MEF cells were infected for 3 h and transfected with empty plasmid ( CTRL ) , hNDP52 , hLC3 ‐ C or hNDP52 and hLC3 ‐ C . Viral production ( E ) and cell mortality ( F ) were assessed 24 h post transfection . Isogenic wt or Atg5 − / − MEFs were infected for 24 h and cell mortality ( that is , fold change relative to mock ‐ infected cells ) ( G ) and viral production ( H ) were assessed . MEF cells were infected and immunoprecipitation experiments were performed 24 h p . i . using antibodies to p62 or nonspecific IgG controls . Immunoprecipitated proteins were revealed with anti ‐ capsid antibody ( I ) .

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

MEF cells were infected for 3 h and transfected with empty plasmid (CTRL), hNDP52, hLC3‐C or hNDP52 and hLC3‐C. Viral production (E) and cell mortality (F) were assessed 24 h post transfection. Isogenic wt or Atg5−/− MEFs were infected for 24 h and cell mortality (that is, fold change relative to mock‐infected cells) (G) and viral production (H) were assessed. MEF cells were infected and immunoprecipitation experiments were performed 24 h p.i. using antibodies to p62 or nonspecific IgG controls. Immunoprecipitated proteins were revealed with anti‐capsid antibody (I).


MEF cells were treated with CTRL or p62 siRNA , then infected for 24 h . Cell mortality ( that is , fold change relative to mock ‐ infected cells ) ( J ) and viral production were assessed . Whole ‐ cell lysates of siRNA ‐ treated cells were immunoblotted for p62 or actin ( K ) .

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MEF cells were treated with CTRL or p62 siRNA, then infected for 24 h. Cell mortality (that is, fold change relative to mock‐infected cells) (J) and viral production were assessed. Whole‐cell lysates of siRNA‐treated cells were immunoblotted for p62 or actin (K).


TRIP6 promotes YAP activity by inhibiting LATS1 / 2 ( A ) Full length ( WT ) , the amino - terminal half ( 1 - 277 ) , or the carboxy - terminal half ( 278 - 476 ) of TRIP6 were tested for binding to LATS2 by immunoprecipitation . FLAG - TRIP6 variants were co - expressed with LATS2 - GFP in HEK293 cells , anti - FLAG or control ( IgG ) antibodies were used to isolate immune complexes . Immune complexes and lysates were probed by western blotting for LATS2 - GFP and FLAG - TRIP6 . Schematic diagram depicts TRIP6 domains ( NES : Nuclear Export Signal ; LIM : LIM domain ; PDZ : PDZ domain binding motif ) .

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

TRIP6 promotes YAP activity by inhibiting LATS1/2 (A) Full length (WT), the amino-terminal half (1-277), or the carboxy-terminal half (278-476) of TRIP6 were tested for binding to LATS2 by immunoprecipitation. FLAG-TRIP6 variants were co-expressed with LATS2-GFP in HEK293 cells, anti-FLAG or control (IgG) antibodies were used to isolate immune complexes. Immune complexes and lysates were probed by western blotting for LATS2-GFP and FLAG-TRIP6. Schematic diagram depicts TRIP6 domains (NES: Nuclear Export Signal; LIM: LIM domain; PDZ: PDZ domain binding motif).


( B ) Lysates from MCF10A cells were subjected to immunoprecipitation using anti - TRIP6 or control ( IgG ) antibodies , and immune complexes and lysates were probed for TRIP6 and LATS1 .

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(B) Lysates from MCF10A cells were subjected to immunoprecipitation using anti-TRIP6 or control (IgG) antibodies, and immune complexes and lysates were probed for TRIP6 and LATS1.


( C ) FLAG - TRIP6 was tested for binding to various LATS2 - GFP deletion mutants as described in part ( A ) . Schematic diagram of LATS2 shows MOB1 binding domain , and the autophosphorylation ( S872 ) and MST1 / 2 phosphorylation sites ( T1041 ) in the kinase domain .

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(C) FLAG-TRIP6 was tested for binding to various LATS2-GFP deletion mutants as described in part (A). Schematic diagram of LATS2 shows MOB1 binding domain, and the autophosphorylation (S872) and MST1/2 phosphorylation sites (T1041) in the kinase domain.


The regions marked in green depict TRIP6 binding sites on LATS2 ( D ) Lysates from HEK293A cells transfected with control or FLAG - TRIP6 plasmid were analyzed by western blotting using the indicated antibodies ( quantification is shown in ( F ) ) .

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The regions marked in green depict TRIP6 binding sites on LATS2 (D) Lysates from HEK293A cells transfected with control or FLAG-TRIP6 plasmid were analyzed by western blotting using the indicated antibodies (quantification is shown in (F)).


( E ) Lysates from control ( WT ) or CRISPR generated TRIP6 null ( TRIP6 - KO ) HEK293A cells were analyzed by western blotting using the indicated antibodies ( quantification shown in ( G ) ) .

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(E) Lysates from control (WT) or CRISPR generated TRIP6 null (TRIP6-KO) HEK293A cells were analyzed by western blotting using the indicated antibodies (quantification shown in (G)).


( F ) The relative levels of LATS1 activating phosphorylation ( pLATS1 - 1079 , 909 ) and YAP S127 inhibitory phosphorylation from ( D ) were measured relative to LATS1 and YAP levels respectively . ( Mean ± SD ; n = 3 ; * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 , T - test )

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(F) The relative levels of LATS1 activating phosphorylation (pLATS1-1079, 909) and YAP S127 inhibitory phosphorylation from (D) were measured relative to LATS1 and YAP levels respectively. (Mean ± SD; n=3; **P≤0.01, ***P≤0.001, T-test)


( G ) The levels of LATS1 activating phosphorylation and YAP inactivating phosphorylation from part ( E ) were quantified ( Mean ± SD ; n = 3 ; * P ≤ 0 . 05 , T - test ) .

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(G) The levels of LATS1 activating phosphorylation and YAP inactivating phosphorylation from part (E) were quantified (Mean ± SD; n=3; *P≤0.05, T-test).


( H ) GFP - MST2 was expressed with or without FLAG - TRIP6 in HEK293 cells and the levels of MST2 , MST2 activating phosphorylation ( pMST2 - T180 ) , and FLAG - TRIP6 were measured by western blotting with the indicated antibodies . ( Mean ± SD ; n = 3 ; n . s . ≥ 0 . 05 , T - test ) .

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(H) GFP-MST2 was expressed with or without FLAG-TRIP6 in HEK293 cells and the levels of MST2, MST2 activating phosphorylation (pMST2-T180), and FLAG-TRIP6 were measured by western blotting with the indicated antibodies. (Mean ± SD; n=3; n.s.≥0.05, T-test).


( I ) TRIP6 was overexpressed in HEK293A cells and the levels of TRIP6 , and YAP target gene expression was analyzed using RT - qPCR . ( Mean ± SD ; n = 3 ; * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * * P ≤ 0 . 0001 , T - test ) .

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(I) TRIP6 was overexpressed in HEK293A cells and the levels of TRIP6, and YAP target gene expression was analyzed using RT-qPCR. (Mean ± SD; n=3; *P≤0.05, **P≤0.01, ****P≤0.0001, T-test).


( J ) The levels of YAP target gene expression was analyzed using RT - qPCR in control ( WT ) and TRIP6 - KO HEK293A cells . ( Mean ± SD ; n = 3 ; * * * P ≤ 0 . 001 , T - test )

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(J) The levels of YAP target gene expression was analyzed using RT-qPCR in control (WT) and TRIP6-KO HEK293A cells. (Mean ± SD; n=3; ***P≤0.001, T-test)


( K ) Control ( WT ) and TRIP6 - KO HEK293A cells were stained for YAP and TRIP6 . Merged image shows YAP ( green ) , TRIP6 ( red ) , and DNA ( blue ) . Quantification of at least 100 cells is shown ( Mean ± SD ; n = 3 ; * * * * P ≤ 0 . 0001 , Fisher ' s test ) . Scale bar = 20µm .

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(K) Control (WT) and TRIP6-KO HEK293A cells were stained for YAP and TRIP6. Merged image shows YAP (green), TRIP6 (red), and DNA (blue). Quantification of at least 100 cells is shown (Mean ± SD; n=3; ****P≤0.0001, Fisher's test). Scale bar=20µm.


( A ) LATS2 - GFP and Myc - MOB1A were overexpressed in HEK293 cells with or without co - overexpression of full length FLAG - TRIP6 and FLAG - TRIP6 1 - 277 . Myc - MOB1A was immunoprecipitated using anti - Myc antibodies and immune complexes were assayed for Myc - MOB1A and LATS2 - GFP levels . Levels of FLAG - TRIP6 , FLAG - TRIP6 1 - 277 , Myc - MOB1A , and LATS2 - GFP in the lysate are also shown . The levels of LATS2 - GFP in immune complexes relative to the level of Myc - MOB1A are shown in the graph ( Mean ± SD ; n = 3 ; * * P ≤ 0 . 01 , T - test )

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

(A) LATS2-GFP and Myc-MOB1A were overexpressed in HEK293 cells with or without co-overexpression of full length FLAG-TRIP6 and FLAG-TRIP6 1-277. Myc-MOB1A was immunoprecipitated using anti-Myc antibodies and immune complexes were assayed for Myc-MOB1A and LATS2-GFP levels. Levels of FLAG-TRIP6, FLAG-TRIP6 1-277, Myc-MOB1A, and LATS2-GFP in the lysate are also shown. The levels of LATS2-GFP in immune complexes relative to the level of Myc-MOB1A are shown in the graph (Mean ± SD; n=3; **P≤0.01, T-test)


( B ) Competitive binding experiments were done using purified recombinant MBP - LATS2 , GST - TRIP6 , and 6His - MOB1A . MBP - LATS2 bound to maltose beads was incubated with GST - TRIP6 with or without increasing amounts of 6His - MOB1A , and the levels of each protein bound to MBP - LATS2 on the beads at the end of the experiment was determined by western blotting . The levels of input proteins are shown ( lysate ) . The binding of 6His - MOB1A and GST - TRIP6 to MBP alone , and the use BSA as a competitor instead of 6His - MOB1A are shown as controls . The numbers at the bottom are referred to in the text .

