Densities ( cells / mm2 ) of BrdU + cells in the OB granule cell layer ( GCL ) 2 days after TAM treatment . n = 4 ( Ctrl ) and 3 ( Ccn1cKO ) , * * p = 0 . 0086 . OB sections stained for BrdU ( yellow ) and nuclei ( blue ) . Scale bar : 50 μm .

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Densities (cells/mm2) of BrdU+ cells in the OB granule cell layer (GCL) 2 days after TAM treatment. n=4 (Ctrl) and 3 (Ccn1cKO), **p=0.0086. OB sections stained for BrdU (yellow) and nuclei (blue). Scale bar: 50 μm.


Densities ( cells / mm2 ) of GFAP + Ki67 + cells in V - SVZ whole - mounts 4 weeks after TAM treatment . Arrowheads point to dividing GFAP + cells . n = 4 , n . s . not significant . Scale bar : 10 μm .

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Densities (cells/mm2) of GFAP+Ki67+ cells in V-SVZ whole-mounts 4 weeks after TAM treatment. Arrowheads point to dividing GFAP+ cells. n=4, n.s. not significant. Scale bar: 10 μm.


Numbers of ASCL1 + cells in V - SVZ coronal sections 4 weeks after TAM treatment . n = 3 , n . s . not significant . Scale bar : 20 μm .

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Numbers of ASCL1+ cells in V-SVZ coronal sections 4 weeks after TAM treatment. n=3, n.s. not significant. Scale bar: 20 μm.


Densities ( cells / mm2 ) of BrdU + cells in the OB GCL 4 weeks after TAM treatment . n = 4 , n . s . not significant . Scale bar : 50 μm .

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Densities (cells/mm2) of BrdU+ cells in the OB GCL 4 weeks after TAM treatment. n=4, n.s. not significant. Scale bar: 50 μm.


Densities ( cells / mm2 ) of GFAP + Ki67 + cells in V - SVZ whole - mounts 16 months after TAM treatment . Arrowheads point to dividing GFAP + cells . n = 4 , n . s . not significant . Scale bar : 10 μm .

Densities (cells/mm2) of GFAP+Ki67+ cells in V-SVZ whole-mounts 16 months after TAM treatment. Arrowheads point to dividing GFAP+ cells. n=4, n.s. not significant. Scale bar: 10 μm.


Densities ( cells / mm2 ) of BrdU + cells in the OB GCL 16 months after TAM treatment . n = 4 , n . s . not significant . Scale bar : 50 μm .

Densities (cells/mm2) of BrdU+ cells in the OB GCL 16 months after TAM treatment. n=4, n.s. not significant. Scale bar: 50 μm.


Numbers of GFAP + BrdU + label - retaining NSCs in the V - SVZ . Arrowheads point to label - retaining NSCs . n = 4 , * p = 0 . 039 . V - SVZ coronal sections stained for GFAP ( cyan ) and BrdU ( red ) . Scale bar : 10 μm .

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Numbers of GFAP+BrdU+ label-retaining NSCs in the V-SVZ. Arrowheads point to label-retaining NSCs. n=4, *p= 0.039. V-SVZ coronal sections stained for GFAP (cyan) and BrdU (red). Scale bar: 10 μm.


Densities ( cells / mm2 ) of GFAP + Ki67 + cells in V - SVZ whole - mounts 24h after TMZ treatment . Arrowheads point to dividing NSCs under regenerative condition . n = 6 , * p = 0 . 0173 . Scale bar : 10 μm .

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Densities (cells/mm2) of GFAP+Ki67+ cells in V-SVZ whole-mounts 24h after TMZ treatment. Arrowheads point to dividing NSCs under regenerative condition. n=6, *p= 0.0173. Scale bar: 10 μm.


Densities ( cells / mm2 ) of DCX + neuroblasts in V - SVZ whole - mounts 3 days after TMZ treatment . n = 3 , * p = 0 . 0179 . V - SVZ whole - mounts stained for DCX . Scale bar : 500 μm .

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Densities (cells/mm2) of DCX+ neuroblasts in V-SVZ whole-mounts 3 days after TMZ treatment. n=3, *p= 0.0179. V-SVZ whole-mounts stained for DCX. Scale bar: 500 μm.


Densities ( cells / mm2 ) of BrdU + cells in the OB GCL labeled 24h after TMZ treatment . n = 5 ( Ctrl ) and 6 ( Ccn1cKO ) , * p = 0 . 0484 . OB sections stained for BrdU ( yellow ) and nuclei ( blue ) . Scale bar : 50 μm .

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Densities (cells/mm2) of BrdU+ cells in the OB GCL labeled 24h after TMZ treatment. n=5 (Ctrl) and 6 (Ccn1cKO), *p= 0.0484. OB sections stained for BrdU (yellow) and nuclei (blue). Scale bar: 50 μm.


Densities ( cells / mm2 ) of GFAP + Ki67 + cells in V - SVZ whole - mounts of aged mice 24h after TMZ treatment . Arrowheads point to dividing NSCs under regenerative condition . n = 3 , * p = 0 . 0321 . Scale bar : 10 μm .

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Densities (cells/mm2) of GFAP+Ki67+ cells in V-SVZ whole-mounts of aged mice 24h after TMZ treatment. Arrowheads point to dividing NSCs under regenerative condition. n=3, *p= 0.0321. Scale bar: 10 μm.


Densities ( cells / mm2 ) of DCX + neuroblasts in V - SVZ whole - mounts of aged mice 3 days after TMZ treatment . n = 3 , n . s . not significant . Scale bar : 500 μm .

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Densities (cells/mm2) of DCX+ neuroblasts in V-SVZ whole-mounts of aged mice 3 days after TMZ treatment. n=3, n.s. not significant. Scale bar: 500 μm.


Densities ( cells / mm2 ) of BrdU + cells in the OB GCL of aged mice labeled 3 days after TMZ treatment . n = 4 ( Ctrl ) and 3 ( Ccn1cKO ) , n . s . not significant . Scale bar : 50 μm .

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Densities (cells/mm2) of BrdU+ cells in the OB GCL of aged mice labeled 3 days after TMZ treatment. n=4 (Ctrl) and 3 (Ccn1cKO), n.s. not significant. Scale bar: 50 μm.


Representative FACS plots showing gating strategy for the analysis of cell types in the adult V - SVZ .

Representative FACS plots showing gating strategy for the analysis of cell types in the adult V-SVZ.


Total number of V - SVZ cells isolated from one mouse . n = 3 , n . s . not significant .

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Total number of V-SVZ cells isolated from one mouse. n=3, n.s. not significant.


Representative FACS plots showing aNSCs ( GFP + EGFR + ) , qNSCs ( GFP + EGFR - ) and C cells ( GFP - EGFR + ) in the adult V - SVZ .

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Representative FACS plots showing aNSCs (GFP+EGFR+), qNSCs (GFP+EGFR-) and C cells (GFP-EGFR+) in the adult V-SVZ.


Percentage of each cell population in the adult V - SVZ . n = 3 , * * p = 0 . 0014 ( GFP + EGFR - ) , * * * p = 0 . 0002 ( GFP + EGFR + ) , * p = 0 . 0111 ( GFP - EGFR + ) , * * * p = 0 . 0006 ( GFP + ) , n . s . not significant .

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Percentage of each cell population in the adult V-SVZ. n=3, **p=0.0014 (GFP+EGFR-), ***p=0.0002 (GFP+EGFR+), *p=0.0111 (GFP-EGFR+), ***p=0.0006 (GFP+), n.s. not significant.


Densities ( cells / mm2 ) of VCAM1 + B1 cells in the adult V - SVZ whole - mounts . n = 4 ( Ctrl + vehicle and Ccn1cKO + vehicle ) and 3 ( Ccn1cKO + erlotinib ) , * * p = 0 . 0021 ( Ccn1cKO + vehicle vs Ccn1cKO + erlotinib ) , * * p = 0 . 0014 ( Ccn1cKO + erlotinib vs Ctrl + vehicle ) , * * * * p < 0 . 0001 .

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Densities (cells/mm2) of VCAM1+ B1 cells in the adult V-SVZ whole-mounts. n=4 (Ctrl + vehicle and Ccn1cKO + vehicle) and 3 (Ccn1cKO + erlotinib), **p=0.0021 (Ccn1cKO + vehicle vs Ccn1cKO + erlotinib), **p=0.0014 (Ccn1cKO + erlotinib vs Ctrl + vehicle), ****p<0.0001.


Densities ( units / mm2 ) of NSC units in the adult V - SVZ whole - mounts . n = 4 ( Ctrl + vehicle and Ccn1cKO + vehicle ) and 3 ( Ccn1cKO + erlotinib ) , * p = 0 . 017 , * * p = 0 . 0061 , * * * p = 0 . 0002 .

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Densities (units/mm2) of NSC units in the adult V-SVZ whole-mounts. n=4 (Ctrl + vehicle and Ccn1cKO + vehicle) and 3 (Ccn1cKO + erlotinib), *p=0.017, **p=0.0061, ***p=0.0002.


V - SVZ whole - mounts stained for γ - tubulin ( cyan ) , GFAP ( red ) and VCAM1 ( white ) . Scale bar : 50 μm .

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V-SVZ whole-mounts stained for γ-tubulin (cyan), GFAP (red) and VCAM1 (white). Scale bar: 50 μm.


A Kinetics of intracellular Ca2 + after EDN stimulation . UACC257 and A2058 ( WT and ENDRB - KO ) were seeded on 96 well plates , loaded with Fluo - 4 dye and stimulated with 100 nM EDN or PBS . Fluo - 4 fluorescence was monitored over ten minutes ( n = 6 ) . Representative example of three independent experiments .

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A Kinetics of intracellular Ca2+ after EDN stimulation. UACC257 and A2058 (WT and ENDRB-KO) were seeded on 96 well plates, loaded with Fluo-4 dye and stimulated with 100 nM EDN or PBS. Fluo-4 fluorescence was monitored over ten minutes (n = 6). Representative example of three independent experiments.


B Time course of AKT and CREB phosphorylation . Melanoma cell lines were stimulated with 100 nM EDN for 2 - 90 min . AKT and CREB phosphorylation and expression was evaluated by Western blot . Representative example of three independent experiments .

