A HeLa - H2B / tub cells were transfected with control or TIAR siRNAs for 48 hours and synchronized by double thymidine ( TT ) block . After release from the block , mitotic cells were counted by fluorescence microscopy ( mean ± SD ; n = 3 ) . Data information : Statistical significance was determined by unpaired Student ' s t - test ; * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 .

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A HeLa-H2B/tub cells were transfected with control or TIAR siRNAs for 48 hours and synchronized by double thymidine (TT) block. After release from the block, mitotic cells were counted by fluorescence microscopy (mean ± SD; n = 3). Data information: Statistical significance was determined by unpaired Student's t-test; *, p < 0.05; **, p < 0.01; ***, p < 0.001.


B Time from S - to M - phase was measured in si - RNA transfected HeLa - H2B / tub cells by time - lapse microscopy following release from TT block ( 4 repeat experiments ; all measurements depicted ) . Data information : Statistical significance was determined by unpaired Student ' s t - test ; * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Time from S- to M-phase was measured in si-RNA transfected HeLa-H2B/tub cells by time-lapse microscopy following release from TT block (4 repeat experiments; all measurements depicted). Data information: Statistical significance was determined by unpaired Student's t-test; *, p < 0.05; **, p < 0.01; ***, p < 0.001.


Western blot analysis of unsynchronized HeLa - H2B / tub cells was carried out to monitor expression of p ( S10 ) - H3 , total H3 and TIAR in control and TIAR - depleted cells ( mean ± SD ; n = 3 ) . Data information : Statistical significance was determined by unpaired Student ' s t - test ; * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 .

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

Western blot analysis of unsynchronized HeLa-H2B/tub cells was carried out to monitor expression of p(S10)-H3, total H3 and TIAR in control and TIAR-depleted cells (mean ± SD; n = 3). Data information: Statistical significance was determined by unpaired Student's t-test; *, p < 0.05; **, p < 0.01; ***, p < 0.001.


D HeLa cells were transfected with control or TIAR si - RNAs , alone or together with Cdc25B siRNA . 72 hours after transfection , p ( S10 ) - H3 - positive cells were quantified by flow cytometry ( mean ± SD ; n = 3 ) . Data information : Statistical significance was determined by unpaired Student ' s t - test ; * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 .

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D HeLa cells were transfected with control or TIAR si-RNAs, alone or together with Cdc25B siRNA. 72 hours after transfection, p(S10)-H3-positive cells were quantified by flow cytometry (mean ± SD; n = 3). Data information: Statistical significance was determined by unpaired Student's t-test; *, p < 0.05; **, p < 0.01; ***, p < 0.001.


HeLadox - YFP - TIARr cells were transfected with control or TIAR siRNAs , and 24h hours later cell were cultured in the abscence or presence of 1 μg / ml doxocycline for 48 hours . The expression of p ( S10 ) - H3 , total H3 and TIAR was measured by Westen blot analysis . The graph shows the quantification of p - H3 levels normalized to total H3 ( mean ± SD , n = 4 ) . Data information : Statistical significance was determined by unpaired Student ' s t - test ; * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 .

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HeLadox-YFP-TIARr cells were transfected with control or TIAR siRNAs, and 24h hours later cell were cultured in the abscence or presence of 1 μg/ml doxocycline for 48 hours. The expression of p(S10)-H3, total H3 and TIAR was measured by Westen blot analysis. The graph shows the quantification of p-H3 levels normalized to total H3 (mean ± SD, n = 4). Data information: Statistical significance was determined by unpaired Student's t-test; *, p < 0.05; **, p < 0.01; ***, p < 0.001.


F HeLadox - YFP - TIARr - RRM123m cells were analyzed as in panel ( E ) ( mean ± SD , n = 4 ) . Data information : Statistical significance was determined by unpaired Student ' s t - test ; * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 .

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F HeLadox-YFP-TIARr-RRM123m cells were analyzed as in panel (E) (mean ± SD, n = 4). Data information: Statistical significance was determined by unpaired Student's t-test; *, p < 0.05; **, p < 0.01; ***, p < 0.001.


G HeLadox - YFP - TIARr - dQRD cells were analyzed as in panel ( E ) ( mean ± SD , n = 4 ) . Data information : Statistical significance was determined by unpaired Student ' s t - test ; * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 .

G HeLadox-YFP-TIARr-dQRD cells were analyzed as in panel (E) (mean ± SD, n = 4). Data information: Statistical significance was determined by unpaired Student's t-test; *, p < 0.05; **, p < 0.01; ***, p < 0.001.


A Following transfection of HeLa cells with control or TIAR siRNAs for 72 hours , mitotic cells were analyzed by IF microscopy and scored for chromatin ( mean ± SE , n = 3 ) . B HeLa cells were analyzed as in ( A ) and scored for spindle defects ( mean ± SE , n = 3 ) . Data information : statistical significance was determined by unpaired Student ' s t - test ; * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 .

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A Following transfection of HeLa cells with control or TIAR siRNAs for 72 hours, mitotic cells were analyzed by IF microscopy and scored for chromatin (mean ± SE, n = 3). B HeLa cells were analyzed as in (A) and scored for spindle defects (mean ± SE, n = 3). Data information: statistical significance was determined by unpaired Student's t-test; *, p < 0.05; **, p < 0.01; ***, p < 0.001.


C HeLa cells were transfected with control or TIAR siRNAs for 4 days , and subjected to colcemid for 2 hours prior to preparation of metaphase spreads . Chromosomes were stained with Hoechst ( red ) and anti - CENP - A antibody ( green ) ; the yellow arrowhead marks a chromosome break .

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C HeLa cells were transfected with control or TIAR siRNAs for 4 days, and subjected to colcemid for 2 hours prior to preparation of metaphase spreads. Chromosomes were stained with Hoechst (red) and anti-CENP-A antibody (green); the yellow arrowhead marks a chromosome break.


D Metaphase spreads were prepared as in ( C ) , and the frequency of cells with chromosomal breaks was quantified ( mean ± SD , n = 3 independent experiments , approximately 30 metaphase spreads were assessed per experiment and condition ) . E Metaphase spreads were prepared as in ( C ) , and the frequency of cells with scattered chromosomes was quantified ( mean ± SD , n = 3 ) . Data information statistical significance was determined by unpaired Student ' s t - test ; * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 .

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D Metaphase spreads were prepared as in (C), and the frequency of cells with chromosomal breaks was quantified (mean ± SD, n = 3 independent experiments, approximately 30 metaphase spreads were assessed per experiment and condition). E Metaphase spreads were prepared as in (C), and the frequency of cells with scattered chromosomes was quantified (mean ± SD, n = 3). Data information statistical significance was determined by unpaired Student's t-test; *, p < 0.05; **, p < 0.01; ***, p < 0.001.


F HeLa cells were transfected with control or TIAR si - RNAs , alone or together with Cdc25B siRNA , and 4 days later subjected to colcemid for 2 hours prior to preparation of metaphase spreads . The frequency of cells with scattered chromatids was quantified ( mean ± SD , n = 3 independent experiments , approximately 30 metaphase spreads were assessed per experiment and condition ) . G Metaphase spreads were prepared as in ( F ) , and the frequency of cells with chromosomal breaks was quantified ( mean ± SD , n = 3 ) . Data information , statistical significance was determined by unpaired Student ' s t - test ; * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 HeLa cells were transfected with control or TIAR si-RNAs, alone or together with Cdc25B siRNA, and 4 days later subjected to colcemid for 2 hours prior to preparation of metaphase spreads. The frequency of cells with scattered chromatids was quantified (mean ± SD, n = 3 independent experiments, approximately 30 metaphase spreads were assessed per experiment and condition). G Metaphase spreads were prepared as in (F), and the frequency of cells with chromosomal breaks was quantified (mean ± SD, n = 3). Data information , statistical significance was determined by unpaired Student's t-test; *, p < 0.05; **, p < 0.01; ***, p < 0.001.


A HeLa cells were transfected with control or TIAR siRNAs for 48 hours prior to treatment with 0 . 4 µM APH . Cells were fixed at regular time intervals , and p ( S10 ) - H3 - positive cells were quantified by flow cytometry ( mean ± SE , n = 3 ) .

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A HeLa cells were transfected with control or TIAR siRNAs for 48 hours prior to treatment with 0.4 µM APH. Cells were fixed at regular time intervals, and p(S10)-H3-positive cells were quantified by flow cytometry (mean ± SE, n = 3).


B HeLa cells transfected with control or TIAR siRNAs were treated with APH for 24 hours . Pan - nuclear γH2AX signals were quantified by HTM ( n = 3 , 2000 cells examined per experiment and condition ) . Each dot represents the signal from one cell , horizontal lines indicate mean values , and the blue area delineates cells above an arbitrarily chosen threshold . Data information : statistical significance was determined by Wilcoxon rank - sum test . ; * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; * * * * , p < 0 . 0001 .

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B HeLa cells transfected with control or TIAR siRNAs were treated with APH for 24 hours. Pan-nuclear γH2AX signals were quantified by HTM (n = 3, 2000 cells examined per experiment and condition). Each dot represents the signal from one cell, horizontal lines indicate mean values, and the blue area delineates cells above an arbitrarily chosen threshold. Data information: statistical significance was determined by Wilcoxon rank-sum test. ; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.


C Chromatin - bound RPA2 signals were quantified by HTM HeLa cells transfected with control or TIAR siRNAs ( n = 3 , 600 cells examined per experiment and condition ) .

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C Chromatin-bound RPA2 signals were quantified by HTM HeLa cells transfected with control or TIAR siRNAs (n = 3, 600 cells examined per experiment and condition).


D HeLa cells were transfected with control or TIAR siRNAs for 72 hours prior to treatment with ATRi ( 5 μM . ) alone or together with Ro3306 ( 5 μM ) for 12 hours . Pan - nuclear γH2AX signals were quantified by HTM ( n = 3 , 1000 cells examined per experiment and condition ) . Data information : statistical significance was determined by Wilcoxon rank - sum test . ; * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; * * * * , p < 0 . 0001 .

