D . Western blot of total , aqueous lipid ( GPI - anchor - containing ) Triton X - 114 extracts derived from wild type HeLa two independent CRISPR CWH43 knockout ( KO ) HeLa lines in which a mutated CWH43 gene encodes a that is truncated near Leu533 CWH43 mRNA are markedly reduced . were transfected to overexpress a control GFP plasmid , a plasmid encoding human wild type Cwh43 with GFP fused to N - terminus , or a plasmid encoding human CWH43 harboring iNPH - associated mutation ( Lys696AsnfsTer23 ) with GFP fused to N - terminus . Western blot was stained using an antibody directed against CD59 , a GPI - anchored .

O O B-EXP_ASSAY I-EXP_ASSAY O O O B-SUBCELLULAR I-SUBCELLULAR O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-CELL_LINE O O B-GENEPROD I-GENEPROD O O O O B-CELL_LINE O O O O O B-GENEPROD O O O O O O O B-SMALL_MOLECULE B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O O B-ORGANISM O O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-ORGANISM B-GENEPROD O B-DISEASE O O O O O O O B-GENEPROD O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O O O B-GENEPROD O O B-SMALL_MOLECULE O O O

D. Western blot analysis of total membrane, aqueous and lipid (GPI-anchor-containing) Triton X-114 extracts derived from wild type HeLa cells and two independent CRISPR CWH43 knockout (KO) HeLa cell lines in which a mutated CWH43 gene encodes a protein that is truncated near Leu533 and CWH43 mRNA and protein are markedly reduced. Cells were transfected to overexpress a control GFP plasmid, a plasmid encoding human wild type Cwh43 with GFP fused to the N-terminus, or a plasmid encoding human CWH43 harboring the iNPH-associated mutation (Lys696AsnfsTer23) with GFP fused to the N-terminus. The Western blot was stained using an antibody directed against CD59, a GPI-anchored protein.


Confocal immunocytochemistry of a single cultured mouse ciliated ependymal . Cilia were visualized using an antibody for acetylated alpha tubulin ( green ) . Cwh43 immunoreactivity was visualized using a specific anti - Cwh43 antibody ( red ) . Scale bar ( left column ) is approximately 4 µm . in column on right represent a Z - stack reconstruction of confocal showing localization of Cwh43 immunoreactivity in cilia of a mouse ependymal . Scale bar ( right column ) is approximately 5 µm .

B-EXP_ASSAY I-EXP_ASSAY O O O O B-ORGANISM B-SUBCELLULAR B-CELL_TYPE O B-SUBCELLULAR O O O O O O B-EXP_ASSAY B-GENEPROD I-GENEPROD O O O O B-GENEPROD B-EXP_ASSAY O O O O O O O B-GENEPROD O O O 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-EXP_ASSAY O B-EXP_ASSAY O B-GENEPROD B-EXP_ASSAY O B-SUBCELLULAR O O B-ORGANISM B-CELL_TYPE O O O O O O O O O O O O

Confocal fluorescence immunocytochemistry images of a single cultured mouse ciliated ependymal cell. Cilia were visualized using an antibody for acetylated alpha tubulin (green). Cwh43 immunoreactivity was visualized using a specific anti-Cwh43 antibody (red). Scale bar (left column) is approximately 4 µm. Images in the column on the right represent a Z-stack reconstruction of confocal images showing localization of Cwh43 immunoreactivity in cilia of a mouse ependymal cell. Scale bar (right column) is approximately 5 µm.


A . Scatter plot comparing ventricular volume from CWH43WT / WT ( wild type , WT ) , heterozygous CWH43WT / M533 , homozygous CWH43M533 / M533 CWH43M533 / A530 mice at 6 months . Ventricular volume was calculated from T2 - weighted MR of mouse brain using a custom automated computer algorithm . Horizontal bars indicate mean of measurements in each column . Statistical significance for each mutant mouse compared to WT was determined using unpaired t - ( P = 0 . 0015 for CWH43WT / M533 ; P = 0 . 0014 for CWH43M533 / M533 ; P = 0 . 006 for CWH43M533 / A530 ) .

O O O O O B-TISSUE B-EXP_ASSAY O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O B-GENEPROD O O B-ORGANISM O O O O B-TISSUE B-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-ORGANISM B-TISSUE O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O

A. Scatter plot comparing ventricular volume from CWH43WT/WT (wild type, WT), heterozygous CWH43WT/M533, homozygous CWH43M533/M533 and CWH43M533/A530 mice at 6 months. Ventricular volume was calculated from T2-weighted MR images of the mouse brain using a custom automated computer algorithm. Horizontal bars indicate the mean of the measurements in each column. Statistical significance for each mutant mouse line compared to WT was determined using the unpaired t-test (P=0.0015 for CWH43WT/M533; P=0.0014 for CWH43M533/M533; P=0.006 for CWH43M533/A530).


D . Box plot showing rotarod performance data for CWH43WT / WT , CWH43WT / M533 , CWH43M533 / M533 CWH43M533 / A530 mice at 7 months of . Data shown are mean , 1st quartile , 3rd quartile , minimum maximum for each group of mice . Statistical significance was determined using unpaired t - . When compared to wild type CWH43WT / WT mice ( n = 10 ) , balance time on rotarod was decreased significantly among heterozygous CWH43WT / M533 mice ( * P = 0 . 04 , n = 8 ) , homozygous CWH43M533 / M533 mice ( * P = 0 . 03 , n = 7 ) , compound heterozygous CWH43M533 / A530 mice ( * P = 0 . 03 , n = 4 ) .

O O O O O B-EXP_ASSAY O O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O O B-GENEPROD O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O B-GENEPROD O O B-ORGANISM O O O O O O O O O B-EXP_ASSAY O O O O O B-GENEPROD O O B-ORGANISM O O O O O O O O O O O O O O B-GENEPROD O O B-ORGANISM O O O O O O O O O O O O O O O B-GENEPROD O O B-ORGANISM O O O O O O O O O O O O O

D. Box plot showing rotarod performance data for CWH43WT/WT, CWH43WT/M533, CWH43M533/M533 and CWH43M533/A530 mice at 7 months of age. Data shown are the mean, 1st quartile, 3rd quartile, minimum and maximum for each group of mice. Statistical significance was determined using the unpaired t-test. When compared to wild type CWH43WT/WT mice (n=10), balance time on the rotarod was decreased significantly among heterozygous CWH43WT/M533 mice (*P=0.04, n=8), homozygous CWH43M533/M533 mice (*P=0.03, n=7), and compound heterozygous CWH43M533/A530 mice (*P=0.03, n=4).


Scanning electron of ependymal surface of lateral ventricle of CWH43 WT CWH43M533 / M533 mice . Scale bar is approximately 40 µm . graph on right quantifies data from electron on left . Data shown are mean ± . * = P = 0 . 004 , n = 4 , unpaired t - .

B-EXP_ASSAY I-EXP_ASSAY O B-CELL_TYPE O O B-TISSUE I-TISSUE O B-GENEPROD O B-GENEPROD O O B-ORGANISM O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O

Scanning electron micrographs of the ependymal surface of the lateral ventricle of CWH43 WT and CWH43M533/M533 mice. Scale bar is approximately 40 µm. The graph on the right quantifies the data from the electron micrographs on the left. Data shown are the mean ± SEM. * = P=0.004, n = 4, unpaired t-test.


Western blot of total , aqueous lipid ( GPI - anchor - containing ) Triton X - 114 extracts derived from wild type , CWH43M533 / M533 CWH43WT / M533mouse brain or kidney . Western blot was stained using an antibody directed against CD59 , a GPI - anchored .

B-EXP_ASSAY I-EXP_ASSAY O O O B-SUBCELLULAR I-SUBCELLULAR O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O B-GENEPROD O O B-GENEPROD O O B-TISSUE O B-TISSUE O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O O O B-GENEPROD O O B-SMALL_MOLECULE O O O

Western blot analysis of total membrane, aqueous and lipid (GPI-anchor-containing) Triton X-114 extracts derived from wild type, CWH43M533/M533 and CWH43WT/M533mouse brain or kidney. The Western blot was stained using an antibody directed against CD59, a GPI-anchored protein.


immunohistochemistry for CD59 in ependymal layer choroid plexus of lateral ventricle from CWH43WT / WT , CWH43M533 / M533 CWH43M533 / A530 mice . Arrowheads point to apical surfaces of ependymal choroid plexus . Nuclei are counterstained using DAPI ( blue ) . Scale bar is approximately 5 µm .

B-EXP_ASSAY O B-GENEPROD O B-CELL_TYPE O B-TISSUE I-TISSUE O B-TISSUE I-TISSUE O B-GENEPROD O O O B-GENEPROD O O B-GENEPROD O O B-ORGANISM O O O O B-SUBCELLULAR I-SUBCELLULAR O B-CELL_TYPE B-TISSUE I-TISSUE O B-SUBCELLULAR O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O O O O

Fluorescence immunohistochemistry for CD59 in the ependymal layer and choroid plexus of the lateral ventricle from CWH43WT/WT, CWH43M533/M533 and CWH43M533/A530 mice. Arrowheads point to apical surfaces of ependymal and choroid plexus cells. Nuclei are counterstained using DAPI (blue). Scale bar is approximately 5 µm.


( B ) Representative of importin β cargo gradients detected by Rango - 3 FLIM / FRET imaging . Scale bars , 20µm .

O O O O O B-GENEPROD I-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O O O O O O

(B) Representative images of importin β cargo gradients detected by Rango-3 FLIM/FRET imaging. Scale bars, 20µm.


( C ) scans of Rango - 3 FRET efficiency ( E ) in 10 µm - wide

O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O

(C) Line scans of Rango-3 FRET efficiency (E) in 10 µm-wide areas


( D - F ) of Rango - 3 E gradients ( top panels ) average Rango - 3 E ( bottom ) in involving untreated oocytes ( controls ) or oocytes injected with RanT24N mRNA , importin β ( 71 - 876 ) or treated with 20 µM IPZ . Data for each panel are from at least 2 separate experiments . Means ± SDs , t - , oocyte numbers indicated in brackets .

O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-EXP_ASSAY O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-CELL_TYPE O O O O B-CELL_TYPE O O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O B-CELL_TYPE O O O O O

(D-F) Quantification of the Rango-3 E gradients (top panels) and average cellular Rango-3 E (bottom) in involving untreated oocytes (controls) or oocytes injected with RanT24N mRNA, importin β(71-876) protein or treated with 20 µM IPZ. Data for each panel are from at least 2 separate experiments. Means ± SDs, t-test, oocyte numbers indicated in brackets.


( G ) Nonlinear single - exponential regression was used to fit average of radial Rango - 3 τdonor scans in control oocyte with one - phase decay model . dashed corresponds to average of spindle pole from chromosomes in same .

O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-EXP_ASSAY O O B-CELL_TYPE O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O B-SUBCELLULAR O O O

(G) Nonlinear single-exponential regression was used to fit the average of radial Rango-3 τdonor line scans in control oocyte with the one-phase decay model. The dashed line corresponds to the average of spindle pole distance from chromosomes in the same cell.


( H ) Immunoblotting in biochemical pull - downs with recombinant Ran added to human DL D1 lysates . A representative of at least 5 repeats .

O O O B-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O B-ORGANISM B-CELL_LINE I-CELL_LINE O O O O O O O O O O

(H) Immunoblotting in biochemical pull-downs with recombinant Ran proteins added to human DL D1 cell lysates. A representative of at least 5 repeats.


