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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

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

D) 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 .

O O O B-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 ) .

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

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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 .

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 O O

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 .

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

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 .

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

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 .

O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O 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). 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 .

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

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 .

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-MEASURED_VAR 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

(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 ) .

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 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

(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 .

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 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 O O O O O

(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 .

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

(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 .

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

(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 .

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

(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 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 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 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 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 .

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 O 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) 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 .

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 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 O O O O O 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) 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) 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 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 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 O O O O O O O O O O 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 (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 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 B-MEASURED_VAR O O O O O O O O O O O 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.


The expression of the 20 genes encoding TFs was measured 24 h after mannitol treatment during the proliferating ( n = 192 plants ) , expanding ( n = 16 plants ) and mature ( n = 16 ) leaf developmental stage . Expression levels in wild - type plants transferred to mannitol - induced stress were compared to those transferred to control conditions at the same developmental stage . Data information : Data are presented as mean ± SEM , n = 4 independent experiments . FC = Fold change . * = FDR < 0 . 05 , Unpaired two - sided Student ' s t - test .

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 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

The expression of the 20 genes encoding TFs was measured 24 h after mannitol treatment during the proliferating (n = 192 plants), expanding (n = 16 plants) and mature (n = 16) leaf developmental stage. Expression levels in wild-type plants transferred to mannitol-induced stress were compared to those transferred to control conditions at the same developmental stage. Data information: Data are presented as mean ± SEM, n = 4 independent experiments. FC = Fold change. * = FDR < 0.05, Unpaired two-sided Student's t-test.


A , B , C , D Based on a threshold of log2 [ fold change ( FC ) ] > 1 , the 20 TFs were categorized into four groups . The first group contains TFs that reached the log2 ( FC ) threshold 40 min after mannitol treatment ( A ) The arrow indicates the initial upregulation of every group . Data information : Data are presented as mean ± SEM . n = 4 independent experiments . FDR values are available in Table EV1 .

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

A, B, C, D Based on a threshold of log2[fold change (FC)] > 1, the 20 TFs were categorized into four groups. The first group contains TFs that reached the log2(FC) threshold 40 min after mannitol treatment (A) The arrow indicates the initial upregulation of every group. Data information: Data are presented as mean ± SEM. n = 4 independent experiments. FDR values are available in Table EV1.


A , B , C , D Based on a threshold of log2 [ fold change ( FC ) ] > 1 , the 20 TFs were categorized into four groups . the second group reached the threshold after 1 h ( B ) The arrow indicates the initial upregulation of every group . Data information : Data are presented as mean ± SEM . n = 4 independent experiments . FDR values are available in Table EV1 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, C, D Based on a threshold of log2[fold change (FC)] > 1, the 20 TFs were categorized into four groups. the second group reached the threshold after 1 h (B) The arrow indicates the initial upregulation of every group. Data information: Data are presented as mean ± SEM. n = 4 independent experiments. FDR values are available in Table EV1.


A , B , C , D Based on a threshold of log2 [ fold change ( FC ) ] > 1 , the 20 TFs were categorized into four groups . the third group after 2 h ( C ) The arrow indicates the initial upregulation of every group . Data information : Data are presented as mean ± SEM . n = 4 independent experiments . FDR values are available in Table EV1 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, C, D Based on a threshold of log2[fold change (FC)] > 1, the 20 TFs were categorized into four groups. the third group after 2 h (C) The arrow indicates the initial upregulation of every group. Data information: Data are presented as mean ± SEM. n = 4 independent experiments. FDR values are available in Table EV1.


A , B , C , D Based on a threshold of log2 [ fold change ( FC ) ] > 1 , the 20 TFs were categorized into four groups . the fourth group after 4 h ( D ) . The arrow indicates the initial upregulation of every group . Data information : Data are presented as mean ± SEM . n = 4 independent experiments . FDR values are available in Table EV1 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, C, D Based on a threshold of log2[fold change (FC)] > 1, the 20 TFs were categorized into four groups. the fourth group after 4 h (D). The arrow indicates the initial upregulation of every group. Data information: Data are presented as mean ± SEM. n = 4 independent experiments. FDR values are available in Table EV1.


