Neuroblastoma cells were treated with indicated concentrations of IBL - 302 for 48 hours E . N - Myc protein expression determined by western blotting . SDHA was used as loading control .

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Neuroblastoma cells were treated with indicated concentrations of IBL-302 for 48 hours E. N-Myc protein expression determined by western blotting. SDHA was used as loading control.


Neuroblastoma cells were treated with indicated concentrations of IBL - 302 for 72 hours F . Cell viability determined by CellTiterGlo .

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Neuroblastoma cells were treated with indicated concentrations of IBL-302 for 72 hours F. Cell viability determined by CellTiterGlo.


Neuroblastoma cells were treated with indicated concentrations of IBL - 302 for 48 hours G . Flow cytometry analyses of Annexin V and PI stainings following treatment with 50nM IBL - 302 H . Quantification of live and dead cells . Dead cells = PI positive , Live cells = PI negative . Dot plots from two independent experiments and error bars represent SEM .

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Neuroblastoma cells were treated with indicated concentrations of IBL-302 for 48 hours G. Flow cytometry analyses of Annexin V and PI stainings following treatment with 50nM IBL-302 H. Quantification of live and dead cells. Dead cells=PI positive, Live cells=PI negative. Dot plots from two independent experiments and error bars represent SEM.


I . Neuroblastoma SK - N - BE ( 2 ) c carrying mice ( n = 5 in each group ) were treated with vehicle ( CTRL ) or 40 mg / kg IBL - 302 for up to 35 days and tumor growth was followed over time . Asterisks indicate each occasion a mouse within that particular group was sacrificed .

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I. Neuroblastoma SK-N-BE(2)c carrying mice (n=5 in each group) were treated with vehicle (CTRL) or 40 mg/kg IBL-302 for up to 35 days and tumor growth was followed over time. Asterisks indicate each occasion a mouse within that particular group was sacrificed.


J . Kaplan - Meier survival curves comparing mice treated with vehicle ( CTRL ) or IBL - 302 . Log - rank test was used to determine statistical significance . p = 0 . 0577 .

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J. Kaplan-Meier survival curves comparing mice treated with vehicle (CTRL) or IBL-302. Log-rank test was used to determine statistical significance. p=0.0577.


A - B . LU - NB - 3 PDX , SK - N - BE ( 2 ) c and SK - N - SH cells were treated with indicated concentrations of cisplatin and / or 0 . 36 μM IBL - 202 , 0 . 05 μM IBL - 301 , or 0 . 05 μM IBL - 302 for 48 hours . Cell viability determined by CellTiterGlo . Graphs show mean values and SEM from three independent experiments . Theoretical additive curves were evaluated by calculating the combination index ( CI ) based on the Bliss Independence model .

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A-B. LU-NB-3 PDX, SK-N-BE(2)c and SK-N-SH cells were treated with indicated concentrations of cisplatin and/or 0.36 μM IBL-202, 0.05 μM IBL-301, or 0.05 μM IBL-302 for 48 hours. Cell viability determined by CellTiterGlo. Graphs show mean values and SEM from three independent experiments. Theoretical additive curves were evaluated by calculating the combination index (CI) based on the Bliss Independence model.


C - D . Cell cycle distribution in LU - NB - 3 PDX cells after treatment with 320 nM cisplatin , 50 nM IBL - 302 or the combination of these , determined by flow cytometry . Bars show mean values from two independent experiments .

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C-D. Cell cycle distribution in LU-NB-3 PDX cells after treatment with 320 nM cisplatin, 50 nM IBL-302 or the combination of these, determined by flow cytometry. Bars show mean values from two independent experiments.


E . Tumor size in individual mice from Neuroblastoma PDX carrying mice ( n = 5 in each group ) were treated with vehicle ( CTRL ) , low - dose ( 20mg / kg ) IBL - 302 , low - dose ( 1mg / kg ) cisplatin , or the combination of low - dose IBL - 302 and low - dose cisplatin for up to 70 days . Tumor size was measured over time . Asterisks indicate each occasion a mouse within that particular group was sacrificed .

