Cell fractionation upon inhibition of DPP9 activity . HeLa cells were incubated for 4 days with the DPP9 inhibitor 1G244 or as control with DMSO . Cells were subjected to fractionation into ' cytosol ' ( supernatant ) and ' mitochondria ' ( pellet ) by differential digitonin lysis and centrifugation . The IMS marker proteins CPOX and cytochrome c were exclusively localized to the pellet fraction at one specific digitonin concentration ( lanes 2 , 5 , 8 , 11 ) irrespective of 1G244 treatment . Likewise , the cytosolic marker protein LDH localized under all conditions to the same fractions . Conversely , endogenous AK2 appeared at this digitonin concentration in the ' cytosol ' fraction but only upon 1G244 treatment ( compare lanes 2 and 5 ) . Thus , inhibition of DPP9 results in appearance of AK2 in the cytosol . Quantification shows loading - control adjusted AK2 - signals of 1G244 - treated ' cytosol ' fraction ( lane 5 ) over DMSO - treated ' cytosol ' fraction ( lane 2 ) . White arrow head indicates the endogenous AK2 appearing in the cytosolic fraction upon 1G244 treatment . Reported values are the mean of 3 independent experiments ; error bars represent ± SD . Student ' s t - test was performed .

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

Cell fractionation upon inhibition of DPP9 activity. HeLa cells were incubated for 4 days with the DPP9 inhibitor 1G244 or as control with DMSO. Cells were subjected to fractionation into 'cytosol' (supernatant) and 'mitochondria' (pellet) by differential digitonin lysis and centrifugation. The IMS marker proteins CPOX and cytochrome c were exclusively localized to the pellet fraction at one specific digitonin concentration (lanes 2,5,8,11) irrespective of 1G244 treatment. Likewise, the cytosolic marker protein LDH localized under all conditions to the same fractions. Conversely, endogenous AK2 appeared at this digitonin concentration in the 'cytosol' fraction but only upon 1G244 treatment (compare lanes 2 and 5). Thus, inhibition of DPP9 results in appearance of AK2 in the cytosol. Quantification shows loading-control adjusted AK2-signals of 1G244-treated 'cytosol' fraction (lane 5) over DMSO-treated 'cytosol' fraction (lane 2). White arrow head indicates the endogenous AK2 appearing in the cytosolic fraction upon 1G244 treatment. Reported values are the mean of 3 independent experiments; error bars represent ±SD. Student's t-test was performed.


Localization of DPP9 - insensitive AK2 - HA variants . Experiment was performed as described in Figure 2C . Compared to the wild type , DPP9 - insensitive and import - competent AK2 - HA variants co - localized with cytosolic markers indicating partial cytosolic localization . The colocalization of AK2 - HA variants with Mitotracker was quantified using Fiji ( Pearson ' s correlation coefficient ) . A one - way ANOVA with post - hoc Tukey test was performed . Numbers of analysed cells are indicated in the plot . * * represents p < 0 . 01 . Data shown in Figures 2C , 5B , S2 and S5 are derived from the same experiments . The black horizontal line represents the median . Data information : Scale bars : 10 μm .

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

Localization of DPP9-insensitive AK2-HA variants. Experiment was performed as described in Figure 2C. Compared to the wild type, DPP9-insensitive and import-competent AK2-HA variants co-localized with cytosolic markers indicating partial cytosolic localization. The colocalization of AK2-HA variants with Mitotracker was quantified using Fiji (Pearson's correlation coefficient). A one-way ANOVA with post-hoc Tukey test was performed. Numbers of analysed cells are indicated in the plot. ** represents p<0.01. Data shown in Figures 2C, 5B, S2 and S5 are derived from the same experiments. The black horizontal line represents the median. Data information: Scale bars: 10 μm.


Localization of AK2C40S - HA upon proteasomal inhibition . Experiment was performed as described in Figure 2C . Upon MG132 treatment , colocalization of AK2C40S - HA with the cytosolic marker increased . A one - way ANOVA with post - hoc Tukey test was performed . Numbers of analysed cells are indicated in the plot . * * * represents p < 0 . 001 . The black horizontal line represents the median . Data information : Scale bars : 10 μm .

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

Localization of AK2C40S-HA upon proteasomal inhibition. Experiment was performed as described in Figure 2C. Upon MG132 treatment, colocalization of AK2C40S-HA with the cytosolic marker increased. A one-way ANOVA with post-hoc Tukey test was performed. Numbers of analysed cells are indicated in the plot. *** represents p<0.001.The black horizontal line represents the median. Data information: Scale bars: 10 μm.


AK2 activity in dependence of its substrate AMP . Heterologously expressed and purified AK2 variants exhibited maximal activity at around 40 µM AMP with an apparent KM of 0 . 012 mM AMP . The profiles for both AK2C40S , C232S and AK2C40S , C42S , C92S , C232S are superimposable . Reported values are the mean of at least 3 independent experiments ; error bars represent ± SD . AK2 activity in dependence of its substrate ADP . Maximal activity was observed at around 1 - 2 mM ADP with an apparent KM of 0 . 12 mM and 0 . 1 mM ADP for AK2C40S , C232S and AK2C40S , C42S , C92S , C232S , respectively . The profiles for both AK2C40S , C232S and AK2C40S , C42S , C92S , C232S are thus highly similar . Reported values are the mean of at least 3 independent experiments ; error bars represent ± SD .

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

AK2 activity in dependence of its substrate AMP. Heterologously expressed and purified AK2 variants exhibited maximal activity at around 40 µM AMP with an apparent KM of 0.012 mM AMP. The profiles for both AK2C40S,C232S and AK2C40S,C42S,C92S,C232S are superimposable. Reported values are the mean of at least 3 independent experiments; error bars represent ±SD.   AK2 activity in dependence of its substrate ADP. Maximal activity was observed at around 1-2 mM ADP with an apparent KM of 0.12 mM and 0.1 mM ADP for AK2C40S,C232S and AK2C40S,C42S,C92S,C232S, respectively. The profiles for both AK2C40S,C232S and AK2C40S,C42S,C92S,C232S are thus highly similar. Reported values are the mean of at least 3 independent experiments; error bars represent ±SD.


Activity assay for AK2 - HA variants isolated from HEK293 cells . Indicated AK2 - HA variants were isolated by native immunoprecipitation from HEK293 cells stably expressing these variants . Then , the AK2 activity assay described in ( B ) was performed . While import - competent AK2 variants exhibited full activity , a cytosolic AK2 variant with the native N - terminus showed very low activity . This activity strongly increased upon incubation with the DPP9 inhibitor 1G244 or upon using DPP9 - insensitive cytosolic AK2 variants . The enzymatic inactive K28Q variants served as negative control indicating that only AK2 - HA - dependent activity ways measured by our assay . Reported values are the mean of 2 independent experiments ; error bars represent ± SD .

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

Activity assay for AK2-HA variants isolated from HEK293 cells. Indicated AK2-HA variants were isolated by native immunoprecipitation from HEK293 cells stably expressing these variants. Then, the AK2 activity assay described in (B) was performed. While import-competent AK2 variants exhibited full activity, a cytosolic AK2 variant with the native N-terminus showed very low activity. This activity strongly increased upon incubation with the DPP9 inhibitor 1G244 or upon using DPP9-insensitive cytosolic AK2 variants. The enzymatic inactive K28Q variants served as negative control indicating that only AK2-HA-dependent activity ways measured by our assay. Reported values are the mean of 2 independent experiments; error bars represent ±SD.


Steady state levels of NDUFB10 , NDUFS5 , NDUFA8 , COA6 , CHCHD2 , and TIMM9 upon incubation with the DPP9 inhibitor 1G244 . Cells were treated for 24h with inhibitor or DMSO as control , and were then analysed by immunoblot against the indicated proteins . Proteins except TIMM9 are stabilized upon DPP9 inhibition . Reported values are the mean of 3 - 5 independent experiments ; error bars represent ± SD . Student ' s t - test was performed . * * represents p < 0 . 01 .

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

Steady state levels of NDUFB10, NDUFS5, NDUFA8, COA6, CHCHD2, and TIMM9 upon incubation with the DPP9 inhibitor 1G244. Cells were treated for 24h with inhibitor or DMSO as control, and were then analysed by immunoblot against the indicated proteins. Proteins except TIMM9 are stabilized upon DPP9 inhibition. Reported values are the mean of 3-5 independent experiments; error bars represent ±SD. Student's t-test was performed. ** represents p<0.01.


Steady state levels of NDUFB10 variants upon incubation with the DPP9 inhibitor 1G244 . Cells were treated for 24h with inhibitor or DMSO as control , and were then analysed by immunoblot against the indicated proteins . NDUFB10C107S is stabilized upon DPP9 inhibition . Reported values are the mean of 4 independent experiments ; error bars represent ± SD . Student ' s t - test was performed for the respective control and treatment samples . * * represents p < 0 . 01 .

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

Steady state levels of NDUFB10 variants upon incubation with the DPP9 inhibitor 1G244. Cells were treated for 24h with inhibitor or DMSO as control, and were then analysed by immunoblot against the indicated proteins. NDUFB10C107S is stabilized upon DPP9 inhibition. Reported values are the mean of 4 independent experiments; error bars represent ±SD. Student's t-test was performed for the respective control and treatment samples. ** represents p<0.01.


Localization of NDUFB10 variants upon 1G244 treatment . Experiment was performed as in ( C ) except that mitochondria were isolated and the fractions analysed by immunoblot against the indicated proteins . Mitochondria contain increased NDUFB10C107S levels after DPP9 inhibition . Reported values are the mean of 2 independent experiments ; error bars represent ± SD . Student ' s t - test was performed . * represents p < 0 . 05 .

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

Localization of NDUFB10 variants upon 1G244 treatment. Experiment was performed as in (C) except that mitochondria were isolated and the fractions analysed by immunoblot against the indicated proteins. Mitochondria contain increased NDUFB10C107S levels after DPP9 inhibition. Reported values are the mean of 2 independent experiments; error bars represent ±SD. Student's t-test was performed. * represents p<0.05.


A Pup survival analysis between P0 - P10 , by sex . Each dot represents the absolute percent of remaining animals per group ( Jag1CTRL n = 211 , male Jag1Ndr / Ndr n = 26 , female Jag1Ndr / Ndr n = 20 , log - rank test , * * * * P < 0 . 0001 ) .

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

A Pup survival analysis between P0-P10, by sex. Each dot represents the absolute percent of remaining animals per group (Jag1CTRL n=211, male Jag1Ndr/Ndr n=26, female Jag1Ndr/Ndr n= 20, log-rank test, ****P<0.0001).


B P10 plasma total bilirubin levels ( n = 6 per group , unpaired t test , * * P = 0 . 0091 ) . C P10 plasma thrombin antithrombin levels ( n = 6 per group , unpaired t test , ns , P = 0 . 2788 ) .

