number of fish counted per group is indicated.


( A ) Gross morphology of smyhc1 mutant adults . Most smyhc1 - / - adult fish display dorsal tail curvature , while smyhc1R673H / + adults have a shortened body axis and variable spinal curves .

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(A) Gross morphology of smyhc1 mutant adults. Most smyhc1-/- adult fish display dorsal tail curvature, while smyhc1R673H/+ adults have a shortened body axis and variable spinal curves.


( B ) Alizarin red staining of bone shows spinal curvature but no bony fusions in the skeleton of smyhc1 - / - adults , in contrast to the compression and fusion of vertebrae seen in smyhc1R673H / + adults . Distal tail regions are highlighted and enlarged .

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(B) Alizarin red staining of bone shows spinal curvature but no bony fusions in the skeleton of smyhc1-/- adults, in contrast to the compression and fusion of vertebrae seen in smyhc1R673H/+ adults. Distal tail regions are highlighted and enlarged.


( C ) Quantification of skeletal phenotype severity in adults . Scale bars represent a length of 5 mm .

(C) Quantification of skeletal phenotype severity in adults. Scale bars represent a length of 5 mm.


( A ) Time course of light - triggered larval movements were manually counted at indicated times between 24 and 48 hpf . The smyhc1 - / - larvae ( n = 36 ) were paralyzed up to 48 hpf . smyhc1R673H / + mutants ( n = 15 ) twitched slightly less than their smyhc1 + / + siblings ( n = 21 ) at 34hpf . The central bands of the boxplots represent the median , the boxes of the boxplots represent the interquartile range ( between the first and third quartile ) , and the whiskers represent the minimum and maximum values , up to 1 . 5 times the interquartile range . Outliers displayed are outside of this range .

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

(A) Time course of light-triggered larval movements were manually counted at indicated times between 24 and 48 hpf. The smyhc1-/- larvae (n = 36) were paralyzed up to 48 hpf. smyhc1R673H/+ mutants (n = 15) twitched slightly less than their smyhc1+/+ siblings (n = 21) at 34hpf. The central bands of the boxplots represent the median, the boxes of the boxplots represent the interquartile range (between the first and third quartile), and the whiskers represent the minimum and maximum values, up to 1.5 times the interquartile range. Outliers displayed are outside of this range.


( B ) Distance travelled during 5 minutes of spontaneous swimming at 6 dpf quantified by motion tracking software ( Noldus Ethovision ) . At 6 dpf , both smyhc1 - / - ( n = 24 ) and smyhc1R673H / + ( n = 11 ) larvae travelled significantly less than smyhc1 + / + larvae ( n = 13 ) . The central bands of the boxplots represent the median , the boxes of the boxplots represent the interquartile range ( between the first and third quartile ) , and the whiskers represent the minimum and maximum values , up to 1 . 5 times the interquartile range . Outliers displayed are outside of this range .

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(B) Distance travelled during 5 minutes of spontaneous swimming at 6 dpf quantified by motion tracking software (Noldus Ethovision). At 6 dpf, both smyhc1-/- (n = 24) and smyhc1R673H/+ (n = 11) larvae travelled significantly less than smyhc1+/+ larvae (n = 13). The central bands of the boxplots represent the median, the boxes of the boxplots represent the interquartile range (between the first and third quartile), and the whiskers represent the minimum and maximum values, up to 1.5 times the interquartile range. Outliers displayed are outside of this range.


( C ) Diagram of swim tunnel used to quantify adult swimming behavior . Water flow was gradually increased until the fish fatigued and were collected at the grate .

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(C) Diagram of swim tunnel used to quantify adult swimming behavior. Water flow was gradually increased until the fish fatigued and were collected at the grate.


( D ) Time spent swimming in swim tunnel before fatigue in adults at 6 months post fertilization ( mpf ) . smyhc1 + / + ( n = 6 ) , smyhc1 - / - ( n = 5 ) , smyhc1R673H / + ( n = 5 ) .

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(D) Time spent swimming in swim tunnel before fatigue in adults at 6 months post fertilization (mpf). smyhc1+/+ (n = 6), smyhc1-/- (n = 5), smyhc1R673H/+ (n = 5).


( A ) Confocal fluorescence images of slow skeletal muscle of 1 dpf larvae , stained with phalloidin - rhodamine to detect actin . Puncta of filamentous actin are indicated with white arrowheads , misshapen myofibers are indicated with white arrows and outlined . ( B ) Confocal microscope fluorescence images of slow skeletal muscle of 3 dpf larvae , stained with phalloidin - rhodamine . Puncta of filamentous actin are indicated with white arrowheads , distorted myofibers are indicated with arrows and outlined , and frayed myofibers are indicated with yellow arrowhead .

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(A) Confocal fluorescence images of slow skeletal muscle of 1 dpf larvae, stained with phalloidin-rhodamine to detect actin. Puncta of filamentous actin are indicated with white arrowheads, misshapen myofibers are indicated with white arrows and outlined. (B) Confocal microscope fluorescence images of slow skeletal muscle of 3 dpf larvae, stained with phalloidin-rhodamine. Puncta of filamentous actin are indicated with white arrowheads, distorted myofibers are indicated with arrows and outlined, and frayed myofibers are indicated with yellow arrowhead.


( C ) Confocal fluorescence images of slow skeletal muscle of 1dpf larvae , stained with phalloidin - rhodamine ( red ) , anti - α - actinin antibodies ( green ) , and DAPI ( blue ) . ( D ) Confocal fluorescence images of slow skeletal muscle of 3dpf larvae , stained with phalloidin - rhodamine ( red ) , anti - α - actinin antibodies ( green ) , and DAPI ( blue ) . Bundle of actin ringed with α - actinin indicated with white - bordered black arrowhead .

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(C) Confocal fluorescence images of slow skeletal muscle of 1dpf larvae, stained with phalloidin-rhodamine (red), anti-α-actinin antibodies (green), and DAPI (blue). (D) Confocal fluorescence images of slow skeletal muscle of 3dpf larvae, stained with phalloidin-rhodamine (red), anti-α-actinin antibodies (green), and DAPI (blue). Bundle of actin ringed with α-actinin indicated with white-bordered black arrowhead.


( E ) Myoseptal intervals of slow skeletal muscle at 1 dpf in smyhc1 + / + ( n = 54 ) , smyhc1 - / - ( n = 31 ) , smyhc1R673H / + ( n = 45 ) , and smyhc1R673H / R673H ( n = 49 ) . Distance between myosepta was measured perpendicular to rostral - caudal body axis at defined mid - body regions in phalloidin stained slow skeletal muscle fluorescence images . The central bands of the boxplots represent the median , the boxes of the boxplots represent the interquartile range ( between the first and third quartile ) , and the whiskers represent the minimum and maximum values , up to 1 . 5 times the interquartile range . Outliers displayed are outside of this range . ( F ) Myoseptal intervals of slow skeletal muscle at 3 dpf in smyhc1 + / + ( n = 51 ) , smyhc1 - / - ( n = 32 ) , smyhc1R673H / + ( n = 64 ) , and smyhc1R673H / R673H ( n = 38 ) . The central bands of the boxplots represent the median , the boxes of the boxplots represent the interquartile range ( between the first and third quartile ) , and the whiskers represent the minimum and maximum values , up to 1 . 5 times the interquartile range . Outliers displayed are outside of this range .

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(E) Myoseptal intervals of slow skeletal muscle at 1 dpf in smyhc1+/+ (n = 54), smyhc1-/- (n = 31), smyhc1R673H/+ (n = 45), and smyhc1R673H/R673H (n = 49). Distance between myosepta was measured perpendicular to rostral-caudal body axis at defined mid-body regions in phalloidin stained slow skeletal muscle fluorescence images. The central bands of the boxplots represent the median, the boxes of the boxplots represent the interquartile range (between the first and third quartile), and the whiskers represent the minimum and maximum values, up to 1.5 times the interquartile range. Outliers displayed are outside of this range. (F) Myoseptal intervals of slow skeletal muscle at 3 dpf in smyhc1+/+ (n = 51), smyhc1-/- (n = 32), smyhc1R673H/+ (n = 64), and smyhc1R673H/R673H (n = 38). The central bands of the boxplots represent the median, the boxes of the boxplots represent the interquartile range (between the first and third quartile), and the whiskers represent the minimum and maximum values, up to 1.5 times the interquartile range. Outliers displayed are outside of this range.