( A ) of chromosomal cargo gradients ( top ) average Rango - 3 E in oocytes injected with increasing concentrations of Ran mutant mRNA concentrations . Data from oocytes imaged side - by - side within one experiment . Means ± SDs , one - side ANOVA , Tukey ' s . Numbers of analyzed oocytes are indicated in brackets in top panel are identical for both panels .

O O O O B-SUBCELLULAR B-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-CELL_TYPE O O O B-EXP_ASSAY O B-GENEPROD O O B-EXP_ASSAY O O O B-CELL_TYPE B-EXP_ASSAY O 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-CELL_TYPE O O O O O O O O O O O O O

(A) Quantification of chromosomal cargo gradients (top) and average Rango-3 E in oocytes injected with increasing concentrations of Ran mutant mRNA concentrations. Data from oocytes imaged side-by-side within one experiment. Means ± SDs, one-side ANOVA, Tukey's test. Numbers of analyzed oocytes are indicated in the brackets in the top panel and are identical for both panels.


( C ) Linescans of estimated Rango - 3 FRET efficiency ( E ) in 10 µm - wide

O O O B-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O

(C) Linescans of the estimated Rango-3 FRET efficiency (E) in 10 µm-wide areas


( D ) of Rango - 3 E gradients ( top panels ) average Rango - 3 E ( bottom ) oocytes that were treated side - by - side by injection of mRNA for Rango - 3 , alongside with buffer ( controls ) or 2000ng / µl mRNA for RanT24N or RanT24N , T42A . Means ± SDs , one - side ANOVA , Tukey ' s , oocyte numbers indicated in brackets .

O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-EXP_ASSAY O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-CELL_TYPE O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O B-CELL_TYPE O O O O O

(D) Quantification of the Rango-3 E gradients (top panels) and average cellular Rango-3 E (bottom) oocytes that were treated side-by-side by the injection of mRNA for Rango-3, alongside with buffer (controls) or 2000ng/µl mRNA for RanT24N or RanT24N, T42A. Means ± SDs, one-side ANOVA, Tukey's test, oocyte numbers indicated in brackets.


( B ) Chromosomal gradients detected by RBP3 FLIM / FRET imaging . Scale bars , 20µm .

O O O B-SUBCELLULAR B-EXP_ASSAY O O B-GENEPROD B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O O O O O O

(B) Chromosomal gradients detected by RBP3 FLIM/FRET imaging. Scale bars, 20µm.


( C ) scans of estimated RBP - 3 in 10 µm - wide

O O O B-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O

(C) Line scans of the estimated RBP-3 in 10 µm-wide areas


( D - F ) of RBP - 3 gradients ( top panels ) average RBP - 3 E ( bottom ) in separate experiments involving untreated oocytes ( controls ) or oocytes injected with RanT24N mRNA , importin β ( 71 - 876 ) or treated with 20µM IPZ . Means ± SDs , t - , oocyte numbers indicated in brackets .

O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O B-CELL_TYPE O O O O B-CELL_TYPE O O B-GENEPROD O O B-GENEPROD I-GENEPROD O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-CELL_TYPE O O O O O

(D-F) Quantification of the RBP-3 gradients (top panels) and average cellular RBP-3 E (bottom) in separate experiments involving untreated oocytes (controls) or oocytes injected with RanT24N mRNA, importin β(71-876) protein or treated with 20µM IPZ. Means ± SDs, t-test, oocyte numbers indicated in brackets.


( G ) Nonlinear single - exponential regression was used to fit average of radial RBP - 3 τdonor scans in control oocyte with one - phase decay model .

O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-EXP_ASSAY O O B-CELL_TYPE O O O O O O O

(G) Nonlinear single-exponential regression was used to fit the average of radial RBP-3 τdonor line scans in control oocyte with the one-phase decay model.


( H ) of oocytes expressing GFP - Ran RCC1 - mCherry from injected mRNAs before FRAP assays . Scale bars , 20 µm .

O O O O B-CELL_TYPE O B-GENEPROD O B-GENEPROD I-GENEPROD O B-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O

(H) Images of oocytes expressing GFP-Ran and RCC1-mCherry from injected mRNAs before FRAP assays. Scale bars, 20 µm.


( I ) RCC1 - mCherry half - time recovery ( right , means ± SDs , t - . ) an immobile fraction ( left , means ± SDs , Mann - Whitney - ) in control IPZ - treated , oocyte numbers indicated in brackets .

O O O B-GENEPROD O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O B-CELL_TYPE O O O O O

(I) RCC1-mCherry half-time fluorescence recovery (right, means ± SDs, t-test.) and an immobile fraction (left, means ± SDs, Mann-Whitney-test) in control and IPZ-treated cells, oocyte numbers indicated in brackets.


( A ) Dose - dependent effects of IPZ on MI spindle assembly are shown by maximum z - projection from confocal time - lapse imaging of oocytes obtained from transgenic histone H2B - EGFP mice stained with SiR - tubulin . Dashed circles indicate oocyte edges ; scale bars , 10 µm .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-SMALL_MOLECULE O B-SUBCELLULAR I-SUBCELLULAR B-EXP_ASSAY O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-CELL_TYPE O O O B-GENEPROD I-GENEPROD O B-GENEPROD B-ORGANISM B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O B-CELL_TYPE O O O O O O O O

(A) Dose-dependent effects of IPZ on MI spindle assembly are shown by maximum z-projection images from confocal time-lapse imaging of oocytes obtained from transgenic histone H2B-EGFP mice stained with SiR-tubulin. Dashed circles indicate oocyte edges; scale bars, 10 µm.


( B - C ) Live - imaging of oocytes was used to quantify effects of IPZ on GVBD ( B ) anaphase I entry ( C ) . Anaphase I was defined as first time point when chromosome segregation was visible . Fisher ' s exact , oocyte numbers are indicated in brackets .

O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-CELL_TYPE O O O O O O B-SMALL_MOLECULE O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR B-EXP_ASSAY O O O O O O O O B-CELL_TYPE O O O O O O

(B-C) Live-cell imaging of oocytes was used to quantify the effects of IPZ on GVBD (B) and anaphase I entry (C). Anaphase I was defined as the first time point when chromosome segregation was visible. Fisher's exact test, oocyte numbers are indicated in brackets.


( D ) Frequency severity of MI spindle disruption induced by increasing doses of IPZ Oocyte numbers are shown at top of bars .

O O O B-EXP_ASSAY O O B-SUBCELLULAR I-SUBCELLULAR O O O O B-EXP_ASSAY O B-SMALL_MOLECULE B-CELL_TYPE O O O O O O O O

(D) Frequency and severity of MI spindle disruption induced by increasing doses of IPZ Oocyte numbers are shown at the top of the bars.


( E ) MI spindle formation in oocytes expressing CDK5RAP2 - EGFP , histone H2B - mCherry stained with SiR - tubulin matured in presence of 15 µM IPZ . Representative still of maximum z - projections are shown . Time in hh : mm is relative to GVBD . Scale bar , 10 µm .

O O O B-SUBCELLULAR I-SUBCELLULAR O O B-CELL_TYPE O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD O B-GENEPROD B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O

(E) MI spindle formation in oocytes expressing CDK5RAP2-EGFP, histone H2B-mCherry and stained with SiR-tubulin matured in the presence of 15 µM IPZ. Representative still images of maximum z-projections are shown. Time in hh:mm is relative to GVBD. Scale bar, 10 µm.


( F ) MT at MTOCs at time of GVBD quantified from SiR - tubulin signal that was normalized to mean SiR - tubulin signal . Mean of MT on MTOCs was arbitrarily centered to 1 in control group ( means ± SDs , Mann - Whitney - ) . Total of 184 MTOC in 17 control oocytes a total of 170 MTOCs in 17 IPZ - treated oocytes were analyzed . ( G ) Time course of MT in of spindle after GVBD . Mean of MT was arbitrarily centered to 1 in control group ( means ± SDs ) . Oocyte numbers are indicated in brackets .

O O O B-SUBCELLULAR O B-SUBCELLULAR O O O B-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O O O O B-SUBCELLULAR O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O B-CELL_TYPE O O O O B-SUBCELLULAR O O B-SMALL_MOLECULE O O B-CELL_TYPE O O O O O O O O O B-SUBCELLULAR O O B-SUBCELLULAR O B-EXP_ASSAY O O O B-SUBCELLULAR O O O O O O O O O O O O O O B-CELL_TYPE O O O O O O

(F) MT intensity at MTOCs at the time of GVBD quantified from SiR-tubulin signal that was normalized to mean cytoplasmic SiR-tubulin signal. Mean of MT intensity on MTOCs was arbitrarily centered to 1 in the control group (means ± SDs, Mann-Whitney-test). Total of 184 MTOC in 17 control oocytes and a total of 170 MTOCs in 17 IPZ-treated oocytes were analyzed. (G) Time course of the MT intensity in the area of spindle after GVBD. Mean of MT intensity was arbitrarily centered to 1 in the control group (means ± SDs). Oocyte numbers are indicated in brackets.


( H ) Oocytes expressing CDK5RAP2 - EGFP , histone H2B - mCherry stained with SiR - tubulin were treated with 15 µM IPZ after being cultured for 5h in control media . Maximum z - projections from confocal live - imaging are shown . Time in hh : mm is relative to GVBD , DMSO or 15 uM IPZ was added at time 5 : 00 . Scale bars , 10 µm .

O O O B-CELL_TYPE O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD O B-GENEPROD B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O

(H) Oocytes expressing CDK5RAP2-EGFP, histone H2B-mCherry and stained with SiR-tubulin were treated with 15 µM IPZ after being cultured for 5h in control media. Maximum intensity z-projections from confocal live-cell imaging are shown. Time in hh:mm is relative to GVBD, DMSO or 15 uM IPZ was added at time 5:00. Scale bars, 10 µm.


( I ) . Spindle volume at 1h metaphase I after IPZ addition . metaphase time was defined as last time point before anaphase or , in IPZ - treated oocytes that failed anaphase entry , as average control metaphase time . Means ± SDs , Mann - Whitney , oocyte numbers are indicated in brackets .

O O O O B-SUBCELLULAR B-EXP_ASSAY O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O B-CELL_TYPE O O O O O O

(I). Spindle volume at 1h and metaphase I after IPZ addition. The metaphase time was defined as the last time point before anaphase or, in the IPZ-treated oocytes that failed anaphase entry, as the average control metaphase time. Means ± SDs, Mann-Whitney test, oocyte numbers are indicated in brackets.


( J ) Time of anaphase I entry in oocytes that were treated with 15 µM IPZ after being cultured for 5h in control media . Oocyte numbers are indicated in brackets .

O O O O O O O O O B-CELL_TYPE O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-CELL_TYPE O O O O O O

(J) Time of anaphase I entry in oocytes that were treated with 15 µM IPZ after being cultured for 5h in control media. Oocyte numbers are indicated in brackets.


( A ) rescue of effects of 15 µM IPZ on MI spindles was examined in mouse oocytes expressing histone H2B - mCherry from microinjected mRNA , co - stained with SiR - tubulin noninjected or injected with mRNAs coding for RanQ69L or hTPX2 . Maximum z - stack projections from confocal live - movies 30 min after GVBD , at metaphase I anaphase I . Time in hh : mm is relative to GVBD . Scale bars , 10 µm .

O O O O O O O O O B-SMALL_MOLECULE O B-SUBCELLULAR I-SUBCELLULAR O O O B-ORGANISM B-CELL_TYPE O B-GENEPROD I-GENEPROD O B-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O

(A) The rescue of the effects of 15 µM IPZ on MI spindles was examined in mouse oocytes expressing histone H2B-mCherry from microinjected mRNA, co-stained with SiR-tubulin and noninjected or injected with mRNAs coding for RanQ69L or hTPX2. Maximum intensity z-stack projections from confocal live-cell movies 30 min after GVBD, at metaphase I and anaphase I. Time in hh: mm is relative to GVBD. Scale bars, 10 µm.