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

B P10 plasma total bilirubin levels (n=6 per group, unpaired t test, **P=0.0091). C P10 plasma thrombin antithrombin levels (n=6 per group, unpaired t test, ns, P=0.2788).


D P10 serum fibrinogen levels ( n = 6 per group , unpaired t test , ns , P = 0 . 7585 ) .

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

D P10 serum fibrinogen levels (n=6 per group, unpaired t test, ns, P=0.7585).


E P10 whole blood prothrombin time ( INR ) ( n = 6 per group , unpaired t test , ns , P > 0 . 9999 ) .

O O O O O 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 P10 whole blood prothrombin time (INR) (n=6 per group, unpaired t test, ns, P>0.9999).


F P10 tail bleeding time ( n = 6 per group , unpaired t test , ns , P = 0 . 8916 ) .

O 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 P10 tail bleeding time (n=6 per group, unpaired t test, ns, P=0.8916).


G - I µCT of P30 skulls . ( G ) Color map depicting wall thickness . Scale bar 3 . 5 mm . ( H ) Skull full thickness volume ( n = 5 - 6 per group , two - way ANOVA . Interaction P = 0 . 7595 ; Sex P = 0 . 1164 , Genotype * * P = 0 . 0012 . Šídák ' s multiple comparisons test : Male Jag1 + / + vs Jag1Ndr / Ndr * P = 0 . 0227 ; : Female Jag1 + / + vs Jag1Ndr / Ndr * P = 0 . 034 ) , ( I ) Compact bone total volume ( n = 5 - 6 per group , two - way ANOVA . Interaction P = 0 . 8631 ; Sex P = 0 . 0901 , Genotype * * P = 0 . 0014 . Šídák ' s multiple comparisons test : Male Jag1 + / + vs Jag1Ndr / Ndr * P = 0 . 0305 ; Female Jag1 + / + vs Jag1Ndr / Ndr * P = 0 . 0307 ) .

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

G-I µCT of P30 skulls. (G) Color map depicting wall thickness. Scale bar 3.5 mm. (H) Skull full thickness volume (n=5-6 per group, two-way ANOVA. Interaction P= 0.7595; Sex P= 0.1164, Genotype **P= 0.0012. Šídák's multiple comparisons test: Male Jag1+/+ vs Jag1Ndr/Ndr *P= 0.0227; : Female Jag1+/+ vs Jag1Ndr/Ndr *P= 0.034), (I) Compact bone total volume (n=5-6 per group, two-way ANOVA. Interaction P= 0.8631; Sex P= 0.0901, Genotype **P= 0.0014. Šídák's multiple comparisons test: Male Jag1+/+ vs Jag1Ndr/Ndr *P= 0.0305; Female Jag1+/+ vs Jag1Ndr/Ndr *P= 0.0307).


J - K ( J ) Provoked vascular accidents induced by portal vein resin injection , with ( K ) resin leakage quantification outside of blood vessels ( red ) in adult mice ( n = 6 per group , unpaired t test , ns , P = 0 . 1088 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-K (J) Provoked vascular accidents induced by portal vein resin injection, with (K) resin leakage quantification outside of blood vessels (red) in adult mice (n=6 per group, unpaired t test, ns, P=0.1088).


L Adult brain Evans blue assay ( Jag1CTRL n = 7 , Jag1Ndr / Ndr n = 8 , unpaired t test , ns , P = 0 . 3807 ) .

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

L Adult brain Evans blue assay (Jag1CTRL n=7, Jag1Ndr/Ndr n=8, unpaired t test, ns, P=0.3807).


M Hemorrhagic P3 Jag1Ndr / Ndr brain ( n = 2 / 83 Jag1Ndr / Ndr mice , sex n . d . ) .

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

M Hemorrhagic P3 Jag1Ndr/Ndr brain (n= 2/83 Jag1Ndr/Ndr mice, sex n.d.).


N Hemorrhagic P15 Jag1Ndr / Ndr retina . Scale bar 20 µm . Sex n . d .

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

N Hemorrhagic P15 Jag1Ndr/Ndr retina. Scale bar 20 µm. Sex n.d.


O Red blood cells outside P10 Jag1Ndr / Ndr retinal arteriole . Scale bar 10 µm .

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

O Red blood cells outside P10 Jag1Ndr/Ndr retinal arteriole. Scale bar 10 µm.


( A ) Radial arrangement of arterioles ( magenta ) and venules ( green ) from the optic nerve . Blue arrows label arteriovenous crossings . Magnification of crossing shows compression / narrowing of the underlying vein . Scale bar left and middle panels 50 µm , and right panel 20 µm .

(A) Radial arrangement of arterioles (magenta) and venules (green) from the optic nerve. Blue arrows label arteriovenous crossings. Magnification of crossing shows compression/narrowing of the underlying vein. Scale bar left and middle panels 50 µm, and right panel 20 µm.


( B ) Arteriovenous crossings number per retina ( n = 6 per group , two - way ANOVA with Tukey ' s multiple comparisons test . Interaction P = 0 . 7074 , Sex P = > 0 . 9999 , Genotype * * * * P = < 0 . 0001 , Tukey ' s multiple comparisons test : Male : Jag1 + / + vs . Male : Jag1Ndr / Ndr * P = 0 . 0201 ; Female : Jag1 + / + vs . Female : Jag1Ndr / Ndr * * P = 0 . 0061 ) .

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

(B) Arteriovenous crossings number per retina (n=6 per group, two-way ANOVA with Tukey's multiple comparisons test. Interaction P = 0.7074, Sex P =>0.9999, Genotype ****P= <0.0001, Tukey's multiple comparisons test: Male:Jag1+/+ vs. Male:Jag1Ndr/Ndr *P= 0.0201; Female:Jag1+/+ vs. Female:Jag1Ndr/Ndr **P= 0.0061).


C - D ( C ) Number of major arterioles ( n = 6 per group , two - way ANOVA with Tukey ' s multiple comparisons test . Interaction P = 0 . 4353 , Sex P = 0 . 1995 , Genotype * * * * P = < 0 . 0001 , Tukey ' s multiple comparisons test : Male : Jag1 + / + vs . Male : Jag1Ndr / Ndr * P = 0 . 0146 ; Female : Jag1 + / + vs . Female : Jag1Ndr / Ndr * * P = 0 . 0011 ) and ( D ) venules ( n = 6 per group , two - way ANOVA with Tukey ' s multiple comparisons test . Interaction P = 0 . 044 , Sex P = 0 . 044 , Genotype * * * * P = < 0 . 0001 , Tukey ' s multiple comparisons test : Male : Jag1Ndr / Ndr vs . Female : Jag1Ndr / Ndr * P = 0 . 0302 ; Female : Jag1 + / + vs . Female : Jag1Ndr / Ndr * * * P = 0 . 0002 ) .

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

C-D (C) Number of major arterioles (n=6 per group, two-way ANOVA with Tukey's multiple comparisons test. Interaction P = 0.4353, Sex P =0.1995, Genotype ****P= <0.0001, Tukey's multiple comparisons test: Male:Jag1+/+ vs. Male:Jag1Ndr/Ndr *P= 0.0146; Female:Jag1+/+ vs. Female:Jag1Ndr/Ndr **P= 0.0011) and (D) venules (n=6 per group, two-way ANOVA with Tukey's multiple comparisons test. Interaction P = 0.044, Sex P =0.044, Genotype ****P= <0.0001, Tukey's multiple comparisons test: Male:Jag1Ndr/Ndr vs. Female:Jag1Ndr/Ndr *P=0.0302; Female:Jag1+/+ vs. Female:Jag1Ndr/Ndr ***P= 0.0002).


E - F ( E ) Arterial tortuosity at P30 and 1 year , irrespective of sex ( n = 6 per group , two - way ANOVA , not significant ) . ( F ) Arterial tortuosity in male and female mice , irrespective of age ( n = 5 - 7 , two - way ANOVA , not significant ) . G - H ( G ) Venous tortuosity at P30 and 1 year , irrespective of sex ( n = 6 - 8 per group , two - way ANOVA with Tukey ' s multiple comparisons test . Interaction P = 0 . 1166 , Age P = 0 . 0787 , Genotype * * P = 0 . 0013 . Tukey ' s multiple comparisons test : P30 : Jag1 + / + vs . P30 : Jag1Ndr / Ndr * * P = 0 . 004 ; P30 : Jag1Ndr / Ndr vs . 1 year : Jag1Ndr / Ndr P = 0 . 0802 ; 1 year : Jag1 + / + vs . 1 year : Jag1Ndr / Ndr P = 0 . 5066 ) . ( H ) Venous tortuosity in male and female mice , irrespective of age ( n = 5 - 7 , two - way ANOVA with Tukey ' s multiple comparisons test . Interaction * P = 0 . 0223 , Sex * * P = 0 . 007 , Genotype * * * P = < 0 . 0001 . Tukey ' s multiple comparisons test : Male : Jag1 + / + vs . Male : Jag1Ndr / Ndr P = 0 . 3247 , Male : Jag1Ndr / Ndr vs . Female : Jag1Ndr / Ndr * * P = 0 . 0031 , Female : Jag1 + / + vs . Female : Jag1Ndr / Ndr * * * * P = 0 . 0001 ) .

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

E-F (E) Arterial tortuosity at P30 and 1 year, irrespective of sex (n=6 per group, two-way ANOVA, not significant). (F) Arterial tortuosity in male and female mice, irrespective of age (n=5-7, two-way ANOVA, not significant). G-H (G) Venous tortuosity at P30 and 1 year, irrespective of sex (n=6-8 per group, two-way ANOVA with Tukey's multiple comparisons test. Interaction P = 0.1166, Age P = 0.0787, Genotype **P=0.0013. Tukey's multiple comparisons test: P30:Jag1+/+ vs. P30:Jag1Ndr/Ndr **P=0.004; P30:Jag1Ndr/Ndr vs. 1 year:Jag1Ndr/Ndr P=0.0802; 1 year:Jag1+/+ vs. 1 year:Jag1Ndr/Ndr P= 0.5066). (H) Venous tortuosity in male and female mice, irrespective of age (n=5-7, two-way ANOVA with Tukey's multiple comparisons test. Interaction *P = 0.0223, Sex **P = 0.007, Genotype ***P= <0.0001. Tukey's multiple comparisons test: Male:Jag1+/+ vs. Male:Jag1Ndr/Ndr P= 0.3247, Male:Jag1Ndr/Ndr vs. Female:Jag1Ndr/Ndr **P= 0.0031, Female:Jag1+/+ vs. Female:Jag1Ndr/Ndr ****P= 0.0001).


( J ) Tortuosity of MCA , as assessed by number of diversions per length ( n = 6 , t - test ns ) .

