M . Representative images of P90 Ctrl and Poly I : C male offspring cortices stained for TJs proteins . Claudin - 5 ( top - green ) and ZO - 1 ( bottom - red ) - Scale bar = 10 µm . N . Quantitative analysis of mean intensity of CLDN5 in cortices from P90 male Ctrl ( 75 . 14 ± 1 . 08 ) and Poly I : C ( 67 . 39 ± 2 . 00 ) offspring . Student ' s t test , * p < . 05 . O . Quantitative analysis of the CLDN5 signal volume in cortices from P90 male Ctrl ( 5 . 54 ± 0 . 59 ) and Poly I : C ( 3 . 70 ± 0 . 48 ) offspring . Student ' s t test , * p < . 05 . P . Quantitative analysis of mean intensity of ZO - 1 in cortices from P90 male Ctrl ( 73 . 16 ± 0 . 59 ) and Poly I : C ( 73 . 86 ± 1 . 94 ) offspring . Q . Quantitative analysis of the ZO - 1 signal volume in cortices from P90 male Ctrl ( 23 . 29 ± 1 . 42 ) and Poly I : C ( 16 . 78 ± 2 . 18 ) offspring . Student ' s t 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 images ( n ) . Bars represent mean ± SEM .

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

M. Representative images of P90 Ctrl and Poly I:C male offspring cortices stained for TJs proteins. Claudin-5 (top-green) and ZO-1 (bottom-red) - Scale bar = 10 µm. N. Quantitative analysis of mean intensity of CLDN5 in cortices from P90 male Ctrl (75.14 ± 1.08) and Poly I:C (67.39 ± 2.00) offspring. Student's t test, *p < .05. O. Quantitative analysis of the CLDN5 signal volume in cortices from P90 male Ctrl (5.54 ± 0.59) and Poly I:C (3.70 ± 0.48) offspring. Student's t test, *p < .05. P. Quantitative analysis of mean intensity of ZO-1 in cortices from P90 male Ctrl (73.16 ± 0.59) and Poly I:C (73.86 ± 1.94) offspring. Q. Quantitative analysis of the ZO-1 signal volume in cortices from P90 male Ctrl (23.29 ± 1.42) and Poly I:C (16.78 ± 2.18) offspring. Student's t 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 images (n). Bars represent mean ± SEM.


T . [ Left ] Quantification of in - vivo cerebrovascular permeability to EB of P90 Ctrl ( 0 . 16 ± 0 . 16 ) and Poly I : C - treated ( 0 . 89 ± 0 . 89 ) males injected at P20 . [ Right ] Representative pictures of saline perfused brains 24h after the EB injection accompanied by pseudocolor images - blue pixels are represented as a gradient of light - to - dark blue depending on the intensity of the color in the original photo while the absence of blue tones is represented as green - from Ctrl ( top ) and Poly I : C ( bottom ) male mice . 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 images ( n ) . Bars represent mean ± SEM .

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

T. [Left] Quantification of in-vivo cerebrovascular permeability to EB of P90 Ctrl (0.16 ± 0.16) and Poly I:C-treated (0.89 ± 0.89) males injected at P20. [Right] Representative pictures of saline perfused brains 24h after the EB injection accompanied by pseudocolor images - blue pixels are represented as a gradient of light-to-dark blue depending on the intensity of the color in the original photo while the absence of blue tones is represented as green - from Ctrl (top) and Poly I:C (bottom) male mice. 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 images (n). Bars represent mean ± SEM.


U . Quantitative analysis of the optical density of CLDN5 immunoreactive bands normalized to CANX in cortices from P90 Ctrl ( 0 . 99 ± 0 . 12 ) and Poly I : C ( 0 . 98 ± 0 . 10 ) males treated at P20 . 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 images ( n ) . Bars represent mean ± SEM .

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

U. Quantitative analysis of the optical density of CLDN5 immunoreactive bands normalized to CANX in cortices from P90 Ctrl (0.99 ± 0.12) and Poly I:C (0.98 ± 0.10) males treated at P20. 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 images (n). Bars represent mean ± SEM.


V . Quantitative analysis of the optical density of ZO - 1 immunoreactive bands normalized to CANX in cortices from P90 Ctrl ( 1 . 00 ± 0 . 11 ) and Poly I : C ( 0 . 73 ± 0 . 05 ) males treated at P20 . 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 images ( n ) . Bars represent mean ± SEM .

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

V. Quantitative analysis of the optical density of ZO-1 immunoreactive bands normalized to CANX in cortices from P90 Ctrl (1.00 ± 0.11) and Poly I:C (0.73 ± 0.05) males treated at P20. 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 images (n). Bars represent mean ± SEM.


