Oxidation kinetics experiment to investigate influence of MIA40 on AK2 maturation . Experiment was performed as described in ( D ) , except that 72 h before experiments were transfected with control siRNA or siRNA directed against MIA40 . Endogenous AK2 oxidation depends on MIA40 . Reported values are mean of 3 independent experiments ; error bars represent ± SD . Student ' s t - was performed . * represents p < 0 . 05 , * * represents p < 0 . 01 * * * represents p < 0 . 001 .

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

Oxidation kinetics experiment to investigate the influence of MIA40 on AK2 maturation. Experiment was performed as described in (D), except that 72 h before experiments cells were transfected with control siRNA or siRNA directed against MIA40. Endogenous AK2 oxidation depends on MIA40. Reported values are the mean of 3 independent experiments; error bars represent ±SD. Student's t-test was performed. * represents p<0.05, ** represents p<0.01 and *** represents p<0.001.


Influence of AK2 cysteines on AK2 redox state . expressing single cysteine to serine mutants of AK2 - HA ( isoform A ) were lysed after NEM treatment , analyzed by inverse redox shift using mmPEG12 , SDS - PAGE immunoblot against HA tag . AK2C42S AK2C92S are present in their completely reduced state indicating presence of a disulfide bond between both cysteines .

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

Influence of AK2 cysteines on the AK2 redox state. Cells expressing single cysteine to serine mutants of AK2-HA (isoform A) were lysed after NEM treatment, and analyzed by inverse redox shift assay using mmPEG12, SDS-PAGE and immunoblot against the HA tag. AK2C42S and AK2C92S are present in their completely reduced state indicating presence of a disulfide bond between both cysteines.


Influence of AK2 cysteines on localization . expressing single cysteine to serine mutants of AK2 - HA ( isoform A ) were fixed analyzed by immunofluorescence . colocalization of AK2 - HA variants with Mitotracker was quantified using Cellprofiler ( Pearson ' s correlation coefficient ) . C42 C92 are critical for mitochondrial localization of AK2 . C40S mutant exhibits significantly decreased colocalization with mitochondria . A Kruskall - Wallis rank sum / Dunn - was performed . Numbers of analysed are indicated in plot . * * * represents p - value < 0 . 001 compared to wild - type . black horizontal represents median . Data information : Scale bars : 10 μm .

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 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 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

Influence of AK2 cysteines on cellular localization. Cells expressing single cysteine to serine mutants of AK2-HA (isoform A) were fixed and analyzed by immunofluorescence. The colocalization of AK2-HA variants with Mitotracker was quantified using Cellprofiler (Pearson's correlation coefficient). C42 and C92 are critical for mitochondrial localization of AK2. The C40S mutant exhibits significantly decreased colocalization with mitochondria. A Kruskall-Wallis rank sum test/ Dunn-test was performed. Numbers of analysed cells are indicated in the plot. *** represents p-value <0.001 compared to wild-type. The black horizontal line represents the median. Data information: Scale bars: 10 μm.


Steady state of endogenous AK2 in presence absence of MIA40 . 72 h before experiments , HeLa were transfected with control siRNA or siRNAs directed against MIA40 or ALR . lysates were analysed by immunoblot against indicated . MIA40 depletion led to a strong decrease in AK2 ALR but not of control . ALR depletion did not affect AK2 . Reported values are mean of 3 independent experiments ; error bars represent ± SD . Student ' s t - was performed . * represents p < 0 . 05 , .

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

Steady state levels of endogenous AK2 in presence and absence of MIA40. 72 h before experiments, HeLa cells were transfected with control siRNA or siRNAs directed against MIA40 or ALR. Cell lysates were analysed by immunoblot against the indicated proteins. MIA40 depletion led to a strong decrease in AK2 and ALR levels but not of the control proteins. ALR depletion did not affect AK2 levels. Reported values are the mean of 3 independent experiments; error bars represent ±SD. Student's t-test was performed. * represents p<0.05,.