O O 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) Tortuosity of MCA, as assessed by number of diversions per length (n=6, t-test ns).


( K ) Visualization of MCA with alpha smooth muscle cell actin ( αSMA ) showed stereotype vasculature in 6 of 6 Jag1 + / + animals , but 2 of 6 Jag1Ndr / Ndr animals showed highly divergent MCA architecture ( blue arrows ) .

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

(K) Visualization of MCA with alpha smooth muscle cell actin (αSMA) showed stereotype vasculature in 6 of 6 Jag1+/+ animals, but 2 of 6 Jag1Ndr/Ndr animals showed highly divergent MCA architecture (blue arrows).


( L ) Distances between arterial branches in MCA at P10 ( unpaired t - test , not significant ) .

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

(L) Distances between arterial branches in MCA at P10 (unpaired t-test, not significant).


A - E αSMA coverage of ( A ) P10 , ( B ) P15 , ( C ) P30 , ( D ) 3 - 6 month old and € 1 - year - old retinal arterioles . Brackets in A , B denote perpendicular VSMCs around arteriole . Yellow arrows in A , B , C . indicate VSMCs with an orientation parallel to the blood vessel . Green arrowheads in D denote stenosis . White arrowheads in E label αSMA - negative gaps and white arrows an aneurysm . Cropped images were placed on black background for esthetic purposes .

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

A-E αSMA coverage of (A) P10, (B) P15, (C) P30, (D) 3-6 month old and € 1-year-old retinal arterioles. Brackets in A, B denote perpendicular VSMCs around arteriole. Yellow arrows in A, B, C. indicate VSMCs with an orientation parallel to the blood vessel. Green arrowheads in D denote stenosis. White arrowheads in E label αSMA-negative gaps and white arrows an aneurysm. Cropped images were placed on black background for esthetic purposes.


( F ) Incidence of α SMA negative gaps on arteries by age ( Two - way ANOVA with Sidak ' s multiple comparisons test ) .

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

(F) Incidence of α SMA negative gaps on arteries by age (Two-way ANOVA with Sidak's multiple comparisons test).


( G ) Number of α SMA negative gaps per retina and mouse by age ( Two - way ANOVA with Sidak ' s multiple comparisons test . Dots represent biological replicates / individual retinas / mice . Two - way ANOVA not significant . Šídák ' s multiple comparisons test : 1 year Jag1 + / + vs 1 year Jag1Ndr / Ndr * P = 0 . 0189 ) .

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

(G) Number of α SMA negative gaps per retina and mouse by age (Two-way ANOVA with Sidak's multiple comparisons test. Dots represent biological replicates/individual retinas/mice. Two-way ANOVA not significant. Šídák's multiple comparisons test: 1 year Jag1+/+ vs 1 year Jag1Ndr/Ndr *P= 0.0189).


H Transmission electron microscopy of retinal arterioles . VSMCs color - coded in magenta , EC in green . Scale bar 5 µm , boxed region 1 µm . Blue arrowheads denote the edges of VSMCs .

O O O O O O O O O O O O O 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 Transmission electron microscopy of retinal arterioles. VSMCs color-coded in magenta, EC in green. Scale bar 5 µm, boxed region 1 µm. Blue arrowheads denote the edges of VSMCs.


I - J ( I ) One - year - old retinal arteriolar cCasp3 + apoptotic cells . Scale bar 10 µm . ( J ) cCasp3 + cells per field ( n = 6 per group , unpaired t test , * P = 0 . 0109 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O 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-J (I) One-year-old retinal arteriolar cCasp3+ apoptotic cells. Scale bar 10 µm. (J) cCasp3+ cells per field (n=6 per group, unpaired t test, *P=0.0109).


( K ) Left panel aSMA coverage of MCA of P10 mice . Scale bar 20 µm . Right panel magnification of area where number of nuclei was counted . Nuclei were color - coded blue .

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

(K) Left panel aSMA coverage of MCA of P10 mice. Scale bar 20 µm. Right panel magnification of area where number of nuclei was counted. Nuclei were color-coded blue.


( L ) MCA vascular smooth muscle cell nuclei quantification ( n = 6 per group , unpaired t test , * P = 0 . 0421 ) .

O O O O O 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) MCA vascular smooth muscle cell nuclei quantification (n=6 per group, unpaired t test, *P=0.0421).


( N ) Blood pressure ( BP ) before treatment in male and female Jag1CTRL and Jag1Ndr / Ndr mice . Each dot represents one animal / one biological replicate . ( Two - way ANOVA Interaction P = 0 . 9407 , Sex P = 0 . 4957 , Genotype * P = 0 . 0207 . Šídák ' s multiple comparisons test not significant ) .

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

(N) Blood pressure (BP) before treatment in male and female Jag1CTRL and Jag1Ndr/Ndr mice. Each dot represents one animal/one biological replicate. (Two-way ANOVA Interaction P=0.9407, Sex P=0.4957, Genotype *P= 0.0207. Šídák's multiple comparisons test not significant).


( O ) Blood pressure increase in Jag1CTRL and Jag1Ndr / Ndr mice treated with vehicle or AngII for two weeks ( Jag1 + / + ctrl n = 4 , Jag1 + / + AngII n = 4 , Jag1Ndr / Ndr ctrl n = 5 , Jag1Ndr / Ndr AngII n = 5 , two - way ANOVA , with Sidak ' s multiple comparisons ) .

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

(O) Blood pressure increase in Jag1CTRL and Jag1Ndr/Ndr mice treated with vehicle or AngII for two weeks (Jag1+/+ ctrl n=4, Jag1+/+ AngII n=4, Jag1Ndr/Ndr ctrl n=5, Jag1Ndr/Ndr AngII n=5, two-way ANOVA, with Sidak's multiple comparisons).


( P ) Detection of Evans blue leakage in brain in Jag1CTRL and Jag1Ndr / Ndr mice with or without AngII treatment . ( Jag1 + / + ctrl n = 4 , Jag1 + / + AngII n = 4 , Jag1Ndr / Ndr ctrl n = 5 , Jag1Ndr / Ndr AngII n = 5 , two - way ANOVA , with Sidak ' s multiple comparison . Interaction P = 0 . 9687 , Treatment * * P = 0 . 007 , Genotype P = 0 . 5058 ) .

O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O 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) Detection of Evans blue leakage in brain in Jag1CTRL and Jag1Ndr/Ndr mice with or without AngII treatment. (Jag1+/+ ctrl n=4, Jag1+/+ AngII n=4, Jag1Ndr/Ndr ctrl n=5, Jag1Ndr/Ndr AngII n=5, two-way ANOVA, with Sidak's multiple comparison. Interaction P=0.9687, Treatment **P=0.007, Genotype P=0.5058).


( Q ) One Jag1Ndr / Ndr mouse ( male ) displayed a macroscopic EB leakage , upon AngII treatment .

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

(Q) One Jag1Ndr/Ndr mouse (male) displayed a macroscopic EB leakage, upon AngII treatment.


® Retinal arteriolar αSMA coverage in vehicle or AngII - treated mice . Green arrowheads denote stenosis , blue arrowheads label αSMA - negative gap . ( S ) αSMA - negative gaps in arterioles per retina ( Jag1 + / + ctrl n = 4 , Jag1 + / + AngII n = 4 , Jag1Ndr / Ndr ctrl n = 3 - 5 , Jag1Ndr / Ndr AngII n = 5 , two - way ANOVA , with Sidak ' s multiple comparison . Interaction P = 0 . 1054 , treatment P = 0 . 0815 ; Genotype P = 0 . 0747 . Ší ' ák ' s multiple comparisons test : AngII treated Jag1 + / + vs AngII treated Jag1Ndr / Ndr * P = 0 . 0315 ) . ) .

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

® Retinal arteriolar αSMA coverage in vehicle or AngII-treated mice. Green arrowheads denote stenosis, blue arrowheads label αSMA-negative gap. (S) αSMA-negative gaps in arterioles per retina (Jag1+/+ ctrl n=4, Jag1+/+ AngII n=4, Jag1Ndr/Ndr ctrl n=3-5, Jag1Ndr/Ndr AngII n=5, two-way ANOVA, with Sidak's multiple comparison. Interaction P = 0.1054, treatment P=0.0815; Genotype P=0.0747. Ší'ák's multiple comparisons test: AngII treated Jag1+/+ vs AngII treated Jag1Ndr/Ndr *P= 0.0315).).


A - D ( A ) P30 retina , three CD31 + capillary layers pseudo - colored for superficial , intermediate and deep layers . Scale bar 50 µm . ( B ) P30 SCP branching point number ( n = 6 - 8 per group , unpaired t test , ns , P = 0 . 5346 ) . ( C ) P30 ICP vascular length ( n = 6 per group , unpaired t test , * P = 0 . 0124 ) . ( D ) P30 ICP branching point number ( n = 6 per group , unpaired t test , * * P = 0 . 0057 ) . E - H ( E ) 3 - 6 months old retina , three pseudo - colored CD31 + capillary layers . Scale bar 50 µm . ( F ) Adult SCP branching point number ( n = 6 per group , unpaired t test , ns , P = 0 . 5365 ) . ( G ) Adult ICP vascular length ( n = 6 - 7 per group , unpaired t test , * * P = 0 . 0078 ) . ( H ) Adult ICP branching point number ( n = 6 - 7 per group , unpaired t test , * * P = 0 .

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

A-D (A) P30 retina, three CD31+ capillary layers pseudo-colored for superficial, intermediate and deep layers. Scale bar 50 µm. (B) P30 SCP branching point number (n=6-8 per group, unpaired t test, ns, P=0.5346). (C) P30 ICP vascular length (n=6 per group, unpaired t test, *P=0.0124). (D) P30 ICP branching point number (n=6 per group, unpaired t test, **P=0.0057). E-H (E) 3-6 months old retina, three pseudo-colored CD31+ capillary layers. Scale bar 50 µm. (F) Adult SCP branching point number (n=6 per group, unpaired t test, ns, P=0.5365). (G) Adult ICP vascular length (n=6-7 per group, unpaired t test, **P=0.0078). (H) Adult ICP branching point number (n=6-7 per group, unpaired t test, **P=0.001


I - J ( I ) ICP COLIV and CD31 ( arrowhead labels COLIV + CD31 - capillary ) . Scale bar 50 µm . ( J ) Empty COLIV sleeve length in ICP per field ( n = 6 per group , unpaired t test , ns , P = 0 . 1583 ) .

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

I-J (I) ICP COLIV and CD31 (arrowhead labels COLIV+ CD31- capillary). Scale bar 50 µm. (J) Empty COLIV sleeve length in ICP per field (n=6 per group, unpaired t test, ns, P=0.1583).


( K ) Retinal capillary plexuses in 3 - 6 months old ( top panels ) and one - year - old mice ( bottom panels ) . White arrowheads label discontinuous ICP . Blue arrowhead labels SCP , green ICP and magenta DCP .