( G ) Z - disc intervals ( sarcomere length ) of slow skeletal muscle at 1 dpf in smyhc1 + / + ( n = 380 ) , smyhc1R673H / + ( n = 293 ) , and smyhc1R673H / R673H ( n = 268 ) . Distance between z - discs was measured in anti - α - actinin stained slow skeletal muscle fluorescence images . The central bands of the boxplots represent the median , the boxes of the boxplots represent the interquartile range ( between the first and third quartile ) , and the whiskers represent the minimum and maximum values , up to 1 . 5 times the interquartile range . Outliers displayed are outside of this range . ( H ) Z - disc intervals ( sarcomere length ) of slow skeletal muscle at 3 dpf in smyhc1 + / + ( n = 249 ) , smyhc1 - / - ( n = 278 ) , smyhc1R673H / + ( n = 246 ) , and smyhc1R673H / R673H ( n = 252 ) . The central bands of the boxplots represent the median , the boxes of the boxplots represent the interquartile range ( between the first and third quartile ) , and the whiskers represent the minimum and maximum values , up to 1 . 5 times the interquartile range . Outliers displayed are outside of this range .

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(G) Z-disc intervals (sarcomere length) of slow skeletal muscle at 1 dpf in smyhc1+/+ (n = 380), smyhc1R673H/+ (n = 293), and smyhc1R673H/R673H (n = 268). Distance between z-discs was measured in anti-α-actinin stained slow skeletal muscle fluorescence images. The central bands of the boxplots represent the median, the boxes of the boxplots represent the interquartile range (between the first and third quartile), and the whiskers represent the minimum and maximum values, up to 1.5 times the interquartile range. Outliers displayed are outside of this range. (H) Z-disc intervals (sarcomere length) of slow skeletal muscle at 3 dpf in smyhc1+/+ (n = 249), smyhc1-/- (n = 278), smyhc1R673H/+ (n = 246), and smyhc1R673H/R673H (n = 252). The central bands of the boxplots represent the median, the boxes of the boxplots represent the interquartile range (between the first and third quartile), and the whiskers represent the minimum and maximum values, up to 1.5 times the interquartile range. Outliers displayed are outside of this range.


( A ) Representative morphologies of smyhc1 + / + , smyhc1R673H / + , and smyhc1R673H / R673H larvae at 2 dpf after 24 hour treatment with 0 . 25 % DMSO ( control ) or 25 μM para - aminoblebbistatin . All larvae treated with para - aminoblebbistatin develop a slight dorsal tail curve and pericardial edema regardless of genotype . All para - aminoblebbistatin - treated smyhc1R673H / + ( n = 9 ) and smyhc1R673H / R673H ( n = 3 ) larvae are indistinguishable from smyhc1 + / + unlike the untreated larvae which have markedly curved or distorted body axis . ( B ) Representative images of smyhc1 + / + , smyhc1R673H / + , and smyhc1R673H / R673H larvae at 3dpf after 48 hour treatment with 0 . 25 % DMSO ( control ) or 25 μM para - aminoblebbistatin . All larvae treated with para - aminoblebbistatin develop a slight dorsal tail curve and pericardial edema regardless of genotype . Para - aminoblebbistatin - treated smyhc1R673H / + ( n = 9 ) and smyhc1R673H / R673H ( n = 3 ) larvae are indistinguishable from smyhc1 + / + unlike the untreated larvae which have markedly curved or distorted body axis .

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(A) Representative morphologies of smyhc1+/+, smyhc1R673H/+, and smyhc1R673H/R673H larvae at 2 dpf after 24 hour treatment with 0.25% DMSO (control) or 25 μM para-aminoblebbistatin. All larvae treated with para-aminoblebbistatin develop a slight dorsal tail curve and pericardial edema regardless of genotype. All para-aminoblebbistatin-treated smyhc1R673H/+ (n=9) and smyhc1R673H/R673H (n=3) larvae are indistinguishable from smyhc1+/+ unlike the untreated larvae which have markedly curved or distorted body axis. (B) Representative images of smyhc1+/+, smyhc1R673H/+, and smyhc1R673H/R673H larvae at 3dpf after 48 hour treatment with 0.25% DMSO (control) or 25 μM para-aminoblebbistatin. All larvae treated with para-aminoblebbistatin develop a slight dorsal tail curve and pericardial edema regardless of genotype. Para-aminoblebbistatin-treated smyhc1R673H/+ (n=9) and smyhc1R673H/R673H (n=3) larvae are indistinguishable from smyhc1+/+ unlike the untreated larvae which have markedly curved or distorted body axis.


B Expression levels of GlcNAc in wild - type ( WT ) GAS , ΔgacG , H , I , J , K , and L were measured as the sWGA fluorescence intensity ( normalized to WT GAS ) .

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B Expression levels of GlcNAc in wild-type (WT) GAS, ΔgacG, H, I, J, K, and L were measured as the sWGA fluorescence intensity (normalized to WT GAS).


B , C Accumulation of LC3 - II . ( B ) Representative western blot of LC3 - II during non - infection ( NI ) , WT GAS , ΔgacI , and ΔgacI : : gacI . ( C ) LC3 - II intensity normalized to NI .

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B, C Accumulation of LC3-II. (B) Representative western blot of LC3-II during non-infection (NI), WT GAS, ΔgacI, and ΔgacI::gacI. (C) LC3-II intensity normalized to NI.


D , E Recruitment of ubiquitin in GAS - infected cells . HeLa cells were infected with WT GAS , ΔgacI , and ΔgacI : : gacI for 4 h , fixed , and immunostained for ubiquitin ( FK2 : magenta ) . Cellular and bacterial DNA were stained with DAPI ( cyan ) . ( D ) Representative confocal images and ( E ) percentage of cells with ubiquitin - positive GAS . Scale bar , 10 μm .

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D, E Recruitment of ubiquitin in GAS-infected cells. HeLa cells were infected with WT GAS, ΔgacI, and ΔgacI::gacI for 4 h, fixed, and immunostained for ubiquitin (FK2: magenta). Cellular and bacterial DNA were stained with DAPI (cyan). (D) Representative confocal images and (E) percentage of cells with ubiquitin-positive GAS. Scale bar, 10 μm.


G , H Localization of ubiquitin in sWGA - treated GAS . HeLa WT were infected with the indicated GAS strains masked with sWGA for 4 h , fixed , and immunostained for ubiquitin ( magenta ) . ( G ) Representative confocal images and ( H ) percentages of cell with ubiquitin - positive GAS . Scale bar , 10 μm .

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G, H Localization of ubiquitin in sWGA-treated GAS. HeLa WT were infected with the indicated GAS strains masked with sWGA for 4 h, fixed, and immunostained for ubiquitin (magenta). (G) Representative confocal images and (H) percentages of cell with ubiquitin-positive GAS. Scale bar, 10 μm.


I , J Recruitment of p62 to gacI mutants . HeLa cells were infected with WT GAS , ΔgacI , and ΔgacI : : gacI for 4 h , fixed , and immunostained for p62 ( magenta ) . Cellular and bacterial DNA were stained with DAPI ( cyan ) . ( I ) Representative confocal images and ( J ) percentage of cells with p62 - positive GAS . Scale bar , 10 μm .

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I, J Recruitment of p62 to gacI mutants. HeLa cells were infected with WT GAS, ΔgacI, and ΔgacI::gacI for 4 h, fixed, and immunostained for p62 (magenta). Cellular and bacterial DNA were stained with DAPI (cyan). (I) Representative confocal images and (J) percentage of cells with p62-positive GAS. Scale bar, 10 μm.


A , B Co - localization of GFP - FBXO2 and mCherry - LC3 in GAS - infected cells . HeLa cells transfected with the indicated EmGFP - FBXO ( green ) and mCherry - LC3 ( magenta ) were infected with WT GAS for 2 h ; scale bar , 10 μm . Representative confocal images ( A ) and ( B ) percentages of FBXO - positive cells .

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A, B Co-localization of GFP-FBXO2 and mCherry-LC3 in GAS-infected cells. HeLa cells transfected with the indicated EmGFP-FBXO (green) and mCherry-LC3 (magenta) were infected with WT GAS for 2 h; scale bar, 10 μm. Representative confocal images (A) and (B) percentages of FBXO-positive cells.