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E. Tumor size in individual mice from Neuroblastoma PDX carrying mice (n=5 in each group) were treated with vehicle (CTRL), low-dose (20mg/kg) IBL-302, low-dose (1mg/kg) cisplatin, or the combination of low-dose IBL-302 and low-dose cisplatin for up to 70 days. Tumor size was measured over time. Asterisks indicate each occasion a mouse within that particular group was sacrificed.


F . Neuroblastoma PDX carrying mice ( n = 5 in each group ) were treated with vehicle ( CTRL ) , low - dose ( 20mg / kg ) IBL - 302 , low - dose ( 1mg / kg ) cisplatin , or the combination of low - dose IBL - 302 and low - dose cisplatin for up to 70 days . Tumor size was measured over time . Asterisks indicate each occasion a mouse within that particular group was sacrificed .

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F. Neuroblastoma PDX carrying mice (n=5 in each group) were treated with vehicle (CTRL), low-dose (20mg/kg) IBL-302, low-dose (1mg/kg) cisplatin, or the combination of low-dose IBL-302 and low-dose cisplatin for up to 70 days. Tumor size was measured over time. Asterisks indicate each occasion a mouse within that particular group was sacrificed.


G . Kaplan - Meier survival curves comparing mice treated with vehicle ( CTRL ) , IBL - 302 , cisplatin , or the combination . Log - rank test was used to determine statistical significance . p = 0 . 0014 .

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G. Kaplan-Meier survival curves comparing mice treated with vehicle (CTRL), IBL-302, cisplatin, or the combination. Log-rank test was used to determine statistical significance. p=0.0014.


H . Immunohistochemical staining of N - Myc expression in tumors Scale bars represent 100 μm .

H. Immunohistochemical staining of N-Myc expression in tumors Scale bars represent 100 μm.


LU - NB - 3 PDX cells were treated with 50 nM of IBL - 302 for 48 hours and subsequently analyzed by RNAseq and mass spectrometry . A - C . Heat map of significantly differentially expressed genes across treatment groups from RNAseq data ( FDR < 0 . 1 ) ( A ) . Genes defined as differentially expressed that were also present in MS and phospho - MS are visualized in ( B ) and ( C ) , respectively .

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LU-NB-3 PDX cells were treated with 50 nM of IBL-302 for 48 hours and subsequently analyzed by RNAseq and mass spectrometry. A-C. Heat map of significantly differentially expressed genes across treatment groups from RNAseq data (FDR<0.1) (A). Genes defined as differentially expressed that were also present in MS and phospho-MS are visualized in (B) and (C), respectively.


Dose - response curves representing 72 - h viability measurements for six selected BRAFV600E melanoma cell lines after treatment with vemurafenib . Ranges of estimated IC50 and Emax for the selected lines are shown . Data are represented as mean ± SD .

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Dose-response curves representing 72-h viability measurements for six selected BRAFV600E melanoma cell lines after treatment with vemurafenib. Ranges of estimated IC50 and Emax for the selected lines are shown. Data are represented as mean ± SD.


Pairwise distribution and correlation of estimated IC50 and Emax values for responses ( 72 - h viability ) of 8 cell lines to four RAF inhibitors and one MEK inhibitor . Each dot represents one drug / cell line combination . Sensitive cell lines are arbitrarily defined based on their IC50s ( the most widely used metric for evaluation of drug response ; log10 [ IC50 ] < − 6 . 5 ) .

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Pairwise distribution and correlation of estimated IC50 and Emax values for responses (72-h viability) of 8 cell lines to four RAF inhibitors and one MEK inhibitor. Each dot represents one drug/cell line combination. Sensitive cell lines are arbitrarily defined based on their IC50s (the most widely used metric for evaluation of drug response; log10[IC50] < −6.5).


c - Jun and p - c - Jun ( Ser63 ) changes as measured by RPPA in six melanoma cell lines in response to different doses of RAF and MEK inhibitors for 10 h ( or 5 h in the case of WM115cell line ) and 48 h .