O O O O O O O O O O O O O O O O O O O O O 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) Retinal capillary plexuses in 3-6 months old (top panels) and one-year-old mice (bottom panels). White arrowheads label discontinuous ICP. Blue arrowhead labels SCP, green ICP and magenta DCP.


( L - M ) Vertical sprout number between SCP ( S ) and ICP ( I ) and ICP ( I ) and DCP ( D ) in ( L ) adult ( n = 6 per group , two - way ANOVA , genotype * * * P = 0 . 0004 , Sidak ' s multiple comparisons test S - I ns , P = 0 . 0592 , I - D * * * * P < 0 . 0001 ) and ( M ) one - year - old mice ( n = 8 per group , two - way ANOVA , genotype ns , P = 0 . 4507 , Sidak ' s multiple comparisons test S - I ns , P = 0 . 9483 , I - D ns , P = 0 . 6046 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-M) Vertical sprout number between SCP (S) and ICP (I) and ICP (I) and DCP (D) in (L) adult (n=6 per group, two-way ANOVA, genotype ***P=0.0004, Sidak's multiple comparisons test S-I ns, P=0.0592, I-D ****P<0.0001) and (M) one-year-old mice (n=8 per group, two-way ANOVA, genotype ns, P=0.4507, Sidak's multiple comparisons test S-I ns, P=0.9483, I-D ns, P=0.6046).


N - P ( N ) P10 and ( O ) P40 neurofilament ( NF ) and CD31 staining . Scale bar 50 µm . ( P ) % area positive for NF in P10 and P40 mice ( n = 6 per group , two - way ANOVA : Interaction * * * P = 0 . 0001 ; Age * P = 0 . 0428 ; Genotype * * * P = 0 . 0004 . Sidak ' s multiple comparisons test : P10 Jag1 + / + vs P10 Jag1Ndr / Ndr P = 0 . 9246 ; P40 Jag1 + / + vs P40 Jag1Ndr / Ndr * * * * P = < 0 . 0001 ) .

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

N-P (N) P10 and (O) P40 neurofilament (NF) and CD31 staining. Scale bar 50 µm. (P) % area positive for NF in P10 and P40 mice (n=6 per group, two-way ANOVA: Interaction ***P= 0.0001; Age *P=0.0428; Genotype ***P=0.0004. Sidak's multiple comparisons test: P10 Jag1+/+ vs P10 Jag1Ndr/Ndr P= 0.9246; P40 Jag1+/+ vs P40 Jag1Ndr/Ndr ****P= <0.0001).


A Representative retinographs from a healthy adult , patient with CADASIL , healthy pediatric individual , patient with ALGS and patient with BA . Boxed regions and black arrowheads magnify arteriovenous crossing .

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

A Representative retinographs from a healthy adult, patient with CADASIL, healthy pediatric individual, patient with ALGS and patient with BA. Boxed regions and black arrowheads magnify arteriovenous crossing.


B - F ( B ) Arteriovenous crossings per retina ( n = 14 per group , unpaired t test , ns , P = 0 . 3854 . ( C ) Major arterioles ( n = 14 per group , unpaired t test , ns , P = 0 . 2902 ) and ( D ) venules ( n = 14 per group , unpaired t test , ns , P = 0 . 1536 ) . ( E ) Arterial ( n = 14 per group , unpaired t test , ns , P = 0 . 0759 ) and ( F ) venous tortuosity ( n = 14 per group , unpaired t test , ns , P = 0 . 1050 ) .

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

B-F (B) Arteriovenous crossings per retina (n=14 per group, unpaired t test, ns, P=0.3854. (C) Major arterioles (n=14 per group, unpaired t test, ns, P=0.2902) and (D) venules (n=14 per group, unpaired t test, ns, P=0.1536). (E) Arterial (n=14 per group, unpaired t test, ns, P=0.0759) and (F) venous tortuosity (n=14 per group, unpaired t test, ns, P=0.1050).


G - K ( G ) Arteriovenous crossings per retina ( CTRL n = 5 , ALGS n = 3 , BA n = 4 , one - way ANOVA , ns , P = 0 . 0608 , Sidak ' s multiple comparisons ALGS vs . CTRL * P = 0 . 0492 , ALGS vs . BA ns , P = 0 . 0999 ) . ( H ) Major arterioles ( CTRL n = 5 , ALGS n = 5 , BA n = 4 , one - way ANOVA , * * P = 0 . 0026 , Sidak ' s multiple comparisons ALGS vs . CTRL * * P = 0 . 0039 , ALGS vs . BA ns , P = 0 . 9799 ) and ( I ) venules ( CTRL n = 5 , ALGS n = 5 , BA n = 4 , one - way ANOVA , * * * P = 0 . 0008 , Sidak ' s multiple comparisons ALGS vs . CTRL * * P = 0 . 0011 , ALGS vs . BA ns , P > 0 . 9999 ) . ( J ) Arterial ( CTRL n = 5 , ALGS n = 5 , BA n = 4 , one - way ANOVA , * P = 0 . 0486 , Sidak ' s multiple comparisons ALGS vs . CTRL * P = 0 . 0392 , ALGS vs . BA ns , P = 0 . 1204 ) and ( K ) venous tortuosity ( CTRL n = 5 , ALGS n = 5 , BA n = 4 , one - way ANOVA , * * P = 0 . 009 , Sidak ' s multiple comparisons ALGS vs . CTRL * * P = 0 . 0076 , ALGS vs . BA * P = 0 . 0327 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-K (G) Arteriovenous crossings per retina (CTRL n=5, ALGS n=3, BA n=4, one-way ANOVA, ns, P=0.0608, Sidak's multiple comparisons ALGS vs. CTRL *P=0.0492, ALGS vs. BA ns, P=0.0999). (H) Major arterioles (CTRL n=5, ALGS n=5, BA n=4, one-way ANOVA, **P=0.0026, Sidak's multiple comparisons ALGS vs. CTRL **P=0.0039, ALGS vs. BA ns, P=0.9799) and (I) venules (CTRL n=5, ALGS n=5, BA n=4, one-way ANOVA, ***P=0.0008, Sidak's multiple comparisons ALGS vs. CTRL **P=0.0011, ALGS vs. BA ns, P>0.9999). (J) Arterial (CTRL n=5, ALGS n=5, BA n=4, one-way ANOVA, *P=0.0486, Sidak's multiple comparisons ALGS vs. CTRL *P=0.0392, ALGS vs. BA ns, P=0.1204) and (K) venous tortuosity (CTRL n=5, ALGS n=5, BA n=4, one-way ANOVA, **P=0.009, Sidak's multiple comparisons ALGS vs. CTRL **P=0.0076, ALGS vs. BA *P=0.0327).


( A ) Upper panels - Immunofluorescence imaging of PRR7 with Shank ( synaptic ) and MAP2 ( dendritic ) markers in DIV21 hippocampal neurons . Lower panels - Single plane apotome images of nuclei ( DAPI ) show PRR7 can be present ( arrow ) or absent ( arrowhead ) from nuclei .

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

(A) Upper panels- Immunofluorescence imaging of PRR7 with Shank (synaptic) and MAP2 (dendritic) markers in DIV21 hippocampal neurons. Lower panels- Single plane apotome images of nuclei (DAPI) show PRR7 can be present (arrow) or absent (arrowhead) from nuclei.


( B ) Synaptic and dendritic distribution of PRR7 and pre ( Bassoon ) and postsynaptic ( PSD95 ) markers ( arrowheads - triple colocalization ) .

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

(B) Synaptic and dendritic distribution of PRR7 and pre (Bassoon) and postsynaptic (PSD95) markers (arrowheads-triple colocalization).


( C ) PRR7 - GFP expressed in 17DIV hippocampal neurons is present in dendritic spines ( arrows ) . Scale bar is 15 µm .

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

(C) PRR7-GFP expressed in 17DIV hippocampal neurons is present in dendritic spines (arrows). Scale bar is 15 µm.


( D ) PRR7 distribution across subcellular fractions ( TOT = whole lysate , Syn = synaptosomes , Nuc = nuclei ) isolated from rat hippocampal tissue . Fib ( Fibrillarin ) , SNAP25 and PSD95 were used as nuclear , presynaptic and postsynaptic markers , respectively .

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

(D) PRR7 distribution across subcellular fractions (TOT = whole lysate, Syn = synaptosomes, Nuc = nuclei) isolated from rat hippocampal tissue. Fib (Fibrillarin), SNAP25 and PSD95 were used as nuclear, presynaptic and postsynaptic markers, respectively.


( A ) Immunocytochemistry analyses show that glutamate stimulation ( Glut , 100 µM 5 min + 25 min washout ) increased PRR7 in the nucleus . Cycloheximide ( 100 µM ) added 30 min prior to glutamate stimulation did not affect this increase ( Cont = 100 ± 12 . 5 % , Glut = 202 . 1 ± 15 . 2 % ; + cycloheximide Cont = 104 . 9 ± 3 . 6 , Glut = 231 . 4 ± 10 . 9 ) ( n = ~ 30 - 50 cells ) .

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

(A) Immunocytochemistry analyses show that glutamate stimulation (Glut, 100 µM 5 min + 25 min washout) increased PRR7 in the nucleus. Cycloheximide (100 µM) added 30 min prior to glutamate stimulation did not affect this increase (Cont=100±12.5%, Glut=202.1±15.2%; +cycloheximide Cont=104.9±3.6, Glut=231.4±10.9) (n=~30-50 cells).


( B ) Decreased synaptic PRR7 immunostaining following glutamate stimulation in DIV 21 hippocampal neurons . Epoxomicin ( 50 nM ) added 20 min prior to stimulation did not affect this decrease ( Cont = 100 ± 3 . 6 % , Glut = 32 . 3 ± 1 . 3 % ; + epoxomicin Cont = 99 . 8 ± 4 . 6 , Glut = 42 . 9 ± 1 . 1 ) . ( n = 14 - 17 dendrites , > 1200 puncta for each group ) . Statistical analyses for ( A ) and ( B ) were performed using the Mann Whitney test .

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

(B) Decreased synaptic PRR7 immunostaining following glutamate stimulation in DIV 21 hippocampal neurons. Epoxomicin (50 nM) added 20 min prior to stimulation did not affect this decrease (Cont=100±3.6%, Glut=32.3±1.3%; +epoxomicin Cont=99.8±4.6, Glut=42.9±1.1). (n=14-17 dendrites, >1200 puncta for each group). Statistical analyses for (A) and (B) were performed using the Mann Whitney test.