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D HeLa cells were transfected with control or TIAR siRNAs for 72 hours prior to treatment with ATRi (5 μM.) alone or together with Ro3306 (5 μM) for 12 hours. Pan-nuclear γH2AX signals were quantified by HTM (n = 3, 1000 cells examined per experiment and condition). Data information: statistical significance was determined by Wilcoxon rank-sum test. ; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.


E HeLa cells were transfected with control or TIAR siRNAs were treated with ATRi for 48 hours , and the sub - G1 population was quantified by flow cytometry following propidium iodide staining ( mean ± SD , n = 3 ) . Data information : statistical significance was determined by unpaired Student ' s t - test ; * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; * * * * , p < 0 . 0001 .

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E HeLa cells were transfected with control or TIAR siRNAs were treated with ATRi for 48 hours, and the sub-G1 population was quantified by flow cytometry following propidium iodide staining (mean ± SD, n = 3). Data information: statistical significance was determined by unpaired Student's t-test; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.


F HeLa cells transfected with control or TIAR siRNAs were treated with APH , alone or in combination with Gö6976 ( 1µg / ml ) or UCN - 01 ( 300 nM ) . After 24 hours , p ( S10 ) - H3 - positive cells were quantified by flow cytometry , and expressed relative to the values in the untreated condition ( mean ± SE , n = 3 ) . Data information : statistical significance was determined by unpaired Student ' s t - test ; * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; * * * * , p < 0 . 0001 .

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F HeLa cells transfected with control or TIAR siRNAs were treated with APH, alone or in combination with Gö6976 (1µg/ml) or UCN-01 (300 nM). After 24 hours, p(S10)-H3-positive cells were quantified by flow cytometry, and expressed relative to the values in the untreated condition (mean ± SE, n = 3). Data information: statistical significance was determined by unpaired Student's t-test; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.


A HeLa cells , untreated or treated for 24 hours with 0 . 4 µM APH , were fixed with methanol and analyzed by IF microscopy after staining with anti - p ( S10 ) - H3 antibody , anti - TIAR antibody and DAPI . Yellow arrows mark focal accumulation of TIAR in GMGs .

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

A HeLa cells, untreated or treated for 24 hours with 0.4 µM APH, were fixed with methanol and analyzed by IF microscopy after staining with anti-p(S10)-H3 antibody, anti-TIAR antibody and DAPI. Yellow arrows mark focal accumulation of TIAR in GMGs.


B IF microscopy was performed as in ( A ) to determine the intensity of the TIAR signal within GMGs ( mean ± SD , n = 3 independent experiments , approximately 100 GMGs in 25 cells were analyzed per experiment and condition ) . C IF microscopy was performed as in ( A ) to determine the area of the TIAR signal within GMGs ( mean ± SD , n = 3 ) . Data information statistical significance was determined by unpaired Student ' s t - test ; * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 .

B IF microscopy was performed as in (A) to determine the intensity of the TIAR signal within GMGs (mean ± SD, n = 3 independent experiments, approximately 100 GMGs in 25 cells were analyzed per experiment and condition). C IF microscopy was performed as in (A) to determine the area of the TIAR signal within GMGs (mean ± SD, n = 3). Data information statistical significance was determined by unpaired Student's t-test; *, p < 0.05; **, p < 0.01; ***, p < 0.001.


D IF microscopy was performed as in ( A ) to quantify the percentage of late G2 / prophase cells containing GMGs ( mean ± SD , n = 3 independent experiments , 40 cells were analyzed per experiment and condition ) . Data information statistical significance was determined by unpaired Student ' s t - test ; * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 .

D IF microscopy was performed as in (A) to quantify the percentage of late G2/prophase cells containing GMGs (mean ± SD, n = 3 independent experiments, 40 cells were analyzed per experiment and condition). Data information statistical significance was determined by unpaired Student's t-test; *, p < 0.05; **, p < 0.01; ***, p < 0.001.


E HeLa cells were processed for IF microscopy as in ( A ) , images depict different cell cycle phases .

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E HeLa cells were processed for IF microscopy as in (A), images depict different cell cycle phases.


HeLa cell were treated for 16 hours with ( F ) the CDK1 inhibitor Ro3306 ( 9 µM ) prior to fixation with methanol and staining with anti - TIAR antibody and DAPI . The graphs show the percentage of late G2 / prophase cells containing GMGs ( mean ± SD , n = 3 independent experiments , 40 cells were analyzed per experiment and condition ) . Data information : statistical significance was determined by unpaired Student ' s t - test ; * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 .

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HeLa cell were treated for 16 hours with (F) the CDK1 inhibitor Ro3306 (9 µM) prior to fixation with methanol and staining with anti-TIAR antibody and DAPI. The graphs show the percentage of late G2/prophase cells containing GMGs (mean ± SD, n = 3 independent experiments, 40 cells were analyzed per experiment and condition). Data information: statistical significance was determined by unpaired Student's t-test; *, p < 0.05; **, p < 0.01; ***, p < 0.001.


HeLa cell were treated for 16 hours with ( G ) ICFR - 193 ( 1 μM ) prior to fixation with methanol and staining with anti - TIAR antibody and DAPI . The graphs show the percentage of late G2 / prophase cells containing GMGs ( mean ± SD , n = 3 independent experiments , 40 cells were analyzed per experiment and condition ) .

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

HeLa cell were treated for 16 hours with (G) ICFR-193 (1 μM) prior to fixation with methanol and staining with anti-TIAR antibody and DAPI. The graphs show the percentage of late G2/prophase cells containing GMGs (mean ± SD, n = 3 independent experiments, 40 cells were analyzed per experiment and condition).


A - G HeLa cells were treated with 0 . 4 µM APH for 24 hours , fixed and processed for IF microscopy after staining with anti - TIAR antibody , DAPI in combination with ( A ) anti - PCNA - 1 antibody , ( B ) anti - FANCD2 antibody , ( C ) anti - BRCA1 antibody , ( D ) anti - PRP19 antibody , ( E ) anti - Sm ( Y12 ) antibody , ( F ) anti - HuR antibody and ( G ) pS2 - RPOL2 ( H5 ) antibody . Intensity profiles along the white line in the merged image are presented on the right side ; yellow and green arrows mark GMGs .

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A-G HeLa cells were treated with 0.4 µM APH for 24 hours, fixed and processed for IF microscopy after staining with anti-TIAR antibody, DAPI in combination with (A) anti-PCNA-1 antibody, (B) anti-FANCD2 antibody, (C) anti-BRCA1 antibody, (D) anti-PRP19 antibody, (E) anti-Sm(Y12) antibody, (F) anti-HuR antibody and (G) pS2-RPOL2 (H5) antibody. Intensity profiles along the white line in the merged image are presented on the right side; yellow and green arrows mark GMGs.


A HeLa cells were treated with 0 . 4 µM APH for 24 hours , fixed and processed for IF microscopy after staining with anti - TIAR and anti - CDK1 antibodies . Intensity profiles along the white line in the merged image are presented on the right side ; yellow arrows mark GMGs .

A HeLa cells were treated with 0.4 µM APH for 24 hours, fixed and processed for IF microscopy after staining with anti-TIAR and anti-CDK1 antibodies. Intensity profiles along the white line in the merged image are presented on the right side; yellow arrows mark GMGs.


B IF microscopy analysis as in ( A ) using anti - Cyclin B antibody .

B IF microscopy analysis as in (A) using anti-Cyclin B antibody.


C HeLa cells transfected with control or TIAR siRNAs were treated with 0 . 4 µM APH 24 hours prior to fixation . Cells were processed for IF microscopy after staining with anti - PRP19 and anti - CDK1 antibodies . Intensity profiles from staining of the same cells with anti - TIAR and anti - CDK1 antibodies are depicted on the right side .

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C HeLa cells transfected with control or TIAR siRNAs were treated with 0.4 µM APH 24 hours prior to fixation. Cells were processed for IF microscopy after staining with anti-PRP19 and anti-CDK1 antibodies. Intensity profiles from staining of the same cells with anti-TIAR and anti-CDK1 antibodies are depicted on the right side.


D CDK1 was immunoprecipitated from HeLa cells transfected with control or TIAR siRNAs , tested for in vitro kinase activity using recombinant histone H1 as substrate , and visualized by Western blot analysis and autoradiography . The mean CDK1 activity ± SE was quantified from n = 5 experiments . Data information statistical significance was determined by unpaired Student ' s t - test . ; * , p < 0 . 05 ; * * * , p < 0 . 001 .

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D CDK1 was immunoprecipitated from HeLa cells transfected with control or TIAR siRNAs, tested for in vitro kinase activity using recombinant histone H1 as substrate, and visualized by Western blot analysis and autoradiography. The mean CDK1 activity ± SE was quantified from n = 5 experiments. Data information statistical significance was determined by unpaired Student's t-test. ; *, p < 0.05; ***, p < 0.001.


E CDK2 activity was analyzed following immunoprecipitation as in ( D ) ( mean ± SE , n = 3 ) . Data information statistical significance was determined by unpaired Student ' s t - test . * , p < 0 . 05 ; * * * , p < 0 . 001 .

E CDK2 activity was analyzed following immunoprecipitation as in (D) (mean ± SE, n = 3). Data information statistical significance was determined by unpaired Student's t-test. *, p < 0.05; ***, p < 0.001.


F HeLa cells were transfected with control or TIAR siRNAs for 72 hours before measuring expression levels of p ( S22 ) - Lamin A by Western blot analysis ( mean ± SE , n = 3 ) . Data information statistical significance was determined by unpaired Student ' s t - test . ; * , p < 0 . 05 ; * * * , p < 0 . 001 .

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F HeLa cells were transfected with control or TIAR siRNAs for 72 hours before measuring expression levels of p(S22)-Lamin A by Western blot analysis (mean ± SE, n = 3). Data information statistical significance was determined by unpaired Student's t-test. ; *, p < 0.05; ***, p < 0.001.


G p ( S22 ) - Lamin A levels were analyzed by HTM in si - RNA transfected HeLa cells ( n = 3 , 1000 cells examined per experiment and condition ) . Data information statistical significance was determined by Wilcoxon rank - sum test ; * , p < 0 . 05 ; * * * , p < 0 . 001 .

