C : Cal27 JHU029 were transfected with control or mimic miR - 7 relative expression of ELDR was analyzed by qRT - PCR . 18S rRNA was used as an internal control . n = 2 biological replicates ; n = 3 technical replicates . Data are represented as mean ± SD ,

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

C: Cal27 and JHU029 cells were transfected with control or mimic miR-7 and relative expression of ELDR was analyzed by qRT-PCR. 18S rRNA was used as an internal control. n = 2 biological replicates; n= 3 technical replicates. Data are represented as the mean ± SD,


D : Cal27 JHU029 were transfected with miR - 7 mimic , ELDR plasmid or both miR - 7 ELDR plasmid . lysates were analyzed by Western blot for EGFR expression using specific antibody . was reprobed with an antibody against Actin as an internal control . Representative bands were cropped from same gel . Right panels show quantitative representation of Western blot band intensities . n = 3 biological replicates . Data are represented as mean ± SD ,

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

D: Cal27 and JHU029 cells were transfected with miR-7 mimic, ELDR plasmid or both miR-7 and ELDR plasmid. Cell lysates were analyzed by Western blot analysis for EGFR expression using specific antibody. The membrane was reprobed with an antibody against Actin as an internal control. Representative bands were cropped from same gel. Right panels show quantitative representation of Western blot band intensities. n = 3 biological replicates. Data are represented as the mean ± SD,


E : Relative expression of miR - 7 in Cal27 JHU029 compared to NOK analyzed by qRT - PCR . U47 was used as an internal control . n = 3 biological replicates 3 technical replicates . Data are represented as mean ± SD ,

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

E: Relative expression of miR-7 in Cal27 and JHU029 cells compared to NOK cell line analyzed by qRT-PCR. U47 was used as an internal control. n = 3 biological replicates and 3 technical replicates. Data are represented as the mean ± SD,


A : was measured in control siELDR treated mice . n = 5 per

A: Body weight was measured in control and siELDR treated mice. n=5 animal per group


B : Representative of tumors in control treatment groups .

B: Representative images of tumors in control and treatment groups.


C : Tumors were measured using a slide caliper volumes were calculated . Arrow indicates starting point siELDR treatment . n = 5 per group volume presented as mean ±

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C: Tumors were measured using a slide caliper and tumor volumes were calculated. Arrow indicates starting point siELDR treatment. n=5 animal per group and tumor volume presented as the mean ± SD


D : ELDR knockdown was examined by analyzing expression of ELDR in treated tumors compared to control tumors . 18S rRNA gene was used as internal control . n = 5 per group 3 technical replicates from each .

O O O O O O O O O O O O O O O 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: ELDR knockdown was examined by analyzing expression of ELDR in treated tumors compared to the control tumors. 18S rRNA gene was used as internal control. n=5 animal per group and 3 technical replicates from each tumor.


E : Control or siELDR treated lysates were subjected to Western blot for EGFR Cyclin E1expression using specific antibodies representative bands are shown . blot was reprobed with an antibody against Actin for normalization . Right panels show quantitation . n = 3 biological replicates from each

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

E: Control or siELDR treated tumor lysates were subjected to Western blot analysis for EGFR and Cyclin E1expression using specific antibodies and representative bands are shown. The blot was reprobed with an antibody against Actin for normalization. Right panels show quantitation. n= 3 biological replicates from each tumor


F : OSCC - PDX tissue was implanted subcutaneously into flank of NSG mice . When volume reached > 70 mm3 , mice were divided into two groups . 10µg of siELDR or control oligoes were injected intratumorally as described above . was measured in control siELDR treated mice . n = 5 per

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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: OSCC-PDX tissue was implanted subcutaneously into the flank of NSG mice. When the tumor volume reached >70 mm3, mice were divided into two groups. 10µg of siELDR or control oligoes were injected intratumorally as described above. Body weight was measured in control and siELDR treated mice. n=5 animal per group


G : Representative of OSCC - PDX tumors in control treatment groups .

G: Representative images of OSCC-PDX tumors in control and treatment groups.


H : PDX tumors were measured using a slide caliper volumes were calculated . n = 5 per group volume presented as mean ±

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

H: PDX tumors were measured using a slide caliper and tumor volumes were calculated. n=5 animal per group and tumor volume presented as the mean ± SD


I : ELDR knockdown was examined by analyzing expression of ELDR in treated OSCC - PDX tumors compared to control tumors . 18S gene was used as internal control . n = 5 per group 3 technical replicates presented as mean ±

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

I: ELDR knockdown was examined by analyzing expression of ELDR in treated OSCC-PDX tumors compared to the control tumors. 18S gene was used as internal control. n=5 animal per group and 3 technical replicates presented as the mean ± SD


Immunohistochemistry performed on 12 month - old THY - Tau22 mice showing AT100 - positive neurons ( green ) , CBP - positive nuclei ( red ) DAPI - stained nuclei ( blue ) in CA1 region of dorsal hippocampus . A representative image is shown with a focus . Scale bars : 40µm 20µm as noted . ( n = 5 mice ) . Arrows depict neurons bearing neurofibrillary tangles do not display CBP immunostaining . star depicts a ghost tangle ( AT100 - positive neuron no nucleus )

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

Immunohistochemistry performed on 12 month-old THY-Tau22 mice showing AT100-positive neurons (green), CBP-positive nuclei (red) and DAPI-stained nuclei (blue) in the CA1 region of the dorsal hippocampus. A representative image is shown with a focus. Scale bars: 40µm and 20µm as noted. (n=5 mice). Arrows depict neurons bearing neurofibrillary tangles and do not display CBP immunostaining. The star depicts a ghost tangle (AT100-positive neuron and no nucleus)


Western blot analyses perfomed in dorsal hippocampus of 12 month - old THY - Tau22 mice compared to - matched controls . NeuN , actin , tubulin total H2B are not changed . Phosphorylated tau on serine 404 ( Tau - Ph404 ) attest for samples from tauopathic mice . represents ration of level detected on total amount of on membranes , with WT arbitrarily set at 1 ( fold induction ) . Bar graphs are mean ± . n = 5 - 7 / group as noted , multiple t , CBP * p = 0 . 003 GFAP * * p = 0 . 0001 for THY - Tau vs . WT

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

Western blot analyses perfomed in the dorsal hippocampus of 12 month-old THY-Tau22 mice compared to age-matched controls. NeuN, actin, tubulin and total H2B levels are not changed. Phosphorylated tau on serine 404 (Tau-Ph404) attest for samples from tauopathic mice. Quantification represents the ration of the protein level detected on the total amount of proteins on the membranes, with WT arbitrarily set at 1 (fold induction). Bar graphs are mean ± SEM. n=5-7/group as noted, multiple t tests, CBP * p=0.003 and GFAP **p=0.0001 for THY-Tau vs. WT mice


- sequencing data performed in hippocampus of 8 month - old mice . Functional analyses ( DAVID GOTERM ) performed on significantly deregulated genes in THY - Tau22 mice compared to WT mice . Representative genes are presented below . | log2 Fold Change | > 0 . 2 ; * indicates FDR < 0 . 05 . IEG , Immediate Early Genes . Predicted promoter motif ( right ) analyses performed with GREAT . Groups : WT mice ( n = 3 ) , THY - Tau22 mice ( n = 2 )

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

RNA-sequencing data performed in the hippocampus of 8 month-old mice. Functional analyses (DAVID GOTERM) performed on significantly deregulated genes in THY-Tau22 mice compared to WT mice. Representative genes are presented below. |log2 Fold Change| > 0.2; * indicates FDR<0.05. IEG, Immediate Early Genes. Predicted promoter motif (right) analyses performed with GREAT. Groups: WT mice (n=3), THY-Tau22 mice (n=2)


Long - term spatial memory testing : Mice were injected 3 times ( 1 per week ) with Vehicle ( WT mice saline , WT VEH , n = 17 ) , Vehicle ( THY - Tau22 mice CSP , 500 μg / mouse , TAU VEH , n = 10 ) or Molecule ( THY - Tau22 mice CSP - TTK21 , 500 μg / mice TAU MOL , n = 13 ) before training of spatial memory in Morris water maze ( MWM ) ; retention ( Probe ) was tested 10 days after last training session . Acquisition ( Escape latencies , seconds ) retention performances ( Time in target quadrant , seconds ) are shown for 3 groups of mice . groups of mice displayed significant acquisition of platform location ( Day effect , F ( 4 , 148 ) = 26 . 45 , p < 0 . 001 ; Group Group X Day effects , ns ) , but only TAU VEH exhibited impaired retention . CSP - TTK21 treatment fully restored ability of THY - Tau22 mice to remember platform location . Bar graphs are mean ± . Student ' s t to a constant value , # when compared to random ( dotted , 15sec ) WT VEH , t ( 16 ) = 4 . 6323 , # p = 0 . 0002 ; TAU VEH , t ( 9 ) = 0 . 3606 , p = 0 . 7267 ; TAU MOL , t ( 12 ) = 3 . 945 , # p = 0 . 0019 . One - way ANOVA ; F ( 2 , 37 ) = 3 . 55 ; p = 0 . 03 ; * in different comparisons : TAU MOL vs . TAU VEH , * p = 0 . 0166 ; WT VEH vs . TAU VEH , * p = 0 . 040 ; TAU MOL vs WT VEH , non significant p = 0 . 437 ) . TQ , Target Quadrant ; O , Other , corresponds to mean of 3 other