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c-Jun and p-c-Jun(Ser63) changes as measured by RPPA in six melanoma cell lines in response to different doses of RAF and MEK inhibitors for 10 h (or 5 h in the case of WM115cell line) and 48 h.


B , C Synergistic apoptosis induction in four cell lines ( WM115 , WM1552C , LOXIMVI , and COLO858 ) treated for 72 h with combinations of vemurafenib and JNK - IN - 8 . ( B ) Dose - response profiles for apoptosis induction with vemurafenib and JNK - IN - 8 combination .

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B, C Synergistic apoptosis induction in four cell lines (WM115, WM1552C, LOXIMVI, and COLO858) treated for 72 h with combinations of vemurafenib and JNK-IN-8. (B) Dose-response profiles for apoptosis induction with vemurafenib and JNK-IN-8 combination.


C Synergistic apoptosis induction in four cell lines ( WM115 , WM1552C , LOXIMVI , and COLO858 ) treated for 72 h with combinations of vemurafenib and JNK - IN - 8 . ( C ) Excess over the predicted Bliss independence ( EOBI ) calculated for different combined doses of vemurafenib and JNK - IN - 8 .

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C Synergistic apoptosis induction in four cell lines (WM115, WM1552C, LOXIMVI, and COLO858) treated for 72 h with combinations of vemurafenib and JNK-IN-8. (C) Excess over the predicted Bliss independence (EOBI) calculated for different combined doses of vemurafenib and JNK-IN-8.


D c - Jun expression in WM115 cells transfected with JUN siRNA relative to no RNA and non - targeting controls quantified in triplicate 48 h after transfection .

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D c-Jun expression in WM115 cells transfected with JUN siRNA relative to no RNA and non-targeting controls quantified in triplicate 48 h after transfection.


E , F Apoptosis in WM115 cells with or without 48 h JUN knockdown after 96 - h treatment with increasing doses of

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E, F Apoptosis in WM115 cells with or without 48 h JUN knockdown after 96-h treatment with increasing doses of vemurafenib


E , F Apoptosis in WM115 cells with or without 48 h JUN knockdown after 96 - h treatment with increasing doses of selumetinib ( F )

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E, F Apoptosis in WM115 cells with or without 48 h JUN knockdown after 96-h treatment with increasing doses of selumetinib (F)


Time - dose - response plots indicating changes in RPPA measurements for p27 versus p - histone H3 for two selected cell lines ( C32 and WM115 ) after exposure to PLX4720 . Mean values of four biological replicates are shown . Protein levels represent log2 fold change of each signal ( at a specific dose and time ) relative to a DMSO - treated control .

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Time-dose-response plots indicating changes in RPPA measurements for p27 versus p-histone H3 for two selected cell lines (C32 and WM115) after exposure to PLX4720. Mean values of four biological replicates are shown. Protein levels represent log2 fold change of each signal (at a specific dose and time) relative to a DMSO-treated control.


B - D Time - dose - response plots indicating changes in RPPA measurements for pERK ( Thr202 / Tyr204 ) versus pAKT ( Ser473 ) for two selected cell lines ( C32 and WM115 ) after exposure to PLX4720 . Mean values of four biological replicates are shown . Protein levels represent log2 fold change of each signal ( at a specific dose and time ) relative to a DMSO - treated control .

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B-D Time-dose-response plots indicating changes in RPPA measurements for pERK(Thr202/Tyr204) versus pAKT(Ser473) for two selected cell lines (C32 and WM115) after exposure to PLX4720. Mean values of four biological replicates are shown. Protein levels represent log2 fold change of each signal (at a specific dose and time) relative to a DMSO-treated control.


Time - dose - response plots indicating changes in RPPA measurements for pS6 ( Ser235 / 236 ) versus total c - Jun levels for two selected cell lines ( C32 and WM115 ) after exposure to PLX4720 . Mean values of four biological replicates are shown . Protein levels represent log2 fold change of each signal ( at a specific dose and time ) relative to a DMSO - treated control .