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

A. qPCR analyses of GD9+6h male embryos for: Pecam1 (Ctrl = 1.00 ± 0.17 N=8, Poly I:C = 0.89 ± 0.23 N=13), CD248 (Ctrl = 1.00 ± 0.20 N=6, Poly I:C = 1.35 ± 0.31 N=11), Vegfa (Ctrl = 1.00 ± 0.19 N=8, Poly I:C = 0.15 ± 0.04 N=8, Student's t test, ***p < .001), Pdfgb (Ctrl = 1.00 ± 0.23 N=5, Poly I:C = 1.10 ± 0.18 N=13), Tgfb1 (Ctrl = 1.00 ± 0.21 N=7, Poly I:C = 1.76 ± 0.22 N=10, Mann-Whitney U test, *p < .05), Tgfb2 (Ctrl = 1.00 ± 0.28 N=6, Poly I:C = 1.37 ± 0.28 N=13), Tgfb3 (Ctrl = 1.00 ± 0.51 N=6, Poly I:C = 0.73 ± 0.19 N=10), Foxf2 (Ctrl = 1.00 ± 0.45 N=8, Poly I:C = 0.46 ± 0.18 N=13) mRNAs. B. qPCR analyses of E17 male cortices for: Pecam1 (Ctrl = 1.00 ± 0.63 N=10, Poly I:C = 3.07 ± 1.58 N=8; Mann-Whitney U test, *p < .05), CD248 (Ctrl = 1.00 ± 0.23 N=10, Poly I:C = 3.33 ± 0.79 N=9; Mann-Whitney U test, **p < .01), Vegfa (Ctrl = 1.00 ± 0.10 N=8, Poly I:C = 1.70 ± 0.29 N=5; Mann-Whitney U test, *p < .05), Pdfgb (Ctrl = 1.00 ± 0.75 N=9, Poly I:C = 2.05 ± 0.96 N=9; Mann-Whitney U test, *p < .05), Tgfb1 (Ctrl = 1.00 ± 0.16 N=7, Poly I:C = 15.27 ± 5.75 N=7; Mann-Whitney U test, ***p < .001), Tgfb2 (Ctrl = 1.00 ± 0.16 N=8, Poly I:C = 3.82 ± 1.05 N=6; Mann-Whitney U test, *p < .05), Tgfb3 (Ctrl = 1.00 ± 0.10 N=9, Poly I:C = 10.58 ± 6.73 N=8; Mann-Whitney U test, ***p < .001); Foxf2 (Ctrl = 1.00 ± 0.06 N=5, Poly I:C = 2.60 ± 0.57 N=8; Mann-Whitney U test, *p < .05) mRNAs. C. qPCR analyses of P90 male cortices for: Pecam1 (Ctrl = 1.00 ± 0.18 N=8, Poly I:C = 0.85 ± 0.08 N=8), CD248 (Ctrl = 1.00 ± 0.20 N=9, Poly I:C = 0.93 ± 0.11 N=8), Vegfa (Ctrl = 1.00 ± 0.07 N=8, Poly I:C = 0.75 ± 0.06 N=8; Mann-Whitney U test, *p < .05); Pdfgb (Ctrl = 1.00 ± 0.20 N=8, Poly I:C = 1.11 ± 0.17 N=4), Tgfb1 (Ctrl = 1.00 ± 0.29 N=5, Poly I:C = 2.37 ± 0.47 N=7; Mann-Whitney U test, *p < .05); Tgfb2 (Ctrl = 1.00 ± 0.29 N=4, Poly I:C = 1.19 ± 0.26 N=7), Tgfb3 (Ctrl = 1.00 ± 0.18 N=10, Poly I:C = 1.78 ± 0.50 N=11) mRNAs. D. qPCR analyses of GD9+6h female embryos for: Pecam1 (Ctrl = 1.00 ± 0.27 N=10, Poly I:C = 2.22 ± 0.50 N=6), CD248 (Ctrl = 1.00 ± 0.27 N=10, Poly I:C = 2.32 ± 0.57 N=6; Mann-Whitney U test, *p < .05), Vegfa (Ctrl = 1.00 ± 0.38 N=8, Poly I:C = 0.29 ± 0.03 N=5; Mann-Whitney U test, *p < .05), Pdfgb (Ctrl = 1.00 ± 0.36 N=10, Poly I:C = 1.31 ± 0.47 N=6), Tgfb1 (Ctrl = 1.00 ± 0.16 N=8, Poly I:C = 1.63 ± 0.42 N=7), Tgfb2 (Ctrl = 1.00 ± 0.33 N=9, Poly I:C = 1.75 ± 0.29 N=6; Mann-Whitney U test, *p < .05), Tgfb3 (Ctrl = 1.00 ± 0.29 N=9, Poly I:C = 1.33 ± 0.51 N=6), Foxf2 (Ctrl = 1.00 ± 0.28 N=9, Poly I:C = 1.71 ± 0.52 N=6) mRNAs. E. qPCR analyses of E17 female cortices for: Pecam1 (Ctrl = 1.00 ± 0.34 N=6, Poly I:C = 0.27 ± 0.08 N=8), CD248 (Ctrl = 1.00 ± 0.27 N=8, Poly I:C = 