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

(B) Competitive binding experiments were done using purified recombinant MBP-LATS2, GST-TRIP6, and 6His-MOB1A. MBP-LATS2 bound to maltose beads was incubated with GST-TRIP6 with or without increasing amounts of 6His-MOB1A, and the levels of each protein bound to MBP-LATS2 on the beads at the end of the experiment was determined by western blotting. The levels of input proteins are shown (lysate). The binding of 6His-MOB1A and GST-TRIP6 to MBP alone, and the use BSA as a competitor instead of 6His-MOB1A are shown as controls. The numbers at the bottom are referred to in the text.


( A ) MCF10A cells were infected with lentivirus carrying control shRNA ( shEGFP ) , or a mix of two different shRNA against TRIP6 ( shTRIP6 - 1 and shTRIP6 - 4 ) and were stained for TRIP6 and LATS1 . Merged images show LATS1 ( green ) , TRIP6 ( red ) and DNA ( blue ) ( quantification of LATS1 localization at cell - cell junctions is shown in Figure EV3D ) . Scale bar = 20µm .

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

(A) MCF10A cells were infected with lentivirus carrying control shRNA (shEGFP), or a mix of two different shRNA against TRIP6 (shTRIP6-1 and shTRIP6-4) and were stained for TRIP6 and LATS1. Merged images show LATS1 (green), TRIP6 (red) and DNA (blue) (quantification of LATS1 localization at cell-cell junctions is shown in Figure EV3D). Scale bar=20µm.


( B ) LATS1 and LATS2 were knocked down using siLATS1 and siLATS2 SMARTPools in MCF10A cells . MCF10A control cells were treated with control siRNA ( siControl ) against fire fly luciferase . Cells were stained for TRIP6 and LATS1 as in ( A ) ( quantification of TRIP6 localization at cell - cell junctions is shown in Figure EV3E ) . Scale bar = 20µm .

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 B-MEASURED_VAR O B-MEASURED_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 O O O O

(B) LATS1 and LATS2 were knocked down using siLATS1 and siLATS2 SMARTPools in MCF10A cells. MCF10A control cells were treated with control siRNA (siControl) against fire fly luciferase. Cells were stained for TRIP6 and LATS1 as in (A) (quantification of TRIP6 localization at cell-cell junctions is shown in Figure EV3E). Scale bar=20µm.


( C ) MCF10A cells were grown to high density , and were stained for TRIP6 and LATS1 as in ( A ) . Scale bar = 20µm .

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(C) MCF10A cells were grown to high density, and were stained for TRIP6 and LATS1 as in (A). Scale bar=20µm.


( D ) Cells were grown as in ( C ) , then lysed and anti - TRIP6 antibodies were used to isolate immune complexes . Immune complexes and lysates were probed by western blotting for LATS1 and TRIP6 . Quantification is shown . ( Mean ± SD ; n = 3 ; * P ≤ 0 . 05 , T - test ) .

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(D) Cells were grown as in (C), then lysed and anti-TRIP6 antibodies were used to isolate immune complexes. Immune complexes and lysates were probed by western blotting for LATS1 and TRIP6. Quantification is shown. (Mean ± SD; n=3; *P≤0.05, T-test).


( E ) MCF10A cells grown at high density on PDMS membranes and were stretched ( or not ) at 17 % elongation for 2 hours and lysed while under tension . Anti - TRIP6 antibodies were used to isolate immune complexes . Immune complexes and lysates were probed by western blotting for LATS1 and TRIP6 . Quantification is shown . ( Mean ± SD ; n = 3 ; * P ≤ 0 . 05 , T - test ) .

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

(E) MCF10A cells grown at high density on PDMS membranes and were stretched (or not) at 17% elongation for 2 hours and lysed while under tension. Anti-TRIP6 antibodies were used to isolate immune complexes. Immune complexes and lysates were probed by western blotting for LATS1 and TRIP6. Quantification is shown. (Mean ± SD; n=3; *P≤0.05, T-test).


( F ) Cells were treated as in ( E ) , fixed while under tension , and stained for TRIP6 and LATS1 as in ( A ) . Scale bar = 20µm .

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(F) Cells were treated as in (E), fixed while under tension, and stained for TRIP6 and LATS1 as in (A). Scale bar=20µm.


( G ) MCF10A cells infected with lentivirus carrying control shRNA ( shEGFP ) , or a mix of two different shRNA against TRIP6 ( shTRIP6 - 1 and shTRIP6 - 4 ) , were grown at high density on PDMS membranes and were stretched or not ( only stretched cells shown ) at 17 % elongation for 2 hours , fixed while under tension , and were stained for TRIP6 and LATS1 as in ( A ) . Scale bar = 20µm .

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(G) MCF10A cells infected with lentivirus carrying control shRNA (shEGFP), or a mix of two different shRNA against TRIP6 (shTRIP6-1 and shTRIP6-4), were grown at high density on PDMS membranes and were stretched or not (only stretched cells shown) at 17% elongation for 2 hours, fixed while under tension, and were stained for TRIP6 and LATS1 as in (A). Scale bar=20µm.


( H ) Cells were treated as in ( G ) and YAP target gene ( CTGF and Cyr61 ) and TRIP6 expression were analyzed using RT - qPCR . ( Mean ± SD ; n = 3 ; * P ≤ 0 . 05 , * * P ≤ 0 . 01 , T - test ) .

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(H) Cells were treated as in (G) and YAP target gene (CTGF and Cyr61) and TRIP6 expression were analyzed using RT-qPCR. (Mean ± SD; n=3; *P≤0.05, **P≤0.01, T-test).


( I ) TRIP6 expression levels in ( H ) in control ( shEGFP ) and TRIP6 ( shTRIP6 ) knockdown cells just prior to stretch were analyzed using RT - qPCR . ( Mean ± SD ; n = 3 ; * P ≤ 0 . 05 , T - test ) .

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(I) TRIP6 expression levels in (H) in control (shEGFP) and TRIP6 (shTRIP6) knockdown cells just prior to stretch were analyzed using RT-qPCR. (Mean ± SD; n=3; *P≤0.05, T-test).


( A ) MCF10A cells were either not treated or treated separately with Latrunculin B , Blebbistatin , EGTA , serum starvation , and Y27632 , and were stained for TRIP6 and LATS1 . Merged images show LATS1 ( green ) , TRIP6 ( red ) and DNA ( blue ) . Scale bar = 20µm .

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(A) MCF10A cells were either not treated or treated separately with Latrunculin B, Blebbistatin, EGTA, serum starvation, and Y27632, and were stained for TRIP6 and LATS1. Merged images show LATS1 (green), TRIP6 (red) and DNA (blue). Scale bar=20µm.


( B ) Cells were treated as in ( A ) , then lysed and anti - TRIP6 antibodies were used to isolate immune complexes . Immune complexes and lysates were probed by western blotting for LATS1 and TRIP6 .

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(B) Cells were treated as in (A), then lysed and anti-TRIP6 antibodies were used to isolate immune complexes. Immune complexes and lysates were probed by western blotting for LATS1 and TRIP6.


( A ) MCF10A cells were lysed and anti - TRIP6 or control ( IgG ) antibodies were used to isolate immune complexes . Immune complexes and lysates were probed by western blotting for vinculin and TRIP6 .

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(A) MCF10A cells were lysed and anti-TRIP6 or control (IgG) antibodies were used to isolate immune complexes. Immune complexes and lysates were probed by western blotting for vinculin and TRIP6.


( B ) MCF10A cells were either not treated ( control ) or treated separately by growth to high density , serum starvation , Latrunculin B , Blebbistatin , EGTA , or Y27632 treatment , and stained using anti - vinculin antibodies by immunofluorescence . Scale bar = 20µm .

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(B) MCF10A cells were either not treated (control) or treated separately by growth to high density, serum starvation, Latrunculin B, Blebbistatin, EGTA, or Y27632 treatment, and stained using anti-vinculin antibodies by immunofluorescence. Scale bar=20µm.


( C ) MCF10A cells grown at high density on PDMS membranes and were stretched ( or not ) at 17 % elongation for 2 hours and fixed while under tension and stained for vinculin . Scale bar = 20µm .

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(C) MCF10A cells grown at high density on PDMS membranes and were stretched (or not) at 17% elongation for 2 hours and fixed while under tension and stained for vinculin. Scale bar=20µm.


( D ) Vinculin was knocked down using two different siRNAs or control siRNA in MCF10A cells and the cell lysates were probed by western blotting for phospho - LATS1 ( T1079 and S909 ) , phospho - YAP ( S127 ) , LATS1 , YAP , vinculin , and tubulin antibodies and the relative levels quantified ( Mean ± SD ; n = 3 ; * * P ≤ 0 . 01 , T - test ) .

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(D) Vinculin was knocked down using two different siRNAs or control siRNA in MCF10A cells and the cell lysates were probed by western blotting for phospho-LATS1 (T1079 and S909), phospho-YAP (S127), LATS1, YAP, vinculin, and tubulin antibodies and the relative levels quantified (Mean ± SD; n=3; **P≤0.01, T-test).


( E ) Vinculin was knocked down as described in ( D ) and cells were stained for YAP and vinculin . Merged image shows YAP ( green ) , vinculin ( red ) , and DNA ( blue ) . Quantification of at least 100 cells is shown ( Mean ± SD ; n = 3 ; * * * P ≤ 0 . 001 , Fisher ' s test ) . Scale bar = 20µm .

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(E) Vinculin was knocked down as described in (D) and cells were stained for YAP and vinculin. Merged image shows YAP (green), vinculin (red), and DNA (blue). Quantification of at least 100 cells is shown (Mean ± SD; n=3; ***P≤0.001, Fisher's test). Scale bar=20µm.


( F ) Vinculin was knocked down as described in ( D ) and the levels of vinculin and YAP target gene expression was analyzed using RT - qPCR ( Mean ± SD ; n = 3 ; * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 , T - test ) .

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(F) Vinculin was knocked down as described in (D) and the levels of vinculin and YAP target gene expression was analyzed using RT-qPCR (Mean ± SD; n=3; ***P≤0.001, ****P≤0.0001, T-test).