G p(S22)-Lamin A levels were analyzed by HTM in si-RNA transfected HeLa cells (n = 3, 1000 cells examined per experiment and condition). Data information statistical significance was determined by Wilcoxon rank-sum test; *, p < 0.05; ***, p < 0.001.


( A ) BI ‐ 1 WT and KO MEFs cells were stably transduced with retroviruses expressing cytochrome b5 - GFP to visualize the ER ( green ) . Then cells were stained with lysotracker ( red ) and observed with a confocal microscope . Nucleus was stained with Hoechst ( blue ) . Scale bar : 30 μm .

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(A) BI‐1 WT and KO MEFs cells were stably transduced with retroviruses expressing cytochrome b5-GFP to visualize the ER (green). Then cells were stained with lysotracker (red) and observed with a confocal microscope. Nucleus was stained with Hoechst (blue). Scale bar: 30 μm.


( B ) BI ‐ 1 WT and KO MEFs cells stimulated with EBSS for 3 h to induce autophagy . Acidic vesicles were visualized with a confocal microscope after lysotracker staining . Data represent the results of three independent experiments . Scale bar : 50 μm .

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(B) BI‐1 WT and KO MEFs cells stimulated with EBSS for 3 h to induce autophagy. Acidic vesicles were visualized with a confocal microscope after lysotracker staining. Data represent the results of three independent experiments. Scale bar: 50 μm.


( C ) ( a ) Epifluorescence imaging of BI ‐ 1 KO cells stained with lysotracker ; ( b ) electron micrograph of the same field of cells shown in ( a ) ; ( c - e ) , magnification of lysotracker ‐ positive vesicles in BI ‐ 1 KO cells exposed to EBSS for 3 h and visualized with a fluorescent microscope ( c ) and the same field subsequently imaged by EM ( d ) . Overlapping images are presented ( e ) . Left panel : analysis of vesicular structures by EM with morphologies resembling early ( f ) , intermediate ( g ) and late ( h ) autophagy vesicles .

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(C) (a) Epifluorescence imaging of BI‐1 KO cells stained with lysotracker; (b) electron micrograph of the same field of cells shown in (a); (c-e), magnification of lysotracker‐positive vesicles in BI‐1 KO cells exposed to EBSS for 3 h and visualized with a fluorescent microscope (c) and the same field subsequently imaged by EM (d). Overlapping images are presented (e). Left panel: analysis of vesicular structures by EM with morphologies resembling early (f), intermediate (g) and late (h) autophagy vesicles.


( D ) Left panel : the distribution of endogenous LC3 was monitored by immunofluorescence and confocal microscopy in BI ‐ 1 WT and KO MEFs cells at basal conditions ( NT ) or after exposure to EBSS for 3 h . Scale bar : left 15 μm and right 10 μm . Right panel : quantification of the number cells containing three or more LC3 ‐ positive vesicles ( N = 160 cells ) . Mean and standard deviation are presented ( N = 4 ) . Student ' s t ‐ test was used to analyse statistical significance , * * P0 . 001 and * P0 . 01 .

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(D) Left panel: the distribution of endogenous LC3 was monitored by immunofluorescence and confocal microscopy in BI‐1 WT and KO MEFs cells at basal conditions (NT) or after exposure to EBSS for 3 h. Scale bar: left 15 μm and right 10 μm. Right panel: quantification of the number cells containing three or more LC3‐positive vesicles (N=160 cells). Mean and standard deviation are presented (N=4). Student's t‐test was used to analyse statistical significance, **P0.001 and *P0.01.


( E ) BI ‐ 1 WT and KO cells were transiently transfected with an expression vector for a monomeric ‐ tandem LC3 - RFP - GFP construct . After 24 h , cells were exposed to EBSS for 3 h . Autophagy fluxes were monitored in living cells by visualizing the distribution of LC3 ‐ positive dots in the red and green channels using a confocal microscope . Scale bar : 10 μm . Right panel : quantification of the ratio between red and yellow dots per cell is presented . Mean and standard error of the analysis of 15 cells are shown .

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(E) BI‐1 WT and KO cells were transiently transfected with an expression vector for a monomeric‐tandem LC3-RFP-GFP construct. After 24 h, cells were exposed to EBSS for 3 h. Autophagy fluxes were monitored in living cells by visualizing the distribution of LC3‐positive dots in the red and green channels using a confocal microscope. Scale bar: 10 μm. Right panel: quantification of the ratio between red and yellow dots per cell is presented. Mean and standard error of the analysis of 15 cells are shown.


( A ) BI ‐ 1 WT and KO MEFs were treated with EBSS ( left panel ) or glucose / serum ‐ free RPMI media ( right panel ) for the indicated time points . Then , levels of LC3 were determined by western blot analysis . LC3 ‐ I and LC3 ‐ II forms are indicated . Hsp90 levels were assessed as loading control .

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(A) BI‐1 WT and KO MEFs were treated with EBSS (left panel) or glucose/serum‐free RPMI media (right panel) for the indicated time points. Then, levels of LC3 were determined by western blot analysis. LC3‐I and LC3‐II forms are indicated. Hsp90 levels were assessed as loading control.


( B ) Quantification of LC3 ‐ II levels relative to Hsp90 expression was performed in several experiments performed as presented in ( A ) .

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(B) Quantification of LC3‐II levels relative to Hsp90 expression was performed in several experiments performed as presented in (A).


( C ) Cells were pre ‐ treated with a cocktail of lysosomal inhibitors ( 200 nM bafilomycin A1 , 10 μg / ml pepstatin , and E64d ; left panel ) or 10 mM 3 ‐ methyladenine ( 3 ‐ MA ; right panel ) for 12 h and then exposed to starvation . LC3 levels monitored by western blot ( D ) and quantification of LC3 ‐ II levels relative to Hsp90 were performed in the experimental conditions described in ( C ) .

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(C)Cells were pre‐treated with a cocktail of lysosomal inhibitors (200 nM bafilomycin A1, 10 μg/ml pepstatin, and E64d; left panel) or 10 mM 3‐methyladenine (3‐MA; right panel) for 12 h and then exposed to starvation. LC3 levels monitored by western blot (D) and quantification of LC3‐II levels relative to Hsp90 were performed in the experimental conditions described in (C).


( E ) Basal autophagy flux was monitored in cells treated with a cocktail of lysosomal inhibitors ( Lys . Inh . ) in the presence of normal cell culture media . Right panel : quantification of independent experiments is presented .

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(E) Basal autophagy flux was monitored in cells treated with a cocktail of lysosomal inhibitors (Lys. Inh.) in the presence of normal cell culture media. Right panel: quantification of independent experiments is presented.


( F ) BI ‐ 1 KO cells were stably transduced with retroviruses expressing BI ‐ 1 - GFP or empty vector , and then levels of LC3 ‐ II were assessed over time by western blot after exposure to EBSS . Right panel : as control , the levels of BI ‐ 1 - GFP were monitored by western blot . Hsp90 levels were used as loading control . In ( B , D and E ) mean and standard deviation are presented . Two ‐ way ANOVA was applied to analyse statistical significance . In parenthesis , the number of independent experiments for each time point is indicated . Student ' s t ‐ test was also used in ( E ) to analyse the statistical significance between each time point ( * P0 . 001 ) . In ( B , D and E ) , normalization was performed as a ratio with the LC3 ‐ II / Hsp90 normalized levels from non ‐ treated BI ‐ 1 WT cells .

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(F) BI‐1 KO cells were stably transduced with retroviruses expressing BI‐1-GFP or empty vector, and then levels of LC3‐II were assessed over time by western blot after exposure to EBSS. Right panel: as control, the levels of BI‐1-GFP were monitored by western blot. Hsp90 levels were used as loading control. In (B, D and E) mean and standard deviation are presented. Two‐way ANOVA was applied to analyse statistical significance. In parenthesis, the number of independent experiments for each time point is indicated. Student's t‐test was also used in (E) to analyse the statistical significance between each time point (*P0.001). In (B, D and E), normalization was performed as a ratio with the LC3‐II/Hsp90 normalized levels from non‐treated BI‐1 WT cells.


( A ) Left panel : BI ‐ 1 WT and KO MEFs cells were incubated in EBSS , and then cell viability was monitored using the MTS assay . Right panel : a similar experiment was performed after treating cells with the indicated concentration of tunicamycin for 24 h . Mean and standard deviation are presented of triplicates representative of three independent experiments .

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(A) Left panel: BI‐1 WT and KO MEFs cells were incubated in EBSS, and then cell viability was monitored using the MTS assay. Right panel: a similar experiment was performed after treating cells with the indicated concentration of tunicamycin for 24 h. Mean and standard deviation are presented of triplicates representative of three independent experiments.


( B ) BI ‐ 1 WT and KO cells were treated with three different starvation stimuli for 6 and 24 h . Cell death was determined after propidium iodide ( PI ) staining and FACS analysis . In addition , cells were treated with 100 ng / ml tunicamycin ( Tm ) for 24 h . Mean and standard deviation are presented of one experiment performed in triplicates .

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(B) BI‐1 WT and KO cells were treated with three different starvation stimuli for 6 and 24 h. Cell death was determined after propidium iodide (PI) staining and FACS analysis. In addition, cells were treated with 100 ng/ml tunicamycin (Tm) for 24 h. Mean and standard deviation are presented of one experiment performed in triplicates.


( C ) Cells were exposed to EBSS for 6 h or 1 μg / ml Tm for 2 h , and then replated in normal cell culture media . After 5 days , cell viability was monitored by staining with crystal violet . Data are representative of three independent experiments .

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(C) Cells were exposed to EBSS for 6 h or 1 μg/ml Tm for 2 h, and then replated in normal cell culture media. After 5 days, cell viability was monitored by staining with crystal violet. Data are representative of three independent experiments.


( D ) BI ‐ 1 WT MEFs were stably transduced with lentiviral expression vectors to deliver an shRNA against bi ‐ 1 mRNA or control mRNA ( luciferase shRNA ) . Cell survival was measured after treatment of cells as described in ( B ) . Mean and standard deviation of an experiment made by triplicate , representative of two independent experiments .

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(D) BI‐1 WT MEFs were stably transduced with lentiviral expression vectors to deliver an shRNA against bi‐1 mRNA or control mRNA (luciferase shRNA). Cell survival was measured after treatment of cells as described in (B). Mean and standard deviation of an experiment made by triplicate, representative of two independent experiments.