( C ) Quantification of nuclear PRR7 immunostaining in cultured hippocampal neurons . Treatments ( APV [ 50 µM , APV - ( 2R ) - amino - 5 - phosphonovaleric acid ] , CNQX [ 40 µM , 6 - cyano - 7 nitroquinoxaline - 2 , 3 - dione ] , TTX [ 1 µM , tetrodotoxin ] , NMDA [ 50 µM , N - Methyl - D - Aspartate ] ) as indicated . Only APV treatment blocked the glutamate dependent increase in nuclear PRR7 ( 108 . 1 ± 19 % ) . Statistical significance was determined using one - way ANOVA in conjugation with Sidak post - hoc test .

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 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

(C) Quantification of nuclear PRR7 immunostaining in cultured hippocampal neurons. Treatments (APV [50 µM, APV-(2R)-amino-5-phosphonovaleric acid], CNQX [40 µM, 6-cyano-7 nitroquinoxaline-2,3-dione], TTX [1 µM, tetrodotoxin], NMDA [50 µM, N-Methyl-D-Aspartate]) as indicated. Only APV treatment blocked the glutamate dependent increase in nuclear PRR7 (108.1 ± 19%). Statistical significance was determined using one-way ANOVA in conjugation with Sidak post-hoc test.


( D ) PRR7 - GFP accumulates in the nucleus of hippocampal neurons upon NMDA treatment . Depicted are representative averaged confocal images ( 30x30 µm2 ) from optical sections ( 300 nm step size ) of PRR7 - GFP expressing neurons ( DIV17 ) before and after bleaching . Prior to stimulation there was substantial PRR7 - GFP in the nucleus due to basal neuronal activity ( upper panel ) . This was reduced by incubation overnight with MK801 ( 10 µM ) and TTX ( 1 µM ) ( lower panel ) . NMDA ( 50 µM ) stimulation , but not MK - 801 , led to recovery of nuclear PRR7 - GFP fluorescence following bleaching of nuclear ROIs at t0 ` ( blue arrow in E ) . ( E ) Quantification of changes in nuclear GFP fluorescent intensities were normalized to time point t0 ` and corrected for bleaching . No significant differences in PRR7 - GFPFRAP were found in control ( MK801 ) conditions . GFP intensities are displayed via look - up - table ( LUT ) . Data are represented as mean ± SEM ; n = 9 cells ( NMDA ) and n = 5 cells ( MK801 ) . Analysis was done using one - way ANOVA using Bonferroni post - hoc correction .

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

(D) PRR7-GFP accumulates in the nucleus of hippocampal neurons upon NMDA treatment. Depicted are representative averaged confocal images (30x30 µm2) from optical sections (300 nm step size) of PRR7-GFP expressing neurons (DIV17) before and after bleaching. Prior to stimulation there was substantial PRR7-GFP in the nucleus due to basal neuronal activity (upper panel). This was reduced by incubation overnight with MK801 (10 µM) and TTX (1 µM) (lower panel). NMDA (50 µM) stimulation, but not MK-801, led to recovery of nuclear PRR7-GFP fluorescence following bleaching of nuclear ROIs at t0`(blue arrow in E).(E) Quantification of changes in nuclear GFP fluorescent intensities were normalized to time point t0` and corrected for bleaching. No significant differences in PRR7-GFPFRAP were found in control (MK801) conditions. GFP intensities are displayed via look-up-table (LUT). Data are represented as mean±SEM; n=9 cells (NMDA) and n=5 cells (MK801). Analysis was done using one-way ANOVA using Bonferroni post-hoc correction.


( F ) PRR7 - mEos translocates from distal dendrites to the nucleus upon NMDA treatment . ( Left panels ) Confocal maximal intensity projection images of hippocampal neurons expressing PRR7 - mEos before and after photoconversion of distal dendrites ( ROIs ) through the image z - stack from from green ( 488nm ) to red ( 568nm ) using a UV laser ( 405 nm ) . ( Right panels ) Averaged intensity projection images from nuclear planes showing accumulation of photoconverted PRR7 - mEos ( 568nm ) in the nucleus following NMDA stimulation ( 50 µM - 30min ) . ( G ) Paired quantification of changes in background corrected fluorescent intensities of nuclear PRR7 - mEos at 0 min and 30 min following NMDA stimulation or MK801 treatment . ( Average increase ; NMDA = 617 . 8 ± 47 . 7 % , n = 11 cells , p < 0 . 0005 ; MK801 = 206 . 1 ± 13 . 6 % , n = 5 cells , n . s . ) . Statistical significance was determined by two - way ANOVA using Sidak ' s correction for multiple comparisons .

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

(F) PRR7-mEos translocates from distal dendrites to the nucleus upon NMDA treatment. (Left panels) Confocal maximal intensity projection images of hippocampal neurons expressing PRR7-mEos before and after photoconversion of distal dendrites (ROIs) through the image z-stack from from green (488nm) to red (568nm) using a UV laser (405 nm). (Right panels) Averaged intensity projection images from nuclear planes showing accumulation of photoconverted PRR7-mEos (568nm) in the nucleus following NMDA stimulation (50 µM- 30min).(G) Paired quantification of changes in background corrected fluorescent intensities of nuclear PRR7-mEos at 0 min and 30 min following NMDA stimulation or MK801 treatment. (Average increase; NMDA=617.8±47.7%, n=11 cells, p<0.0005; MK801=206.1±13.6%, n=5 cells, n.s.). Statistical significance was determined by two-way ANOVA using Sidak's correction for multiple comparisons.


( A ) Co - Immunoprecipitations ( Co - IPs ) of PRR7 with GluN1 , PSD95 and GluN2B from rat brain lysates . FT = flowthrough , IgG = normal IgGs .

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

(A) Co-Immunoprecipitations (Co-IPs) of PRR7 with GluN1, PSD95 and GluN2B from rat brain lysates. FT = flowthrough, IgG = normal IgGs.


( B ) Co - IPs from HEK293 cells of c - terminal myc - tagged PRR7 with GluN1 in the absence of other NMDAR subunits or PSD95 .

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

(B) Co-IPs from HEK293 cells of c-terminal myc-tagged PRR7 with GluN1 in the absence of other NMDAR subunits or PSD95.


( C ) Co - IPs from HEK293 cells transfected with tagged NMDA or AMPA receptor subunits and PRR7 ( myc - tagged GluN2B and GluA2 and flag - tagged PRR7 ) .

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

(C) Co-IPs from HEK293 cells transfected with tagged NMDA or AMPA receptor subunits and PRR7 (myc-tagged GluN2B and GluA2 and flag-tagged PRR7).


( D ) ( top ) Schematics of the different PRR7 constructs used for co - IP ( FL = full length ) . ( bottom ) PRR7 deletion constructs and NMDAR subunits were expressed in HEK293 cells and Co - IPs performed as indicated .

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

(D) (top) Schematics of the different PRR7 constructs used for co-IP (FL= full length). (bottom) PRR7 deletion constructs and NMDAR subunits were expressed in HEK293 cells and Co-IPs performed as indicated.


( E ) Co - IPs of PRR7 with GluN1 from high - density primary neuronal cultures following treatment with NMDA ( 100µM , 30 min , representative of n = 4 ) .

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

(E) Co-IPs of PRR7 with GluN1 from high-density primary neuronal cultures following treatment with NMDA (100µM, 30 min, representative of n=4).


( A ) Western blots of lysates of primary neurons transduced with control lentiviruses ( NT ) or lentiviruses to knockdown ( KD ) or overexpress ( OE ) PRR7 . PRR7 knockdown reduced ( 75 . 6 ± 6 . 9 % ) while PRR7 overexpression ( OE ) increased ( 157 . 6 ± 22 . 8 % ) c - Jun levels compared to controls . No change was detected in c - Fos levels . One - way ANOVA test was used to analyze results with Dunett ' s post hoc test ( n = 16 gels for c - Jun and 4 gels for c - Fos ) .

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

(A) Western blots of lysates of primary neurons transduced with control lentiviruses (NT) or lentiviruses to knockdown (KD) or overexpress (OE) PRR7. PRR7 knockdown reduced (75.6 ± 6.9%) while PRR7 overexpression (OE) increased (157.6 ± 22.8%) c-Jun levels compared to controls. No change was detected in c-Fos levels. One-way ANOVA test was used to analyze results with Dunett's post hoc test (n=16 gels for c-Jun and 4 gels for c-Fos).


( B ) Control or PRR7 transfected HEK293 cells were treated with cycloheximide for the indicated time points and c - Jun protein levels were measured by Western blotting . PRR7 expression significantly stabilized c - Jun levels at 120 min ( 110 . 0 ± 7 . 5 % vs control 68 . 7 ± 7 . 9 % ) and 240 minutes ( 83 . 6 ± 5 . 2 % vs control 43 . 7 ± 3 . 4 % ) . Values for c - Jun were normalized to their respective 0 min time point ( n = 4 gels for each group performed blind ) . T - tests between similar time points were used to analyze results .

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

(B) Control or PRR7 transfected HEK293 cells were treated with cycloheximide for the indicated time points and c-Jun protein levels were measured by Western blotting. PRR7 expression significantly stabilized c-Jun levels at 120 min (110.0 ± 7.5% vs control 68.7 ± 7.9%) and 240 minutes (83.6 ± 5.2% vs control 43.7 ± 3.4%). Values for c-Jun were normalized to their respective 0 min time point (n=4 gels for each group performed blind). T-tests between similar time points were used to analyze results.


( C ) PRR7 , FBW7 and / or a control protein ( PSD95 ) were transfected into HEK293 cells and c - Jun ubiquitination was measured by immunoprecipitating c - Jun and blotting for ubiquitin . Ubiquitination of c - Jun in the presence of FBW7 ( 178 . 3 ± 26 . 9 % ) was significantly reduced in the presence of PRR7 ( 103 . 7 ± 20 . 6 % ) but not a control protein ( PSD95 ) ( n = 5 gels for all groups ) .

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

(C) PRR7, FBW7 and/or a control protein (PSD95) were transfected into HEK293 cells and c-Jun ubiquitination was measured by immunoprecipitating c-Jun and blotting for ubiquitin. Ubiquitination of c-Jun in the presence of FBW7 (178.3 ± 26.9%) was significantly reduced in the presence of PRR7 (103.7 ± 20.6%) but not a control protein (PSD95) (n=5 gels for all groups).


( D ) Western blots of c - Junubiquitination in hippocampal primary neuronal cultures . PRR7 knockdown ( KD ) elevated ( 149 ± 10 . 62 % , n = 6 gels ) c - Junubiquitination level in comparison to control neurons ( NT ) . Statistical significance was determined by t - test .

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

(D) Western blots of c-Junubiquitination in hippocampal primary neuronal cultures. PRR7 knockdown (KD) elevated (149±10.62%, n=6 gels) c-Junubiquitination level in comparison to control neurons (NT). Statistical significance was determined by t-test.