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

Long-term spatial memory testing: Mice were injected 3 times (1 per week) with Vehicle (WT mice saline, WT VEH, n=17), Vehicle (THY-Tau22 mice CSP, 500 μg /mouse, TAU VEH, n=10) or Molecule (THY-Tau22 mice CSP-TTK21, 500 μg/mice TAU MOL, n=13) before training of spatial memory in the Morris water maze (MWM); retention (Probe test) was tested 10 days after the last training session. Acquisition (Escape latencies, seconds) and retention performances (Time in target quadrant, seconds) are shown for the 3 groups of mice. All groups of mice displayed significant acquisition of the platform location (Day effect, F(4,148)= 26.45, p<0.001; Group and Group X Day effects, ns), but only TAU VEH exhibited impaired retention. CSP-TTK21 treatment fully restored the ability of THY-Tau22 mice to remember the platform location. Bar graphs are mean ± SEM. Student's t test to a constant value, # when compared to random (dotted line, 15sec) WT VEH, t(16)=4.6323, #p=0.0002; TAU VEH, t(9)=0.3606, p=0.7267; TAU MOL, t(12)=3.945, #p=0.0019. One-way ANOVA; F(2,37)= 3.55; p= 0.03; * in the different comparisons: TAU MOL vs. TAU VEH, *p=0.0166; WT VEH vs. TAU VEH, *p= 0.040; TAU MOL vs WT VEH, non significant p=0.437). TQ, Target Quadrant; O, Other, corresponds to the mean of the 3 other quadrants


timeline of GFP - lentivirus CSP or CSP - TTK21 ( one injection , 500 μg / mouse ) injection is shown . ( Left ) total number of spines was significantly decreased in TAU VEH compared to WT VEH mice . TAU MOL hippocampi showed a significant increase in total number of spines compared to TAU VEH ( One - way ANOVA p = 0 . 0011 , post hoc Holm Sidak ' s multiple comparisons F ( 2 , 243 ) = 7 . 03 ; WT VEH vs . TAU VEH , p < 0 . 0009 ; TAU MOL vs . TAU VEH , p = 0 . 0455 ) . ( Middle ) on spine type , number of head spines ( Mushrooms , thins ) was significantly lower in both TAU VEH TAU MOL than in WT VEH controls ( One - way ANOVA p = 0 . 0018 , post hoc Holm Sidak ' s multiple comparisons F ( 2 , 243 ) = 6 . 467 ; WT VEH vs . TAU VEH , p = 0 . 0013 ; WT VEH vs . TAU MOL , p = 0 . 0272 . Stubby spine density was significantly increased in TAU MOL compared to TAU VEH mice ( One - way ANOVA F ( 2 , 243 ) = 4 . 311 , p = 0 . 0145 , post hoc Holm Sidak ' s multiple comparisons : TAU MOL vs . TAU VEH , p = 0 . 0109 ) , as number of filopodia ( One - way ANOVA F ( 2 , 243 ) = 3 . 845 , p = 0 . 0227 , post hoc Holm Sidak ' s multiple comparisons TAU MOL vs . TAU VEH , p = 0 . 0179 ) . ( Right ) Typical are presented showing a dendrite fragment for each condition . arrowhead depicts stubby spines . Scale bar , 2 μm . Number of dendritc segments : WT VEH , n = 67 ; TAU VEH , n = 93 , TAU MOL , n = 87 ; Number of neurons : WT VEH , n = 20 ; TAU VEH , n = 28 , TAU MOL , n = 16 ; Number of mice : WT VEH , n = 2 ; TAU VEH , n = 3 , TAU MOL , n = 3

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

The timeline of GFP-lentivirus and CSP or CSP-TTK21 (one injection, 500 μg/mouse) injection is shown. (Left) The total number of spines was significantly decreased in TAU VEH compared to WT VEH mice. TAU MOL hippocampi showed a significant increase in the total number of spines compared to TAU VEH (One-way ANOVA p=0.0011, post hoc Holm Sidak's multiple comparisons test F(2,243) = 7.03; WT VEH vs. TAU VEH, p < 0.0009; TAU MOL vs. TAU VEH, p=0.0455). (Middle) Based on spine type, the number of head spines (Mushrooms, thins) was significantly lower in both TAU VEH and TAU MOL than in WT VEH controls (One-way ANOVA p=0.0018, post hoc Holm Sidak's multiple comparisons test F(2,243) = 6.467; WT VEH vs. TAU VEH, p=0.0013; WT VEH vs. TAU MOL, p=0.0272. Stubby spine density was significantly increased in TAU MOL compared to TAU VEH mice (One-way ANOVA F(2,243) = 4.311, p = 0.0145, post hoc Holm Sidak's multiple comparisons test: TAU MOL vs. TAU VEH, p=0.0109), as the number of filopodia (One-way ANOVA F(2,243) = 3.845, p = 0.0227, post hoc Holm Sidak's multiple comparisons test TAU MOL vs. TAU VEH, p=0.0179). (Right) Typical images are presented showing a dendrite fragment for each condition. White arrowhead depicts stubby spines. Scale bar, 2 μm. Number of dendritc segments: WT VEH, n=67; TAU VEH, n=93, TAU MOL, n=87; Number of neurons: WT VEH, n=20; TAU VEH, n=28, TAU MOL, n=16; Number of mice: WT VEH, n=2; TAU VEH, n=3, TAU MOL, n=3


CSP - TTK21 injection into THY - Tau22 mice rescues mature dendritic spines formation in response to learning . ( Top left ) time of injections is shown : Mice were injected 3 times ( 1 per week ) with Vehicle ( WT mice , WT VEH , NaCl 0 . 9 % ) , Vehicle ( THY - Tau22 mice ( TAU VEH ) , CSP 500 μg / mouse ) or Molecule ( THY - Tau22 mice ( TAU MOL ) , 500 μg / mice ) either trained over a 4 - day acquisition period in MWM ( " learning " group ) or left in their home cage ( " basal " group ) . Mushroom - shaped spines were counted in dorsal CA1 , four days post - training . ( Bottom left ) number of mature spines was significantly increased by learning in WT VEH TAU MOL mice . Bar graphs are mean ± . ( Two - way ANOVA ; Learning effect , F ( 1 , 139 ) = 54 . 18 ; p < 0 . 0001 ; # # # post hoc Holm Sidak ' s multiple comparisons : Learning vs . Basal in WT VEH ( p = 0 . 0001 ) in TAU MOL mice ; ( p = 0 . 0001 ) . After learning , WT VEH TAU MOL mice displayed significantly higher number of mature spines than TAU VEH mice ( Genotype X Treatment effect , F ( 2 , 139 ) = 9 . 704 ; p = 0 . 0001 ; * * * post hoc Holm Sidak ' s multiple comparisons : TAU MOL vs . TAU VEH ( p = 0 . 0001 ) , WT VEH vs . TAU VEH ( p = 0 . 0001 ) . ( Right ) Typical examples of a dendritic fragment bearing mushroom spines ( arrows ) are shown for each sub - group in response to learning . Number of dendritc segments : Learning : WT VEH , n = 27 ; TAU VEH , n = 27 , TAU MOL , n = 30 ; WT VEH _ HC , n = 27 ; Number of mice : WT VEH , n = 3 ; TAU VEH , n = 3 , TAU MOL , n = 3 . Basal : WT VEH , n = 27 ; TAU VEH , n = 7 , TAU MOL , n = 15 ; Number of mice : WT VEH , n = 3 ; TAU VEH , n = 3 , TAU MOL , n = 2

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

CSP-TTK21 injection into THY-Tau22 mice rescues mature dendritic spines formation in response to learning. (Top left) The time line of injections is shown: Mice were injected 3 times (1 per week) with Vehicle (WT mice, WT VEH, NaCl 0.9%), Vehicle (THY-Tau22 mice (TAU VEH), CSP 500 μg /mouse) or Molecule (THY-Tau22 mice (TAU MOL), 500 μg/mice) and either trained over a 4-day acquisition period in the MWM ("learning" group) or left in their home cage ("basal" group). Mushroom-shaped spines were counted in dorsal CA1, four days post-training. (Bottom left) The number of mature spines was significantly increased by learning in WT VEH and TAU MOL mice. Bar graphs are mean ± SEM. (Two-way ANOVA; Learning effect, F(1, 139)=54.18; p< 0.0001; ### post hoc Holm Sidak's multiple comparisons test: Learning vs. Basal in WT VEH (p=0.0001) and in TAU MOL mice; (p=0.0001). After learning, WT VEH and TAU MOL mice displayed significantly higher number of mature spines than TAU VEH mice (Genotype X Treatment effect, F(2,139)= 9.704; p=0.0001; *** post hoc Holm Sidak's multiple comparisons test: TAU MOL vs. TAU VEH (p=0.0001), WT VEH vs. TAU VEH (p=0.0001). (Right) Typical examples of a dendritic fragment bearing mushroom spines (arrows) are shown for each sub-group in response to learning. Number of dendritc segments: Learning: WT VEH, n=27; TAU VEH, n=27, TAU MOL, n=30; WT VEH_HC, n=27; Number of mice: WT VEH, n=3; TAU VEH, n=3, TAU MOL, n=3. Basal: WT VEH, n=27; TAU VEH, n=7, TAU MOL, n=15; Number of mice: WT VEH, n=3; TAU VEH, n=3, TAU MOL, n=2


Mice were injected 3 times ( 1 per week ) with saline ( WT VEH ) , CSP ( Vehicle , VEH ) or CSP - TTK21 ( Molecule , MOL ) ( 500 μg / mice ; THY - Tau22 mice ( TAU ) before euthanasia . Long Term Depression measurements were performed on hippocampal slices . ( Top left ) Examples of analog traces recorded 10 min before ( a ) 55 min after LTD induction ( b ; dotted ) in three groups of mice . ( Bottom left ) Time course of LTD ; LTD is expressed as a change in fEPSP ( field excitatory postsynaptic potentials ) slope over time . After 20 - min baseline recording , a low - frequency stimulation ( LFS , 2Hz for 10 min ) was applied ( arrow ) . Recording was stopped during 10 - min conditioning stimulation resumed after completion of LFS . LFS induced a strong depression of fEPSP slope , which recovered partially to reach a stable level of depression about 20 min after stimulation . ( Right ) Average depression measured in last 10 min of LTD . LTD was significantly different in TAU VEH ( 88 . 4 ± 4 . 1 % of baseline , n = 10 ) compared to controls ( WT VEH , 71 . 1 ± 4 . 4 % , n = 9 ) ( F ( 1 , 17 ) = 8 . 8 , p = 0 . 008 ) . CSP - TTK21 treatment restored LTD to control ( 64 . 9 ± 5 . 2 % , n = 10 ) ( WT VEH vs . TAU MOL : F ( 1 , 17 ) = 0 . 83 , p = 0 . 37 , ns ; TAU VEH vs . TAU MOL : F ( 1 , 18 ) = 13 . 2 . p = 0 . 0019 ) . Bar graphs are mean ±

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

Mice were injected 3 times (1 per week) with saline (WT VEH), CSP (Vehicle, VEH) or CSP-TTK21 (Molecule, MOL) (500 μg/mice; THY-Tau22 mice (TAU) before euthanasia. Long Term Depression measurements were performed on hippocampal slices. (Top left) Examples of analog traces recorded 10 min before (a) and 55 min after LTD induction (b; dotted line) in the three groups of mice. (Bottom left) Time course of LTD; LTD is expressed as a percent change in fEPSP (field excitatory postsynaptic potentials) slope over time. After the 20-min baseline recording, a low-frequency stimulation (LFS, 2Hz for 10 min) was applied (arrow). Recording was stopped during the 10-min conditioning stimulation and resumed after completion of LFS. LFS induced a strong depression of the fEPSP slope, which recovered partially to reach a stable level of depression about 20 min after stimulation. (Right) Average depression measured in the last 10 min of LTD. LTD was significantly different in TAU VEH (88.4 ± 4.1% of the baseline, n=10) compared to controls (WT VEH, 71.1 ± 4.4%, n=9) (F(1,17)=8.8, p=0.008). CSP-TTK21 treatment restored LTD to control levels (64.9 ± 5.2%, n=10) (WT VEH vs. TAU MOL: F(1,17)=0.83, p=0.37, ns; TAU VEH vs. TAU MOL: F(1,18)=13.2. p=0.0019). Bar graphs are mean ± SEM