1.04 ± 0.28 N=9), Vegfa (Ctrl = 1.00 ± 0.70 N=5, Poly I:C = 0.32 ± 0.13 N=5), Pdfgb (Ctrl = 1.00 ± 0.38 N=6, Poly I:C = 0.19 ± 0.07 N=9), Tgfb1 (Ctrl = 1.00 ± 0.33 N=5, Poly I:C = 0.09 ± 0.03 N=6), Tgfb2 (Ctrl = 1.00 ± 0.32 N=7, Poly I:C = 0.45 ± 0.11 N=8), Tgfb3 (Ctrl = 1.00 ± 0.38 N=7, Poly I:C = 0.40 ± 0.20 N=9), Foxf2 (Ctrl = 1.00 ± 0.18 N=5, Poly I:C = 1.04 ± 0.28 N=8) mRNAs. F. qPCR analyses of P90 female cortices for: Pecam1 (Ctrl = 1.00 ± 0.15 N=5, Poly I:C = 0.69 ± 0.15 N=5), CD248 (Ctrl = 1.00 ± 0.13 N=5, Poly I:C = 0.58 ± 0.09 N=5), Vegfa (Ctrl = 1.00 ± 0.38 N=6, Poly I:C = 1.53 ± 0.38 N=8), Pdfgb (Ctrl = 1.00 ± 0.16 N=9, Poly I:C = 1.77 ± 0.35 N=17), Tgfb1 (Ctrl = 1.00 ± 0.26 N=13, Poly I:C = 1.30 ± 0.23 N=14), Tgfb2 (Ctrl = 1.00 ± 0.27 N=11, Poly I:C = 0.59 ± 0.06 N=4), Tgfb3 (Ctrl = 1.00 ± 0.45 N=11, Poly I:C = 0.51 ± 0.06 N=4) mRNAs. G. qPCR analyses of GD9+6h male embryos for Il1α (Ctrl = 1.00 ± 0.29 N=7, Poly I:C = 2.10 ± 0.57 N=17), Il1β (Ctrl = 1.00 ± 0.39 N=6, Poly I:C = 10.84 ± 4.70 N=6; Mann-Whitney U test, **p < .01), Il1rn (Ctrl = 1.00 ± 0.27 N=7, Poly I:C = 3.60 ± 1.58 N=11), Il6 (Ctrl = 1.00 ± 0.17 N=6, Poly I:C = 0.82 ± 0.12 N=7) mRNAs. H. qPCR analyses of E17 male cortices for: Il1α (Ctrl = 1.00 ± 0.27 N=9, Poly I:C = 1.77 ± 0.53 N=11), Il1β (Ctrl = 1.00 ± 0.21 N=7, Poly I:C = 1.33 ± 0.33 N=8), Il1rn (Ctrl = 1.00 ± 0.25 N=10, Poly I:C = 0.82 ± 0.40 N=18), Il6 (Ctrl = 1.00 ± 0.24 N=7, Poly I:C = 2.17 ± 0.43 N=8) mRNAs. I. qPCR analyses of P90 male cortices for: Il1α (Ctrl = 1.00 ± 0.24 N=8, Poly I:C = 1.51 ± 0.26 N=10), Il1β (Ctrl = 1.00 ± 0.27 N=8, Poly I:C = 1.19 ± 0.34 N=8), Il1rn (Ctrl = 1.00 ± 0.31 N=18, Poly I:C = 1.30 ± 0.50 N=14), Il6 (Ctrl = 1.00 ± 0.27 N=7, Poly I:C = 2.40 ± 0.70 N=8) mRNAs. J. qPCR analyses of GD9+6h female embryos for: Il1α (Ctrl = 1.00 ± 0.93 N=6, Poly I:C = 0.03 ± 0.00 N=4), Il1β (Ctrl = 1.00 ± 0.61 N=5, Poly I:C = 0.17 ± 0.04 N=7), Il1rn (Ctrl = 1.00 ± 0.25 N=9, Poly I:C = 0.33 ± 0.11 N=7), Il6 (Ctrl = 1.00 ± 0.48 N=7, Poly I:C = 0.59 ± 0.24 N=5) mRNAs. K. qPCR analyses of E17 female cortices for: Il1α (Ctrl = 1.00 ± 0.31 N=5, Poly I:C = 1.38 ± 0.20 N=12), Il1β (Ctrl = 1.00 ± 0.30 N=5, Poly I:C = 1.05 ± 0.35 N=7), Il1rn (Ctrl = 1.00 ± 0.55 N=6, Poly I:C = 0.45 ± 0.22 N=6), Il6 (Ctrl = 1.00 ± 0.26 N=7, Poly I:C = 0.94 ± 0.14 N=8) mRNAs. L. qPCR analyses of P90 female cortices for Il1α (Ctrl = 1.00 ± 0.08 N=5, Poly I:C = 1.32 ± 0.11 N=9), Il1β (Ctrl = 1.00 ± 0.13 N=5, Poly I:C = 0.65 ± 0.09 N=8), Il1rn (Ctrl = 1.00 ± 0.61 N=4, Poly I:C = 0.85 ± 0.29 N=7), Il6 (Ctrl = 1.00 ± 0.11 N=9, Poly I:C = 1.28 ± 0.20 N=20) mRNAs. Values are normalized over control (dashed line). 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). Bars represent mean ± SEM.