Stability of endogenous AK2 in presence absence of MIA40 . 72 h before experiments HeLa , were transfected with control siRNA or siRNAs directed against MIA40 . were labeled for 10 min with [ 35S ] - methionine chased with cold methionine for indicated times . chase was stopped by lysis , AK2 was isolated by denaturing immunoprecipitation . Eluates were analyzed by SDS - PAGE autoradiography . Stability of endogenous AK2 is significantly decreased upon depletion of MIA40 . Reported values are mean of 3 independent experiments ; error bars represent ± SD . Student ' s t - was performed ; * represents p - value < 0 . 05 compared to wild - type .

O 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 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-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 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

Stability of endogenous AK2 in the presence and absence of MIA40. 72 h before experiments HeLa, cells were transfected with control siRNA or siRNAs directed against MIA40. Cells were pulse labeled for 10 min with [35S]-methionine and chased with cold methionine for the indicated times. The chase was stopped by cell lysis, and AK2 was isolated by denaturing immunoprecipitation. Eluates were analyzed by SDS-PAGE and autoradiography. Stability of endogenous AK2 is significantly decreased upon depletion of MIA40. Reported values are the mean of 3 independent experiments; error bars represent ±SD. Student's t-test was performed; * represents p-value <0.05 compared to wild-type.


Stability of AK2 - HA cysteine variant . Experiment was performed as in ( B ) except that expressing AK2C40S , C42S , C92S - HA were analysed by IP against HA . AK2C40S , C42S , C92S - HA is unstable but becomes stabilized by application of proteasomal inhibitor MG132 . Reported values are mean of 3 independent experiments ; error bars represent ± SD . Student ' s t - 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 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 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

Stability of cytosolic AK2-HA cysteine variant. Experiment was performed as in (B) except that cells expressing AK2C40S,C42S,C92S-HA were analysed by IP against HA. AK2C40S,C42S,C92S-HA is unstable but becomes stabilized by application of the proteasomal inhibitor MG132. Reported values are the mean of 3 independent experiments; error bars represent ±SD. Student's t-test was performed. * represents p<0.05,.


Steady state of AK2 - HA cysteine variants . AK2C40S , C42S , C92S - HA AK2WT - HA were expressed in presence or absence of proteasomal inhibitor MG132 . at steady state were analysed by immunoblot . variants are stabilized by proteasomal inhibition . Reported values are mean of 3 ( AK2WT - HA ) or 5 ( AK2C40S , C42S , C92S - HA ) independent experiments ; error bars represent ± SD . ANOVA / Tukey ' s post - hoc was performed . * * * represents p < 0 . 001 .

O O O B-MEASURED_VAR 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 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 O 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 AK2-HA cysteine variants. AK2C40S,C42S,C92S-HA and AK2WT-HA were expressed in the presence or absence of proteasomal inhibitor MG132. Protein levels at steady state were analysed by immunoblot. All variants are stabilized by proteasomal inhibition. Reported values are the mean of 3 (AK2WT-HA) or 5 (AK2C40S,C42S,C92S-HA) independent experiments; error bars represent ±SD. ANOVA/Tukey's post-hoc test was performed. *** represents p<0.001.


Steady state of AK2 - HA cysteine variant . 72 h before experiments HEK293 were transfected with control siRNA or siRNAs directed against proteasomal subunits PSMB3 PSMC5 . AK2C40S , C42S , C92S - HA was stabilized by decreased proteasome . Reported values are mean of 4 independent experiments ; error bars represent ± SD . Student ' s t - was performed . * represents p < 0 . 05 .

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

Steady state levels of AK2-HA cysteine variant. 72 h before experiments HEK293 cells were transfected with control siRNA or siRNAs directed against the proteasomal subunits PSMB3 and PSMC5. AK2C40S,C42S,C92S-HA was stabilized by decreased proteasome levels. Reported values are the mean of 4 independent experiments; error bars represent ±SD. Student's t-test was performed. * represents p<0.05.