( E ) BI ‐ 1 KO cells were stably transduced with retroviruses expressing BI ‐ 1 - GFP or empty vector , and then exposed to EBSS for indicated time points . Cell viability was monitored after PI staining by FACS . Mean and standard deviation are presented of triplicates representative of two independent experiments .

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(E) BI‐1 KO cells were stably transduced with retroviruses expressing BI‐1-GFP or empty vector, and then exposed to EBSS for indicated time points. Cell viability was monitored after PI staining by FACS. Mean and standard deviation are presented of triplicates representative of two independent experiments.


( A ) BI ‐ 1 WT and KO MEFs were treated with EBSS for 2 h , and the levels of phospho ‐ p70S6k were determined by western blot analysis . Total p70S6k is also shown .

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(A) BI‐1 WT and KO MEFs were treated with EBSS for 2 h, and the levels of phospho‐p70S6k were determined by western blot analysis. Total p70S6k is also shown.


( B ) LC3 was monitored by immunofluorescence in cells treated with EBSS for 3 h in the presence or absence of 10 μM JNK inhibitor SP600125 . Mean and standard deviation are presented ( N = 3 ) . Student ' s t ‐ test was used to analyse statistical significance , * P0 . 001 and * * P0 . 0001 .

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(B) LC3 was monitored by immunofluorescence in cells treated with EBSS for 3 h in the presence or absence of 10 μM JNK inhibitor SP600125. Mean and standard deviation are presented (N=3). Student's t‐test was used to analyse statistical significance, *P0.001 and **P0.0001.


( C ) BI ‐ 1 WT and KO cells were treated with EBSS for indicated time points , in the presence or absence of 10 μM of the JNK inhibitor SP600125 . Levels of phosphorylation of JNK ( pJNK ) and LC3 ‐ II were determined by western blot . The levels of total JNK and Hsp90 are shown as control ( N = 4 ) . Image was assembled from cropped lanes of the same western blot analysis of the same gel .

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(C) BI‐1 WT and KO cells were treated with EBSS for indicated time points, in the presence or absence of 10 μM of the JNK inhibitor SP600125. Levels of phosphorylation of JNK (pJNK) and LC3‐II were determined by western blot. The levels of total JNK and Hsp90 are shown as control (N=4). Image was assembled from cropped lanes of the same western blot analysis of the same gel.


( D ) BI ‐ 1 WT and KO cells were treated with EBSS for indicated time points in the presence or absence of 10 μM SP600125 , and the electrophoretic shift associated with BCL ‐ 2 phosphorylation was monitored by western blot .

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(D) BI‐1 WT and KO cells were treated with EBSS for indicated time points in the presence or absence of 10 μM SP600125, and the electrophoretic shift associated with BCL‐2 phosphorylation was monitored by western blot.


( E ) BI ‐ 1 WT and KO cells were co ‐ transfected with expression vectors for Beclin ‐ 1 - MYC , BI ‐ 1 - HA , and BCL ‐ XL - FLAG . After 24 h , cells were treated with EBSS for 2 h or left untreated . The association of MYC ‐ tagged expressed Beclin ‐ 1 and BCL ‐ XL - FLAG was assessed by immunoprecipitation of Beclin ‐ 1 followed by western blot analysis .

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(E) BI‐1 WT and KO cells were co‐transfected with expression vectors for Beclin‐1-MYC, BI‐1-HA, and BCL‐XL-FLAG. After 24 h, cells were treated with EBSS for 2 h or left untreated. The association of MYC‐tagged expressed Beclin‐1 and BCL‐XL-FLAG was assessed by immunoprecipitation of Beclin‐1 followed by western blot analysis.


( F ) 293T cells were co ‐ transfected with expression vectors for Beclin ‐ 1 - MYC , BI ‐ 1 - HA , and BCL ‐ XL - FLAG . Cell extracts were prepared in CHAPS buffer and Beclin ‐ 1 - MYC immunoprecipitated , and the possible co ‐ precipitation of BI ‐ 1 - HA , and BCL ‐ XL - FLAG was assessed by western blot analysis ( N = 3 ) . Asterisks indicate BI ‐ 1 oligomers .

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(F) 293T cells were co‐transfected with expression vectors for Beclin‐1-MYC, BI‐1-HA, and BCL‐XL-FLAG. Cell extracts were prepared in CHAPS buffer and Beclin‐1-MYC immunoprecipitated, and the possible co‐precipitation of BI‐1-HA, and BCL‐XL-FLAG was assessed by western blot analysis (N=3). Asterisks indicate BI‐1 oligomers.


( G ) ( G ) 293T cells were co ‐ transfected as described in ( F ) and BI ‐ 1 - HA was immunoprecipitated , and the possible co ‐ precipitation of Beclin ‐ 1 - MYC , and BCL ‐ XL - FLAG determined by western blot analysis ( N = 3 ) . Figure source data can be found with the Supplementary Information .

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(G) (G) 293T cells were co‐transfected as described in (F) and BI‐1-HA was immunoprecipitated, and the possible co‐precipitation of Beclin‐1-MYC, and BCL‐XL-FLAG determined by western blot analysis (N=3). Figure source data can be found with the Supplementary Information.


( A ) BI ‐ 1 KO MEFs were stably transduced with lentiviral vectors expressing an shRNA against beclin ‐ 1 ( shBeclin ‐ 1 ) or ire1α ( shIRE1α ) mRNA or luciferase ( shLuc ) as control . The levels of LC3 , Beclin ‐ 1 , IRE1α and Hsp90 were monitored by western blot .

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(A) BI‐1 KO MEFs were stably transduced with lentiviral vectors expressing an shRNA against beclin‐1 (shBeclin‐1) or ire1α (shIRE1α) mRNA or luciferase (shLuc) as control. The levels of LC3, Beclin‐1, IRE1α and Hsp90 were monitored by western blot.


( B ) BI ‐ 1 KO MEFs were stably transduced with lentiviral vectors as described in A . Cells were exposed to EBSS for 3 h , and then LC3 levels were analysed by western blot . Image was assembled from cropped lanes of the same western blot analysis .

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(B) BI‐1 KO MEFs were stably transduced with lentiviral vectors as described in A. Cells were exposed to EBSS for 3 h, and then LC3 levels were analysed by western blot. Image was assembled from cropped lanes of the same western blot analysis.


( C ) Endogenous LC3 distribution was visualized using immunofluorescence and confocal microscopy in BI ‐ 1 KO / shLuc and BI ‐ 1 KO / shBeclin ‐ 1 cells . Quantification represents the visualization of at least 180 cells . Student ' s t ‐ test was used to analyse statistical significance . Mean and standard deviation are presented , * P0 . 001 , NS : non ‐ significant .

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(C) Endogenous LC3 distribution was visualized using immunofluorescence and confocal microscopy in BI‐1 KO/shLuc and BI‐1 KO/shBeclin‐1 cells. Quantification represents the visualization of at least 180 cells. Student's t‐test was used to analyse statistical significance. Mean and standard deviation are presented, *P0.001, NS: non‐significant.


( D ) LC3 was visualized and quantified in BI ‐ 1 KO / shLuc and BI ‐ 1 KO / shIRE1α cells described in ( B ) by immunofluorescence and confocal microscopy analysis .

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(D) LC3 was visualized and quantified in BI‐1 KO/shLuc and BI‐1 KO/shIRE1α cells described in (B) by immunofluorescence and confocal microscopy analysis.


( E ) BI ‐ 1 KO cells were transiently transfected with a TRAF2 dominant ‐ negative ( TRAF2 ‐ DN ) construct or empty vector ( mock ) , and after 48 h cells were stimulated with EBSS and the levels of LC3 ‐ II and Hsp90 were monitored by western blot . Right panel : quantification of relative LC3 ‐ II levels normalized with Hsp90 and non ‐ treated cells . Mean and standard deviation are presented .

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(E) BI‐1 KO cells were transiently transfected with a TRAF2 dominant‐negative (TRAF2‐DN) construct or empty vector (mock), and after 48 h cells were stimulated with EBSS and the levels of LC3‐II and Hsp90 were monitored by western blot. Right panel: quantification of relative LC3‐II levels normalized with Hsp90 and non‐treated cells. Mean and standard deviation are presented.


( F ) 293T cells were co ‐ transfected with expression vectors for HA ‐ tagged IRE1α ( IRE1 - HA ) , TRAF2 - FLAG , and MYC ‐ tagged BI ‐ 1 ( BI1 - MYC ) . After 48 h of transfection , HA ‐ tagged proteins were immunoprecipitated and the possible interaction with TRAF2 was analysed by western blot . Right panel : the percentage of TRAF2 dissociation from IRE1α by the presence or absence of BI ‐ 1 was quantified and normalized with the expression levels observed in the inputs . For comparison , the co ‐ IP signal observed in the absence of BI ‐ 1 was normalized as 100 % co ‐ IP in each independent experiment ( N = 3 ) . Mean and standard deviation are presented , * P0 . 05 . Figure source data can be found with the Supplementary Information .

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(F) 293T cells were co‐transfected with expression vectors for HA‐tagged IRE1α (IRE1-HA), TRAF2-FLAG, and MYC‐tagged BI‐1 (BI1-MYC). After 48 h of transfection, HA‐tagged proteins were immunoprecipitated and the possible interaction with TRAF2 was analysed by western blot. Right panel: the percentage of TRAF2 dissociation from IRE1α by the presence or absence of BI‐1 was quantified and normalized with the expression levels observed in the inputs. For comparison, the co‐IP signal observed in the absence of BI‐1 was normalized as 100% co‐IP in each independent experiment (N=3). Mean and standard deviation are presented, *P0.05. Figure source data can be found with the Supplementary Information.


( A ) The expression of dBI ‐ 1 was knocked down in Drosophila melanogaster . Then , relative expression levels of dBI ‐ 1 mRNA were monitored by semiquantitative PCR . Actin levels were monitored as control .

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(A) The expression of dBI‐1 was knocked down in Drosophila melanogaster. Then, relative expression levels of dBI‐1 mRNA were monitored by semiquantitative PCR. Actin levels were monitored as control.