M . ELISA quantification of total TGF - β1 in E17 male embryos brains as pg / mL ( Ctrl = 132 . 6 ± 18 . 14 , Poly I : C = 129 . 6 ± 8 . 62 ) . N . ELISA quantification of active free TGF - β1 in E17 male embryos brains as pg / mL ( Ctrl = undetectable , Poly I : C = 0 . 92 ± 0 . 25 ) . O . ELISA quantification of total TGF - β1 in E17 female embryos brains as pg / mL ( Ctrl = 177 . 6 ± 10 . 88 , Poly I : C = 134 . 4 ± 13 . 59 ) . P . ELISA quantification of active free TGF - β1 in E17 female embryos brains as pg / mL ( undetectable ) . 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 ) . Bars represent mean ± SEM .

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

M. ELISA quantification of total TGF-β1 in E17 male embryos brains as pg/mL (Ctrl = 132.6 ± 18.14, Poly I:C = 129.6 ± 8.62). N. ELISA quantification of active free TGF-β1 in E17 male embryos brains as pg/mL (Ctrl = undetectable, Poly I:C = 0.92 ± 0.25). O. ELISA quantification of total TGF-β1 in E17 female embryos brains as pg/mL (Ctrl = 177.6 ± 10.88, Poly I:C = 134.4 ± 13.59). P. ELISA quantification of active free TGF-β1 in E17 female embryos brains as pg/mL (undetectable). 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). Bars represent mean ± SEM.


Q - S . Western Blotting quantification of SMAD proteins in E17 male cortices for ( Q ) pSMAD1 - 5 ( Ctrl = 0 . 99 ± 0 . 21 , Poly I : C = 2 . 67 ± 0 . 46 ) ; Mann - Whitney U test , * p < . 05 , ( R ) pSMAD2 ( Ctrl = 1 . 00 ± 0 . 04 , Poly I : C = 0 . 88 ± 0 . 24 ) , ( S ) SMAD4 ( Ctrl = 1 . 00 ± 0 . 12 , Poly I : C = 0 . 96 ± 0 . 21 ) . Protein levels were normalized to GAPDH . T - V . Western Blotting quantification of SMAD proteins in E17 female cortices for ( T ) pSMAD1 - 5 ( Ctrl = 1 . 00 ± 0 . 02 , Poly I : C = 0 . 70 ± 0 . 18 ) , ( U ) pSMAD2 ( Ctrl = 1 . 00 ± 0 . 01 , Poly I : C = 1 . 09 ± 0 . 12 ) , ( V ) SMAD4 ( Ctrl = 1 . 00 ± 0 . 16 , Poly I : C = 1 . 46 ± 0 . 57 . Protein levels were normalized to GAPDH . 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 ) . Bars represent mean ± SEM .

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

Q-S. Western Blotting quantification of SMAD proteins in E17 male cortices for (Q) pSMAD1-5 (Ctrl = 0.99 ± 0.21, Poly I:C = 2.67 ± 0.46); Mann-Whitney U test, *p < .05, (R) pSMAD2 (Ctrl = 1.00 ± 0.04, Poly I:C = 0.88 ± 0.24), (S) SMAD4 (Ctrl = 1.00 ± 0.12, Poly I:C = 0.96 ± 0.21). Protein levels were normalized to GAPDH. T-V. Western Blotting quantification of SMAD proteins in E17 female cortices for (T) pSMAD1-5 (Ctrl = 1.00 ± 0.02, Poly I:C = 0.70 ± 0.18), (U) pSMAD2 (Ctrl = 1.00 ± 0.01, Poly I:C = 1.09 ± 0.12), (V) SMAD4(Ctrl = 1.00 ± 0.16, Poly I:C = 1.46 ± 0.57. Protein levels were normalized to GAPDH. 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). Bars represent mean ± SEM.


A . TEER of the in vitro BBB models composed by a monolayer of ECs sorted from E17 Male Ctrl ( 1 . 00 ± 0 . 02 ) , Male Poly I : C ( 0 . 92 ± 0 . 04 ) , Female Ctrl ( 1 . 15 ± 0 . 04 ) , Female Poly I : C ( 1 . 00 ± 0 . 07 ) offspring . Data are normalized to male Ctrl . 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 Poly I : C males = blu bars with blu border . Cells derived from Ctrl females = white bars with purple border . Cells derived from Poly I : C females = purple bars with purple border . Numbers in bars indicate the number of animals ( N ) or independent cultures / pictures . Bars represent mean ± SEM .

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

A. TEER of the in vitro BBB models composed by a monolayer of ECs sorted from E17 Male Ctrl (1.00 ± 0.02), Male Poly I:C (0.92 ± 0.04), Female Ctrl (1.15 ± 0.04), Female Poly I:C (1.00 ± 0.07) offspring. Data are normalized to male Ctrl. 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 Poly I:C males = blu bars with blu border. Cells derived from Ctrl females = white bars with purple border. Cells derived from Poly I:C females = purple bars with purple border. Numbers in bars indicate the number of animals (N) or independent cultures/pictures. Bars represent mean ± SEM.