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B Time course of AKT and CREB phosphorylation. Melanoma cell lines were stimulated with 100 nM EDN for 2-90 min. AKT and CREB phosphorylation and expression was evaluated by Western blot. Representative example of three independent experiments.


D High reproducibility in the UACC257 and A2058 datasets . All CV values were grouped according to cell line to represent dataset specific CV distributions . Numbers and lines indicate the median .

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D High reproducibility in the UACC257 and A2058 datasets. All CV values were grouped according to cell line to represent dataset specific CV distributions. Numbers and lines indicate the median.


A Phosphopeptide MS1 intensities in UACC257 samples were analysed by PCA . Colours indicate experimental groups and shading is mapped to time .

A Phosphopeptide MS1 intensities in UACC257 samples were analysed by PCA. Colours indicate experimental groups and shading is mapped to time.


C Comparison of the kinetics of the 242 shared target phosphopeptides . Log2 FC of EDN / Ctr is plotted from top to bottom for each phosphopeptide for both cell lines with increases shown in green and decreases shown in red .

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C Comparison of the kinetics of the 242 shared target phosphopeptides. Log2 FC of EDN/Ctr is plotted from top to bottom for each phosphopeptide for both cell lines with increases shown in green and decreases shown in red.


A , B EDN target phosphopeptides for ( A ) UACC257 and ( B ) A2058 were segregated into six clusters each , using c - means clustering ( MFuzz ) . Each line represents one phosphopeptide and cluster membership is indicated by colour . Most likely kinases for each phosphosite were predicted using NetworKIN and enrichment of kinase substrates was calculated for each cluster . Enriched kinases are indicated above each cluster . Note that substrates of these kinases only account for a fraction of all phosphosites in a cluster .

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A,B EDN target phosphopeptides for (A) UACC257 and (B) A2058 were segregated into six clusters each, using c-means clustering (MFuzz). Each line represents one phosphopeptide and cluster membership is indicated by colour. Most likely kinases for each phosphosite were predicted using NetworKIN and enrichment of kinase substrates was calculated for each cluster. Enriched kinases are indicated above each cluster. Note that substrates of these kinases only account for a fraction of all phosphosites in a cluster.


C Average MS1 intensity z - score kinetics for 217 predicted CaMKII - α sites that are shared between both cell lines . For comparison , average ( n = 6 ) calcium abundance kinetics are shown .

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C Average MS1 intensity z-score kinetics for 217 predicted CaMKII-α sites that are shared between both cell lines. For comparison, average (n = 6) calcium abundance kinetics are shown.


Predicted kinases are indicated on top of each panel for which one of their marker substrate phosphorylations was assayed . UACC257 cells were serum starved and stimulated with PBS or EDN at 100 nM for 60 min ( 5 min for CaMKII ) . Ten μg protein from RIPA lysates were analysed by Western blot . Kinase marker substrate phosphorylations and corresponding loading controls are shown . For each kinase , two independent replicates were performed and are shown . Each gel also had control lanes with increasing concentration of kinase inhibitors to support phospho band

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Predicted kinases are indicated on top of each panel for which one of their marker substrate phosphorylations was assayed. UACC257 cells were serum starved and stimulated with PBS or EDN at 100 nM for 60 min (5 min for CaMKII). Ten μg protein from RIPA lysates were analysed by Western blot. Kinase marker substrate phosphorylations and corresponding loading controls are shown. For each kinase, two independent replicates were performed and are shown. Each gel also had control lanes with increasing concentration of kinase inhibitors to support phospho band identity


The effect of kinase inhibitors on EDN induced cell migration of UACC257 cells was analysed using a scratch assay . Scratches were made in UACC257 monolayers . Serum was removed and cells were treated with PBS or 100 nM EDN in combination with DMSO or kinase inhibitors at 0 . 1 μM or 1 μM in triplicates . A Example pictures showing the effect of EDN on migration . Cell free areas are delimited by blue lines . Migration was quantified as the change in cell free area between 0 h and 48 h . B The four inhibitors that were effective at 1 µM are shown in detail , i . e . every point corresponds to one well . C Abundance of E - Cadherin ( normalized to GAPDH and Actin ) was assessed by LC - SRM after 48 h treatment with EDN or kinase inhibitors at 1 µM . Only the single inhibitor which could reverse EDN induced E - Cadherin repression is shown . * p < 0 . 05 two - sided unpaired t - test .

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The effect of kinase inhibitors on EDN induced cell migration of UACC257 cells was analysed using a scratch assay. Scratches were made in UACC257 monolayers. Serum was removed and cells were treated with PBS or 100 nM EDN in combination with DMSO or kinase inhibitors at 0.1 μM or 1 μM in triplicates. A Example pictures showing the effect of EDN on migration. Cell free areas are delimited by blue lines. Migration was quantified as the change in cell free area between 0 h and 48 h. B The four inhibitors that were effective at 1 µM are shown in detail, i.e. every point corresponds to one well. C Abundance of E-Cadherin (normalized to GAPDH and Actin) was assessed by LC-SRM after 48 h treatment with EDN or kinase inhibitors at 1 µM. Only the single inhibitor which could reverse EDN induced E-Cadherin repression is shown. * p < 0.05 two-sided unpaired t-test.


( A - B ) Coronal brain section , bregma : - 2 . 88 from The Mouse Brain Atlas shows the site of injection in the CA1 cell body layer of the right hippocampus ( A ) . 8 h post - injection , majority of the injected Fib - Tau - 1N3R - ATTO550 ( red ) is taken up by the corpus callosum of the injected side ( B ) . Images are shown at low ( left , 10X ) and high ( right , 63X ) magnification . Data information : Scale bar , 100µm in the left panel in ( B ) , 10µm everywhere else .

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(A-B) Coronal brain section, bregma: - 2.88 from The Mouse Brain Atlas shows the site of injection in the CA1 cell body layer of the right hippocampus (A). 8 h post-injection, majority of the injected Fib-Tau-1N3R-ATTO550 (red) is taken up by the corpus callosum of the injected side (B). Images are shown at low (left, 10X) and high (right, 63X) magnification. Data information: Scale bar, 100µm in the left panel in (B), 10µm everywhere else.


Representative images of the distribution of Fib - Tau ( red ) 8 h and 24 h after injection . Dendrites are immunolabeled using MAP2 antibody ( green ) . 8 h after injection , both diffused and clustered distribution of exogenous Fib - Tau is observed in stratum oriens and CA1 cell body region No exogenous Fib - Tau is detected in the adjoining cortex , stratum radiatum and entrorhinal cortex 24 h after injection , clustered distribution is primarily restricted to the pyramidal ( CA ) cell body layer ( D ) . Uptake of Fib - Tau is seen in some cells in both stratum oriens and pyramidal cell body layer ( D ) . Paired - t - test performed between ' 8 / 24h - right ' and ' 8 / 24h - left ' ; Unpaired t - test performed between ' 8h - left ' and ' 24h - left ' columns ( n is number of animals : 6 for 8h and 5 for 24h ) ; * * * p < 0 . 001 , * * p < 0 . 01 , * p < 0 . 05 , ns = not significant . Note : The images presented in panels C and D were independently acquired images under non - identical exposure setting at low magnification for display purpose Data information : Scale bar ,

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Representative images of the distribution of Fib-Tau (red) 8 h and 24 h after injection. Dendrites are immunolabeled using MAP2 antibody (green). 8 h after injection, both diffused and clustered distribution of exogenous Fib-Tau is observed in stratum oriens and CA1 cell body region No exogenous Fib-Tau is detected in the adjoining cortex, stratum radiatum and entrorhinal cortex 24 h after injection, clustered distribution is primarily restricted to the pyramidal (CA) cell body layer (D). Uptake of Fib-Tau is seen in some cells in both stratum oriens and pyramidal cell body layer (D). Paired-t-test performed between '8/24h-right' and '8/24h-left'; Unpaired t-test performed between '8h-left' and '24h-left' columns (n is number of animals: 6 for 8h and 5 for 24h); ***p<0.001, **p<0.01, *p<0.05, ns= not significant. Note: The images presented in panels C and D were independently acquired images under non-identical exposure setting at low magnification for display purpose Data information: Scale bar, 10µm


Representative images of the distribution of Fib - Tau ( red ) 8 h and 24 h after injection . Dendrites are immunolabeled using MAP2 antibody ( green ) . 8 h after injection , both diffused and clustered distribution of exogenous Fib - Tau is observed in stratum oriens The number of Fib - Tau clusters per µm2 within stratum oriens decreases between 8 h and 24 h post - injection ( G ) . A similar decrease in the total fluorescence ( clusters + diffused ) is observed indicating the diffusion and / or clearance of exogenous Fib - Tau ( H ) . Paired - t - test performed between ' 8 / 24h - right ' and ' 8 / 24h - left ' ; Unpaired t - test performed between ' 8h - left ' and ' 24h - left ' columns ( n is number of animals : 6 for 8h and 5 for 24h ) ; * * * p < 0 . 001 , * * p < 0 . 01 , * p < 0 . 05 , ns = not significant . Panels ( E and F ) are shown for same exposure and are used for quantification . Data information : Scale bar

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Representative images of the distribution of Fib-Tau (red) 8 h and 24 h after injection. Dendrites are immunolabeled using MAP2 antibody (green). 8 h after injection, both diffused and clustered distribution of exogenous Fib-Tau is observed in stratum oriens The number of Fib-Tau clusters per µm2 within stratum oriens decreases between 8 h and 24 h post-injection (G). A similar decrease in the total fluorescence (clusters + diffused) is observed indicating the diffusion and/or clearance of exogenous Fib-Tau (H). Paired-t-test performed between '8/24h-right' and '8/24h-left'; Unpaired t-test performed between '8h-left' and '24h-left' columns (n is number of animals: 6 for 8h and 5 for 24h); ***p<0.001, **p<0.01, *p<0.05, ns= not significant. Panels (E and F) are shown for same exposure and are used for quantification. Data information: Scale bar 10µm


