F . TEER ( Ω . cm2 ) was monitored over 72h treatment with TGF - β1 normalized to baseline in barrier models composed by a monolayer of bEND . 3 alone ( 72h TGF - β1 = 89 . 85 ± 2 . 05 ) or co - cultured with sorted PCs from ( 72h TGF - β1 = 88 . 83 ± 1 . 93 ) or ( 72h TGF - β1 = 89 . 02 ± 2 . 22 ) . insert shows values at 72h ( bars ) normalized to 0h values ( dashed ) . Wilcoxon signed - rank , * p < . 05 . Data information : prenatally exposed to vehicle ( Ctrl ) or Poly I : C on GD9 were dissected , cortex derived vascular were used to build in - vitro barrier models . In Fig . 3 F blue purple dotted lines represent derived from brain respectively .

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

F. TEER (Ω.cm2) was monitored over 72h treatment with TGF-β1 and normalized to baseline in barrier models composed by a monolayer of bEND.3 alone (72h TGF-β1 = 89.85 ± 2.05) or co-cultured with sorted PCs from Male (72h TGF-β1 = 88.83 ± 1.93) or Female (72h TGF-β1 = 89.02 ± 2.22) embryos. The insert shows the values at 72h (bars) normalized to the 0h values (dashed line). Wilcoxon signed-rank test, *p < .05. Data information: Embryos prenatally exposed to vehicle (Ctrl) or Poly I:C on GD9 were dissected, and cortex derived vascular cells were used to build in-vitro barrier models. In Fig.3 F blue and purple dotted lines represent cells derived from male and female brain respectively.


G . 10 kDa Dextran permeability after 72h TGF - β1 treatment of in vitro BBBs prototypes composed by a monolayer made of sorted ECs from NT ( 0 . 43 ± 0 . 02 ) , TGF - β1 ( 0 . 43 ± 0 . 03 ) ; NT ( 0 . 45 ± 0 . 00 ) , TGF - β1 ( 0 . 47 ± 0 . 01 ) . Data information : prenatally exposed to vehicle ( Ctrl ) or Poly I : C on GD9 were dissected , cortex derived vascular were used to build in - vitro barrier models . derived from Ctrl = bars with blu border . derived from Ctrl = bars with purple border . Numbers in bars indicate number of ( N ) or independent cultures / pictures . Bars represent mean ± .

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

G. 10 kDa Dextran permeability after 72h TGF-β1 treatment of the in vitro BBBs prototypes composed by a monolayer made of sorted ECs from Male NT (0.43 ± 0.02), Male TGF-β1 (0.43 ± 0.03); Female NT (0.45 ± 0.00), Female TGF-β1 (0.47 ± 0.01). Data information: Embryos prenatally exposed to vehicle (Ctrl) or Poly I:C on GD9 were dissected, and cortex derived vascular cells were used to build in-vitro barrier models. Cells derived from Ctrl males = white bars with blu border. Cells derived from Ctrl females = white bars with purple border. Numbers in bars indicate the number of animals (N) or independent cultures/pictures. Bars represent mean ± SEM.


H . 10 kDa Dextran permeability 72h after TGF - β1 treatment of NVU prototypes composed by a monolayer made of bEND . 3 alone ( NT , solid black bar = 0 . 45 ± 0 . 01 , TGF - β1 , dotted black bar = 0 . 49 ± 0 . 00 ) co - cultured with sorted PCs from ( NT = 0 . 45 ± 0 . 04 , TGF - β1 = 0 . 48 ± 0 . 00 ) , or ( NT = 0 . 49 ± 0 . 03 , TGF - β1 = 0 . 53 ± 0 . 02 ) . Student ' s t , * p < . 05 . Data information : prenatally exposed to vehicle ( Ctrl ) or Poly I : C on GD9 were dissected , cortex derived vascular were used to build in - vitro barrier models . bEend . 3 = bars with black border . derived from Ctrl = bars with blu border . derived from Ctrl = bars with purple border . Numbers in bars indicate number of ( N ) or independent cultures / pictures . Bars represent mean ± .

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

H. 10 kDa Dextran permeability 72h after TGF-β1 treatment of NVU prototypes composed by a monolayer made of bEND.3 alone (NT, solid black bar = 0.45 ± 0.01, TGF-β1, dotted black bar = 0.49 ± 0.00) and co-cultured with sorted PCs from Male (NT = 0.45 ± 0.04, TGF-β1 = 0.48 ± 0.00), or Female (NT = 0.49 ± 0.03, TGF-β1 = 0.53 ± 0.02) embryos. Student's t test, *p < .05. Data information: Embryos prenatally exposed to vehicle (Ctrl) or Poly I:C on GD9 were dissected, and cortex derived vascular cells were used to build in-vitro barrier models. bEend.3 cells = bars with black border. Cells derived from Ctrl males = white bars with blu border. Cells derived from Ctrl females = white bars with purple border. Numbers in bars indicate the number of animals (N) or independent cultures/pictures. Bars represent mean ± SEM.