C Heatmap of the significant regulations upon induction of overexpression of the five members of the core network , the activators ( green ) ERF6 , ERF59 , ERF98 and the repressors ( red ) ERF8 and ERF9 . Color code represents FDR - corrected p - values with thresholds at FDR = 0 . 01 , 0 . 05 and 0 . 1 . Data information : In A , data are extrapolated from estimated averages , n = 3 independent experiments , FDR - corrected p - values < 0 . 1 ( mixed model analysis , user - defined Wald tests ) . The thickness of the arrows represents the FDR value . In B , data are presented as averages , n = 3 independent experiments . The intensity of the color of the arrows represents the strength of the regulation according to the TEA values and the thickness the FDR value of the nCounter Nanostring experiment . In C , data are represented as FDR - corrected p - values , n = 3 independent experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Heatmap of the significant regulations upon induction of overexpression of the five members of the core network, the activators (green) ERF6, ERF59, ERF98 and the repressors (red) ERF8 and ERF9. Color code represents FDR-corrected p-values with thresholds at FDR=0.01, 0.05 and 0.1. Data information: In A, data are extrapolated from estimated averages, n = 3 independent experiments, FDR-corrected p-values < 0.1 (mixed model analysis, user-defined Wald tests). The thickness of the arrows represents the FDR value. In B, data are presented as averages, n = 3 independent experiments. The intensity of the color of the arrows represents the strength of the regulation according to the TEA values and the thickness the FDR value of the nCounter Nanostring experiment. In C, data are represented as FDR-corrected p-values, n = 3 independent experiments.


A At 22 days after stratification ( DAS ) , leaf series of the LOF lines were made and the rosette area was calculated as the sum of the area of all individual leaves ( n = 10 plants ) . The rosette area is presented relative to the corresponding wild type .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 At 22 days after stratification (DAS), leaf series of the LOF lines were made and the rosette area was calculated as the sum of the area of all individual leaves (n = 10 plants). The rosette area is presented relative to the corresponding wild type.


B Average area of the individual leaves of rap2 . 6L and myb51 , two knock - out lines with a smaller average rosette area .

O O 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 Average area of the individual leaves of rap2.6L and myb51, two knock-out lines with a smaller average rosette area.


C Rosette area of two or three independent GOF lines per TF ( calculated as the projected area or the sum of the leaves , n = 10 plants ) germinated and grown on DEX - containing medium . Measurements were performed at 22 DAS relative to the control line . " Independent line 1 " is the line with the highest overexpression level .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Rosette area of two or three independent GOF lines per TF (calculated as the projected area or the sum of the leaves, n = 10 plants) germinated and grown on DEX-containing medium. Measurements were performed at 22 DAS relative to the control line. "Independent line 1" is the line with the highest overexpression level.


D A representative picture of the rosette of GOF lines with significant growth phenotypes in both independent lines at 22 DAS . Scale bar is 1 cm .

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

D A representative picture of the rosette of GOF lines with significant growth phenotypes in both independent lines at 22 DAS. Scale bar is 1 cm.


E , F Pavement cell number and area of the third leaf at 22 DAS of LOF ( E ) that showed a significant rosette area phenotype . Data information : In A and C , data are presented as mean ± SEM , n = 3 independent experiments , * = P < 0 . 05 ( Tukey ' s test ) . In B , data are presented as mean ± SEM , n = 3 independent experiments , * = P < 0 . 05 ( mixed model , partial F - tests ) . In E and F , data are presented as mean ± SEM , n = 3 independent experiments , . = P < 0 . 1 , * = P < 0 . 05 , * * = P < 0 . 01 , * * * = P < 0 . 001 ( Tukey ' s 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, F Pavement cell number and area of the third leaf at 22 DAS of LOF (E) that showed a significant rosette area phenotype. Data information: In A and C, data are presented as mean ± SEM, n = 3 independent experiments, *=P<0.05 (Tukey's test). In B, data are presented as mean ± SEM, n = 3 independent experiments, *=P<0.05 (mixed model, partial F-tests). In E and F, data are presented as mean ± SEM, n = 3 independent experiments, . = P<0.1, * = P<0.05, ** = P<0.01, *** = P<0.001 (Tukey's test).


E , F Pavement cell number and area of the third leaf at 22 DAS of GOF lines ( F ) that showed a significant rosette area phenotype . Data information : In A and C , data are presented as mean ± SEM , n = 3 independent experiments , * = P < 0 . 05 ( Tukey ' s test ) . In B , data are presented as mean ± SEM , n = 3 independent experiments , * = P < 0 . 05 ( mixed model , partial F - tests ) . In E and F , data are presented as mean ± SEM , n = 3 independent experiments , . = P < 0 . 1 , * = P < 0 . 05 , * * = P < 0 . 01 , * * * = P < 0 . 001 ( Tukey ' s 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, F Pavement cell number and area of the third leaf at 22 DAS of GOF lines (F) that showed a significant rosette area phenotype. Data information: In A and C, data are presented as mean ± SEM, n = 3 independent experiments, *=P<0.05 (Tukey's test). In B, data are presented as mean ± SEM, n = 3 independent experiments, *=P<0.05 (mixed model, partial F-tests). In E and F, data are presented as mean ± SEM, n = 3 independent experiments, . = P<0.1, * = P<0.05, ** = P<0.01, *** = P<0.001 (Tukey's test).