( A - C ) Time and concentration dependent clustering of Fib - Tau in primary neurons . Representative images are shown for certain conditions to illustrate Fib - Tau clustering time dependence ( A , top - row ) and concentration dependence ( A , bottom - row ) . Quantification of the number of Fib - Tau clusters per µm2 ( B ) or fluorescence intensity of clusters At low concentrations ( up to 0 . 72nM ) the density of clusters increased with time ( between 10 and 60 min ) but the increase in intensity was small . At high concentrations of Fib - Tau ( ≥ 1 . 8nM ) , both density and size increased with increasing time . Box - plot represents : median , interquartile range and 10 - 90 % distribution ; One - way ANOVA with Dunnett post - hoc test , number of images analysed from 3 cultures ( from left to right : 25 , 25 , 25 , 25 , 70 , 45 , 45 , 45 , 45 , 45 , 30 , 40 , 40 , 40 , 40 and 40 images ) . Data information : * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; ns = not significant . Scale bar

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(A-C) Time and concentration dependent clustering of Fib-Tau in primary neurons. Representative images are shown for certain conditions to illustrate Fib-Tau clustering time dependence (A, top-row) and concentration dependence (A, bottom-row). Quantification of the number of Fib-Tau clusters per µm2 (B) or fluorescence intensity of clusters At low concentrations (up to 0.72nM) the density of clusters increased with time (between 10 and 60 min) but the increase in intensity was small. At high concentrations of Fib-Tau (≥1.8nM), both density and size increased with increasing time. Box-plot represents: median, interquartile range and 10-90% distribution; One-way ANOVA with Dunnett post-hoc test, number of images analysed from 3 cultures (from left to right: 25, 25, 25, 25, 70, 45, 45, 45, 45, 45, 30, 40, 40, 40, 40 and 40 images). Data information: *p<0.05; **p<0.01; ***p<0.001; ns= not significant. Scale bar 2µm


( D - F ) Single Particle Tracking using Quantum Dots ( SPT - QD ) of biotin tagged Fib - Tau . Representative single molecule trajectories of Fib - Tau following 10 or 60 min exposure are shown ( D ) . Note after 60min exposure ( 0 . 36nM ) , single molecules are more confined suggesting they are trapped and clustered . Quantification of diffusion coefficient ( E ) and explored area ( F , extracted from mean squared displacement ( MSD ) , see methods ) shows that both these parameters decrease after 60min exposure to Fib - Tau . Unpaired t - test , n is averaged value per cells imaged in 3 experiments ( 10 min : 22 , 60 min : 23 ) . Data information : * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; ns = not significant . Scale bar ,

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(D-F) Single Particle Tracking using Quantum Dots (SPT-QD) of biotin tagged Fib-Tau. Representative single molecule trajectories of Fib-Tau following 10 or 60 min exposure are shown (D). Note after 60min exposure (0.36nM), single molecules are more confined suggesting they are trapped and clustered. Quantification of diffusion coefficient (E) and explored area (F, extracted from mean squared displacement (MSD), see methods) shows that both these parameters decrease after 60min exposure to Fib-Tau. Unpaired t-test, n is averaged value per cells imaged in 3 experiments (10 min: 22, 60 min: 23). Data information: *p<0.05; **p<0.01; ***p<0.001; ns= not significant. Scale bar, 2µm


( G ) Neurons were exposed for 60 min to Fib - Tau ( 0 . 36nM ) labeled both with biotin and ATTO488 ( red ) . Cell surface exposed biotin was labelled using streptavidin - 550 ( green ) followed by live imaging . Note that most of the clusters of ATTO488 ( red ) are co - labelled with Streptavidin 550 ( green ) indicating that the clusters are at the cell surface . Data information : * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; ns = not significant . Scale bar ,

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(G) Neurons were exposed for 60 min to Fib-Tau (0.36nM) labeled both with biotin and ATTO488 (red). Cell surface exposed biotin was labelled using streptavidin-550 (green) followed by live imaging. Note that most of the clusters of ATTO488 (red) are co-labelled with Streptavidin 550 (green) indicating that the clusters are at the cell surface. Data information: *p<0.05; **p<0.01; ***p<0.001; ns= not significant. Scale bar, 5µm


( H - J ) Clearance of Tau clusters from neurons . Neurons were exposed ( 0 . 36nM ) to ATTO 550 labeled Fib - Tau for 10 min and the unbound fibrils were washed . Cells were fixed immediately ( 10min ) or allowed to recover in culture medium for 60 min . Two representative images ( H ) and quantifications ( I , J ) show that following 60 min recovery most of the Tau clusters disappear / dissociate as indicated by a decrease in their density . Box - plot represents : median , interquartile range and 10 - 90 % distribution , Unpaired t - test , n is number of images analyzed from 3 cultures ( 49 images ) . Data information : * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; ns = not significant . Scale bar

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(H-J) Clearance of Tau clusters from neurons. Neurons were exposed (0.36nM) to ATTO 550 labeled Fib-Tau for 10 min and the unbound fibrils were washed. Cells were fixed immediately (10min) or allowed to recover in culture medium for 60 min. Two representative images (H) and quantifications (I, J) show that following 60 min recovery most of the Tau clusters disappear/dissociate as indicated by a decrease in their density. Box-plot represents: median, interquartile range and 10-90% distribution, Unpaired t-test, n is number of images analyzed from 3 cultures (49 images). Data information: *p<0.05; **p<0.01; ***p<0.001; ns= not significant. Scale bar 2µm


( A ) Super - resolution STORM images of neuronal membrane after exposure to ATTO647N labeled Fib - Tau ( 0 . 36nM ) . Primary neurons were fixed 10 , 60 , 120 and 240 min after exposure to ATTO647N labeled Fib - Tau . Images rendered with a pixel size of 5nm show both clustered and diffused ( non - clustered ) distribution of exogenous Fib - Tau molecules . Scale bar , 1µm . ( B - F ) Quantification of various parameters from the super - resolution images . Averaged values per image is plotted . Increased " total detections per µm2 " ( B ) and " cluster - density " ( C ) of Fib - Tau bound to neurons with increasing exposure time is observed . No change in the " size of cluster " ( D ) or " number of molecules within a cluster " ( E ) is seen with increased exposure time . The proportion of single molecule events detected within clusters remained nearly constant ( ~ 50 % ) in all conditions ( F ) indicating an equilibrium between clustered and non - clustered Fib - Tau single molecules . Unpaired t - test to compare difference with 10 min , n is number of images ( 8 images ) . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; ns = not significant .

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(A) Super-resolution STORM images of neuronal membrane after exposure to ATTO647N labeled Fib-Tau (0.36nM). Primary neurons were fixed 10, 60, 120 and 240 min after exposure to ATTO647N labeled Fib-Tau. Images rendered with a pixel size of 5nm show both clustered and diffused (non-clustered) distribution of exogenous Fib-Tau molecules. Scale bar, 1µm. (B-F) Quantification of various parameters from the super-resolution images. Averaged values per image is plotted. Increased "total detections per µm2" (B) and "cluster-density" (C) of Fib-Tau bound to neurons with increasing exposure time is observed. No change in the "size of cluster" (D) or "number of molecules within a cluster" (E) is seen with increased exposure time. The proportion of single molecule events detected within clusters remained nearly constant (~ 50%) in all conditions (F) indicating an equilibrium between clustered and non-clustered Fib-Tau single molecules. Unpaired t-test to compare difference with 10 min, n is number of images (8 images). *p<0.05; **p<0.01; ***p<0.001; ns= not significant.


( D ) List of synaptic and plasma membrane proteins with extracellular domains significantly enriched in pull - downs from neurons exposed to Fib - Tau . For each identified protein , the name of the protein , the gene name , the p - value ( t - test with Benjamini - Hochberg correction ) , and the fold change corresponding to the ratio of spectral counts between exposed neuron and control samples are given . In an independent analysis , after 10 min exposure of neurons to biotinylated Fib - Tau , a cross - linking step was performed during 20 min using 1mM of DTSSP added in the culture medium , in order to cross - link the protein complexes formed at the cell surface using a membrane impermeable cross - linker . After cross - linking , proteins were analyzed and identified exactly as non - cross - linked samples . Proteins identified with at least 2 peptides are labeled " + " , the other are labeled " - " .

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(D) List of synaptic and plasma membrane proteins with extracellular domains significantly enriched in pull-downs from neurons exposed to Fib-Tau. For each identified protein, the name of the protein, the gene name, the p-value (t-test with Benjamini-Hochberg correction), and the fold change corresponding to the ratio of spectral counts between exposed neuron and control samples are given. In an independent analysis, after 10 min exposure of neurons to biotinylated Fib-Tau, a cross-linking step was performed during 20 min using 1mM of DTSSP added in the culture medium, in order to cross-link the protein complexes formed at the cell surface using a membrane impermeable cross-linker. After cross-linking, proteins were analyzed and identified exactly as non-cross-linked samples. Proteins identified with at least 2 peptides are labeled "+", the other are labeled "-".


( E ) . Co - immunoprecipitation of exogenous biotin - labelled Fib - Tau with α3 - NKA , GluA2 and GluN1 . α3 - NKA , GluA2 and GluN1 were immunoprecipitated using specific antibodies as described in the methods section . The presence of Fib - Tau in the immunoprecipitate was assessed using a slot blot apparatus and nitrocellulose membranes probed with streptavidin - HRP . A 2 . 4 - , 2 . 3 - , and 1 . 8 - fold enrichment in Tau band - intensity is observed in α3 - NKA , GluA2 and GluN1 immunoprecipitates , respectively , compared to controls performed with pre - immune goat or rabbit IgGs . Co - immunoprecipitates of exogenous biotin - labelled Fib - Tau with anti α3 - NKA , GluA2 and GluN1 specific antibodies were also subjected to SDS - PAGE and Western blot analysis . The presence of Fib - Tau in the immunoprecipitates was assessed by probing the nitrocellulose membranes with streptavidin - HRP . Fib - Tau co - immunoprecipitates with α3 - NKA and GluA2 - AMPA receptor but not with GluN1 - NMDA receptor .