( B ) ( B ) LC3 levels were monitored in control ( Da ‐ Gal4 > huLC3 : GFP ) or dBI ‐ 1 RNAi larvae ( Da ‐ Gal4 > huLC3 : GFP , Dcr2 , dBI ‐ 1i ) under basal or fasting conditions . Then , huLC3 - GFP levels were analysed by western blot . In addition , dBI ‐ 1i larvae were treated with 100 μM SP600125 ( added in the growing media ) .

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(B) (B) LC3 levels were monitored in control (Da‐Gal4>huLC3:GFP) or dBI‐1 RNAi larvae (Da‐Gal4>huLC3:GFP, Dcr2, dBI‐1i) under basal or fasting conditions. Then, huLC3-GFP levels were analysed by western blot. In addition, dBI‐1i larvae were treated with 100 μM SP600125 (added in the growing media).


( C ) Left panel : the presence of LC3 ‐ positive vesicles ( white arrowheads ) was monitored by confocal microscopy in control ( Da ‐ Gal4 > huLC3 > GFP ) and dBI ‐ 1 knockdown pupae ( Da ‐ Gal4 > huLC3 : GFP , Dcr2 , dBI ‐ 1i ) after 6 h of puparium formation . The organization of actin cytoskeleton was monitored by staining with phalloidin ( Ph , red ) . Nucleus was stained with Topro ( blue ) . Scale bar : left 20 μm and right 11 μm . Right panel : overexpression of dBI ‐ 1 ( dBI ‐ 1EY03662 ) delays salivary gland degradation . Overexpression of dBI ‐ 1 was confirmed by semiquantitative RT - PCR . Actin levels were monitored as loading control . Right panel : wild ‐ type control and dBI ‐ 1EY03662 pupae were analysed at 14 h after puparium formation . Superficial and internal confocal planes of the cells are presented . Scale bar : 40 μm .

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(C) Left panel: the presence of LC3‐positive vesicles (white arrowheads) was monitored by confocal microscopy in control (Da‐Gal4>huLC3>GFP) and dBI‐1 knockdown pupae (Da‐Gal4>huLC3:GFP, Dcr2, dBI‐1i) after 6 h of puparium formation. The organization of actin cytoskeleton was monitored by staining with phalloidin (Ph, red). Nucleus was stained with Topro (blue). Scale bar: left 20 μm and right 11 μm. Right panel: overexpression of dBI‐1 (dBI‐1EY03662) delays salivary gland degradation. Overexpression of dBI‐1 was confirmed by semiquantitative RT-PCR. Actin levels were monitored as loading control. Right panel: wild‐type control and dBI‐1EY03662 pupae were analysed at 14 h after puparium formation. Superficial and internal confocal planes of the cells are presented. Scale bar: 40 μm.


( D ) Wild ‐ type control and dBI ‐ 1EY03662 larvae were cultured in fasting conditions for different periods of time and fat body stained with lysotracker ( red ) and Hoechst ( blue ) . Then , lysosomal content in the fat body was visualized by epifluorescence microscopy . The percentage of cells presenting lysotracker ‐ positive stain is indicated . Scale bar : 50 μm .

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(D) Wild‐type control and dBI‐1EY03662 larvae were cultured in fasting conditions for different periods of time and fat body stained with lysotracker (red) and Hoechst (blue). Then, lysosomal content in the fat body was visualized by epifluorescence microscopy. The percentage of cells presenting lysotracker‐positive stain is indicated. Scale bar: 50 μm.


( E ) Control or dBI ‐ 1 knockdown adult flies were exposed to nutrient starvation and then animal viability was monitored over time for several days . In all , 100 individuals were monitored in each condition . Data represent mean and standard error ( N = 3 ) . Two ‐ way ANOVA was used to analyse statistical significance between groups .

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(E) Control or dBI‐1 knockdown adult flies were exposed to nutrient starvation and then animal viability was monitored over time for several days. In all, 100 individuals were monitored in each condition. Data represent mean and standard error (N=3). Two‐way ANOVA was used to analyse statistical significance between groups.


( F ) Second instar dBI ‐ 1 RNAi or control larvae were grown in food supplemented with 25 μg / ml Tm dissolved in DMSO or 0 . 5 % DMSO as control . The number of individual reaching the adult fly stage was evaluated . Mean and standard error are presented ( N = 3 ) , * * P0 . 01 .

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(F) Second instar dBI‐1 RNAi or control larvae were grown in food supplemented with 25 μg/ml Tm dissolved in DMSO or 0.5% DMSO as control. The number of individual reaching the adult fly stage was evaluated. Mean and standard error are presented (N=3), **P0.01.


( A ) The levels of LC3 , Atg5 / Atg12 complex , Beclin ‐ 1 , Bcl ‐ 2 , Bcl ‐ XL , IRE1α and Hsp90 were monitored in liver protein extracts of bi ‐ 1 + / + and bi ‐ 1 − / − at 6 ‐ month ‐ old mice . Each well represents independent mice .

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(A) The levels of LC3, Atg5/Atg12 complex, Beclin‐1, Bcl‐2, Bcl‐XL, IRE1α and Hsp90 were monitored in liver protein extracts of bi‐1+/+ and bi‐1−/− at 6‐month‐old mice. Each well represents independent mice.


( B ) A similar analysis was performed for indicated markers in kidney protein extracts as described in liver .

(B) A similar analysis was performed for indicated markers in kidney protein extracts as described in liver.


( C ) bi ‐ 1 + / + and bi ‐ 1 − / − mice were injected with 50 ng / ml of tunicamycin by intraperitoneal injection ( N = 3 ) and then LC3 levels were monitored in liver protein extracts by western blot analysis . Right panel : quantification of relative LC3 ‐ II levels , * * P0 . 005 .

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

(C) bi‐1+/+ and bi‐1−/− mice were injected with 50 ng/ml of tunicamycin by intraperitoneal injection (N=3) and then LC3 levels were monitored in liver protein extracts by western blot analysis. Right panel: quantification of relative LC3‐II levels, **P0.005.


A , B Regression plot showing the association between baseline AV45 PET ( x - axis ) and annual AV45 PET change ( y - axis ) across the entire sample ( N = 300 ) . The vertical dashed line represents the Aβ - positivity threshold of AV45 PET SUVR > 0 . 079 ( A ) . Regression plot for the association between baseline AV45 PET and annual AV45 PET change stratified by baseline CSF sTREM2 levels ( B ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, B Regression plot showing the association between baseline AV45 PET (x-axis) and annual AV45 PET change (y-axis) across the entire sample (N=300). The vertical dashed line represents the Aβ-positivity threshold of AV45 PET SUVR > 0.079 (A). Regression plot for the association between baseline AV45 PET and annual AV45 PET change stratified by baseline CSF sTREM2 levels (B).


Boxplots ( central band = median , boxes = Quartiles , whiskers = 1 . 5 * interquartile range ) , illustrating the differences in GE180 PET ( A ) compared between wild type C57BL / 6 mice ( n = 43 ) and APPNL - G - G ( n = 15 ) .

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Boxplots (central band = median, boxes = Quartiles, whiskers = 1.5 * interquartile range), illustrating the differences in GE180 PET (A) compared between wild type C57BL/6 mice (n=43) and APPNL-G-G (n=15).


B Boxplots ( central band = median , boxes = Quartiles , whiskers = 1 . 5 * interquartile range ) , illustrating the differences in the rate of change in Florbetaben PET ( B ) compared between wild type C57BL / 6 mice ( n = 43 ) and APPNL - G - G ( n = 15 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Boxplots (central band = median, boxes = Quartiles, whiskers = 1.5 * interquartile range), illustrating the differences in the rate of change in Florbetaben PET (B) compared between wild type C57BL/6 mice (n=43) and APPNL-G-G (n=15).


C The regression plot shows lower rate of change in Florbetaben PET to be associated with lower GE180 PET at baseline , i . e . at 5 months of age ( C ) . The shaded area corresponds to the 95 % CI of the regression line .

O O O O O O O O O O O O O O O O O O O O O O O O O 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 The regression plot shows lower rate of change in Florbetaben PET to be associated with lower GE180 PET at baseline, i.e. at 5 months of age (C). The shaded area corresponds to the 95% CI of the regression line.


A - C Coronal ( top row ) and axial ( bottom row ) slices of average 18 - F GE - 180 TSPO ( microglia ) tracer and 18F - florbetaben amyloid - PET split up by group ( A - C ) : C57BL / 6 ( A ) , APPNL - G - F mice subgroup with a low ( < median ) GE - 180 PET ( B ) , and the APPNL - G - F mice subgroup with a high ( > median ) GE - 180 PET ( C ) . APPNL - G - F mice with low baseline GE180 PET levels showed faster increase in Florbetaben PET between 5 and 10 months ( B ) compared to those at high baseline level of GE - 180 PET ( C ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-C Coronal (top row) and axial (bottom row) slices of average 18-F GE-180 TSPO (microglia) tracer and 18F-florbetaben amyloid-PET split up by group (A - C): C57BL/6 (A), APPNL-G-F mice subgroup with a low (< median) GE-180 PET (B), and the APPNL-G-F mice subgroup with a high (> median) GE-180 PET (C). APPNL-G-F mice with low baseline GE180 PET levels showed faster increase in Florbetaben PET between 5 and 10 months (B) compared to those at high baseline level of GE-180 PET (C).


Effects of sTREM2 on AV1451 - PET - assessed tau pathology stratified by Braak - stages shown as boxplots ( central band = median , boxes = Quartiles , whiskers = 1 . 5 * interquartile range ) .

Effects of sTREM2 on AV1451-PET-assessed tau pathology stratified by Braak-stages shown as boxplots (central band = median, boxes = Quartiles, whiskers = 1.5 * interquartile range).


( A ) Vero cell was inoculated with BALF sample , and the CPEs were observed at 4 - dpi .

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(A) Vero cell was inoculated with BALF sample, and the CPEs were observed at 4-dpi.


( B ) RNAs were extracted from the cell culture supernatant , and detected using a commercial kit targeting the ORF 1ab ( red ) and N ( blue ) genes of SARS - CoV - 2 .