( G ) Vinculin was knocked down in MCF10A cells as described in ( D ) , grown at high density on PDMS membranes and cells were stretched ( or not ) at 17 % elongation for 2 hours and the levels of vinculin and YAP target gene expression was analyzed using RT - qPCR ( Mean ± SD ; n = 3 ; * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 , T - test ) .

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(G) Vinculin was knocked down in MCF10A cells as described in (D), grown at high density on PDMS membranes and cells were stretched (or not) at 17% elongation for 2 hours and the levels of vinculin and YAP target gene expression was analyzed using RT-qPCR (Mean ± SD; n=3; ***P≤0.001, ****P≤0.0001, T-test).


( A ) Vinculin was knocked down as described ( Figure 5D ) in MCF10A cells and cells were stained for LATS1 and TRIP6 . Merged images show LATS1 ( green ) , TRIP6 ( red ) and DNA ( blue ) . Scale bar = 20µm .

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(A) Vinculin was knocked down as described (Figure 5D) in MCF10A cells and cells were stained for LATS1 and TRIP6. Merged images show LATS1 (green), TRIP6 (red) and DNA (blue). Scale bar=20µm.


( B ) Vinculin was knocked down as described ( Figure 5D ) and TRIP6 immune complexes and lysates were probed by western blotting for LATS1 , TRIP6 , and vinculin and the relative levels quantified ( Mean ± SD ; n = 3 ; * * P ≤ 0 . 01 , T - test ) .

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(B) Vinculin was knocked down as described (Figure 5D) and TRIP6 immune complexes and lysates were probed by western blotting for LATS1, TRIP6, and vinculin and the relative levels quantified (Mean ± SD; n=3; **P≤0.01, T-test).


( C ) MCF10A cells were subjected to different treatments as described ( Figure 4A ) and TRIP6 immune complexes and lysates were probed by western blotting for vinculin and TRIP6 .

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(C) MCF10A cells were subjected to different treatments as described (Figure 4A) and TRIP6 immune complexes and lysates were probed by western blotting for vinculin and TRIP6.


( D ) Quantification of TRIP6 - vinculin binding from part ( C ) is shown . ( Mean ± SD ; n = 3 ; * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 , T - test ) .

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(D) Quantification of TRIP6-vinculin binding from part (C) is shown. (Mean ± SD; n=3; *P≤0.05, **P≤0.01, ***P≤0.001, T-test).


( E , F ) MCF10A cells were treated separately ( or not ) with Latrunculin B ( E ) and by serum starvation ( F ) and LATS1 immune complexes and lysates were probed by western blotting for vinculin , LATS1 , and TRIP6 . Quantification of LATS1 - TRIP6 - vinculin binding is shown . ( Mean ± SD ; n = 3 ; * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 , T - test ) .

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(E, F) MCF10A cells were treated separately (or not) with Latrunculin B (E) and by serum starvation (F) and LATS1 immune complexes and lysates were probed by western blotting for vinculin, LATS1, and TRIP6. Quantification of LATS1-TRIP6-vinculin binding is shown. (Mean ± SD; n=3; *P≤0.05, **P≤0.01, ***P≤0.001, T-test).


( G ) Wild type and T12 mutant of vinculin were co - expressed with LATS2 and FLAG - TRIP6 and anti - FLAG antibody was used to isolate immune complexes . Immune complexes and lysates were probed by western blotting for vinculin , LATS2 and FLAG - TRIP6 . Quantification of LATS2 - TRIP6 - vinculin ( WT or T12 ) binding is shown . ( Mean ± SD ; n = 3 ; * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 , T - test ) .

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(G) Wild type and T12 mutant of vinculin were co-expressed with LATS2 and FLAG-TRIP6 and anti-FLAG antibody was used to isolate immune complexes. Immune complexes and lysates were probed by western blotting for vinculin, LATS2 and FLAG-TRIP6. Quantification of LATS2-TRIP6-vinculin (WT or T12) binding is shown. (Mean ± SD; n=3; *P≤0.05, **P≤0.01, ***P≤0.001, T-test).


( H ) Vinculin was knocked down as described ( Figure 5D ) in MCF10A cells grown at high density on PDMS membranes . The membranes were stretched at 17 % elongation for 2 hours and fixed while under tension and stained for TRIP6 . Merged images show TRIP6 ( red ) and DNA ( blue ) . Scale bar = 20µm .

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(H) Vinculin was knocked down as described (Figure 5D) in MCF10A cells grown at high density on PDMS membranes. The membranes were stretched at 17% elongation for 2 hours and fixed while under tension and stained for TRIP6. Merged images show TRIP6 (red) and DNA (blue). Scale bar=20µm.


a , Survival curves of wild type ( n = 27 ) and Cdc14 DKO ( n = 51 ) mice during the first 6 weeks of life . ( * * * , p < 0 . 001 ; Log - rank , Mantel - Cox test ) . b , Representative images of postnatal day 9 Cdc14 DKO and wild - type littermates . c , Weight of Cdc14 DKO and wild - type littermates during the first 3 weeks of life ( n = 4 per genotype ) . Data information : Bars indicate mean ± SEM . p > 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 , Student ' s t - test with Welch ' s correction .

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a, Survival curves of wild type (n=27) and Cdc14 DKO (n=51) mice during the first 6 weeks of life. (***, p<0.001; Log-rank, Mantel-Cox test). b, Representative images of postnatal day 9 Cdc14 DKO and wild-type littermates. c, Weight of Cdc14 DKO and wild-type littermates during the first 3 weeks of life (n=4 per genotype). Data information: Bars indicate mean ± SEM. p>0.05; **, p<0.01; ***, p<0.001, Student's t-test with Welch's correction.


d , Representative macroscopic pictures of brain dorsal view of wild type ( WT ) and Cdc14 KO mice at postnatal 0 , 5 and 9 days ( P0 , P5 and P9 ) . Scale bar , 2 mm . The bar plots show the quantification of brain width , length and area , respectively . Data information : Bars indicate mean ± SEM . , * , p > 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 , Student ' s t - test with Welch ' s correction . Non - significant data ( p > 0 . 05 ) are not labeled . In data show the individual data , average and SEM from 3 WT and 3 DKO mice .

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d, Representative macroscopic pictures of brain dorsal view of wild type (WT) and Cdc14 KO mice at postnatal 0, 5 and 9 days (P0, P5 and P9). Scale bar, 2 mm. The bar plots show the quantification of brain width, length and area, respectively. Data information: Bars indicate mean ± SEM. , *, p>0.05; **, p<0.01; ***, p<0.001, Student's t-test with Welch's correction. Non-significant data (p>0.05) are not labeled.In data show the individual data, average and SEM from 3 WT and 3 DKO mice.


e , Representative cresyl violet staining images of midsagittal sections of cerebellar vermis of WT and Cdc14 KO mice at P0 , P5 and P9 . Fissures underdeveloped in mutant samples are labeled with arrowheads . Scale bar , 700 μm . The bar plots show the quantification of the cerebellar vermis area , the number of folia and the average length of the fissures , respectively . Data information : Bars indicate mean ± SEM . p > 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 , Student ' s t - test with Welch ' s correction . Non - significant data ( p > 0 . 05 ) are not labeled . In data show the individual data , average and SEM from 3 WT and 3 DKO mice .

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e, Representative cresyl violet staining images of midsagittal sections of cerebellar vermis of WT and Cdc14 KO mice at P0, P5 and P9. Fissures underdeveloped in mutant samples are labeled with arrowheads. Scale bar, 700 μm. The bar plots show the quantification of the cerebellar vermis area, the number of folia and the average length of the fissures, respectively. Data information: Bars indicate mean ± SEM. p>0.05; **, p<0.01; ***, p<0.001, Student's t-test with Welch's correction. Non-significant data (p>0.05) are not labeled.In data show the individual data, average and SEM from 3 WT and 3 DKO mice.


f , Detailed cresyl violet staining images of midsagittal sections of cerebellar vermis of the same samples . The molecular layer ( ML ) , internal granule cell layer ( IGL ) and external granule cell layer ( EGL ) are indicated . The width of these layers is shown in the bar plots . Data information : Bars indicate mean ± SEM . * , p > 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 , Student ' s t - test with Welch ' s correction . Non - significant data ( p > 0 . 05 ) are not labeled . In data show the individual data , average and SEM from 3 WT and 3 DKO mice . In f , each point represents the average value of 9 different random measurements across distinctive folia for each mouse .

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f, Detailed cresyl violet staining images of midsagittal sections of cerebellar vermis of the same samples. The molecular layer (ML), internal granule cell layer (IGL) and external granule cell layer (EGL) are indicated. The width of these layers is shown in the bar plots. Data information: Bars indicate mean ± SEM. *, p>0.05; **, p<0.01; ***, p<0.001, Student's t-test with Welch's correction. Non-significant data (p>0.05) are not labeled.In data show the individual data, average and SEM from 3 WT and 3 DKO mice. In f, each point represents the average value of 9 different random measurements across distinctive folia for each mouse.


g , Representative images of misdigital sections stained with DAPI ( blue ) , Calbindin ( green ) and GFAP ( red ) of cerebellar vermis of wild - type ad Cdc14 DKO mice at 9 days after birth . Scale bar , 50 μm . Plots show the quantification of Purkinje cell dendritic length ( average distance of 30 random measurements ) and the number of cells per 100 μm from 3 different fields from 3 different mice in each plot . Data information : Bars indicate mean ± SEM . , * , p > 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 , Student ' s t - test with Welch ' s correction .

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g, Representative images of misdigital sections stained with DAPI (blue), Calbindin (green) and GFAP (red) of cerebellar vermis of wild-type ad Cdc14 DKO mice at 9 days after birth. Scale bar, 50 μm. Plots show the quantification of Purkinje cell dendritic length (average distance of 30 random measurements) and the number of cells per 100 μm from 3 different fields from 3 different mice in each plot. Data information: Bars indicate mean ± SEM. , *, p>0.05; **, p<0.01; ***, p<0.001, Student's t-test with Welch's correction.


a . Confocal microscopy analysis of wild - type and Cdc14 DKO embryonic stem cells ( ESCs ) subjected to neural differentiation . Neuronal marker TUJ1 ( green ) , glial marker GFAP ( red ) and nuclear marker DAPI ( blue ) are stained and quantified after 18 days in differentiation media . Each dot represents the ratio between TUJ or GFAP and DAPI area in an image containing at least 100 cells from 4 independent experiments with two different clones . * * * * p - value < 0 . 0001 . Student ' s t - test . Scale bar 50 μm .