Steady state of AK2C40S , C42S , C92S - HA in presence absence of DPP8 DPP9 . 72 h before experiments , HEK293 stably expressing AK2C40S , C42S , C92S - HA were transfected with control siRNA or siRNAs directed against DPP8 or / DPP9 . lysates were analysed by immunoblot against indicated . DPP9 depletion led to an increase in AK2C40S , C42S , C92S - HA , while DPP8 depletion did not . Combined depletion of DPP8 DPP9 increased AK2C40S , C42S , C92S - HA even further . This indicates that DPP9 is main peptidase DPP8 can serve as its backup . black arrow heads indicate endogenous HA - tagged AK2 , respectively . gray arrow head indicates DPP9 band . Reported values are mean of 3 independent experiments ; error bars represent ± SD . ANOVA / Tukey ' s post - hoc was performed . * represents p < 0 . 05 , , # # represent p < 0 . 01 * * * , # # # represent p < 0 . 001 .

O O O B-MEASURED_VAR O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O B-MEASURED_VAR 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 B-CONTROLLED_VAR 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 B-CONTROLLED_VAR I-CONTROLLED_VAR O B-MEASURED_VAR 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 B-MEASURED_VAR O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 AK2C40S,C42S,C92S-HA in presence and absence of DPP8 and DPP9. 72 h before experiments, HEK293 cells stably expressing AK2C40S,C42S,C92S-HA were transfected with control siRNA or siRNAs directed against DPP8 or/and DPP9. Cell lysates were analysed by immunoblot against the indicated proteins. DPP9 depletion led to an increase in AK2C40S,C42S,C92S-HA levels, while DPP8 depletion did not. Combined depletion of DPP8 and DPP9 increased AK2C40S,C42S,C92S-HA levels even further. This indicates that DPP9 is the main peptidase and DPP8 can serve as its backup. White and black arrow heads indicate the endogenous and HA-tagged AK2, respectively. The gray arrow head indicates the DPP9 band. Reported values are the mean of 3 independent experiments; error bars represent ±SD. ANOVA/Tukey's post-hoc test was performed. * represents p<0.05, ,## represent p<0.01 and ***,### represent p<0.001.


Steady state of AK2C40S , C42S , C92S - HA upon incubation with DPP9 inhibitor 1G244 ( Wu et al . , 2009 ) . As ( B ) except that were treated for 16 h with inhibitor or DMSO as control . AK2C40S , C42S , C92S - HA were strongly increased upon DPP8 / 9 inhibition . Reported values are mean of 4 independent experiments ; error bars represent ± SD . Student ' s t - was performed . * * * represents p < 0 . 001 .

O O O B-MEASURED_VAR 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 B-MEASURED_VAR 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

Steady state levels of AK2C40S,C42S,C92S-HA upon incubation with the DPP9 inhibitor 1G244 (Wu et al., 2009). As (B) except that cells were treated for 16 h with inhibitor or DMSO as control. AK2C40S,C42S,C92S-HA levels were strongly increased upon DPP8/9 inhibition. Reported values are the mean of 4 independent experiments; error bars represent ±SD. Student's t-test was performed. *** represents p<0.001.


Steady state of endogenous AK2 in presence absence of DPP9 MIA40 . 72 h before experiments HeLa were transfected with control siRNA or siRNAs directed against MIA40 / or DPP9 . lysates were analysed by immunoblot against indicated . If import of endogenous AK2 was prevented by depletion of MIA40 , then concomitant depletion of DPPs increased its . Strikingly , depletion of DPP9 alone also increased of endogenous AK2 . Reported values are mean of 3 independent experiments ; error bars represent ± SD . ANOVA / Tukey ' s post - hoc was performed . * * represents p < 0 . 01 .

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

Steady state levels of endogenous AK2 in presence and absence of DPP9 and MIA40. 72 h before experiments HeLa cells were transfected with control siRNA or siRNAs directed against MIA40 and/or DPP9. Cell lysates were analysed by immunoblot against the indicated proteins. If import of endogenous AK2 was prevented by depletion of MIA40, then concomitant depletion of DPPs increased its levels. Strikingly, depletion of DPP9 alone also increased the levels of endogenous AK2. Reported values are the mean of 3 independent experiments; error bars represent ±SD. ANOVA/Tukey's post-hoc test was performed. ** represents p<0.01.