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

(B) RNAs were extracted from the cell culture supernatant, and detected using a commercial kit targeting the ORF 1ab (red) and N (blue) genes of SARS-CoV-2.


Testing the convalescent plasma IgG antibody ( C ) using the purified SARS - CoV - 2 particles . The control plasmas 1 and 2 were obtained from a patient recovered from influenza A virus infection and a healthy volunteer , respectively . The control monoclonal antibody is a human monoclonal antibody specific to influenza A virus generated by our laboratory . The PBS control was serum - free .

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

Testing the convalescent plasma IgG antibody (C) using the purified SARS-CoV-2 particles. The control plasmas 1 and 2 were obtained from a patient recovered from influenza A virus infection and a healthy volunteer, respectively. The control monoclonal antibody is a human monoclonal antibody specific to influenza A virus generated by our laboratory. The PBS control was serum-free.


Testing the SARA - CoV - 2 - RBD specific human monoclonal antibodies ( D ) using the purified SARS - CoV - 2 particles . The control plasmas 1 and 2 were obtained from a patient recovered from influenza A virus infection and a healthy volunteer , respectively . The control monoclonal antibody is a human monoclonal antibody specific to influenza A virus generated by our laboratory . The PBS control was serum - free .

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

Testing the SARA-CoV-2-RBD specific human monoclonal antibodies (D) using the purified SARS-CoV-2 particles. The control plasmas 1 and 2 were obtained from a patient recovered from influenza A virus infection and a healthy volunteer, respectively. The control monoclonal antibody is a human monoclonal antibody specific to influenza A virus generated by our laboratory. The PBS control was serum-free.


( E ) Viruses were detected by IFA using the patient ' s plasma , and plasma from a healthy control was used as negative control . Scale bar , 100 μm .

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(E) Viruses were detected by IFA using the patient's plasma, and plasma from a healthy control was used as negative control. Scale bar, 100 μm.


( A ) . Image of negative stained SARS - CoV - 2 . Nail - like spikes can be clearly seen .

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(A). Image of negative stained SARS-CoV-2. Nail-like spikes can be clearly seen.


Enlarged view of virion boxed in ( A ) . ( C ) Zoom - in view of a spike boxed in ( B ) . The shape is depicted by red dot line . Length , the diameter of stem and spike ' s head are 23nm , 4nm and 7nm , respectively .

Enlarged view of virion boxed in (A). (C) Zoom-in view of a spike boxed in (B). The shape is depicted by red dot line. Length, the diameter of stem and spike's head are 23nm, 4nm and 7nm, respectively.


( A ) and ( B ) . Cryo - EM images of SARS - CoV - 2 . ( C ) . Zoom - in view of the virion showed in ( A ) . Envelope and nucleocapsid are indicated by green and blue respectively , remarkable spikes are indicated by red triangles . ( D ) . Zoom - in views of the two virions showed in ( B ) , remarkable spikes are indicated by red triangles . ( E ) . Zoom - in view of the spike indicated by yellow triangle in ( C ) . The shape is depicted by yellow dot lines . ( F ) . Zoom - in view of the spike indicated by yellow triangle in ( D ) . The shape is depicted by yellow dot lines .

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(A) and (B). Cryo-EM images of SARS-CoV-2. (C). Zoom-in view of the virion showed in (A). Envelope and nucleocapsid are indicated by green and blue respectively, remarkable spikes are indicated by red triangles. (D). Zoom-in views of the two virions showed in (B), remarkable spikes are indicated by red triangles. (E). Zoom-in view of the spike indicated by yellow triangle in (C). The shape is depicted by yellow dot lines. (F). Zoom-in view of the spike indicated by yellow triangle in (D). The shape is depicted by yellow dot lines.


( A ) Levels of Piwil1 ( Miwi ) , Piwil2 ( Mili ) and Piwil4 ( Miwi2 ) transcripts in RNA seq . data from undifferentiated aNPCs ( Days of differentiation - DIF - 0 ) and differentiating neuroblasts ( DIF 4 - 14 ) ; insets in left and right panels show the same data with smaller scales in the ordinate axes . Data information : data are expressed as mean ± SEM , n = 3 biological replicates .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Levels of Piwil1 (Miwi), Piwil2 (Mili) and Piwil4 (Miwi2) transcripts in RNA seq. data from undifferentiated aNPCs (Days of differentiation -DIF- 0) and differentiating neuroblasts (DIF 4-14); insets in left and right panels show the same data with smaller scales in the ordinate axes. Data information: data are expressed as mean ± SEM, n = 3 biological replicates.


( B ) Western blot ( inset ) and quantification ( bar graph ) of Mili protein abundance in DIF0 aNPCs and differentiating neuroblasts upon viral - induced neurogenesis ( DIF4 - 14 ) . Data information : data are expressed as mean ± SEM , n = 3 biological replicates . * p < 0 . 05 , * * p < 0 . 01 , * * * * p < 0 . 0001 as assessed by one - way ANOVA with Bonferroni

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(B) Western blot (inset) and quantification (bar graph) of Mili protein abundance in DIF0 aNPCs and differentiating neuroblasts upon viral-induced neurogenesis (DIF4-14). Data information: data are expressed as mean ± SEM, n = 3 biological replicates. *p < 0.05, **p < 0.01, **** p < 0.0001 as assessed by one-way ANOVA with Bonferroni test


( C , D ) Western blot ( insets ) and quantification ( bar graphs ) of Miwi and Mili protein abundance in lysates from postnatal mouse testis , hippocampus and undifferentiated aNPCs cultures . Data information : data are expressed as mean ± SEM , n = 3 biological replicates . * p < 0 . 05 , * * p < 0 . 01 , * * * * p < 0 . 0001 as assessed by one - way ANOVA with Bonferroni

(C, D) Western blot (insets) and quantification (bar graphs) of Miwi and Mili protein abundance in lysates from postnatal mouse testis, hippocampus and undifferentiated aNPCs cultures. Data information: data are expressed as mean ± SEM, n = 3 biological replicates. *p < 0.05, **p < 0.01, **** p < 0.0001 as assessed by one-way ANOVA with Bonferroni test


( E ) Western blot ( inset ) and quantification ( bar graph ) of Mili protein abundance in lysates from cultured mouse hippocampal neurons and undifferentiated aNPCs . Data information : data are expressed as mean ± SEM , n = 3 biological replicates . * p < 0 . 05 , * * p < 0 . 01 , * * * * p < 0 . 0001 as assessed by two - tailed Student ' s t - test ( in E ) .

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(E) Western blot (inset) and quantification (bar graph) of Mili protein abundance in lysates from cultured mouse hippocampal neurons and undifferentiated aNPCs. Data information: data are expressed as mean ± SEM, n = 3 biological replicates. *p < 0.05, **p < 0.01, **** p < 0.0001 as assessed by two-tailed Student's t-test (in E).


( F ) Representative immunofluorescence micrograph of Mili ( white ) and Td + NSCs ( red ) in hippocampal subgranular zone ( SGZ ) ; arrows indicate Td + Mili + double - positive cells . Data information : In F : GCL , granular cell layer ; H , Hilus . The scale bars represent 10 µm .

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(F) Representative immunofluorescence micrograph of Mili (white) and Td+ NSCs (red) in hippocampal subgranular zone (SGZ); arrows indicate Td+ Mili + double-positive cells. Data information: In F: GCL, granular cell layer; H, Hilus. The scale bars represent 10 µm.


( G ) Scheme of the experiment ( top ) and Mili mRNA expression in sorted Td + and Td - cells after in vivo transduction with split - Cre viruses in the hippocampus ( bottom ) . Data information : data are expressed as mean ± SEM , n = 3 biological replicates . * p < 0 . 05 , * * p < 0 . 01 , * * * * p < 0 . 0001 as assessed by one - way ANOVA with Bonferroni test G )

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(G) Scheme of the experiment (top) and Mili mRNA expression in sorted Td+ and Td- cells after in vivo transduction with split-Cre viruses in the hippocampus (bottom). Data information: data are expressed as mean ± SEM, n = 3 biological replicates. *p < 0.05, **p < 0.01, **** p < 0.0001 as assessed by one-way ANOVA with Bonferroni test G)


( E ) Mili mRNA expression ( left bar graph ) ; western blot ( inset ) and quantification ( right bar graph ) of Mili protein abundance in aNPCs upon viral transduction of scrambled shRNA ( Control ) or shRNA targeting Mili ( Mili KD ) . Data information : data are expressed as mean ± SEM unless differently indicated , n = 3 biological replicates ∗ p < 0 . 05 , ∗ ∗ p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , as assessed by two - tailed Student ' s t - test .

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(E) Mili mRNA expression (left bar graph); western blot (inset) and quantification (right bar graph) of Mili protein abundance in aNPCs upon viral transduction of scrambled shRNA (Control) or shRNA targeting Mili (Mili KD). Data information: data are expressed as mean ± SEM unless differently indicated, n = 3 biological replicates ∗p < 0.05, ∗∗p < 0.01, *** p < 0.001, **** p < 0.0001, as assessed by two-tailed Student's t-test.


Western blot ( G ) of the endogenous piCS after co - immunoprecipitation ( IP ) with endogenous Mili ( IP Mili ) , or control IgG ( IP IgG ) in lysates of DIF4 aNPCs .

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

Western blot (G) of the endogenous piCS after co-immunoprecipitation (IP) with endogenous Mili (IP Mili), or control IgG (IP IgG) in lysates of DIF4 aNPCs.


( C ) Mili mRNA expression ( left bar graph ) ; western blot ( inset ) and quantification ( right bar graph ) of Mili protein abundance in lysates from the DG of mouse hippocampi 48 hours after the injection of scrambled ( Control ) or GapmeR1 against Mili ( Mili KD ) . Data information : data are expressed as mean ± SEM , n = 3 ( C , biological replicates . ∗ p < 0 . 05 , ∗ ∗ p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , as assessed by two - tailed Student ' s t - test .

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(C) Mili mRNA expression (left bar graph); western blot (inset) and quantification (right bar graph) of Mili protein abundance in lysates from the DG of mouse hippocampi 48 hours after the injection of scrambled (Control) or GapmeR1 against Mili (Mili KD). Data information: data are expressed as mean ± SEM, n = 3 (C, biological replicates. ∗p < 0.05, ∗∗p < 0.01, *** p < 0.001, **** p < 0.0001, as assessed by two-tailed Student's t-test.