I . FACS quantitative data of normalized MFI of CD146 ( NT = 100 . 00 ± 1 . 14 , TGF - β1 = 54 . 08 ± 9 . 42 ; Student ' s t , * p < . 05 ) representative histogram of untreated ( solid gray ) TGF - β1 treated ( hollow black bold ) . J . FACS quantitative data of normalized MFI of Claudin - 5 ( CLDN5 ; NT = 100 . 00 ± 8 . 94 , TGF - β1 = 65 . 22 ± 5 . 29 ; Student ' s t , * * p < . 01 ) representative histogram of untreated ( solid gray ) TGF - β1 treated ( hollow black bold ) . Data information : bEend . 3 = bars with black border . black dotted represent bEnd . 3 cultures . Numbers in bars indicate number of ( N ) or independent cultures / pictures . Bars represent mean ± .

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

I. FACS quantitative data of the normalized MFI of CD146 (NT = 100.00 ± 1.14, TGF-β1 = 54.08 ± 9.42; Student's t test, *p < .05) and representative histogram of untreated (solid gray) and TGF-β1 treated cells (hollow black bold line). J. FACS quantitative data of the normalized MFI of Claudin-5 (CLDN5; NT = 100.00 ± 8.94, TGF-β1 = 65.22 ± 5.29; Student's t test, **p < .01) and representative histogram of untreated (solid gray) and TGF-β1 treated cells (hollow black bold line). Data information: bEend.3 cells = bars with black border. The black dotted line represent bEnd.3 cultures. Numbers in bars indicate the number of animals (N) or independent cultures/pictures. Bars represent mean ± SEM.


K . Representative of monolayers of endothelial isolated from brains stained for CD146 ( red ) CLDN5 ( green ) under control conditions ( NT ) or upon treatment with different cytokines . Scale bar = 50 µm . L . Quantitative of CD146 immunofluorescence mean measured in brain EC monolayers under control conditions or after 72h treatments with different cytokines : NT = 100 . 00 ± 6 . 55 , TGF - β1 = 94 . 03 ± 2 . 01 , IL - 1β + TGF - β1 = 84 . 87 ± 4 . 59 , IL - 1β = 92 . 34 ± 2 . 65 ; Kruskal - Wallis , * p < . 05 . M . Quantitative of Claudin - 5 immunofluorescence mean in brain EC monolayers under control conditions or after 72h treatments with different cytokines : NT = 99 . 76 ± 5 . 19 , TGF - β1 = 86 . 74 ± 6 . 76 , IL - 1β + TGF - β1 = 51 . 64 ± 3 . 31 , IL - 1β = 107 . 3 ± 7 . 37 ; Ordinary one - way ANOVA , * * p < . 01 , * * * * p < . 0001 . N . Quantitative of Claudin - 5 immunofluorescence ( µm2 ) in brain EC monolayers under control conditions or after 72h treatments with different cytokines : NT = 39781 ± 2342 , TGF - β1 = 31707 ± 5939 , IL - 1β + TGF - β1 = 5415 ± 1037 , IL - 1β = 46303 ± 2986 ; Ordinary one - way ANOVA , * p < . 05 , * * p < . 01 , * * * * p < . 0001 . Data information : Cortex - derived - EC isolated from mice at 2 mouths were used to build in - vitro barrier models analyzed . derived from Ctrl = bars with blu border . Numbers in bars indicate number of ( N ) or independent cultures / pictures . Bars represent mean ± .

O O O O O O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Representative images of monolayers of primary endothelial cells isolated from male brains and stained for CD146 (red) and CLDN5 (green) under control conditions (NT) or upon treatment with different cytokines. Scale bar = 50 µm. L. Quantitative analysis of CD146 immunofluorescence mean intensity measured in primary male brain EC monolayers under control conditions or after 72h treatments with different cytokines: NT = 100.00 ± 6.55, TGF-β1 = 94.03 ± 2.01, IL-1β + TGF-β1 = 84.87 ± 4.59, IL-1β = 92.34 ± 2.65; Kruskal-Wallis, *p < .05. M. Quantitative analysis of Claudin-5 immunofluorescence mean intensity in primary male brain EC monolayers under control conditions or after 72h treatments with different cytokines: NT = 99.76 ± 5.19, TGF-β1 = 86.74 ± 6.76, IL-1β + TGF-β1 = 51.64 ± 3.31, IL-1β = 107.3 ± 7.37; Ordinary one-way ANOVA, **p < .01, ****p < .0001. N. Quantitative analysis of Claudin-5 immunofluorescence area (µm2) in primary male brain EC monolayers under control conditions or after 72h treatments with different cytokines: NT = 39781 ± 2342, TGF-β1 = 31707 ± 5939, IL-1β + TGF-β1 = 5415 ± 1037, IL-1β = 46303 ± 2986; Ordinary one-way ANOVA, *p < .05, **p < .01, ****p < .0001. Data information: Cortex-derived-EC isolated from male mice at 2 mouths were used to build in-vitro barrier models and analyzed. Cells derived from Ctrl males = white bars with blu border. Numbers in bars indicate the number of animals (N) or independent cultures/pictures. Bars represent mean ± SEM.