Steady state of endogenous AK2 upon incubation with DPP9 inhibitor 1G244 . As ( D ) except that were treated for 3 or 5 days with inhibitor or DMSO as control . Endogenous AK2 were increased upon DPP8 / 9 inhibition . Reported values are mean of 5 ( 3 d ) 6 ( 5 d ) independent experiments ; error bars represent ± SD . Student ' s t - was performed . * represents p < 0 . 05 , * * * represents p < 0 . 001 .

O O O O B-MEASURED_VAR 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 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 O O

Steady state levels of endogenous AK2 upon incubation with the DPP9 inhibitor 1G244. As (D) except that cells were treated for 3 or 5 days with inhibitor or DMSO as control. Endogenous AK2 levels were increased upon DPP8/9 inhibition. Reported values are the mean of 5 (3 d) and 6 (5 d) independent experiments; error bars represent ±SD. Student's t-test was performed. * represents p<0.05, and *** represents p<0.001.


Steady state of AK2 - HA variants with mutated N - termini upon incubation with either 1G244 or vehicle control . HEK293 stably expressing AK2 - HA variants were lysed subjected to immunoblot against indicated . of DPP8 / 9 - insensitive AK2 variants are strongly increased . Reported values are mean of 4 independent experiments ; error bars represent ± SD . ANOVA / Tukey ' s post - hoc was performed . * * represents p < 0 . 01 # # # , * * * represents p < 0 . 001 . Asterisk denominate statistical comparison with respective control , hash tag represents comparison to N - WT .

O O O B-MEASURED_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 B-CONTROLLED_VAR O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 AK2-HA variants with mutated N-termini upon incubation with either 1G244 or vehicle control. HEK293 cells stably expressing AK2-HA variants were lysed and subjected to immunoblot against the indicated proteins. Levels of DPP8/9-insensitive AK2 variants are strongly increased. Reported values are the mean of 4 independent experiments; error bars represent ±SD. ANOVA/Tukey's post-hoc test was performed. ** represents p<0.01 and ###, *** represents p<0.001. Asterisk denominate statistical comparison with respective control, and hash tag represents comparison to N-WT.


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

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

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 wild type , DPP9 - insensitive import - competent AK2 - HA variants co - localized with markers indicating partial localization . colocalization of AK2 - HA variants with Mitotracker was quantified using Fiji ( Pearson ' s correlation coefficient ) . A one - way ANOVA with post - hoc Tukey was performed . Numbers of analysed are indicated in plot . * * represents p < 0 . 01 . Data shown in Figures 2C , 5B , S2 S5 are derived from same experiments . black horizontal represents 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 marker increased . A one - way ANOVA with post - hoc Tukey was performed . Numbers of analysed are indicated in plot . * * * represents p < 0 . 001 . black horizontal represents 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 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

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 purified AK2 variants exhibited maximal activity at around 40 µM AMP with an apparent KM of 0 . 012 mM AMP . profiles for both AK2C40S , C232S AK2C40S , C42S , C92S , C232S are superimposable . Reported values are 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 0 . 1 mM ADP for AK2C40S , C232S AK2C40S , C42S , C92S , C232S , respectively . profiles for both AK2C40S , C232S AK2C40S , C42S , C92S , C232S are thus highly similar . Reported values are mean of at least 3 independent experiments ; error bars represent ± SD .

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

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

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 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 B-MEASURED_VAR 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 B-CONTROLLED_VAR O 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

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

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

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

O O O B-MEASURED_VAR 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 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

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 fractions analysed by immunoblot against indicated . Mitochondria contain increased NDUFB10C107S after DPP9 inhibition . Reported values are mean of 2 independent experiments ; error bars represent ± SD . Student ' s t - 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 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

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 between P0 - P10 , by sex . Each dot represents absolute of remaining per group ( Jag1CTRL n = 211 , Jag1Ndr / Ndr n = 26 , Jag1Ndr / Ndr n = 20 , log - rank , * * * * P < 0 . 0001 ) .

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 B-CONTROLLED_VAR 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 ( n = 6 per group , unpaired t , * * P = 0 . 0091 ) . C P10 plasma thrombin antithrombin ( n = 6 per group , unpaired t , ns , P = 0 . 2788 ) .