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Time-dose-response plots indicating changes in RPPA measurements for pS6(Ser235/236) versus total c-Jun levels for two selected cell lines (C32 and WM115) after exposure to PLX4720. Mean values of four biological replicates are shown. Protein levels represent log2 fold change of each signal (at a specific dose and time) relative to a DMSO-treated control.


Covariate single - cell analysis of Ki - 67 ( left ) and pRb ( Ser807 / 811 ) ( right ) versus p - cJun ( Ser73 ) in WM1552C cells before and 24 h after exposure to 0 . 8 μM vemurafenib . Density scatter plots were generated using signal intensities for individual cells as measured by immunofluorescence microscopy . The vertical lines were used to gate p - cJunHigh versus p - cJunLow cells . The horizontal lines were used to gate Ki - 67High versus Ki - 67Low cells , and pRbHigh versus pRbLow cells .

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Covariate single-cell analysis of Ki-67 (left) and pRb(Ser807/811) (right) versus p-cJun(Ser73) in WM1552C cells before and 24 h after exposure to 0.8 μM vemurafenib. Density scatter plots were generated using signal intensities for individual cells as measured by immunofluorescence microscopy. The vertical lines were used to gate p-cJunHigh versus p-cJunLow cells. The horizontal lines were used to gate Ki-67High versus Ki-67Low cells, and pRbHigh versus pRbLow cells.


Analysis of drug dose - dependent changes in proportion of pRbLow / p - cJunHigh and pRbHigh / p - cJunHigh subpopulations in four melanoma cell lines ( WM115 , WM1552C , LOXIMVI , COLO858 ) after exposure to vemurafenib for 24 h . These subpopulations were gated as shown in ( A ) . Data are represented as mean ± SD for two

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Analysis of drug dose-dependent changes in proportion of pRbLow/p-cJunHigh and pRbHigh/p-cJunHigh subpopulations in four melanoma cell lines (WM115, WM1552C, LOXIMVI, COLO858) after exposure to vemurafenib for 24 h. These subpopulations were gated as shown in (A). Data are represented as mean ± SD for two replicates


Covariate single - cell analysis of pRb ( Ser807 / 811 ) versus pS6 ( Ser235 / 236 ) following 24 - h treatment of WM1552C cells with different doses of vemurafenib alone ( top ) and vemurafenib and JNK - IN - 8 together ( bottom ) . Drugs were added at a 1 : 1 ratio , each at indicated concentrations when used in combination . Density scatter plots were generated using signal intensities for individual cells as measured by immunofluorescence microscopy . The horizontal and vertical lines were used to gate pS6High versus pS6Low cells , and pRbHigh versus pRbLow cells , respectively .

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Covariate single-cell analysis of pRb(Ser807/811) versus pS6(Ser235/236) following 24-h treatment of WM1552C cells with different doses of vemurafenib alone (top) and vemurafenib and JNK-IN-8 together (bottom). Drugs were added at a 1:1 ratio, each at indicated concentrations when used in combination. Density scatter plots were generated using signal intensities for individual cells as measured by immunofluorescence microscopy. The horizontal and vertical lines were used to gate pS6High versus pS6Low cells, and pRbHigh versus pRbLow cells, respectively.


Selected immunofluorescence images of pS6 ( Ser235 / 236 ) and Hoechst staining in WM1552C cells in a DMSO - treated control and 24 h after exposure to 5 μM of vemurafenib , JNK - IN - 8 , and their combination .

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Selected immunofluorescence images of pS6(Ser235/236) and Hoechst staining in WM1552C cells in a DMSO-treated control and 24 h after exposure to 5 μM of vemurafenib, JNK-IN-8, and their combination.


Analysis of the changes in proportion of pS6Highcell population in three melanoma cell lines ( LOXIMVI , WM115 and WM1552C ) as a function of drug concentration for single - drug vemurafenib and JNK - IN - 8 treatments and their combination treatment . Drugs were added at a 1 : 1 ratio , each at indicated concentrations when used in combination . pS6High population of cells was gated as indicated in ( C ) . Data are represented as mean ± SD for two replicates . Data comparison between vemurafenib treatment and vemurafenib / JNK - IN - 8 combined treatment was made by using two - way analysis of variance ( ANOVA ) .