Volcano plot showing that 2756 genes are differentially regulated between THY - Tau22 WT mice during learning . log2 ( Fold - Change ) was estimated by DESeq2 . FDR < 0 . 05 | log2 Fold Change | > 0 . 2 . Red dots correspond to genes with adjusted p - value < 0 . 05 . Numbers in corners represent number of down - regulated ( left ) up - regulated ( right ) genes . C representing expression of z - score of 2756 deregulated genes in experimental conditions . Color - coding was performed according to z - score of normalized reads divided by gene . Clustering was performed using Unweighted Pair Group Method with Arithmetic mean method Pearson " s . Groups : WT VEH _ HC ( n = 3 ) , WT VEH ( n = 2 ) ; TAU VEH ( n = 2 ) ; TAU

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

Volcano plot showing that 2756 genes are differentially regulated between THY-Tau22 and WT mice during learning. The log2(Fold-Change) was estimated by DESeq2. FDR<0.05 and |log2 Fold Change| > 0.2. Red dots correspond to genes with adjusted p-value <0.05. Numbers in the corners represent the number of down-regulated (left) and up-regulated (right) genes. C Heatmap representing expression of z-score of the 2756 deregulated genes in all experimental conditions. Color-coding was performed according to the z-score of the normalized reads counts divided by gene length. Clustering was performed using the Unweighted Pair Group Method with Arithmetic mean method and the Pearson"s distance. Groups: WT VEH_HC (n=3), WT VEH (n=2); TAU VEH (n=2); TAU MOL


Volcano plot showing that 1756 genes are differentially regulated between treated - THY - Tau22 WT mice during learning . Same parameters as in

Volcano plot showing that 1756 genes are differentially regulated between treated-THY-Tau22 and WT mice during learning. Same parameters as in B


F , G Volcano plots showing differentially regulated genes by molecule in THY - Tau22 mice ( F ) by learning in WT mice ( G ) . Same parameters as in

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F, G Volcano plots showing differentially regulated genes by the molecule in THY-Tau22 mice (F) and by learning in WT mice (G). Same parameters as in B


representing expression z - score of 98 significantly over - expressed genes in TAU MOL vs . TAU VEH comparison , for experimental conditions . Color - coding was performed according to z - score of normalized reads divided by gene . Clustering was performed using Unweighted Pair Group Method with Arithmetic mean method Pearson " s

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Heatmap representing expression z-score of the 98 significantly over-expressed genes in the TAU MOL vs. TAU VEH comparison, for all experimental conditions. Color-coding was performed according to the z-score of the normalized reads counts divided by gene length. Clustering was performed using the Unweighted Pair Group Method with Arithmetic mean method and the Pearson"s dis


- seq data showing expression of several immediate early genes ( IEGs ) : Nr4a1 , Arc , Egr1 , Dusp1 , Fos Junb . Most of down - regulated IEGs in tauopathic compared to WT mice present a significant induction in THY - Tau22 mice after CSP - TTK21 injection . Adjusted p - values corresponding to p < 0 . 05 are indicated by * , for WT VEH vs . WT VEH _ HC , $ for TAU VEH vs . WT VEH # , for TAU MOL vs . TAU

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

RNA-seq data showing expression of several immediate early genes (IEGs) : Nr4a1, Arc, Egr1, Dusp1, Fos and Junb. Most of the down-regulated IEGs in tauopathic compared to WT mice present a significant induction in THY-Tau22 mice after CSP-TTK21 injection. Adjusted p-values corresponding to p<0.05 are indicated by *, for WT VEH vs. WT VEH_HC, $ for TAU VEH vs. WT VEH and #, for TAU MOL vs. TAU VEH


- seq data showing that CSP - TTK21 treatment of tauopathic mice restored expression of plasticity / memory - relevant target genes : Klotho ( Kl ) , Neurotensin ( Nts ) . Adjusted p values corresponding to p < 0 . 05 are indicated by * , for WT VEH vs . WT VEH _ HC , $ for TAU VEH vs . WT VEH # , for TAU MOL vs . TAU VEH . F RT - qPCR validations performed in a different cohort of mice from - seq study ( n = 4 - 5 / group ) . One - way ANOVA with uncorrected Fisher " s . Klotho , Kl : F ( 3 , 16 ) = 2 . 949 , p = 0 . 0643 ; * learning p = 0 . 0145 for WT VEH vs . WT VEH _ HC , $ pathology p = 0 . 0395 for WT VEH vs . TAU VEH . Nts : F ( 3 , 14 ) = 4 . 290 , p = 0 . 0241 ; $ pathology p = 0 . 0036 for WT VEH vs . TAU VEH , # molecule p = 0 . 0081 for TAU MOL vs .

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

RNA-seq data showing that CSP-TTK21 treatment of tauopathic mice restored the expression of plasticity/memory-relevant target genes : Klotho (Kl), and Neurotensin (Nts). Adjusted p values corresponding to p<0.05 are indicated by *, for WT VEH vs. WT VEH_HC, $ for TAU VEH vs. WT VEH and #, for TAU MOL vs. TAU VEH. F RT-qPCR validations performed in a different cohort of mice from the RNA-seq study (n=4-5/group). One-way ANOVA with uncorrected Fisher"s test. Klotho, Kl: F(3,16)=2.949, p=0.0643; * learning p=0.0145 for WT VEH vs. WT VEH_HC, $ pathology p=0.0395 for WT VEH vs. TAU VEH. Nts: F(3,14)=4.290, p=0.0241; $ pathology p=0.0036 for WT VEH vs. TAU VEH, # molecule p=0.0081 for TAU MOL vs. TA


Western blot for Klotho Neurotensin expression in different experimental conditions . Immunoblot results are shown ( n = 5 / group ) . Bar graphs represent of as of control group WT VEH _ HC , arbitrarily set at 100 % . Each detected was normalized to corresponding amount of total in gels or nitrocellulose . Klotho Neurotensin were further normalized to level of actin . One - way ANOVA with uncorrected Fisher " s . Klotho : F ( 3 , 16 ) = 5 . 192 , p = 0 . 0107 , * learning p = 0 . 0117 for WT VEH vs . WT VEH _ HC , $ pathology p = 0 . 0027 for WT VEH vs . TAU VEH , # molecule p = 0 . 0282 for TAU MOL vs . TAU VEH . Nts : F ( 3 , 16 ) = 3 . 748 , p = 0 . 0326 , $ pathology p = 0 . 0200 for WT VEH vs . TAU VEH , # molecule p = 0 . 0079 for TAU MOL vs .

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

Western blot for Klotho and Neurotensin expression in the different experimental conditions. Immunoblot results are shown (n=5/group). Bar graphs represent the quantification of the protein levels as percentage of the control group WT VEH_HC, arbitrarily set at 100%. Each detected protein was normalized to the corresponding amount of total proteins in the gels or the nitrocellulose membrane. Klotho and Neurotensin were further normalized to the level of actin. One-way ANOVA with uncorrected Fisher"s test. Klotho: F(3,16)=5.192, p=0.0107, * learning p=0.0117 for WT VEH vs. WT VEH_HC, $ pathology p=0.0027 for WT VEH vs. TAU VEH, # molecule p=0.0282 for TAU MOL vs. TAU VEH. Nts: F(3,16)=3.748, p=0.0326, $ pathology p=0.0200 for WT VEH vs. TAU VEH, # molecule p=0.0079 for TAU MOL vs. TA


A Functional annotation charts ( Benjamini , p < 0 . 05 ) using using DAVID ( GOTERM _ Component ) perfomed on 82 significantly down - regulated genes by CSP - TTK21 treatment in tauopathic mice ( TAU MOL vs . TAU VEH ) , showing a significant association with neuronal terms . Significance is indicated as - log10 ( Benjamini p _ value ) . B representing expression z - score of 82 significantly down - regulated genes in TAU MOL vs . TAU VEH comparison , for experimental conditions . Color - coding was performed according to z - score of normalized reads divided by gene . Clustering was performed using Unweighted Pair Group Method with Arithmetic mean method Pearson " s

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A Functional annotation charts (Benjamini, p<0.05) using using DAVID (GOTERM_Cellular Component) perfomed on the 82 significantly down-regulated genes by CSP-TTK21 treatment in tauopathic mice (TAU MOL vs. TAU VEH), showing a significant association with neuronal terms. Significance is indicated as -log10(Benjamini p_value). B Heatmap representing expression z-score of the 82 significantly down-regulated genes in the TAU MOL vs. TAU VEH comparison, for all experimental conditions. Color-coding was performed according to the z-score of the normalized reads counts divided by gene length. Clustering was performed using the Unweighted Pair Group Method with Arithmetic mean method and the Pearson"s dis


C - seq data showing expression of several genes belonging to « synapse » annotation ( DAVID ) : Grik4 , Homer3 Sv2b , which are significantly down - regulated by CSPTTK21 treatment in THY - Tau22 mice . Adjusted p values corresponding to p < 0 . 05 are indicated by * for WT VEH vs . WT VEH _ HC , $ for TAU VEH vs . WT VEH # , for TAU MOL vs . TAU

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 RNA-seq data showing expression of several genes belonging to « synapse » annotation (DAVID): Grik4, Homer3 and Sv2b, which are significantly down-regulated by CSPTTK21 treatment in THY-Tau22 mice. Adjusted p values corresponding to p<0.05 are indicated by * for WT VEH vs. WT VEH_HC, $ for TAU VEH vs. WT VEH and #, for TAU MOL vs. TAU VEH


Mean H2Bac profiles at TSS relative to gene expression obtained from - seq data . Expressed genes were separated into 4 groups ( Q1 to Q4 , 6483 genes / group ) , Q1 representing less expressed genes ( 0 - 25 % ) Q4 , most expressed genes ( 75 - 100 % ) . Profiles were established with SeqMiner centered on TSS ± 2Kb . C Mean H2Bac profiles established with SeqMiner for genes at TSS ( top ) gene bodies ( bottom ) for 4th quartile ( most expressed genes ) in two replicates ( rep1 rep2 ) . Significance ( Benjamini , p < 0 . 05 ) of annotations associated with both TSS ( green ) or Gene profiles ( red ) are shown ( DAVID , KEGG Pathways )

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Mean H2Bac profiles at TSS relative to gene expression obtained from RNA-seq data. Expressed genes were separated into 4 groups (Q1 to Q4, 6483 genes/group), Q1 representing the less expressed genes (0-25%) and Q4, the most expressed genes (75-100%). Profiles were established with SeqMiner centered on TSS ±2Kb. C Mean H2Bac profiles established with SeqMiner for all genes at TSS (top) and gene bodies (bottom) for the 4th quartile (most expressed genes) in the two replicates (rep1 and rep2). Significance (Benjamini, p<0.05) of the annotations associated with both TSS (green) or Gene profiles (red) are shown (DAVID, KEGG Pathways)