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a. Confocal microscopy analysis of wild-type and Cdc14 DKO embryonic stem cells (ESCs) subjected to neural differentiation. Neuronal marker TUJ1 (green), glial marker GFAP (red) and nuclear marker DAPI (blue) are stained and quantified after 18 days in differentiation media. Each dot represents the ratio between TUJ or GFAP and DAPI area in an image containing at least 100 cells from 4 independent experiments with two different clones. **** p-value<0.0001. Student's t-test. Scale bar 50 μm.


c , Confocal microscopy analysis of WT and Cdc14 DKO ESC subjected to neural differentiation . Neural progenitor marker Nestin was stained and quantified after 6 days in differentiation media . Each dot represents the ratio between Nestin and DAPI area in an image containing at least 100 cells . * * * * , p < 0 . 0001 ; Students ' t - test .

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c, Confocal microscopy analysis of WT and Cdc14 DKO ESC subjected to neural differentiation. Neural progenitor marker Nestin was stained and quantified after 6 days in differentiation media. Each dot represents the ratio between Nestin and DAPI area in an image containing at least 100 cells. ****, p<0.0001; Students' t-test.


d , Western blot analysis of CDC14B - GFP co - immunoprecipitations . HeLa cells were transfected with UTF1 and GFP or CDC14B - GFP . Cytoplasmic and nuclear soluble proteins were extracted with RIPA , and nucleolar and chromatin - bound proteins were extracted with high salt RIPA . Immunoprecipitations with anti - GFP antibody were performed . Total protein extracts and immunoprecipitates were loaded and UTF ( upper panel ) and GFP ( lower panel ) were blotted .

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d, Western blot analysis of CDC14B-GFP co-immunoprecipitations. HeLa cells were transfected with UTF1 and GFP or CDC14B-GFP. Cytoplasmic and nuclear soluble proteins were extracted with RIPA, and nucleolar and chromatin-bound proteins were extracted with high salt RIPA. Immunoprecipitations with anti-GFP antibody were performed. Total protein extracts and immunoprecipitates were loaded and UTF (upper panel) and GFP (lower panel) were blotted.


e , Immunoprecipitation of UTF1 upon co - transfection with CDC14B . Total protein was extracted with high - salt RIPA and immunoprecipitations with anti - UTF1 antibody were performed .

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e, Immunoprecipitation of UTF1 upon co-transfection with CDC14B. Total protein was extracted with high-salt RIPA and immunoprecipitations with anti-UTF1 antibody were performed.


f , Expression of active CDC14B , but not a phosphatase - dead ( PD ) isoform , reduces slow - mobility bands of UTF1 . Vinculin was used as a loading control .

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f, Expression of active CDC14B, but not a phosphatase-dead (PD) isoform, reduces slow-mobility bands of UTF1. Vinculin was used as a loading control.


h , In vitro kinase assay of recombinant human UTF1 with ERK2 , CDK1 - Cyclin B1 , CDK1 - Cyclin A2 or in absence of kinases ( C ) .

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h, In vitro kinase assay of recombinant human UTF1 with ERK2, CDK1-Cyclin B1, CDK1-Cyclin A2 or in absence of kinases (C).


j , In vitro phosphatase assay of UTF1 . Three different fragments of UTF1 were phosphorylated by CDK1 - Cyclin A2 and subsequently exposed to wild - type or a catalytically inactive form ( PD ) of CDC14B .

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j, In vitro phosphatase assay of UTF1. Three different fragments of UTF1 were phosphorylated by CDK1-Cyclin A2 and subsequently exposed to wild-type or a catalytically inactive form (PD) of CDC14B.


a , Quantitative RT - PCR in wild - type ( WT ) or Cdc14 DKO ESCs of the indicated transcripts during neural differentiation . n = 3 independent experiments ( ± S . E . M . ) ; * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; Student ' s t - test .

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a, Quantitative RT-PCR in wild-type (WT) or Cdc14 DKO ESCs of the indicated transcripts during neural differentiation. n=3 independent experiments (±S.E.M.); *, p<0.05; **, p<0.01; ***, p<0.001; Student's t-test.


b , Expression of a CDC14 - GFP fusion protein in ESCs before ( T0 ) and after three days ( T3 ) in differentiation conditions . The arrowheads indicate the concentration of CDC14 - GFP in the nucleolus before but not after the induction of differentiation . Scale bars , 5 μM . The plot shows the ratio between nucleolar vs . total nuclear URF1 intensity at T0 ( n = 26 cells ) or T3 ( n = 31 ) in cells from two separate experiments . * * * , p < 0 . 001 , Student ' s t - test with Welch ' s correction .

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b, Expression of a CDC14-GFP fusion protein in ESCs before (T0) and after three days (T3) in differentiation conditions. The arrowheads indicate the concentration of CDC14-GFP in the nucleolus before but not after the induction of differentiation. Scale bars, 5 μM. The plot shows the ratio between nucleolar vs. total nuclear URF1 intensity at T0 (n= 26 cells) or T3 (n=31) in cells from two separate experiments. ***, p<0.001, Student's t-test with Welch's correction.


c , Confocal microscopy analysis of UTF1 intensity in WT and Cdc14 DKO ESCs at time 0 ( T0 ) and after 1 day ( T1 ) in differentiation media . The quantification of UTF1 fluorescence mean intensity ( FMI ) per cell is shown in the plot to the right . Figures and horizontal bars indicate mean ± S . E . M . ( n = 1269 WT T0 cells , n = 2726 WT T1 , n = 974 Cdc14 DKO T0 , n = 1173 Cdc14 DKO T1 ) . * * , p < 0 . 01 ; * * * * , p < 0 . 0001 ; Student ' s t - test with Welch ' s correction .

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c, Confocal microscopy analysis of UTF1 intensity in WT and Cdc14 DKO ESCs at time 0 (T0) and after 1 day (T1) in differentiation media. The quantification of UTF1 fluorescence mean intensity (FMI) per cell is shown in the plot to the right. Figures and horizontal bars indicate mean ± S.E.M. (n=1269 WT T0 cells, n=2726 WT T1, n=974 Cdc14 DKO T0, n=1173 Cdc14 DKO T1). **, p<0.01; ****, p<0.0001; Student's t-test with Welch's correction.


d , Western blot analysis of UTF1 expression in WT and Cdc14 DKO ESCs at time 0 and after 3 days in differentiation media .

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d, Western blot analysis of UTF1 expression in WT and Cdc14 DKO ESCs at time 0 and after 3 days in differentiation media.


e , Western blot analysis of UTF1 expression in WT and Cdc14 DKO ESCs in absence or presence of the kinase inhibitors roscovitine ( R ) or trametinib ( T ) .

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e, Western blot analysis of UTF1 expression in WT and Cdc14 DKO ESCs in absence or presence of the kinase inhibitors roscovitine (R) or trametinib (T).


f , Immunoblot analysis of UTF1 protein stability after transfection of HeLa cells with UTF1 and GFP or CDC14B - GFP . Cells were treated with cycloheximide ( CHX ) and total proteins were detected at different time points as indicated . Cyclin B1 and β - catenin are used as controls .

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f, Immunoblot analysis of UTF1 protein stability after transfection of HeLa cells with UTF1 and GFP or CDC14B-GFP. Cells were treated with cycloheximide (CHX) and total proteins were detected at different time points as indicated. Cyclin B1 and β-catenin are used as controls.


g , Western blot analysis of UTF1 WT or mutants in three ( UTF1 - 3KR ) or five ( UTF1 - 5KR ) lysines in absence or presence of trametinib ( T ) or roscovitine ( R ) .

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g, Western blot analysis of UTF1 WT or mutants in three (UTF1-3KR) or five (UTF1-5KR) lysines in absence or presence of trametinib (T) or roscovitine (R).


h , Immunoblot of UTF1 mutants in SPOP , SIAH or SPOP + SIAH potential binding sites , as well as S48D and S54D phosphorylation sites . Actin was used as a loading control .

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h, Immunoblot of UTF1 mutants in SPOP, SIAH or SPOP+SIAH potential binding sites, as well as S48D and S54D phosphorylation sites. Actin was used as a loading control.


A . Fura2 340 / 380 time traces from M - CSF - differentiated macrophages derived from conditionally - immortalised macrophage precursor cell lines ( M - MØP ) of Plcg2P522 mice ( P522 , blue ) and Plcg2R522 mice ( R522 , red ) . One set of cell lines , generated from male mice , is shown . Cells were exposed to 5µg / ml anti - FcγRII / III along with 20µM EGTA and 2µM Ionomycin as indicated . Data shows the mean ± SD of 3 independent experiments analysed by two - way ANOVA with Sidak post - hoc tests ( * * = p < 0 . 01 ; * * * * = p < 0 . 0001 ) . B . Fura2 340 / 380 time traces from primary microglia derived from the cortex of Plcg2R522 mice ( blue : P522 ) and Plcg2R522 mice ( red : R522 ) with or without pre - exposure for 2 hours with Edelfosine ( 10µM ) . Cells were exposed to 5µg / ml anti - FcγRII / III along with EGTA and 2µM Ionomycin . Data shows the mean ± SD of 3 independent experiments analysed by two - way ANOVA with Sidak post - hoc tests ( * * = p < 0 . 01 ; * * * = p < 0 . 001 ; * * * * = p < 0 . 0001 ) . 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 B-CONTROLLED_VAR 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 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 B-CONTROLLED_VAR 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 B-CONTROLLED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A. Fura2 340/380 time traces from M-CSF-differentiated macrophages derived from conditionally-immortalised macrophage precursor cell lines (M-MØP) of Plcg2P522 mice (P522, blue) and Plcg2R522 mice (R522, red). One set of cell lines, generated from male mice, is shown. Cells were exposed to 5µg/ml anti-FcγRII/III along with 20µM EGTA and 2µM Ionomycin as indicated. Data shows the mean±SD of 3 independent experiments analysed by two-way ANOVA with Sidak post-hoc tests (** = p<0.01; **** = p< 0.0001). B. Fura2 340/380 time traces from primary microglia derived from the cortex of Plcg2R522 mice (blue: P522) and Plcg2R522 mice (red: R522) with or without pre-exposure for 2 hours with Edelfosine (10µM). Cells were exposed to 5µg/ml anti-FcγRII/III along with EGTA and 2µM Ionomycin. Data shows the mean±SD of 3 independent experiments analysed by two-way ANOVA with Sidak post-hoc tests (** = p<0.01; *** = p<0.001; **** = p< 0.0001). C


C - E . M - MOPS ( C ) , primary mouse microglia ( D ) and hIPSC ( E ) ( blue : P522 and red : R522 ) were loaded with Fluo - 8 Ca2 + indicator and examined for peak changes in fluoresce after exposure to DOPS - liposomes ( 25 μg / ml ) . Data information : Data shown as the mean ± SD of 3 independent experiments . The data were analysed by two - way ANOVA with Sidak ' s post - tests ( * = p < 0 . 05 , * * = p < 0 . 01 , * * * = p < 0 . 001 ) .