B . Permeability to 10 kDa Dextran of the in vitro BBB models composed by a monolayer of sorted ECs from Male Ctrl ( 1 . 00 ± 0 . 06 ) , Male Poly I : C ( 1 . 13 ± 0 . 02 ) , Female Ctrl ( 0 . 97 ± 0 . 05 ) , Female Poly I : C ( 0 . 95 ± 0 . 01 ) offspring . Mann - Whitney U test , * p < . 05 . Data are normalized to male Ctrl . 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 Poly I : C males = blu bars with blu border . Cells derived from Ctrl females = white bars with purple border . Cells derived from Poly I : C females = purple bars with purple border . Numbers in bars indicate the number of animals ( N ) or independent cultures / pictures . Bars represent mean ± SEM .

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

B. Permeability to 10 kDa Dextran of the in vitro BBB models composed by a monolayer of sorted ECs from Male Ctrl (1.00 ± 0.06), Male Poly I:C (1.13 ± 0.02), Female Ctrl (0.97 ± 0.05), Female Poly I:C (0.95 ± 0.01) offspring. Mann-Whitney U test, *p < .05. Data are normalized to male Ctrl. 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 Poly I:C males = blu bars with blu border. Cells derived from Ctrl females = white bars with purple border. Cells derived from Poly I:C females = purple bars with purple border. Numbers in bars indicate the number of animals (N) or independent cultures/pictures. Bars represent mean ± SEM.


C . TEER of barriers composed of bEND . 3 alone ( 1 . 00 ± 0 . 00 ) or co - cultured with sorted PCs from Male Ctrl ( 1 . 17 ± 0 . 07 ) , Male Poly I : C ( 0 . 95 ± 0 . 05 ) , Female Ctrl ( 1 . 15 ± 0 . 08 ) , Female Poly I : C ( 1 . 15 ± 0 . 08 ) offspring . Mann - Whitney U test , * p < . 05 . Data are normalized to bEnd . 3 . 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 Poly I : C males = blu bars with blu border . Cells derived from Ctrl females = white bars with purple border . Cells derived from Poly I : C females = purple bars with purple border . Numbers in bars indicate the number of animals ( N ) or independent cultures / pictures . Bars represent mean ± SEM .

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

C. TEER of barriers composed of bEND.3 alone (1.00 ± 0.00) or co-cultured with sorted PCs from Male Ctrl (1.17 ± 0.07), Male Poly I:C (0.95 ± 0.05), Female Ctrl (1.15 ± 0.08), Female Poly I:C (1.15 ± 0.08) offspring. Mann-Whitney U test, *p < .05. Data are normalized to bEnd.3. 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 Poly I:C males = blu bars with blu border. Cells derived from Ctrl females = white bars with purple border. Cells derived from Poly I:C females = purple bars with purple border. Numbers in bars indicate the number of animals (N) or independent cultures/pictures. Bars represent mean ± SEM.


D . Permeability to 10 kDa Dextran of barriers composed by a monolayer of bEND . 3 alone ( 1 . 00 ± 0 . 06 ) or co - cultured with sorted PCs from Male Ctrl ( 0 . 47 ± 0 . 09 ) , Male Poly I : C ( 0 . 46 ± 0 . 07 ) , Female Ctrl ( 0 . 46 ± 0 . 11 ) , Female Poly I : C ( 0 . 65 ± 0 . 05 ) offspring . One sample t test , * * p < . 01 . Data are normalized to bEnd . 3 . 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 Poly I : C males = blu bars with blu border . Cells derived from Ctrl females = white bars with purple border . Cells derived from Poly I : C females = purple bars with purple border . Numbers in bars indicate the number of animals ( N ) or independent cultures / pictures . Bars represent mean ± SEM .

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

D. Permeability to 10 kDa Dextran of barriers composed by a monolayer of bEND.3 alone (1.00 ± 0.06) or co-cultured with sorted PCs from Male Ctrl (0.47 ± 0.09), Male Poly I:C (0.46 ± 0.07), Female Ctrl (0.46 ± 0.11), Female Poly I:C (0.65 ± 0.05) offspring. One sample t test, **p < .01. Data are normalized to bEnd.3. 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 Poly I:C males = blu bars with blu border. Cells derived from Ctrl females = white bars with purple border. Cells derived from Poly I:C females = purple bars with purple border. Numbers in bars indicate the number of animals (N) or independent cultures/pictures. Bars represent mean ± SEM.


E . TEER ( Ω . cm2 ) was monitored over 72h treatment with TGF - β1 and normalized to baseline in BBB models composed by a monolayer of sorted ECs from Male ( 72h TGF - β1 = 82 . 85 ± 1 . 56 ) or Female ( 72h TGF - β1 = 85 . 92 ± 2 . 03 ) embryos . The insert shows the values at 72h ( bars ) normalized to the 0h values ( dashed line ) . One sample t test , * * * p < . 001 , * * * * p < . 0001 . 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 E blue and purple dotted lines represent cells derived from male and female 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 O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. TEER (Ω.cm2) was monitored over 72h treatment with TGF-β1 and normalized to baseline in BBB models composed by a monolayer of sorted ECs from Male (72h TGF-β1 = 82.85 ± 1.56) or Female (72h TGF-β1 = 85.92 ± 2.03) embryos. The insert shows the values at 72h (bars) normalized to the 0h values (dashed line). One sample t test, ***p < .001, ****p < .0001. 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 E blue and purple dotted lines represent cells derived from male and female brain respectively.


