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

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


( 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 .

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(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 the average of radial RBP - 3 τdonor line scans in control oocyte with the one - phase decay model .

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

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(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 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 .

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(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 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 .

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

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

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

(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 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 .

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(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 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 .

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(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 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 .

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(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 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 .

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(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 .

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(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 ) 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 .

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(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 ) 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 .

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(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 ) 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 .

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

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

(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 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 .

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

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


( 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 .

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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 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 .

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(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 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 .

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

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(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 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 .

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(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 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 .

(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 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 .

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

(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 levels 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-CONTROLLED_VAR O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O B-MEASURED_VAR O O O O 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 ) 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 ) .

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

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(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 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 .

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(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 - body bioluminescence imaging . Shown are the images acquired two weeks after primary tumor resection .

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(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 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 ) .

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(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 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 ) .

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(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 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 ) .

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(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 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 ) .

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(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 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 ) .

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(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 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 .

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(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 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 .

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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 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 .

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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 ) 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 .

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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 ) 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 .

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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 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 .

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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 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 )

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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 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 .

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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 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 ) .

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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 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 .

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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 levels without affecting YAP subcellular localisation . Quantification of YAP protein levels is shown below , with statistical significance in an unpaired 2 - tailed t - test .

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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 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 .

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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 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

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 ) .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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(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 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 )

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(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 ) and for cell nuclei ( DAPI ) ( n = 3 - 5 biological replicates ) . Scale bar , 50 μm .

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(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 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 .

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(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 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 .

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(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 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 ) .

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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 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 .

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(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 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 ) .

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(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 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 .

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(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 ) 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 .

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(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 ) 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 .

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(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 - / - ) . 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 .

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(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.


( H ) Representative immunostaining for whole - cell NSF and GluA2 in tau + / + , tau - / - and tau - transfected tau - / - ( tau - / - . tau ) neurons at 2 h post - cLTP / cLTD induction or treatment with control artificial cerebrospinal fluid ( aCSF ) . ( n = 3 - 6 biological replicates per condition with 10 - 20 cells each ) . Scale bar , 10 μm . Arrows indicate areas of marked co - distribution . ( I ) Co - localisation analysis of NSF and GluA2 upon chemical LTP / LTD induction . Note significantly higher proportional co - distribution of NSF and GluA2 in tau - expressing ( tau + / + and tau - / - . tau ) cells upon cLTP induction . ( n = 3 - 6 cells 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 .

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(H) Representative immunostaining for whole-cell NSF and GluA2 in tau+/+, tau-/- and tau-transfected tau-/- (tau-/-.tau) neurons at 2 h post-cLTP/cLTD induction or treatment with control artificial cerebrospinal fluid (aCSF). (n = 3-6 biological replicates per condition with 10-20 cells each). Scale bar, 10 μm. Arrows indicate areas of marked co-distribution. (I) Co-localisation analysis of NSF and GluA2 upon chemical LTP/LTD induction. Note significantly higher proportional co-distribution of NSF and GluA2 in tau-expressing (tau+/+ and tau-/-.tau) cells upon cLTP induction. (n = 3-6 cells 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.


( B ) Examples of sagittal sections of tau - / - and AAV . syn - hTau - injected tau - / - ( tau - / - . AAVtau ) mice showing localized AAV - mediated expression of eGFP or tau and position of infusion cannulas ending in the hippocampus ( dashed line ) . GluA1 , immunolabelling for GluA1 AMPA receptor subunit . Scale bar , 50 μm .

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(B) Examples of sagittal sections of tau-/- and AAV.syn-hTau-injected tau-/- (tau-/-.AAVtau) mice showing localized AAV-mediated expression of eGFP or tau and position of infusion cannulas ending in the hippocampus (dashed line). GluA1, immunolabelling for GluA1 AMPA receptor subunit. Scale bar, 50 μm.