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C-E. M-MOPS (C), primary mouse microglia (D) and hIPSC (E) (blue: P522 and red: R522) were loaded with Fluo-8 Ca2+ indicator and examined for peak changes in fluoresce after exposure to DOPS-liposomes (25 μg/ml). Data information: Data shown as the mean±SD of 3 independent experiments. The data were analysed by two-way ANOVA with Sidak's post-tests (*=p<0.05, **=p<0.01, ***=p<0.001).


A - F . Phagocytotic activity of R522 and P522 M - MOP and microglia was assessed using pHrodo Red E . coli and zymosan BioParticles . Phagocytosis arbitrary units ( A . U ) describes the amount of bioparticle cellular fluorescence emission at each time point . E . coli uptake in M - MOP ( A ) , primary mouse microglia ( B ) , hIPSC - derived microglia ( C ) . Zymosan uptake in M - MOP ( D ) , primary mouse microglia ( E ) and hIPSC - derived microglia ( F ) . For hIPSC - derived microglia , 3 isogenic P522 and 3 isogenic R522 clones were examined with at least 6 wells in 3 independent experiments . All microglia and M - MOP data shows the mean ± SD of 3 independent experiments were analysed by two - way ANOVA using Sidak multiple comparison test . * = p < 0 . 05 , * * = p < 0 . 01 , * * * = p < 0 . 001 , * * * * = p < 0 . 0001 . ( blue : P522 ; red : R522 )

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A-F. Phagocytotic activity of R522 and P522 M-MOP and microglia was assessed using pHrodo Red E. coli and zymosan BioParticles. Phagocytosis arbitrary units (A.U) describes the amount of bioparticle cellular fluorescence emission at each time point. E.coli uptake in M-MOP (A), primary mouse microglia (B), hIPSC-derived microglia (C). Zymosan uptake in M-MOP (D), primary mouse microglia (E) and hIPSC-derived microglia (F). For hIPSC-derived microglia, 3 isogenic P522 and 3 isogenic R522 clones were examined with at least 6 wells in 3 independent experiments. All microglia and M-MOP data shows the mean±SD of 3 independent experiments were analysed by two-way ANOVA using Sidak multiple comparison test. *=p<0.05, **=p<0.01, ***=p<0.001, ****=p<0.0001. (blue: P522; red: R522)


A - F . Endocytic activity of R522 and P522 M - MOP and microglia was assessed using FITC soluble Aβ1 - 42 oligomers and pHrodo Red Dexran ( 10 , 000 MW ) . Endocytosis arbitrary units ( A . U ) describes the amount of bioparticle cellular fluorescence emission at each time point . Aβ1 - 42 oligomer uptake in M - MOP ( A ) , primary microglia ( B ) , and hIPSC - derived microglia ( C ) . Dextran uptake in M - MOP ( D ) , primary microglia ( E ) and hIPSC - derived microglia ( F ) . For hIPSC - derived microglia , 3 isogenic P522 and 3 isogenic R522 clones were examined with at least 6 wells in 3 independent experiments . All microglia and M - MOP data shows the mean ± SD of 3 independent experiments were analysed by two - way ANOVA using Sidak ' s multiple comparison test . * = p < 0 . 05 , * * = p < 0 . 01 , * * * = p < 0 . 001 . ( blue : P522 ; red : R522 )

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A-F. Endocytic activity of R522 and P522 M-MOP and microglia was assessed using FITC soluble Aβ1-42 oligomers and pHrodo Red Dexran (10,000 MW). Endocytosis arbitrary units (A.U) describes the amount of bioparticle cellular fluorescence emission at each time point. Aβ1-42 oligomer uptake in M-MOP (A), primary microglia (B), and hIPSC-derived microglia (C). Dextran uptake in M-MOP (D), primary microglia (E) and hIPSC-derived microglia (F). For hIPSC-derived microglia, 3 isogenic P522 and 3 isogenic R522 clones were examined with at least 6 wells in 3 independent experiments. All microglia and M-MOP data shows the mean±SD of 3 independent experiments were analysed by two-way ANOVA using Sidak's multiple comparison test. *=p<0.05, **=p<0.01, ***=p<0.001. (blue: P522; red: R522)


level of PIP2 in M - MOPS was examined by measuring immunofluorescence from images at set time points after exposure after exposure to 5µg / ml anti - FcγRII / III ( 2 . 4G2 ) , 50 ng / ml LPS or oligomers of 40 µM Aβ1 - 42 Data shows the mean ± SD of 3 independent experiments were analysed by 2 way ANOVA with Sidak ' s multiple comparison * = p < 0 . 05 , * * = p < 0 . 01 , * * * = p < 0 . 001 , * * * * = p < 0 . 0001 . ( blue : P522 ; red : R522 ) .

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level of PIP2 in M-MOPS was examined by measuring immunofluorescence from images at set time points after exposure after exposure to 5µg/ml anti-FcγRII/III (2.4G2), 50 ng/ml LPS or oligomers of 40 µM Aβ1-42 Data shows the mean±SD of 3 independent experiments were analysed by 2 way ANOVA with Sidak's multiple comparison *=p<0.05, **=p<0.01, ***=p<0.001, ****=p<0.0001. (blue: P522; red: R522).


A - C . A mass ELISA was used to detect specific PIP species after exposure to anti - FcγRII / III ( 2 . 4G2 ) with or without 3 - a - aminocholestane ( SHIP 1 inhibitor ) , SF1670 ( PTEN inhibitor ) and LY294002 ( PI - 3K inhibitor ) . In M - MOP cells ( left panel ) and primary microglia ( right panel ) PI ( 4 , 5 ) P2 ( A ) , PI ( 3 , 4 ) P2 ( B ) and PIP3 ( C ) were detected . All data shows the mean ± SD of 3 independent experiments were analysed by 2 way ANOVA ( log - transformed data was used in C ) with Sidak ' s multiple comparison tests performed * = p < 0 . 05 , * * = p < 0 . 01 , * * * = p < 0 . 001 , * * * * = p < 0 . 0001 . ( blue : P522 ; red : R522 )

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A-C. A mass ELISA was used to detect specific PIP species after exposure to anti-FcγRII/III (2.4G2) with or without 3-a-aminocholestane (SHIP 1 inhibitor), SF1670 (PTEN inhibitor) and LY294002 (PI-3K inhibitor). In M-MOP cells (left panel) and primary microglia (right panel) PI(4,5)P2 (A), PI(3,4)P2 (B) and PIP3 (C) were detected. All data shows the mean±SD of 3 independent experiments were analysed by 2 way ANOVA (log-transformed data was used in C) with Sidak's multiple comparison tests performed *=p<0.05, **=p<0.01, ***=p<0.001, ****=p<0.0001. (blue: P522; red: R522)


A - C . Iba1 was used as a marker for microglia and PIP2 levels were detected by measuring the intensity of florescence per cell . The average fluorescence was calculated above background in the cortex and hippocampus at 2 ( A ) , 6 ( B ) and 9 ( C ) months . All data shows the mean ± SD of 3 independent experiments analysed using 2 way ANOVA with Sidak ' s multiple comparison tests performed * * = p < 0 . 01 , * * * * = p < 0 . 0001 .

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A-C. Iba1 was used as a marker for microglia and PIP2 levels were detected by measuring the intensity of florescence per cell. The average fluorescence was calculated above background in the cortex and hippocampus at 2 (A), 6 (B) and 9 (C) months. All data shows the mean±SD of 3 independent experiments analysed using 2 way ANOVA with Sidak's multiple comparison tests performed **=p<0.01, ****=p<0.0001.


Western blot analysis of lysates from cerebellum of control and FXS patients ' ' FXS ' ' ( n = 4 ) . Extract of pCI - hDGKk - transfected Hela cells ( hDGKk ) was used as antibody specificity control and size marker . Representative images of immunoblots probed with antibodies against the indicated proteins are shown . GAPDH was the loading control . Quantification of Western blots is shown on right . Protein amounts of DGKk are normalized to GAPDH and presented as fold change relative to control .

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Western blot analysis of lysates from cerebellum of control and FXS patients ''FXS'' (n = 4). Extract of pCI-hDGKk-transfected Hela cells (hDGKk) was used as antibody specificity control and size marker. Representative images of immunoblots probed with antibodies against the indicated proteins are shown. GAPDH was the loading control. Quantification of Western blots is shown on right. Protein amounts of DGKk are normalized to GAPDH and presented as fold change relative to control.