Differentially regulated H2Bac peaks were analyzed with SICER significant Fold Changes ( FC > 1 ; FDR < 0 . 001 ) between comparisons are shown for pathology ( TAU VEH vs . WT VEH , blue ) effect of molecule ( TAU MOL vs . TAU VEH , red ) . Fold Change is shown as log2 ( Fold Change ) ( X - axis ) significance , as - log10 ( FDR ) ( Y - axis )

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Differentially regulated H2Bac peaks were analyzed with SICER and significant Fold Changes (FC>1; FDR<0.001) between comparisons are shown for the pathology (TAU VEH vs. WT VEH, blue) and the effect of the molecule (TAU MOL vs. TAU VEH, red). Fold Change is shown as log2 (Fold Change) (X-axis) and significance, as -log10(FDR) (Y-axis)


Mean H2Bac H3K27ac profiles established with SeqMiner for 1624 decreased peaks obtained in comparison TAU VEH vs . WT VEH . H2Bac but not H3K27ac peaks show differential

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Mean H2Bac and H3K27ac profiles established with SeqMiner for the 1624 decreased peaks obtained in the comparison TAU VEH vs. WT VEH. H2Bac but not H3K27ac peaks show differential regulation


Time series Like plot . For every significant H2Bac decreased regions ( 1624 ) in WT VEH vs TAU VEH , normalized read were computed for each of different conditions ( WT VEH , TAU VEH , TAU MOL ) . Fold changes are referring to WT VEH . Y - axis : Fold change . X - axis :

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

Time series Like plot. For every significant H2Bac decreased regions (1624) in WT VEH vs TAU VEH, normalized read counts were computed for each of the different conditions (WT VEH, TAU VEH, TAU MOL). Fold changes are all referring to WT VEH. Y-axis: Fold change. X-axis: Conditions


A Mean enrichment profiles performed with SeqMiner presenting genomic distribution of H2Bac H3K27ac reads obtained in ChIP - seq experiments in different experimental groups , established along putative neuronal CBP enhancers ( ± 4Kb ) ( Kim et al . , 2010 ) . In dorsal hippocampus , H2Bac are enriched at CBP enhancers in WT WEH ( blue ) , decreased in tauopathic mice ( TAU VEH , pink ) induced by CSP - TTK21 treatment ( TAU MOL , green ) . B Mean profiles were separated in two sets of loci , including 17817 peaks highly enriched in H2Bac showing a differential regulation in experimental groups 23331 peaks that were poorly enriched in H2Bac not

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

A Mean enrichment profiles performed with SeqMiner presenting the genomic distribution of H2Bac and H3K27ac reads obtained in ChIP-seq experiments in the different experimental groups, established along putative neuronal CBP enhancers (± 4Kb) (Kim et al., 2010). In the dorsal hippocampus, H2Bac levels are enriched at CBP enhancers in WT WEH (blue), decreased in tauopathic mice (TAU VEH, pink) and induced by CSP-TTK21 treatment (TAU MOL, green). B Mean profiles were separated in two sets of loci, including 17817 peaks highly enriched in H2Bac and showing a differential regulation in the experimental groups and 23331 peaks that were poorly enriched in H2Bac and not regulated


Mean profiles established with SeqMiner for genes at TSS for H2Bac ( Left ) H3K27ac ( Right ) . H2Bac but not H3K24ac presents a differential regulation at

Mean profiles established with SeqMiner for all genes at TSS for H2Bac (Left) and H3K27ac (Right). H2Bac but not H3K24ac presents a differential regulation at TSS


Mean H2Bac profiles established with SeqMiner on differentially regulated genes obtained in transcriptomic experiments ( - seq ) , either decreased ( left ) or increased ( right ) . ' basal ' H2Bac TSS enrichment state of these genes is differentially regulated whether genes are decreased or increased in response to

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Mean H2Bac profiles established with SeqMiner on the differentially regulated genes obtained in transcriptomic experiments (RNA-seq), either decreased (left) or increased (right). The 'basal' H2Bac TSS enrichment state of these genes is differentially regulated whether the genes are decreased or increased in response to learning


UCSC genome browser view of c - fos genomic locus with H3K27ac ChIP - seq ( blue ) , H2Bac ChIP - seq ( green ) Input ( black ) signals . Blue shading indicates locations of c - fos enhancers ( Joo et al . , 2016 ) ; gray shading indicates location of promoter . Differential peaks were analyzed with SICER . This set of data shows that H2Bac were significantly decreased at e1 ( FDR = 1 . 95 . 10 - 08 ) , e2 ( FDR = 0 . 0005 ) e5 ( FDR = 2 . 44 . 10 - 05 ) enhancers in THY - Tau22 compared to WT mice , significantly enriched at e1 ( FDR = 4 . 01 . 10 - 08 ) regions encompassing e3 , e4 TSS ( FDR = 7 . 53 . 10 - 07 ) e5 ( FDR = 0 . 001 ) after CSP - TTK21 treatment . Expanded View Figure

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

UCSC genome browser view of the c-fos genomic locus with H3K27ac ChIP-seq (blue), H2Bac ChIP-seq (green) and Input (black) signals. Blue shading indicates locations of the c-fos enhancers (Joo et al., 2016); gray shading indicates location of the promoter. Differential peaks were analyzed with SICER. This set of data shows that H2Bac levels were significantly decreased at e1 (FDR=1.95.10-08), e2 (FDR=0.0005) and e5 (FDR= 2.44.10-05) enhancers in THY-Tau22 compared to WT mice, and significantly enriched at e1 (FDR= 4.01.10-08) the regions encompassing e3, e4 and TSS (FDR=7.53.10-07) and e5 (FDR= 0.001) after CSP-TTK21 treatment. Expanded View Figure Legend


Immunoblotting of SPATC1L in testicular ( TC ) , testicular sperm ( TS ) , epididymal sperm ( S ) . An anti - ADAM2 antibody was used as a control .

Immunoblotting of SPATC1L in testicular cells (TC), testicular sperm (TS), and epididymal sperm (S). An anti-ADAM2 antibody was used as a control.


Expression of SPATC1L in human testis lysates ( TL ) mature sperm ( S ) . An anti - α - tubulin antibody was used as a control .

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Expression of SPATC1L in human testis lysates (TL) and mature sperm (S). An anti-α-tubulin antibody was used as a control.


Developmental expression pattern of SPATC1L in testis , examined by immunoblotting using total lysates obtained from prepubertal adult mice ( days 7 , 14 - 21 , 28 , 35 , 42 , 49 , 56 ) . An anti - α - tubulin antibody was used as a control .

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Developmental expression pattern of SPATC1L in testis, examined by immunoblotting using total lysates obtained from prepubertal and adult male mice (days 7, 14-21, 28, 35, 42, 49, and 56). An anti-α-tubulin antibody was used as a control.


Localization of SPATC1L in germ during spermatogenesis . Isolated spermatogenic were immunostained for SPATC1L ( red ) γ - tubulin ( green ) . γ - Tubulin was used as a centrosome marker , were counterstained with dye Hoechst 33342 ( blue ) . of are shown at top merged are shown in middle . Insets are magnified iamges . Sc , spermatocyte ; RS , round spermatid ; ES , elongating spermatid ; TS , testicular sperm ; MS , epididymal mature sperm ; BF , overlay with bright field image .

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Localization of SPATC1L in germ cells during spermatogenesis. Isolated spermatogenic cells were immunostained for SPATC1L (red) and γ-tubulin (green). γ-Tubulin was used as a centrosome marker, and cells were counterstained with the nuclear dye Hoechst 33342 (blue). Fluorescence images of individual staining are shown at the top and merged images are shown in the middle. Insets are magnified iamges. Sc, spermatocyte; RS, round spermatid; ES, elongating spermatid; TS, testicular sperm; MS, epididymal mature sperm; BF, overlay with bright field image.


Immunoprecipitation of SPATC1L in testis using an anti - SPATC1L antibody . IgG was used as a control . In , input ; IgG , normal rabbit serum .

Immunoprecipitation of SPATC1L in the testis using an anti-SPATC1L antibody. IgG was used as a control. In, input; IgG, normal rabbit serum.


Interaction between SPATC1L PKA subunits . Samples precipitated with an anti - SPATC1L antibody were analyzed by immunoblotting with anti - RIα anti - Cα antibodies . Immunoprecipitation of SPATC1L was confirmed by immunoblotting with anti - SPATC1L antibody . IgG was used as a control . IP , immunoprecipitation ; In , input ; IgG , immunoglobulin G from normal rabbit serum .

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Interaction between SPATC1L and PKA subunits. Samples precipitated with an anti-SPATC1L antibody were analyzed by immunoblotting with anti-RIα and anti-Cα antibodies. Immunoprecipitation of SPATC1L was confirmed by immunoblotting with anti-SPATC1L antibody. IgG was used as a control. IP, immunoprecipitation; In, input; IgG, immunoglobulin G from normal rabbit serum.


Expression of PKA subunits during spermatogenesis , examined by immunoblotting for RIα Cα in testicular ( TC ) , testicular sperm ( TS ) , epididymal sperm ( S ) . Anti - ADAM2 anti - α - tubulin antibodies were used as controls .

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Expression of PKA subunits during spermatogenesis, examined by immunoblotting for RIα and Cα in testicular cells (TC), testicular sperm (TS), and epididymal sperm (S). Anti-ADAM2 and anti-α-tubulin antibodies were used as controls.


Localization of SPATC1L RIα during spermatogenesis . Isolated spermatogenic were immunostained for SPATC1L ( red ) RIα ( green ) , counterstained with dye Hoechst 33342 ( blue ) . Overlay with bright field image is shown at right in each type . TC , testicular ( spermatocyte ) ; RS , round spermatid ; ES , elongating spermatid ; TS , testicular sperm . Scale bar = 5 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

Localization of SPATC1L and RIα during spermatogenesis. Isolated spermatogenic cells were immunostained for SPATC1L (red) and RIα (green), and counterstained with the nuclear dye Hoechst 33342 (blue). Overlay with bright field image is shown at right in each cell type. TC, testicular cell (spermatocyte); RS, round spermatid; ES, elongating spermatid; TS, testicular sperm. Scale bar = 5 μm.


Mapping of SPATC1L binding site on RIα by in vitro binding using purified GST - SPATC1L His - RIα consisting of regions 1 - 6 . Purified GST only was used as a control . Binding was confirmed by immunoprecipitation using an anti - SPATC1L antibody followed by immunoblot analyses of RIα , detected using an anti - His antibody . In , input ; Sup , supernatant ; IP , immunoprecipitated .

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

Mapping of the SPATC1L binding site on RIα by in vitro binding assay using purified GST-SPATC1L and His-RIα proteins consisting of regions 1-6. Purified GST protein only was used as a control. Binding was confirmed by immunoprecipitation using an anti-SPATC1L antibody followed by immunoblot analyses of RIα, detected using an anti-His antibody. In, input; Sup, supernatant; IP, immunoprecipitated protein.