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(E). Co-immunoprecipitation of exogenous biotin-labelled Fib-Tau with α3-NKA, GluA2 and GluN1. α3-NKA, GluA2 and GluN1 were immunoprecipitated using specific antibodies as described in the methods section. The presence of Fib-Tau in the immunoprecipitate was assessed using a slot blot apparatus and nitrocellulose membranes probed with streptavidin-HRP. A 2.4-, 2.3-, and 1.8- fold enrichment in Tau band-intensity is observed in α3-NKA, GluA2 and GluN1 immunoprecipitates, respectively, compared to controls performed with pre-immune goat or rabbit IgGs. Co-immunoprecipitates of exogenous biotin-labelled Fib-Tau with anti α3-NKA, GluA2 and GluN1 specific antibodies were also subjected to SDS-PAGE and Western blot analysis. The presence of Fib-Tau in the immunoprecipitates was assessed by probing the nitrocellulose membranes with streptavidin-HRP. Fib-Tau co-immunoprecipitates with α3-NKA and GluA2-AMPA receptor but not with GluN1-NMDA receptor.


( A , B ) Primary neurons were exposed to Fib - Tau - ATTO550 ( 0 . 36nM , red ) for 10 ( top row ) or 60 min ( bottom row ) and excitatory ( Homer , green ) and inhibitory synapses ( Gephyrin , blue ) were immuno - labelled . Fib - Tau clusters co - localized and / or apposed to Homer ( arrowheads ) but not Gephyrin ( A ) . Quantitative analysis shows that 10 to 20 % of Fib - Tau clusters are localized at Homer containing synapses ( B ) . Box - plot represents : median , interquartile range and 10 - 90 % distribution , Unpaired t - test to compare difference between 10 and 60 min , n is number of images analyzed from 3 - experiments ( 10 min : 49 ; 60 min : 50 ) . Scale bar , 5µm .

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(A, B) Primary neurons were exposed to Fib-Tau-ATTO550 (0.36nM, red) for 10 (top row) or 60 min (bottom row) and excitatory (Homer, green) and inhibitory synapses (Gephyrin, blue) were immuno-labelled. Fib-Tau clusters co-localized and/or apposed to Homer (arrowheads) but not Gephyrin (A). Quantitative analysis shows that 10 to 20% of Fib-Tau clusters are localized at Homer containing synapses (B). Box-plot represents: median, interquartile range and 10-90% distribution, Unpaired t-test to compare difference between 10 and 60 min, n is number of images analyzed from 3-experiments (10 min: 49; 60 min: 50). Scale bar, 5µm.


Exposure of neurons to Fib - Tau - ATTO550 ( 0 . 36nM , 60 min , red ) and immuno - labelling of excitatory synapse ( Anti - Rabbit - Homer or Anti - Mouse - PSD , blue ) and α3 - NKA or GluA1 - AMPA or GluA2 - AMPA or GluN1 - NMDA or GluN2B - NMDA subunits ( green , post - permeabilization ) . Arrows indicate excitatory synapses where Fib - Tau and α3 - NKA / AMPA / NMDA are co - localized ( C ) . Mann - Whitney - test , n is number of images analyzed from 3 - 4 experiments ( α3 - NKA : 75 ; GluA1 : 95 ; GluA2 : 50 ; GluN1 / N2B : 45 ) .

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Exposure of neurons to Fib-Tau-ATTO550 (0.36nM, 60 min, red) and immuno-labelling of excitatory synapse (Anti-Rabbit-Homer or Anti-Mouse-PSD, blue) and α3-NKA or GluA1-AMPA or GluA2-AMPA or GluN1-NMDA or GluN2B-NMDA subunits (green, post-permeabilization). Arrows indicate excitatory synapses where Fib-Tau and α3-NKA/AMPA/NMDA are co-localized (C). Mann-Whitney-test, n is number of images analyzed from 3-4 experiments (α3-NKA: 75; GluA1: 95; GluA2: 50; GluN1/N2B: 45).


Exposure of neurons to Fib - Tau - ATTO550 ( 0 . 36nM , 60 min , red ) and immuno - labelling of excitatory synapse ( Anti - Rabbit - Homer or Anti - Mouse - PSD , blue ) and α3 - NKA or GluA1 - AMPA or GluA2 - AMPA or GluN1 - NMDA or GluN2B - NMDA subunits ( green , post - permeabilization ) . Quantification of the fluorescence intensity ( indicating size of clusters of synaptic α3 - NKA / AMPA / NMDA spots ( obtained after thresholding ) following exposure to Fib - Tau showed a reduction in the size of α3 - NKA and increase in the size of GluA2 - subunit containing AMPA receptors ( D ) . Box - plot represents : median , interquartile range and 10 - 90 % distribution , * * p < 0 . 01 ; ns = not significant . Scale bar , 5µm .

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Exposure of neurons to Fib-Tau-ATTO550 (0.36nM, 60 min, red) and immuno-labelling of excitatory synapse (Anti-Rabbit-Homer or Anti-Mouse-PSD, blue) and α3-NKA or GluA1-AMPA or GluA2-AMPA or GluN1-NMDA or GluN2B-NMDA subunits (green, post-permeabilization). Quantification of the fluorescence intensity (indicating size of clusters of synaptic α3-NKA/AMPA/NMDA spots (obtained after thresholding) following exposure to Fib-Tau showed a reduction in the size of α3-NKA and increase in the size of GluA2-subunit containing AMPA receptors (D). Box-plot represents: median, interquartile range and 10-90% distribution, **p<0.01; ns= not significant. Scale bar, 5µm.


( A - C ) SPT - QD of pHluorin tagged AMPA ( GluA1 and GluA2 ) and NMDA ( GluN1 and GluN2B ) receptor subunits using GFP - antibody pre - coupled to QD655 . Primary neurons were exposed or not to Fib - Tau ( 0 . 36nM , 60 min ) . Cumulative frequency of diffusion coefficient of all trajectories ( extracted from MSD plot , inset , A ) show a slow - down in diffusion of GluA2 - subunit but not GluA1 , GluN1 and GluN2B subunits ( A ) . To compute statistical differences , the diffusion coefficient of all trajectories per movie ( from 3 independent experiments ) were averaged ( B ) . To measure statistical differences in MSD , the explored area ( area under curve during 600 - 900ms of MSD plot ) per movie ( from 3 independent experiments ) were averaged ( C ) . Decreased diffusion coefficient and area explored for GluA2 - AMPA was observed indicating increased confinement concomitant with increased clustering ( Fig 5C ) following exposure to Fib - Tau . Mann - Whitney - test , n is number of movies ( left to right ) : 14 , 15 , 14 , 13 , 15 , 15 , 15 , and 15 . * * p < 0 . 01 ; ns = not significant .

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(A-C) SPT-QD of pHluorin tagged AMPA (GluA1 and GluA2) and NMDA (GluN1 and GluN2B) receptor subunits using GFP-antibody pre-coupled to QD655. Primary neurons were exposed or not to Fib-Tau (0.36nM, 60 min). Cumulative frequency of diffusion coefficient of all trajectories (extracted from MSD plot, inset, A) show a slow-down in diffusion of GluA2-subunit but not GluA1, GluN1 and GluN2B subunits (A). To compute statistical differences, the diffusion coefficient of all trajectories per movie (from 3 independent experiments) were averaged (B). To measure statistical differences in MSD, the explored area (area under curve during 600-900ms of MSD plot) per movie (from 3 independent experiments) were averaged (C). Decreased diffusion coefficient and area explored for GluA2-AMPA was observed indicating increased confinement concomitant with increased clustering (Fig 5C) following exposure to Fib-Tau. Mann-Whitney-test, n is number of movies (left to right): 14, 15, 14, 13, 15, 15, 15, and 15. **p<0.01; ns= not significant.


( A ) Single Particle Tracking using PALM ( SPT - PALM ) was performed on α3 - NKA - Dendra expressing primary neurons . A representative image shows an epifluorescence image and trajectories ( strands of different colors ) of minimum 5 points in length . Scale bars : 1 µm in panel ( A )

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(A) Single Particle Tracking using PALM (SPT-PALM) was performed on α3-NKA-Dendra expressing primary neurons. A representative image shows an epifluorescence image and trajectories (strands of different colors) of minimum 5 points in length. Scale bars: 1 µm in panel (A)


( B , C ) Extraction of diffusion coefficient using mean squared displacement ( MSD ) approach using minimum 5 - point trajectory length . Spine and shaft were visually identified . An upward shift of MSD curve , indicating reduced confinement was observed following exposure to Fib - Tau ( 0 . 36nM , 60 min ) ( B ) . As expected , an increased diffusion coefficient ( i . e . increased mobility ) ( C ) of α3 - NKA is observed in dendritic shaft following exposure to Fib - Tau . Mann - Whitney - test , n = 21 cells , obtained from 4 independent experiments ; Each dot represents average diffusion coefficient of all trajectories value per cell . Data information : * p < 0 . 05 ; ns = not significant .

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(B, C) Extraction of diffusion coefficient using mean squared displacement (MSD) approach using minimum 5-point trajectory length. Spine and shaft were visually identified. An upward shift of MSD curve, indicating reduced confinement was observed following exposure to Fib-Tau (0.36nM, 60 min) (B). As expected, an increased diffusion coefficient (i.e. increased mobility) (C) of α3-NKA is observed in dendritic shaft following exposure to Fib-Tau. Mann-Whitney-test, n=21 cells, obtained from 4 independent experiments; Each dot represents average diffusion coefficient of all trajectories value per cell. Data information: *p<0.05; ns= not significant.


Extraction of the number of diffusive states of α3 - NKA and the transition rates between these diffusive states using thousands of short ( minimum 2 - point ) trajectories using Hidden Markov Model The occupancy time of α3 - NKA in bound - state also decreased whereas that in free - state increased upon neuron exposure to Fib - Tau ( E ) . Box - plot represents : median , interquartile range and entire distribution , Mann - Whitney - test on averaged value per movie from 4 experiments ( n = 21 cells ) . Data information : * p < 0 . 05 ; ns = not significant .