O . Graph showing TEER measured by volt - ohmmeter at 72h normalized in NT ( 100 . 00 ± 7 . 46 ) IL - 1β + TGF - β1 ( 49 . 93 ± 10 . 96 ) treated brain EC cultures . Student ' s t , * * p < . 01 . Data are normalized to t0 . Data information : Cortex - derived - EC isolated from mice at 2 mouths were used to build in - vitro barrier models analyzed . derived from Ctrl = bars with blu border . Numbers in bars indicate number of ( N ) or independent cultures / pictures . Bars represent mean ± .

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

O. Graph showing TEER measured by volt-ohmmeter at 72h normalized in NT (100.00 ± 7.46) and IL-1β + TGF-β1 (49.93 ± 10.96) treated primary male brain EC cultures. Student's t test, **p < .01. Data are normalized to t0. Data information: Cortex-derived-EC isolated from male mice at 2 mouths were used to build in-vitro barrier models and analyzed. Cells derived from Ctrl males = white bars with blu border. Numbers in bars indicate the number of animals (N) or independent cultures/pictures. Bars represent mean ± SEM.


P . Permeability to 3kDa dextran quantified by spectrophotometry in NT ( 1 . 00 ± 0 . 10 ) IL - 1β + TGF - β1 ( 1 . 92 ± 0 . 33 ) treated brain EC cultures . Student ' s t , * p < . 05 . Q . Permeability to 10kDa dextran quantified by spectrophotometry in NT ( 1 . 00 ± 0 . 11 ) IL - 1β + TGF - β1 ( 1 . 80 ± 0 . 26 ) treated brain EC cultures . Student ' s t , * p < . 05 . Data information : Cortex - derived - EC isolated from mice at 2 mouths were used to build in - vitro barrier models analyzed . derived from Ctrl = bars with blu border . Numbers in bars indicate number of ( N ) or independent cultures / pictures . Bars represent mean ± .

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

P. Permeability to 3kDa dextran quantified by fluorescence spectrophotometry in NT (1.00 ± 0.10) and IL-1β + TGF-β1 (1.92 ± 0.33) treated primary male brain EC cultures. Student's t test, *p < .05. Q. Permeability to 10kDa dextran quantified by fluorescence spectrophotometry in NT (1.00 ± 0.11) and IL-1β + TGF-β1 (1.80 ± 0.26) treated primary male brain EC cultures. Student's t test, *p < .05. Data information: Cortex-derived-EC isolated from male mice at 2 mouths were used to build in-vitro barrier models and analyzed. Cells derived from Ctrl males = white bars with blu border. Numbers in bars indicate the number of animals (N) or independent cultures/pictures. Bars represent mean ± SEM.


A . [ left ] Representative dot plots showing gating strategy for E17 samples to distinguish NVU . were pre - gated on single live ( Zombie Aqua negative ) . Live were pre - gated on CD45 ( CD45 - ) . [ right ] Brain Pericytes ( PC ) were defined as CD146 + CD31 - brain Endothelial ( EC ) as CD31 + . B . [ left ] of between PC number to EC numbers for ( blue ) ( purple ) E17 offspring cortex samples . Ctrl = 1 . 10 ± 0 . 08 , Poly I : C = 1 . 34 ± 0 . 09 , Ctrl = 1 . 02 ± 0 . 08 , Poly I : C = 1 . 12 ± 0 . 09 . Mann - Whitney U , * p < . 05 . Bars represent average of ± , numbers in bars indicate number of . [ right up ] Percentages of ECs in Ctrl ( 0 . 79 ± 0 . 05 ) , Poly I : C ( 0 . 90 ± 0 . 09 ) , Ctrl ( 0 . 65 ± 0 . 07 ) , Poly I : C ( 0 . 80 ± 0 . 07 ) samples [ right down ] PCs in Ctrl ( 0 . 60 ± 0 . 05 ) , Poly I : C ( 0 . 57 ± 0 . 06 ) , Ctrl ( 0 . 53 ± 0 . 07 ) , Poly I : C ( 0 . 62 ± 0 . 06 ) samples . C . [ left ] Gating strategy used to analyze P30 samples : single , live ( zombie Aqua negative ) , CD45 - . [ right ] PCs are identified as CD146 + CD13 + CD31 - ; ECs are CD13 - CD31 + D . [ left ] of EC / PC number for P30 offspring cortex samples , obtained by manual gating . Ctrl = 1 . 78 ± 0 . 29 , Poly I : C = 1 . 74 ± 0 . 32 , Ctrl = 1 . 49 ± 0 . 11 , Poly I : C = 1 . 60 ± 0 . 19 . [ right up ] Percentages of ECs in Ctrl ( 17 . 07 ± 1 . 88 ) , Poly I : C ( 17 . 52 ± 1 . 90 ) , Ctrl ( 14 . 58 ± 0 . 66 ) , Poly I : C ( 15 . 73 ± 1 . 07 ) samples , [ right down ] of PCs in Ctrl ( 10 . 55 ± 1 . 89 ) , Poly I : C ( 10 . 67 ± 1 . 04 ) , Ctrl ( 10 . 07 ± 0 . 85 ) , Poly I : C ( 10 . 20 ± 0 . 79 Data information : prenatally exposed to vehicle ( Ctrl ) or Poly I : C on GD9 were dissected , cortex single - suspension was analyzed by multi - color flow cytometry . Ctrl = bars with blu border . Poly I : C = blu bars with blu border . Ctrl = bars with purple border . Poly I : C = purple bars with purple border . Bars represent mean ± , numbers in bars indicate number of .