( B - C ) frequency of GVBD ( B ) anaphase I entry ( C ) quantified by live confocal imaging of oocytes stained treated Fisher ' s exact , oocyte numbers are shown in brackets . ( D ) Changes in spindle volume over time after GVBD . Means ± SDs , mixed - design ANOVA , oocyte numbers are shown in brackets .

O O O O O B-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-CELL_TYPE B-EXP_ASSAY O O O O O O B-CELL_TYPE O O O O O O O O O O O B-SUBCELLULAR B-EXP_ASSAY O O O B-EXP_ASSAY O O O O O O O O O O B-CELL_TYPE O O O O O O

(B-C) The frequency of GVBD (B) and anaphase I entry (C) quantified by live confocal imaging of oocytes stained and treated Fisher's exact test, oocyte numbers are shown in brackets. (D) Changes in spindle volume over time after GVBD. Means ± SDs, mixed-design ANOVA, oocyte numbers are shown in brackets.


( A ) effects of RanT24N , T42A , RanT24N on MI spindle assembly observed in maximum z - projection from confocal time - lapse imaging of oocytes expressing CDK5RAP2 - EGFP , histone H2B - mCherry stained with SiR - tubulin . Time in hh : mm is relative to GVBD . Scale bar , 10 µm . ( B , C ) Live - imaging of oocytes as in ( A ) was used to quantify effects of Ran mutant . Means ± SDs . , Mann - Whitney , oocyte numbers are indicated in brackets .

O O O O O B-GENEPROD O O O B-GENEPROD O B-SUBCELLULAR I-SUBCELLULAR B-EXP_ASSAY O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-CELL_TYPE O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD O B-GENEPROD B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-CELL_TYPE O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O B-CELL_TYPE O O O O O O

(A) The effects of RanT24N, T42A, and RanT24N on MI spindle assembly observed in maximum z-projection images from confocal time-lapse imaging of oocytes expressing CDK5RAP2-EGFP, histone H2B-mCherry and stained with SiR-tubulin. Time in hh:mm is relative to GVBD. Scale bar, 10 µm. (B, C) Live-cell imaging of oocytes as in (A) was used to quantify the effects of Ran mutant. Means ± SDs., Mann-Whitney test, oocyte numbers are indicated in brackets.


( D ) MT at MTOCs at time of GVBD quantified from SiR - tubulin signal that was normalized to mean SiR - tubulin signal . Mean of MT on MTOCs was arbitrarily centered to 1 in control group . Means ± SDs , Kruskal - Wallis , Dunn ' s . 25 control oocytes with 264 MTOCs in total , 10 RanT24N - expression oocytes with 70 MTOCs in total 19 RanT24N , T42A - expressing oocytes with 96 MTOCs in total were analyzed .

O O O B-SUBCELLULAR O B-SUBCELLULAR O O O B-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O O O O B-SUBCELLULAR O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O B-CELL_TYPE O O B-SUBCELLULAR O O O O B-GENEPROD O O B-CELL_TYPE O O B-SUBCELLULAR O O O B-GENEPROD O O O O B-CELL_TYPE O O B-SUBCELLULAR O O O O O

(D) MT intensity at MTOCs at the time of GVBD quantified from SiR-tubulin signal that was normalized to mean cytoplasmic SiR-tubulin signal. Mean of MT intensity on MTOCs was arbitrarily centered to 1 in the control group. Means ± SDs, Kruskal-Wallis, and Dunn's tests. 25 control oocytes with 264 MTOCs in total, 10 RanT24N-expression oocytes with 70 MTOCs in total and 19 RanT24N, T42A-expressing oocytes with 96 MTOCs in total were analyzed.


( E ) Time course of MT in of spindle after GVBD . Mean of MT was arbitrarily centered to 1 in control group . Means ± SDs , mixed - design ANOVA , oocyte numbers are shown in brackets .

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(E) Time course of the MT intensity in the area of spindle after GVBD. Mean of MT intensity was arbitrarily centered to 1 in the control group. Means ± SDs, mixed-design ANOVA, oocyte numbers are shown in brackets.


( F ) Frequency severity of MI spindle in oocytes expressing RanT24N or RanT24N , T42A . Oocyte numbers are shown in brackets .

O O O B-EXP_ASSAY O O B-SUBCELLULAR I-SUBCELLULAR O B-CELL_TYPE O B-GENEPROD O B-GENEPROD O O O B-CELL_TYPE O O O O O O

(F) Frequency and severity of MI spindle phenotypes in the oocytes expressing RanT24N or RanT24N, T42A. Oocyte numbers are shown in brackets.


( G , H ) of of oocytes ( G ) with chromosome segregation errors in anaphase I ( H ) with defective MTOC coalescence during metaphase I . Means ± SDs , oocyte numbers are shown in brackets .

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(G, H) Quantification of the percentage of oocytes (G) with chromosome segregation errors in anaphase I and (H) with defective MTOC coalescence during metaphase I. Means ± SDs, oocyte numbers are shown in brackets.


( I ) Detection of MTs pericentrin - containing MTOCs by IF in control RanT24N , T42A - expressing oocytes . Arrow indicates unaligned chromosome . ( J ) of chromosomal spread in oocytes depicted in ( I ) , Means ± SDs , Mann - Whitney , oocyte numbers indicated in brackets .

O O O O O B-SUBCELLULAR B-GENEPROD O O B-SUBCELLULAR O B-EXP_ASSAY O O B-GENEPROD O O O O B-CELL_TYPE O O O O B-SUBCELLULAR O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-CELL_TYPE O O O O O O O O O O O O O O B-CELL_TYPE O O O O O

(I) Detection of MTs and pericentrin-containing MTOCs by IF in control and RanT24N, T42A - expressing oocytes. Arrow indicates unaligned chromosome. (J) Quantification of chromosomal spread area in oocytes depicted in (I), Means ± SDs, Mann-Whitney test, oocyte numbers indicated in brackets.


( A ) . Maximum z - stack projections of confocal IF ( stain , indirect IF for tubulin TPX2 ) in untreated oocytes ( control ) , oocytes expressing RanT24N or injected with importin β ( 71 - 876 ) . representative of at least 5 experiments is shown .

O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD O O O B-CELL_TYPE O O O O B-CELL_TYPE O B-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O

(A). Maximum intensity z-stack projections of confocal IF images (DNA stain, indirect IF for tubulin and TPX2) in untreated oocytes (control), oocytes expressing RanT24N or injected with importin β(71-876). The representative of at least 5 experiments is shown.


( B - D ) Maximum z - stack projections of IF in control importin β ( 71 - 876 ) - injected were used to quantify spindle vs . whole - fraction ( B ) ratios of average signal in spindle vs . cytoplasm for tubulin ( C ) TPX2 ( D ) . Means ± SDs , with t - in ( B , C ) Mann - Whitney in ( D ) , oocyte numbers indicated in brackets .

O O O O O O O O O O O B-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O B-SUBCELLULAR O O B-SUBCELLULAR O B-GENEPROD O O O B-GENEPROD O 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-CELL_TYPE O O O O O

(B-D) Maximum intensity z-stack projections of IF images in control and importin β(71-876)-injected cells were used to quantify the spindle vs. whole-cell area fraction (B) and the ratios of the average signal in the spindle vs. cytoplasm for the tubulin (C) and TPX2 (D) staining. Means ± SDs, with t-test in (B, C) and Mann-Whitney test in (D), oocyte numbers indicated in brackets.


( E ) Ratios of spindle - associated fractions of TPX2 vs . tubulin were calculated in maximum z - stack projections of IF . Means ± SDs , Mann - Whitney , oocyte numbers indicated in brackets .

O O O O O B-SUBCELLULAR O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O B-EXP_ASSAY O O O O O O O O O B-CELL_TYPE O O O O O

(E) Ratios of the spindle-associated fractions of TPX2 vs. tubulin were calculated in maximum intensity z-stack projections of IF images. Means ± SDs, Mann-Whitney test, oocyte numbers indicated in brackets.


( F ) Maximum z - stack projections of IF were used to quantify number of ectopic MT asters . Means ± SDs , ANOVA with Kruskal - Wallis Dunn ' s , oocyte numbers indicated in brackets .

O O O O O O O O O B-EXP_ASSAY O O O O B-EXP_ASSAY O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O B-CELL_TYPE O O O O O

(F) Maximum intensity z-stack projections of IF images were used to quantify the number of ectopic MT asters. Means ± SDs, ANOVA with Kruskal-Wallis and Dunn's tests, oocyte numbers indicated in brackets.


( G ) Maximum z - stack projections of confocal IF stained for , pericentrin , tubulin ( directly labeled antibody ) . A representative example of at least 2 experiments is shown . Scale bar , 20 µm . ( H - I ) number of pericentrin foci were quantified in maximum - z - stack projections of IF stained as in ( G ) in at least 2 separate experiments . Means ± SDs , ANOVA with Kruskal - Wallis Dunn ' s .

O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O O O O 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-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O

(G) Maximum intensity z-stack projections of confocal IF images stained for DNA, pericentrin, and tubulin (directly labeled primary antibody). A representative example of at least 2 experiments is shown. Scale bar, 20 µm. (H-I) The number and size of pericentrin foci were quantified in maximum-intensity z-stack projections of IF images stained as in (G) in at least 2 separate experiments. Means ± SDs, ANOVA with Kruskal-Wallis and Dunn's tests.


( A ) 22 Tie1 - binding antibodies were screened for phospho - AKT in an in vitro Ang1 - stimulation experiment . Only one clone ( AB - Tie1 - 39 ) reduced AKT phosphorylation indicating suppressed Tie2 signaling ( n = 1 experiment ) .

O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O B-GENEPROD O B-EXP_ASSAY O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD B-EXP_ASSAY O O B-GENEPROD O O O O O O O O

(A) 22 Tie1-binding antibodies were screened for phospho-AKT levels in an in vitro Ang1-stimulation experiment. Only one clone (AB-Tie1-39) reduced AKT phosphorylation indicating suppressed Tie2 signaling (n = 1 experiment).


( C ) growth curves show delayed growth upon treatment with AB - Tie1 - 39 antibody as compared to IgG treatment ( mean ± , n = 9 mice ) . * , P < 0 . 05 ; * * * , P < 0 . 001 ( 2 - way ANNOVA ) .

O O O B-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O B-SUBCELLULAR O O O O O O O O B-ORGANISM O O 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) Tumor growth curves show delayed primary tumor growth upon treatment with AB-Tie1-39 antibody as compared to IgG treatment (mean ± SEM, n = 9 mice). *, P<0.05; ***, P<0.001 (2-way ANNOVA test).


( D ) Representative immunofluorescence of sections stained with CD31 ( EC - specific marker ) Desmin ( mural - specific marker ) . Scale bars = 200 μm .

O O O O B-EXP_ASSAY O O B-EXP_ASSAY O B-GENEPROD O B-CELL_TYPE O O O O B-GENEPROD O O O O O O O O O O O O O

(D) Representative immunofluorescence images of tumor sections stained with CD31 (EC-specific marker) and Desmin (mural cell-specific marker). Scale bars = 200 μm.


( E ) Dot plots show quantitation of intratumoral vessel density , mural coverage using Desmin aSMA co - , necrosis ( mean ± SD , n = 9 mice ) . comparisons were rendered non - significant according to two - Mann - Whitney U .