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Analysis of the changes in proportion of pS6Highcell population in three melanoma cell lines (LOXIMVI, WM115 and WM1552C) as a function of drug concentration for single-drug vemurafenib and JNK-IN-8 treatments and their combination treatment. Drugs were added at a 1:1 ratio, each at indicated concentrations when used in combination. pS6High population of cells was gated as indicated in (C). Data are represented as mean ± SD for two replicates. Data comparison between vemurafenib treatment and vemurafenib/JNK-IN-8 combined treatment was made by using two-way analysis of variance (ANOVA).


Fraction of c - JunHighcells ( as measured by single - cell ( immunofluorescence microscopy ) after 48 h JUN knockdown followed by 24 - h treatment with vemurafenib . Fold changes are shown relative to control - treated cells . Data are presented as mean ± SD .

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Fraction of c-JunHighcells (as measured by single-cell (immunofluorescence microscopy) after 48 h JUN knockdown followed by 24-h treatment with vemurafenib. Fold changes are shown relative to control-treated cells. Data are presented as mean ± SD.


Single - cell pS6 ( Ser235 / 236 ) levels in the c - JunHigh and c - JunLow fractions of cells ( as measured by single - cell multiplex immunofluorescence microscopy ) after 48 h of JUN knockdown and 24 - h treatment with 0 . 32 μM vemurafenib . Single - cell pS6 data are presented as box - and - whisker plots with median signal intensities and interquartile ranges ; bars extending to 1 . 5 × the interquartile range are shown for each condition as a measure of variance . P - values were calculated using a two - sided nonparametric Mann - Whitney U - test .

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Single-cell pS6(Ser235/236) levels in the c-JunHigh and c-JunLow fractions of cells (as measured by single-cell multiplex immunofluorescence microscopy) after 48 h of JUN knockdown and 24-h treatment with 0.32 μM vemurafenib. Single-cell pS6 data are presented as box-and-whisker plots with median signal intensities and interquartile ranges; bars extending to 1.5× the interquartile range are shown for each condition as a measure of variance. P-values were calculated using a two-sided nonparametric Mann-Whitney U-test.


Pairwise Spearman ' s correlation between the 1 - h changes in pERK ( Thr202 / Tyr204 ) levels as measured by immunofluorescence microscopy and 72 - h relative viability both represented by the z - score - scaled AUC of the seven dose - response curves for four RAF inhibitors and one MEK inhibitor across 8 / 10 cell lines investigated in this study .

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Pairwise Spearman's correlation between the 1-h changes in pERK(Thr202/Tyr204) levels as measured by immunofluorescence microscopy and 72-h relative viability both represented by the z-score-scaled AUC of the seven dose-response curves for four RAF inhibitors and one MEK inhibitor across 8/10 cell lines investigated in this study.


Comparison of the level of 1 - h pERK inhibition for 8 cell lines treated with SB590885 versus PLX4720 .

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Comparison of the level of 1-h pERK inhibition for 8 cell lines treated with SB590885 versus PLX4720.


Comparison of the 72 - h measured relative viability AUC for 8 / 10 cell lines treated with SB590885 versus

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Comparison of the 72-h measured relative viability AUC for 8/10 cell lines treated with SB590885 versus PLX4720


Comparison of the level of 1 - h pERK inhibition between two cell lines ( C32 and WM1552C ) treated with four RAF inhibitors and one MEK inhibitor .

Comparison of the level of 1-h pERK inhibition between two cell lines (C32 and WM1552C) treated with four RAF inhibitors and one MEK inhibitor.


Comparison of the 72 - h measured relative viability AUC between two cell lines ( C32 and WM1552C ) treated with four RAF inhibitors and one MEK inhibitor .

Comparison of the 72-h measured relative viability AUC between two cell lines (C32 and WM1552C) treated with four RAF inhibitors and one MEK inhibitor.