E , F Localization of EmGFP - FBXO2 in GAS - infected cells . HeLa cells transfected with GFP - FBXO2 ( green ) were infected with the indicated GAS strains for 2 or 4 h at an MOI of 100 ; scale bar , 10 μm . Representative confocal images at 4 h of WT GAS , ΔgacI , and ΔgacI : : gacI ( E ) and ( F ) percentages of FBXO2 ‐ positive cells .

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E, F Localization of EmGFP-FBXO2 in GAS-infected cells. HeLa cells transfected with GFP-FBXO2 (green) were infected with the indicated GAS strains for 2 or 4 h at an MOI of 100; scale bar, 10 μm. Representative confocal images at 4 h of WT GAS, ΔgacI, and ΔgacI::gacI (E) and (F) percentages of FBXO2‐positive cells.


B , HeLa cells transfected the indicated GFP - FBXO2 mutants ( green ) were infected with WT GAS for 4 h ; scale bar , 10 μm . Representative confocal images ( B )

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B, HeLa cells transfected the indicated GFP-FBXO2 mutants (green) were infected with WT GAS for 4 h; scale bar, 10 μm. Representative confocal images (B)


E , F Recruitment of FBXO2 to intracellular GAS in SKP1 / CUL1 / ROC1 knockdown cells . HeLa cells transfected with GFP - FBXO2 ( green ) and the indicated siRNAs were infected with GAS , fixed at 4 h ; scale bar , 10 μm . Representative confocal images ( E ) and percentages of FBXO2 localized to GAS ( F ) .

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E, F Recruitment of FBXO2 to intracellular GAS in SKP1/CUL1/ROC1 knockdown cells. HeLa cells transfected with GFP-FBXO2 (green) and the indicated siRNAs were infected with GAS, fixed at 4 h; scale bar, 10 μm. Representative confocal images (E) and percentages of FBXO2 localized to GAS (F).


D HeLa WT or FBXO2 - KO cells transfected with mCherry - Gal3 ( magenta ) were infected with WT GAS for 2 or 4 h , and percentages of Gal3 - positive GAS - containing cells were quantified .

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D HeLa WT or FBXO2-KO cells transfected with mCherry-Gal3 (magenta) were infected with WT GAS for 2 or 4 h, and percentages of Gal3-positive GAS-containing cells were quantified.


E - J Localization of ubiquitin , LC3 and p62 in FBXO2 KO cells . HeLa WT or FBXO2 - KO cells were infected with WT GAS for 4 h , fixed , and immunostained for ubiquitin ( FK2 : magenta ) or LC3 ( magenta ) or p62 ( magenta ) . Representative confocal images ( E , G , and I ) and percentages of cells with ubiquitin , LC3 , or p62 - positive GAS ( F , H , and J ) .

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E - J Localization of ubiquitin, LC3 and p62 in FBXO2 KO cells. HeLa WT or FBXO2-KO cells were infected with WT GAS for 4 h, fixed, and immunostained for ubiquitin (FK2: magenta) or LC3 (magenta) or p62 (magenta). Representative confocal images (E, G, and I) and percentages of cells with ubiquitin, LC3, or p62 -positive GAS (F, H, and J).


M , N GlcNAc recognition of FBXO2 required for GAS xenophagy . Non - transfected HeLa WT cells and HeLa FBXO2 - KO cells transfected GFP , GFP - FBXO2 , GFP - FBXO2 YW / AA were infected with GAS WT for 4 h , fixed , and immunostained for ubiquitin . Percentages of cells with LC3 ( M ) or ubiquitin ( N ) - positive GAS were quantified .

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M, N GlcNAc recognition of FBXO2 required for GAS xenophagy. Non-transfected HeLa WT cells and HeLa FBXO2-KO cells transfected GFP, GFP-FBXO2, GFP-FBXO2 YW/AA were infected with GAS WT for 4 h, fixed, and immunostained for ubiquitin. Percentages of cells with LC3 (M) or ubiquitin (N) -positive GAS were quantified.


A - C Recruitment of LC3 and ubiquitin to intracellular GAS in SKP1 / CUL1 / ROC1 knockdown cells . HeLa cells transfected with mCherry - LC3 ( magenta ) and the indicated siRNAs were infected with GAS for 4 h . Cells were immunostained for ubiquitin ( FK2 ) ; scale bar , 10 μm . Representative confocal images ( A ) and percentages of cells with ubiquitin ( B ) and LC3 ( C ) - positive GAS .

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A-C Recruitment of LC3 and ubiquitin to intracellular GAS in SKP1/CUL1/ROC1 knockdown cells. HeLa cells transfected with mCherry-LC3 (magenta) and the indicated siRNAs were infected with GAS for 4 h. Cells were immunostained for ubiquitin (FK2); scale bar, 10 μm. Representative confocal images (A) and percentages of cells with ubiquitin (B) and LC3 (C) -positive GAS.


D , E Neddylated cullin inhibitor : MLN4924 - treated HeLa cells were infected with WT GAS , and fixed at 4 h . Cells were immunostained for ubiquitin ( FK2 : magenta ) or LC3 ( magenta ) ; scale bar , 10 μm . Representative images and quantification of cells with ubiquitin - positive GAS ( D ) and LC3 - positive GAS ( E ) .

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D, E Neddylated cullin inhibitor: MLN4924-treated HeLa cells were infected with WT GAS, and fixed at 4 h. Cells were immunostained for ubiquitin (FK2: magenta) or LC3 (magenta); scale bar, 10 μm. Representative images and quantification of cells with ubiquitin-positive GAS (D) and LC3-positive GAS (E).


F Lysates from HeLa cells infected with GAS were immunoprecipitated with an anti - lipoteichoic acid antibody and Protein G - Magnetic Beads . Immunoprecipitates were analyzed by immunoblotting .

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F Lysates from HeLa cells infected with GAS were immunoprecipitated with an anti-lipoteichoic acid antibody and Protein G-Magnetic Beads. Immunoprecipitates were analyzed by immunoblotting.


( A ) The graphs depict the Aβ profiles generated by WT or mutant PSEN1 MEF cell lines transduced with adenovirus encoding APPC99 . ELISA quantified secreted Aβ37 , Aβ38 , Aβ40 and Aβ42 peptide levels are shown as % of total Aβ ( sum of the four peptides ) . The blue , orange , green and purple dotted lines indicate the levels of Aβ37 , Aβ38 , Aβ40 and Aβ42 peptides in the WT Aβ profile , respectively . Data are presented as mean ± SD , N ≥ 3 independent experiments .

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(A) The graphs depict the Aβ profiles generated by WT or mutant PSEN1 MEF cell lines transduced with adenovirus encoding APPC99. ELISA quantified secreted Aβ37, Aβ38, Aβ40 and Aβ42 peptide levels are shown as % of total Aβ (sum of the four peptides). The blue, orange, green and purple dotted lines indicate the levels of Aβ37, Aβ38, Aβ40 and Aβ42 peptides in the WT Aβ profile, respectively. Data are presented as mean ± SD, N ≥ 3 independent experiments.


( B ) GSEC processivity levels for the cleavage of APPC99 determined in WT and mutant PSEN1 / GSEC cell lines . The mutant Aβ ( 37 + 38 ) / ( 40 + 42 ) ratios are shown as % of the WT cell line . Data presented as mean ± SD , N ≥ 3 independent experiments . As reference , a dotted line was drawn to indicate 10 % ( WT levels ) . One - way ANOVA followed by Dunnett ' s post - hoc test with comparison to WT was used to determine statistical significance ( P < 0 . 05 ) ; * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 .

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(B) GSEC processivity levels for the cleavage of APPC99 determined in WT and mutant PSEN1/GSEC cell lines. The mutant Aβ(37+38)/(40+42) ratios are shown as % of the WT cell line. Data presented as mean ± SD, N ≥ 3 independent experiments. As reference, a dotted line was drawn to indicate 10% (WT levels). One-way ANOVA followed by Dunnett's post-hoc test with comparison to WT was used to determine statistical significance (P < 0.05); **P < 0.01, ***P < 0.001, ****P < 0.0001.


( A ) Aβ profiles generated by WT or mutant PSEN1 MEFs lines treated with 1 µM GSM III . The chemical structure of the bicyclic heterocycle GSM III is depicted . Data are presented as mean ± SD , N ≥ 3 independent experiments . The blue , orange , green and purple dotted lines indicate the levels of Aβ37 , Aβ38 , Aβ40 and Aβ42 peptides in the WT Aβ profile , respectively .