O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD I-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O

(E) Dot plots show quantitation of intratumoral vessel density, mural cell coverage using Desmin and aSMA co-staining, and tumor necrosis (mean ± SD, n = 9 mice). All comparisons were rendered non-significant according to two-tailed Mann-Whitney U test.


( A ) Following a neoadjuvant treatment regimen with IgG or AB - Tie1 - 39 , mice were monitored by whole - bioluminescence imaging . Shown are acquired two after resection .

O O O O O O O O O B-SUBCELLULAR O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-ORGANISM O O O B-TISSUE I-TISSUE B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O

(A) Following a neoadjuvant treatment regimen with IgG or AB-Tie1-39, mice were monitored by whole-body bioluminescence imaging. Shown are the images acquired two weeks after primary tumor resection.


( B ) Dot plots quantifying total photon flux during in vivo whole as well as ex vivo lung lymph node bioluminescence imaging ( mean ± SD , nIgG = 8 , nAB - Tie1 - 39 = 9 mice ) . * , P < 0 . 05 ; * * , P < 0 . 01 ( two - Mann - Whitney U ) .

O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-TISSUE O O O O O B-TISSUE I-TISSUE I-TISSUE B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O B-ORGANISM O O O 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) Dot plots quantifying total photon flux during in vivo whole body as well as ex vivo lung and lymph node bioluminescence imaging (mean ± SD, nIgG = 8, nAB-Tie1-39 = 9 mice). *, P<0.05; **, P<0.01 (two-tailed Mann-Whitney U test).


( D ) Kaplan - Meier graph comparing survival between mice treated with either IgG or AB - Tie1 - 39 ( nIgG = 10 , nAB - Tie1 - 39 = 9 mice ) . * * , P < 0 . 01 ( Log - rank ( Mantel - Cox ) ) .

O O O O O O O O B-EXP_ASSAY O B-ORGANISM O O O B-SUBCELLULAR O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O

(D) Kaplan-Meier graph comparing percent survival between mice treated with either IgG or AB-Tie1-39 (nIgG = 10, nAB-Tie1-39 = 9 mice). **, P<0.01 (Log-rank (Mantel-Cox) test).


( B ) Representative of whole lungs are shown on left . Scale bars = 5 mm . Quantitation of number of lung metastatic foci is plotted on right ( mean ± SD , nIgG = 9 , nAB - Tie1 - 39 = 8 mice ) . * , P < 0 . 05 ( two - Mann - Whitney U ) .

O O O O O O B-TISSUE O O O O O O O O O O O O O B-EXP_ASSAY O B-TISSUE I-TISSUE I-TISSUE O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O B-ORGANISM O O O O O O O O O O O O O O O O O O

(B) Representative images of the whole lungs are shown on the left. Scale bars = 5 mm. Quantitation of the number of lung metastatic foci is plotted on the right (mean ± SD, nIgG = 9, nAB-Tie1-39 = 8 mice). *, P<0.05 (two-tailed Mann-Whitney U test).


( C ) EC were seeded on gelatin - coated Transwell inserts . RFP - labeled LLC were allowed to transmigrate for 8 h through endothelial layer . Thereafter , transmigrated were counted . dot plot presents normalized data ( mean ± SD , n = 9 inserts from three independent experiments ) . * * * , P < 0 . 001 ( two - Mann - Whitney U ) .

O O O B-CELL_TYPE O O O B-SMALL_MOLECULE O O O O O B-GENEPROD O O B-CELL_LINE O O O B-EXP_ASSAY O O O O B-TISSUE I-TISSUE O O O B-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O O O O O O O O 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) EC were seeded on gelatin-coated Transwell inserts. RFP-labeled LLC cells were allowed to transmigrate for 8 h through the endothelial layer. Thereafter, transmigrated tumor cells were counted. The dot plot presents the normalized data (mean ± SD, n = 9 inserts from three independent experiments). ***, P<0.001 (two-tailed Mann-Whitney U test).


( D ) LLC - bearing mice were pretreated with IgG or AB - Tie1 - 39 . On day 14 , lung tissue lysates were immunoblotted with anti - phospho - TIE2 , total - TIE2 ACTB ( upper panel ; n = 6 mice ) . Densitometric quantitation of blots presented in upper panel is shown ( mean ± SD , n = 6 mice ) . * * , P < 0 . 01 ( two - Mann - Whitney U ) .

O O O B-CELL_LINE O O B-ORGANISM O O O B-SUBCELLULAR O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-TISSUE O O O B-EXP_ASSAY O O O O O B-GENEPROD O O O B-GENEPROD I-GENEPROD O O O O O O O B-ORGANISM O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O

(D) LLC tumor-bearing mice were pretreated with IgG or AB-Tie1-39. On day 14, lung tissue lysates were immunoblotted with anti-phospho-TIE2, total-TIE2 and ACTB (upper panel; n = 6 mice). Densitometric quantitation of the blots presented in the upper panel is shown (mean ± SD, n = 6 mice). **, P<0.01 (two-tailed Mann-Whitney U test).


( E ) LLC - bearing mice were pretreated with IgG or AB - Tie1 - 39 . On day 14 , lung EC were FACS - sorted used for global gene expression ( n = 4 mice ) . On left , a shows significantly - regulated genes ( red outline = AB - Tie1 - 39 - treated ; black outline = IgG - treated ) . On right , top significantly regulated bio - functions in AB - Tie1 - 39 - treated as compared to IgG - treated mice in Ingenuity Pathway .

O O O B-CELL_LINE O O B-ORGANISM O O O B-SUBCELLULAR O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-TISSUE B-CELL_TYPE O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-ORGANISM O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-SUBCELLULAR O O B-ORGANISM O B-EXP_ASSAY I-EXP_ASSAY O

(E) LLC tumor-bearing mice were pretreated with IgG or AB-Tie1-39. On day 14, lung EC were FACS-sorted and used for global gene expression (n = 4 mice). On the left, a heatmap shows significantly- regulated genes (red outline = AB-Tie1-39-treated; black outline = IgG-treated). On the right, top significantly regulated bio-functions in AB-Tie1-39-treated as compared to IgG-treated mice in Ingenuity Pathway Analysis.


A ) Murine small intestines immunostained for proliferation marker Ki67 , apoptosis marker caspase 3 ( Cas3 ) , or YAP from 4 control ( Yapfl / fl Tazfl / fl ) 4 YAP / TAZ double conditional knockout ( Villin - CreERt Yapfl / fl Tazfl / fl ) treated with tamoxifen . Tissues were harvested analyzed at day 7 after first tamoxifen injection . Loss of YAP / TAZ leads to a moderate increase in apoptosis ( Cas3 + ) in crypt base , quantified on right , but no overall effect on of small intestine . Arrows point to apoptotic . B ) Murine large intestines immunostained for proliferation marker Ki67 , apoptosis marker caspase 3 ( Cas3 ) , or YAP from control ( Yapfl / fl Tazfl / fl ) YAP / TAZ double conditional knockout ( Villin - CreERt Yapfl / fl Tazfl / fl ) treated with tamoxifen . Tissues were harvested analyzed at day 7 after first tamoxifen injection . Loss of YAP / TAZ leads to an increased number of apoptotic ( Cas3 + ) within transverse folds of ascending colon .

O O B-ORGANISM B-TISSUE I-TISSUE B-EXP_ASSAY O B-EXP_ASSAY O B-GENEPROD O B-EXP_ASSAY O B-GENEPROD I-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O O B-GENEPROD O O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD I-GENEPROD O O B-GENEPROD O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE O O O O B-GENEPROD O B-GENEPROD O O O O O O B-EXP_ASSAY O B-GENEPROD O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O B-TISSUE I-TISSUE O O O O B-EXP_ASSAY O O O B-ORGANISM B-TISSUE I-TISSUE B-EXP_ASSAY O B-EXP_ASSAY O B-GENEPROD O B-EXP_ASSAY O B-GENEPROD I-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD I-GENEPROD O O B-GENEPROD O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE O O O O B-GENEPROD O B-GENEPROD O O O O O O B-EXP_ASSAY O B-GENEPROD O O O O O O O B-TISSUE O

A) Murine small intestines immunostained for the proliferation marker Ki67, apoptosis marker cleaved caspase 3 (Cas3), or YAP from 4 control (Yapfl/fl Tazfl/fl) and 4 YAP/TAZ double conditional knockout (Villin-CreERt Yapfl/fl Tazfl/fl) animals treated with tamoxifen. Tissues were harvested and analyzed at day 7 after first tamoxifen injection. Loss of YAP/TAZ leads to a moderate increase in apoptosis (Cas3+) in crypt base stem cells, quantified on the right, but no overall effect on the morphology of the small intestine. Arrows point to apoptotic cells. B) Murine large intestines immunostained for proliferation marker Ki67, apoptosis marker cleaved caspase 3 (Cas3), or YAP from control (Yapfl/fl Tazfl/fl) and YAP/TAZ double conditional knockout (Villin-CreERt Yapfl/fl Tazfl/fl) animals treated with tamoxifen. Tissues were harvested and analyzed at day 7 after first tamoxifen injection. Loss of YAP/TAZ leads to an increased number of apoptotic (Cas3+) cells within the transverse folds of the ascending colon.


C ) Murine small intestines immunostained for YAP or caspase 3 ( Cas3 ) from control ( Villin - CreERt ) YAP / TAZ double conditional knockout ( Villin - CreERt Yapfl / fl Tazfl / fl ) treated with tamoxifen . Tissues were harvested analyzed at day 7 after first tamoxifen injection . Loss of YAP / TAZ leads to a mild increase in apoptosis ( Cas3 + ) in crypt base , but no overall effect on of small intestine . Arrows point to ( YAP ) , while arrows point to apoptotic ( Cas3 ) . N > 10 per genotype . D ) Murine large intestines immunostained for YAP or caspase 3 ( Cas3 ) from control ( Villin - CreERt ) YAP / TAZ double conditional knockout ( Villin - CreERt Yapfl / fl Tazfl / fl ) treated with tamoxifen . Tissues were harvested analyzed at day 7 after first tamoxifen injection . Loss of YAP / TAZ leads to increased apopotosis ( Cas3 + ) severe morphological defects in transverse folds of ascending colon , but does not affect of remaining colon regions . Quantitatively , 75 % of n = 4 showed this severe phenotype .

O O B-ORGANISM B-TISSUE I-TISSUE B-EXP_ASSAY O B-GENEPROD O B-GENEPROD I-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD I-GENEPROD O O B-GENEPROD O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE O O O O B-GENEPROD O B-GENEPROD O O O O O O B-EXP_ASSAY O B-GENEPROD O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O B-TISSUE I-TISSUE O O O O O B-GENEPROD O O O O O O B-EXP_ASSAY O B-GENEPROD O O O O O O O O O O B-ORGANISM B-TISSUE I-TISSUE B-EXP_ASSAY O B-GENEPROD O B-GENEPROD I-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD I-GENEPROD O O B-GENEPROD O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE O O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O O O B-EXP_ASSAY O O O O O O B-TISSUE O O O O O O O B-TISSUE O O O O O O O O O O O O O B-EXP_ASSAY O

C) Murine small intestines immunostained for YAP or cleaved caspase 3 (Cas3) from control (Villin-CreERt) and YAP/TAZ double conditional knockout (Villin-CreERt Yapfl/fl Tazfl/fl) animals treated with tamoxifen. Tissues were harvested and analyzed at day 7 after first tamoxifen injection. Loss of YAP/TAZ leads to a mild increase in apoptosis (Cas3+) in crypt base stem cells, but no overall effect on the morphology of the small intestine. Arrows point to stem cells (YAP nuclear staining), while arrows point to apoptotic cells (Cas3 staining). N > 10 animals per genotype. D) Murine large intestines immunostained for YAP or cleaved caspase 3 (Cas3 ) from control (Villin-CreERt) and YAP/TAZ double conditional knockout (Villin-CreERt Yapfl/fl Tazfl/fl) animals treated with tamoxifen. Tissues were harvested and analyzed at day 7 after first tamoxifen injection. Loss of YAP/TAZ leads to increased apopotosis (Cas3+) and severe morphological defects in the transverse folds of the ascending colon, but does not affect morphology of the remaining colon regions. Quantitatively, 75% of n = 4 animals showed this severe phenotype.