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Extraction of the number of diffusive states of α3-NKA and the transition rates between these diffusive states using thousands of short (minimum 2-point) trajectories using Hidden Markov Model The occupancy time of α3-NKA in bound-state also decreased whereas that in free-state increased upon neuron exposure to Fib-Tau (E). Box-plot represents: median, interquartile range and entire distribution, Mann-Whitney-test on averaged value per movie from 4 experiments (n=21 cells). Data information: *p<0.05; ns= not significant.


Extraction of the number of diffusive states of α3 - NKA and the transition rates between these diffusive states using thousands of short ( minimum 2 - point ) trajectories using Hidden Markov Model Dwell - time of α3 - NKA is also reduced in bound state indicating destabilization of native α3 - NKA - complexes following interaction with Fib - Tau ( F ) . Box - plot represents : median , interquartile range and entire distribution , Mann - Whitney - test on averaged value per movie from 4 experiments ( n = 21 cells ) . Data information : * p < 0 . 05 ; ns = not significant .

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Extraction of the number of diffusive states of α3-NKA and the transition rates between these diffusive states using thousands of short (minimum 2-point) trajectories using Hidden Markov Model Dwell-time of α3-NKA is also reduced in bound state indicating destabilization of native α3-NKA-complexes following interaction with Fib-Tau (F). Box-plot represents: median, interquartile range and entire distribution, Mann-Whitney-test on averaged value per movie from 4 experiments (n=21 cells). Data information: *p<0.05; ns= not significant.


( G - H ) Cell - surface biotinylation on primary neurons exposed or not to Fib - Tau ( 1 . 8nM , 60 min ) . The amount of α - NKA at cell surface decreased upon exposure of neurons to Fib - Tau ( G ) . Quantification was performed by normalizing the western blot band intensity with input . Paired - t - test , 3 independent experiments . Data information : * p < 0 . 05 ; ns = not significant .

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(G-H) Cell-surface biotinylation on primary neurons exposed or not to Fib-Tau (1.8nM, 60 min). The amount of α-NKA at cell surface decreased upon exposure of neurons to Fib-Tau (G). Quantification was performed by normalizing the western blot band intensity with input. Paired-t-test, 3 independent experiments. Data information: *p<0.05; ns= not significant.


( I ) Paraformaldehyde fixation α3 - NKA - pHluorin ( green , top row ) reveals endocytic spots otherwise not visible in live neurons ( green , bottom row ) . Many Fib - Tau - ATTO647 ( red ) clusters overlapped ( arrow ) with α3 - NKA - pHluorin spots ( exposure , 0 . 36nM , 60 min ) . Scale bars

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(I) Paraformaldehyde fixation α3-NKA-pHluorin (green, top row) reveals endocytic spots otherwise not visible in live neurons (green, bottom row). Many Fib-Tau-ATTO647 (red) clusters overlapped (arrow) with α3-NKA-pHluorin spots (exposure, 0.36nM, 60 min). Scale bars 2µm


( J ) The majority of the overlapping Fib - Tau - ATTO647 clusters ( red ) and α3 - NKA - pHluorin spots ( green ) localized on top of Clathrin - mRFP ( blue ) puncta . Scale bars :

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(J) The majority of the overlapping Fib-Tau-ATTO647 clusters (red) and α3-NKA-pHluorin spots (green) localized on top of Clathrin-mRFP (blue) puncta. Scale bars: 2µm


DIV21 neurons were co - exposed to fixed concentration of Fib - Tau ( 0 . 36nM , 60min , ATTO550 labelled , red ) and increasing concentrations ( given in monomer - equivalent ) of α - Syn fibrils [ in total 5 - experiments were performed with either ATTO647 labeled α - Syn ( shown here in green ) or unlabeled α - Syn A concentration - dependent increase in the binding and clustering of α - Syn fibrils is seen . Notably , Fib - Tau clustering dramatically increased with increasing concentrations of α -

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DIV21 neurons were co-exposed to fixed concentration of Fib-Tau (0.36nM, 60min, ATTO550 labelled, red) and increasing concentrations (given in monomer-equivalent) of α-Syn fibrils [in total 5-experiments were performed with either ATTO647 labeled α-Syn (shown here in green) or unlabeled α-Syn A concentration-dependent increase in the binding and clustering of α-Syn fibrils is seen. Notably, Fib-Tau clustering dramatically increased with increasing concentrations of α-Syn


DIV21 neurons were co - exposed to fixed concentration of Fib - Tau ( 0 . 36nM , 60min , ATTO550 labelled , red ) and increasing Aβ ( given in monomer - equivalent ) of Aβ ( B ) fibrils [ in total 5 - experiments were performed with either ATTO647 labeled Aβ ( shown here in green ) or unlabeled Aβ fibrils ] . concentration - dependent increase in the binding and clustering of Aβ fibrils is seen . Notably , Fib - Tau clustering dramatically increased with increasing concentrations of α - Syn , but not Aβ

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DIV21 neurons were co-exposed to fixed concentration of Fib-Tau (0.36nM, 60min, ATTO550 labelled, red) and increasing Aβ (given in monomer-equivalent) of Aβ (B) fibrils [in total 5-experiments were performed with either ATTO647 labeled Aβ (shown here in green) or unlabeled Aβ fibrils]. concentration-dependent increase in the binding and clustering of Aβ fibrils is seen. Notably, Fib-Tau clustering dramatically increased with increasing concentrations of α-Syn, but not Aβ fibrils


DIV21 neurons were co - exposed to fixed concentration of Fib - Tau ( 0 . 36nM , 60min , ATTO550 labelled , red ) and increasing concentrations ( given in monomer - equivalent ) of α - Syn fibrils [ in total 5 - experiments were performed with either ATTO647 labeled α - Syn or unlabeled α - Syn A concentration - dependent increase in the binding and clustering of both α - Syn fibrils is seen . One - Way ANOVA with Dunnett ' s post - hoc test was performed to compare the different condition to the reference condition ( 100nM ) . The number of images analyzed is 27 , 25 , 25 , 26 and 28 in ( C ) , from left to right . The data originate from 3 to 4 independent experiments . Data information : Box - plot represents : median , interquartile range and 10 - 90 % distribution , Mann - Whitney - test is performed , n = 40 images from 4 - experiments , Scale bars , 5µm . * p < 0 . 05 , * * * p < 0 . 001 ; ns = not significant .

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DIV21 neurons were co-exposed to fixed concentration of Fib-Tau (0.36nM, 60min, ATTO550 labelled, red) and increasing concentrations (given in monomer-equivalent) of α-Syn fibrils [in total 5-experiments were performed with either ATTO647 labeled α-Syn or unlabeled α-Syn A concentration-dependent increase in the binding and clustering of both α-Syn fibrils is seen. One-Way ANOVA with Dunnett's post-hoc test was performed to compare the different condition to the reference condition (100nM).The number of images analyzed is 27, 25, 25, 26 and 28 in (C) , from left to right. The data originate from 3 to 4 independent experiments. Data information: Box-plot represents: median, interquartile range and 10-90% distribution, Mann-Whitney-test is performed, n=40 images from 4-experiments, Scale bars, 5µm. *p<0.05, ***p<0.001; ns= not significant.


DIV21 neurons were co - exposed to fixed concentration of Fib - Tau ( 0 . 36nM , 60min , ATTO550 labelled , red ) and increasing concentrations ( given in monomer - equivalent ) of Aβ fibrils [ in total 5 - experiments were performed with either ATTO647 labeled Aβ or unlabeled Aβ fibrils A concentration - dependent increase in the binding and clustering of Aβ fibrils is seen . One - Way ANOVA with Dunnett ' s post - hoc test was performed to compare the different condition to the reference condition ( 100nM ) . The number of images analyzed is 18 , 50 , 50 , 50 , 25 in ( D ) , from left to right . The data originate from 3 to 4 independent experiments . Data information : Box - plot represents : median , interquartile range and 10 - 90 % distribution , Mann - Whitney - test is performed , n = 40 images from 4 - experiments , Scale bars , 5µm . * p < 0 . 05 , * * * p < 0 . 001 ; ns = not significant .

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DIV21 neurons were co-exposed to fixed concentration of Fib-Tau (0.36nM, 60min, ATTO550 labelled, red) and increasing concentrations (given in monomer-equivalent) of Aβ fibrils [in total 5-experiments were performed with either ATTO647 labeled Aβ or unlabeled Aβ fibrils A concentration-dependent increase in the binding and clustering of Aβ fibrils is seen. One-Way ANOVA with Dunnett's post-hoc test was performed to compare the different condition to the reference condition (100nM).The number of images analyzed is 18, 50, 50, 50, 25 in (D), from left to right. The data originate from 3 to 4 independent experiments. Data information: Box-plot represents: median, interquartile range and 10-90% distribution, Mann-Whitney-test is performed, n=40 images from 4-experiments, Scale bars, 5µm. *p<0.05, ***p<0.001; ns= not significant.


Notably , Fib - Tau clustering dramatically increased with increasing concentrations of α - Syn , but not Aβ fibrils The quantification in ( E ) includes all 5 - experiments ( n = number of images analyzed , left to right : 58 , 54 , 50 , 62 , 63 , 65 , 43 , 35 , 60 , 60 , 60 , 35 ; One - Way ANOVA with Dunnette ' s post - hoc test was performed to compare the size of Fib - Tau clusters ) . Data information : Box - plot represents : median , interquartile range and 10 - 90 % distribution , Mann - Whitney - test is performed , n = 40 images from 4 - experiments , Scale bars , 5µm . * p < 0 . 05 , * * * p < 0 . 001 ; ns = not significant .

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Notably, Fib-Tau clustering dramatically increased with increasing concentrations of α-Syn, but not Aβ fibrils The quantification in (E) includes all 5-experiments (n = number of images analyzed, left to right: 58, 54, 50, 62, 63, 65, 43, 35, 60, 60, 60, 35; One-Way ANOVA with Dunnette's post-hoc test was performed to compare the size of Fib-Tau clusters). Data information: Box-plot represents: median, interquartile range and 10-90% distribution, Mann-Whitney-test is performed, n=40 images from 4-experiments, Scale bars, 5µm. *p<0.05, ***p<0.001; ns= not significant.