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

A. [left] Representative dot plots showing the gating strategy for E17 samples to distinguish NVU cells. The cells were pre-gated on single cells and live cells (Zombie Aqua negative). Live cells were pre-gated on CD45 (CD45-). [right] Brain Pericytes (PC) were defined as CD146+CD31- cells and brain Endothelial Cells (EC) as CD31+cells. B. [left] Quantification of the ratio between PC number to EC numbers for male (blue) and female (purple) E17 offspring cortex samples. Male Ctrl = 1.10 ± 0.08, Male Poly I:C = 1.34 ± 0.09, Female Ctrl = 1.02 ± 0.08, Female Poly I:C = 1.12 ± 0.09. Mann-Whitney U test, *p < .05. Bars represent average ratio of individual embryos ± SEM, numbers in bars indicate the number of embryos. [right up] Percentages of ECs in Male Ctrl (0.79 ± 0.05), Male Poly I:C (0.90 ± 0.09), Female Ctrl (0.65 ± 0.07), Female Poly I:C (0.80 ± 0.07) samples and [right down] PCs in Male Ctrl (0.60 ± 0.05), Male Poly I:C (0.57 ± 0.06), Female Ctrl (0.53 ± 0.07), Female Poly I:C (0.62 ± 0.06) samples. C. [left] Gating strategy used to analyze P30 samples: single cells, live (zombie Aqua negative), CD45- cells. [right] PCs are identified as CD146+CD13+CD31-; ECs are CD13-CD31+ D. [left] Quantification of EC/PC number ratio for male and female P30 offspring cortex samples, obtained by manual gating. Male Ctrl = 1.78 ± 0.29, Male Poly I:C = 1.74 ± 0.32, Female Ctrl = 1.49 ± 0.11, Female Poly I:C = 1.60 ± 0.19. [right up] Percentages of ECs in Male Ctrl (17.07 ± 1.88), Male Poly I:C (17.52 ± 1.90), Female Ctrl (14.58 ± 0.66), Female Poly I:C (15.73 ± 1.07) samples, and [right down] percentage of PCs in Male Ctrl (10.55 ± 1.89), Male Poly I:C (10.67 ± 1.04), Female Ctrl (10.07 ± 0.85), Female Poly I:C (10.20 ± 0.79 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.


E . Normalized of live ( CD45 - ) EdU - positive at E17 in Ctrl ( 1 . 00 ± 0 . 05 ) , Poly I : C ( 0 . 62 ± 0 . 11 ) , Ctrl ( 0 . 61 ± 0 . 04 ) , Poly I : C ( 0 . 59 ± 0 . 11 ) samples . Mann - Whitney U Kruskal - Wallis , * p < . 05 , * * p < . 001 . F . of PCs EdU - positive at E17 in Ctrl ( 7 . 88 ± 1 . 57 ) , Poly I : C ( 4 . 55 ± 0 . 70 ) , Ctrl ( 7 . 03 ± 1 . 04 ) , Poly I : C ( 4 . 93 ± 0 . 83 ) samples . Student ' s t , * p < . 05 . G . of ECs EdU - positive at E17 in Ctrl ( 4 . 30 ± 0 . 71 ) , Poly I : C ( 2 . 80 ± 0 . 53 ) , Ctrl ( 2 . 84 ± 0 . 43 ) , Poly I : C ( 2 . 29 ± 0 . 77 ) samples . H . Normalized of live ( CD45 - ) EdU - positive at P30 in Ctrl ( 1 . 16 ± 0 . 14 ) , Poly I : C ( 0 . 66 ± 0 . 16 ) , Ctrl ( 1 . 00 ± 0 . 08 ) , Poly I : C ( 0 . 91 ± 0 . 13 ) samples . Mann - Whitney U , * p < . 05 . I . of PCs EdU - positive at P30 in Ctrl ( 0 . 80 ± 0 . 24 ) , Poly I : C ( 0 . 24 ± 0 . 06 ) , Ctrl ( 0 . 26 ± 0 . 08 ) , Poly I : C ( 0 . 38 ± 0 . 13 ) samples . Mann - Whitney U , * p < . 05 . J . of ECs EdU - positive at P30 in Ctrl ( 0 . 58 ± 0 . 10 ) , Poly I : C ( 0 . 44 ± 0 . 19 ) , Ctrl ( 0 . 38 ± 0 . 22 ) , Poly I : C ( 0 . 25 ± 0 . 06 ) samples . Data information : Ctrl = bars with blu border . Poly I : C = blu bars with blu border . Ctrl = bars with purple border . Poly I : C = purple bars with purple border . Bars represent mean ± , numbers in bars indicate number of .