A ) Murine small ( top ) large ( bottom ) intestines isolated from control ( Cre negative ) Lats1flox / flox Lats2flox / flox Villin - CreERt Lats1flox / flox Lats2flox / flox treated with tamoxifen to induce homozygous deletion of Lats1 / 2 ( dKO ) . Immunostaining for YAP Ki67 shows a gradient of YAP expression along crypt - villus axis in controls , with YAP Ki67 positive restricted to crypt base ( representative from n = 5 mice ) . Tamoxifen treated ( 3 days i . p . ) Villin - CreERt Lats1flox / flox Lats2flox / flox double homozygous mouse intestines show an enlarged crypt compartment after 7 days with strongly YAP immunostaining in epithelial an expanded proliferative zone marked by Ki67 positive . Note gradient of YAP expression is maintained along crypt - villus axis ( representative from n = 5 mice for each genotype ) . ( Right ) Immunostaining for Paneth marker Lyz reveals loss of this marker from crypt base . ( Bottom right ) Q - PCR of Wnt pathway target genes reveals that Lats1 / 2 dKO causes a mild reduction in Lgr5 expression , with complete loss of Olfm4 .

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A) Murine small (top) and large (bottom) intestines isolated from control (Cre negative) Lats1flox/flox Lats2flox/flox animals and Villin-CreERt Lats1flox/flox Lats2flox/flox animals treated with tamoxifen to induce homozygous deletion of Lats1/2 (dKO). Immunostaining for YAP and Ki67 shows a gradient of YAP expression along the crypt-villus axis in controls, with nuclear YAP and Ki67 positive cells restricted to the crypt base (representative images from n=5 mice). Tamoxifen treated (3 days i.p.) Villin-CreERt Lats1flox/flox Lats2flox/flox double homozygous mouse intestines show an enlarged crypt compartment after 7 days with strongly nuclear YAP immunostaining in all epithelial cells and an expanded proliferative zone marked by Ki67 positive cells. Note the gradient of YAP expression levels is maintained along the crypt-villus axis (representative images from n=5 mice for each genotype). (Right) Immunostaining for the Paneth cell marker Lyz reveals loss of this marker from the crypt base. (Bottom right) Q-PCR analysis of Wnt pathway target genes reveals that Lats1/2 dKO causes a mild reduction in Lgr5 expression, with complete loss of Olfm4.


B ) Axin2 mRNA expression was measured by RNAscope ( red ) found to be increased in both uniformity of along crypt - villus axis in Lats1 / 2 dKO after 3 days post i . p . injection ( dpi ) with tamoxifen to induce homozygous deletion of Lats1 Lats2 . At 7dpi , Axin2 mRNA remain uniform along crypt - villus axis in Lats1 / 2 dKO , although their total level has declined compared to 3dpi .

O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY O O O O O O O O O O O O B-CELL_TYPE O B-TISSUE O O B-GENEPROD O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O B-GENEPROD I-GENEPROD O O O O B-GENEPROD B-EXP_ASSAY O O O B-CELL_TYPE O B-TISSUE O O B-GENEPROD O O O O O O O O O O O O O O

B) Axin2 mRNA expression was measured by RNAscope (red) and found to be increased in both intensity and uniformity of staining along the crypt-villus axis in Lats1/2 dKO after 3 days post i.p. injection (dpi) with tamoxifen to induce the homozygous deletion of Lats1 and Lats2. At 7dpi, Axin2 mRNA levels remain uniform along the crypt-villus axis in Lats1/2 dKO animals, although their total level has declined compared to 3dpi.


D ) Q - PCR of YAP - TEAD their target genes Ctgf Cyr61 reveals that Lats1 / 2 dKO causes a strong induction of Ctgf Cyr61 expression , as expected , as well as a strong induction of common Wnt YAP target gene CyclinD1 .

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O B-GENEPROD I-GENEPROD O O B-GENEPROD O O O O O O O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O B-GENEPROD I-GENEPROD O O B-GENEPROD O

D) Q-PCR analysis of YAP-TEAD and their target genes Ctgf and Cyr61 reveals that Lats1/2 dKO causes a strong induction of Ctgf and Cyr61 expression, as expected, as well as a strong induction of the common Wnt and YAP target gene CyclinD1.


B ) RNAscope in situ hybridisation of TEAD1 / 2 / 4 mRNA expression from control ( Cre negative ) Lats1flox / flox Lats2flox / flox Villin - CreERt Lats1flox / flox Lats2flox / flox treated with tamoxifen to induce homozygous deletion of Lats1 / 2 ( dKO ) . Tamoxifen treated ( 3 days i . p . ) Villin - CreERt Lats1flox / flox Lats2flox / flox double homozygous mouse intestines show elevated expression of TEAD1 TEAD4 in both small intestine ( top ) colon ( bottom ) . Tissues were harvested analyzed at day 7 after first tamoxifen injection . ( n = 5 for each genotype )

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O B-GENEPROD I-GENEPROD O O B-GENEPROD O O O O B-SMALL_MOLECULE O O O O O B-GENEPROD O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD O O B-GENEPROD O O O O B-ORGANISM B-TISSUE O O O O B-GENEPROD I-GENEPROD O O B-TISSUE I-TISSUE O O O B-TISSUE O O O O B-TISSUE O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O

B) RNAscope in situ hybridisation analysis of TEAD1/2/4 mRNA expression from control (Cre negative) Lats1flox/flox Lats2flox/flox animals and Villin-CreERt Lats1flox/flox Lats2flox/flox animals treated with tamoxifen to induce homozygous deletion of Lats1/2 (dKO). Tamoxifen treated (3 days i.p.) Villin-CreERt Lats1flox/flox Lats2flox/flox double homozygous mouse intestines show elevated expression of TEAD1 and TEAD4 in both the small intestine (top) and colon (bottom). Tissues were harvested and analyzed at day 7 after first tamoxifen injection. (n=5 animals for each genotype)


C ) Q - PCR reveals a progressive increase in YAP - TEAD target gene Ctgf in Lats1 / 2 dKO intestines at 3 - days 7 - days post i . p . injection with tamoxifen , as well as similarly increased TEAD1 TEAD4 expression , confirming that TEAD1 TEAD4 are YAP - responsive genes . Notably , Axin2 TEAD2 exhibit a mild but progressive decline over 3 to 7 days after Lats1 / 2 deletion , consistent with Axin2 being a known Wnt - specific target gene , with Wnt signalling remaining active in Lats1 / 2 dKO intestines .

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O B-TISSUE O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O B-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O B-GENEPROD O O O O B-GENEPROD O O O B-TISSUE O

C) Q-PCR analysis reveals a progressive increase in the YAP-TEAD target gene Ctgf in Lats1/2 dKO intestines at 3-days and 7-days post i.p. injection with tamoxifen, as well as similarly increased TEAD1 and TEAD4 expression, confirming that TEAD1 and TEAD4 are YAP-responsive genes. Notably, Axin2 and TEAD2 exhibit a mild but progressive decline over 3 to 7 days after Lats1/2 deletion, consistent with Axin2 being a known Wnt-specific target gene, and with Wnt signalling remaining active in Lats1/2 dKO intestines.


A ) RNAscope in situ hybridisation of TEAD1 / 2 / 4 mRNA expression from control ( wild - type ) ApcMin mutant tumours from small intestine ( top ) colon ( bottom ) . three genes were strongly induced within ApcMin mutant tumours , with TEAD2 being most strongly induced in colon ApcMin tumours ( n = 3 with multiple tumours within small large intestines ) .

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-GENEPROD O O O B-TISSUE I-TISSUE O O O B-TISSUE O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O B-TISSUE B-GENEPROD O O O O O O O O O O B-TISSUE I-TISSUE O O

A) RNAscope in situ hybridisation analysis of TEAD1/2/4 mRNA expression from control (wild-type) and ApcMin mutant tumours from the small intestine (top) and colon (bottom). All three genes were strongly induced within the ApcMin mutant tumours, with TEAD2 being most strongly induced in colon ApcMin tumours (n=3 animals with multiple tumours within the small and large intestines).


C ) Antibody for TEAD4 reveals a gradient of expression along crypt - villus axis in both small large intestine , with uniformly high expression in colorectal adenomas , similar to SOX9 .

O O O O B-GENEPROD O O O O O O B-CELL_TYPE O B-TISSUE O O O O B-TISSUE I-TISSUE O O O O O O B-DISEASE I-DISEASE O O O B-GENEPROD O

C) Antibody staining for TEAD4 reveals a gradient of expression along the crypt-villus axis in both the small and large intestine, with uniformly high expression in colorectal adenomas, similar to SOX9.


B ) YAP immunostaining of 3 - day old wild type organoids treated with DMSO ( Control ) or Porcupine inhibitor LGK974 at 5µM . Wnt signalling inhibition upon Porcupine inhibitor treatment decreases YAP without affecting YAP subcellular localisation . of YAP is shown below , with statistical significance in an unpaired 2 - t - .

O O B-GENEPROD B-EXP_ASSAY O O O O O O O B-TISSUE O O B-SMALL_MOLECULE O O O O B-GENEPROD O B-SMALL_MOLECULE O O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O O O O O O O O O O O O O

B) YAP immunostaining of 3-day old wild type organoids treated with DMSO (Control) or Porcupine inhibitor LGK974 at 5µM. Wnt signalling inhibition upon Porcupine inhibitor treatment decreases YAP levels without affecting YAP subcellular localisation. Quantification of YAP protein levels is shown below, with statistical significance in an unpaired 2-tailed t-test.


C ) Wnt activation in Apc5 homozygous mutant organoids drives a drastic shift in organoid from budding structures to cystic spheres increases YAP level . Scale bar is 250µm . n > 10 organoids in each experiment . of YAP from crypt villus regions of control organoids as well as from regions bounded by rectangles in Apc5 organoids are shown on right , with statistical significance in an unpaired 2 - t - .

O O B-GENEPROD O O B-GENEPROD O O B-TISSUE O O O O O B-TISSUE O O O O O O O B-GENEPROD B-EXP_ASSAY O O O O O O O O O B-TISSUE O O O O O B-GENEPROD O B-CELL_TYPE B-TISSUE O O O B-TISSUE O O O O O O O O O B-GENEPROD B-TISSUE O O O O O O O O O O O O O O O O

C) Wnt activation in Apc5 homozygous mutant organoids drives a drastic shift in organoid morphology from budding structures to cystic spheres and increases the YAP protein level. Scale bar is 250µm. n > 10 organoids in each experiment. Quantification of YAP protein levels from crypt and villus regions of control organoids as well as from the regions bounded by rectangles in Apc5 organoids are shown on the right, with statistical significance in an unpaired 2-tailed t-test.