A perfect co - localization between Fib - Tau and α - Syn clusters is also observed . The proportion of Fib - Tau clusters co - localizing with α - Syn fibrils increased with an increasing concentration of α - Syn fibrils reaching 60 - 70 % overlap A weaker , concentration independent , overlap ( 10 - 20 % ) was observed with Aβ fibrils The quantification in ( F ) is derived from 3 to 4 independent experiments . The number of images analyzed is 27 , 25 , 25 , 26 , 28 for α - Syn and 18 , 50 , 50 , 50 , 25 for Aβ from left to right . One - Way ANOVA with Dunnett ' s post - hoc test was performed to compare the co - localization differences from 100nM concentration . Box - plot represents : median , interquartile range and 10 - 90 % distribution . Data information : Box - plot represents : median , interquartile range and 10 - 90 % distribution , Mann - Whitney - test is performed , n = 40 images from 4 - experiments , Scale bars , 5µm . * p < 0 . 05 , * * * p < 0 . 001 ; ns = not significant .

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A perfect co-localization between Fib-Tau and α-Syn clusters is also observed. The proportion of Fib-Tau clusters co-localizing with α-Syn fibrils increased with an increasing concentration of α-Syn fibrils reaching 60-70% overlap A weaker, concentration independent, overlap (10-20%) was observed with Aβ fibrils The quantification in (F) is derived from 3 to 4 independent experiments. The number of images analyzed is 27, 25, 25, 26, 28 for α-Syn and 18, 50, 50, 50, 25 for Aβ from left to right. One-Way ANOVA with Dunnett's post-hoc test was performed to compare the co-localization differences from 100nM concentration. Box-plot represents: median, interquartile range and 10-90% distribution. Data information: Box-plot represents: median, interquartile range and 10-90% distribution, Mann-Whitney-test is performed, n=40 images from 4-experiments, Scale bars, 5µm. *p<0.05, ***p<0.001; ns= not significant.


( G - I ) Primary neurons were co - exposed to Fib - Tau ( 0 . 36nM , ATTO550 labelled ) and Fib - α - Syn ( 50nM , monomer - equivalent , unlabeled ) for 60min and immunolabelled for homer ( G ) or α3 - NKA ( H ) . Quantification of Fib - Tau clusters co - localized with homer or α3 - NKA ( I ) . Note an increased co - localization of Fib - Tau with homer in presence of Fib - α - Syn . Data information : Box - plot represents : median , interquartile range and 10 - 90 % distribution , Mann - Whitney - test is performed , n = 40 images from 4 - experiments , Scale bars , 5µm . * p < 0 . 05 , * * * p < 0 . 001 ; ns = not significant .

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(G-I) Primary neurons were co-exposed to Fib-Tau (0.36nM, ATTO550 labelled) and Fib-α-Syn (50nM, monomer-equivalent, unlabeled) for 60min and immunolabelled for homer (G) or α3-NKA (H). Quantification of Fib-Tau clusters co-localized with homer or α3-NKA (I). Note an increased co-localization of Fib-Tau with homer in presence of Fib-α-Syn. Data information: Box-plot represents: median, interquartile range and 10-90% distribution, Mann-Whitney-test is performed, n=40 images from 4-experiments, Scale bars, 5µm. *p<0.05, ***p<0.001; ns= not significant.


( B ) The 39 - kDa catalytic domain ( 8 - kDa subdomain not shown for clarity ) is colored by subdomain : fingers ( salmon ) , palm ( yellow ) and thumb ( purple ) . The polymerase is in complex with a 1 - nt gapped 16 - mer oligo ( grey ) containing a templating 8 - oxo - dG ( green ) . Key residues involved in stabilizing the templating base ( Y505 and R517 ) are shown in black .

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(B) The 39-kDa catalytic domain (8-kDa subdomain not shown for clarity) is colored by subdomain: fingers (salmon), palm (yellow) and thumb (purple). The polymerase is in complex with a 1-nt gapped 16-mer oligo (grey) containing a templating 8-oxo-dG (green). Key residues involved in stabilizing the templating base (Y505 and R517) are shown in black.


( C ) ( Left panel ) 8 - oxo - dG ( green ) preferentially adopts the pro - mutagenic syn - conformation . A simulated annealing Fo - Fc omit density map is shown for 8 - oxo - dG ( contoured at 3σ ) . ( Right panel ) 8 - oxo - dG ( anti ) ( black , modeled using 2PFO ) was overlayed with the Fo - Fc omit density map . In the anti - conformation , the C8 - carbonyl would directly clash with the 5 ′ - phosphate ( red - dotted line ) .

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(C) (Left panel) 8-oxo-dG (green) preferentially adopts the pro-mutagenic syn-conformation. A simulated annealing Fo-Fc omit density map is shown for 8-oxo-dG (contoured at 3σ). (Right panel) 8-oxo-dG(anti) (black, modeled using 2PFO)was overlayed with the Fo-Fc omit density map. In the anti-conformation, the C8-carbonyl would directly clash with the 5′-phosphate (red-dotted line).


( D ) 8 - oxo - dG ( syn ) ( green ) is stabilized by multiple interactions , including Tyr505 and Arg517 ( grey ) .

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(D) 8-oxo-dG(syn) (green) is stabilized by multiple interactions, including Tyr505 and Arg517 (grey).


( B ) Overview of the 8 - oxo - dG : dATP insertion complex . The 39 - kDa catalytic domain ( 8 - kDa subdomain not shown for clarity ) is colored by subdomain : fingers ( salmon ) , palm ( yellow ) and thumb ( purple ) . The polymerase is in complex with a 1 - nt gapped 16 - mer oligo ( grey ) containing a templating 8 - oxo - dG ( green ) . The primer terminal ddC and the incoming ddCTP are shown in cyan and magenta , respectively . Key residues that form the nascent base pair binding pocket ( N513 and R517 ) and are involved in catalysis ( D427 , D429 and D490 ) are shown in black . The catalytic metal A and the nucleotide binding metal B are shown in red and neon , respectively .

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(B) Overview of the 8-oxo-dG:dATP insertion complex. The 39-kDa catalytic domain (8-kDa subdomain not shown for clarity) is colored by subdomain: fingers (salmon), palm (yellow) and thumb (purple). The polymerase is in complex with a 1-nt gapped 16-mer oligo (grey) containing a templating 8-oxo-dG (green). The primer terminal ddC and the incoming ddCTP are shown in cyan and magenta, respectively. Key residues that form the nascent base pair binding pocket (N513 and R517) and are involved in catalysis (D427, D429 and D490) are shown in black. The catalytic metal A and the nucleotide binding metal B are shown in red and neon, respectively.


( C ) 8 - oxo - dG ( green ) establishes a Watson - Crick pair with ddCTP ( magenta ) . Binding of the 8 - oxo - dG in the anti - conformation is only possible because the C8 - carbonyl is far enough away not to clash with the 5 ′ - phosphate . Asn513 and Arg517 ( purple ) interact with the minor groove of the nascent base pair . The base pair geometry ( C1 ′ distance and λ angles ) is indicated at the bottom of the figure . A simulated annealing Fo - Fc omit density map for the pair is also shown ( contoured at 3σ ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 8-oxo-dG (green) establishes a Watson-Crick pair with ddCTP (magenta). Binding of the 8-oxo-dG in the anti-conformation is only possible because the C8-carbonyl is far enough away not to clash with the 5′-phosphate. Asn513 and Arg517 (purple) interact with the minor groove of the nascent base pair. The base pair geometry (C1′ distance and λ angles) is indicated at the bottom of the figure. A simulated annealing Fo-Fc omit density map for the pair is also shown (contoured at 3σ).


( D ) Overlay of the primer terminus and active site with an undamaged reference structure ( 2PFO , black ) . The catalytic aspartic acids are shown in yellow , the primer terminus in cyan and the incoming dATP in magenta . A water molecule ( red ) and Mg2 + ( neon ) are occupying the metal A and metal B sites , respectively .

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

(D) Overlay of the primer terminus and active site with an undamaged reference structure (2PFO, black). The catalytic aspartic acids are shown in yellow, the primer terminus in cyan and the incoming dATP in magenta. A water molecule (red) and Mg2+ (neon) are occupying the metal A and metal B sites, respectively.


( B ) Overview of the 8 - oxo - dG : dATP insertion complex . The 39 - kDa catalytic domain ( 8 - kDa subdomain not shown for clarity ) is colored by subdomain : fingers ( salmon ) , palm ( yellow ) and thumb ( purple ) . The polymerase is in complex with a 1 - nt gapped 16 - mer oligo ( grey ) containing a templating 8 - oxo - dG ( green ) . The primer terminal ddC and the incoming dATP are shown in cyan and magenta , respectively . Key residues that form the nascent base pair binding pocket ( N513 and R517 ) and are involved in catalysis ( D427 , D429 and D490 ) are shown in black . The catalytic metal A and the nucleotide binding metal B are shown in red and neon , respectively .

(B) Overview of the 8-oxo-dG:dATP insertion complex. The 39-kDa catalytic domain (8-kDa subdomain not shown for clarity) is colored by subdomain: fingers (salmon), palm (yellow) and thumb (purple). The polymerase is in complex with a 1-nt gapped 16-mer oligo (grey) containing a templating 8-oxo-dG (green). The primer terminal ddC and the incoming dATP are shown in cyan and magenta, respectively. Key residues that form the nascent base pair binding pocket (N513 and R517) and are involved in catalysis (D427, D429 and D490) are shown in black. The catalytic metal A and the nucleotide binding metal B are shown in red and neon, respectively.


( C ) 8 - oxo - dG ( green ) establishes a Hoogsteen base pair with dATP ( magenta ) . Asn513 and Arg517 ( purple ) interact with the minor groove of the nascent base pair . The base pair geometry ( C1 ′ distance and λ angles ) is indicated at the bottom of the figure . A simulated annealing Fo - Fc omit density map for the pair is also shown ( contoured at 3σ ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 8-oxo-dG (green) establishes a Hoogsteen base pair with dATP (magenta). Asn513 and Arg517 (purple) interact with the minor groove of the nascent base pair. The base pair geometry (C1′ distance and λ angles) is indicated at the bottom of the figure. A simulated annealing Fo-Fc omit density map for the pair is also shown (contoured at 3σ).