PKA activity after overexpression of SPATC1L or SPATC1LΔC . Graphs show average PKA activity of Spatc1l - transfected ( bar ) Spatc1lΔC - transfected ( black bar ) in presence or absence of cAMP , expressed relative to control ( mock ) as a . Representative signals observed in are shown above . overexpression of SPATC1L was confirmed in every experiment by immunoblotting ( data not shown ) .

O O O O O O O O O O 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

PKA activity after overexpression of SPATC1L or SPATC1LΔC. Graphs show the average PKA activity of Spatc1l-transfected cells (white bar) and Spatc1lΔC-transfected cells (black bar) in the presence or absence of cAMP, expressed relative to control cells (mock) as a percentage. Representative signals observed in the assay are shown above. The overexpression of SPATC1L was confirmed in every experiment by immunoblotting (data not shown).


amount of Cα associated with RIα in presence or absence of SPATC1LΔC . After IP of RIα in overexpressing SPATC1LΔC or control , immunoblot was performed using anti - Cα antibody . Graph shows densitometric of Cα in relation to immunoprecipitated RIα in immunoblot . In , input ; IgG , immunoglobulin G from normal rabbit serum .

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The amount of Cα associated with RIα in the presence or absence of SPATC1LΔC. After IP of RIα in cells overexpressing SPATC1LΔC or control, immunoblot analysis was performed using anti-Cα antibody. Graph shows densitometric analysis of Cα in relation to immunoprecipitated RIα in immunoblot analysis . In, input; IgG, immunoglobulin G from normal rabbit serum.


PCR genotyping of WT ( Spatc1l + / + ) , heterozygous ( Spatc1l + / - ) homozygous ( Spatc1l - / - ) mice .

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PCR genotyping analysis of WT (Spatc1l+/+), heterozygous (Spatc1l+/-) and homozygous (Spatc1l-/-) mice.


SPATC1L expression in testes obtained from WT , Spatc1l + / - , Spatc1l - / - mice . An anti - GAPDH antibody was used as a control . Graph shows densitometric of SPATC1L in relation to GAPDH in immunoblot .

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SPATC1L expression levels in testes obtained from WT, Spatc1l+/-, and Spatc1l-/- mice. An anti-GAPDH antibody was used as a control. Graph shows densitometric analysis of SPATC1L in relation to GAPDH in immunoblot analysis.


of that became pregnant after mating with WT ( bar ) , Spatc1l + / - ( gray bar ) , Spatc1l - / - ( black bar ) mice . mice used : WT , n = 5 ; Spatc1l + / - , n = 6 ; Spatc1l - / - , n = 7 . WT mice used for mating with indicated genotype : WT , n = 30 ; Spatc1l + / - , n = 36 ; Spatc1l - / - , n = 42 . Vaginal plugs were observed in mated .

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Percentage of females that became pregnant after mating with WT (white bar), Spatc1l+/- (gray bar), and Spatc1l-/- (black bar) male mice. Male mice used: WT, n = 5; Spatc1l+/-, n = 6; Spatc1l-/-, n = 7. WT female mice used for mating with the indicated genotype: WT, n = 30; Spatc1l+/-, n = 36; Spatc1l-/-, n = 42. Vaginal plugs were observed in all mated females.


Average litter of pups produced by mated with WT ( bar ) , Spatc1l + / - ( gray bar ) , Spatc1l - / - ( black bar ) mice .

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Average litter size of pups produced by females mated with WT (white bar), Spatc1l+/- (gray bar), and Spatc1l-/- (black bar) male mice.


Macroscopic appearance of adult testes from an 8 - week - old WT ( Spatc1l + / + ) , heterozygous ( Spatc1l + / - ) homozygous ( Spatc1l - / - ) mice . Scale bar = 2 mm .

Macroscopic appearance of adult testes from an 8-week-old WT (Spatc1l+/+), heterozygous (Spatc1l+/-) and homozygous (Spatc1l-/-) mice. Scale bar = 2 mm.


Comparison of testis in WT ( bar ) , Spatc1l + / - ( gray bar ) , Spatc1l - / - ( black bar ) mice . organs were trimmed of fat weighed . average testis / ratios for WT , Spatc1l + / - , Spatc1l - / - were 0 . 0034 ± 0 . 0001 , 0 . 0038 ± 0 . 0003 , 0 . 0031 ± 0 . 0002 , respectively . Data are presented as means ± s . d . ( n = 7 ) .

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Comparison of testis weight in WT (white bar), Spatc1l+/- (gray bar), and Spatc1l-/- (black bar) mice. The organs were trimmed of fat and weighed. The average testis weight/body weight ratios for WT, Spatc1l+/-, and Spatc1l-/- males were 0.0034±0.0001, 0.0038±0.0003, and 0.0031±0.0002, respectively. Data are presented as means ± s.d. (n = 7).


Macroscopic appearance of adult epididymis from WT , Spatc1l + / - , Spatc1l - / - mice . Scale bar = 2 mm .

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Macroscopic appearance of the adult epididymis from WT, Spatc1l+/-, and Spatc1l-/- mice. Scale bar = 2 mm.


Epididymis in WT ( bar ) , Spatc1l + / - ( gray bar ) , Spatc1l - / - ( black bar ) mice . average epididymis / ratios for WT , Spatc1l + / - , Spatc1l - / - were 0 . 001 ± 0 . 0001 , 0 . 001 ± 0 . 0001 , 0 . 001 ± 0 . 00008 , respectively . Data are presented as means ± s . d . ( n = 7 ) .

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Epididymis weight in WT (white bar), Spatc1l+/- (gray bar), and Spatc1l-/- (black bar) mice. The average epididymis weight/body weight ratios for WT, Spatc1l+/-, and Spatc1l-/- males were 0.001±0.0001, 0.001±0.0001, and 0.001±0.00008, respectively. Data are presented as means ± s.d. (n = 7).


Histological of hematoxylin eosin - stained sections of seminiferous tubules prepared from testes of WT KO mice . Scale bar = 100 μm ( upper ) 50 μm ( lower ) .

Histological analysis of hematoxylin and eosin-stained sections of seminiferous tubules prepared from testes of WT and KO mice. Scale bar = 100 μm (upper) and 50 μm (lower).


Number of mature epididymal sperm from WT ( bar ) , Spatc1l + / - ( gray bar ) , Spatc1l - / - ( black bar ) mice . Sperm were collected from vas deferens . average number of mature sperm in WT , Spatc1l + / - , Spatc1l - / - was ( 4 . 06 ± 0 . 40 ) × 106 , ( 3 . 7 ± 0 . 7 ) × 106 , ( 3 . 93 ± 0 . 90 ) × 106 , respectively . Data are presented as means ± s . d . ( n = 5 ) .

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Number of mature epididymal sperm from WT (white bar), Spatc1l+/- (gray bar), and Spatc1l-/- (black bar) mice. Sperm were collected from the vas deferens. The average number of mature sperm in WT, Spatc1l+/-, and Spatc1l-/- males was (4.06 ± 0.40) × 106, (3.7 ± 0.7) × 106, and (3.93 ± 0.90) × 106, respectively. Data are presented as means ± s.d. (n = 5).


Morphological analyses of epididymal sperm in WT , He KO mice . Epididymal sperm were observed under an inverted microscope . arrows indicate separated tails , black arrowheads indicate separated heads arrowheads indicate folded heads . Scale bar = 50 μm .

Morphological analyses of epididymal sperm in WT, He and KO mice. Epididymal sperm were observed under an inverted microscope. The arrows indicate separated tails, black arrowheads indicate separated heads and white arrowheads indicate folded heads. Scale bar = 50 μm.


of headless sperm from cauda epididymides of WT ( black ) , Spatc1l + / - ( gray ) , Spatc1l - / - ( ) mice . average of headless sperm in WT , Spatc1l + / - , Spatc1l - / - was 6 . 3 , 52 . 9 100 , respectively . Each dot represents of headless sperm in an mouse , medians are represented as horizontal lines . ( n = 13 , * P < 0 . 001 , Student ' s t - ) .

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Percentage of headless sperm from cauda epididymides of WT (black), Spatc1l+/- (gray), and Spatc1l-/- (white) mice. The average percentage of headless sperm in WT, Spatc1l+/-, and Spatc1l-/- males was 6.3, 52.9 and 100, respectively. Each dot represents percentage of headless sperm in an individual mouse, and medians are represented as horizontal lines. (n=13, *P < 0.001, Student's t-test).


Immunostaining of sperm heads tails in WT KO mice . Sperm tails were observed by immunostaining for α - tubulin antibody ( red ) , counterstaining with dye Hoechst 33342 ( blue ) . Sperm were also observed by differential interference contrast ( DIC ) microscopy . Scale bars = 20 μm ( upper ) 5 μm ( lower ) .

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Immunostaining analysis of sperm heads and tails in WT and KO mice. Sperm tails were observed by immunostaining for α-tubulin antibody (red), and counterstaining with the nuclear dye Hoechst 33342 (blue). Sperm were also observed by differential interference contrast (DIC) microscopy. Scale bars = 20 μm (upper) and 5 μm (lower).


Morphological analyses of Spatc1l - KO epididymal sperm by . Sperm were obtained from vas deferens of 8 - week - old WT ( Spatc1l + / + ) KO ( Spatc1l - / - ) mice . Sperm neck regions are magnified in lower panels . AC , acrosomal cap ; MP , mid piece ; BP , basal plates . Scale bar = 2 μm ( upper ) 0 . 5 μm ( lower ) .

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Morphological analyses of Spatc1l-KO epididymal sperm by SEM. Sperm were obtained from the vas deferens of 8-week-old WT (Spatc1l+/+) and KO (Spatc1l-/-) mice. Sperm neck regions are magnified in lower panels. AC, acrosomal cap; MP, mid piece; BP, basal plates. Scale bar = 2 μm (upper) and 0.5 μm (lower).


analyses of flagella in WT KO testicular sperm . represent ultrastructures of cross - sections of midpiece ( upper ) end piece ( lower ) of WT KO testicular sperm . Ax , axonemal microtubules ; Mt , mitochondrial sheath ; ODF , outer dense . Scale bars = 200 nm ( upper ) 100 nm ( lower ) .

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TEM analyses of flagella in WT and KO testicular sperm. TEM images represent ultrastructures of cross-sections of the midpiece (upper) and end piece (lower) of WT and KO testicular sperm. Ax, axonemal microtubules; Mt, mitochondrial sheath; ODF, outer dense fibers. Scale bars = 200 nm (upper) and 100 nm (lower).


analyses of development of connecting piece in WT ( left ) KO ( right ) sperm . represent ultrastructures of longitudinal sections of elongating spermatids ( ES ) testicular sperm ( TS ) in WT KO mice . An , annulus ; BP , basal plates ; Cp , capitulum ; Mc , manchette ; Mt , mitochondria ; Nu , nucleus ; PCV , proximal centriolar vault ; PM , plasma ; SC , segmented column . Scale bar = 500 nm .