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(A) Aβ profiles generated by WT or mutant PSEN1 MEFs lines treated with 1 µM GSM III. The chemical structure of the bicyclic heterocycle GSM III is depicted. Data are presented as mean ± SD, N ≥ 3 independent experiments. The blue, orange, green and purple dotted lines indicate the levels of Aβ37, Aβ38, Aβ40 and Aβ42 peptides in the WT Aβ profile, respectively.


( B ) Fold change in GSEC processivity of APPC99 estimated by the Aβ ( 37 + 38 ) / ( 40 + 42 ) ratio and presented as % of the WT response . The data are presented as mean ± SD , N ≥ 3 . The symbol # marks mutations that fully abolished the GSEC response towards GSM III , As reference , a dotted line was drawn to indicate 10 % ( WT levels ) . One - way ANOVA followed by Dunnett ' s post - hoc test with comparison to WT was used to determine statistical significance ( P < 0 . 05 ) . * * P < 0 . 0001 , * * * * P < 0 . 0001 ( F ( DFn , DFd ) : F ( 32 , 101 ) = 112 . 5 .

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(B) Fold change in GSEC processivity of APPC99 estimated by the Aβ(37+38)/(40+42) ratio and presented as % of the WT response. The data are presented as mean ± SD, N ≥ 3. The symbol # marks mutations that fully abolished the GSEC response towards GSM III, As reference, a dotted line was drawn to indicate 10% (WT levels). One-way ANOVA followed by Dunnett's post-hoc test with comparison to WT was used to determine statistical significance (P < 0.05). **P < 0.0001, ****P < 0.0001 (F(DFn, DFd): F (32, 101) = 112.5.


( D ) Aβ profiles generated by WT or mutant PSEN1 cell lines overexpressing APPC99 and treated with increasing concentrations of GSM III . Data are presented as mean ± SD , N = 2 independent experiments .

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(D) Aβ profiles generated by WT or mutant PSEN1 cell lines overexpressing APPC99 and treated with increasing concentrations of GSM III. Data are presented as mean ± SD, N = 2 independent experiments.


( E ) GSEC processivity estimated by the Aβ ( 37 + 38 ) / ( 40 + 42 ) ratio was determined for the dose - response analysis ( presented in panel D ) and shown as % of DMSO . Data are presented as mean ± SD .

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(E) GSEC processivity estimated by the Aβ(37+38)/(40+42) ratio was determined for the dose-response analysis (presented in panel D) and shown as % of DMSO. Data are presented as mean ± SD.


( A ) Aβ profiles generated by WT and indicated mutant MEF lines in the presence of vehicle ( 0 . 1 % DMSO ) , 1 µM GSM III Data are presented as mean ± SD , N ≥ 3 independent experiments . One - way ANOVA followed by Dunnett ' s post - hoc test with comparison to WT was used to determine statistical significance ( P < 0 . 05 ) . * P < 0 . 01 , * * P < 0 . 0001 , * * * P < 0 . 001 , * * * * P < 0 . 0001 ( F ( DFn , DFd ) : F ( 3 , 45 ) = 56 . 9 .

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(A) Aβ profiles generated by WT and indicated mutant MEF lines in the presence of vehicle (0.1% DMSO), 1 µM GSM III Data are presented as mean ± SD, N ≥ 3 independent experiments. One-way ANOVA followed by Dunnett's post-hoc test with comparison to WT was used to determine statistical significance (P < 0.05). *P < 0.01, **P < 0.0001, ***P < 0.001, ****P < 0.0001 (F(DFn, DFd): F (3, 45) = 56.9.


( B ) WT and mutant GSEC processivity estimated by the Aβ ( 37 + 38 ) / ( 40 + 42 ) ratio and presented as % of WT . Data presented as mean ± SD , N ≥ 3 independent experiments . One - way ANOVA followed by Dunnett ' s post - hoc test with comparison to WT was used to determine statistical significance ( P < 0 . 05 ) . * P < 0 . 01 , * * P < 0 . 0001 , * * * P < 0 . 001 , * * * * P < 0 . 0001 ( F ( DFn , DFd ) : F ( 3 , 45 ) = 56 . 9 .

(B) WT and mutant GSEC processivity estimated by the Aβ (37+38)/(40+42) ratio and presented as % of WT. Data presented as mean ± SD, N ≥ 3 independent experiments. One-way ANOVA followed by Dunnett's post-hoc test with comparison to WT was used to determine statistical significance (P < 0.05). *P < 0.01, **P < 0.0001, ***P < 0.001, ****P < 0.0001 (F(DFn, DFd): F (3, 45) = 56.9.


Aβ profiles generated by WT and indicated mutant MEF lines in the presence of vehicle ( 0 . 1 % DMSO ) , 1 µM GSM III or ( C ) 1 µM GSM II . Data are presented as mean ± SD , N ≥ 3 independent experiments . One - way ANOVA followed by Dunnett ' s post - hoc test with comparison to WT was used to determine statistical significance ( P < 0 . 05 ) . * P < 0 . 01 , * * P < 0 . 0001 , * * * P < 0 . 001 , * * * * P < 0 . 0001 ( F ( DFn , DFd ) : F ( 3 , 45 ) = 56 . 9 .

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Aβ profiles generated by WT and indicated mutant MEF lines in the presence of vehicle (0.1% DMSO), 1 µM GSM III or (C) 1 µM GSM II. Data are presented as mean ± SD, N ≥ 3 independent experiments. One-way ANOVA followed by Dunnett's post-hoc test with comparison to WT was used to determine statistical significance (P < 0.05). *P < 0.01, **P < 0.0001, ***P < 0.001, ****P < 0.0001 (F(DFn, DFd): F (3, 45) = 56.9.


Analysis of mutant APP substrates with regards to their effects on the response of GSEC to the imidazole - based GSM III . Aβ profiles secreted by HEK293T cells transiently expressing WT or mutant APPC99 substrates in presence of vehicle ( DMSO ) or 1 µM GSM III were analysed by ELISA ( D ) ELISA - based Aβ profiles are presented as % of total Aβ ( Aβ37 + Aβ38 + Aβ40 + Aβ42 ) ( mean ± SD , N ≥ 3 ) ; One - way ANOVA followed by Dunnett ' s post - hoc test with comparison to WT was used to determine statistical significance ; * * * * P < 0 . 0001 . F ( DFn , DFd ) : F ( 4 , 17 ) = 71 . 68 .

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Analysis of mutant APP substrates with regards to their effects on the response of GSEC to the imidazole-based GSM III. Aβ profiles secreted by HEK293T cells transiently expressing WT or mutant APPC99 substrates in presence of vehicle (DMSO) or 1 µM GSM III were analysed by ELISA (D) ELISA-based Aβ profiles are presented as % of total Aβ (Aβ37+ Aβ38+ Aβ40+ Aβ42) (mean ± SD, N ≥ 3); One-way ANOVA followed by Dunnett's post-hoc test with comparison to WT was used to determine statistical significance; ****P < 0.0001. F(DFn, DFd): F (4, 17) = 71.68.


Analysis of mutant APP substrates with regards to their effects on the response of GSEC to the imidazole - based GSM III . Aβ profiles secreted by HEK293T cells transiently expressing WT or mutant APPC99 substrates in presence of vehicle ( DMSO ) or 1 µM GSM III were analysed by mass spectrometry ( MS ) . ( E ) APP mutation - driven effects on GSEC processivity according to the Aβ ( 37 + 38 ) / ( 40 + 42 ) ratio are presented as % of the WT condition ( mean ± SD ) . One - way ANOVA followed by Dunnett ' s post - hoc test with comparison to WT was used to determine statistical significance ; * * * * P < 0 . 0001 . F ( DFn , DFd ) : F ( 4 , 17 ) = 71 . 68 .

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Analysis of mutant APP substrates with regards to their effects on the response of GSEC to the imidazole-based GSM III. Aβ profiles secreted by HEK293T cells transiently expressing WT or mutant APPC99 substrates in presence of vehicle (DMSO) or 1 µM GSM III were analysed by mass spectrometry (MS). (E) APP mutation-driven effects on GSEC processivity according to the Aβ(37+38)/(40+42) ratio are presented as % of the WT condition (mean ± SD). One-way ANOVA followed by Dunnett's post-hoc test with comparison to WT was used to determine statistical significance; ****P < 0.0001. F(DFn, DFd): F (4, 17) = 71.68.