A ) Src family kinase inhibition via Dasatinib treatment in control organoids enforces YAP localisation after 4h of treatment in organoids cultured for 3d . n > 10 organoids in each experiment . B ) of of YAP per control crypt bud as shown in panel ( A ) , error bars show 1 SD ( * * * * p < 0 . 0001 ) . n > 10 organoids per condition ( top ) . of / of YAP per in Apc5 mutant organoids as shown in panel ( C ) . n > 10 organoids per condition ( bottom ) . Statistical was performed with an unpaired student ' s t - . C

O O B-GENEPROD O O O O B-SMALL_MOLECULE O O O B-TISSUE O B-GENEPROD B-EXP_ASSAY O O O O O B-TISSUE O O O O O O O B-TISSUE O O O O O O O O B-GENEPROD O O B-CELL_TYPE O O O O O 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-TISSUE O O O O O O O O O B-GENEPROD O O B-GENEPROD O B-TISSUE O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O

A) Src family kinase inhibition via Dasatinib treatment in control organoids enforces YAP cytoplasmic localisation after 4h of treatment in organoids cultured for 3d. n > 10 organoids in each experiment. B) Quantification of the percentage of nuclear YAP cells per control crypt bud as shown in panel (A), error bars show 1 SD (**** p <0.0001). n > 10 organoids per condition (top). Quantification of the nuclear/cytoplasmic ratio of YAP per cell in Apc5 mutant organoids as shown in panel (C). n > 10 organoids per condition (bottom). Statistical analysis was performed with an unpaired student's t-test. C


C ) Src family kinase inhibition via Dasatinib or eCF506 treatment in Apc5 organoids causes relocalisation of YAP to cytoplasm . n > 10 organoids in each experiment . Q - PCR of YAP - TEAD target gene Ctgf reveals a strong inhibition of mRNA expression upon treatment with eCF506 ( relative expression , error bars = 1 SD ) . Statistical was performed with an unpaired student ' s t - .

O O B-GENEPROD O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-GENEPROD B-TISSUE O O O B-GENEPROD O B-SUBCELLULAR O O O O B-TISSUE O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O

C) Src family kinase inhibition via Dasatinib or eCF506 treatment in Apc5 organoids causes relocalisation of YAP to the cytoplasm. n > 10 organoids in each experiment. Q-PCR analysis of the YAP-TEAD target gene Ctgf reveals a strong inhibition of mRNA expression upon treatment with eCF506 (relative expression, error bars = 1 SD). Statistical analysis was performed with an unpaired student's t-test.


A ) Mouse intestinal organoids immunostained for YAP ( green ) . Note mostly localisation of YAP at 48h in organoid . Irradiation with 4Gy of X - rays drives strong localisation of YAP in many , which is reversible by treatment with Src family kinase inhibitor Dasatinib . Dasatinib treated organoids exhibit increased after irradiation , as indicated by pyknotic nuclei ( arrow ) . n > 10 organoids in each experiment . This experiment employs same control batch of 2 - 3d organoids also shown in Figure 6A .

O O B-ORGANISM B-TISSUE I-TISSUE B-EXP_ASSAY O B-GENEPROD O O O O O O B-EXP_ASSAY O B-GENEPROD O O O B-TISSUE O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O O O O O O O O O B-GENEPROD O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-TISSUE O O O O O O O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O B-TISSUE O O O O O O

A) Mouse intestinal organoids immunostained for YAP (green). Note the mostly cytoplasmic localisation of YAP at 48h in the organoid. Irradiation with 4Gy of X-rays drives strong nuclear localisation of YAP in many cells, which is reversible by treatment with the Src family kinase inhibitor Dasatinib. The Dasatinib treated organoids exhibit increased cell death after irradiation, as indicated by pyknotic nuclei (white arrow). n > 10 organoids in each experiment. This experiment employs the same control batch of 2-3d organoids also shown in Figure 6A.


B ) Control VillinCreERt mouse small intestine immunostained for YAP shows a gradient of expression with mostly localisation . Irradiation with 14Gy of X - rays drives strong localisation of YAP along entire crypt - villus axis . Deletion of Src within VillinCreERt Srcflox / flox intestines prevents YAP localisation normal regeneration after 14Gy irradiation . Arrows point to villar with YAP localisation . Lower panels show high - mag views of a different example than those shown in upper panels .

O O O B-GENEPROD B-ORGANISM B-TISSUE I-TISSUE B-EXP_ASSAY O B-GENEPROD O O O O O O O B-EXP_ASSAY O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O O B-CELL_TYPE O B-TISSUE O O O O B-GENEPROD O B-GENEPROD I-GENEPROD O O B-TISSUE O B-GENEPROD B-EXP_ASSAY O O O O O O O O O O O B-GENEPROD B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O

B) Control VillinCreERt mouse small intestine immunostained for YAP shows a gradient of expression with mostly cytoplasmic localisation. Irradiation with 14Gy of X-rays drives strong nuclear localisation of YAP along the entire crypt-villus axis. Deletion of Src within VillinCreERt Srcflox/flox intestines prevents YAP nuclear localisation and normal regeneration after 14Gy irradiation. Arrows point to villar cells with nuclear YAP localisation. Lower panels show high-mag views of a different example than those shown in upper panels.


A ) Control irradiated murine small intestines analysed with RNAscope in situ hybridisation for Myc mRNA ( black arrows point to mRNA granules in control ) by immunostaining for Sox9 reveals an expansion in expression domain of both genes . localisation of beta - catenin is readily detectable after irradiation . n = 3 samples per condition , shown in insets , error bars = 1 SD . Statistical was performed with an unpaired student ' s t - . Similar results were obtained at 3dpi 5dpi .

O O O O B-ORGANISM B-TISSUE I-TISSUE O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O 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) Control and irradiated murine small intestines analysed with RNAscope in situ hybridisation for Myc mRNA (black arrows point to mRNA granules in the control) and by immunostaining for Sox9 protein reveals an expansion in the expression domain of both genes. Nuclear localisation of beta-catenin is readily detectable after irradiation. n = 3 samples per condition, quantification shown in the insets, error bars = 1 SD. Statistical analysis was performed with an unpaired student's t-test. Similar results were obtained at 3dpi and 5dpi.


B ) Q - PCR of YAP , TEADs their target genes Ctgf Cyr61 over a 4 days post irradiation time course at 12Gy . Note strong upregulation of TEAD4 , Ctgf , Cyr61 . In contrast , Axin2 , TEAD2 Sox9 mRNA expression are only moderately altered , confirming that Wnt signalling remains active upon irradiation - induced YAP - TEAD activation . n = 4 biological replicates per time point . Statistical was performed with unpaired t - compared to control 0Gy condition , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD I-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Q-PCR analysis of YAP, TEADs and their target genes Ctgf and Cyr61 over a 4 days post irradiation time course at 12Gy. Note the strong upregulation of TEAD4, Ctgf, and Cyr61. In contrast, Axin2, TEAD2 and Sox9 mRNA expression levels are only moderately altered, confirming that Wnt signalling remains active upon irradiation-induced YAP-TEAD activation. n = 4 biological replicates per time point. Statistical analysis was performed with unpaired t-test compared to the control 0Gy condition, * p<0.05, ** p<0.01, ***p<0.001.


C ) Control irradiated murine small intestines analysed by immunostaining for Sox9 reveals that treatment with Porcupine inhibitor LGK974 strongly reduces Sox9 expression domain induced by 3 days after 12Gy irradiation . Phosphorylated Src immunostaining reveals an upregulation by 3 days after irradiation which is also lost upon treatment with Porcupine inhibitor LGK974 ( n = 4 in each condition ) .

O O O O B-ORGANISM B-TISSUE I-TISSUE O O B-EXP_ASSAY O B-GENEPROD O O O O B-GENEPROD O B-SMALL_MOLECULE O O B-GENEPROD O O O O O O O O O O B-EXP_ASSAY B-GENEPROD B-EXP_ASSAY O O O O O O O O O O O O O O O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O

C) Control and irradiated murine small intestines analysed by immunostaining for Sox9 protein reveals that treatment with Porcupine inhibitor LGK974 strongly reduces the Sox9 expression domain induced by 3 days after 12Gy irradiation. Phosphorylated Src immunostaining reveals an upregulation by 3 days after irradiation which is also lost upon treatment with Porcupine inhibitor LGK974 (n= 4 animals in each condition).


D ) Q - PCR of Wnt YAP target gene expression in intestinal crypts isolated at 1 day after 12Gy irradiation . Note inhibition of Axin2 , Sox9 , YAP , Ctgf , Cyr61 , TEAD family expression upon treatment with Porcupine inhibitor LGK974 . mild reduction of Sox9 mRNA expression by LGK974 at 1 day after irradiation ( 1dpi ) becomes more pronounced at 3 days after irradiation ( 3dpi ) . n = 4 biological replicates per condition , statistical was performed with Student ' s 2 - t - , error bars = 1 SD , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD O B-EXP_ASSAY I-EXP_ASSAY O B-TISSUE B-CELL_TYPE O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O B-SMALL_MOLECULE O O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Q-PCR analysis of Wnt and YAP target gene expression in intestinal crypts isolated at 1 day after 12Gy irradiation. Note the inhibition of Axin2, Sox9, YAP, Ctgf, Cyr61, and TEAD family expression upon treatment with Porcupine inhibitor LGK974. The mild reduction of Sox9 mRNA expression by LGK974 at 1 day after irradiation (1dpi) becomes more pronounced at 3 days after irradiation (3dpi). n = 4 biological replicates per condition, statistical analysis was performed with Student's 2-tailed t-test, error bars = 1 SD, * p<0.05, ** p<0.01, ***p<0.001.


A . Volcano plots of genes that are differentially expressed in infected relative to mock - infected at 12 hpi 24 hpi in ileum organoids . statistical significance ( - log10 of adjusted p - value ) is shown as a function of log2 fold change . MAST were used to generate P ‐ values , bonferroni multiple hypotheses correction was used to compute FDR values . Labeled dots in panels are gene names of selected differentially expressed genes between compared two populations .

O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O O B-EXP_ASSAY O O O O O O B-TISSUE I-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY I-EXP_ASSAY O O O O O O

A. Volcano plots of genes that are differentially expressed in infected cells relative to mock-infected cells at 12 hpi and 24 hpi in ileum organoids. The statistical significance (-log10 of the adjusted p-value) is shown as a function of the log2 fold change. MAST tests were used to generate P‐values, bonferroni multiple hypotheses correction was used to compute FDR values. Labeled dots in all panels are gene names of selected differentially expressed genes between the compared two populations.


F . Multiplex in situ hybridization of ACE2 SARS - CoV - 2 of mock - infected infected 2D ileum organoids at 12 hpi 24 hpi . arrows point at SARS - CoV - 2 - infected . A representative image is shown .

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O B-EXP_ASSAY I-EXP_ASSAY O B-TISSUE I-TISSUE O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O B-EXP_ASSAY O O O B-EXP_ASSAY O O O

F. Multiplex in situ RNA hybridization of ACE2 and SARS-CoV-2 of mock-infected and infected 2D ileum organoids at 12 hpi and 24 hpi. White arrows point at SARS-CoV-2-infected cells. A representative image is shown.


Volcano plots displaying genes that are differentially expressed in immature enterocytes 2 upon SARS - CoV - 2 infection of colon organoids . infected vs . mock infected statistical significance ( - log10 adjusted p - value ) is shown as a function of log2 fold change . MAST were used to generate P ‐ values , bonferroni multiple hypotheses correction was used to compute FDR values . Labeled dots in blue in panels are gene names of selected differentially expressed genes between compared two populations . Labeled dots in red in panels are gene corresponding to interferon if detected .