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

B 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 ( n = 6 per group , unpaired t , 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

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 , 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

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 , 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

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 : Jag1 + / + vs Jag1Ndr / Ndr * P = 0 . 0227 ; : 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 : Jag1 + / + vs Jag1Ndr / Ndr * P = 0 . 0305 ; 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 outside of blood vessels ( red ) in adult mice ( n = 6 per group , unpaired t , 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

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 ( Jag1CTRL n = 7 , Jag1Ndr / Ndr n = 8 , unpaired t , ns , P = 0 . 3807 ) .

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

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 outside P10 Jag1Ndr / Ndr retinal arteriole . Scale bar 10 µm .

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

(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 . Interaction P = 0 . 7074 , Sex P = > 0 . 9999 , Genotype * * * * P = < 0 . 0001 , Tukey ' s multiple comparisons : : Jag1 + / + vs . : Jag1Ndr / Ndr * P = 0 . 0201 ; : Jag1 + / + vs . : 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 B-CONTROLLED_VAR 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

(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 . Interaction P = 0 . 4353 , Sex P = 0 . 1995 , Genotype * * * * P = < 0 . 0001 , Tukey ' s multiple comparisons : : Jag1 + / + vs . : Jag1Ndr / Ndr * P = 0 . 0146 ; : Jag1 + / + vs . : Jag1Ndr / Ndr * * P = 0 . 0011 ) ( D ) venules ( n = 6 per group , two - way ANOVA with Tukey ' s multiple comparisons . Interaction P = 0 . 044 , Sex P = 0 . 044 , Genotype * * * * P = < 0 . 0001 , Tukey ' s multiple comparisons : : Jag1Ndr / Ndr vs . : Jag1Ndr / Ndr * P = 0 . 0302 ; : Jag1 + / + vs . : 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 B-CONTROLLED_VAR 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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

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 1 year , irrespective of sex ( n = 6 per group , two - way ANOVA , not significant ) . ( F ) Arterial tortuosity in mice , irrespective of ( n = 5 - 7 , two - way ANOVA , not significant ) . G - H ( G ) Venous tortuosity at P30 1 year , irrespective of sex ( n = 6 - 8 per group , two - way ANOVA with Tukey ' s multiple comparisons . Interaction P = 0 . 1166 , P = 0 . 0787 , Genotype * * P = 0 . 0013 . Tukey ' s multiple comparisons : 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 mice , irrespective of ( n = 5 - 7 , two - way ANOVA with Tukey ' s multiple comparisons . Interaction * P = 0 . 0223 , Sex * * P = 0 . 007 , Genotype * * * P = < 0 . 0001 . Tukey ' s multiple comparisons : : Jag1 + / + vs . : Jag1Ndr / Ndr P = 0 . 3247 , : Jag1Ndr / Ndr vs . : Jag1Ndr / Ndr * * P = 0 . 0031 , : Jag1 + / + vs . : 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 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 B-CONTROLLED_VAR 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 B-CONTROLLED_VAR 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 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 ( n = 6 , t - ns ) .

(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 actin ( αSMA ) showed stereotype vasculature in 6 of 6 Jag1 + / + , but 2 of 6 Jag1Ndr / Ndr 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 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 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 - , not significant ) .

(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 € 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 blood vessel . Green arrowheads in D denote stenosis . arrowheads in E label αSMA - negative gaps arrows an aneurysm . Cropped 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 B-MEASURED_VAR 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 ( Two - way ANOVA with Sidak ' s multiple comparisons ) .

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

(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 mouse by ( Two - way ANOVA with Sidak ' s multiple comparisons . Dots represent biological replicates / retinas / mice . Two - way ANOVA not significant . Šídák ' s multiple comparisons : 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 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 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

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 . Scale bar 10 µm . ( J ) cCasp3 + per field ( n = 6 per group , unpaired t , * 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

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 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

(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 nuclei ( n = 6 per group , unpaired t , * 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

(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 Jag1CTRL Jag1Ndr / Ndr mice . Each dot represents one / one biological replicate . ( Two - way ANOVA Interaction P = 0 . 9407 , Sex P = 0 . 4957 , Genotype * P = 0 . 0207 . Šídák ' s multiple comparisons not significant ) .