( C ) Cued fear conditioning memory in tau + / + , tau - / - and tau - / - . AAVtau mice upon bilateral infusion of TAT - NSF or TAT - Scr . Due to infusion of Tat - fusion peptides occurring under brief isoflurane anaesthesia , a general anaesthesia ( GA ) control is included . Note that infusion of TAT - NSF lowers memory performance to comparable levels in tau + / + , tau - / - and tau - / - . AAVtau mice . ( n = 2 - 5 biological replicates ) Data information : Values are mean ± 95 % confidence intervals . Adjusted p - values : * * * p < 0 . 001 , * * p < 0 . 01 , * p < 0 . 05 , ns , not significant , ANOVA with Sidak ' s test .

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(C) Cued fear conditioning memory in tau+/+, tau-/- and tau-/-.AAVtau mice upon bilateral infusion of TAT-NSF or TAT-Scr. Due to infusion of Tat-fusion peptides occurring under brief isoflurane anaesthesia, a general anaesthesia (GA) control is included. Note that infusion of TAT-NSF lowers memory performance to comparable levels in tau+/+, tau-/- and tau-/-.AAVtau mice. (n = 2-5 biological replicates) Data information: Values are mean ± 95% confidence intervals. Adjusted p-values: ***p < 0.001, **p < 0.01, *p < 0.05, ns, not significant, ANOVA with Sidak's test.


( D ) Novel object preference ( fold preference over familiar object ) in tau + / + , tau - / - and tau - / - . AAVtau mice upon bilateral infusion of TAT - NSF or TAT - Scr ( n = 2 - 5 biological replicates ) . Due to infusion of Tat - fusion peptides occurring under brief isoflurane anaesthesia , a general anaesthesia ( GA ) control is included ( n = 2 for GA ) . Note that infusion of TAT - NSF lowers object discrimination to comparable levels in tau + / + , tau - / - and tau - / - . AAVtau mice . Data information : Values are mean ± 95 % confidence intervals . Adjusted p - values : * * * p < 0 . 001 , * * p < 0 . 01 , * p < 0 . 05 , ns , not significant , ANOVA with Sidak ' s test .

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(D) Novel object preference (fold preference over familiar object) in tau+/+, tau-/- and tau-/-.AAVtau mice upon bilateral infusion of TAT-NSF or TAT-Scr (n = 2-5 biological replicates). Due to infusion of Tat-fusion peptides occurring under brief isoflurane anaesthesia, a general anaesthesia (GA) control is included (n = 2 for GA). Note that infusion of TAT-NSF lowers object discrimination to comparable levels in tau+/+, tau-/- and tau-/-.AAVtau mice. Data information: Values are mean ± 95% confidence intervals. Adjusted p-values: ***p < 0.001, **p < 0.01, *p < 0.05, ns, not significant, ANOVA with Sidak's test.


( E ) ATPase assay of recombinant NSF upon incubation with tau coimmunoprecipitates ( tau5 ) from cortical lysates of indicated genotypes , including from tau - transgenic mice ( ALZ17 , Tau58 / 2 ) ( n = 5 - 8 biological replicates ) . IgG , control immunoprecipitate with non - specific IgG from tau + / + lysate . Data information : Values are mean ± 95 % confidence intervals . Adjusted p - values : * * * p < 0 . 001 , * * p < 0 . 01 , * p < 0 . 05 , ns , not significant , ANOVA with Sidak ' s test .

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(E) ATPase assay of recombinant NSF upon incubation with tau coimmunoprecipitates (tau5) from cortical lysates of indicated genotypes, including from tau-transgenic mice (ALZ17, Tau58/2) (n = 5-8 biological replicates). IgG, control immunoprecipitate with non-specific IgG from tau+/+ lysate. Data information: Values are mean ± 95% confidence intervals. Adjusted p-values: ***p < 0.001, **p < 0.01, *p < 0.05, ns, not significant, ANOVA with Sidak's test.