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 .

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

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 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 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 ) 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 .

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 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-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 O O O O O 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 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 .

O 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 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-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 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

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

O 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 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 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 .

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 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 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 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 .

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

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

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

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 + 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 .

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

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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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

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


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

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

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

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


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

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

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


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

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

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


( a ) Representative image of a Control ( left ) and Myo1cKO ( right ) embryo . Top : max projections of embryos stained with Phalloidin ( green ) and DAPI ( gray ) . Bottom : magnifications at dashed rectangles of Phalloidin ( grey ) with white arrows pointing at fragments . Data information : Mann - Whitney test p values are indicated . Scale bars , 10 µm .

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

(a) Representative image of a Control (left) and Myo1cKO (right) embryo. Top: max projections of embryos stained with Phalloidin (green) and DAPI (gray). Bottom: magnifications at dashed rectangles of Phalloidin (grey) with white arrows pointing at fragments. Data information: Mann-Whitney test p values are indicated. Scale bars, 10 µm.


( b - c ) Number of fragments without DNA ( b ) per embryo and mean radius ( c ) in Control ( grey ) and Myo1cKO ( salmon ) embryos ( left , Control n = 20 , Myo1cKO n = 20 ) . Boxplot shows median , upper and lower quartiles , min and max values . Whisker plot shows median , upper and lower quartiles . Data information : Mann - Whitney test p values are indicated . Scale bars , 10 µm .

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

(b-c) Number of fragments without DNA (b) per embryo and mean radius (c) in Control (grey) and Myo1cKO (salmon) embryos (left, Control n = 20, Myo1cKO n = 20). Boxplot shows median, upper and lower quartiles, min and max values. Whisker plot shows median, upper and lower quartiles. Data information: Mann-Whitney test p values are indicated. Scale bars, 10 µm.


( d ) Representative images of a time - lapse of Myo1cKO embryo undergoing fragmentation outside of division ( top ) and during division ( bottom ) . Max projections of embryos expressing mTmG ( grey ) and mCherry - EB3 ( grey ) imaged with light sheet microscopy are shown . White arrows point at the forming fragments . ( e ) Ratio of fragmentation events during and outside of division ( n = 13 cells from 7 embryos ) . Data information : Mann - Whitney test p values are indicated . Scale bars , 10 µm .

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

(d) Representative images of a time-lapse of Myo1cKO embryo undergoing fragmentation outside of division (top) and during division (bottom). Max projections of embryos expressing mTmG (grey) and mCherry-EB3 (grey) imaged with light sheet microscopy are shown. White arrows point at the forming fragments. (e) Ratio of fragmentation events during and outside of division (n = 13 cells from 7 embryos). Data information: Mann-Whitney test p values are indicated. Scale bars, 10 µm.


( a ) Representative images of Control ( left ) and Myo1cKO ( right ) embryos expressing mTmG and mCherry - EB3 ( grey ) shown as max projections . White arrows point at the mitotic spindle . ( b - c ) Spindle persistence in Control ( n = 6 ) and Myo1cKO ( n = 9 ) embryos ( b ) and in non - fragmenting and fragmenting cells of Myo1cKO embryos ( c , n = 42 and 12 cells from 7 Myo1cKO embryos ) . Spindle persistence measures the time from appearance to disassembly of the spindle . Data information : Mann - Whitney test p values are indicated . Boxplots show median , upper and lower quartiles , min and max values . Scale bars , 10 µm .

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

(a) Representative images of Control (left) and Myo1cKO (right) embryos expressing mTmG and mCherry-EB3 (grey) shown as max projections. White arrows point at the mitotic spindle. (b-c) Spindle persistence in Control (n = 6) and Myo1cKO (n = 9) embryos (b) and in non-fragmenting and fragmenting cells of Myo1cKO embryos (c, n = 42 and 12 cells from 7 Myo1cKO embryos). Spindle persistence measures the time from appearance to disassembly of the spindle. Data information: Mann-Whitney test p values are indicated. Boxplots show median, upper and lower quartiles, min and max values. Scale bars, 10 µm.


( f ) Representative images of embryos treated with DMSO ( left ) and 37 . 5 µM Ciliobrevin D ( right ) , stained with Phalloidin . Top : Maximum projection . Bottom : magnifications of a single plane at dashed rectangles of Phalloidin with white arrows pointing at fragments . Data information : Mann - Whitney test p values are indicated . Boxplots show median , upper and lower quartiles , min and max values . Scale bars , 10 µm .

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

(f) Representative images of embryos treated with DMSO (left) and 37.5 µM Ciliobrevin D (right), stained with Phalloidin. Top: Maximum projection. Bottom: magnifications of a single plane at dashed rectangles of Phalloidin with white arrows pointing at fragments. Data information: Mann-Whitney test p values are indicated. Boxplots show median, upper and lower quartiles, min and max values. Scale bars, 10 µm.