( F ) MS - based profiles show the proportion of individual Aβ peptides on the total immunoprecipitated pool . Data shown as mean of 3 independent experiments .

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(F) MS-based profiles show the proportion of individual Aβ peptides on the total immunoprecipitated pool. Data shown as mean of 3 independent experiments.


( B ) Aβ profiles generated by WT or mutant PSEN1 cell lines transduced with APPC99 . Dotted lines indicate the levels of Aβ peptides ( % ) in WT profiles . Data presented as mean ± SD , N ≥ 3 independent experiments .

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(B) Aβ profiles generated by WT or mutant PSEN1 cell lines transduced with APPC99. Dotted lines indicate the levels of Aβ peptides (%) in WT profiles. Data presented as mean ± SD, N ≥ 3 independent experiments.


( C ) Pocket filling PSEN1 V236W and Y106W - V236W substitutions lead to increased Aβ ( 37 + 38 ) / ( 40 + 42 ) ratio , indicating the activation of the sequential GSEC - mediated cleavage of APPC99 . Data presented as mean ± SD , N ≥ 3 independent experiments .

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(C) Pocket filling PSEN1 V236W and Y106W-V236W substitutions lead to increased Aβ(37+38)/(40+42) ratio, indicating the activation of the sequential GSEC-mediated cleavage of APPC99. Data presented as mean ± SD, N ≥ 3 independent experiments.


( D ) Aβ profile analysis in dose - response experiments for GSM III revealed no modulation of the PSEN1 V236W mutant cell line . Data are presented as mean ± SD , N = 2 independent experiments . For comparison purposes , WT and V236W mutant Aβ profiles are presented in panel 6B .

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(D) Aβ profile analysis in dose-response experiments for GSM III revealed no modulation of the PSEN1 V236W mutant cell line. Data are presented as mean ± SD, N = 2 independent experiments. For comparison purposes, WT and V236W mutant Aβ profiles are presented in panel 6B.


( E ) GSEC activity assay using purified GSEC and an APP - based fluorogenic peptide as substrate Upper panel : In the non - cleaved state the Dpn ( 2 , 4 - dinitrophenyl ) quencher group suppresses fluorescent emission from the Nma ( N - methyl - o - aminobenzoic acid ) fluorophore through Förster resonance energy transfer ( FRET ) . Upon GSEC cleavage , the Nma group emits at λ = 430 nm a fluorescent signal upon excitation at λ = 355 nm . Lower panel : Specific activities for the purified WT or mutant GSECs ( bearing the indicated substitutions in PSEN1 ) treated with DMSO ( vehicle ) , 10 µM GSMIII or GSM II . Data are normalized to the WT GSEC activity in DMSO ( vehicle ) . Data are shown as mean ± SD , N ≥ 3 independent experiments . Significance is shown for meaningful comparisons ; two - way ANOVA with comparison to WT was used to determine statistical significance ; * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 , ns : not significant .

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(E) GSEC activity assay using purified GSEC and an APP-based fluorogenic peptide as substrate Upper panel: In the non-cleaved state the Dpn (2,4-dinitrophenyl) quencher group suppresses fluorescent emission from the Nma (N-methyl-o-aminobenzoic acid) fluorophore through Förster resonance energy transfer (FRET). Upon GSEC cleavage, the Nma group emits at λ = 430 nm a fluorescent signal upon excitation at λ = 355 nm. Lower panel: Specific activities for the purified WT or mutant GSECs (bearing the indicated substitutions in PSEN1) treated with DMSO (vehicle), 10 µM GSMIII or GSM II. Data are normalized to the WT GSEC activity in DMSO (vehicle). Data are shown as mean ± SD, N ≥ 3 independent experiments. Significance is shown for meaningful comparisons; two-way ANOVA with comparison to WT was used to determine statistical significance; **P < 0.01, ***P < 0.001, ****P < 0.0001, ns: not significant.


( F ) Transition state analogue inhibitor L - 685 , 458 ( Inhibitor X ) treatment of WT , V236W or Y240W mutant PSEN1 cell lines . Inhibitory profiles and derived IC50 values ( table ) indicate that the PSEN1 - V236W significantly increases the affinity for the inhibitor . Dotted line indicates 50 % inhibition . Data presented as mean ± SD , N = 3 independent experiments .

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(F) Transition state analogue inhibitor L-685,458 (Inhibitor X) treatment of WT, V236W or Y240W mutant PSEN1 cell lines. Inhibitory profiles and derived IC50 values (table) indicate that the PSEN1-V236W significantly increases the affinity for the inhibitor. Dotted line indicates 50% inhibition. Data presented as mean ± SD, N = 3 independent experiments.


( H ) Ang - 2 dose and time - dependently activates p - EphrinB , p - TIE2 and p - AKT in HUVEC ; immunoblotting results ; NA3VO4 : sodium orthovanadate .

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(H) Ang-2 dose and time-dependently activates p-EphrinB, p-TIE2 and p-AKT in HUVEC; immunoblotting results; NA3VO4: sodium orthovanadate.


( I ) Tumor vessels in B16F10 tumors are mostly p - TIE2 ( Tyr992 ) + and pSHP2 ( Tyr542 ) + ; confocal images ( scale bars : 200μm ) .

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(I) Tumor vessels in B16F10 tumors are mostly p-TIE2 (Tyr992)+ and pSHP2(Tyr542)+; confocal images (scale bars: 200μm).


( A ) Representative tissue immunostaining of human melanoma , colon carcinoma and colon adenoma ; confocal images from serial sections ( scale bars : 50μm tissues ) . Arrowheads point to CD31 + / p - EphrinB + ; CD31 + / p - SHP2 + ; CD31 + / p - Tie2 + and CD31 + / Ang2 + endothelial cells .

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(A) Representative tissue immunostaining of human melanoma, colon carcinoma and colon adenoma; confocal images from serial sections (scale bars: 50μm tissues). Arrowheads point to CD31+/p-EphrinB+; CD31+/p-SHP2+; CD31+/p-Tie2+ and CD31+/Ang2+ endothelial cells.


( B ) Gene expression in tumor ( black bar ) and control ( white bar ) ; top panel : melanoma ( GSE7553 , primary and metastatic ; tumor n = 54 ; control skin n = 5 ) ; bottom panel : colon cancer ( TCGA , primary and metastatic ; tumor n = 269 ; control n = 41 . * * P < 0 . 01 , * * * * P < 0 . 0001 ; P values from Mann Whitney U - test .

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(B) Gene expression in tumor (black bar) and control (white bar); top panel: melanoma (GSE7553, primary and metastatic; tumor n=54; control skin n=5); bottom panel: colon cancer (TCGA, primary and metastatic; tumor n=269; control n=41. **P<0.01, ****P<0.0001; P values from Mann Whitney U-test.


( C ) Impact of high and low ( 75 % quartile ) mRNA levels on the survival probability of patients with primary melanoma ; Kaplan - Meier analysis using TCGA primary melanoma datasets ( n = 103 ) ; P values from Mann Whitney U - test . ( D ) Impact of high and low ( median ) mRNA levels on the survival probability of patients with colon cancer ; Kaplan - Meier analysis using TCGA colon cancer dataset ( n = 262 ) .

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(C) Impact of high and low (75% quartile) mRNA levels on the survival probability of patients with primary melanoma; Kaplan-Meier analysis using TCGA primary melanoma datasets (n=103); P values from Mann Whitney U-test. (D) Impact of high and low (median) mRNA levels on the survival probability of patients with colon cancer; Kaplan-Meier analysis using TCGA colon cancer dataset (n=262).


( C , D ) The JAK inhibitor Tofacitinib ( Tofa , 50nM ) enhances the proliferation of HUVEC and BMEC compared to control ( C ) and reduces endogenous p - STAT3 levels in HUVEC and BMEC compared to control ( D ) after 72 - hour culture . SHP099 ( 5μM HUVEC ; 20μM BMEC ) reduces cell proliferation and increases p - STAT3 in HUVEC and BMEC compared to control ; error bars : ± S . D . Results of immunoblotting are representative of 3 independent blots .