( E ) Representative immunofluorescence micrograph of postnatal hippocampal sections immunostained for GFAP at 30 dpi of scrambled ( Control ) and GapmeR1 against Mili ( Mili KD ) ; Right panels : Fold change in GFAP fluorescence intensity level ( upper graph ) in a hippocampal region of interest ( ROI ) of 500µm2 in brain slices upon Mili KD compared to Control ; Gfap mRNA levels ( lower graph ) in the DG from mouse hippocampi 48 hours after the injection of scrambled ( Control ) or GapmeR1 ( Mili KD ) . Data information : data are expressed as mean ± SEM , n = 3 E mRNA and 5 ( E biological replicates . ∗ p < 0 . 05 , ∗ ∗ p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , as assessed by two - tailed Student ' s t - test . SGZ , subgranular zone . The scale bars represent 100 µm ( E ,

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

(E) Representative immunofluorescence micrograph of postnatal hippocampal sections immunostained for GFAP at 30 dpi of scrambled (Control) and GapmeR1 against Mili (Mili KD); Right panels: Fold change in GFAP fluorescence intensity level (upper graph) in a hippocampal region of interest (ROI) of 500µm2 in brain slices upon Mili KD compared to Control; Gfap mRNA levels (lower graph) in the DG from mouse hippocampi 48 hours after the injection of scrambled (Control) or GapmeR1 (Mili KD). Data information: data are expressed as mean ± SEM, n = 3 E mRNA and 5 (E biological replicates. ∗p < 0.05, ∗∗p < 0.01, *** p < 0.001, **** p < 0.0001, as assessed by two-tailed Student's t-test. SGZ, subgranular zone. The scale bars represent 100 µm (E,


( F ) ( Left ) representative immunofluorescence micrograph of postnatal hippocampal sections immunostained for GFAP ( green ) , BrdU ( red ) , NeuN ( white ) and nuclear DNA ( blue ) at 30 dpi of scrambled ( Control ) or GapmeR1 against Mili ( Mili KD ) ; ( right graphs ) percentages of NeuN + BrdU + ( white arrowheads in the images ) , or GFAP + BrdU + ( yellow arrowheads in the images ) double - positive cells over total BrdU + cells . Data information : data are expressed as mean ± SEM , n = F ) biological replicates . ∗ p < 0 . 05 , ∗ ∗ p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , as assessed by two - tailed Student ' s t - test . GCL , granular cell layer , SGZ , subgranular zone . The scale bars represent 100 µm F ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) (Left) representative immunofluorescence micrograph of postnatal hippocampal sections immunostained for GFAP (green), BrdU (red), NeuN (white) and nuclear DNA (blue) at 30 dpi of scrambled (Control) or GapmeR1 against Mili (Mili KD); (right graphs) percentages of NeuN+BrdU+ (white arrowheads in the images), or GFAP+BrdU+ (yellow arrowheads in the images) double-positive cells over total BrdU+ cells. Data information: data are expressed as mean ± SEM, n = F) biological replicates. ∗p < 0.05, ∗∗p < 0.01, *** p < 0.001, **** p < 0.0001, as assessed by two-tailed Student's t-test. GCL, granular cell layer, SGZ, subgranular zone. The scale bars represent 100 µm F).


( B ) Representative bright - field microscopy images ( left ) and quantification ( right ) of ß - galactosidase + aNPCs as percent of total cells upon Mili KD , or control 48 h after induction of spontaneous differentiation . Data information : data are expressed as mean ± SEM , n = 5 ( B biological replicates p < 0 . 05 , ∗ ∗ p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , as assessed by two - tailed Student ' s t - test . The scale bars represent 50 µm ( B ,

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(B) Representative bright-field microscopy images (left) and quantification (right) of ß-galactosidase+ aNPCs as percent of total cells upon Mili KD, or control 48 h after induction of spontaneous differentiation. Data information: data are expressed as mean ± SEM, n = 5 (B biological replicates p < 0.05, ∗∗p < 0.01, *** p < 0.001, **** p < 0.0001, as assessed by two-tailed Student's t-test. The scale bars represent 50 µm (B,


( C ) Representative fluorescence microscopy images ( left ) and quantification ( right ) of control or Mili KD neuroblasts 48h after spontaneous differentiation , immunostained with anti - BrdU ( white ) and Ki67 ( purple ) antibodies . ( Right ) Percentage of BrdU + and Ki67 - cells over BrdU + cells . Data information : data are expressed as mean ± SEM , n = C ) biological replicates p < 0 . 05 , ∗ ∗ p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , as assessed by two - tailed Student ' s t - test . The scale bars represent 50 µm C )

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(C) Representative fluorescence microscopy images (left) and quantification (right) of control or Mili KD neuroblasts 48h after spontaneous differentiation, immunostained with anti-BrdU (white) and Ki67 (purple) antibodies. (Right) Percentage of BrdU+ and Ki67- cells over BrdU+ cells. Data information: data are expressed as mean ± SEM, n = C) biological replicates p < 0.05, ∗∗p < 0.01, *** p < 0.001, **** p < 0.0001, as assessed by two-tailed Student's t-test. The scale bars represent 50 µm C)


F , Chow fed hIGFREO had similar body mass to WT , however , hIGFREO did not gain as much weight as WT after 8 weeks of HFD ( n = 6 - 10 mice per group ) .

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F, Chow fed hIGFREO had similar body mass to WT, however, hIGFREO did not gain as much weight as WT after 8 weeks of HFD (n=6-10 mice per group).


G , Representative images of difference in fat and water distribution shown by magnetic resonance ( MR ) imaging in hIGFREO and WT . Scale bar = 1cm .

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G, Representative images of difference in fat and water distribution shown by magnetic resonance (MR) imaging in hIGFREO and WT. Scale bar = 1cm.


hIGFREO glucose intolerance measured by glucose tolerance test ( n = 5 - 7 mice per group ) .

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hIGFREO glucose intolerance measured by glucose tolerance test (n=5-7 mice per group).


G , H , Percentage change in plasma levels of triglycerides after an olive oil oral gavage was reduced over the 3hr period post gavage in hIGFREO compared to WT and shown as area under the curve ( n = 10 - 12 mice per group ) .

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G, H, Percentage change in plasma levels of triglycerides after an olive oil oral gavage was reduced over the 3hr period post gavage in hIGFREO compared to WT and shown as area under the curve (n=10-12 mice per group).


hIGFREO , food consumption using indirect calorimeter assessment after HFD compared to wildtype littermates ( WT ) after HFD . ( n = 4 per genotype ) .

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hIGFREO , food consumption using indirect calorimeter assessment after HFD compared to wildtype littermates (WT) after HFD. (n=4 per genotype).


D , Adipose expression of browning markers are also no different in white epididymal adipose tissue and brown adipose tissue compared to WT ( n = 6 per genotype ) .

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D, Adipose expression of browning markers are also no different in white epididymal adipose tissue and brown adipose tissue compared to WT (n= 6 per genotype).


Faith ' s phylogenetic diversity ( PD ) was used to measure the fecal microbial diversity and demonstrates a significant difference between human IGF - 1R endothelial overexpressing mice ( hIGFREO ) mice and wildtype littermates ( WT ) mice after high fat diet feeding ( n = 4 - 5 mice per group ) . Chao - 1 analysis was used to measure the fecal microbial diversity and abundance and demonstrates a significant difference between hIGFREO and WT ( n = 4 - 5 mice per group ) .

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Faith's phylogenetic diversity (PD) was used to measure the fecal microbial diversity and demonstrates a significant difference between human IGF-1R endothelial overexpressing mice (hIGFREO) mice and wildtype littermates (WT) mice after high fat diet feeding (n=4-5 mice per group). Chao-1 analysis was used to measure the fecal microbial diversity and abundance and demonstrates a significant difference between hIGFREO and WT (n=4-5 mice per group).


B , Bi , human IGF - 1R endothelial overexpressing mice ( hIGFREO ) had comparable weight gain as wildtype littermates ( WT ) after 8 weeks of HFD + antibiotics ( ABs ) when compared to WT . Bii , both hIGFREO and WT gained significant weight compared to chow fed mice ( n = 7 = 9 mice per group ) .

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B, Bi, human IGF-1R endothelial overexpressing mice (hIGFREO) had comparable weight gain as wildtype littermates (WT) after 8 weeks of HFD + antibiotics (ABs) when compared to WT. Bii, both hIGFREO and WT gained significant weight compared to chow fed mice (n=7=9 mice per group).


D , E , There was no difference in hIGFREO and WT glucose tolerance ( as measured by glucose tolerance test and area under the curve ( AUC ) ) ( n = 7 - 9 mice per group ) .

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D, E, There was no difference in hIGFREO and WT glucose tolerance (as measured by glucose tolerance test and area under the curve (AUC)) (n=7-9 mice per group).


Characteristic micrographs for both data sets , respectively ( left and right ) .

Characteristic micrographs for both data sets, respectively (left and right).


g - h ) Representative β - catenin ChIP - qPCR of indicated target genes in wt and TCF1 / LEF1 long isoform double KO ( DLKO ) clones ( g ) and Kaiso KO clones ( h ) in RPMI8402 cells . Enrichment of each ChIP is normalized to its respective negative IgG control , indicated by a red - dashed line . DLKO1 , DLKO2 , KKO1 and KKO2 designate independent single - cell derived clones . Graph represents the mean and sd of 3 technical replicates .

g-h) Representative β-catenin ChIP-qPCR of indicated target genes in wt and TCF1/LEF1 long isoform double KO (DLKO) clones (g) and Kaiso KO clones (h) in RPMI8402 cells. Enrichment of each ChIP is normalized to its respective negative IgG control, indicated by a red-dashed line. DLKO1, DLKO2, KKO1 and KKO2 designate independent single-cell derived clones. Graph represents the mean and sd of 3 technical replicates.


e ) Relative mRNA expression of β - catenin target genes in the RPMI8402 cell line ( right panel ) after knockdown of β - catenin ( left panel ) compared to control . Graph represents the mean and sd of 6 independent experiments . f ) Relative mRNA expression of β - catenin target genes in the RPMI8402 cell line after treatment with the β - catenin inhibitor ICG - 001 ( 10µM , overnight ) . Graph represents the mean and sd of 3 independent experiments . g ) Relative mRNA expression of β - catenin target genes in the RPMI8402 cell lines ( right panel ) after knockdown of ZBTB33 / Kaiso ( left panel ) compared to control . Graph represents the mean and sd of 3 independent experiments . Data information : Statistical significance was determined by two - sided Student ' s t test .