( D ) Overlay of the primer terminus and active site with an undamaged reference structure ( 2PFO , black ) . The catalytic aspartic acids are shown in yellow , the primer terminus in cyan and the incoming dATP in magenta . A water molecule ( red ) and Mg2 + ( neon ) are occupying the metal A and B sites , respectively .

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

(D) Overlay of the primer terminus and active site with an undamaged reference structure (2PFO, black). The catalytic aspartic acids are shown in yellow, the primer terminus in cyan and the incoming dATP in magenta. A water molecule (red) and Mg2+ (neon) are occupying the metal A and B sites, respectively.


( A and B ) Overlay of the nascent base pair in the insertion structures with an undamaged reference structure ( 2PFO , black ) . Binding of 8 - oxo - dG ( green ) in both the anti - ( A ) or syn - ( B ) conformation involves a repositioning of the 5 ′ - phosphate ( red arrow ) . Furthermore , the Arg514 side chain ( purple ) that normally stacks with the templating base and interacts with the 5 ′ - phosphate ( blue - dotted line ) , also repositions ( black arrow ) . In both structures , Arg514 interacts with the C6 - carbonyl of the templating 8 - oxo - dG ( anti ) ( A ) or the C6 - amino group of the incoming dATP ( B ) . The incoming nucleotide is shown in magenta .

(A and B) Overlay of the nascent base pair in the insertion structures with an undamaged reference structure (2PFO, black). Binding of 8-oxo-dG (green) in both the anti- (A) or syn-(B) conformation involves a repositioning of the 5′-phosphate (red arrow). Furthermore, the Arg514 side chain (purple) that normally stacks with the templating base and interacts with the 5′-phosphate (blue-dotted line), also repositions (black arrow). In both structures, Arg514 interacts with the C6-carbonyl of the templating 8-oxo-dG(anti) (A) or the C6-amino group of the incoming dATP (B). The incoming nucleotide is shown in magenta.


( B ) Overview of the 8 - oxo - dG : dC extension complex . The 39 - kDa catalytic domain ( 8 - kDa subdomain not shown for clarity ) is colored by subdomain : fingers ( salmon ) , palm ( yellow ) and thumb ( purple ) . The polymerase is in complex with a 1 - nt gapped 16 - mer oligo ( grey ) and an incoming dUMPNPP ( magenta ) . The primer terminal dC and 8 - oxo - dG are shown in cyan and magenta , respectively . Key residues that form the primer terminal base pair binding pocket ( N513 , R517 and Glu529 ) and are involved in catalysis ( D427 , D429 and D490 ) are shown in black . The nucleotide binding metal ( B ) is shown in neon .

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

(B) Overview of the 8-oxo-dG:dC extension complex. The 39-kDa catalytic domain (8-kDa subdomain not shown for clarity) is colored by subdomain: fingers (salmon), palm (yellow) and thumb (purple). The polymerase is in complex with a 1-nt gapped 16-mer oligo (grey) and an incoming dUMPNPP (magenta). The primer terminal dC and 8-oxo-dG are shown in cyan and magenta, respectively. Key residues that form the primer terminal base pair binding pocket (N513, R517 and Glu529) and are involved in catalysis (D427, D429 and D490) are shown in black. The nucleotide binding metal (B) is shown in neon.


( C ) 8 - oxo - dG ( orange ) establishes a Watson - Crick base pair with dC ( cyan ) at the primer terminus . Tyr505 , Arg517 , and Glu529 ( purple ) interact with the minor groove of the base pair . The base pair geometry ( C1 ′ distance and λ angles ) is indicated at the bottom of the figure . A simulated annealing Fo - Fc omit density map for the pair is also shown ( contoured at 3σ ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 8-oxo-dG (orange) establishes a Watson-Crick base pair with dC (cyan) at the primer terminus. Tyr505, Arg517, and Glu529 (purple) interact with the minor groove of the base pair. The base pair geometry (C1′ distance and λ angles) is indicated at the bottom of the figure. A simulated annealing Fo-Fc omit density map for the pair is also shown (contoured at 3σ).


( D ) Overlay of the primer terminus and active site with an undamaged reference structure ( 2PFO , black ) . The catalytic aspartic acids are shown in yellow , incoming dUMPNPP in magenta , and primer terminal dC in cyan . Expectedly , Mg2 + ( neon ) is occupying the metal B site . However , the metal A is absent in the structure , resulting in a non - catalytic position of the 3 ′ - OH . This is consistent with other structures containing an incoming non - hydrolyzable analog .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Overlay of the primer terminus and active site with an undamaged reference structure (2PFO, black). The catalytic aspartic acids are shown in yellow, incoming dUMPNPP in magenta, and primer terminal dC in cyan. Expectedly, Mg2+ (neon) is occupying the metal B site. However, the metal A is absent in the structure, resulting in a non-catalytic position of the 3′-OH. This is consistent with other structures containing an incoming non-hydrolyzable analog.


( B ) Overview of the 8 - oxo - dG : dC extension complex . The 39 - kDa catalytic domain ( 8 - kDa subdomain not shown for clarity ) is colored by subdomain : fingers ( salmon ) , palm ( yellow ) and thumb ( purple ) . The polymerase is in complex with a 1 - nt gapped 16 - mer oligo ( grey ) and an incoming dUMPNPP ( magenta ) . The primer terminal dA and 8 - oxo - dG are shown in cyan and magenta , respectively . Key residues that form the primer terminal base pair binding pocket ( N513 and R517 ) and are involved in catalysis ( D427 , D429 and D490 ) are shown in black . The nucleotide binding metal ( B ) is shown in neon .

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

(B) Overview of the 8-oxo-dG:dC extension complex. The 39-kDa catalytic domain (8-kDa subdomain not shown for clarity) is colored by subdomain: fingers (salmon), palm (yellow) and thumb (purple). The polymerase is in complex with a 1-nt gapped 16-mer oligo (grey) and an incoming dUMPNPP (magenta). The primer terminal dA and 8-oxo-dG are shown in cyan and magenta, respectively. Key residues that form the primer terminal base pair binding pocket (N513 and R517) and are involved in catalysis (D427, D429 and D490) are shown in black. The nucleotide binding metal (B) is shown in neon.


( C ) 8 - oxo - dG ( orange ) establishes a Hoogsteen base pair with dA ( cyan ) at the primer terminus . Tyr505 and Arg517 ( purple ) interact with the minor groove of the base pair . The base pair geometry ( C1 ′ distance and λ angles ) is indicated at the bottom of the figure . A simulated annealing Fo - Fc omit density map for the pair is also shown ( contoured at 3σ ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 8-oxo-dG (orange) establishes a Hoogsteen base pair with dA (cyan) at the primer terminus. Tyr505 and Arg517 (purple) interact with the minor groove of the base pair. The base pair geometry (C1′ distance and λ angles) is indicated at the bottom of the figure. A simulated annealing Fo-Fc omit density map for the pair is also shown (contoured at 3σ).


( D ) Overlay of the primer terminus and active site with an undamaged reference structure ( 2PFO , black ) . The catalytic aspartic acids are shown in yellow , incoming dUMPNPP in magenta , and primer terminal dA in cyan . Expectedly , Mg2 + ( neon ) is occupying the metal B site . However , the metal A is absent in the structure , resulting in a non - catalytic position of the 3 ′ - OH . This is consistent with other structures containing an incoming non - hydrolyzable analog .

(D) Overlay of the primer terminus and active site with an undamaged reference structure (2PFO, black). The catalytic aspartic acids are shown in yellow, incoming dUMPNPP in magenta, and primer terminal dA in cyan. Expectedly, Mg2+ (neon) is occupying the metal B site. However, the metal A is absent in the structure, resulting in a non-catalytic position of the 3′-OH. This is consistent with other structures containing an incoming non-hydrolyzable analog.


( A ) Glu529 ( purple ) forms a hydrogen bond with the C2 - amino group of the primer terminal templating 8 - oxo - dG ( orange ) in the anti - conformation . ( B ) This interaction is abolished when 8 - oxo - dG ( orange ) adopts the syn - conformation . An overlay with the 8 - oxo - dG ( anti ) - containing structure reveals that Glu529 ( black ) would likely clash ( red - dotted line ) with the C8 - carbonyl of 8 - oxo - dG ( syn ) . Repositioning of Glu529 ( purple ) places the side chain 4 . 5 Å away from the C8 - carbonyl . ( C ) Comparison of catalytic efficiencies for extension past an 8 - oxo - dG ( anti ) : dC , 8 - oxo - dG ( syn ) : dA and dG : dC base pairs at the primer terminus using WT and E529A Pol λ .

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

(A) Glu529 (purple) forms a hydrogen bond with the C2-amino group of the primer terminal templating 8-oxo-dG (orange) in the anti-conformation. (B) This interaction is abolished when 8-oxo-dG (orange) adopts the syn-conformation. An overlay with the 8-oxo-dG(anti)-containing structure reveals that Glu529 (black) would likely clash (red-dotted line) with the C8-carbonyl of 8-oxo-dG(syn). Repositioning of Glu529 (purple) places the side chain 4.5 Å away from the C8-carbonyl. (C) Comparison of catalytic efficiencies for extension past an 8-oxo-dG(anti):dC, 8-oxo-dG(syn):dA and dG:dC base pairs at the primer terminus using WT and E529A Pol λ.


( A ) Undamaged structure containing a templating dG at the primer terminus ( 2PFO ) was overlayed with a structure containing an 8 - oxo - dG : dC base pair ( black ) or an 8 - oxo - dG : dA mispair ( red ) at the primer terminus . ( B ) Undamaged structure containing a templating dA at the primer terminus ( 1XSN ) was overlayed with a structure containing an 8 - oxo - dG : dC base pair ( black ) or an 8 - oxo - dG : dA mispair ( red ) at the primer terminus .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Undamaged structure containing a templating dG at the primer terminus (2PFO) was overlayed with a structure containing an 8-oxo-dG:dC base pair (black) or an 8-oxo-dG:dA mispair (red) at the primer terminus. (B) Undamaged structure containing a templating dA at the primer terminus (1XSN) was overlayed with a structure containing an 8-oxo-dG:dC base pair (black) or an 8-oxo-dG:dA mispair (red) at the primer terminus.