( d ) Top : Representative images of a time - lapse of a Myo1cKO embryo expressing Myh9 - GFP ( green ) and mTmG ( magenta ) while labelled with SiRDNA ( grey ) undergoing fragmentation during mitosis shown as max projections . Bottom : magnifications of Myh9 - GFP ( grey ) at dashed rectangles showing cortical accumulation ( middle ) followed by fragmentation ( right ) . Data information Boxplots show median , upper and lower quartiles , min and max values . Scale bars , 10 µm .

O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O 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) Top: Representative images of a time-lapse of a Myo1cKO embryo expressing Myh9-GFP (green) and mTmG (magenta) while labelled with SiRDNA (grey) undergoing fragmentation during mitosis shown as max projections. Bottom: magnifications of Myh9-GFP (grey) at dashed rectangles showing cortical accumulation (middle) followed by fragmentation (right). Data information Boxplots show median, upper and lower quartiles, min and max values. Scale bars, 10 µm.


( e ) Control ( grey ) and Myo1cKO ( salmon ) embryos Myh9 - GFP Ic / Iref values at DNA - cortex distances < 20 µm and > 20 µm ( Control embryos n = 10 , cells n = 35 ; Myo1cKO embryos n = 9 , cells n = 26 ) Data information : Kruskal - Wallis and pairwise Mann - Whitney tests ( e ) p values are indicated . Boxplots show median , upper and lower quartiles , min and max values . Scale bars , 10 µm .

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

(e) Control (grey) and Myo1cKO (salmon) embryos Myh9-GFP Ic/Iref values at DNA-cortex distances < 20 µm and > 20 µm (Control embryos n = 10, cells n = 35; Myo1cKO embryos n = 9, cells n = 26) Data information: Kruskal-Wallis and pairwise Mann-Whitney tests (e) p values are indicated. Boxplots show median, upper and lower quartiles, min and max values. Scale bars, 10 µm.


( f ) Comparison of the increase in Myh9 - GFP intensity in Control and Myo1cKO embryos when DNA is in close proximity to the cortex . The ratio Ic0 - 20 / Ic > 20 is calculated from Myh9 - GFP intensities at the contact region when DNA - cortex distances are < 20 µm ( Ic0 - 20 ) divided by intensities when distances are > 20 µm ( Ic > 20 ) ( Control embryos n = 10 , cells n = 33 ; Myo1cKO n = 9 , cells n = 26 ) . Data information : Mann - Whitney test p values are indicated . Boxplots show median , upper and lower quartiles , min and max values . Scale bars , 10 µm .

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

(f) Comparison of the increase in Myh9-GFP intensity in Control and Myo1cKO embryos when DNA is in close proximity to the cortex. The ratio Ic0-20/Ic>20 is calculated from Myh9-GFP intensities at the contact region when DNA-cortex distances are < 20 µm (Ic0-20) divided by intensities when distances are > 20 µm (Ic>20) (Control embryos n = 10, cells n = 33; Myo1cKO n = 9, cells n = 26). Data information: Mann-Whitney test p values are indicated. Boxplots show median, upper and lower quartiles, min and max values. Scale bars, 10 µm.


( a ) Representative images of Control ( left ) and Myo1cKO ( right ) embryos labelled with SiRDNA ( grey ) and expressing a reporter of Cdc42 activity ( yellow ) in one blastomere , which is undergoing mitosis , shown as max projections . The second blastomere is outlined with a dashed line . White arrows point at the accumulation of active Cdc42 at the cortex in close proximity to DNA . ( b ) The ratio of active Cdc42 Ic / Iref was compared between < 20 µm and > 20 µm DNA - cortex distances for Control ( dark yellow ) and Myo1cKO ( yellow ) embryos ( Control embryos n = 10 ; Myo1cKO embryos n = 10 ) . Data information : Kruskal - Wallis and pairwise Mann - Whitney tests ( b Boxplots show median , upper and lower quartiles , min and max values . Scale bars , 10 µm .

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 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-MEASURED_VAR 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 B-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 O O O O O 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 Control (left) and Myo1cKO (right) embryos labelled with SiRDNA (grey) and expressing a reporter of Cdc42 activity (yellow) in one blastomere, which is undergoing mitosis, shown as max projections. The second blastomere is outlined with a dashed line. White arrows point at the accumulation of active Cdc42 at the cortex in close proximity to DNA. (b) The ratio of active Cdc42 Ic/Iref was compared between < 20 µm and > 20 µm DNA-cortex distances for Control (dark yellow) and Myo1cKO (yellow) embryos (Control embryos n = 10; Myo1cKO embryos n = 10). Data information: Kruskal-Wallis and pairwise Mann-Whitney tests (b Boxplots show median, upper and lower quartiles, min and max values. Scale bars, 10 µm.