A The expression level of two GA - degradation and two GA - biosynthesis enzymes in expanding leaf tissue ( third leaf at 15 DAS ) of wild - type plants 20 min , 40 min , 1 h , 2 h , 4 h , 8 h , 12 h , 16 h , and 24 h after mannitol treatment . The fold changes ( FC ) were calculated relative to control conditions . Data information : In A , data are presented as mean ± SEM , n = 4 independent experiments , * = FDR < 0 . 05 ( unpaired two - sided Student ' s 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 O 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

A The expression level of two GA-degradation and two GA-biosynthesis enzymes in expanding leaf tissue (third leaf at 15 DAS) of wild-type plants 20 min, 40 min, 1 h, 2 h, 4 h, 8 h, 12 h, 16 h, and 24 h after mannitol treatment. The fold changes (FC) were calculated relative to control conditions.Data information: In A, data are presented as mean ± SEM, n = 4 independent experiments, *= FDR<0.05 (unpaired two-sided Student's t-test)


B The expression of GA2 - OX6 and GA20 - OX1 in expanding leaf tissue ( third leaf - 15 DAS ) , 8 h after transfer to DEX - containing medium to induce overexpression . The FC was calculated relative to control conditions . Data information : In B , data are presented as mean ± SEM , n = 3 independent experiments , * = FDR < 0 . 1 ( mixed model analysis , user - defined Wald tests ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 The expression of GA2-OX6 and GA20-OX1 in expanding leaf tissue (third leaf - 15 DAS), 8 h after transfer to DEX-containing medium to induce overexpression. The FC was calculated relative to control conditions.Data information: In B, data are presented as mean ± SEM, n = 3 independent experiments, * = FDR < 0.1 (mixed model analysis, user-defined Wald tests).


C The effect of ERF6 , ERF9 , ERF98 and WRKY15 on the GA2 - OX6 promoter determined with transient expression assays . The relative luminescence was calculated relative to the control , 35S : : GUS ( n = 4 biological repeats ) . Data information : In C , data are presented as mean ± SEM , n = 3 independent experiments .

C The effect of ERF6, ERF9, ERF98 and WRKY15 on the GA2-OX6 promoter determined with transient expression assays. The relative luminescence was calculated relative to the control, 35S::GUS (n = 4 biological repeats). Data information: In C, data are presented as mean ± SEM, n = 3 independent experiments.


A At 22 DAS , leaf series were made of the LOF lines ( n = 10 plants ) grown under control conditions or mild osmotic stress conditions , and the reduction under mild osmotic stress was calculated . The rosette area reduction is presented relative to the corresponding wild type . Data information : In A and C , data are presented as mean ± SEM , n = 3 independent experiments , * = P < 0 . 05 ( Tukey ' s 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 At 22 DAS, leaf series were made of the LOF lines (n = 10 plants) grown under control conditions or mild osmotic stress conditions, and the reduction under mild osmotic stress was calculated. The rosette area reduction is presented relative to the corresponding wild type.Data information: In A and C, data are presented as mean ± SEM, n = 3 independent experiments, *=P<0.05 (Tukey's test).


B Leaf series of stz and erf11 mutants grown on control and mild osmotic stress conditions . Data information : In B , data are presented as mean ± SEM , n = 3 independent experiments , * = P < 0 . 05 ( mixed model , partial F - tests ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Leaf series of stz and erf11 mutants grown on control and mild osmotic stress conditions.Data information: In B, data are presented as mean ± SEM, n = 3 independent experiments, *=P<0.05 (mixed model, partial F-tests).


C The rosette area of two or three independent GOF lines ( n = 10 plants ) germinated on DEX - containing control or mild osmotic stress medium was measured at 22 DAS and the reduction by mild osmotic stress was calculated relative to the control line . " Independent line 1 " is the line with the highest overexpression level . Data information : In A and C , data are presented as mean ± SEM , n = 3 independent experiments , * = P < 0 . 05 ( Tukey ' s 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 rosette area of two or three independent GOF lines (n = 10 plants) germinated on DEX-containing control or mild osmotic stress medium was measured at 22 DAS and the reduction by mild osmotic stress was calculated relative to the control line. "Independent line 1" is the line with the highest overexpression level. Data information: In A and C, data are presented as mean ± SEM, n = 3 independent experiments, *=P<0.05 (Tukey's test).


A , B , C , D , E The effect of the individual and the combination of two TFs on the expression of target genes ERF11 ( A ) The relative luminescence was calculated relative to the control , 35S : : GUS ( n = 4 biological repeats ) . Green represents activation , red repression and gray absence of regulation . Data information : Data are presented as mean ± SEM , n = 3 independent experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, C, D, E The effect of the individual and the combination of two TFs on the expression of target genes ERF11 (A) The relative luminescence was calculated relative to the control, 35S::GUS (n = 4 biological repeats). Green represents activation, red repression and gray absence of regulation. Data information: Data are presented as mean ± SEM, n = 3 independent experiments.