Pairwise Spearman ' s correlation between the 24 - h changes in pS6 ( Ser235 / 236 ) levels as measured by immunofluorescence microscopy and 72 - h relative viability both represented by the z - score - scaled AUC of the dose - response curves for four RAF inhibitors and one MEK inhibitor across 8 / 10 cell lines investigated in this study .

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Pairwise Spearman's correlation between the 24-h changes in pS6(Ser235/236) levels as measured by immunofluorescence microscopy and 72-h relative viability both represented by the z-score-scaled AUC of the dose-response curves for four RAF inhibitors and one MEK inhibitor across 8/10 cell lines investigated in this study.


Pairwise Spearman ' s correlation between the 24 - h p - cJun levels as measured by immunofluorescence microscopy and 72 - h relative viability after treatment with PLX4720 for 8 cell lines . p - cJun levels are averaged over five doses ( 0 . 1 - 5 μM ) , and viability is represented by the AUC of the dose - response curves .

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Pairwise Spearman's correlation between the 24-h p-cJun levels as measured by immunofluorescence microscopy and 72-h relative viability after treatment with PLX4720 for 8 cell lines. p-cJun levels are averaged over five doses (0.1-5 μM), and viability is represented by the AUC of the dose-response curves.


Comparison of the level of 24 - h pS6 inhibition for 8 cell lines treated with SB590885 versus PLX4720 .

Comparison of the level of 24-h pS6 inhibition for 8 cell lines treated with SB590885 versus PLX4720.


Comparison of the level of 24 - h pS6 inhibition between two cell lines ( C32 and WM1552C ) treated with four RAF inhibitors and one MEK

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

Comparison of the level of 24-h pS6 inhibition between two cell lines (C32 and WM1552C) treated with four RAF inhibitors and one MEK inhibitor


A . 3D rendering of the cerebrovascular tree in brain slices from male P90 Ctrl and Poly I : C MIA offspring stained with intravascular DiI ( red ) . Scale bar = 50 µm . B . DiI - positive blood vessels geometry analysis - quantification of blood vessel number , diameter ( mean and cumulative frequency distribution ) , length , number of branch segments and average branch length - of P90 male Ctrl and Poly I : C MIA offspring . Vessel number for Ctrl ( 20 . 93 ± 1 . 73 ) and Poly I : C ( 27 . 41 ± 1 . 99 ) MIA offspring , Mann - Whitney U test , * * p < . 01 . Mean diameter for Ctrl ( 5 . 58 ± 0 . 01 ) and Poly I : C ( 4 . 96 ± 0 . 01 ) MIA offspring , Mann - Whitney U test , * * * * p < . 0001 . Total length of vessel per image for Ctrl ( 6 . 12 ± 0 . 76 ) and Poly I : C ( 9 . 83 ± 1 . 48 ) MIA offspring , Mann - Whitney U test , * * p < . 01 . Number of branches for Ctrl ( 9 . 60 ± 0 . 54 ) and Poly I : C ( 15 . 80 ± 1 . 21 ) MIA offspring , Mann - Whitney U test , * p < . 05 . Average branch length for Ctrl ( 34 . 07 ± 1 . 35 ) and Poly I : C ( 33 . 47 ± 1 . 67 ) MIA offspring . C . 3D rendering of the cerebrovascular tree in brain slices from female P90 Ctrl and Poly I : C MIA offspring stained with intravascular DiI ( red ) . Scale bar = 50 µm . D . DiI - positive blood vessels geometry analysis - quantification of blood vessel number , diameter ( mean and cumulative frequency distribution ) , length , number of branch segments and average branch length - of P90 female Ctrl and Poly I : C MIA offspring . Vessel number for Ctrl ( 9 . 97 ± 1 . 18 ) and Poly I : C ( 8 . 61 ± 0 . 78 ) MIA offspring . Mean diameter for Ctrl ( 5 . 77 ± 0 . 03 ) and Poly I : C ( 6 . 16 ± 0 . 02 ) MIA offspring , Mann - Whitney U test , * * * * p < . 0001 . Total length of vessel per image for Ctrl ( 3 . 23 ± 0 . 50 ) and Poly I : C ( 2 . 97 ± 0 . 30 ) MIA offspring . Number of branches for Ctrl ( 10 . 50 ± 0 . 87 ) and Poly I : C ( 7 . 24 ± 0 . 44 ) MIA offspring . Average branch length for Ctrl ( 38 . 06 ± 2 . 17 ) and Poly I : C ( 46 . 93 ± 1 . 49 ) MIA offspring , Mann - Whitney U test , * * * p < . 001 . 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 . In Fig . 1 B and D black dotted lines represent the Ctrl while the blue and purple solid line represent the male Poly I : C and female Poly I : C offspring respectively . 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 B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O