( c ) Representative images of Myo1cKO embryos expressing LifeAct - GFP ( green ) alone ( left ) or together with dominant negative Cdc42 ( DNCdc42 , right ) labelled with SiRDNA ( grey ) shown as max projections . White arrows point at the cortex in close proximity to DNA . ( d ) LifeAct - GFP Ic / Iref values at DNA - cortex distances < 20 µm and > 20 µm for Myo1cKO ( salmon ) and Myo1cKO + DNCdc42 ( yellow ) embryos ( Myo1cKO embryos n = 8 , cells n = 19 ; Myo1cKO + DNCdc42 embryos n = 12 , cells n = 30 ) . Data information : Kruskal - Wallis and pairwise Mann - Whitney tests d ) Boxplots show median , upper and lower quartiles , min and max values . Scale bars , 10 µm .

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

(c) Representative images of Myo1cKO embryos expressing LifeAct-GFP (green) alone (left) or together with dominant negative Cdc42 (DNCdc42, right) labelled with SiRDNA (grey) shown as max projections. White arrows point at the cortex in close proximity to DNA. (d) LifeAct-GFP Ic/Iref values at DNA-cortex distances < 20 µm and > 20 µm for Myo1cKO (salmon) and Myo1cKO + DNCdc42 (yellow) embryos (Myo1cKO embryos n = 8, cells n = 19; Myo1cKO + DNCdc42 embryos n = 12, cells n = 30). Data information: Kruskal-Wallis and pairwise Mann-Whitney tests d) Boxplots show median, upper and lower quartiles, min and max values. Scale bars, 10 µm.


( e ) Representative images of a GFP ( left ) and Ect2 - GFP ( right ) expressing embryo stained with Phalloidin ( green ) and DAPI ( grey ) shown as max projections . White arrows indicate fragments . ( f ) Number of fragments without DNA per embryo in GFP ( grey ) and Ect2 - GFP ( blue ) embryos ( GFP n = 14 ; Ect2 - GFP n = 18 ) . Data information Mann - Whitney test ( f ) p values are indicated . Boxplots show median , upper and lower quartiles , min and max values . Scale bars , 10 µm .

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

(e) Representative images of a GFP (left) and Ect2-GFP (right) expressing embryo stained with Phalloidin (green) and DAPI (grey) shown as max projections. White arrows indicate fragments. (f) Number of fragments without DNA per embryo in GFP (grey) and Ect2-GFP (blue) embryos (GFP n = 14; Ect2-GFP n = 18). Data information Mann-Whitney test (f) p values are indicated. Boxplots show median, upper and lower quartiles, min and max values. Scale bars, 10 µm.


( A ) Total lysates of different mouse organs underwent immunoblotting analysis using anti - Pdia4 and anti - actin antibodies . Data information : Data from 3 experiments are presented as the mean ± SD .

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

(A) Total lysates of different mouse organs underwent immunoblotting analysis using anti-Pdia4 and anti-actin antibodies. Data information: Data from 3 experiments are presented as the mean ± SD.


( C ) Min6 cells were treated with glucose and palmitate at the indicated dosages . Total lysate underwent immunoblotting analysis using the indicated antibodies . ( D ) Human islets were treated with glucose and palmitate at the indicated dosages , followed by immunoblotting analysis . ( Data information : Data from 3 experiments are presented as the mean ± SD .

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

(C) Min6 cells were treated with glucose and palmitate at the indicated dosages. Total lysate underwent immunoblotting analysis using the indicated antibodies. (D) Human islets were treated with glucose and palmitate at the indicated dosages, followed by immunoblotting analysis. ( Data information: Data from 3 experiments are presented as the mean ± SD.


( G ) Sera from wild - type ( WT ) BKS and Leprdb / db mice , aged 4 , 8 , and 18 weeks , B6 mice , fed with a normal diet ( ND ) and a high fat diet ( HFD ) for 4 weeks , and humans , healthy volunteers and diabetic patients ( T2D ) , were quantified for Pdia4 level using an anti - Pdia4 ELISA kit . Data information : Data from 3 experiments more are presented as the mean ± SD . One - way ANOVA test was used for statistical analysis of differences between groups , and P ( * ) < 0 . 05 ; P ( * * ) < 0 . 01 and P ( * * * ) < 0 . 001 are considered statistically significant .

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 B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Sera from wild-type (WT) BKS and Leprdb/db mice, aged 4, 8, and 18 weeks, B6 mice, fed with a normal diet (ND) and a high fat diet (HFD) for 4 weeks, and humans, healthy volunteers and diabetic patients (T2D), were quantified for Pdia4 level using an anti-Pdia4 ELISA kit. Data information: Data from 3 experiments more are presented as the mean ± SD. One-way ANOVA test was used for statistical analysis of differences between groups, and P (*) < 0.05; P (**) < 0.01 and P (***) < 0.001 are considered statistically significant.


( H ) Immunoblotting analysis of Pdia4 and markers in the cytosolic ( Cyto ) , nuclear ( Nuc ) , membrane ( Mem ) , mitochondrial ( Mito ) , and ER compartments of Ming 6 cells using the indicated antibodies . Data information : Data from 3 experiments are presented as the mean ± SD .

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

(H) Immunoblotting analysis of Pdia4 and markers in the cytosolic (Cyto), nuclear (Nuc), membrane (Mem), mitochondrial (Mito), and ER compartments of Ming 6 cells using the indicated antibodies. Data information: Data from 3 experiments are presented as the mean ± SD.