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

E. Normalized percentage of live(CD45-) EdU-positive cells at E17 in Male Ctrl (1.00 ± 0.05), Male Poly I:C (0.62 ± 0.11), Female Ctrl (0.61 ± 0.04), Female Poly I:C (0.59 ± 0.11) samples. Mann-Whitney U test and Kruskal-Wallis test, *p < .05, **p < .001. F. Percentage of PCs EdU-positive cells at E17 in Male Ctrl (7.88 ± 1.57), Male Poly I:C (4.55 ± 0.70), Female Ctrl (7.03 ± 1.04), Female Poly I:C (4.93 ± 0.83) samples. Student's t test, *p < .05. G. Percentage of ECs EdU-positive cells at E17 in Male Ctrl (4.30 ± 0.71), Male Poly I:C (2.80 ± 0.53), Female Ctrl (2.84 ± 0.43), Female Poly I:C (2.29 ± 0.77) samples. H. Normalized percentage of live(CD45-) EdU-positive cells at P30 in Male Ctrl (1.16 ± 0.14), Male Poly I:C (0.66 ± 0.16), Female Ctrl (1.00 ± 0.08), Female Poly I:C (0.91 ± 0.13) samples. Mann-Whitney U test, *p < .05. I. Percentage of PCs EdU-positive cells at P30 in Male Ctrl (0.80 ± 0.24), Male Poly I:C (0.24 ± 0.06), Female Ctrl (0.26 ± 0.08), Female Poly I:C (0.38 ± 0.13) samples. Mann-Whitney U test, *p < .05. J. Percentage of ECs EdU-positive cells at P30 in Male Ctrl (0.58 ± 0.10), Male Poly I:C (0.44 ± 0.19), Female Ctrl (0.38 ± 0.22), Female Poly I:C (0.25 ± 0.06) samples. 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. Bars represent mean ± SEM, numbers in bars indicate the number of animals.


K . [ left ] CD146 normalized MFI of EC at P30 in Ctrl ( 100 . 00 ± 8 . 27 ) , Poly I : C ( 65 . 73 ± 3 . 63 ) , Ctrl ( 57 . 08 ± 14 . 10 ) , Poly I : C ( 43 . 07 ± 8 . 34 ) . Mann - Whitney U Ordinary one - way ANOVA , * * p < . 01 . [ right ] Representative histograms CD146 expression on ECs in Ctrl ( blue hollow ) , Poly I : C ( solid blue ) , Ctrl ( purple hollow ) , Poly I : C ( solid purple ) samples . Data information : prenatally exposed to vehicle ( Ctrl ) or Poly I : C on GD9 were dissected , cortex single - suspension was analyzed by multi - color flow cytometry . Ctrl = bars with blu border . Poly I : C = blu bars with blu border . Ctrl = bars with purple border . Poly I : C = purple bars with purple border . Bars represent mean ± , numbers in bars indicate number of .

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

K. [left] CD146 normalized MFI of the EC cells at P30 in Male Ctrl (100.00 ± 8.27), Male Poly I:C (65.73 ± 3.63), Female Ctrl (57.08 ± 14.10), Female Poly I:C (43.07 ± 8.34). Mann-Whitney U test and Ordinary one-way ANOVA, **p < .01. [right] Representative histograms CD146 expression on ECs 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.


L . Percentages of PC CD146 + + , CD146 + CD146 - respectively at P30 in Ctrl ( 26 . 32 / 36 . 75 / 23 . 48 ± 2 . 84 / 3 . 31 / 4 . 62 ) , Poly I : C ( 27 . 53 / 42 . 40 / 20 . 60 ± 2 . 25 / 0 . 58 / 1 . 94 ) , Ctrl ( 29 . 05 / 40 . 98 / 2 . 19 ± 1 . 99 / 1 . 32 / 2 . 19 ) , Poly I : C ( 27 . 17 / 46 . 60 / 17 . 10 ± 3 . 09 / 3 . 86 / 1 . 96 ) samples . Data information : CD13 positive pericytes were represented ad CD146 - ( greed bars ) , CD146 + ( green bars ) CD46 + + ( green bars ) . Bars represent mean ± , numbers in bars indicate number of .

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

L. Percentages of PC CD146++, CD146+ and CD146- cells respectively at P30 in Male Ctrl (26.32/36.75/23.48 ± 2.84/3.31/4.62), Male Poly I:C (27.53/42.40/20.60 ± 2.25/0.58/1.94), Female Ctrl (29.05/40.98/2.19 ± 1.99/1.32/2.19), Female Poly I:C (27.17/46.60/17.10 ± 3.09/3.86/1.96) samples. Data information: CD13 positive pericytes were represented ad CD146- (dark greed bars), CD146+ (green bars) and CD46++ (light green bars). Bars represent mean ± SEM, numbers in bars indicate the number of animals.