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(C,D) The JAK inhibitor Tofacitinib (Tofa, 50nM) enhances the proliferation of HUVEC and BMEC compared to control (C) and reduces endogenous p-STAT3 levels in HUVEC and BMEC compared to control (D) after 72-hour culture. SHP099 (5μM HUVEC; 20μM BMEC) reduces cell proliferation and increases p-STAT3 in HUVEC and BMEC compared to control; error bars: ± S.D. Results of immunoblotting are representative of 3 independent blots.


( E , F ) The MAP kinases inhibitor PD098059 ( PD0980 , 10μM ) and SHP099 ( 5μM HUVEC ; 20μM BMEC ) inhibit HUVEC and BMEC proliferation compared to control ( E ) and reduce p - ERK1 / 2 levels by immunoblotting after 72 - hour culture ( F ) . Results of proliferation in panels C and E ( % of control ) from triplicate cultures are representative of 3 - 4 experiments ; * P < 0 . 05 , * * P < 0 . 01 * * * P < 0 . 001 ; two - tailed Student ' s t - test ; error bars : ± S . D . Results of immunoblotting are representative of 3 independent blots .

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(E,F) The MAP kinases inhibitor PD098059 (PD0980, 10μM) and SHP099 (5μM HUVEC; 20μM BMEC) inhibit HUVEC and BMEC proliferation compared to control (E) and reduce p-ERK1/2 levels by immunoblotting after 72-hour culture (F). Results of proliferation in panels C and E (% of control) from triplicate cultures are representative of 3-4 experiments; *P<0.05, **P<0.01 ***P<0.001; two-tailed Student's t-test; error bars: ± S.D. Results of immunoblotting are representative of 3 independent blots.


( G , H ) The ERK1 / 2 inhibitors AZD6244 and Tamnitinib dose - dependently reduce B16F10 cell proliferation ( G ) ; representative experiment of 3 independent experiments ; the results reflect the means of 5 replicate / condition ; error bars : ± S . D . ; and levels of endogenous p - STAT3 and p - ERK in B16F10 cells ( representative of 3 blots ) ( H ) .

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(G,H) The ERK1/2 inhibitors AZD6244 and Tamnitinib dose-dependently reduce B16F10 cell proliferation (G); representative experiment of 3 independent experiments; the results reflect the means of 5 replicate/condition; error bars: ± S.D.; and levels of endogenous p-STAT3 and p-ERK in B16F10 cells (representative of 3 blots) (H).


( A - C ) SHP099 ( A , 75mg / kg ; B , C : 100mg / kg ) reduces B16F10 tumor growth in syngeneic mice . Panels depict tumor growth from initiation of treatment to endpoint ( left ) and tumor weight ( right ) at endpoint ; no . mice / group : 6 - 11 control ; 7 - 9 SHP099 .

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(A-C) SHP099 (A, 75mg/kg; B, C: 100mg/kg) reduces B16F10 tumor growth in syngeneic mice. Panels depict tumor growth from initiation of treatment to endpoint (left) and tumor weight (right) at endpoint; no. mice/group: 6-11 control; 7-9 SHP099.


( E ) " Vascular sleeves " ( Collagen IV + CD31 - ) identified in a representative SHP099 - treated tumor . Representative confocal images from 6 tumors ( scale bar : 50μm ) . ( F ) Reduced CD31 + vascular area in SHP099 - treated tumors compared to controls ( n = 6 / group ) ; quantification by ImageJ . ( G ) The ratio of Collagen IV + / CD31 + area is increased in SHP099 - treated tumors compared to controls ( n = 6 / group ) ; quantification by ImageJ ; Coll : collagen . Data information : Error bars : ± S . D . ; P values from two - tailed Student ' s t - test ; * P < 0 . 05 , * * P < 0 . 01 and * * * P < 0 . 001 .

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(E) "Vascular sleeves" (Collagen IV+ CD31-) identified in a representative SHP099-treated tumor. Representative confocal images from 6 tumors (scale bar: 50μm). (F) Reduced CD31+ vascular area in SHP099-treated tumors compared to controls (n=6/group); quantification by ImageJ. (G) The ratio of Collagen IV+/CD31+ area is increased in SHP099-treated tumors compared to controls (n=6/group); quantification by ImageJ; Coll: collagen. Data information: Error bars: ± S.D.; P values from two-tailed Student's t-test; *P<0.05, **P<0.01 and ***P<0.001.


( H ) Cleaved caspase - 3 detection in a CD31 + vessel ( representative of 6 SHP099 - treated tumors ) ; arrowheads point to cleaved caspase - 3 + CD31 + cells . Cleaved caspase - 3 + tumor cell is marked by * . Confocal images ( scale bar : 10μm ) . ( I ) Increased cleaved caspase - 3 fluorescence intensity in CD31 + cells from SHP099 - treated tumors compared to controls ( n = 3 / group ) ; at least 100 cells counted / sample . ( J ) Ki67 + proliferating cells in representative of 3 SHP099 - treated and 3 control tumors ; CD31 identifies the vessels ; DAPI identifies cell nuclei . Confocal images ( scale bar : 50μm ) . ( K ) Quantification of proliferating cells within SHP099 and control ( n = 4 / group ) tumors by ImageJ . Data information : Error bars : ± S . D . ; P values from two - tailed Student ' s t - test ; * P < 0 . 05 , * * P < 0 . 01 and * * * P < 0 . 001 .

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(H) Cleaved caspase-3 detection in a CD31+ vessel (representative of 6 SHP099-treated tumors); arrowheads point to cleaved caspase-3+CD31+ cells. Cleaved caspase-3+ tumor cell is marked by *. Confocal images (scale bar: 10μm). (I) Increased cleaved caspase-3 fluorescence intensity in CD31+ cells from SHP099-treated tumors compared to controls (n=3/group); at least 100 cells counted/sample. (J) Ki67+ proliferating cells in representative of 3 SHP099-treated and 3 control tumors; CD31 identifies the vessels; DAPI identifies cell nuclei. Confocal images (scale bar: 50μm). (K) Quantification of proliferating cells within SHP099 and control (n=4/group) tumors by ImageJ. Data information: Error bars: ± S.D.; P values from two-tailed Student's t-test; *P<0.05, **P<0.01 and ***P<0.001.


( L ) Hypoxic tumor identified Hypoxyprobe ( green ) representative of 3 SHP099 - treated tumors ; confocal images ( scale bar : 100μm ) , left ; quantification of hypoxic areas in control ( n = 3 ) and SHP099 - treated ( n = 3 ) tumors by ImageJ . Data information : Error bars : ± S . D . ; P values from two - tailed Student ' s t - test ; * P < 0 . 05 , * * P < 0 . 01 and * * * P < 0 . 001 .

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(L) Hypoxic tumor identified Hypoxyprobe (green) representative of 3 SHP099-treated tumors; confocal images (scale bar: 100μm), left; quantification of hypoxic areas in control (n=3) and SHP099-treated (n=3) tumors by ImageJ. Data information: Error bars: ± S.D.; P values from two-tailed Student's t-test; *P<0.05, **P<0.01 and ***P<0.001.


( M ) Representative control ( of 5 ) and SHP099 - treated ( of 5 ) B16F10 tumor sections through the maximum diameter ; H & E staining ( scale bar : 10mm ) . ( N ) Cleaved caspase - 3 + fluorescence intensity in SHP099 and control tumors ( n = 3 / group ) . Data information : Error bars : ± S . D . ; P values from two - tailed Student ' s t - test ; * P < 0 . 05 , * * P < 0 . 01 and * * * P < 0 . 001 .

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(M) Representative control (of 5) and SHP099-treated (of 5) B16F10 tumor sections through the maximum diameter; H&E staining (scale bar: 10mm). (N) Cleaved caspase-3+ fluorescence intensity in SHP099 and control tumors (n=3/group). Data information: Error bars: ± S.D.; P values from two-tailed Student's t-test; *P<0.05, **P<0.01 and ***P<0.001.


( B ) Extravasation of FITC - dextran ( green ) in tumors established in NSG mice treated with SHP099 ; scale bar ( 50μm ) . Representative images from 3 mice / group . ( C ) Reduced CD31 + vascular area in SHP099 - treated tumors compared to controls ( n = 5 / group ) ; ImageJ quantification . P values from two - tailed Student ' s t - test ; * P < 0 . 05 . Error bars : ± S . D .