( E ) Western blots showing knockdown ( KD ) or overexpression ( OE ) of PRR7 in primary hippocampal neurons do not alter levels of other FBW7 targets mTOR or Myc .

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

(E) Western blots showing knockdown (KD) or overexpression (OE) of PRR7 in primary hippocampal neurons do not alter levels of other FBW7 targets mTOR or Myc.


( A ) Co - IPs of PRR7 with c - Jun from lysates of HEK293 cells overexpressing PRR7 .

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

(A) Co-IPs of PRR7 with c-Jun from lysates of HEK293 cells overexpressing PRR7.


( B ) Pulldown assays in vitro using purified proteins showed a direct interaction between PRR7 and c - Jun .

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

(B) Pulldown assays in vitro using purified proteins showed a direct interaction between PRR7 and c-Jun.


( C ) Co - IPs of PRR7 with FBW7 from lysates of HEK293 cells overexpressing PRR7 and FBW7 .

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

(C) Co-IPs of PRR7 with FBW7 from lysates of HEK293 cells overexpressing PRR7 and FBW7.


( D ) Pulldown assays in vitro using purified PRR7 and FBW7 .

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

(D) Pulldown assays in vitro using purified PRR7 and FBW7.


( E ) Co - IPs of endogenous PRR7 and FBW7 from lysates of ratbrain .

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

(E) Co-IPs of endogenous PRR7 and FBW7 from lysates of ratbrain.


( F ) Co - IPs of PRR7 with c - Jun and FBW7 from lysates of HEK293 cells overexpressing PRR7 and FBW7 . FBW7 expression increased the amount of c - Jun pulled down with PRR7 ( n = 4 gels for each panel , representative blots ) . Lower bar graphs - quantitation for Western blots show c - Jun / FBW7 binding is 41 . 4 ± 13 . 9 % greater in the presence of PRR7 ( normalized for FBW7 expression ) . Similarly , c - Jun / PRR7 binding is 97 . 4 ± 38 . 1 % greater in the presence of FBW7 ( normalized for PRR7 expression ) . Statistical significance was calculated using unpaired t - tests .

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

(F) Co-IPs of PRR7 with c-Jun and FBW7 from lysates of HEK293 cells overexpressing PRR7 and FBW7. FBW7 expression increased the amount of c-Jun pulled down with PRR7 (n=4 gels for each panel, representative blots). Lower bar graphs- quantitation for Western blots show c-Jun/FBW7 binding is 41.4±13.9% greater in the presence of PRR7 (normalized for FBW7 expression). Similarly, c-Jun/PRR7 binding is 97.4±38.1% greater in the presence of FBW7 (normalized for PRR7 expression). Statistical significance was calculated using unpaired t-tests.


( A ) Western blots of phospho - c - Jun in primary neuronal cultures . Quantification of phospho - c - Jun represents a ratio of phospho - c - Jun to total c - Jun and all ratios were normalized to NT . PRR7 knockdown ( KD ) decreased , while PRR7 overexpression ( OE ) increased phospho - c - Jun levels ( black bars ) . Glutamate stimulation ( gray bars ) increased phospho - c - Jun / c - Jun ratio in NT but not in KD or OE neurons ( n = 7 gels for all groups ) . Significance was determined using two - way ANOVA with Sidak post - hoc test .

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

(A) Western blots of phospho-c-Jun in primary neuronal cultures. Quantification of phospho-c-Jun represents a ratio of phospho-c-Jun to total c-Jun and all ratios were normalized to NT. PRR7 knockdown (KD) decreased, while PRR7 overexpression (OE) increased phospho-c-Jun levels (black bars). Glutamate stimulation (gray bars) increased phospho-c-Jun/c-Jun ratio in NT but not in KD or OE neurons (n=7 gels for all groups). Significance was determined using two-way ANOVA with Sidak post-hoc test.


( B ) AP - 1 driven luciferase luminescence assays . PRR7 expression in HEK293 cells increased AP - 1 activity ( 190 . 6 ± 13 . 9 % , n = 12 ; RLU = relative light units ) . Statistical significance was determined using t - tests .

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

(B) AP-1 driven luciferase luminescence assays. PRR7 expression in HEK293 cells increased AP-1 activity (190.6 ± 13.9%, n=12; RLU= relative light units). Statistical significance was determined using t-tests.


( D ) Quantitation of cell death using 30 minutes of PI in - situ staining following 48h of WT - PRR7 - GFP or mNLS - PRR7 - GFP overexpression in primary hippocampal neuronal cultures ( n = 24 cells per group ) . Images represent a max projection of five z - stack sections across the cell body .

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

(D) Quantitation of cell death using 30 minutes of PI in-situ staining following 48h of WT-PRR7-GFP or mNLS-PRR7-GFP overexpression in primary hippocampal neuronal cultures (n=24 cells per group). Images represent a max projection of five z-stack sections across the cell body.


( E ) NMDA treatment ( 25 min , 100 µM followed by 14h chase ) caused 83 . 6 ± 3 . 5 % cell death in control neurons compared to 60 . 3 ± 4 . 6 % cell death in neurons with PRR7 expression knocked down . A c - Jun inhibitory peptide reduced NMDA - mediated cell death to 66 . 2 ± 6 . 0 % in control neurons and to 55 . 6 ± 5 . 3 % in neurons knocked down for PRR7 , but this difference was not statistically significant ( n = 5 independent experiments ) . Two - way ANOVA was used in conjugation with Tukey post - hoc test . ( Left ) Representative images showing transduced neurons ( green - GFP ) and PI staining ( red ) .

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

(E) NMDA treatment (25 min, 100 µM followed by 14h chase) caused 83.6±3.5% cell death in control neurons compared to 60.3±4.6% cell death in neurons with PRR7 expression knocked down. A c-Jun inhibitory peptide reduced NMDA-mediated cell death to 66.2±6.0% in control neurons and to 55.6±5.3% in neurons knocked down for PRR7, but this difference was not statistically significant (n=5 independent experiments). Two-way ANOVA was used in conjugation with Tukey post-hoc test. (Left) Representative images showing transduced neurons (green- GFP) and PI staining (red).


( F ) Quantification of cell death in response to NMDA stimulation in the presence or absence of PRR7 and either WT c - Jun or a dominant negative c - Jun4A ( 4A ) . Overexpression of WT c - Jun caused extensive neuronal cell death ( 84 . 8 ± 2 . 4 % ) that was reduced in neurons knocked down for PRR7 ( 43 . 8 ± 9 . 2 ) . Overexpressing 4A conferred neuroprotection to NMDA excitotoxicity ( 50 . 8 ± 7 . 9 % ) but was not additive with PRR7 knockdown ( 42 . 3 ± 8 . 1 % ) ( n = 3 independent experiments ) . Significance was determined using two - way ANOVA in conjugation with Tukey post - hoc test . Images are representative 10X widefield micrographs showing transduced neurons ( green - GFP ) and PI staining ( red ) . Data is presented as mean ± sem . All % values represent comparisons to control .

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

(F) Quantification of cell death in response to NMDA stimulation in the presence or absence of PRR7 and either WT c-Jun or a dominant negative c-Jun4A (4A). Overexpression of WT c-Jun caused extensive neuronal cell death (84.8±2.4%) that was reduced in neurons knocked down for PRR7 (43.8±9.2). Overexpressing 4A conferred neuroprotection to NMDA excitotoxicity (50.8±7.9%) but was not additive with PRR7 knockdown (42.3±8.1%) (n=3 independent experiments). Significance was determined using two-way ANOVA in conjugation with Tukey post-hoc test. Images are representative 10X widefield micrographs showing transduced neurons (green- GFP) and PI staining (red). Data is presented as mean±sem. All % values represent comparisons to control.


C - D . ID2 ubiquitination on FANCI results in increased rate of FANCD2 ubiquitination ( B ) , whereas ID2 ubiquitination on FANCD2 results in increased rate of FANCI ubiquitination ( C ) . Protein complexes were assembled in vitro on ice in the presence of dsDNA ( 32 bp ) and their in vitro ubiquitination at 30o C was subsequently monitored in a time - course : at indicative time - points , aliquots of the reaction were removed and analysed by western blotting using FANCD2 and V5 antibodies ( Top ) . For each protein complex , data - points from three replicate experiments ( three technical replicates ) were used in fitting to a one - phase association model ( Bottom ) .

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

C-D. ID2 ubiquitination on FANCI results in increased rate of FANCD2 ubiquitination (B), whereas ID2 ubiquitination on FANCD2 results in increased rate of FANCI ubiquitination (C). Protein complexes were assembled in vitro on ice in the presence of dsDNA (32 bp) and their in vitro ubiquitination at 30o C was subsequently monitored in a time-course: at indicative time-points, aliquots of the reaction were removed and analysed by western blotting using FANCD2 and V5 antibodies (Top). For each protein complex, data-points from three replicate experiments (three technical replicates) were used in fitting to a one-phase association model (Bottom).


A . USP1 - UAF1 - mediated deubiquitination of V5 - FANCI and FANCD2 was assessed in the absence or presence of DNA ( 51 bp ) , when ubiquitinated versions of these proteins were in isolation ( IUb and D2Ub ) or within singly / doubly ubiquitinated ID2 complexes ( IUbD2 , IUbD2Ub and ID2Ub ) . At indicated time - points , aliquots of each reaction were removed and analysed by western blotting using FANCD2 and V5 antibodies .

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

A. USP1-UAF1-mediated deubiquitination of V5-FANCI and FANCD2 was assessed in the absence or presence of DNA (51 bp), when ubiquitinated versions of these proteins were in isolation (IUb and D2Ub) or within singly/doubly ubiquitinated ID2 complexes (IUbD2, IUbD2Ub and ID2Ub). At indicated time-points, aliquots of each reaction were removed and analysed by western blotting using FANCD2 and V5 antibodies.


C . Deletion of N - terminus ( ∆ N ) of USP1 ( residues 1 - 54 ) results in greatly reduced FANCD2 deubiquitination . Assays were performed in triplicate ( technical replicates ) as in A , but all reactions contained DNA . Left : Western blotting of reaction products at zero and 30 minutes using FANCD2 and V5 antibodies . Right : Residual FANCI and FANCD2 ubiquitination following USP1 - UAF1 treatment for 30 min . Replicate residual ubiquitination values and statistically significant changes ( one - way ANOVA test with Bonferroni correction ) are shown . * * * p < 0 . 001 .

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 B-MEASURED_VAR O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O 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. Deletion of N-terminus (∆N) of USP1 (residues 1-54) results in greatly reduced FANCD2 deubiquitination. Assays were performed in triplicate (technical replicates) as in A, but all reactions contained DNA. Left: Western blotting of reaction products at zero and 30 minutes using FANCD2 and V5 antibodies. Right: Residual FANCI and FANCD2 ubiquitination following USP1-UAF1 treatment for 30 min. Replicate residual ubiquitination values and statistically significant changes (one-way ANOVA test with Bonferroni correction) are shown. ***p<0.001.