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TEM analyses of the development of the connecting piece in WT (left) and KO (right) sperm. TEM images represent ultrastructures of longitudinal sections of elongating spermatids (ES) and testicular sperm (TS) in WT and KO mice. An, annulus; BP, basal plates; Cp, capitulum; Mc, manchette; Mt, mitochondria; Nu, nucleus; PCV, proximal centriolar vault; PM, plasma membrane; SC, segmented column. Scale bar = 500 nm.


Ultrastructures of epididymal sperm in KO mice . represent ultrastructures of separated sperm head ( upper ) tail ( lower ) in epididymis of KO mice . in boxes are enlarged at right . BP , basal plates ; Cp , capitulum ; SC , segmented column . Scale bar = 500 nm .

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Ultrastructures of epididymal sperm in KO mice. TEM images represent ultrastructures of separated sperm head (upper) and tail (lower) in the epididymis of KO mice. Images in the boxes are enlarged at right. BP, basal plates; Cp, capitulum; SC, segmented column. Scale bar = 500 nm.


of morphologically abnormal KO sperm . Detached heads abnormally located to midpiece of sperm flagella were observed by ( left ) confocal differential interference contrast ( DIC ) microscopy ( right ) . PM , plasma . Scale bars = 500 nm ( left ) 5 μm ( right ) .

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TEM analysis of morphologically abnormal KO sperm. Detached heads abnormally located to the midpiece of sperm flagella were observed by TEM (left) and confocal differential interference contrast (DIC) microscopy (right). PM, plasma membrane. Scale bars = 500 nm (left) and 5 μm (right).


PKA activities in testicular sperm from WT ( + / + ) KO ( - / - ) mice . Graph shows average PKA activity in testicular sperm from KO mice ( black bar ) relative to that from WT mice ( bar ) , expressed as a .

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PKA activities in testicular sperm from WT (+/+) and KO (-/-) mice. Graph shows average PKA activity in testicular sperm from KO mice (black bar) relative to that from WT mice (white bar), expressed as a percentage.


Representative ProQ Diamond - stained 2D gel of isolated from testes in WT ( left ) KO ( right ) mice . Testes lysates were separated by 2DE , phosphoproteins were stained with ProQ diamond . in boxes are enlarged in insets shown below . Circles with numbers indicate spots with significantly changed volume relative to those on WT gels .

Representative ProQ Diamond-stained 2D gel images of isolated proteins from testes in WT (left) and KO (right) mice. Testes lysates were separated by 2DE, and phosphoproteins were stained with ProQ diamond. Images in the boxes are enlarged in insets shown below. Circles with numbers indicate spots with significantly changed volume relative to those on WT gels.


Comparative phosphorylation intensities of selected spots in 2D gels . intensities of selected spots in KO gels were compared with intensities in WT gels . Average intensities of indicated spot numbers ( # ) in WT ( bar ) KO ( black bar ) : # 1205 , 1092 . 2 ( WT ) 244 . 98 ( KO ) ; # 1207 , 729 . 9 ( WT ) 140 . 43 ( KO ) ; # 3303 , 1165 . 5 ( WT ) 279 . 5 ( KO ) ; # 4302 , 2725 . 8 ( WT ) 1 ( KO ) ; # 4405 , 984 . 5 ( WT ) 1 ( KO ) . An value of 1 indicates no spot , but is provided for calculation of fold change .

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Comparative phosphorylation intensities of selected spots in 2D gels. The intensities of selected spots in KO gels were compared with intensities in WT gels. Average intensities of the indicated spot numbers (#) in WT (white bar) and KO (black bar): #1205, 1092.2 (WT) and 244.98 (KO); #1207, 729.9 (WT) and 140.43 (KO); #3303, 1165.5 (WT) and 279.5 (KO); #4302, 2725.8 (WT) and 1 (KO); #4405, 984.5 (WT) and 1 (KO). An intensity value of 1 indicates no spot, but is provided for calculation of fold change.


Immunoblot analyses of five candidate in testis ( E ) sperm ( F ) of WT KO mice . Anti - GAPDH , anti - ADAM2 , anti - α - tubulin antibodies were used as controls . Loss of SPATC1L in KO testis was confirmed by immunoblotting with anti - SPATC1L antibody .

Immunoblot analyses of five candidate proteins in testis (E) and sperm (F) of WT and KO mice. Anti-GAPDH, anti-ADAM2, and anti-α-tubulin antibodies were used as controls. Loss of SPATC1L in KO testis was confirmed by immunoblotting with anti-SPATC1L antibody.


Localization of SPATC1L ( red ) CAPZB ( green ) in testicular sperm . Overlay with bright field image is shown at right . arrow indicates spatial overlap between SPATC1L CAPZB . Scale bar = 5 μm .

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Localization of SPATC1L (red) and CAPZB (green) in testicular sperm. Overlay with bright field image is shown at right. The white arrow indicates the spatial overlap between SPATC1L and CAPZB. Scale bar = 5 μm.


amount of CAPZB in mature sperm from WT ( + / + ) KO ( - / - ) mice . Graph shows densitometric of CAPZB shown in immunoblot analyses ( Figure 7F ) , expressed relative to WT as a . density of CAPZB in immunoblot was calculated in relation to density of α - Tubulin .

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The amount of CAPZB in mature sperm from WT (+/+) and KO (-/-) mice. Graph shows densitometric analysis of CAPZB shown in immunoblot analyses (Figure 7F), expressed relative to WT as a percentage. The density of CAPZB in immunoblot analysis was calculated in relation to density of α-Tubulin.


amount of CAPZB in mock Spatc1lΔC - transfected . Anti - GAPDH antibody was used as control . Overexpression of SPATC1L in TM4 was confirmed by immunoblotting with anti - SPATC1L antibody . Graph shows densitometric of CAPZB in pcDNA3 . 1 vector - transfected control ( mock ; bar ) SPATC1LΔC - transfected ( black bar ) , expressed relative to control ( mock ) as a . density of CAPZB in immunoblot was calculated in relation to density of GAPDH .

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The amount of CAPZB in mock and Spatc1lΔC-transfected cells. Anti-GAPDH antibody was used as control. Overexpression of SPATC1L in TM4 cells was confirmed by immunoblotting with anti-SPATC1L antibody. Graph shows densitometric analysis of CAPZB in pcDNA3.1 vector-transfected control cells (mock; white bar) and SPATC1LΔC-transfected cells (black bar), expressed relative to control cells (mock) as a percentage. The density of CAPZB in immunoblot analysis was calculated in relation to density of GAPDH.


Phosphorylation of CPAZB3 by cAMP - dependent kinase ( PKA ) . CAPZB3 was incubated with PKA 32P , estimated by autoradiography . amount of CAPZB3 was quantified by Coomassie Brilliant Blue ( CBB ) ( lower ) .

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Phosphorylation of CPAZB3 by cAMP-dependent protein kinase (PKA). CAPZB3 was incubated with PKA and 32P, and estimated by autoradiography. The amount of CAPZB3 protein was quantified by Coomassie Brilliant Blue (CBB) staining (lower).


Phosphorylation of CPAZB3 in presence or absence of SPATC1L . Phosphorylation of purified CAPZB3 was labeled with 32P via PKA in presence or absence of SPATC1L , estimated by autoradiography . amount of CAPZB3 was quantified by Coomassie Brilliant Blue ( CBB ) ( lower ) . Graph shows densitometric of phopho - CAPZB3 ( p - CAPZB3 ) in reactions without ( control ; bar ) or with SPATC1L ( black bar ) , expressed relative to control as a .

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Phosphorylation of CPAZB3 in the presence or absence of SPATC1L. Phosphorylation of purified CAPZB3 was labeled with 32P via PKA in the presence or absence of SPATC1L, and estimated by autoradiography. The amount of CAPZB3 protein was quantified by Coomassie Brilliant Blue (CBB) staining (lower). Graph shows densitometric analysis of phopho-CAPZB3 (p-CAPZB3) in reactions without (control; white bar) or with SPATC1L (black bar), expressed relative to control as a percentage.


Observation of filamentous actin ( F - actin ) in presence ( lower ) or absence ( upper ) of SPATC1L . F - actin in pEGFP vector - transfected control ( mock ; upper , green ) overexpressing SPATC1LΔC ( lower , green ) was visualized by using Phalloidin ( red ) . bar shows of transfected . Scale bar = 50 μm .

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Observation of filamentous actin (F-actin) in the presence (lower) or absence (upper) of SPATC1L. F-actin in pEGFP vector-transfected control cells (mock; upper, green) and cells overexpressing SPATC1LΔC (lower, green) was visualized by staining using Phalloidin (red). White bar shows the length of transfected cell. Scale bar = 50 μm.


of overexpressing SPATC1LΔC compared with control . Graph shows average of measured in pEGFP vector - transfected control ( mock ; bar ) Spatc1lΔC - transfected ( black bar ) , expressed relative to control ( mock ) as a .

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The length of cell overexpressing SPATC1LΔC compared with control. Graph shows the average of measured cell length in pEGFP vector-transfected control cells (mock; white bar) and Spatc1lΔC-transfected cells (black bar), expressed relative to control cells (mock) as a percentage.


2 - NBD - glucose uptake in murine pancreatic endothelial ( MS1 ) human umbilical vein endothelial ( HUVEC ) treated for 2 h with VEGF - B167 ( B167 ) or VEGF - B186 ( B186 ) compared to control treated . Data presented as mean ± from representative experiments performed in triplicates . Statistical evaluation using one - way ANOVA Dunnett ´ s multiple comparisons , p - value : * < 0 . 05 , * * < 0 . 01 , * * * < 0 . 001 ( compared to untreated control ) . Scale bar , 20 μm .

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2-NBD-glucose uptake in murine pancreatic endothelial cells (MS1) and primary human umbilical vein endothelial cells (HUVEC) treated for 2 h with VEGF-B167 (B167) or VEGF-B186 (B186) compared to control treated cells. Data presented as mean ± SEM from representative experiments performed in triplicates. Statistical evaluation using one-way ANOVA and Dunnett´s multiple comparisons test, p-value: * <0.05, ** <0.01, *** <0.001 (compared to untreated control). Scale bar, 20 μm.


Trans - 2 - NBD - glucose transport kinetics in MS1 treated for 2 h with VEGF - B186 . Data presented as mean ± from three experiments . Statistical evaluation using t - , p - value : * * < 0 . 01 ( compared to untreated control ) .

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Trans-cellular 2-NBD-glucose transport kinetics in MS1 cells treated for 2 h with VEGF-B186. Data presented as mean ± SEM from three individual experiments. Statistical evaluation using t-test, p-value: ** <0.01 (compared to untreated control).