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(B) Extravasation of FITC-dextran (green) in tumors established in NSG mice treated with SHP099; scale bar (50μm). Representative images from 3 mice/group. (C) Reduced CD31+ vascular area in SHP099-treated tumors compared to controls (n=5/group); ImageJ quantification. P values from two-tailed Student's t-test; *P<0.05. Error bars: ± S.D.


E ) SHP099 reduces weight of control ( p - LKO vector ) and SHP2 - depleted ( shSHP2 ) B16F10 tumors compared to untreated controls : p - LKO untreated ( n = 5 ) , p - LKO treated with SHP099 ( n = 9 ) , shSHP2 untreated ( n = 5 ) , and shSHP2 treated with SHP099 ( n = 13 ) . P values by analysis of variance with Dunnett ' s multiple comparison test ; * * * P < 0 . 001 . ( F ) Persistent depletion of SHP2 in tumors induced by shSHP2 - B16F10 cells ( n = 5 ) compared to control tumors induced by p - LKO - B16F10 ( n = 5 ) ; tumors removed from mice 18 days after cell inoculation .

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E) SHP099 reduces weight of control (p-LKO vector) and SHP2-depleted (shSHP2) B16F10 tumors compared to untreated controls: p-LKO untreated (n=5), p-LKO treated with SHP099 (n=9), shSHP2 untreated (n=5), and shSHP2 treated with SHP099 (n=13). P values by analysis of variance with Dunnett's multiple comparison test; ***P<0.001. (F) Persistent depletion of SHP2 in tumors induced by shSHP2-B16F10 cells (n=5) compared to control tumors induced by p-LKO-B16F10 (n=5); tumors removed from mice 18 days after cell inoculation.


( N ) Representative popliteal lymph nodes from control and SHP099 - treated mice with microscopic evidence of metastases ; H & E stain ; scale bars 500 μm ( low magnification ) 20 μm ( high magnification ) .

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(N) Representative popliteal lymph nodes from control and SHP099-treated mice with microscopic evidence of metastases; H&E stain; scale bars 500 μm (low magnification) 20 μm (high magnification).


( C , D ) p - TIE2 in tumor vessels from control and AMG386 - treated mice ; representative confocal images ; arrowheads point to CD31 + / p - TIE2 - endothelial cells ( scale bars : 50μm ) ( C ) , and quantification ( D ) of p - TIE - 2 / CD31 + in control ( n = 3 ) and AMG386 treated ( n = 3 ) tumors ( 1 , 200 CD31 + cells / group ) . P values from two - tailed Student ' s t - test ; * * * P < 0 . 001 . ( E , F ) p - EphrinB ( E ) in representative tumor vessels from control ( n = 3 ) and AMG386 - treated ( n = 3 ) tumors ; confocal images ( E ) , ( scale bar : 50μm ) and quantification of p - EphrinB / CD31 + ( F ) ; P values from two - tailed Student ' s t - test ; * * * P < 0 . 001 .

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(C,D) p-TIE2 in tumor vessels from control and AMG386-treated mice; representative confocal images; arrowheads point to CD31+/p-TIE2- endothelial cells (scale bars: 50μm) (C), and quantification (D) of p-TIE-2/CD31+ in control (n=3) and AMG386 treated (n=3) tumors (1,200 CD31+ cells/group). P values from two-tailed Student's t-test; ***P<0.001. (E,F) p-EphrinB (E) in representative tumor vessels from control (n=3) and AMG386-treated (n=3) tumors; confocal images (E), (scale bar: 50μm) and quantification of p-EphrinB/CD31+ (F); P values from two-tailed Student's t-test; ***P<0.001.


( G ) Vessel perfusion visualized by FITC - dextran ( green ) ; CD31 ( red ) identifies the endothelium ; DAPI ( blue ) identifies the nuclei ; representative tumor confocal images ( scale bar : 50μm ) of 3 mice / group ; arrow heads point to FITC - dextran extravasation ; asterisks to extravasated erythrocytes .

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(G) Vessel perfusion visualized by FITC-dextran (green); CD31 (red) identifies the endothelium; DAPI (blue) identifies the nuclei; representative tumor confocal images (scale bar:50μm) of 3 mice/group; arrow heads point to FITC-dextran extravasation; asterisks to extravasated erythrocytes.


( H ) Quantification of cleaved caspase - 3 in the tumor vasculature ( CD31 + cells ) ; n = 3 tumors / group ; number of CD31 + cells counted : control ( n = 311 ) ; SHP099 ( n = 425 ) ; AMG386 ( n = 451 ) ; combined SHP099 + AMG386 ( n = 484 ) . Error bars : ± S . D . ; P values by analysis of variance with Dunnett ' s multiple comparison test ; * * P < 0 . 01 , * * * P < 0 . 001 .

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(H) Quantification of cleaved caspase-3 in the tumor vasculature (CD31+ cells); n=3 tumors/group; number of CD31+ cells counted: control (n=311); SHP099 (n=425); AMG386 (n=451); combined SHP099+AMG386 (n=484). Error bars: ± S.D.; P values by analysis of variance with Dunnett's multiple comparison test; **P<0.01, ***P<0.001.


( A - D ) Nuclear p - STAT3 ( A , B ) and p - ERK1 / 2 ( C , D ) in tumor CD31 + endothelial cells of mice treated for 14 days with SHP099 ( 200 mg / kg ) , AMG386 ( 5 . 6mg / kg ) or the combination of SHP099 ( 200 mg / kg ) + AMG386 ( 5 . 6mg / kg ) . Representative ( 3 tumors / group ) immunostaining ( A , C ) and quantification of staining intensity / CD31 + cell ( B , D ) ; horizontal line : mean intensity / group ; n = 3 tumors / group ; number of CD31 + cells counted in B : control ( n = 180 ) , SHP099 ( n = 308 ) , AMG386 ( n = 300 ) , combined SHP099 + AMG386 ( n = 322 ) ; number of CD31 + cells counted in D : control ( n = 1 , 733 ) , SHP099 ( n = 821 ) , AMG386 ( n = 1 , 399 ) , combined SHP099 + AMG386 ( n = 783 ) . Scale bars : A , 50μm ; C , 10μm .

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(A-D) Nuclear p-STAT3 (A,B) and p-ERK1/2 (C,D) in tumor CD31+ endothelial cells of mice treated for 14 days with SHP099 (200 mg/kg), AMG386 (5.6mg/kg) or the combination of SHP099 (200 mg/kg) + AMG386 (5.6mg/kg). Representative (3 tumors/group) immunostaining (A,C) and quantification of staining intensity/CD31+ cell (B, D); horizontal line: mean intensity/group; n=3 tumors/group; number of CD31+ cells counted in B: control (n=180), SHP099 (n=308), AMG386 (n=300), combined SHP099+AMG386 (n=322); number of CD31+ cells counted in D: control (n=1,733), SHP099 (n=821), AMG386 (n=1,399), combined SHP099+AMG386 (n=783). Scale bars: A, 50μm; C, 10μm.


FOXO1 detection in tumor CD31 + endothelial cells after treatment with SHP099 ( 200 mg / kg ) , AMG386 ( 5 . 6mg / kg ) or the combination of SHP099 ( 200 mg / kg ) + AMG386 ( 5 . 6mg / kg ) . Representative ( 3 tumors / group ) confocal

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FOXO1 detection in tumor CD31+ endothelial cells after treatment with SHP099 (200 mg/kg), AMG386 (5.6mg/kg) or the combination of SHP099 (200 mg/kg) + AMG386 (5.6mg/kg). Representative (3 tumors/group) confocal images


( C ) Vessel perfusion visualized by FITC - dextran ( green ) ; CD31 identifies endothelial cells ( red ) ; DAPI ( blue ) identifies the nuclei ; representative ( of 3 mice / group ) confocal images ( scale bar : 50μm ) ; arrow heads point to FITC - dextran extravasation .

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(C) Vessel perfusion visualized by FITC-dextran (green); CD31 identifies endothelial cells (red); DAPI (blue) identifies the nuclei; representative (of 3 mice/group) confocal images (scale bar: 50μm); arrow heads point to FITC-dextran extravasation.


( H ) Representative images depicting Ki67 + cells in control and treated MC38 tumors ; representative images ( scale bar : 50μm ) of 4 tumors / group .

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(H) Representative images depicting Ki67+ cells in control and treated MC38 tumors; representative images (scale bar: 50μm) of 4 tumors/group.