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

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

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

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

O. [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 . of PDGFrB - positive pericyte coverage at P90 in Ctrl ( 66 . 15 ± 3 . 09 ) Poly I : C ( 55 . 08 ± 3 . 10 ) . Student ' s t , * p < . 05 . Data information : Ctrl = bars with blu border . Poly I : C = blu bars with blu border . Bars represent mean ± , numbers in bars indicate number of .

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

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

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

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

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

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 of brain acute bleedings detected through hemoglobin in P30 Ctrl Poly I : C MIA offspring . Scale bar = 500 µm , enlargement scale bar = 50 µm . B . Representative of brain acute bleedings detected through hemoglobin in P90 Ctrl Poly I : C MIA offspring . Scale bar = 500 µm , enlargement scale bar = 50 µm . C . of hemoglobin ( Hb ) positive signal in brain of P30 P90 MIA offspring , expressed as of . 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 , * * * p < . 001 . Data information : Ctrl = bars with blu border . Poly I : C = blu bars with blu border . Numbers in bars indicate number of ( N ) slices ( n ) . Bars represent mean ± .

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

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 in P30 Ctrl Poly I : C MIA offspring brains . Brain volume is rendered gold - transparent with positive signal for color - coded by surface dimension . Scale bar = 100 µm .

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

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

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

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 of P30 MIA offspring , respectively in Coronal ( Ctrl = 1 . 19 ± 0 . 04 , Poly I : C = 0 . 94 ± 0 . 01 ; Mann - Whitney U , * * p < . 01 ) , Axial ( Ctrl = 1 . 16 ± 0 . 08 , Poly I : C = 1 . 03 ± 0 . 03 ) , Sagittal ( Ctrl = 1 . 09 ± 0 . 09 , Poly I : C = 1 . 01 ± 0 . 05 ) planes . Data information : Ctrl = bars with blu border . Poly I : C = blu bars with blu border . Numbers in bars indicate number of ( N ) slices ( n ) . Bars represent mean ± .

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

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

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

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 at 0 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 sawdust . J . of buried marble ( n ° ) by P90 Ctrl ( 3 . 33 ± 1 . 67 ) , Poly I : C ( 5 . 72 ± 2 . 10 ) , Ctrl ( 3 . 83 ± 1 . 72 ) Poly I : C ( 3 . 00 ± 2 . 00 ) mice . Student ' s t , * * p < . 01 . Data information : Ctrl = bars with blu border . Poly I : C = blu bars with blu border . Ctrl = bars with purple border . Poly I : C = purple bars with purple border . Numbers in bars indicate number of ( N ) slices ( n ) . Bars represent mean ± .

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

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

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

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 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 F59 antibody ( green ) ( Elworthy et al , 2008 ) . smyhc1 + / + , smyhc1R673H / + , smyhc1R673H / R673H larvae display Smyhc1 in muscle at 24hpf . smyhc1 - / - larvae at 24 do not stain positively for Smyhc1 . Scale bar represents 50 µm .

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

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

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

(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 ) predicted mendelian ( 1 : 2 : 1 ) experimental of genotypes of progeny from smyhc1R673H / + in - crosses demonstrates survival at indicated ages . This 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 of smyhc1 mutant adults . Most smyhc1 - / - adult fish display dorsal tail curvature , while smyhc1R673H / + adults have a shortened axis 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 of bone shows spinal curvature but no bony fusions in skeleton of smyhc1 - / - adults , in contrast to compression fusion of vertebrae seen in smyhc1R673H / + adults . Distal tail regions are highlighted 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 ) of skeletal phenotype severity in adults . Scale bars represent a of 5 mm .

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


( A ) Time course of - triggered larval movements were manually counted at indicated times between 24 48 hpf . 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 . central bands of boxplots represent median , boxes of boxplots represent interquartile range ( between first third quartile ) , whiskers represent minimum maximum values , up to 1 . 5 times 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 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

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

(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 fish fatigued were collected at 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 of slow skeletal muscle of 1 dpf larvae , stained with phalloidin - rhodamine to detect actin . of filamentous actin are indicated with arrowheads , misshapen myofibers are indicated with arrows outlined . ( B ) Confocal microscope of slow skeletal muscle of 3 dpf larvae , stained with phalloidin - rhodamine . of filamentous actin are indicated with arrowheads , distorted myofibers are indicated with arrows outlined , 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 of slow skeletal muscle of 1dpf larvae , stained with phalloidin - rhodamine ( red ) , anti - α - actinin antibodies ( green ) , DAPI ( blue ) . ( D ) Confocal of slow skeletal muscle of 3dpf larvae , stained with phalloidin - rhodamine ( red ) , anti - α - actinin antibodies ( green ) , DAPI ( blue ) . Bundle of actin ringed with α - actinin indicated with - bordered black arrowhead .

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

(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 ) , smyhc1R673H / R673H ( n = 49 ) . between myosepta was measured perpendicular to rostral - caudal axis at defined mid - regions in phalloidin stained slow skeletal muscle . central bands of boxplots represent median , boxes of boxplots represent interquartile range ( between first third quartile ) , whiskers represent minimum maximum values , up to 1 . 5 times 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 ) , smyhc1R673H / R673H ( n = 38 ) . central bands of boxplots represent median , boxes of boxplots represent interquartile range ( between first third quartile ) , whiskers represent minimum maximum values , up to 1 . 5 times interquartile range . Outliers displayed are outside of this range .