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e) Relative mRNA expression of β-catenin target genes in the RPMI8402 cell line (right panel) after knockdown of β-catenin (left panel) compared to control. Graph represents the mean and sd of 6 independent experiments. f) Relative mRNA expression of β-catenin target genes in the RPMI8402 cell line after treatment with the β-catenin inhibitor ICG-001 (10µM, overnight). Graph represents the mean and sd of 3 independent experiments. g) Relative mRNA expression of β-catenin target genes in the RPMI8402 cell lines (right panel) after knockdown of ZBTB33/Kaiso (left panel) compared to control. Graph represents the mean and sd of 3 independent experiments. Data information: Statistical significance was determined by two-sided Student's t test.


c ) Kaplan - Meier curves representing disease free ( DSF , top panel ) and overall survival ( OS , bottom panel ) probability for groups of patients P1 ( N = 7 ) , P2 ( N = 5 ) , P3 ( N = 4 ) , P4 ( N = 9 ) and P5 ( N = 15 ) obtained in a ) . Time is represented in years . Statistical significance among groups was determined by log - rank test .

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c) Kaplan-Meier curves representing disease free (DSF, top panel) and overall survival (OS, bottom panel) probability for groups of patients P1 (N = 7), P2 (N = 5), P3 (N = 4), P4 (N = 9) and P5 (N = 15) obtained in a). Time is represented in years. Statistical significance among groups was determined by log-rank test.


a ) Effect of Vincristine ( VCR ) treatment on cell viability of RPMI8402 cells transduced with sh β - catenin or sh - control . Dose - response curves represent the logistic fitting and individual points correspond to mean ± SD of three independent experiments . * * * p ≤ 0 . 001 of IC50 comparison with respect to sh control Data information : For all applicable figure panels , data represent mean ± SD of three independent experiments . Statistical significance was determined by two - sided Student ' s t test for a ( at day 0 )

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a) Effect of Vincristine (VCR) treatment on cell viability of RPMI8402 cells transduced with sh β-catenin or sh-control. Dose-response curves represent the logistic fitting and individual points correspond to mean ± SD of three independent experiments. *** p ≤ 0.001 of IC50 comparison with respect to sh control Data information: For all applicable figure panels, data represent mean ± SD of three independent experiments. Statistical significance was determined by two-sided Student's t test for a (at day 0)


Effect of VCR ( b ) treatment in the presence or absence of increasing doses of the β - catenin inhibitor ICG - 001 . Dose - response curves represent the logistic fitting and individual points correspond to mean ± SD of three independent experiments . * * * * p ≤ 0 . 0001 , * * * p ≤ 0 . 001 , * * p ≤ 0 . 01 , * p ≤ 0 . 05 of IC50 comparison with respect VCR - treated cells . Data information : For all applicable figure panels , data represent mean ± SD of three independent experiments . Statistical significance was determined by two - sided Student ' s t test for , b , ( at day 0 )

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Effect of VCR (b) treatment in the presence or absence of increasing doses of the β-catenin inhibitor ICG-001. Dose-response curves represent the logistic fitting and individual points correspond to mean ± SD of three independent experiments. **** p ≤ 0.0001, *** p ≤ 0.001, ** p ≤ 0.01, * p ≤ 0.05 of IC50 comparison with respect VCR-treated cells. Data information: For all applicable figure panels, data represent mean ± SD of three independent experiments. Statistical significance was determined by two-sided Student's t test for , b, (at day 0)


Methotrexate ( MTX ) ( c left ) , L - Asparaginase ( L - ASP ) ( c central ) or Cytarabine ( Ara - C ) ( c right ) treatment in the presence or absence of increasing doses of the β - catenin inhibitor ICG - 001 . Dose - response curves represent the logistic fitting and individual points correspond to mean ± SD of three independent experiments . * * * * p ≤ 0 . 0001 , * * * p ≤ 0 . 001 , * * p ≤ 0 . 01 , * p ≤ 0 . 05 of IC50 Data information : For all applicable figure panels , data represent mean ± SD of three independent experiments . Statistical significance was determined by two - sided Student ' s t test for c , ( at day 0 )

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Methotrexate (MTX) (c left), L-Asparaginase (L-ASP) (c central) or Cytarabine (Ara-C) (c right) treatment in the presence or absence of increasing doses of the β-catenin inhibitor ICG-001. Dose-response curves represent the logistic fitting and individual points correspond to mean ± SD of three independent experiments. **** p ≤ 0.0001, *** p ≤ 0.001, ** p ≤ 0.01, * p ≤ 0.05 of IC50 Data information: For all applicable figure panels, data represent mean ± SD of three independent experiments. Statistical significance was determined by two-sided Student's t test for c, (at day 0)


c ) Percentage of RPMI8402 cells PB , BM , spleen and liver at sacrifice . RPMI8402 cells identified as human CD45 + HLA - ABC + CD5 + by flow cytometry and gated on DAPI negative cells . Control : animals left untreated ( n = 2 ) ; Chemo + DMSO : animals receiving chemotherapy and vehicle ( n = 4 ) ; Chemo + ICG - 001 : animals receiving chemotherapy and the β - catenin inhibitor ICG - 001 ( n = 4 ) Data information : For all applicable figure panels , data from Chemo + DMSO and Chemo + ICG - 001 groups are mean ± SD and dots represent individual values per animal . Control group data is only represented by individual values per animal . Statistical significance between Chemo + DMSO and Chemo + ICG - 001 groups was determined by two - sided Student ' s t test .

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c) Percentage of RPMI8402 cells PB, BM, spleen and liver at sacrifice. RPMI8402 cells identified as human CD45+HLA-ABC+CD5+ by flow cytometry and gated on DAPI negative cells. Control: animals left untreated (n = 2); Chemo + DMSO: animals receiving chemotherapy and vehicle (n = 4); Chemo + ICG-001: animals receiving chemotherapy and the β-catenin inhibitor ICG-001 (n = 4) Data information: For all applicable figure panels, data from Chemo + DMSO and Chemo + ICG-001 groups are mean ± SD and dots represent individual values per animal. Control group data is only represented by individual values per animal. Statistical significance between Chemo + DMSO and Chemo + ICG-001 groups was determined by two-sided Student's t test.


g ) H & E staining of representative images per each mouse treated as indicated . h ) Percentage of liver area covered by cellular colonies per 10X micrograph . 4 different images were quantified per animal and mean values per animal are represented by dots . Control : animals left untreated ( n = 2 ) ; Chemo + DMSO : animals receiving chemotherapy and vehicle ( n = 4 ) ; Chemo + ICG - 001 : animals receiving chemotherapy and the β - catenin inhibitor ICG - 001 ( n = 4 ) Data information : For all applicable figure panels , data from Chemo + DMSO and Chemo + ICG - 001 groups are mean ± SD and dots represent individual values per animal . Control group data is only represented by individual values per animal . Statistical significance between Chemo + DMSO and Chemo + ICG - 001 groups was determined by two - sided Student ' s t test .

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g) H&E staining of representative images per each mouse treated as indicated. h) Percentage of liver area covered by cellular colonies per 10X micrograph. 4 different images were quantified per animal and mean values per animal are represented by dots. Control: animals left untreated (n = 2); Chemo + DMSO: animals receiving chemotherapy and vehicle (n = 4); Chemo + ICG-001: animals receiving chemotherapy and the β-catenin inhibitor ICG-001 (n = 4) Data information: For all applicable figure panels, data from Chemo + DMSO and Chemo + ICG-001 groups are mean ± SD and dots represent individual values per animal. Control group data is only represented by individual values per animal. Statistical significance between Chemo + DMSO and Chemo + ICG-001 groups was determined by two-sided Student's t test.


Representative micrographs of a time - lapse microscopy of K and KM non - regressed tumour cells ( H2B - GFP green ) . Top : Mitotic cell with a chromatin bridge ( yellow arrow ) . Bottom : Mitotic cell with misalignment and cytokinesis failure resulting in binucleation ( yellow arrow ) .

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Representative micrographs of a time-lapse microscopy of K and KM non-regressed tumour cells (H2B-GFP green). Top: Mitotic cell with a chromatin bridge (yellow arrow). Bottom: Mitotic cell with misalignment and cytokinesis failure resulting in binucleation (yellow arrow).


Percentage of mitotic errors in K and KM primary tumours and in K and KM non - regressed tumours . Percentage of cells in K and KM primary tumours and in K and KM non - regressed tumours with the indicated mitotic errors . Scale bar 20 µm .

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Percentage of mitotic errors in K and KM primary tumours and in K and KM non-regressed tumours. Percentage of cells in K and KM primary tumours and in K and KM non-regressed tumours with the indicated mitotic errors. Scale bar 20 µm.


Somatic copy number alterations in each individual Kras and Kras / Mad2 non - regressed tumours . 7 K non - regressed and 16 KM non - regressed tumours were sequenced . Focal deletion ( DEL , green ) , Focal amplification ( AMP , light green ) , whole chromosome gain ( WCG , dark grey ) and loss ( WCL , light grey ) , partial chromosome gain ( PCG , dark blue ) and loss ( PCL , light blue ) and gross chromosomal rearrangement ( GCR , red ) .

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Somatic copy number alterations in each individual Kras and Kras/Mad2 non-regressed tumours. 7 K non-regressed and 16 KM non-regressed tumours were sequenced. Focal deletion (DEL, green), Focal amplification (AMP, light green), whole chromosome gain (WCG, dark grey) and loss (WCL, light grey), partial chromosome gain (PCG, dark blue) and loss (PCL, light blue) and gross chromosomal rearrangement (GCR, red).