A. 3D rendering of the cerebrovascular tree in brain slices from male P90 Ctrl and Poly I:C MIA offspring stained with intravascular DiI (red). Scale bar = 50 µm. B. DiI-positive blood vessels geometry analysis - quantification of blood vessel number, diameter (mean and cumulative frequency distribution), length, number of branch segments and average branch length - of P90 male Ctrl and Poly I:C MIA offspring. Vessel number for Ctrl (20.93 ± 1.73) and Poly I:C (27.41 ± 1.99) MIA offspring, Mann-Whitney U test, **p < .01. Mean diameter for Ctrl (5.58 ± 0.01) and Poly I:C (4.96 ± 0.01) MIA offspring, Mann-Whitney U test, ****p < .0001. Total length of vessel per image for Ctrl (6.12 ± 0.76) and Poly I:C (9.83 ± 1.48) MIA offspring, Mann-Whitney U test, **p < .01. Number of branches for Ctrl (9.60 ± 0.54) and Poly I:C (15.80 ± 1.21) MIA offspring, Mann-Whitney U test, *p < .05. Average branch length for Ctrl (34.07 ± 1.35) and Poly I:C (33.47 ± 1.67) MIA offspring. C. 3D rendering of the cerebrovascular tree in brain slices from female P90 Ctrl and Poly I:C MIA offspring stained with intravascular DiI (red). Scale bar = 50 µm. D. DiI-positive blood vessels geometry analysis - quantification of blood vessel number, diameter (mean and cumulative frequency distribution), length, number of branch segments and average branch length - of P90 female Ctrl and Poly I:C MIA offspring. Vessel number for Ctrl (9.97 ± 1.18) and Poly I:C (8.61 ± 0.78) MIA offspring. Mean diameter for Ctrl (5.77 ± 0.03) and Poly I:C (6.16 ± 0.02) MIA offspring, Mann-Whitney U test, ****p < .0001. Total length of vessel per image for Ctrl (3.23 ± 0.50) and Poly I:C (2.97 ± 0.30) MIA offspring. Number of branches for Ctrl (10.50 ± 0.87) and Poly I:C (7.24 ± 0.44) MIA offspring. Average branch length for Ctrl (38.06 ± 2.17) and Poly I:C (46.93 ± 1.49) MIA offspring, Mann-Whitney U test, ***p < .001. 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. In Fig.1 B and D black dotted lines represent the Ctrl while the blue and purple solid line represent the male Poly I:C and female Poly I:C offspring respectively. Numbers in bars indicate the number of animals (N) and images (n). Bars represent mean ± SEM.


E . Quantification of in - vivo brain permeability at P30 in male Ctrl ( 0 . 00 ± 0 . 00 ) and Poly I : C ( 3 . 68 ± 1 . 98 ) treated offspring , Mann - Whitney U test , p = 0 . 060 . 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 B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E. Quantification of in-vivo brain permeability at P30 in male Ctrl (0.00 ± 0.00) and Poly I:C (3.68 ± 1.98) treated offspring, Mann-Whitney U test, p=0.060. 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.