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

(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 Jag1Ndr / Ndr mice treated with vehicle or AngII for two ( 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 I-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR 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 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 I-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O 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

(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 ( ) 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

(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 : AngII treated Jag1 + / + vs AngII treated Jag1Ndr / Ndr * P = 0 . 0315 ) . ) .

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® 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 deep layers . Scale bar 50 µm . ( B ) P30 SCP branching point number ( n = 6 - 8 per group , unpaired t , ns , P = 0 . 5346 ) . ( C ) P30 ICP vascular ( n = 6 per group , unpaired t , * P = 0 . 0124 ) . ( D ) P30 ICP branching point number ( n = 6 per group , unpaired t , * * 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 , ns , P = 0 . 5365 ) . ( G ) Adult ICP vascular ( n = 6 - 7 per group , unpaired t , * * P = 0 . 0078 ) . ( H ) Adult ICP branching point number ( n = 6 - 7 per group , unpaired t , * * P = 0 .

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

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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 ) one - year - old mice ( bottom panels ) . arrowheads label discontinuous ICP . Blue arrowhead labels SCP , green ICP magenta DCP .

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

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

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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 , with CADASIL , healthy pediatric , with ALGS with BA . Boxed regions black arrowheads magnify arteriovenous crossing .

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

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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 ) ( 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 ) ( 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 ) .

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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 ) MAP2 ( dendritic ) markers in DIV21 hippocampal neurons . Lower panels - Single plane apotome of nuclei ( DAPI ) show PRR7 can be present ( arrow ) or absent ( arrowhead ) from nuclei .

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

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(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 .

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(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 PSD95 were used as , presynaptic postsynaptic markers , respectively .

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(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 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 ) .

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(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 for each group ) . Statistical analyses for ( A ) ( B ) were performed using Mann Whitney .

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(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 ) of PRR7 immunostaining in cultured hippocampal neurons . Treatments ( APV [ 50 µM , APV - ( 2R ) - - 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 glutamate dependent increase in PRR7 ( 108 . 1 ± 19 % ) . Statistical significance was determined using one - way ANOVA in conjugation with Sidak post - hoc .

(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 nucleus of hippocampal neurons upon NMDA treatment . Depicted are representative averaged confocal ( 30x30 µm2 ) from optical sections ( 300 nm step ) of PRR7 - GFP expressing neurons ( DIV17 ) before after bleaching . Prior to stimulation there was substantial PRR7 - GFP in nucleus due to basal neuronal activity ( upper panel ) . This was reduced by incubation overnight with MK801 ( 10 µM ) TTX ( 1 µM ) ( lower panel ) . NMDA ( 50 µM ) stimulation , but not MK - 801 , led to recovery of PRR7 - GFP following bleaching of ROIs at t0 ` ( blue arrow in E ) . ( E ) of changes in GFP intensities were normalized to time point t0 ` 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 ± ; n = 9 ( NMDA ) n = 5 ( MK801 ) . was done using one - way ANOVA using Bonferroni post - hoc correction .

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

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(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 GluN2B from rat brain lysates . FT = flowthrough , IgG = normal IgGs .

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(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 of c - terminal myc - tagged PRR7 with GluN1 in absence of other NMDAR subunits or PSD95 .

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(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 transfected with tagged NMDA or AMPA receptor subunits PRR7 ( myc - tagged GluN2B GluA2 flag - tagged PRR7 ) .

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(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 different PRR7 constructs used for co - IP ( FL = full ) . ( bottom ) PRR7 deletion constructs NMDAR subunits were expressed in HEK293 Co - IPs performed as indicated .

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(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 neuronal cultures following treatment with NMDA ( 100µM , 30 min , representative of n = 4 ) .

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(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 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 compared to controls . No change was detected in c - Fos . One - way ANOVA was used to analyze results with Dunett ' s post hoc ( n = 16 gels for c - Jun 4 gels for c - Fos ) .

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(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 were treated with cycloheximide for indicated time points c - Jun were measured by Western blotting . PRR7 expression significantly stabilized c - Jun at 120 min ( 110 . 0 ± 7 . 5 % vs control 68 . 7 ± 7 . 9 % ) 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 - between similar time points were used to analyze results .

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

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(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 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 - .