C - D ) Immunoblots and quantification of lysates from Cos - 1 cells transfected with plasmid pCI - mDGKk - HA or non - transfected ( NT ) and pre - transfected 24h before with siRNA control ( siC ) or against FMRP ( siFMR1 ) . GAPDH was used as a loading control . For quantification , the DGKk and FMRP signals were normalized against GAPDH signal and presented relative to the signal for siC treated cells ( C ) . Immunoblots and quantification of lysates from Cos - 1 cells transfected with pCI ( empty plasmid ) , pCI - HA - ∆ N - DGKk , or pCI - mDGKk - HA , and pre - treated with siRNA control ( siC ) or against FMRP ( siFMR1 ) ( D ) . Quantifications as in C .

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C-D) Immunoblots and quantification of lysates from Cos-1 cells transfected with plasmid pCI-mDGKk-HA or non-transfected (NT) and pre-transfected 24h before with siRNA control (siC) or against FMRP (siFMR1). GAPDH was used as a loading control. For quantification, the DGKk and FMRP signals were normalized against GAPDH signal and presented relative to the signal for siC treated cells (C). Immunoblots and quantification of lysates from Cos-1 cells transfected with pCI (empty plasmid), pCI-HA-∆N-DGKk, or pCI-mDGKk-HA, and pre-treated with siRNA control (siC) or against FMRP (siFMR1) (D). Quantifications as in C.


A ) HeLa cells transfected with indicated plasmids expressing mouse ( m ) or human ( h ) DGKk were analyzed by immunofluorescence confocal microscopy for HA tag ( red ) and Dapi staining ( blue ) . ∆ C1 - hDGKk construct bears deletion of phorbol ester C1 domain ( 328 - 449 ) .

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A) HeLa cells transfected with indicated plasmids expressing mouse (m) or human (h) DGKk were analyzed by immunofluorescence confocal microscopy for HA tag (red) and Dapi staining (blue). ∆C1-hDGKk construct bears deletion of phorbol ester C1 domain (328-449).


B ) Immunoblots and quantification of lysates from Cos - 1 cells transfected with plasmid pCI - HA - ∆ N - DGKk , untransfected ( NT ) , or plasmid pCI control , with the indicated amount of plasmid ( µg ) and after 24h , incubated with puromycin ( 20µg / ml , 30 min ) to measure rates of protein synthesis . GAPDH was used as a loading control . 0 indicates no puromycin treatment , - / + indicates treatment without or with DGK inhibitor ( DGKi ) 3 µM R59022 and 0 . 2 µM R59949 at 6 µM , 15 min . Densitogram of puromycin incorporation is presented as change relative to mock transfected conditions .

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B) Immunoblots and quantification of lysates from Cos-1 cells transfected with plasmid pCI-HA-∆N-DGKk, untransfected (NT), or plasmid pCI control, with the indicated amount of plasmid (µg) and after 24h, incubated with puromycin (20µg/ml, 30 min) to measure rates of protein synthesis. GAPDH was used as a loading control. 0 indicates no puromycin treatment, -/+ indicates treatment without or with DGK inhibitor (DGKi) 3 µM R59022 and 0.2 µM R59949 at 6 µM, 15 min. Densitogram of puromycin incorporation is presented as change relative to mock transfected conditions.


C ) Representative immunofluorescence staining of cortical neuron cultures transduced at 8 DIV ( days in vitro ) with 10e9 VG / ml of AAVRh10 - GFP or AAVRh10 - ∆ N - DGKk and assessed after 5 days using anti - MAP2 and anti - HA for ∆ N - DGKk or direct 488 nm excitation for GFP . DAPI was used to visualize nuclei on merged images .

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C) Representative immunofluorescence staining of cortical neuron cultures transduced at 8 DIV (days in vitro) with 10e9 VG/ml of AAVRh10-GFP or AAVRh10-∆N-DGKk and assessed after 5 days using anti-MAP2 and anti-HA for ∆N-DGKk or direct 488 nm excitation for GFP. DAPI was used to visualize nuclei on merged images.


D ) Representative immunoblots of lysates from WT and Fmr1 - KO cortical neurons transduced with AAVRh10 - ∆ N - DGKk ( AAV ∆ N - DGKk ) , at the indicated titers ( VG / ml culture volume ) and quantification of phosphorylation and total levels of eIF4E . GAPDH was used as a loading control . For quantification , the phospho - protein signal was normalized first against total protein signal and is presented relative to the signal for WT culture .

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D) Representative immunoblots of lysates from WT and Fmr1-KO cortical neurons transduced with AAVRh10-∆N-DGKk (AAV ∆N-DGKk), at the indicated titers (VG/ml culture volume) and quantification of phosphorylation and total levels of eIF4E. GAPDH was used as a loading control. For quantification, the phospho-protein signal was normalized first against total protein signal and is presented relative to the signal for WT culture.


B ) Viral titers ( viral genome copy VG per cell ) determined by qPCR in cortical , hippocampal and rest of brain areas of Fmr1 - KO mice treated with saline solution ( Vehicle ) , AAVPHP . eB - ∆ N - DGKk ( PHP . eB ) , AAVRh10 - ∆ N - DGKk ( Rh10 ) 8 weeks after injections . Data are mean ± SEM . Each dot represents an individual mouse .

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B) Viral titers (viral genome copy VG per cell) determined by qPCR in cortical, hippocampal and rest of brain areas of Fmr1-KO mice treated with saline solution (Vehicle), AAVPHP.eB-∆N-DGKk (PHP.eB), AAVRh10-∆N-DGKk (Rh10) 8 weeks after injections. Data are mean ± SEM. Each dot represents an individual mouse.


C ) Immunoblots and quantification of ∆ N - DGKk protein in lysates from brain areas of mice treated as in B . GAPDH was used as a loading control . Densitogram of ∆ N - DGKk expression is presented as change relative to vehicle conditions .

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C) Immunoblots and quantification of ∆N-DGKk protein in lysates from brain areas of mice treated as in B. GAPDH was used as a loading control. Densitogram of ∆N-DGKk expression is presented as change relative to vehicle conditions.


D ) Representative coronal brain sections processed for detection of ∆ N - DGKk using immunohistochemistry on Fmr1 - KO mice treated with indicated treatment , 8 weeks post - injections , counter stained with eosin hematoxylin . Adjacent sections were immunolabelled with NeuN . 3 mice per genotype were processed . The sections shown are between Bregma levels - 1 . 50 mm and - 1 . 80 mm . Scale bar is 2 mm . Magnifications of regions of cortex ( c ) , hippocampus ( h ) , and striatum ( s ) are shown in side panels , scale bar 200 µm .

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D) Representative coronal brain sections processed for detection of ∆N-DGKk using immunohistochemistry on Fmr1-KO mice treated with indicated treatment, 8 weeks post-injections, counter stained with eosin hematoxylin. Adjacent sections were immunolabelled with NeuN. 3 mice per genotype were processed. The sections shown are between Bregma levels -1.50 mm and -1.80 mm. Scale bar is 2 mm. Magnifications of regions of cortex (c), hippocampus (h), and striatum (s) are shown in side panels, scale bar 200 µm.


E ) Total phosphatidic acid ( PA ) level measure by mass spectrometry in cortex of WT mice treated with saline solution ( WT - S ) and Fmr1 - KO mice treated with saline ( Fmr1 - S ) , AAVPHP . eB - ∆ N - DGKk ( Fmr1 - PHP . eB ) , AAVRh10 - ∆ N - DGKk ( Fmr1 - Rh10 ) 8 weeks after injections . Data are expressed as mol % of total lipids and represented as median with interquartile range with minimum and maximum values . Statistics : one - way ANOVA and Tukey ' s multiple comparisons test , n = 8 individual animals , except for WT - S and Fmr1 - S n = 11 . * * P < 0 . 01 , * * * P < 0 . 001 .

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E) Total phosphatidic acid (PA) level measure by mass spectrometry in cortex of WT mice treated with saline solution (WT-S) and Fmr1-KO mice treated with saline (Fmr1-S), AAVPHP.eB-∆N-DGKk (Fmr1-PHP.eB), AAVRh10-∆N-DGKk (Fmr1-Rh10) 8 weeks after injections. Data are expressed as mol % of total lipids and represented as median with interquartile range with minimum and maximum values. Statistics: one-way ANOVA and Tukey's multiple comparisons test, n=8 individual animals, except for WT-S and Fmr1-S n=11. **P < 0.01, ***P < 0.001.


F ) Total diacylglycerol ( DAG ) level in cortex measured as in E ) .

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F) Total diacylglycerol (DAG) level in cortex measured as in E).


A ) Elevated Plus Maze . Percentage of time spent in open arms and number of entries in open , closed and total ( open + closed ) arms . ( n = 12 for time in open arm , n = 24 for arm entries ) .

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A) Elevated Plus Maze. Percentage of time spent in open arms and number of entries in open, closed and total (open+closed) arms. (n = 12 for time in open arm, n=24 for arm entries).


B ) Novel object recognition . Locomotor activity ( distance in m ) in the whole arena and percentage of time spent in the center during 15 min habituation . Locomotor activity during the acquisition and retention trials . Recognition index . ( n = 24 ) .

B) Novel object recognition. Locomotor activity (distance in m) in the whole arena and percentage of time spent in the center during 15 min habituation. Locomotor activity during the acquisition and retention trials. Recognition index. (n = 24).


C ) Social recognition . Number of entries in the two side compartments during preference session ( left ) , social preference ( percentage of exploration of a congener vs an object ) ( center ) and social memory ( percentage of exploration of a novel vs familiar congener ) ( right ) . ( n = 24 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Social recognition. Number of entries in the two side compartments during preference session (left), social preference (percentage of exploration of a congener vs an object) (center) and social memory (percentage of exploration of a novel vs familiar congener) (right). (n=24).


A ) Time - dependent GSK - 3 inhibition of VP2 . 51 at 1 and 2 μM .

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A) Time-dependent GSK-3 inhibition of VP2.51 at 1 and 2 μM.


B ) Kinetic GSK - 3 inhibition data determined for VP2 . 51 . ATP concentrations in the reaction mixture varied from 1 to 50 μM . Compound concentrations used are depicted in the plot , and the concentration of GS - 2 was kept constant at 12 . 5 μM . Each point is the mean of two different experiments , each one analyzed in duplicate .