F . Quantification of Evans Blue ( EB ) analysis of in - vivo brain permeability at P90 ( top ) and representative pictures ( bottom ) of perfused brains from Male Ctrl ( left ; 0 . 00 ± 0 . 00 ) and Poly I : C ( right ; 20 . 32 ± 10 . 95 ) offspring . Mann - Whitney U test , * p < . 05 . In accompanying 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 . 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 B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F. Quantification of Evans Blue (EB) analysis of in-vivo brain permeability at P90 (top) and representative pictures (bottom) of perfused brains from Male Ctrl (left; 0.00 ± 0.00) and Poly I:C (right; 20.32 ± 10.95) offspring. Mann-Whitney U test, *p < .05. In accompanying 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. 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.


G . Quantitative analysis of the optical density of Claudin - 5 ( CLDN5 ) immunoreactive bands normalized to Calnexin ( CANX ) in cortices from P90 male Ctrl ( 0 . 99 ± 0 . 09 ) and Poly I : C ( 0 . 58 ± 0 . 05 ) offspring . 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 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 B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

G. Quantitative analysis of the optical density of Claudin-5 (CLDN5) immunoreactive bands normalized to Calnexin (CANX) in cortices from P90 male Ctrl (0.99 ± 0.09) and Poly I:C (0.58 ± 0.05) offspring. 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 images (n). Bars represent mean ± SEM.


H . Quantitative analysis of the optical density of ZO - 1 immunoreactive bands normalized by α - Tubulin ( αTUB ) in cortices from P90 male Ctrl ( 1 . 00 ± 0 . 04 ) and Poly I : C ( 0 . 75 ± 0 . 08 ) 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 O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

H. Quantitative analysis of the optical density of ZO-1 immunoreactive bands normalized by α-Tubulin (αTUB) in cortices from P90 male Ctrl (1.00 ± 0.04) and Poly I:C (0.75 ± 0.08) 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.


I . Quantification of in - vivo brain permeability at P30 in female Ctrl ( 0 . 58 ± 0 . 17 ) and Poly I : C ( 0 . 31 ± 0 . 14 ) treated offspring . Data information : 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 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 B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

I. Quantification of in-vivo brain permeability at P30 in female Ctrl (0.58 ± 0.17) and Poly I:C (0.31 ± 0.14) treated offspring. Data information: 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 images (n). Bars represent mean ± SEM.


J . Quantification of Evans Blue ( EB ) analysis of in - vivo brain permeability at P90 ( top ) and representative pictures ( bottom ) of perfused brains from Female Ctrl ( left ; 0 . 70 ± 0 . 47 ) and Poly I : C ( right ; 0 . 10 ± 0 . 10 ) offspring . In accompanying 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 . Data information : 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 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 B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

J. Quantification of Evans Blue (EB) analysis of in-vivo brain permeability at P90 (top) and representative pictures (bottom) of perfused brains from Female Ctrl (left; 0.70 ± 0.47) and Poly I:C (right; 0.10 ± 0.10) offspring. In accompanying 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. Data information: 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 images (n). Bars represent mean ± SEM.


K . Quantitative analysis of the optical density of Claudin - 5 ( CLDN5 ) immunoreactive bands normalized by CANX in cortices from P90 female Ctrl ( 1 . 00 ± 0 . 16 ) and Poly I : C ( 1 . 39 ± 0 . 13 ) offspring . Data information : 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 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 B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

K. Quantitative analysis of the optical density of Claudin-5 (CLDN5) immunoreactive bands normalized by CANX in cortices from P90 female Ctrl (1.00 ± 0.16) and Poly I:C (1.39 ± 0.13) offspring. Data information: 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 images (n). Bars represent mean ± SEM.


L . Quantitative analysis of the optical density of ZO - 1 immunoreactive bands normalized by αTUB in cortices from P90 female Ctrl ( 1 . 00 ± 0 . 11 ) and Poly I : C ( 1 . 19 ± 0 . 19 ) offspring . Data information : 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 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 B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

L. Quantitative analysis of the optical density of ZO-1 immunoreactive bands normalized by αTUB in cortices from P90 female Ctrl (1.00 ± 0.11) and Poly I:C (1.19 ± 0.19) offspring. Data information: 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 images (n). Bars represent mean ± SEM.


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

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

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

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

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

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

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

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

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

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

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

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

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

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.