A , B , C , D , E The effect of the individual and the combination of two TFs on the expression of target genes ERF5 ( B ) The relative luminescence was calculated relative to the control , 35S : : GUS ( n = 4 biological repeats ) . Green represents activation , red repression and gray absence of regulation . Data information : Data are presented as mean ± SEM , n = 3 independent experiments .

A, B, C, D, E The effect of the individual and the combination of two TFs on the expression of target genes ERF5 (B) The relative luminescence was calculated relative to the control, 35S::GUS (n = 4 biological repeats). Green represents activation, red repression and gray absence of regulation. Data information: Data are presented as mean ± SEM, n = 3 independent experiments.


A , B , C , D , E The effect of the individual and the combination of two TFs on the expression of target genes MYB51 ( C ) The relative luminescence was calculated relative to the control , 35S : : GUS ( n = 4 biological repeats ) . Green represents activation , red repression and gray absence of regulation . Data information : Data are presented as mean ± SEM , n = 3 independent experiments .

A, B, C, D, E The effect of the individual and the combination of two TFs on the expression of target genes MYB51 (C) The relative luminescence was calculated relative to the control, 35S::GUS (n = 4 biological repeats). Green represents activation, red repression and gray absence of regulation. Data information: Data are presented as mean ± SEM, n = 3 independent experiments.


A , B , C , D , E The effect of the individual and the combination of two TFs on the expression of target genes , RAP2 . 6L ( D ) The relative luminescence was calculated relative to the control , 35S : : GUS ( n = 4 biological repeats ) . Green represents activation , red repression and gray absence of regulation . Data information : Data are presented as mean ± SEM , n = 3 independent experiments .

A, B, C, D, E The effect of the individual and the combination of two TFs on the expression of target genes, RAP2.6L (D) The relative luminescence was calculated relative to the control, 35S::GUS (n = 4 biological repeats). Green represents activation, red repression and gray absence of regulation. Data information: Data are presented as mean ± SEM, n = 3 independent experiments.


A , B , C , D , E The effect of the individual and the combination of two TFs on the expression of target genes ERF6 ( E ) . The relative luminescence was calculated relative to the control , 35S : : GUS ( n = 4 biological repeats ) . Green represents activation , red repression and gray absence of regulation . Data information : Data are presented as mean ± SEM , n = 3 independent experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, C, D, E The effect of the individual and the combination of two TFs on the expression of target genes ERF6 (E). The relative luminescence was calculated relative to the control, 35S::GUS (n = 4 biological repeats). Green represents activation, red repression and gray absence of regulation. Data information: Data are presented as mean ± SEM, n = 3 independent experiments.


The gain - of - function lines of all members of the core network were crossed either with each other , referred to as double cross , or with the control line ( GFP - GR ) , referred to as single cross . The projected rosette area of the F1 double and single cross and the GFP - GR , germinated and grown on DEX , was measured at 22 DAS . A representative picture of the single and double crosses is depicted . The values represent the relative increase or decrease in rosette size of the double crosses compared to their parental single crosses . The outcome of every double cross is classified into three groups : additive ( green connections ) , negative ( blue connection ) and synergistic ( orange connections ) phenotype . The precise measurements can be found in Fig EV2 .

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

The gain-of-function lines of all members of the core network were crossed either with each other, referred to as double cross, or with the control line (GFP-GR), referred to as single cross. The projected rosette area of the F1 double and single cross and the GFP-GR, germinated and grown on DEX, was measured at 22 DAS. A representative picture of the single and double crosses is depicted. The values represent the relative increase or decrease in rosette size of the double crosses compared to their parental single crosses. The outcome of every double cross is classified into three groups: additive (green connections), negative (blue connection) and synergistic (orange connections) phenotype. The precise measurements can be found in Fig EV2.


C . Imaging of SPBs ( Cnm67 - RFP ) and Spc72 - GFP or Mpc70 - GFP in control and PHSL1 - CDC20 cells . Top , time - lapse series . Bottom , meiotic events were quantified in cells synchronized in silico to SPB separation at entry into metaphase I ( t = 0 ) . Graphs show overlays of SPC72 - GFP and MPC70 - GFP strains . Data information : data are representative of four independent experiments .

C. Imaging of SPBs (Cnm67-RFP) and Spc72-GFP or Mpc70-GFP in control and PHSL1-CDC20 cells. Top, time-lapse series. Bottom, meiotic events were quantified in cells synchronized in silico to SPB separation at entry into metaphase I (t = 0). Graphs show overlays of SPC72-GFP and MPC70-GFP strains. Data information: data are representative of four independent experiments.


D . Imaging of SPBs ( Cnm67 - RFP ) and Spc72 - GFP or Mpc70 - GFP in PHSL1 - CDC20 strains containing the indicated mutations . Meiotic events were analyzed as in ( C ) . Data information : data are representative of two independent experiments .

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

D. Imaging of SPBs (Cnm67-RFP) and Spc72-GFP or Mpc70-GFP in PHSL1-CDC20 strains containing the indicated mutations. Meiotic events were analyzed as in (C). Data information: data are representative of two independent experiments.