( F ) ATPase assay of recombinant NSF upon incubation with tau coimmunoprecipitates ( tau5 ) from brain lysates of healthy controls ( HC ) , mild cognitive impairment ( MCI ) , and Alzheimer ' s disease ( AD ) ( n = 3 - 4 biological replicates ) . IgG , control immunoprecipitate . Data information : Values are mean ± 95 % confidence intervals . Adjusted p - values : * * * p < 0 . 001 , * * p < 0 . 01 , * p < 0 . 05 , ns , not significant , ANOVA with Sidak ' s test .

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(F) ATPase assay of recombinant NSF upon incubation with tau coimmunoprecipitates (tau5) from brain lysates of healthy controls (HC), mild cognitive impairment (MCI), and Alzheimer's disease (AD) (n = 3-4 biological replicates). IgG, control immunoprecipitate. Data information: Values are mean ± 95% confidence intervals. Adjusted p-values: ***p < 0.001, **p < 0.01, *p < 0.05, ns, not significant, ANOVA with Sidak's test.


( B ) Electrophoretic mobility shift assay ( EMSA ) with increasing concentration ( 0 , 15nM , 30nM , 60nM , 1 . 25μM and 2 . 5μM ) of GST - FOXD3 and fixed concentration ( 50nM ) of 5 ' - Cy5 labelled dsDNA oligonucleotides ( 35 bp each ) from Sox15 promoter ( positive control ) , MSR , MERVL - LTR , MERVL - int , L1MdA and Hprt promoter ( negative control ) . The experiment was repeated 3 times . ( D ) ChIP - qPCR enrichment of FOXD3 in mESCs using primers specific for MSR , MERVL - LTR , MERVL - int ( including and excluding the FOXD3 cognate motif ) , L1MdA promoter sequences . Sox15 promoter and Actb promoter primers are used as positive and negative control respectively . Data is represented as percentage of input and the average of 3 biological replicates is plotted . Error bar indicates standard error of the mean ( SEM ) . Asterisks indicate statistically significant differences compared to no antibody control levels ( * : p < 0 . 05 , paired t - test ) .

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(B) Electrophoretic mobility shift assay (EMSA) with increasing concentration (0, 15nM, 30nM, 60nM, 1.25μM and 2.5μM) of GST-FOXD3 and fixed concentration (50nM) of 5'-Cy5 labelled dsDNA oligonucleotides (35 bp each) from Sox15 promoter (positive control), MSR, MERVL-LTR, MERVL-int, L1MdA and Hprt promoter (negative control). The experiment was repeated 3 times. (D) ChIP-qPCR enrichment of FOXD3 in mESCs using primers specific for MSR, MERVL-LTR, MERVL-int (including and excluding the FOXD3 cognate motif), L1MdA promoter sequences. Sox15 promoter and Actb promoter primers are used as positive and negative control respectively. Data is represented as percentage of input and the average of 3 biological replicates is plotted. Error bar indicates standard error of the mean (SEM). Asterisks indicate statistically significant differences compared to no antibody control levels (*: p< 0.05, paired t-test).


( B ) Validation of select upregulated repeats in Foxd3 KO cells by RT - qPCR . The data is plotted as average fold change relative to control , after normalization to Gapdh . Error bars indicate SEM ( n = 3 biological replicates ) . Asterisks indicate statistically significant differences when compared to control ( * * : p < 0 . 005 , * : p < 0 . 05 , paired t - test ) .

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(B) Validation of select upregulated repeats in Foxd3 KO cells by RT-qPCR. The data is plotted as average fold change relative to control, after normalization to Gapdh. Error bars indicate SEM (n=3 biological replicates). Asterisks indicate statistically significant differences when compared to control (**: p<0.005, *: p<0.05, paired t-test).


( C ) Immunofluorescence analysis of the MERVL - gag protein in control and Foxd3 KO cells . Nuclei are labelled with DAPI . The percentage of counted cells ( ~ 150 cells per experiment ) exhibiting immunofluorescence signal is indicated . Scale bar represents 10 μm , n = 3 biological replicates .