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

(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 ) of slow skeletal muscle at 1 dpf in smyhc1 + / + ( n = 380 ) , smyhc1R673H / + ( n = 293 ) , smyhc1R673H / R673H ( n = 268 ) . between z - discs was measured in anti - α - actinin stained slow skeletal muscle . central bands of boxplots represent median , boxes of boxplots represent interquartile range ( between first third quartile ) , whiskers represent minimum maximum values , up to 1 . 5 times interquartile range . Outliers displayed are outside of this range . ( H ) Z - disc intervals ( sarcomere ) of slow skeletal muscle at 3 dpf in smyhc1 + / + ( n = 249 ) , smyhc1 - / - ( n = 278 ) , smyhc1R673H / + ( n = 246 ) , smyhc1R673H / R673H ( n = 252 ) . central bands of boxplots represent median , boxes of boxplots represent interquartile range ( between first third quartile ) , whiskers represent minimum maximum values , up to 1 . 5 times 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 / + , smyhc1R673H / R673H larvae at 2 dpf after 24 hour treatment with 0 . 25 % DMSO ( control ) or 25 μM para - aminoblebbistatin . larvae treated with para - aminoblebbistatin develop a slight dorsal tail curve pericardial edema regardless of genotype . para - aminoblebbistatin - treated smyhc1R673H / + ( n = 9 ) smyhc1R673H / R673H ( n = 3 ) larvae are indistinguishable from smyhc1 + / + unlike untreated larvae which have markedly curved or distorted axis . ( B ) Representative of smyhc1 + / + , smyhc1R673H / + , smyhc1R673H / R673H larvae at 3dpf after 48 hour treatment with 0 . 25 % DMSO ( control ) or 25 μM para - aminoblebbistatin . larvae treated with para - aminoblebbistatin develop a slight dorsal tail curve pericardial edema regardless of genotype . Para - aminoblebbistatin - treated smyhc1R673H / + ( n = 9 ) smyhc1R673H / R673H ( n = 3 ) larvae are indistinguishable from smyhc1 + / + unlike untreated larvae which have markedly curved or distorted 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 of GlcNAc in wild - type ( WT ) , ΔgacG , H , I , J , K , L were measured as sWGA ( normalized to WT ) .

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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 , ΔgacI , ΔgacI : : gacI . ( C ) LC3 - II 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 - infected . HeLa were infected with WT , ΔgacI , ΔgacI : : gacI for 4 h , fixed , immunostained for ubiquitin ( FK2 : magenta ) . were stained with DAPI ( cyan ) . ( D ) Representative confocal ( E ) of with ubiquitin - positive . 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 . HeLa WT were infected with indicated strains masked with sWGA for 4 h , fixed , immunostained for ubiquitin ( magenta ) . ( G ) Representative confocal ( H ) percentages of with ubiquitin - positive . 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 were infected with WT , ΔgacI , ΔgacI : : gacI for 4 h , fixed , immunostained for p62 ( magenta ) . were stained with DAPI ( cyan ) . ( I ) Representative confocal ( J ) of with p62 - positive . 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 mCherry - LC3 in - infected . HeLa transfected with indicated EmGFP - FBXO ( green ) mCherry - LC3 ( magenta ) were infected with WT for 2 h ; scale bar , 10 μm . Representative confocal ( A ) ( B ) percentages of FBXO - positive .

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

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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 transfected indicated GFP - FBXO2 mutants ( green ) were infected with WT for 4 h ; scale bar , 10 μm . Representative confocal ( 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 in SKP1 / CUL1 / ROC1 knockdown . HeLa transfected with GFP - FBXO2 ( green ) indicated siRNAs were infected with , fixed at 4 h ; scale bar , 10 μm . Representative confocal ( E ) percentages of FBXO2 localized to ( 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 transfected with mCherry - Gal3 ( magenta ) were infected with WT for 2 or 4 h , percentages of Gal3 - positive - containing 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 p62 in FBXO2 KO . HeLa WT or FBXO2 - KO were infected with WT for 4 h , fixed , immunostained for ubiquitin ( FK2 : magenta ) or LC3 ( magenta ) or p62 ( magenta ) . Representative confocal ( E , G , I ) percentages of with ubiquitin , LC3 , or p62 - positive ( F , H , 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 xenophagy . Non - transfected HeLa WT HeLa FBXO2 - KO transfected GFP , GFP - FBXO2 , GFP - FBXO2 YW / AA were infected with WT for 4 h , fixed , immunostained for ubiquitin . Percentages of with LC3 ( M ) or ubiquitin ( N ) - positive 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 ubiquitin to intracellular in SKP1 / CUL1 / ROC1 knockdown . HeLa transfected with mCherry - LC3 ( magenta ) indicated siRNAs were infected with for 4 h . were immunostained for ubiquitin ( FK2 ) ; scale bar , 10 μm . Representative confocal ( A ) percentages of with ubiquitin ( B ) LC3 ( C ) - positive .