A . Imaging of SPBs ( Cnm67 - RFP ) and Spc72 - GFP or Mpc70 - GFP in PHSL1 - CDC20 ama1 ∆ control cells and spo13 mutants . Top , time - lapse series . Bottom , meiotic events were quantified in cells synchronized in silico to SPB separation at entry into metaphase I ( t = 0 ) . Graphs show overlays of SPC72 - GFP and MPC70 - GFP strains . Data information : data are representative of two independent experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Imaging of SPBs (Cnm67-RFP) and Spc72-GFP or Mpc70-GFP in PHSL1-CDC20 ama1∆ control cells and spo13 mutants. Top, time-lapse series. Bottom, meiotic events were quantified in cells synchronized in silico to SPB separation at entry into metaphase I (t = 0). Graphs show overlays of SPC72-GFP and MPC70-GFP strains. Data information: data are representative of two independent experiments.


C . Imaging of SPBs ( Cnm67 - RFP ) and Spc72 - GFP or Mpc70 - GFP in PHSL1 - CDC20 ama1 ∆ control cells and cells containing clb1 ∆ or cdc28 - as1 , which have been treated with 1NM - PP1 at metaphase I ( 8 h in SPM ) . Meiotic events ( bottom ) were analyzed as in ( A ) . Data information : data are representative of two independent experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Imaging of SPBs (Cnm67-RFP) and Spc72-GFP or Mpc70-GFP in PHSL1-CDC20 ama1∆ control cells and cells containing clb1∆ or cdc28-as1, which have been treated with 1NM-PP1 at metaphase I (8 h in SPM). Meiotic events (bottom) were analyzed as in (A). Data information: data are representative of two independent experiments.


A . Imaging of SPBs ( Cnm67 - RFP ) and Mpc70 - GFP in PHSL1 - CDC20 ama1 ∆ cells containing SPC72 , spc72 - 7 , or spc72 - 7 plus hrr25 - as . Cells were shifted from 24 to 36 ° C at 4 h in SPM and treated with 1NM - PP1 to inhibit Hrr25 - as . Top , time - lapse series . Bottom , Mpc70 loading to SPBs was quantified in cells synchronized in silico to SPB separation at entry into metaphase I ( t = 0 ) . Data information : data are representative of three independent experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Imaging of SPBs (Cnm67-RFP) and Mpc70-GFP in PHSL1-CDC20 ama1∆ cells containing SPC72, spc72-7, or spc72-7 plus hrr25-as. Cells were shifted from 24 to 36°C at 4 h in SPM and treated with 1NM-PP1 to inhibit Hrr25-as. Top, time-lapse series. Bottom, Mpc70 loading to SPBs was quantified in cells synchronized in silico to SPB separation at entry into metaphase I (t = 0). Data information: data are representative of three independent experiments.


C . Imaging of SPBs ( Cnm67 - RFP ) and Mpc70 - GFP in PHSL1 - CDC20 ama1 ∆ spo13 ∆ control cells and cells expressing an additional copy of SPC72 from the PEST promoter at 4 h in SPM . Mpc70 loading ( bottom ) was analyzed as in ( A ) . PEST - SPC72 expression delays the time of Mpc70 loading by 98 min ( 95 % CI , 67 - 129 ; P < 0 . 0001 ; Welch ' s t - test ) . Data information : data are representative of three independent experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Imaging of SPBs (Cnm67-RFP) and Mpc70-GFP in PHSL1-CDC20 ama1∆ spo13∆ control cells and cells expressing an additional copy of SPC72 from the PEST promoter at 4 h in SPM. Mpc70 loading (bottom) was analyzed as in (A). PEST-SPC72 expression delays the time of Mpc70 loading by 98 min (95% CI, 67-129; P < 0.0001; Welch's t-test). Data information: data are representative of three independent experiments.


( B ) Analysis of cells containing wild - type Spo13 , Spo13 - mD , or Spo13 - mD - m2 . Meiotic events were quantified in cells synchronized in silico to SPB reduplication at entry into metaphase II ( t = 0 ) . Graphs show overlays of SPC72 - GFP and MPC70 - GFP strains . spo13 - mD delays Spc72 removal and Mpc70 loading by 30 min ( 95 % CI , 26 - 34 ; P < 0 . 0001 ) and 60 min ( 95 % CI , 55 - 66 ; P < 0 . 0001 ; Welch ' s t - test ) , respectively . ( C ) Analysis of cells expressing Clb1 - mDK and different versions of Spo13 . Meiotic events were quantified as in ( B ) . spo13 - mD plus PEST - clb1 - mDK delays Spc72 removal and Mpc70 loading by 72 min ( 95 % CI , 63 - 80 ; P < 0 . 0001 ) and 114 min ( 95 % CI , 99 - 129 ; P < 0 . 0001 ; Welch ' s t - test ) , respectively .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Analysis of cells containing wild-type Spo13, Spo13-mD, or Spo13-mD-m2. Meiotic events were quantified in cells synchronized in silico to SPB reduplication at entry into metaphase II (t = 0). Graphs show overlays of SPC72-GFP and MPC70-GFP strains. spo13-mD delays Spc72 removal and Mpc70 loading by 30 min (95% CI, 26-34; P < 0.0001) and 60 min (95% CI, 55-66; P < 0.0001; Welch's t-test), respectively. (C) Analysis of cells expressing Clb1-mDK and different versions of Spo13. Meiotic events were quantified as in (B). spo13-mD plus PEST-clb1-mDK delays Spc72 removal and Mpc70 loading by 72 min (95% CI, 63-80; P < 0.0001) and 114 min (95% CI, 99-129; P < 0.0001; Welch's t-test), respectively.