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B) Kinetic GSK-3 inhibition data determined for VP2.51. ATP concentrations in the reaction mixture varied from 1 to 50 μM. Compound concentrations used are depicted in the plot, and the concentration of GS-2 was kept constant at 12.5 μM. Each point is the mean of two different experiments, each one analyzed in duplicate.


C ) Kinase profiling of VP2 . 51 on human recombinant kinases . The percentage ( % ) of kinase activity after the treatment with a fixed concentration ( 10 μM ) of the compound is shown .

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C) Kinase profiling of VP2.51 on human recombinant kinases. The percentage (%) of kinase activity after the treatment with a fixed concentration (10 μM) of the compound is shown.


A ) Evolution of rat weight of different treatment groups ( 12 animals in each group ) : control , L - BMAA ( 300 mg / kg / day during 5 consecutive days treated at weaning ) , VP2 . 51 ( 2 , 5 mg / kg during 15 days ) with a group treated at PT1 and another at PT30 , L - BMAA ( with the same treatment as the group of L - BMAA ) + VP2 . 51 ( with the same treatment groups VP2 . 51 ) with a group treated at PT1 and another at PT30 .

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A) Evolution of rat weight of different treatment groups (12 animals in each group): control, L-BMAA (300 mg/kg/day during 5 consecutive days treated at weaning), VP2.51 (2,5 mg/kg during 15 days) with a group treated at PT1 and another at PT30, L-BMAA (with the same treatment as the group of L-BMAA) + VP2.51 (with the same treatment groups VP2.51) with a group treated at PT1 and another at PT30.


B ) . Evolution of neurological evaluation in the treatment assay with VP2 . 51 ( 12 animals in each group ) . The control group and the groups treated exclusively with VP2 . 51 ( at PT1 and PT30 ) are not shown because its assessment coincides with the horizontal axis . The time when the inhibitor is added is indicated with arrows .

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B). Evolution of neurological evaluation in the treatment assay with VP2.51 (12 animals in each group). The control group and the groups treated exclusively with VP2.51 (at PT1 and PT30) are not shown because its assessment coincides with the horizontal axis. The time when the inhibitor is added is indicated with arrows.


A ) Analysis of LC3 - II and P - mTOR by western - blot in the motor cortex of control and treated animals . Proteins were detected by Western blotting using β - tubulin for calibration of sample loading . The protein bands were quantified by Image Multigauge v3 . 0 software . Immunoblots are representative of one of three experiments with similar results . These results were quantitated in arbitrary units and were represented in the accompanying graphs as mean ± SEM . * , * * Indicate differences of p - value < 0 . 05 < 0 . 01 with the control group , respectively . # , # # # indicate differences of p - value < 0 . 05 < 0 . 001 with the group of animals treated with L - BMAA , respectively .

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A) Analysis of LC3-II and P-mTOR by western-blot in the motor cortex of control and treated animals. Proteins were detected by Western blotting using β-tubulin for calibration of sample loading. The protein bands were quantified by Image Multigauge v3.0 software. Immunoblots are representative of one of three experiments with similar results. These results were quantitated in arbitrary units and were represented in the accompanying graphs as mean ± SEM. *, ** Indicate differences of p-value <0.05 <0.01 with the control group, respectively. #, ### indicate differences of p-value <0.05 <0.001 with the group of animals treated with L-BMAA, respectively.


B ) Analysis of LC3 - II and P - mTOR by western - blot in the lumbar spinal cord of control and treated animals . Proteins were detected by Western blotting using β - tubulin for calibration of sample loading . The protein bands were quantified by Image Multigauge v3 . 0 software . Immunoblots are representative of one of three experiments with similar results . These results were quantitated in arbitrary units and were represented in the accompanying graphs as mean ± SEM . * , * * * Indicate differences of p - value < 0 . 05 < 0 . 001 with the control group , respectively . # # Indicate differences of p - value < 0 . 01 with the group of animals treated with L - BMAA .

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

B) Analysis of LC3-II and P-mTOR by western-blot in the lumbar spinal cord of control and treated animals. Proteins were detected by Western blotting using β-tubulin for calibration of sample loading. The protein bands were quantified by Image Multigauge v3.0 software. Immunoblots are representative of one of three experiments with similar results. These results were quantitated in arbitrary units and were represented in the accompanying graphs as mean ± SEM. *, *** Indicate differences of p-value <0.05 <0.001 with the control group, respectively. ## Indicate differences of p-value <0.01 with the group of animals treated with L-BMAA.


B ) Cell viability measured in the experiment with or without inhibitors .

B) Cell viability measured in the experiment with or without inhibitors.


( A ) Relaxed DNA ( 3 nM ) , condensin ( 0 , 60 , 120 , 240 nM ) and Topo I ( 1 unit ) were mixed and incubated with or without ATP ( 1 mM ) at 30ºC for 30 min . DNA electrophoresis contained 0 . 1 µg / ml chloroquine . In all gels , N denotes nicked circles ; Lkº , the input Lk distribution of relaxed DNA ; and LkC , the Lk distribution restrained by condensin . ( B ) 2D - gel of the preceding samples in lane 1 ( relaxed DNA , R ) , and lanes 5 and 6 ( condensin 120 nM ± ATP ) . Electrophoresis contained 0 . 1 and 1 µg / ml chloroquine in the first and second dimension , respectively . The histogram shows the relative intensity ( i ) of individual topoisomers of the Lk distributions resolved in lane 1 ( green ) and lane 6 ( red ) . Lkº and LkC denote the midpoint of each Lk distribution ; and ∆ Lk , the difference ( Lk units ) between them . ( C ) Experiment conducted as in A , but reducing the concentration of DNA ( 0 . 3 nM ) and condensin ( 0 , 1 . 5 , 3 , 6 nM ) . DNA electrophoresis contained 0 . 4 µg / ml chloroquine . ( D ) 2D - gel of the preceding sample in lane 1 ( relaxed DNA , R ) , and lanes 5 and 6 ( condensin 3 nM ± ATP ) . Electrophoresis contained 0 . 4 and 1 µg / ml chloroquine in the first and second dimension , respectively . The histogram shows relative Lk intensities ( i ) of lanes 1 and 6 , indicating Lkº , LkC and ∆ Lk .

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

(A) Relaxed DNA (3 nM), condensin (0, 60, 120, 240 nM) and Topo I (1 unit) were mixed and incubated with or without ATP (1 mM) at 30ºC for 30 min. DNA electrophoresis contained 0.1 µg/ml chloroquine. In all gels, N denotes nicked circles; Lkº, the input Lk distribution of relaxed DNA; and LkC, the Lk distribution restrained by condensin. (B) 2D-gel of the preceding samples in lane 1 (relaxed DNA, R), and lanes 5 and 6 (condensin 120 nM ± ATP). Electrophoresis contained 0.1 and 1 µg/ml chloroquine in the first and second dimension, respectively. The histogram shows the relative intensity (i) of individual topoisomers of the Lk distributions resolved in lane 1 (green) and lane 6 (red). Lkº and LkC denote the midpoint of each Lk distribution; and ∆Lk, the difference (Lk units) between them. (C) Experiment conducted as in A, but reducing the concentration of DNA (0.3 nM) and condensin (0, 1.5, 3, 6 nM). DNA electrophoresis contained 0.4 µg/ml chloroquine. (D) 2D-gel of the preceding sample in lane 1(relaxed DNA, R), and lanes 5 and 6 (condensin 3 nM ± ATP). Electrophoresis contained 0.4 and 1 µg/ml chloroquine in the first and second dimension, respectively. The histogram shows relative Lk intensities (i) of lanes 1 and 6, indicating Lkº, LkC and ∆Lk.


( E ) Experiment conducted as in A , but mixing DNA ( 1 nM ) with intermediate condensin concentrations ( 10 to 100 nM ) . DNA electrophoresis contained 0 . 2 µg / ml chloroquine . The length of DNA ( bp ) available per condensin complex in each reaction is indicated .

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(E) Experiment conducted as in A, but mixing DNA (1 nM) with intermediate condensin concentrations (10 to 100 nM). DNA electrophoresis contained 0.2 µg/ml chloroquine. The length of DNA (bp) available per condensin complex in each reaction is indicated.


( A ) Relaxed DNA ( 0 . 3 nM ) , condensin ( 3 nM ) and Topo I ( 1 unit ) were incubated at 30ºC for indicated time periods ( 0 to 90 min ) in the presence of different concentrations of ATP ( 1 , 0 . 25 , 0 . 1 mM ) .

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(A) Relaxed DNA (0.3 nM), condensin (3 nM) and Topo I (1 unit) were incubated at 30ºC for indicated time periods (0 to 90 min) in the presence of different concentrations of ATP (1, 0.25, 0.1 mM).


( B ) Plot of ∆ Lk values ( mean ± SD , 3 technical replicates ) constrained in the preceding experiment .

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

(B) Plot of ∆Lk values (mean ±SD, 3 technical replicates) constrained in the preceding experiment.


( C ) DNA , condensin and Topo I ( mixed as in A ) were incubated at 30ºC without nucleotides for 10 min ( lane 1 ) , with ATP 1mM for 10 min ( lane 2 ) , AMPPNP 2 mM for 10 min ( lane 3 ) , and AMPPNP 2mM for 10 min followed by ATP 1 mM for 10 min ( lane 4 ) . ( D ) DNA , condensin and Topo I ( mixed as in A ) were incubated at 30ºC without nucleotides for 10 min ( lane 1 ) , with Apyrase for 10 min ( lane 2 ) , Apyrase and ATP 1mM for 10 min ( lane 3 ) , ATP 1mM for 10 min ( lane 4 ) , no nucleotide for 10 min followed by Apyrase for 60 min ( lane 5 ) , ATP 1mM for 10 min followed by Apyrase for 60 min ( lane 6 ) .

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(C) DNA, condensin and Topo I (mixed as in A) were incubated at 30ºC without nucleotides for 10 min (lane 1), with ATP 1mM for 10 min (lane 2), AMPPNP 2 mM for 10 min (lane 3), and AMPPNP 2mM for 10 min followed by ATP 1 mM for 10 min (lane 4). (D) DNA, condensin and Topo I (mixed as in A) were incubated at 30ºC without nucleotides for 10 min (lane 1), with Apyrase for 10 min (lane 2), Apyrase and ATP 1mM for 10 min (lane 3), ATP 1mM for 10 min (lane 4), no nucleotide for 10 min followed by Apyrase for 60 min (lane 5), ATP 1mM for 10 min followed by Apyrase for 60 min (lane 6).