O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-CELL_TYPE I-CELL_TYPE O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM B-EXP_ASSAY O B-TISSUE I-TISSUE O B-EXP_ASSAY O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O

Volcano plots displaying the genes that are differentially expressed in immature enterocytes 2 upon SARS-CoV-2 infection of colon organoids. infected vs. mock infected cells The statistical significance (-log10 adjusted p-value) is shown as a function of the log2 fold change. MAST tests were used to generate P‐values, bonferroni multiple hypotheses correction was used to compute FDR values. Labeled dots in blue in all panels are gene names of selected differentially expressed genes between the compared two populations. Labeled dots in red in all panels are gene corresponding to interferon if detected.


A . Human ileum - derived organoids were seeded in 2D on iBIDi chamber slides . 12 24 hpi were fixed amount of SARS - CoV - 2 infected ( red ) induction of ISG15 ( ) was analyzed by single molecule FISH . Nuclei were visualized with DAPI ( blue ) . arrows indicate SARS - CoV - 2 positive . N = 3 biological samples . Representative image is shown . Scale bar = 100 um .

O O B-ORGANISM B-TISSUE O O B-TISSUE O O O O O O O O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM B-EXP_ASSAY O O O O O B-GENEPROD O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-SUBCELLULAR O O O B-SMALL_MOLECULE O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O B-EXP_ASSAY O O O O O O O O O

A. Human ileum-derived organoids were seeded in 2D on iBIDi chamber slides. 12 and 24 hpi cells were fixed and the amount of SARS-CoV-2 infected cells (red) and the induction of ISG15 (white) was analyzed by single molecule RNA FISH. Nuclei were visualized with DAPI (blue). White arrows indicate SARS-CoV-2 positive cells. N=3 biological samples. Representative image is shown. Scale bar=100 um.


D . T84 IRF3 knock - out were infected with SARS - CoV - 2 or human astrovirus 1 at an MOI = 3 . 24 hpi , were incubated in presence or absence of 2000 IU / mL of IFNβ1 . 12 h post - treatment was harvested induction of ISG15 was analyzed by q - RT - PCR normalized to housekeeping gene TBP . N = 3 biological replicates . Error bar indicates standard deviation . Statistics were determined by unpaired t - .

O O B-CELL_LINE B-GENEPROD O O O O B-EXP_ASSAY O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O

D. T84 IRF3 knock-out cells were infected with SARS-CoV-2 or human astrovirus 1 at an MOI=3. 24 hpi, cells were incubated in the presence or absence of 2000 IU/mL of IFNβ1. 12 h post-treatment RNA was harvested and the induction of ISG15 was analyzed by q-RT-PCR and normalized to the housekeeping gene TBP. N=3 biological replicates. Error bar indicates standard deviation. Statistics were determined by unpaired t-test.


E . T84 IRF3 knock - out were infected with SARS - CoV - 2 or human astrovirus 1 at an MOI = 3 . 24 hpi , were fixed stained for either SARS - CoV - 2 N or for human astrovirus 1 ( HAstV1 ) capsid . N = 3 biological replicates . Representative image is shown . Scale bar = 100 um .

O O B-CELL_LINE B-GENEPROD O O O O B-EXP_ASSAY O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O B-EXP_ASSAY O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM B-GENEPROD O O B-ORGANISM I-ORGANISM I-ORGANISM O B-ORGANISM O B-GENEPROD O O O O O O O O B-EXP_ASSAY O O O O O O O O O

E. T84 IRF3 knock-out cells were infected with SARS-CoV-2 or human astrovirus 1 at an MOI=3. 24 hpi, cells were fixed and stained for either the SARS-CoV-2 N protein or for human astrovirus 1 (HAstV1) capsid protein. N=3 biological replicates. Representative image is shown. Scale bar=100 um.


G . T84 MX - 1 mcherry were infected with SARS - CoV - 2 at an MOI = 3 or MOI = 1 . 24 hpi were incubated in presence or absence of 2000 IU / mL of IFNβ1 . 12 h post - treatment were fixed stained for SARS - CoV - 2 N . number of MX - 1 positive , SARS - CoV - 2 positive double positive was quantified . N = 3 biological replicates were performed . Error bar indicates standard deviation .

O O B-CELL_LINE B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-EXP_ASSAY O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM 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-GENEPROD O O O O O O O O B-EXP_ASSAY O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM B-GENEPROD O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O

G. T84 MX-1 mcherry were infected with SARS-CoV-2 at an MOI=3 or MOI=1. 24 hpi cells were incubated in the presence or absence of 2000 IU/mL of IFNβ1. 12 h post-treatment cells were fixed and stained for SARS-CoV-2 N protein. The number of MX-1 positive, SARS-CoV-2 positive and double positive cells was quantified. N=3 biological replicates were performed. Error bar indicates standard deviation.


( D ) Immunofluorescence of neurons expressing BioID2 - tau or BioID2 ( myc ) or non - transduced control . were incubated in biotin medium ( 50 μM ) or vehicle medium for 30 h prior to fixation probed for biotin ( streptavidin , SA ) nuclei ( DAPI ) ( n = 3 biological replicates ) . BioID2 - tau incorporated biotin co - localise in axons somata . Scale bar , 25 μm .

O O O B-EXP_ASSAY O B-CELL_TYPE O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SUBCELLULAR O B-SMALL_MOLECULE O O O O O O O O O O O B-GENEPROD O B-SMALL_MOLECULE B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O

(D) Immunofluorescence of neurons expressing BioID2-tau or BioID2 (myc) or non-transduced control cells. Cells were incubated in biotin medium (50 μM) or vehicle medium for 30 h prior to fixation and probed for biotin (streptavidin, SA) and cell nuclei (DAPI) (n = 3 biological replicates). BioID2-tau and incorporated biotin co-localise in axons and somata. Scale bar, 25 μm.


( B ) Immunoblot of combined brain lysates from mice ( P35 ) expressing BioID2 - tau or BioID2 control ( n = 4 biological replicates ) probed for myc - tagged BioID2 BioID2 - tau . Controls : lysates from ( 1 ) BioID2 - or ( 2 ) BioID2 - tau - expressing ( 3 ) non - transduced mouse brain . Loading , glyceraldehyde - 3 - phosphate dehydrogenase ( Gapdh ) . * , unspecific band . Relative confirmed similar expression of BioID2 BioID2 - tau . Means ± S . E . M . ns , not significant ( Student ' s t - )

O O O B-EXP_ASSAY O O B-TISSUE O O B-ORGANISM O O O O O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O B-ORGANISM B-TISSUE O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O

(B) Immunoblot of combined brain lysates from mice (P35) expressing BioID2-tau or BioID2 control (n = 4 biological replicates) probed for myc-tagged BioID2 and BioID2-tau. Controls: lysates from (1) BioID2- or (2) BioID2-tau-expressing and (3) non-transduced mouse brain. Loading, glyceraldehyde-3-phosphate dehydrogenase (Gapdh). *, unspecific band. Relative quantification confirmed similar expression levels of BioID2 and BioID2-tau. Means ± S.E.M. ns, not significant (Student's t-test)


( C ) Immunofluorescence of P35 mouse brain expressing BioID2 - tau . Hippocampal Cornu ammonis 1 ( CA1 ) region was probed for BioID2 - tau ( myc ) , for incorporated biotin ( streptavidin , SA ) for nuclei ( DAPI ) ( n = 3 - 5 biological replicates ) . Scale bar , 50 μm .

O O O B-EXP_ASSAY O O B-ORGANISM B-TISSUE O O O B-GENEPROD O B-TISSUE I-TISSUE I-TISSUE I-TISSUE O B-TISSUE O O O O O O O B-GENEPROD O B-GENEPROD O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-SUBCELLULAR O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O

(C) Immunofluorescence of P35 mouse brain expressing BioID2-tau. Hippocampal Cornu ammonis 1 (CA1) region was probed for BioID2-tau (myc), for incorporated biotin (streptavidin, SA) and for cell nuclei (DAPI) (n = 3-5 biological replicates). Scale bar, 50 μm.


( A ) Confocal immunofluorescence for endogenous tau NSF in murine hippocampal neurons ( DIV15 ) . Colocalization of tau NSF is apparent in perinuclear compartments ( arrows ) neurites ( insets i - iii ) . Scale bar , 25 μm . Inset of magnified neurite with partial overlap of tau NSF signals ( arrowheads ) . Scale bar , 3 μm . Co - distribution ( R Ch1 - Ch2 ) are indicated .

O O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD I-GENEPROD O B-ORGANISM B-CELL_TYPE I-CELL_TYPE O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD O O O B-SUBCELLULAR O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O

(A) Confocal immunofluorescence for endogenous tau and NSF in primary murine hippocampal neurons (DIV15). Colocalization of tau and NSF is apparent in perinuclear compartments (arrows) and neurites (insets i-iii). Scale bar, 25 μm. Inset of magnified neurite with partial overlap of tau and NSF signals (arrowheads). Scale bar, 3 μm. Co-distribution (R Ch1-Ch2) are indicated.


( B - C ) Co - immunoprecipitation ( Co - IP ) of tau NSF from mouse brain . ( B ) Endogenous transgenic tau were immunoprecipitated from mouse brain lysates of tau - / - , ALZ17tg or ALZ17non - tg mice using anti - tau antibody followed by immunoblot probed for NSF , tau α - actin . Endogenous NSF was co - immunoprecipitated with transgenic endogenous tau from ALZ17tg or ALZ17non - tg but not from tau - / - with . GAPDH , input loading control ( n = 3 biological replicates ) . Recombinant tau NSF served as controls . ( C ) Densitometric of co - IP results in ( B ) . Data information : Values are means ± S . E . M ( normalized to control ) . Adjusted p - values : * * * p < 0 . 001 , * * p < 0 . 01 , * p < 0 . 05 , ns , not significant , ANOVA with Sidak ' s .

O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD I-GENEPROD O B-ORGANISM B-TISSUE O O O O O O B-GENEPROD O B-EXP_ASSAY O B-ORGANISM B-TISSUE O O B-GENEPROD O O O O O O O O O B-ORGANISM O O O B-GENEPROD O O O B-EXP_ASSAY O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD I-GENEPROD O O O O O O O B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-C) Co-immunoprecipitation (Co-IP) of tau and NSF from mouse brain. (B) Endogenous and transgenic tau were immunoprecipitated from mouse brain lysates of tau-/-, ALZ17tg or ALZ17non-tg mice using anti-tau antibody followed by immunoblot probed for NSF, tau and α-actin. Endogenous NSF was co-immunoprecipitated with transgenic and endogenous tau from ALZ17tg or ALZ17non-tg but not from tau-/- with. GAPDH, input loading control (n = 3 biological replicates). Recombinant tau and NSF served as controls. (C) Densitometric quantification of co-IP results in (B). Data information: Values are means ± S.E.M (normalized to control). Adjusted p-values: ***p < 0.001, **p < 0.01, *p < 0.05, ns, not significant, ANOVA with Sidak's test.


Co - IP of HA - tagged tau with NSF from 293T . ( E ) Co - IP of NSF with haemagglutinin ( HA ) - tagged tau variants from cultured . HA - tagged tau variants NSF were expressed in 293T followed by immunoprecipitation using HA antibody immunoblot probed for NSf , tau GAPDH . ( n = 3 biological replicates ) .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O B-GENEPROD O B-GENEPROD O B-CELL_LINE O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O B-CELL_LINE O O B-EXP_ASSAY O B-GENEPROD O B-EXP_ASSAY O O B-GENEPROD O B-GENEPROD I-GENEPROD O O O O O O O O O

Co-IP of HA-tagged tau with NSF from 293T cells. (E) Co-IP of NSF with haemagglutinin (HA)-tagged tau variants from cultured cells. HA-tagged tau variants and NSF were expressed in 293T cells followed by immunoprecipitation using HA antibody and immunoblot probed for NSf, tau and GAPDH. (n = 3 biological replicates).