C . Cdc5 interacts with Ime2 . Cdc5 - ha3 , Ime2 , and Spo13 were detected by immunoblotting in whole - cell extracts and α - Ha immunoprecipitates from the indicated strains . Data information : data are representative of two independent experiments .

O O O O O O O O O O O O O O O O O O O O O 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. Cdc5 interacts with Ime2. Cdc5-ha3, Ime2, and Spo13 were detected by immunoblotting in whole-cell extracts and α-Ha immunoprecipitates from the indicated strains. Data information: data are representative of two independent experiments.


D . Cdc5 ' s polo - box domain ( PBD ) binds to Ime2 . Ha3 - tagged versions of the wild - type PBD or the phosphopeptide - binding mutant PBD - FAA were expressed at t = 5 . 5 h in SPM in cells depleted of Cdc20 and Cdc5 . PBD - ha3 and Ime2 were detected by immunoblotting in whole - cell extracts and α - Ha immunoprecipitates . Data information : data are representative of two independent experiments .

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

D. Cdc5's polo-box domain (PBD) binds to Ime2. Ha3-tagged versions of the wild-type PBD or the phosphopeptide-binding mutant PBD-FAA were expressed at t = 5.5 h in SPM in cells depleted of Cdc20 and Cdc5. PBD-ha3 and Ime2 were detected by immunoblotting in whole-cell extracts and α-Ha immunoprecipitates. Data information: data are representative of two independent experiments.


E . Cdc5 - dependent modification of Ime2 . PHSL1 - CDC20 ama1 ∆ control cells and cells containing spo13 ∆ or cdc5 - as were treated with CMK ( to inhibit Cdc5 - as ) at t = 8 h in SPM ( arrow heads ) . The panel shows immunoblot analysis of whole - cell extracts . C , sample from proliferating cells . Increased gel mobility of Ndt80 at t ≥ 10 h confirms inhibition of Cdc5 - as . The arrow marks the modified form of Ime2 . Data information : data are representative of two independent experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Cdc5-dependent modification of Ime2. PHSL1-CDC20 ama1∆ control cells and cells containing spo13∆ or cdc5-as were treated with CMK (to inhibit Cdc5-as) at t = 8 h in SPM (arrow heads). The panel shows immunoblot analysis of whole-cell extracts. C, sample from proliferating cells. Increased gel mobility of Ndt80 at t ≥ 10 h confirms inhibition of Cdc5-as. The arrow marks the modified form of Ime2. Data information: data are representative of two independent experiments.


C , D . Immunoblot analysis of whole - cell extracts . C , sample from proliferating cells . ( C ) Protein levels in the cultures shown in ( A ) . ( D ) Protein levels in the cultures shown in ( 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

C, D. Immunoblot analysis of whole-cell extracts. C, sample from proliferating cells. (C) Protein levels in the cultures shown in (A). (D) Protein levels in the cultures shown in (B).


A , B . Mitotic cdc20 - 3 cells were shifted to 36 ° C for 80 min . At t = 0 , cells were treated with estradiol to induce expression from the PEST promoter . Top , time - lapse series from the imaging of SPBs ( Cnm67 - RFP ) and Spc72 - GFP . Frame width , 19 μm . Bottom , quantification of cells with Spc72 - GFP at SPBs . ( A ) Expression of Ime2 - ∆ C induces removal of Spc72 from SPBs , which is inhibited by co - expression of Spo13 . ( B ) Expression of Cdc5 plus Ime2 - ∆ C causes rapid removal of Spc72 , which is inhibited by co - expression of Spo13 . Data information : data are representative of three independent experiments .

O O O O 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 O O O O O O O O O O O O O O O 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. Mitotic cdc20-3 cells were shifted to 36°C for 80 min. At t = 0, cells were treated with estradiol to induce expression from the PEST promoter. Top, time-lapse series from the imaging of SPBs (Cnm67-RFP) and Spc72-GFP. Frame width, 19 μm. Bottom, quantification of cells with Spc72-GFP at SPBs. (A) Expression of Ime2-∆C induces removal of Spc72 from SPBs, which is inhibited by co-expression of Spo13. (B) Expression of Cdc5 plus Ime2-∆C causes rapid removal of Spc72, which is inhibited by co-expression of Spo13. Data information: data are representative of three independent experiments.