( A ) Gross morphology of control ( Ctrl ) , Sas - 4 − / − , Sas - 4 − / − 53bp1 − / − , and Sas - 4 − / − Usp28 − / − embryos at E9 . 5 . At least five embryos were considered per genotype and all the double mutant embryos exhibited the rescue criteria mentioned in the text . Scale bar = 500 μm .

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

(A) Gross morphology of control (Ctrl), Sas-4−/−, Sas-4−/− 53bp1−/−, and Sas-4−/− Usp28−/− embryos at E9.5. At least five embryos were considered per genotype and all the double mutant embryos exhibited the rescue criteria mentioned in the text. Scale bar = 500 μm.


( B , C ) Immunostaining for p53 ( B ) and Cleaved - Caspase3 ( Cl - CASP3 , C ) on transverse sections of Ctrl , Sas - 4 − / − , Sas - 4 − / − 53bp1 − / − , and Sas - 4 − / − Usp28 − / − embryos at E9 . 5 . The area shown encompasses the ventral neural tube and surrounding mesenchyme . Asterisks in ( B ) denote non - specific staining of blood cells . Scale bar = 50 μm . ( D , E ) Quantifications of the p53 ( D ) and Cl - CASP3 ( E ) fluorescence signals shown in ( B ) and ( C ) , respectively . Three embryos per genotype were used for the quantifications . * * * p < 0 . 001 , * p < 0 . 05 ( two - tailed student ' s T - test , one - way ANOVA with Tukey ' s multiple comparisons tests gave similar results ) . Bars represent mean ± s . d . ( D ) Ctrl : 1 . 00 ± 0 . 06 ( n = 1770 cells ) ; Sas - 4 − / − : 6 . 84 ± 0 . 34 ( n = 275 ) , Sas - 4 − / − 53bp1 − / − : 1 . 85 ± 0 . 75 ( n = 690 ) , Sas - 4 − / − Usp28 − / − : 1 . 50 ± 0 . 48 ( n = 975 ) . ( E ) Ctrl : 1 . 00 ± 0 . 70 ( n = 1690 ) ; Sas - 4 − / − : 7 . 88 ± 2 . 62 ( n = 175 ) ; Sas - 4 − / − 53bp1 − / − : 1 . 43 ± 0 . 71 ( n = 1030 ) ; Sas - 4 − / − Usp28 − / − : 1 . 57 ± 0 . 44 ( n = 950 ) .

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

(B, C) Immunostaining for p53 (B) and Cleaved-Caspase3 (Cl-CASP3, C) on transverse sections of Ctrl, Sas-4−/−, Sas-4−/− 53bp1−/−, and Sas-4−/− Usp28−/− embryos at E9.5. The area shown encompasses the ventral neural tube and surrounding mesenchyme. Asterisks in (B) denote non-specific staining of blood cells. Scale bar = 50 μm. (D, E) Quantifications of the p53 (D) and Cl-CASP3 (E) fluorescence signals shown in (B) and (C), respectively. Three embryos per genotype were used for the quantifications. *** p < 0.001, *p<0.05 (two-tailed student's T-test, one-way ANOVA with Tukey's multiple comparisons tests gave similar results). Bars represent mean ± s.d. (D) Ctrl: 1.00 ± 0.06 (n=1770 cells); Sas-4−/−: 6.84 ± 0.34 (n=275), Sas-4−/− 53bp1−/−: 1.85 ± 0.75 (n=690), Sas-4−/− Usp28−/−: 1.50 ± 0.48 (n=975). (E) Ctrl: 1.00 ± 0.70 (n=1690); Sas-4−/−: 7.88 ± 2.62 (n=175); Sas-4−/− 53bp1−/−: 1.43 ± 0.71 (n=1030); Sas-4−/− Usp28−/−: 1.57 ± 0.44 (n=950).


( A , B ) Whole - mount immunostaining and quantification for p53 on Ctrl and Sas - 4 − / − embryos at E7 . 5 ( A ) and E6 . 5 ( B ) . Representative sagittal planes are shown and the dotted lines demarcate the epiblast . Quantification of nuclear p53 fluorescence intensity in the epiblast is shown below , normalized to control embryos in the same batch at E7 . 5 ( 4 embryos ) and at E6 . 5 ( 9 embryos ) . * * * p < 0 . 001 ( two - tailed student ' s T - test ) . Bars represent mean ± s . d . Scale bars = 100 μm . ( A ) Ctrl : 1 . 04 ± 0 . 00 ( n = 495 cells ) ; Sas - 4 - / - : 1 . 65 ± 0 . 19 ( n = 850 ) . ( B ) Ctrl : 1 . 00 ± 0 . 18 ( n = 1665 ) ; Sas - 4 - / - : 1 . 16 ± 0 . 44 ( n = 1330 )

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

(A, B) Whole-mount immunostaining and quantification for p53 on Ctrl and Sas-4−/− embryos at E7.5 (A) and E6.5 (B). Representative sagittal planes are shown and the dotted lines demarcate the epiblast. Quantification of nuclear p53 fluorescence intensity in the epiblast is shown below, normalized to control embryos in the same batch at E7.5 (4 embryos) and at E6.5 (9 embryos). *** p < 0.001 (two-tailed student's T-test). Bars represent mean ± s.d. Scale bars = 100 μm. (A) Ctrl: 1.04 ± 0.00 (n=495 cells); Sas-4-/-: 1.65 ± 0.19 (n=850). (B) Ctrl: 1.00 ± 0.18 (n=1665); Sas-4-/-: 1.16 ± 0.44 (n=1330)


( C , D ) Immunostaining for 53BP1 and USP28 on sagittal sections of WT embryos at E6 . 5 ( C ) or sagittal planes of whole - mount immunostaining at E5 . 5 ( D ) . Quantification of the fluorescent signals in the epiblast ( Ep ) versus visceral endoderm ( VE ) are shown below . Four embryos per genotype were used for the quantifications . * * * * p < 0 . 0001 , * * * p < 0 . 001 ( two - tailed student ' s T - test ) . Bars represent mean ± s . d . Scale bar = 50 μm . ( C ) 53BP1 in Ep : 1 . 00 ± 0 . 10 ( n = 225 cells ) ; 53BP1 in VE : 0 . 37 ± 0 . 06 ( n = 105 ) ; USP28 in Ep : 1 . 00 ± 0 . 08 ( n = 235 ) ; USP28 in VE : 0 . 17 ± 0 . 05 ( n = 90 ) . ( D ) 53BP1 in Ep : 1 . 00 ± 0 . 24 ( n = 225 ) ; 53BP1 in VE : 0 . 68 ± 0 . 14 ( n = 115 ) ; USP28 in Ep : 1 . 00 ± 0 . 23 ( n = 280 ) ; USP28 in VE : 0 . 28 ± 0 . 00 ( n = 110 ) .

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

(C, D) Immunostaining for 53BP1 and USP28 on sagittal sections of WT embryos at E6.5 (C) or sagittal planes of whole-mount immunostaining at E5.5 (D). Quantification of the fluorescent signals in the epiblast (Ep) versus visceral endoderm (VE) are shown below. Four embryos per genotype were used for the quantifications. **** p < 0.0001, ***p<0.001 (two-tailed student's T-test). Bars represent mean ± s.d. Scale bar = 50 μm. (C) 53BP1 in Ep: 1.00 ± 0.10 (n=225 cells); 53BP1 in VE: 0.37 ± 0.06 (n=105); USP28 in Ep: 1.00 ± 0.08 (n=235); USP28 in VE: 0.17 ± 0.05 (n=90). (D) 53BP1 in Ep: 1.00 ± 0.24 (n=225); 53BP1 in VE: 0.68 ± 0.14 (n=115); USP28 in Ep: 1.00 ± 0.23 (n=280); USP28 in VE: 0.28 ± 0.00 (n=110).


( A ) Immunostaining for the centrosome marker γ - tubulin ( TUBG ) on Ctrl and Sas - 4 − / − primary mESCs . The bottom panels are magnifications of the areas marked in the top panels . Scale bars = 20 μm ( top ) and 10 μm ( bottom ) .

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

(A) Immunostaining for the centrosome marker γ-tubulin (TUBG) on Ctrl and Sas-4−/− primary mESCs. The bottom panels are magnifications of the areas marked in the top panels. Scale bars = 20 μm (top) and 10 μm (bottom).


( B ) Immunostaining and quantification for p53 on Ctrl and Sas - 4 − / − primary mESCs in pluripotent and partially differentiated ( diff . ) conditions . The quantification of p53 fluorescence intensities was normalized to Ctrl ( Four independent experiments ) . * * p < 0 . 01 , * p < 0 . 05 ( two - tailed student ' s T - test , the comparison of the genotypes in the " Pluripotent " condition was not significant using the one - way ANOVA with Tukey ' s multiple comparisons tests ) . Bars represent mean ± s . d . Scale bar = 50 μm . Pluripotent Ctrl : 1 . 00 ± 0 . 02 ( n = 2030 cells ) ; pluripotent Sas - 4 - / - : 1 . 17 ± 0 . 10 ( n = 2170 ) ; partially diff . Ctrl : 0 . 62 ± 0 . 13 ( n = 2745 ) ; partially diff . Sas - 4 - / - : 1 . 12 ± 0 . 24 ( n = 2000 ) .

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

(B) Immunostaining and quantification for p53 on Ctrl and Sas-4−/− primary mESCs in pluripotent and partially differentiated (diff.) conditions. The quantification of p53 fluorescence intensities was normalized to Ctrl (Four independent experiments). ** p < 0.01, * p < 0.05 (two-tailed student's T-test, the comparison of the genotypes in the "Pluripotent" condition was not significant using the one-way ANOVA with Tukey's multiple comparisons tests). Bars represent mean ± s.d. Scale bar = 50 μm. Pluripotent Ctrl: 1.00 ± 0.02 (n=2030 cells); pluripotent Sas-4-/-: 1.17 ± 0.10 (n=2170); partially diff. Ctrl: 0.62 ± 0.13 (n=2745); partially diff. Sas-4-/-: 1.12 ± 0.24 (n=2000).