( F ) Microscale thermophoretic ( MST ) measurement of direct tau NSF interaction . MST binding curve of recombinant hexameric NT - 647 - NHS - labelled NSF ( 27 pM ) recombinant tau ( 18 mM - 0 . 275 nM ) . Dissociation constant : KD = 146 ± 70 nM ( sigmoidal curve fit , R2 = 0 . 844 ) . ( n = 5 technical replicates ) . Data information : Values are means ± S . E . M ( normalized to control ) . Adjusted p - values : * * * p < 0 . 001 , * * p < 0 . 01 , * p < 0 . 05 , ns , not significant , ANOVA with Sidak ' s .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O O B-GENEPROD I-GENEPROD B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Microscale thermophoretic (MST) measurement of direct tau and NSF interaction. MST binding curve of recombinant hexameric NT-647-NHS-labelled NSF (27 pM) and recombinant tau (18 mM - 0.275 nM). Dissociation constant: KD = 146 ± 70 nM (sigmoidal curve fit, R2 = 0.844). (n = 5 technical replicates). Data information: Values are means ± S.E.M (normalized to control). Adjusted p-values: ***p < 0.001, **p < 0.01, *p < 0.05, ns, not significant, ANOVA with Sidak's test.


( G ) Inhibition of NSF ATPase activity with increasing concentrations of tau . NSF activity ( 70 nM ) was assessed alone or in presence of NSF - inhibiting peptide TAT - NSF or control TAT - Scramble peptides ( 140 nM ) ; or co - incubated with recombinant tau ( 8 nM , 16 nM , 24 nM ) , or heat - denatured tau ( dTau , 8 nM ) or albumin ( Alb , 16 nM ) . Data were fitted to Michaelis - Menten model to determine rate constants ( R2 = 0 . 904 - 0 . 994 for conditions ; n = 3 technical replicates ) .

O O O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(G) Inhibition of NSF ATPase activity with increasing concentrations of tau. NSF activity (70 nM) was assessed alone or in the presence of NSF-inhibiting peptide TAT-NSF or control TAT-Scramble peptides (140 nM); or co-incubated with recombinant tau protein (8 nM, 16 nM, 24 nM), or heat-denatured tau (dTau, 8 nM) or albumin (Alb, 16 nM). Data were fitted to Michaelis-Menten model to determine rate constants (R2 = 0.904 - 0.994 for all conditions; n = 3 technical replicates).


( H ) Rate constants derived from kinetic data in i . ( n = 3 technical replicates ) ATP hydrolysis is similar in NSF NSF incubated with control peptide TAT - Scr , albumin , or denatured tau , yet lower with increasing concentrations of tau or after addition of NSF - inhibiting peptide TAT - NSF . Data information : Values are means ± S . E . M ( normalized to control ) . Adjusted p - values : * * * p < 0 . 001 , * * p < 0 . 01 , * p < 0 . 05 , ns , not significant , ANOVA with Sidak ' s .

O O O O O O O B-EXP_ASSAY O O O O O O O O O O O B-SMALL_MOLECULE O O O O B-GENEPROD I-GENEPROD O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Rate constants derived from kinetic data in i. (n = 3 technical replicates) ATP hydrolysis is similar in NSF and NSF incubated with control peptide TAT-Scr, albumin, or denatured tau, yet lower with increasing concentrations of tau or after addition of NSF-inhibiting peptide TAT-NSF. Data information: Values are means ± S.E.M (normalized to control). Adjusted p-values: ***p < 0.001, **p < 0.01, *p < 0.05, ns, not significant, ANOVA with Sidak's test.


( A ) Impaired AMPA - type glutamate receptor surface expression in cultured tau - / - neurons . Cortical tau + / + or tau - / - neurons were surface biotinylated after incubation with non - competitive NMDAR antagonist MK - 801 ( 50 μM , 3 min ) / or GABAAR antagonist bicuculline ( 30 μM , 10 min ) at 37 ° C . Surface GluA1 were detected by immunoblot after with streptavidin ( SA ) enrichment . Input was probed for total GluA1 , NSF , tau βIII - tubulin . ( n = 3 biological replicates ) Input SA - enrichment from non - surface biotinylated neurons served as negative controls . ( B ) Reduced neuronal surface GluA1 in tau - / - neurons . of tau + / + tau - / - surface GluA1 upon treatment SA - enrichment shown in ( A ) . Data information : Values are means ± S . E . M ( normalized to control ) . Adjusted p - values : * * * * p < 0 . 0001 , * * * p < 0 . 001 , * * p < 0 . 01 , * p < 0 . 05 , ns , not significant , ANOVA with Sidak ' s .

O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD O O O B-CELL_TYPE O B-TISSUE B-GENEPROD O O O O B-GENEPROD O O O B-CELL_TYPE O O O O O O O O O B-GENEPROD O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O O O O O B-GENEPROD O O O B-EXP_ASSAY O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-CELL_TYPE O O O O O O O O O B-CELL_TYPE O B-GENEPROD O B-GENEPROD O O O B-CELL_TYPE O O B-GENEPROD O O O B-GENEPROD O O O O B-GENEPROD O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Impaired AMPA-type glutamate receptor surface expression in cultured tau-/- neurons. Cortical tau+/+ or tau-/- neurons were surface biotinylated after incubation with non-competitive NMDAR antagonist MK-801 (50 μM, 3 min) and/or GABAAR antagonist bicuculline (30 μM, 10 min) at 37°C. Surface GluA1 levels were detected by immunoblot after with streptavidin (SA) enrichment. Input was probed for total cellular GluA1, NSF, tau and βIII-tubulin. (n = 3 biological replicates) Input and SA-enrichment from non-surface biotinylated neurons served as negative controls. (B) Reduced neuronal surface GluA1 in tau-/- neurons. Quantification of tau+/+ and tau-/- surface GluA1 upon treatment and SA-enrichment shown in (A). Data information: Values are means ± S.E.M (normalized to control). Adjusted p-values: ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05, ns, not significant, ANOVA with Sidak's test.


( C ) surface GluA1 total NSF immunostaining in mouse hippocampal neurons ( DIV15 ) . Tau + / + , tau - / - neurons tau - / - expressing human tau ( tau - / - . AAVtau ) were treated with either vehicle ( Veh ; DMSO ) or MK - 801 ( 50 μM ) for 3 min . Representative confocal of whole ( left ) neurites ( right ) are shown ( n = 3 biological replicates ) . NSF , surface GluA1 nuclei ( DAPI ) were visualized . Scale bar , 25 μm . ( D - E ) of neurite surface GluA1 neurite NSF relative to tau + / + Veh control . Reduced surface GluA1 ( D ) total NSF ( n = 10 - 12 neurites from 3 biological replicates ) ( E ) localized to neurites in tau - / - neurons treated with MK801 ( n = 21 - 23 neurites from 3 biological replicates ) . Data information : Values are means ± S . E . M ( normalized to control ) . Adjusted p - values : * * * * p < 0 . 0001 , * * * p < 0 . 001 , * * p < 0 . 01 , * p < 0 . 05 , ns , not significant , ANOVA with Sidak ' s .

O O O O B-GENEPROD O B-GENEPROD B-EXP_ASSAY O B-ORGANISM B-CELL_TYPE I-CELL_TYPE O O O O B-GENEPROD O O O O B-GENEPROD O O O B-CELL_TYPE B-GENEPROD O O O O B-ORGANISM B-GENEPROD O B-GENEPROD O O O O B-ORGANISM O O O O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O B-EXP_ASSAY O O O O O B-SUBCELLULAR O O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD B-SUBCELLULAR O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-SUBCELLULAR O B-GENEPROD B-SUBCELLULAR B-GENEPROD O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O B-SUBCELLULAR O O O O O O O O B-EXP_ASSAY O B-SUBCELLULAR O B-GENEPROD O O O B-CELL_TYPE O O B-SMALL_MOLECULE O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Cell surface GluA1 and total NSF immunostaining in primary mouse hippocampal neurons (DIV15). Tau+/+, tau-/- neurons and tau-/- expressing human tau (tau-/-.AAVtau) were treated with either vehicle (Veh; DMSO) or MK-801 (50 μM) for 3 min. Representative confocal images of whole cell (left) and neurites (right) are shown (n = 3 biological replicates). NSF, surface GluA1 and cell nuclei (DAPI) were visualized. Scale bar, 25 μm. (D-E) Quantification of neurite surface GluA1 and neurite NSF relative to tau+/+ Veh control. Reduced surface GluA1 (D) and total NSF (n = 10-12 neurites from 3 biological replicates) (E) localized to neurites in tau-/- neurons treated with MK801 (n = 21-23 neurites from 3 biological replicates). Data information: Values are means ± S.E.M (normalized to control). Adjusted p-values: ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05, ns, not significant, ANOVA with Sidak's test.


( F ) Changes in secondary dendrite GluA2 expression in neurons ( DIV18 ) upon cLTP / cLTD induction . Tau - / - neurons were transfected with tau at DIV12 ( = tau - / - . tau ) or mock transfected ( tau - / - ) . were stained for GluA2 , dendritic marker Map2 , NSF at 2 h post - cLTP / cLTD induction or treatment with control artificial cerebrospinal fluid ( aCSF ) . Dendritic GluA2 cluster was on GluA2 - Map2 binary map . Representative insets from n = 10 - 20 dendrites per condition from 2 mice per genotype . Scale bar , 3 μm . ( G ) of GluA2 mean relative to Map2 normalised to aCSF controls . Secondary dendrite GluA2 increased in tau - expressing ( tau + / + tau - / - . tau ) neurons upon cLTP induction . ( n = 10 - 19 dendrites from 3 biological replicates ) . Data information : Values are means ± S . E . M ( normalized to control ) . Adjusted p - values : * * * * p < 0 . 0001 , * * * p < 0 . 001 , * * p < 0 . 01 , * p < 0 . 05 , ns , not significant , ANOVA with Sidak ' s .

O O O O O O B-SUBCELLULAR B-GENEPROD B-EXP_ASSAY O B-CELL_TYPE O O O O O O O O O B-GENEPROD O O O B-CELL_TYPE O O O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O B-EXP_ASSAY O B-GENEPROD O B-SUBCELLULAR O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O B-TISSUE I-TISSUE I-TISSUE O B-TISSUE O O B-SUBCELLULAR B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O B-SUBCELLULAR O O O O B-ORGANISM O O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O B-SUBCELLULAR B-GENEPROD O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O O O O B-GENEPROD O B-CELL_TYPE O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Changes in secondary dendrite GluA2 expression in neurons (DIV18) upon cLTP/cLTD induction. Tau-/- neurons were transfected with tau at DIV12 (= tau-/-.tau) or mock transfected (tau-/-). Cells were stained for GluA2, dendritic marker Map2, and NSF at 2 h post-cLTP/cLTD induction or treatment with control artificial cerebrospinal fluid (aCSF). Dendritic GluA2 cluster analysis was based on GluA2-Map2 binary intensity map. Representative insets from n = 10-20 dendrites per condition from 2 mice per genotype. Scale bar, 3 μm. (G) Quantification of GluA2 mean intensity relative to Map2 area and normalised to aCSF controls. Secondary dendrite GluA2 increased in tau-expressing (tau+/+ and tau-/-.tau) neurons upon cLTP induction. (n = 10-19 dendrites from 3 biological replicates). Data information: Values are means ± S.E.M (normalized to control). Adjusted p-values: ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05, ns, not significant, ANOVA with Sidak's test.