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(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 hippocampal neurons do not alter of other FBW7 targets mTOR or Myc .

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(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 overexpressing PRR7 .

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


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

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(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 overexpressing PRR7 FBW7 .

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


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

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


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

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


( F ) Co - IPs of PRR7 with c - Jun FBW7 from lysates of HEK293 overexpressing PRR7 FBW7 . FBW7 expression increased 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 presence of PRR7 ( normalized for FBW7 expression ) . Similarly , c - Jun / PRR7 binding is 97 . 4 ± 38 . 1 % greater in presence of FBW7 ( normalized for PRR7 expression ) . Statistical significance was calculated using unpaired t - .

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

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(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 increased AP - 1 activity ( 190 . 6 ± 13 . 9 % , n = 12 ; RLU = relative units ) . Statistical significance was determined using t - .

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(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 using 30 minutes of PI in - situ following 48h of WT - PRR7 - GFP or mNLS - PRR7 - GFP overexpression in hippocampal neuronal cultures ( n = 24 per group ) . represent a max projection of five z - stack sections across .

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(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 % in control neurons compared to 60 . 3 ± 4 . 6 % in neurons with PRR7 expression knocked down . A c - Jun inhibitory peptide reduced NMDA - mediated to 66 . 2 ± 6 . 0 % in control neurons 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 . ( Left ) Representative showing transduced neurons ( green - GFP ) PI ( red ) .

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(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 ) of in response to NMDA stimulation in presence or absence of PRR7 either WT c - Jun or a dominant negative c - Jun4A ( 4A ) . Overexpression of WT c - Jun caused extensive neuronal ( 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 . are representative 10X widefield showing transduced neurons ( green - GFP ) PI ( red ) . Data is presented as mean ± . % values represent comparisons to control .

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

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

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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 reactions contained . Left : Western blotting of reaction products at zero 30 minutes using FANCD2 V5 antibodies . Right : Residual FANCI FANCD2 ubiquitination following USP1 - UAF1 treatment for 30 min . Replicate residual ubiquitination values statistically significant changes ( one - way ANOVA with Bonferroni correction ) are shown . * * * p < 0 . 001 .

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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 P244 ) participating in extended FANCD2 - ubiquitin interface ( IUbD2 - IUbD2Ub - 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 ) in presence of , in absence or presence of wild - type ( D2WT ) or mutant ( D2H209A , VP243AA ) FANCD2 . Replicate residual ubiquitination values ( three technical replicates ) statistically significant changes ( t - one - way ANOVA with Bonferroni correction ) are shown .

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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 ) its deubiquitination by USP1 - UAF1 ( incubation with 100 nM USP1 - UAF1 for 20 minutes at room ) , 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 ) , fold change in residual FANCI ubiquitination was determined by normalisation to mean of IUb - DL680 at 20 nM FANCD2 ( average from six values , for both D2WT D2H209A , VP243AA ) . Mean ( with range ) fold - increase values statistically significant changes upon FANCD2 mutation for each FANCD2 ( t - two - way ANOVA with Bonferroni correction ) are shown .

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

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(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 blood - brain barrier permeability , i . e . , extravasation is indicative of leakage from intravascular lumen . Sulfo - NHS - Biotin was confined to capillaries of meninges cortex ( as shown with arrows ) , there was no evidence of extravasation . Scale bar = 100 μm .

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(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 K79 in HBMEC with or without EGFR inhibitor ( gefitinib ) .

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


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

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(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 K79 infection at 15 min 60 min post infection .

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


( G ) EGFR expression in EGFR knockout HBMEC using CRISPR / Cas9 ( left panel ) relative invasion frequency of 8 meningitis isolates of GBS strains in EGFR knockout 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 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 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 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 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 ) recovered from blood brain of EGFR conditional knockout ( n = 5 ) control mice ( n = 5 ) 1 h after intravenous inoculation with K79 .

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

(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 infected with GBS K79 infection for 60 min . EGFR phosphorylation in control 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 B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O B-MEASURED_VAR O O O 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 K79 in HBMEC with or without SphK1 SphK2 inhibitors . 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 , p value of HB56 is 0 . 0045 .

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