( E ) DNA , condensin and Topo I ( mixed as in A ) were incubated at 30ºC without nucleotides for 20 min ( lane 1 ) , with ATP 1mM for 20 min ( lane 2 ) , AMPPNP 2 mM for 20 min ( lane 3 ) , ATP 1mM for 10 min followed by AMPPNP 2 mM for 10 min ( lane 4 ) . ( F ) 2D - gel of the samples in E ( lanes 1 to 4 ) and histogram of Lk intensities ( i ) of lanes 1 and 4 , indicating Lkº , LkC and ∆ Lk .

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(E) DNA, condensin and Topo I (mixed as in A) were incubated at 30ºC without nucleotides for 20 min (lane 1), with ATP 1mM for 20 min (lane 2), AMPPNP 2 mM for 20 min (lane 3), ATP 1mM for 10 min followed by AMPPNP 2 mM for 10 min (lane 4). (F) 2D-gel of the samples in E (lanes 1 to 4) and histogram of Lk intensities (i) of lanes 1 and 4, indicating Lkº, LkC and ∆Lk.


( D ) Relaxed DNA ( 0 . 3 nM ) , condensin ( 3 nM ) and Topo I were mixed with / without nuclease P1 and incubated at 30ºC with no nucleotide for 30 min , ATP 1mM for 30 min , AMPPNP 2 mM for 30 min , ATP for 20 min followed by AMPPNP for 10 min or ATP for 20 min followed by Apyrase for 10 min .

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(D) Relaxed DNA (0.3 nM), condensin (3 nM) and Topo I were mixed with/without nuclease P1 and incubated at 30ºC with no nucleotide for 30 min, ATP 1mM for 30 min, AMPPNP 2 mM for 30 min, ATP for 20 min followed by AMPPNP for 10 min or ATP for 20 min followed by Apyrase for 10 min.


( E ) Experiment conducted as in D , but using ( - ) supercoiled DNA and without Topo I . Following the incubation with nucleotides for 30 min , nuclease P1 was added for 5 min .

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(E) Experiment conducted as in D, but using (-) supercoiled DNA and without Topo I. Following the incubation with nucleotides for 30 min, nuclease P1 was added for 5 min.


( A ) Relaxed DNA ( 0 . 3 nM ) , condensin ( 3 nM ) and Topo I ( 1 unit ) were mixed with ss - oligos or ds - oligos ( 100 and 500 nM ) . Following incubation for 10 min at 30ºC , ATP ( 1 mM ) was added and incubations continued for 10 min . ( B ) Experiment as in A , but first adding ATP for 10 min and afterwards the oligonucleotides for 10 min . ( C ) Experiment as in B , but after the 10 min incubation with oligonucleotides , AMPPNP ( 2 mM ) was added and incubations continued for 10 min .

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(A) Relaxed DNA (0.3 nM), condensin (3 nM) and Topo I (1 unit) were mixed with ss-oligos or ds-oligos (100 and 500 nM). Following incubation for 10 min at 30ºC, ATP (1 mM) was added and incubations continued for 10 min. (B) Experiment as in A, but first adding ATP for 10 min and afterwards the oligonucleotides for 10 min. (C) Experiment as in B, but after the 10 min incubation with oligonucleotides, AMPPNP (2 mM) was added and incubations continued for 10 min.


( D ) Relaxed DNA ( 0 . 3 nM ) , condensin ( 3 nM ) and Topo I ( 1 unit ) were incubated at 30ºC for 10 min . Reactions were then split into thirds to which NaCl concentration was raised to 50 , 150 and 300 mM . Salt - resistant condensin - DNA complexes were immobilized to His - Tag magnetic beads and the fractions of free ( F ) and bound ( B ) DNA recovered . The plot shows the fractions of bound DNA ( mean ± SD , 3 technical replicates ) . ( E ) As in D , but containing AMPPNP ( 2 mM ) . ( F ) As in D , but containing ATP ( 1 mM ) . ( G ) As in D , but containing ATP ( 1 mM ) for 10 min followed by AMPPNP ( 2 mM ) for 10 min . ( H ) As in D , but containing ATP ( 1 mM ) for 10 min followed by Apyrase for 10 min .

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(D) Relaxed DNA (0.3 nM), condensin (3 nM) and Topo I (1 unit) were incubated at 30ºC for 10 min. Reactions were then split into thirds to which NaCl concentration was raised to 50, 150 and 300 mM. Salt-resistant condensin-DNA complexes were immobilized to His-Tag magnetic beads and the fractions of free (F) and bound (B) DNA recovered. The plot shows the fractions of bound DNA (mean ±SD, 3 technical replicates). (E) As in D, but containing AMPPNP (2 mM). (F) As in D, but containing ATP (1 mM). (G) As in D, but containing ATP (1 mM) for 10 min followed by AMPPNP (2 mM) for 10 min. (H) As in D, but containing ATP (1 mM) for 10 min followed by Apyrase for 10 min.


( B ) Relaxed DNA ( 0 . 3 nM ) and condensin ( 3 nM ) were mixed with Topo I or Topo II ( 1 unit ) . Upon addition of ATP ( 1 mM ) , incubations proceeded at 30ºC for 30 min . ( C ) DNA , condensin , and Topo II were mixed as in B . Upon addition of ATP ( 1 mM ) , incubations proceeded at 30ºC for 10 min . Topo I was added to one half of the mixtures and incubations continued for 10 min . ( D ) DNA , condensin , and Topo II were mixed as in B . Following incubation at 30ºC for 10 min with or without ATP , DNA was recovered and nicked to reveal the occurrence of DNA knots . K3 to K6 denote knots with 3 to 6 irreducible DNA crossings .

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

(B) Relaxed DNA (0.3 nM) and condensin (3 nM) were mixed with Topo I or Topo II (1 unit). Upon addition of ATP (1 mM), incubations proceeded at 30ºC for 30 min. (C) DNA, condensin, and Topo II were mixed as in B. Upon addition of ATP (1 mM), incubations proceeded at 30ºC for 10 min. Topo I was added to one half of the mixtures and incubations continued for 10 min. (D) DNA, condensin, and Topo II were mixed as in B. Following incubation at 30ºC for 10 min with or without ATP, DNA was recovered and nicked to reveal the occurrence of DNA knots. K3 to K6 denote knots with 3 to 6 irreducible DNA crossings.


( F ) Nicked DNA ( 0 . 3 nM ) was mixed without or with condensin ( 3 nM ) in the presence of ATP ( 1 mM ) . Following incubations at 30ºC for 20 min , mixtures were supplemented with T4 DNA Ligase and Topo I ( as indicated ) and incubation continued for 10 min .

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(F) Nicked DNA (0.3 nM) was mixed without or with condensin (3 nM) in the presence of ATP (1 mM). Following incubations at 30ºC for 20 min, mixtures were supplemented with T4 DNA Ligase and Topo I (as indicated) and incubation continued for 10 min.


( B ) Relaxed DNA ( 0 . 3 nM ) was mixed with condensin ( 3 nM ) . Following incubation in absence or presence of the indicated nucleotides ( ATP 1mM for 30 min , AMPPNP 2 mM for 30 min , ATP for 20 min followed by AMPPNP for 10 min or ATP for 20 min followed by Apyrase for 10 min ) , TopA was added to one half of each reaction and Topo I to the other half . Incubations continued for 10 min .

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(B) Relaxed DNA (0.3 nM) was mixed with condensin (3 nM). Following incubation in absence or presence of the indicated nucleotides (ATP 1mM for 30 min, AMPPNP 2 mM for 30 min, ATP for 20 min followed by AMPPNP for 10 min or ATP for 20 min followed by Apyrase for 10 min), TopA was added to one half of each reaction and Topo I to the other half. Incubations continued for 10 min.


( D ) Experiment conducted as in B but starting with ( - ) supercoiled DNA instead of relaxed DNA . Following incubation in presence of the indicated nucleotides ( as in B ) Topo I was added to one half of each mixture and incubations continued for 10 min . ( E ) Experiment conducted as in D but adding TopA instead of Topo I .

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

(D) Experiment conducted as in B but starting with (-) supercoiled DNA instead of relaxed DNA. Following incubation in presence of the indicated nucleotides (as in B) Topo I was added to one half of each mixture and incubations continued for 10 min. (E) Experiment conducted as in D but adding TopA instead of Topo I.


( A ) Relaxed DNA ( 0 . 3 nM ) , ∆ Ycg1 condensin ( 3 nM ) and Topo I were incubated at 30ºC with no nucleotide for 30 min , ATP 1mM for 30 min , AMPPNP 2 mM for 30 min , ATP for 20 min followed by AMPPNP for 10 min or ATP for 20 min followed by Apyrase for 10 min . The plot ( mean ± SD , 3 technical replicates ) compares the ∆ Lk values restrained by the condensin holo - complex and the ∆ Ycg1 condensin .

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

(A) Relaxed DNA (0.3 nM), ∆Ycg1 condensin (3 nM) and Topo I were incubated at 30ºC with no nucleotide for 30 min, ATP 1mM for 30 min, AMPPNP 2 mM for 30 min, ATP for 20 min followed by AMPPNP for 10 min or ATP for 20 min followed by Apyrase for 10 min. The plot (mean ±SD, 3 technical replicates) compares the ∆Lk values restrained by the condensin holo-complex and the ∆Ycg1 condensin.


( B ) Salt resistance of the ∆ Ycg1 condensin - DNA complex . Experiments were conducted as in Fig 4D - H . The plot ( mean ± SD , 3 technical replicates ) compares the fractions of DNA bound to the holo - complex and to the ∆ Ycg1 tetramer .

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

(B) Salt resistance of the ∆Ycg1 condensin-DNA complex. Experiments were conducted as in Fig 4D-H. The plot (mean ±SD, 3 technical replicates) compares the fractions of DNA bound to the holo-complex and to the ∆Ycg1 tetramer.