Alanine mutagenesis of key FANCD2 ' s residues ( H209 , V243 and P244 ) participating in the extended FANCD2 - ubiquitin interface ( IUbD2 - DNA and IUbD2Ub - DNA structures ) , results in enhanced FANCI deubiquitination by USP1 - UAF1 . C . FANCI deubiquitination by USP1 - UAF1 ( incubation with 50 nM USP1 - UAF1 for 30 minutes at room temperature ) in the presence of DNA and , in the absence or presence of wild - type ( D2WT ) or mutant ( D2H209A , VP243AA ) FANCD2 . Replicate residual ubiquitination values ( three technical replicates ) and statistically significant changes ( t - test and one - way ANOVA test with Bonferroni correction ) are shown .

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

Alanine mutagenesis of key FANCD2's residues (H209, V243 and P244) participating in the extended FANCD2-ubiquitin interface (IUbD2-DNA and IUbD2Ub-DNA structures), results in enhanced FANCI deubiquitination by USP1-UAF1. C. FANCI deubiquitination by USP1-UAF1 (incubation with 50 nM USP1-UAF1 for 30 minutes at room temperature) in the presence of DNA and, in the absence or presence of wild-type (D2WT) or mutant (D2H209A, VP243AA) FANCD2. Replicate residual ubiquitination values (three technical replicates) and statistically significant changes (t-test and one-way ANOVA test with Bonferroni correction) are shown.


D . FANCI was ubiquitinated with DyLight - 680 labelled ubiquitin ( IUb - DL680 ) and its deubiquitination by USP1 - UAF1 ( incubation with 100 nM USP1 - UAF1 for 20 minutes at room temperature ) , was assessed at increasing concentrations ( 20 , 200 or 200 nM ) of , either wild - type ( D2WT ) , or mutant ( D2H209A , VP243AA ) FANCD2 . Experiment was repeated three times ( technical replicates ) , and the fold change in residual FANCI ubiquitination was determined by normalisation to the mean intensity of IUb - DL680 at 20 nM FANCD2 ( average from six values , for both D2WT and D2H209A , VP243AA ) . Mean ( with range ) fold - increase values and statistically significant changes upon FANCD2 mutation for each FANCD2 concentration ( t - test and two - way ANOVA test with Bonferroni correction ) are shown .

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

D. FANCI was ubiquitinated with DyLight-680 labelled ubiquitin (IUb-DL680) and its deubiquitination by USP1-UAF1 (incubation with 100 nM USP1-UAF1 for 20 minutes at room temperature), was assessed at increasing concentrations (20, 200 or 200 nM) of, either wild-type (D2WT), or mutant (D2H209A, VP243AA) FANCD2. Experiment was repeated three times (technical replicates), and the fold change in residual FANCI ubiquitination was determined by normalisation to the mean intensity of IUb-DL680 at 20 nM FANCD2 (average from six values, for both D2WT and D2H209A, VP243AA). Mean (with range) fold-increase values and statistically significant changes upon FANCD2 mutation for each FANCD2 concentration (t-test and two-way ANOVA test with Bonferroni correction) are shown.


( B ) Wild type mice received strain GFP - K79 via the tail vein and 1 hr later , animals were perfused and the meninges , cortex and choroid plexus were obtained for demonstration of intravenously injected bacteria . The arrows represent bacteria co - localized with capillaries ( e . g . , within capillaries ) and arrowheads represent bacteria outside the capillaries ( e . g . , exited from the capillaries ) . A few bacteria were successfully passed through the meningeal and cortex capillaries , but no bacteria were demonstrated in the choroid plexus at this time .

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

(B) Wild type mice received strain GFP-K79 via the tail vein and 1 hr later, animals were perfused and the meninges, cortex and choroid plexus were obtained for demonstration of intravenously injected bacteria. The arrows represent bacteria co-localized with capillaries (e.g., within capillaries) and arrowheads represent bacteria outside the capillaries (e.g., exited from the capillaries). A few bacteria were successfully passed through the meningeal and cortex capillaries, but no bacteria were demonstrated in the choroid plexus at this time.


( C ) Sulfo - NHS - biotin was administered via intraperitoneal injection 10 min before perfusion for assessing the blood - brain barrier permeability , i . e . , extravasation is indicative of leakage from the intravascular lumen . Sulfo - NHS - Biotin was confined to the capillaries of the meninges and cortex ( as shown with arrows ) , and there was no evidence of extravasation . Scale bar = 100 μm .

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

(C) Sulfo-NHS-biotin was administered via intraperitoneal injection 10 min before perfusion for assessing the blood-brain barrier permeability, i.e., extravasation is indicative of leakage from the intravascular lumen. Sulfo-NHS-Biotin was confined to the capillaries of the meninges and cortex (as shown with arrows), and there was no evidence of extravasation. Scale bar = 100 μm.


( D ) Relative invasion frequency of GBS strain K79 in HBMEC with or without EGFR inhibitor ( gefitinib ) .

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

(D) Relative invasion frequency of GBS strain K79 in HBMEC with or without EGFR inhibitor (gefitinib).


( E ) Bacterial counts recovered from the blood and brain in wild type mice receiving vehicle control ( n = 5 ) or gefitinib ( 10 mg / kg ) ( n = 6 ) , infected with strain K79 for 1 h .

O O O O O O O O O O O O O O O O O O O O O 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) Bacterial counts recovered from the blood and brain in wild type mice receiving vehicle control (n = 5) or gefitinib (10 mg/kg) (n = 6), infected with strain K79 for 1 h.


( F ) Tyrosine phosphorylation of EGFR in HBMEC in response to GBS strain K79 infection at 15 min and 60 min post infection .

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

(F) Tyrosine phosphorylation of EGFR in HBMEC in response to GBS strain K79 infection at 15 min and 60 min post infection.


( G ) EGFR protein expression in EGFR knockout HBMEC using CRISPR / Cas9 ( left panel ) and relative invasion frequency of 8 meningitis isolates of GBS strains in EGFR knockout and control HBMEC ( right panel ) . From left to right , p value of K79 is 0 . 0059 , p value of K160 is 0 . 00024 , p value of K161 is 0 . 00080 , p value of K181 is 0 . 00042 , p value of K226 is 0 . 00011 , p value of K237 is 0 . 00072 , p value of K238 is 0 . 0093 , p value of P539 is 0 . 00013 ( right panel ) .

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

(G) EGFR protein expression in EGFR knockout HBMEC using CRISPR/Cas9 (left panel) and relative invasion frequency of 8 meningitis isolates of GBS strains in EGFR knockout and control HBMEC (right panel). From left to right, p value of K79 is 0.0059, p value of K160 is 0.00024, p value of K161 is 0.00080, p value of K181 is 0.00042, p value of K226 is 0.00011, p value of K237 is 0.00072, p value of K238 is 0.0093, p value of P539 is 0.00013 (right panel).


( H ) GBS strain K79 traversal of HBMEC monolayer was significantly decreased in EGFR knockout HBMEC compare to control HBMEC .

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

(H) GBS strain K79 traversal of HBMEC monolayer was significantly decreased in EGFR knockout HBMEC compare to control HBMEC.


( I ) Bacterial counts recovered from the blood and brain of EGFR conditional knockout ( n = 5 ) and control mice ( n = 5 ) 1 h after intravenous inoculation with strain K79 .

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

(I) Bacterial counts recovered from the blood and brain of EGFR conditional knockout (n = 5) and control mice (n = 5) 1 h after intravenous inoculation with strain K79.


( A ) Tyrosine phosphorylation of EGFR in HBMEC pre - treated with 20 μM S1P2 antagonist ( JTE - 013 ) or vehicle control and infected with GBS strain K79 infection for 60 min . EGFR phosphorylation in control and S1P2 knockout HBMEC infected with K79 .

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

(A) Tyrosine phosphorylation of EGFR in HBMEC pre-treated with 20 μM S1P2 antagonist (JTE-013) or vehicle control and infected with GBS strain K79 infection for 60 min. EGFR phosphorylation in control and S1P2 knockout HBMEC infected with K79.


( B ) Relative invasion frequency of strain K79 in HBMEC with or without SphK1 and SphK2 inhibitors . All the inhibitors were used at 10 μM . From left to right , p value of DJB - V - 39 is 0 . 0047 , p value of SKI is 0 . 0062 , p value of FTY720 is 0 . 0022 , and p value of HB56 is 0 . 0045 .

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

(B) Relative invasion frequency of strain K79 in HBMEC with or without SphK1 and SphK2 inhibitors. All the inhibitors were used at 10 μM. From left to right, p value of DJB-V-39 is 0.0047, p value of SKI is 0.0062, p value of FTY720 is 0.0022, and p value of HB56 is 0.0045.


( C ) S1P2 protein expression in S1P2 knockout HBMEC using CRISPR / Cas9 ( left panel ) . Relative invasion frequency of 8 meningitis isolates of GBS in S1P2 knockout HBMEC . From left to right , p value of K79 is 0 . 0018 , p value of K160 is 0 . 0091 , p value of K161 is 0 . 0054 , p value of K181 is 0 . 00040 , p value of K226 is 0 . 00032 , p value of K237 is 0 . 040 , p value of K238 is 0 . 041 and p value of P539 is 0 . 0025 .

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

(C) S1P2 protein expression in S1P2 knockout HBMEC using CRISPR/Cas9 (left panel). Relative invasion frequency of 8 meningitis isolates of GBS in S1P2 knockout HBMEC. From left to right, p value of K79 is 0.0018, p value of K160 is 0.0091, p value of K161 is 0.0054, p value of K181 is 0.00040, p value of K226 is 0.00032, p value of K237 is 0.040, p value of K238 is 0.041 and p value of P539 is 0.0025.


( D ) GBS strain K79 traversal of HBMEC monolayer was significantly decreased in S1P2 knockout HBMEC compare to control .

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

(D) GBS strain K79 traversal of HBMEC monolayer was significantly decreased in S1P2 knockout HBMEC compare to control.


( E ) Bacterial counts recovered from the blood and brain of wild type ( n = 5 ) and S1P2 - / - mice ( n = 5 ) infected with strain K79 for 1h .

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

(E) Bacterial counts recovered from the blood and brain of wild type (n = 5) and S1P2-/- mice (n = 5) infected with strain K79 for 1h.


( A ) Serine phosphorylation of cPLA2α in response to GBS strain K79 in HBMEC pretreated with gefitinib or vehicle control and in EGFR knockout HBMEC .

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

(A) Serine phosphorylation of cPLA2α in response to GBS strain K79 in HBMEC pretreated with gefitinib or vehicle control and in EGFR knockout HBMEC.