Cardiac glycogen content in 17 old Vegfb + / + ( n = 6 ) , Vegfb + / - ( n = 6 ) Vegfb - / - mice ( n = 5 ) presented as mean ± . Statistical evaluation using one - way ANOVA Dunnett ´ s multiple comparisons , p - value : * < 0 . 05 ( compared to Vegfb + / + ) .

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Cardiac glycogen content in 17 weeks old male Vegfb+/+ (n=6), Vegfb+/- (n=6) and Vegfb-/- mice (n=5) presented as mean ± SEM. Statistical evaluation using one-way ANOVA and Dunnett´s multiple comparisons test, p-value: * <0.05 (compared to Vegfb+/+).


Glucose uptake accumulation ( 18F - DG PET ) in hearts of HFD fed wild - type ( WT ) mice pre - treated for 1 week with 2H10 antibody ( n = 4 ) compared to control antibody treated mice ( n = 4 ) . Data presented as mean ± . Statistical evaluation using two - way ANOVA , p - value : * * * * < 0 . 0001 .

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Glucose uptake and accumulation (18F-DG PET) in hearts of HFD fed wild-type (WT) mice pre-treated for 1 week with 2H10 antibody (n=4) compared to control antibody treated mice (n=4). Data presented as mean ± SEM. Statistical evaluation using two-way ANOVA, p-value: **** <0.0001.


Linear regression of 18F - DG PET data in D assessing differences in initial ( T = 0 . 9 - 6 . 5 min ) glucose uptake rate . Data presented as mean curve slope ( m ) ± . Statistical evaluation comparing slope differences using of covariance ( ANCOVA ) , p - value : * < 0 . 05 .

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Linear regression analysis of the 18F-DG PET data in D assessing differences in initial (T=0.9-6.5 min) glucose uptake rate. Data presented as mean curve slope (m) ± SEM. Statistical evaluation comparing slope differences using analysis of covariance (ANCOVA), p-value: * <0.05.


Relative mRNA expression of Slc2a glucose transporter ( GLUT ) family members ( 1 - 13 ) in cardiac ECs derived from Vegfb + / + ( n = 4 ) Vegfb - / - ( n = 4 ) mice . Data represent normalized PLIER values ( mean ± ) from microarray performed on n = 4 + 4 samples ( one sample / mouse ) .

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Relative mRNA expression of the Slc2a glucose transporter (GLUT) family members (1-13) in cardiac ECs derived from Vegfb+/+ (n=4) and Vegfb-/- (n=4) mice. Data represent normalized PLIER values (mean ± SEM) from microarray analysis performed on n=4+4 RNA samples (one RNA sample/mouse).


mRNA expression of SLC2A transporter ( GLUT ) family members ( 1 - 14 ) in HUVECs . Data presented as mean ± StDev relative to RPL19 expression from 3 independent experiments .

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mRNA expression levels of the SLC2A transporter (GLUT) family members (1-14) in HUVECs. Data presented as mean ± StDev relative to RPL19 expression from 3 independent experiments.


mRNA expression of VEGFB , SLC2A1 , NRP1 FLT1 ( VEGFR1 ) in HUVECs . Data presented as mean ± StDev relative to RPL19 expression from 3 independent experiments .

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mRNA expression levels of VEGFB, SLC2A1, NRP1 and FLT1 (VEGFR1) in HUVECs. Data presented as mean ± StDev relative to RPL19 expression from 3 independent experiments.


2 - NBD - glucose uptake in response to 2 h treatment with VEGF - B186 ( B186 ) in HUVECs exhibiting siRNA - mediated knock - down of FLT1 , NRP1 , SCL2A1 , KDR ( VEGFR2 ) or control siRNA . Data presented as mean ± from three experiments . Statistical evaluation using one - way ANOVA Sidak ´ s multiple comparisons , p - value : * < 0 . 05 ( comparisons made to respective untreated control ) .

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2-NBD-glucose uptake in response to 2 h treatment with VEGF-B186 (B186) in HUVECs exhibiting siRNA-mediated knock-down of FLT1, NRP1, SCL2A1, KDR (VEGFR2) or control siRNA. Data presented as mean ± SEM from three individual experiments. Statistical evaluation using one-way ANOVA and Sidak´s multiple comparisons test, p-value: * <0.05 (comparisons made to respective untreated control).


Representative of GLUT1 ( left panels ) CD31 ( right panels ) immunohistochemistry performed on consecutive sections from paraffin embedded mouse hearts . Scale bar , 100 μm .

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Representative images of GLUT1 (left panels) and CD31 (right panels) immunohistochemistry performed on consecutive sections from paraffin embedded mouse hearts. Scale bar, 100 μm.


mRNA expression from enriched cardiac endothelial ( open columns , n = 4 mice ) or whole heart ( closed columns , n = 3 mice ) . Data presented as mean ± StDev relative to Rpl19 expression . Enrichment factor is calculated as mean cardiac EC / mean whole heart presented in box .

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mRNA expression analysis from enriched cardiac endothelial cells (open columns, n=4 mice) or whole heart (closed columns, n= 3 mice). Data presented as mean ± StDev relative to Rpl19 expression. Enrichment factor is calculated as mean cardiac EC/mean whole heart and presented in the box.


Immunofluorescence labeling of GLUT1 ( ) , early endosomes ( EEA1 , green ) nuclei ( blue ) in HUVECs treated with VEGF - B186 for 10 min , 30 min or 2 h . Scale bar , 10 µm .

Immunofluorescence labeling of GLUT1 (white), early endosomes (EEA1, green) and nuclei (blue) in HUVECs treated with VEGF-B186 for 10 min, 30 min or 2 h. Scale bar, 10 µm.


GLUT1 expression in HUVECs treated with VEGF - B186 for 15 min or 2 h . Data presented as mean ± StDev of 3 - 4 independent experiments relative to β - Actin expression . Statistical evaluation using t - revealed no significant changes . Western blots shown in right panel .

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GLUT1 protein expression in HUVECs treated with VEGF-B186 for 15 min or 2 h. Data presented as mean ± StDev of 3-4 independent experiments relative to β-Actin expression. Statistical evaluation using t-test revealed no significant changes. Western blots shown in right panel.


Sucrose gradient ( 15 - 50 % sucrose ) fractions of HUVEC lysates after 2 h VEGF - B186 stimulation were analyzed with Western blots shown in Appendix Fig S2 . Different sub - compartments were identified by accumulation of specific markers : EEA1 for early endosomes , LIMPII for late endosomes / lysosomes , flotillin for plasma / lipid rafts calnexin for endoplasmic reticulum ( ER ) ( left panels ) . Amount sub - distribution of VEGFR1 , NRP1 GLUT1 in control or VEGF - B186 stimulated HUVECs shown in panels to right . Data represent % relative to total of interest measured from a representative experiment .

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Sucrose gradient (15-50% sucrose) fractions of HUVEC cellular lysates after 2 h VEGF-B186 stimulation were analyzed with Western blots shown in Appendix Fig S2. Different cellular sub-compartments were identified by accumulation of specific protein markers: EEA1 for early endosomes, LIMPII for late endosomes/lysosomes, flotillin for plasma membrane/lipid rafts and calnexin for endoplasmic reticulum (ER) (left panels). Amount and sub-cellular distribution of VEGFR1, NRP1 and GLUT1 protein in control or VEGF-B186 stimulated HUVECs shown in panels to the right. Data represent % protein relative to total protein of interest measured from a representative experiment.


of GLUT1 in % relative to total measured in HUVECs after 2 h VEGF - B186 stimulation in different sucrose fractions ( upper panel ) . Data from two independent experiments are shown . Green arrows mark fractions with increased GLUT1 content red arrows mark fractions with decreased GLUT1 content after VEGF - B186 stimulation . Changes in GLUT1 content in response to VEGF - B treatment are shown in table ( bottom panel ) .

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Quantification of GLUT1 protein in % relative to total measured protein in HUVECs after 2 h VEGF-B186 stimulation in different sucrose fractions (upper panel). Data from two independent experiments are shown. Green arrows mark fractions with increased GLUT1 content and red arrows mark fractions with decreased GLUT1 content after VEGF-B186 stimulation. Changes in GLUT1 content in response to VEGF-B treatment are shown in the table (bottom panel).


Representative confocal ( grey ) epifluorescence ( blue ) of filipin in HUVECs in response to 2 h VEGF - B186 stimulation ( left panels ) . cholesterol content measured by of filipin . Data presented as mean ± from a representative experiment performed in triplicates . Statistical evaluation using t - , p - value : * < 0 . 05 . Scale bars , 10 µm .

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Representative confocal (grey) and epifluorescence (blue) images of filipin staining in HUVECs in response to 2 h VEGF-B186 stimulation (left panels). Cellular cholesterol content measured by quantification of fluorescent filipin staining. Data presented as mean ± SEM from a representative experiment performed in triplicates. Statistical evaluation using t-test, p-value: * <0.05. Scale bars, 10 µm.


Measurement of cholesterol content ( B ) in HUVECs after VEGF - B186 stimulation for different time - points . Data presented as mean ± from representative experiments performed in triplicates . Statistical evaluation using t - , p - value : * < 0 . 05 , * * < 0 . 01 , * * * < 0 . 001 ( compared to respective control ) .

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Measurement of cholesterol content (B) in HUVECs after VEGF-B186 stimulation for different time-points. Data presented as mean ± SEM from representative experiments performed in triplicates. Statistical evaluation using t-test, p-value: * <0.05, ** <0.01, *** <0.001 (compared to respective control).


Measurement of glucose uptake ( C ) FA uptake ( D ) in HUVECs after VEGF - B186 stimulation for different time - points . Data presented as mean ± from representative experiments performed in triplicates . Statistical evaluation using t - , p - value : * < 0 . 05 , * * < 0 . 01 , * * * < 0 . 001 ( compared to respective control ) .

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Measurement of glucose uptake (C) and FA uptake (D) in HUVECs after VEGF-B186 stimulation for different time-points. Data presented as mean ± SEM from representative experiments performed in triplicates. Statistical evaluation using t-test, p-value: * <0.05, ** <0.01, *** <0.001 (compared to respective control).


Colorimetric of non - esterified cholesterol extracted from HUVECs stimulated with VEGF - B186 for 20 min . Data are presented as mean ± of three experiments . Statistical evaluation using t - , p - value : * < 0 . 05 .

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Colorimetric quantification of non-esterified cholesterol extracted from HUVECs stimulated with VEGF-B186 for 20 min. Data are presented as mean ± SEM of three individual experiments. Statistical evaluation using t-test, p-value: * <0.05.


Uptake of a cholesterol tracer ( TopFluor ) in HUVECs after VEGF - B186 stimulation for 20 min or 2 h . Data presented as mean ± from a representative experiment performed in triplicates . Statistical evaluation using one - way ANOVA Dunnett ´ s multiple comparisons , p - value : * * < 0 . 01 , * * * < 0 . 001 ( compared to respective untreated control ) .