( A ) WT and CITK - / - embryonic ( E14 . 5 ) mousecerebral cortex was stained for γ - tubulin ( green ) and DNA ( gray ) . The ventricular plane is marked by a red dashed line , and the spindle axis of apical progenitors is indicated by a white dashed line . The angle between these two lines represents the mitotic angle . Scale bars = 5 μm . ( B ) Quantification of vertical divisions of apical progenitors in WT and CITK - / - mice . n = 3 per each genotype .

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(A) WT and CITK -/- embryonic (E14.5) mousecerebral cortex was stained for γ-tubulin (green) and DNA (gray). The ventricular plane is marked by a red dashed line, and the spindle axis of apical progenitors is indicated by a white dashed line. The angle between these two lines represents the mitotic angle. Scale bars = 5 μm.(B) Quantification of vertical divisions of apical progenitors in WT and CITK -/- mice. n= 3 per each genotype.


( C ) Pregnant CITK + / - females , crossed with CITK + / - males , were injected with BrdU at E13 . 5 ; 24 hours later ( E14 . 5 ) WT and CITK - / - embryonic mouse cerebral cortex were fixed in 4 % PFA , cryosectioned and stained for Ki67 ( red ) , BrdU ( green ) , Tbr2 ( blue ) and DAPI ( gray ) . The different cortical regions are indicated : CP = cortical plate ; IZ = intermediate zone ; SVZ = subventricular zone ; VZ = ventricular zone . Scale bars = 10 μm . White arrows in the inset indicate two Ki67 - cells positive for BrdU and Tbr2 .

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(C) Pregnant CITK +/- females, crossed with CITK +/- males, were injected with BrdU at E13.5; 24 hours later (E14.5) WT and CITK -/- embryonic mouse cerebral cortex were fixed in 4% PFA, cryosectioned and stained for Ki67 (red), BrdU (green), Tbr2 (blue) and DAPI (gray). The different cortical regions are indicated: CP=cortical plate; IZ= intermediate zone; SVZ= subventricular zone; VZ= ventricular zone. Scale bars = 10 μm. White arrows in the inset indicate two Ki67- cells positive for BrdU and Tbr2.


( D ) Quantification of cell cycle exit ( ratio of cells BrdU + / Ki67 − ) in the proliferative regions ( VZ / SVZ ) and in the neuronal layers ( IZ and CP ) of sections prepared as in panel C ( n = 4 per each genotype ) .

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(D) Quantification of cell cycle exit (ratio of cells BrdU+/ Ki67−) in the proliferative regions (VZ/SVZ) and in the neuronal layers (IZ and CP) of sections prepared as in panel C (n=4 per each genotype).


( E ) Sections prepared as in C were stained for TUBB3 ( TuJ ) to reveal post - mitotic neurons and with DAPI . Red arrowheads indicate apoptotic neurons ( defined by pyknotic nuclei ) . Quantification of neuronal distribution in VZ - SVZ versus total neurons population is showed on the right . Scale bars = 10 μm .

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(E) Sections prepared as in C were stained for TUBB3 (TuJ) to reveal post-mitotic neurons and with DAPI. Red arrowheads indicate apoptotic neurons (defined by pyknotic nuclei). Quantification of neuronal distribution in VZ-SVZ versus total neurons population is showed on the right. Scale bars = 10 μm.


( F ) Quantification of the percentage of Tbr2 - positive cells in the BrdU - positive population of the same sections analyzed for panel D ( n = 4 per each genotype ) .

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(F) Quantification of the percentage of Tbr2-positive cells in the BrdU-positive population of the same sections analyzed for panel D (n=4 per each genotype).


( G ) Neuroblasts of wild type and dck alleles immunostained for α - tubulin and Miranda . Note that whereas in wild type cells there is a tight coupling of the mitotic spindle with the polarity axis , in dck alleles the spindle show a more oblique orientation respect to Mira crescent . Scale bar = 5 μm . ( H ) Distribution of spindle angle amplitude ( ° ) in neuroblasts of wild type and ck alleles ( n = 58 ) .

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(G) Neuroblasts of wild type and dck alleles immunostained for α-tubulin and Miranda. Note that whereas in wild type cells there is a tight coupling of the mitotic spindle with the polarity axis, in dck alleles the spindle show a more oblique orientation respect to Mira crescent. Scale bar = 5 μm.(H) Distribution of spindle angle amplitude (°) in neuroblasts of wild type and ck alleles (n=58).


( A ) Selected frames from time - lapse imaging experiments ( see Movies EV1 and EV2 ) showing two dividing cells transfected with either control ( CTRL ) or CITK - specific siRNAs , respectively . Note one of the two daughter cells in the lower panel ( red arrow ) going out of focus as a consequence of oblique division . ( B ) Graphical representation of vertical and oblique divisions . The cleavage plane is indicated by a black dashed line . ( C ) Quantification of divisions showing uneven timing of daughter cell flattening onto the substrate after mitosis ( oblique division ) in CITK siRNA treated HeLa cells compared to control ( n > 50 cells , 3 independent experiments ) .

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(A) Selected frames from time-lapse imaging experiments (see Movies EV1 and EV2) showing two dividing cells transfected with either control (CTRL) or CITK-specific siRNAs, respectively. Note one of the two daughter cells in the lower panel (red arrow) going out of focus as a consequence of oblique division.(B) Graphical representation of vertical and oblique divisions. The cleavage plane is indicated by a black dashed line.(C) Quantification of divisions showing uneven timing of daughter cell flattening onto the substrate after mitosis (oblique division) in CITK siRNA treated HeLa cells compared to control (n > 50 cells, 3 independent experiments).


( D ) Control or CITK - depleted cells were immunostained for γ - tubulin ( red ) and DNA ( blue ) and imaged in z ( 0 . 3 µm - thick sections ) . Upper panel : maximum intensity projections of confocal z - stacks are shown . Lower panel : cross - section ( XZ ) through the two poles of the same cell . ( E ) Distribution of spindle angles ( ° ) in control and in CITK - depleted cells . The values represent the angles between the axis crossing the two poles of metaphase spindles and the coverslip . ( n ≥ 150 cells , 6 independent experiments ) .

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(D) Control or CITK-depleted cells were immunostained for γ-tubulin (red) and DNA (blue) and imaged in z (0.3 µm-thick sections). Upper panel: maximum intensity projections of confocal z-stacks are shown. Lower panel: cross-section (XZ) through the two poles of the same cell.(E) Distribution of spindle angles (°) in control and in CITK-depleted cells. The values represent the angles between the axis crossing the two poles of metaphase spindles and the coverslip. (n ≥ 150 cells, 6 independent experiments).


( F ) Quantification of spindle angles average in control cells and in cells depleted of CITK , ASPM or co - depleted of the two proteins ( n ≥ 150 cells , in at least 3 independent experiments ) .

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(F) Quantification of spindle angles average in control cells and in cells depleted of CITK, ASPM or co-depleted of the two proteins (n ≥ 150 cells, in at least 3 independent experiments).


( A ) HeLa cells pre - extracted for 1 min with 0 . 5 % Triton X - 100 in PHEM buffer and immunostained for CITK ( green ) , γ - tubulin ( red ) and DNA ( blue ) . Scale bars ,

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(A) HeLa cells pre-extracted for 1 min with 0.5% Triton X-100 in PHEM buffer and immunostained for CITK (green), γ-tubulin (red) and DNA (blue). Scale bars, 10μm


( B ) HeLa cells expressing CITK - GFP ( green ) from a BAC transgene treated as in ( A ) and immunostained for γ - tubulin ( red ) and DNA ( blue ) . Scale bars ,

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(B) HeLa cells expressing CITK-GFP (green) from a BAC transgene treated as in (A) and immunostained for γ-tubulin (red) and DNA (blue). Scale bars, 5μm


( C ) Western blot of centrosome - containing fractions from HeLa cells , showing that CITK co - purifies with γ ‑ tubulin .

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(C) Western blot of centrosome-containing fractions from HeLa cells, showing that CITK co-purifies with γ‑tubulin.


( D ) HeLa cells pre - extracted 1 min with 0 . 5 % Triton X - 100 in PHEM buffer and immunostained for CITK ( green ) , ASPM ( red ) and DNA ( blue ) .

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(D) HeLa cells pre-extracted 1 min with 0.5% Triton X-100 in PHEM buffer and immunostained for CITK (green), ASPM (red) and DNA (blue).