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(C) Immunofluorescence analysis of the MERVL-gag protein in control and Foxd3 KO cells. Nuclei are labelled with DAPI. The percentage of counted cells (~150 cells per experiment) exhibiting immunofluorescence signal is indicated. Scale bar represents 10 μm, n= 3 biological replicates.


( E ) Immunofluorescence analysis of 2CLC markers ZSCAN4 and DPPA3 in control and Foxd3 KO cells . Nuclei are labelled with DAPI . The percentage of counted cells ( ~ 150 cells per experiment ) exhibiting the immunofluorescence signal is indicated . Scale bar represents 10 μm , n = 3 biological replicates .

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(E) Immunofluorescence analysis of 2CLC markers ZSCAN4 and DPPA3 in control and Foxd3 KO cells. Nuclei are labelled with DAPI. The percentage of counted cells (~150 cells per experiment) exhibiting the immunofluorescence signal is indicated. Scale bar represents 10 μm, n=3 biological replicates.


( A ) Electrophoretic mobility shift assay ( EMSA ) with increasing concentrations ( 0 , 30nM , 60nM , 1 . 25μM , 2 . 5μM and 5μM ) of recombinant GST - FOXD3 , DNA binding mutants GST - FOXD3 M1 and GST - FOXD3 M2 and fixed concentration ( 50nM ) of 5 ' - Cy5 labelled dsDNA oligonucleotides ( 35 bp each ) representing MERVL and MSR . The experiment was repeated 3 times .

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(A) Electrophoretic mobility shift assay (EMSA) with increasing concentrations (0, 30nM, 60nM, 1.25μM, 2.5μM and 5μM) of recombinant GST-FOXD3, DNA binding mutants GST-FOXD3 M1 and GST- FOXD3 M2 and fixed concentration (50nM) of 5'-Cy5 labelled dsDNA oligonucleotides (35 bp each) representing MERVL and MSR. The experiment was repeated 3 times.


( B ) RT - qPCR analysis of MERVL , MSR and L1MdA in Foxd3 cKO cells , Foxd3 cKO cells over expressing full length FOXD3 or FOXD3 M1 without and with 4 - OHT . Data is represented as fold change relative to respective untreated cells after normalizing to Gapdh . The average data for 3 biological replicates is plotted , error bars represent standard error mean ( SEM ) . Asterisks indicate statistically significant differences . ( * * : p < 0 . 005 , * : p < 0 . 05 paired t - test ) .

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(B) RT-qPCR analysis of MERVL, MSR and L1MdA in Foxd3 cKO cells, Foxd3 cKO cells over expressing full length FOXD3 or FOXD3 M1 without and with 4-OHT. Data is represented as fold change relative to respective untreated cells after normalizing to Gapdh. The average data for 3 biological replicates is plotted, error bars represent standard error mean (SEM). Asterisks indicate statistically significant differences. (**: p< 0.005, *: p<0.05 paired t-test).


( D ) RT - qPCR analysis of , 2C genes Zscan4c and Dppa3 in Foxd3 cKO cells , Foxd3 cKO cells over expressing full length FOXD3 or FOXD3 M1 without and with 4 - OHT . Data is represented as fold change relative to respective untreated cells after normalizing to Gapdh . The average data for 3 biological replicates is plotted , error bars represent standard error mean ( SEM ) . Asterisks indicate statistically significant differences . ( * * : p < 0 . 005 , * : p < 0 . 05 paired t - test ) .

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

(D) RT-qPCR analysis of, 2C genes Zscan4c and Dppa3 in Foxd3 cKO cells, Foxd3 cKO cells over expressing full length FOXD3 or FOXD3 M1 without and with 4-OHT. Data is represented as fold change relative to respective untreated cells after normalizing to Gapdh. The average data for 3 biological replicates is plotted, error bars represent standard error mean (SEM). Asterisks indicate statistically significant differences. (**: p< 0.005, *: p<0.05 paired t-test).