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Uptake of a fluorescent cholesterol tracer (TopFluor) in HUVECs after VEGF-B186 stimulation for 20 min or 2 h. Data presented as mean ± SEM from a representative experiment performed in triplicates. Statistical evaluation using one-way ANOVA and Dunnett´s multiple comparisons test, p-value: ** <0.01, *** <0.001 (compared to respective untreated control).


of major cholesteryl ester ( CE ) species in HUVECs after VEGF - B186 stimulation for 20 min , analyzed by lipid mass spectrometry . Data presented as mean ± StDev of an experiment performed in triplicates .

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Quantification of the major cholesteryl ester (CE) species in HUVECs after VEGF-B186 stimulation for 20 min, analyzed by lipid mass spectrometry. Data presented as mean ± StDev of an experiment performed in triplicates.


Measurement of cholesterol content ( upper panel ) glucose uptake ( lower panel ) in HUVECs treated with VEGF - B186 for 1 h or after 15 min treatment with cholesterol - extracting methyl - beta - cyclodextrin ( MbCD ) . Data presented as mean ± from a representative experiment performed in triplicates . Statistical evaluation using one - way ANOVA Dunnett ´ s multiple comparisons , p - value : * * * < 0 . 001 ( compared to untreated control ) .

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

Measurement of cellular cholesterol content (upper panel) and glucose uptake (lower panel) in HUVECs treated with VEGF-B186 for 1 h or after 15 min treatment with cholesterol-extracting methyl-beta-cyclodextrin (MbCD). Data presented as mean ± SEM from a representative experiment performed in triplicates. Statistical evaluation using one-way ANOVA and Dunnett´s multiple comparisons test, p-value: *** <0.001 (compared to untreated control).


Measurement of cholesterol content ( upper panel ) glucose uptake ( lower panel ) in HUVECs treated with VEGF - B186 for 15 min alone or supplemented with soluble methyl - beta - cyclodextrin complexed with cholesterol ( 5 , 10 or 20 μg / ml cholesterol ) . Data presented as mean ± from a representative experiment performed in triplicates . Statistical evaluation using one - way ANOVA Dunnett ´ s multiple comparisons , p - value : * < 0 . 05 , * * < 0 . 01 , * * * < 0 . 001 ( compared to control w / o cholesterol ) .

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

Measurement of cellular cholesterol content (upper panel) and glucose uptake (lower panel) in HUVECs treated with VEGF-B186 for 15 min alone or supplemented with soluble methyl-beta-cyclodextrin complexed with cholesterol (5, 10 or 20 μg/ml cholesterol). Data presented as mean ± SEM from a representative experiment performed in triplicates. Statistical evaluation using one-way ANOVA and Dunnett´s multiple comparisons test, p-value: * <0.05, ** <0.01, *** <0.001 (compared to control w/o cholesterol).


mRNA expression of lipoprotein receptors in HUVECs . Data presented as mean ± StDev relative to RPL19 expression from 3 independent experiments .

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mRNA expression analysis of lipoprotein receptors in HUVECs. Data presented as mean ± StDev relative to RPL19 expression from 3 independent experiments.


cholesterol content in HUVECs treated with control siRNA or siRNA targeting LDLR or Scavenger receptor B1 ( SCARB1 , SRBI ) followed by 15 min stimulation with VEGF - B186 . Data presented as mean ± from representative experiments performed in triplicates . Statistical evaluation using one - way ANOVA Dunnett ´ s multiple comparisons , p - value : * < 0 . 05 , * * < 0 . 01 , * * * < 0 . 001 ( compared to respective control ) .

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Cellular cholesterol content in HUVECs treated with control siRNA or siRNA targeting LDLR or Scavenger receptor B1 (SCARB1, SRBI) followed by 15 min stimulation with VEGF-B186. Data presented as mean ± SEM from representative experiments performed in triplicates. Statistical evaluation using one-way ANOVA and Dunnett´s multiple comparisons test, p-value: * <0.05, ** <0.01, *** <0.001 (compared to respective control).


glucose uptake ( C ) in HUVECs treated with control siRNA or siRNA targeting LDLR or Scavenger receptor B1 ( SCARB1 , SRBI ) followed by 15 min stimulation with VEGF - B186 . Data presented as mean ± from representative experiments performed in triplicates . Statistical evaluation using one - way ANOVA Dunnett ´ s multiple comparisons , p - value : * < 0 . 05 , * * < 0 . 01 , * * * < 0 . 001 ( compared to respective control ) .

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glucose uptake (C) in HUVECs treated with control siRNA or siRNA targeting LDLR or Scavenger receptor B1 (SCARB1, SRBI) followed by 15 min stimulation with VEGF-B186. Data presented as mean ± SEM from representative experiments performed in triplicates. Statistical evaluation using one-way ANOVA and Dunnett´s multiple comparisons test, p-value: * <0.05, ** <0.01, *** <0.001 (compared to respective control).


cholesterol content of ECs freshly isolated from Ldlr + / + Ldlr - / - mice stimulated for 15 min with VEGF - B186 . Data presented as mean ± from a representative experiment performed in triplicates . Statistical evaluation using one - way ANOVA Tukey ´ s multiple comparisons , p - value : * * * < 0 . 001 ( compared to Ldlr + / + control ) .

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Cellular cholesterol content of ECs freshly isolated from Ldlr+/+ and Ldlr-/- mice stimulated for 15 min with VEGF-B186. Data presented as mean ± SEM from a representative experiment performed in triplicates. Statistical evaluation using one-way ANOVA and Tukey´s multiple comparisons test, p-value: *** <0.001 (compared to Ldlr+/+ control).


cholesterol content ( left panel ) glucose uptake ( right panel ) of HUVECs treated with de novo cholesterol synthesis inhibitor simvastatin . Data presented as mean ± from a representative experiment performed in triplicates . Statistical evaluation using one - way ANOVA Tukey ´ s multiple comparisons , p - value : * < 0 . 05 , * * < 0 . 01 , * * * < 0 . 001 ( compared to untreated control ) . p - value : # < 0 . 05 , # # # < 0 . 001 ( compared to simvastatin control ) .

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Cellular cholesterol content (left panel) and glucose uptake (right panel) of HUVECs treated with de novo cholesterol synthesis inhibitor simvastatin. Data presented as mean ± SEM from a representative experiment performed in triplicates. Statistical evaluation using one-way ANOVA and Tukey´s multiple comparisons test, p-value: * <0.05, ** <0.01, *** <0.001 (compared to untreated control). p-value: # <0.05, ### <0.001 (compared to simvastatin control).


surface biotinylated LDLR in HUVECs after 20 min of VEGF - B167 or VEGF - B186 stimulation . Data presented as mean ± from three independent experiments . Western blot shown below . Statistical evaluation using t - , p - value : * < 0 . 05 ( compared to control ) .

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Cell surface biotinylated LDLR protein levels in HUVECs after 20 min of VEGF-B167 or VEGF-B186 stimulation. Data presented as mean ± SEM from three independent experiments. Western blot shown below. Statistical evaluation using t-test, p-value: * <0.05 (compared to control).


Representative of surface distribution of LDLR by immunofluorescence of non - permeabilized ( , left panels ) , Dil - LDL surface binding ( evaluated at 4 ° C , middle panels ) Dil - LDL uptake ( evaluated at 37 ° C , right panels ) in HUVECs in response to VEGF - B186 stimulation for 10 min . Scale bar , 10 µm .

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Representative images of cell surface distribution of the LDLR by immunofluorescence staining of non-permeabilized cells (white, left panels), Dil-LDL cell surface binding (evaluated at 4 °C, middle panels) and Dil-LDL uptake (evaluated at 37 °C, right panels) in HUVECs in response to VEGF-B186 stimulation for 10 min. Scale bar, 10 µm.


of non - esterified cholesterol content by means of filipin in blood vessel compartment ( podocalyxin + ) in hearts of 15 - week old C57BL / 6 Vegfb + / - ( n = 5 ) Vegfb - / - mice ( n = 7 ) , normalized to wildtype ( Vegfb + / + ) mice ( n = 8 ) . Data presented as box - - whisker plots . Boxes represents lower / upper quartiles with median values indicated with a horizontal . Whiskers represent min - max values . Statistical evaluation using one - way ANOVA Dunnett ´ s multiple comparisons , p - value : * < 0 . 05 , * * < 0 . 01 ( compared to Vegfb + / + mice ) .

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Quantification of non-esterified cholesterol content by means of filipin staining in the blood vessel compartment (podocalyxin+) in hearts of 15-week old male C57BL/6 Vegfb+/- (n=5) and Vegfb-/- mice (n=7), normalized to wildtype (Vegfb+/+) mice (n=8). Data presented as box-and-whisker plots. Boxes represents lower/upper quartiles with the median values indicated with a horizontal line. Whiskers represent min-max values. Statistical evaluation using one-way ANOVA and Dunnett´s multiple comparisons test, p-value: * <0.05, ** <0.01 (compared to Vegfb+/+ mice).


of non - esterified cholesterol content by means of filipin in hearts of 15 - week old C57BL / 6 Vegfb + / - ( n = 5 ) Vegfb - / - ( n = 7 ) mice , normalized to wildtype ( Vegfb + / + ) mice ( n = 8 ) . Data presented as box - - whisker plots . Boxes represents lower / upper quartiles with median values indicated with a horizontal . Whiskers represent min - max values . Statistical evaluation using one - way ANOVA Dunnett ´ s multiple comparisons , p - value : * * < 0 . 01 ( compared to Vegfb + / + mice ) .

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Quantification of non-esterified cholesterol content by means of filipin staining in hearts of 15-week old male C57BL/6 Vegfb+/- (n=5) and Vegfb-/- (n=7) mice, normalized to wildtype (Vegfb+/+) mice (n=8). Data presented as box-and-whisker plots. Boxes represents lower/upper quartiles with the median values indicated with a horizontal line. Whiskers represent min-max values. Statistical evaluation using one-way ANOVA and Dunnett´s multiple comparisons test, p-value: ** <0.01 (compared to Vegfb+/+ mice).


of non - esterified cholesterol content by means of filipin in hearts of 25 - week old CD1 Flt1 + / lacz ( n = 20 ) mice , normalized to wildtype CD1 ( Flt1 + / + ) mice ( n = 16 ) . Data presented as box - - whisker plots . Boxes represents lower / upper quartiles with median values indicated with a horizontal . Whiskers represent min - max values . Statistical evaluation using t - , p - value : * * < 0 . 01 .

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Quantification of non-esterified cholesterol content by means of filipin staining in hearts of 25-week old male CD1 Flt1+/lacz (n=20) mice, normalized to wildtype CD1 (Flt1+/+) mice (n=16). Data presented as box-and-whisker plots. Boxes represents lower/upper quartiles with the median values indicated with a horizontal line. Whiskers represent min-max values. Statistical evaluation using t-test, p-value: ** <0.01.