C . Mitotic cdc20 - 3 cells containing PEST - CDC5 plus PEST - IME2 - ∆ C and / or PEST - NDT80 were shifted to 36 ° C for 30 min and subsequently treated with estradiol ( t = 0 ) . Top , time - lapse series from the imaging of SPBs ( Cnm67 - RFP ) and Mpc70 - GFP . Frame width , 19 μm . Bottom , quantification of cells with Mpc70 - GFP at SPBs . Data information : data are representative of two independent experiments .

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

C. Mitotic cdc20-3 cells containing PEST-CDC5 plus PEST-IME2-∆C and/or PEST-NDT80 were shifted to 36°C for 30 min and subsequently treated with estradiol (t = 0). Top, time-lapse series from the imaging of SPBs (Cnm67-RFP) and Mpc70-GFP. Frame width, 19 μm. Bottom, quantification of cells with Mpc70-GFP at SPBs. Data information: data are representative of two independent experiments.


C . PHSL1 - CDC20 ama1 ∆ cdc28 - as2 control cells and cells containing spo13 ∆ or cdc5 - as were treated with 1Na - PP1 at t = 3 h in SPM to inhibit Cdc28 - as2 . At t = 9 h in SPM , Cdc28 - as2 was activated by washing cells with cSPM lacking 1Na - PP1 ( washout ) . cdc5 - as cells were washed with cSPM plus CMK to activate Cdc28 - as2 and inhibit Cdc5 - as . Top , time - lapse series from the imaging of Mpc70 - GFP and RFP - tubulin . The weak , nuclear signal originates from Ndt80 - GFP . Bottom , quantification of spindle formation and Mpc70 loading to SPBs . D . PHSL1 - CDC20 ama1 ∆ cells containing SPC72 , spc72 - 7 , or spc72 - 7 plus cdc5 - as were shifted from 24 to 36 ° C at t = 4 . 2 h in SPM ( to inactivate Spc72 - 7 , arrow heads ) and treated with CMK at t = 6 h in SPM ( to inhibit Cdc5 - as ) . Top , time - lapse series from the imaging of SPBs ( Cnm67 - RFP ) and Mpc70 - GFP . Bottom , quantification of SPB separation and Mpc70 loading to SPBs . Inhibition of Cdc5 - as in spc72 - 7 cells advances the time of Mpc70 loading by 119 min ( 95 % CI , 76 - 162 ; P < 0 . 0001 ; Welch ' s t - test ) . Data are representative of two independent experiments .

O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-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 O O O O O O O O O O O O O O O O O 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 O O 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. PHSL1-CDC20 ama1∆ cdc28-as2 control cells and cells containing spo13∆ or cdc5-as were treated with 1Na-PP1 at t = 3 h in SPM to inhibit Cdc28-as2. At t = 9 h in SPM, Cdc28-as2 was activated by washing cells with cSPM lacking 1Na-PP1 (washout). cdc5-as cells were washed with cSPM plus CMK to activate Cdc28-as2 and inhibit Cdc5-as. Top, time-lapse series from the imaging of Mpc70-GFP and RFP-tubulin. The weak, nuclear signal originates from Ndt80-GFP. Bottom, quantification of spindle formation and Mpc70 loading to SPBs. D. PHSL1-CDC20 ama1∆ cells containing SPC72, spc72-7, or spc72-7 plus cdc5-as were shifted from 24 to 36°C at t = 4.2 h in SPM (to inactivate Spc72-7, arrow heads) and treated with CMK at t = 6 h in SPM (to inhibit Cdc5-as). Top, time-lapse series from the imaging of SPBs (Cnm67-RFP) and Mpc70-GFP. Bottom, quantification of SPB separation and Mpc70 loading to SPBs. Inhibition of Cdc5-as in spc72-7 cells advances the time of Mpc70 loading by 119 min (95% CI, 76-162; P < 0.0001; Welch's t-test). Data are representative of two independent experiments.


A . Inhibition of Cdk1 at anaphase I . CDC20 - mAR and CDC20 - mAR cdc28 - as1 cells were released from the metaphase I - arrest with CuSO4 at 7 h in SPM ( t = 0 ) and treated with 1NM - PP1 at t = 40 min to inhibit Cdc28 - as1 ( arrows ) . Top , time - lapse series from the imaging of Mpc70 - GFP , SPBs ( Cnm67 - RFP ) , and nuclei ( TetR - RFP ) . Bottom , quantification of meiotic events .