( B ) Serine phosphorylation of cPLA2α in response to GBS strain K79 in control , EGFR knockout and S1P2 knockout HBMEC .

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

(B) Serine phosphorylation of cPLA2α in response to GBS strain K79 in control, EGFR knockout and S1P2 knockout HBMEC.


( C ) Bacterial counts recovered from the blood and brain in wild type mice ( n = 5 ) and CysLT2 - / - mice ( n = 5 ) infected with strain K79 for 1h .

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

(C) Bacterial counts recovered from the blood and brain in wild type mice (n = 5) and CysLT2-/- mice (n = 5) infected with strain K79 for 1h.


( D ) CysLT1 protein expression in CysLT1 knockdown HBMEC using shRNA ( left panel ) and relative invasion frequency of 8 GBS isolates in CysLT1 knockdown and control HBMEC . t - test . From left to right , p value of K79 is 0 . 0039 , p value of K160 is 0 . 0082 , p value of K161 is 0 . 00091 , p value of K181 is 0 . 0087 , p value of K226 is 0 . 00020 , p value of K237 is 0 . 0039 , p value of K238 is 0 . 00066 , p value of P539 is 1 . 76E - 05 ( right panel ) .

O O O B-MEASURED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) CysLT1 protein expression in CysLT1 knockdown HBMEC using shRNA (left panel) and relative invasion frequency of 8 GBS isolates in CysLT1 knockdown and control HBMEC. t-test. From left to right, p value of K79 is 0.0039, p value of K160 is 0.0082, p value of K161 is 0.00091, p value of K181 is 0.0087, p value of K226 is 0.00020, p value of K237 is 0.0039, p value of K238 is 0.00066, p value of P539 is 1.76E-05 (right panel).


( E ) GBS strain K79 traversal of HBMEC monolayer was significantly decreased in CysLT1 knockdown HBMEC compare to control .

(E) GBS strain K79 traversal of HBMEC monolayer was significantly decreased in CysLT1 knockdown HBMEC compare to control.


( F ) Bacterial counts recovered from the blood and brain in wild type mice ( n = 7 ) and CysLT1 - / - mice ( n = 7 ) infected with strain K79 for 1h .

(F) Bacterial counts recovered from the blood and brain in wild type mice (n = 7) and CysLT1-/- mice (n = 7) infected with strain K79 for 1h.


( G ) Ezrin protein expression in ezrin knockdown HBMEC using shRNA ( left panel ) and relative invasion frequency of GBS strain K79 in ezrin knockdown and control HBMEC ( right panel ) .

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

(G) Ezrin protein expression in ezrin knockdown HBMEC using shRNA (left panel) and relative invasion frequency of GBS strain K79 in ezrin knockdown and control HBMEC (right panel).


( H Ezrin phosphorylation in HBMEC pre - treated with CysLT1 antagonist ( montelukast ) and infected with GBS strain K79 for 60 min ( left panel )

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

(H Ezrin phosphorylation in HBMEC pre-treated with CysLT1 antagonist (montelukast) and infected with GBS strain K79 for 60 min (left panel)


I ) ezrin phosphorylation in the homogenates of brain capillaries of CysLT1 - / - and control mice infected with GBS strain K79 for 1h ( right panel ) .

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

I) ezrin phosphorylation in the homogenates of brain capillaries of CysLT1-/- and control mice infected with GBS strain K79 for 1h (right panel).


( J ) Intracellular K79 co - localization with EGFR and ezrin in control and S1P2 knockout HBMEC ( as shown by arrows , cyan ) . Percentage of co - localization was calculated by counting numbers of all GBS and co - localized intracellular GBS from at least three representative fields , and expressing as numbers of co - localized GBS / all GBS x 100 .

O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Intracellular K79 co-localization with EGFR and ezrin in control and S1P2 knockout HBMEC (as shown by arrows, cyan). Percentage of co-localization was calculated by counting numbers of all GBS and co-localized intracellular GBS from at least three representative fields, and expressing as numbers of co-localized GBS/all GBS x 100.


( A ) Survival of wild type mice receiving drug administration for 7 days after infection with GBS strain K79 . Data are presented as a Kaplan - Maier plot with a log rank test used to compare percentage of survival between the groups , CFX group n = 18 , and CFX + MONT group n = 10 * p = 0 . 015 . ( CFX : ceftriaxone ; MONT : montelukast ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Survival of wild type mice receiving drug administration for 7 days after infection with GBS strain K79. Data are presented as a Kaplan-Maier plot with a log rank test used to compare percentage of survival between the groups, CFX group n = 18, and CFX + MONT group n = 10*p = 0.015. (CFX: ceftriaxone; MONT: montelukast).


( B ) Nissl staining of the hippocampus CA1 , CA2 & CA3 regions ( low magnification ) and cortex region ( higher magnification ) ( left ) and histograms showing the number of Nissl - stained bodies ( right ) Scale bar , 50 μm . Data represent the means ± SEM from three independent experiments , with statistical analysis by One - way ANOVA , p value between control group and Ceftriaxone group is 0 . 00021 , p value between Ceftriaxone group and Ceftriaxone + Montelukast group is 0 . 013 .

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

(B) Nissl staining of the hippocampus CA1, CA2 & CA3 regions (low magnification) and cortex region (higher magnification) (left) and histograms showing the number of Nissl-stained bodies (right) Scale bar, 50 μm. Data represent the means ± SEM from three independent experiments, with statistical analysis by One-way ANOVA, p value between control group and Ceftriaxone group is 0.00021, p value between Ceftriaxone group and Ceftriaxone + Montelukast group is 0.013.


( C - D ) Representative immunofluorescence images ( left ) and quantification ( right ) of TUNEL bodies , astrocyte ( GFAP ) , microglia ( Iba1 ) in cortex of wild type mice after 7 days of therapy . Scale bar , 50 μm . Data represent the means ± SEM from three independent experiments , with statistical analysis by One - way ANOVA , p value of TUNEL bodies between CFX group and MONT + CFX group is 0 . 0012 , p value of astrocyte between CFX group and MONT + CFX group is 0 . 0068 , and p value of microglia cells between CFX group and MONT + CFX group is 0 . 0029 . ( E ) Y - maze test for spatial learning and memory of different group of wild type mice . The total duration of time spent at novel arm ( left ) and the percentage of entries to novel arm of Y - maze ( right ) . Data represent the means ± SEM was statistically analyzed by One - way ANOVA , p value of total duration of time spent at novel arm between control group ( n = 6 ) and CFX group ( n = 5 ) is 0 . 00012 , p value between CFX group ( n = 5 ) and MONT + CFX group ( n = 6 ) is 0 . 00017 ; p value of total duration of % of entry to novel arm between control group ( n = 6 ) and CFX group ( n = 5 ) is 0 . 0102 , and p value between CFX group ( n = 5 ) and MONT + CFX group ( n = 6 ) is 0 . 0216 .

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

(C-D) Representative immunofluorescence images (left) and quantification (right) of TUNEL bodies, astrocyte (GFAP), microglia (Iba1) in cortex of wild type mice after 7 days of therapy. Scale bar, 50 μm. Data represent the means ± SEM from three independent experiments, with statistical analysis by One-way ANOVA, p value of TUNEL bodies between CFX group and MONT + CFX group is 0.0012, p value of astrocyte between CFX group and MONT + CFX group is 0.0068, and p value of microglia cells between CFX group and MONT + CFX group is 0.0029. (E) Y-maze test for spatial learning and memory of different group of wild type mice. The total duration of time spent at novel arm (left) and the percentage of entries to novel arm of Y-maze (right). Data represent the means ± SEM was statistically analyzed by One-way ANOVA, p value of total duration of time spent at novel arm between control group (n = 6) and CFX group (n = 5) is 0.00012, p value between CFX group (n = 5) and MONT + CFX group (n = 6) is 0.00017; p value of total duration of % of entry to novel arm between control group (n = 6) and CFX group (n = 5) is 0.0102, and p value between CFX group (n = 5) and MONT + CFX group (n = 6) is 0.0216.


Wip1 represses the gene responses induced by Nodal or BMP signaling in animal caps as analyzed by RT - PCR . ODC serves as a loading control . - RT , control in the absence of reverse transcriptase . WE , whole embryo . Co AC , uninjected control animal caps . ( - ) , no injection of wild - type ( wt ) Wip1 or Wip1 ( D277A ) mRNA . The amount of mRNA injected : wt Wip1 ( 1 ng ) , Wip1 ( D277A ) ( 1 ng ) , Xnr1 ( 50 pg )

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

Wip1 represses the gene responses induced by Nodal or BMP signaling in animal caps as analyzed by RT-PCR. ODC serves as a loading control. -RT, control in the absence of reverse transcriptase. WE, whole embryo. Co AC, uninjected control animal caps. (-), no injection of wild-type (wt) Wip1 or Wip1(D277A) mRNA. The amount of mRNA injected: wt Wip1 (1 ng), Wip1(D277A) (1 ng), Xnr1 (50 pg)


Wip1 represses the gene responses induced by Nodal or BMP signaling in animal caps as analyzed by RT - PCR . ODC serves as a loading control . - RT , control in the absence of reverse transcriptase . WE , whole embryo . Co AC , uninjected control animal caps . ( - ) , no injection of wild - type ( wt ) Wip1 or Wip1 ( D277A ) mRNA . The amount of mRNA injected : wt Wip1 ( 1 ng ) , Wip1 ( D277A ) ( 1 ng ) , BMP4 ( 100 pg ) .

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

Wip1 represses the gene responses induced by Nodal or BMP signaling in animal caps as analyzed by RT-PCR. ODC serves as a loading control. -RT, control in the absence of reverse transcriptase. WE, whole embryo. Co AC, uninjected control animal caps. (-), no injection of wild-type (wt) Wip1 or Wip1(D277A) mRNA. The amount of mRNA injected: wt Wip1 (1 ng), Wip1(D277A) (1 ng), BMP4 (100 pg).


Wip1 represses the gene responses induced by Nodal or BMP signaling in animal caps as analyzed by RT - PCR . ODC serves as a loading control . - RT , control in the absence of reverse transcriptase . WE , whole embryo . Co AC , uninjected control animal caps . ( - ) , no injection of wild - type ( wt ) Wip1 or Wip1 ( D277A ) mRNA . The amount of mRNA injected : wt Wip1 ( 1 ng ) , Wip1 ( D277A ) ( 1 ng )

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

Wip1 represses the gene responses induced by Nodal or BMP signaling in animal caps as analyzed by RT-PCR. ODC serves as a loading control. -RT, control in the absence of reverse transcriptase. WE, whole embryo. Co AC, uninjected control animal caps. (-), no injection of wild-type (wt) Wip1 or Wip1(D277A) mRNA. The amount of mRNA injected: wt Wip1 (1 ng), Wip1(D277A) (1 ng)