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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 were infected with WT , fixed at 4 h . were immunostained for ubiquitin ( FK2 : magenta ) or LC3 ( magenta ) ; scale bar , 10 μm . Representative of with ubiquitin - positive ( D ) LC3 - positive ( 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 infected with were immunoprecipitated with an anti - lipoteichoic acid antibody 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 ) graphs depict Aβ profiles generated by WT or mutant PSEN1 MEF lines transduced with adenovirus encoding APPC99 . ELISA quantified secreted Aβ37 , Aβ38 , Aβ40 Aβ42 peptide are shown as % of total Aβ ( sum of four peptides ) . blue , orange , green purple dotted lines indicate of Aβ37 , Aβ38 , Aβ40 Aβ42 peptides in 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 for cleavage of APPC99 determined in WT mutant PSEN1 / GSEC lines . mutant Aβ ( 37 + 38 ) / ( 40 + 42 ) ratios are shown as % of WT . Data presented as mean ± SD , N ≥ 3 independent experiments . As reference , a dotted was drawn to indicate 10 % ( WT ) . One - way ANOVA followed by Dunnett ' s post - hoc 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 . chemical structure of bicyclic heterocycle GSM III is depicted . Data are presented as mean ± SD , N ≥ 3 independent experiments . blue , orange , green purple dotted lines indicate of Aβ37 , Aβ38 , Aβ40 Aβ42 peptides in 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 Aβ ( 37 + 38 ) / ( 40 + 42 ) presented as % of WT response . data are presented as mean ± SD , N ≥ 3 . symbol # marks mutations that fully abolished GSEC response towards GSM III , As reference , a dotted was drawn to indicate 10 % ( WT ) . One - way ANOVA followed by Dunnett ' s post - hoc 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 lines overexpressing APPC99 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 Aβ ( 37 + 38 ) / ( 40 + 42 ) was determined for dose - response ( presented in panel D ) 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 indicated mutant MEF lines in 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 mutant GSEC processivity estimated by Aβ ( 37 + 38 ) / ( 40 + 42 ) presented as % of WT . Data presented as mean ± SD , N ≥ 3 independent experiments . One - way ANOVA followed by Dunnett ' s post - hoc 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 indicated mutant MEF lines in 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 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.


of mutant APP substrates with regards to their effects on response of GSEC to imidazole - GSM III . Aβ profiles secreted by HEK293T transiently expressing WT or mutant APPC99 substrates in presence of vehicle ( DMSO ) or 1 µM GSM III were analysed by ELISA ( D ) ELISA - 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 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.


of mutant APP substrates with regards to their effects on response of GSEC to imidazole - GSM III . Aβ profiles secreted by HEK293T 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 Aβ ( 37 + 38 ) / ( 40 + 42 ) are presented as % of WT condition ( mean ± SD ) . One - way ANOVA followed by Dunnett ' s post - hoc with comparison to WT was used to determine statistical significance ; * * * * P < 0 . 0001 . F ( DFn , DFd ) : F ( 4 , 17 ) = 71 . 68 .

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

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 - profiles show proportion of Aβ peptides on 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 lines transduced with APPC99 . Dotted lines indicate 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 Y106W - V236W substitutions lead to increased Aβ ( 37 + 38 ) / ( 40 + 42 ) , indicating activation of 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 in dose - response experiments for GSM III revealed no modulation of PSEN1 V236W mutant . Data are presented as mean ± SD , N = 2 independent experiments . For comparison purposes , WT 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 using purified GSEC an APP - fluorogenic peptide as substrate Upper panel : In non - state Dpn ( 2 , 4 - dinitrophenyl ) quencher group suppresses emission from Nma ( N - methyl - o - aminobenzoic acid ) fluorophore through Förster resonance energy transfer ( FRET ) . Upon GSEC cleavage , Nma group emits at λ = 430 nm a signal upon excitation at λ = 355 nm . Lower panel : Specific activities for purified WT or mutant GSECs ( bearing indicated substitutions in PSEN1 ) treated with DMSO ( vehicle ) , 10 µM GSMIII or GSM II . Data are normalized to 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 lines . Inhibitory profiles derived IC50 values ( table ) indicate that PSEN1 - V236W significantly increases affinity for inhibitor . Dotted 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 time - dependently activates p - EphrinB , p - TIE2 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 ) vessels in B16F10 tumors are mostly p - TIE2 ( Tyr992 ) + pSHP2 ( Tyr542 ) + ; confocal ( 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 colon adenoma ; confocal from serial sections ( scale bars : 50μm tissues ) . Arrowheads point to CD31 + / p - EphrinB + ; CD31 + / p - SHP2 + ; CD31 + / p - Tie2 + CD31 + / Ang2 + endothelial .

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

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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 low ( 75 % quartile ) mRNA on survival probability of with melanoma ; Kaplan - Meier using TCGA melanoma datasets ( n = 103 ) ; P values from Mann Whitney U - . ( D ) Impact of high low ( median ) mRNA on survival probability of with colon cancer ; Kaplan - Meier 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).