O O O O O 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-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

A. Inhibition of Cdk1 at anaphase I. CDC20-mAR and CDC20-mAR cdc28-as1 cells were released from the metaphase I-arrest with CuSO4 at 7 h in SPM (t = 0) and treated with 1NM-PP1 at t = 40 min to inhibit Cdc28-as1 (arrows). Top, time-lapse series from the imaging of Mpc70-GFP, SPBs (Cnm67-RFP), and nuclei (TetR-RFP). Bottom, quantification of meiotic events.


( D , E ) representative confocal micrographs and quantification of LC3 - II accumulation in CTNSWT and CTNS - / - cells in presence and absence of 25 nM bafilomycin ( BafA1 ) for 4 hrs , respectively ( n = 3 ) . Scale bars are 20 µm .

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

(D, E) representative confocal micrographs and quantification of LC3-II accumulation in CTNSWT and CTNS-/- cells in presence and absence of 25 nM bafilomycin (BafA1) for 4 hrs, respectively (n = 3). Scale bars are 20 µm.


( F , G , H ) Western blotting and densitometric analyses for LC3 - II / LC3 - I ratio and SQSTM1 protein levels in CTNSWT , CTNS - / - , and CTNSPatient cells cultured in the presence or in the absence of 25 nM BafA1 for 4 h , respectively ( n = 3 ) .

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

(F, G, H) Western blotting and densitometric analyses for LC3-II/LC3-I ratio and SQSTM1 protein levels in CTNSWT, CTNS-/-, and CTNSPatient cells cultured in the presence or in the absence of 25 nM BafA1 for 4 h, respectively (n = 3).


( I , J ) Representative confocal micrographs and quantification of DQ - BSA and BSA in CTNSWT , CTNS - / - , and CTNSPatient cells , respectively ( n = 7 - 14 quantified images ) . Scale bars are 20 µm .

(I, J) Representative confocal micrographs and quantification of DQ-BSA and BSA in CTNSWT, CTNS-/-, and CTNSPatient cells, respectively (n = 7-14 quantified images). Scale bars are 20 µm.


( D ) List of metabolites that were shared and significantly altered in both the CTNS - / - and CTNSPatient cells compared to control cells ( n = 6 ) .

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

(D) List of metabolites that were shared and significantly altered in both the CTNS-/- and CTNSPatient cells compared to control cells (n = 6).


( I ) List of proteins that were significantly upregulated and downregulated in CTNS - / - cells compared to control cells ( n = 3 ) . AKGDH ; Alpha - ketoglutarate dehydrogenase , LIPA ; Lysosomal acid lipase , ACP2 ; Lysosomal acid phosphatase , CTSS ; Cathepsin S , CTSC ; Dipeptidyl peptidase , IGF2R ; Cation - independent mannose - 6 - phosphate receptor , SORT1 ; Sortilin , CAT ; Catalase , CTSA ; Lysosomal protective protein , and SOD ; Superoxide dismutase .

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

(I) List of proteins that were significantly upregulated and downregulated in CTNS-/- cells compared to control cells (n = 3). AKGDH; Alpha-ketoglutarate dehydrogenase, LIPA; Lysosomal acid lipase, ACP2; Lysosomal acid phosphatase, CTSS; Cathepsin S, CTSC; Dipeptidyl peptidase, IGF2R; Cation-independent mannose-6-phosphate receptor, SORT1; Sortilin, CAT; Catalase, CTSA; Lysosomal protective protein, and SOD; Superoxide dismutase.


( B , C , D ) ROS production ( ROS / mg protein ) in CTNSWT , CTNS ‑ / - , and CTNSPatient cells upon starvation for 4 hrs in the presence and absence of DMKG ( 4 hrs ) , respectively ( n = 3 ) .

O 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 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

(B, C, D) ROS production (ROS/mg protein) in CTNSWT, CTNS‑/-, and CTNSPatient cells upon starvation for 4 hrs in the presence and absence of DMKG (4 hrs), respectively (n = 3).


Representative confocal micrographs ( scale bars are 20 µm ) of caspase 3 / 7 activation in DMKG ( 2 mM ) - treated CTNSWT , CTNS ‑ / - , and CTNSPatient cells for 24 hrs ( n = 3 ) .

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

Representative confocal micrographs (scale bars are 20 µm) of caspase 3/7 activation in DMKG (2 mM)-treated CTNSWT, CTNS‑/-, and CTNSPatient cells for 24 hrs (n = 3).


( H , I ) Western blotting and densitometric analyses for LC3 - II / LC3 - I ratio in CTNSWT , CTNS ‑ / - , and CTNSPatient cells cultured in the presence or in the absence of BafA1 ( 25 nM ) and DMKG ( 2 mM ) for 4 hrs , respectively ( n = 3 ) .

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

(H, I) Western blotting and densitometric analyses for LC3-II/LC3-I ratio in CTNSWT, CTNS‑/-, and CTNSPatient cells cultured in the presence or in the absence of BafA1 (25 nM) and DMKG (2 mM) for 4 hrs, respectively (n = 3).