( I ) Merged confocal show distribution of Rac1 specific GEF TIAM ( magenta ) in HeLa before ( 0 ) , after optogenetic recruitment at indicated time points . CIBN - Sec61β ( green ) was used to recruit Cry2 - TIAM to ER with 488 nm . ( J ) Graph shows number of SNAP - Sec16A marked ERES in HeLa before ( 0 ) after ( 30 , 60 90 seconds ) recruitment of TIAM to ER from 2 independent experiments . Whiskers indicate min - max range central band indicates median of data . Number of = 9 .

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(I) Merged confocal images show distribution of Rac1 specific GEF TIAM (magenta) in HeLa cells before (0), and after optogenetic recruitment at indicated time points. CIBN-Sec61β (green) was used to recruit Cry2-TIAM to the ER with 488 nm laser. (J) Graph shows the number of SNAP-Sec16A marked ERES in HeLa cells before (0) and after (30, 60 and 90 seconds) recruitment of TIAM to the ER from 2 independent experiments. Whiskers indicate min-max range and central band indicates the median of all data. Number of cells = 9.


( A B ) Representative immunofluorescence showing Sec31A labelled ERES in HeLa treated with DMSO ( Ctrl ) , 0 . 5 µM cytochalasin D ( CytoD ) 0 . 5 µM latrunculin A ( LatA ) for 15 30 min ( A ) . Scale bar : 5 µm . number of ERES per in HeLa treated with different doses of CytoD LatA for 30 min are displayed as % of control ± standard deviation ( 90 - 100 from 3 experiments ) ( B ) .

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(A and B) Representative immunofluorescence images showing Sec31A labelled ERES in HeLa cells treated with DMSO (Ctrl), 0.5 µM cytochalasin D (CytoD) and 0.5 µM latrunculin A (LatA) for 15 and 30 min (A). Scale bar: 5 µm. The number of ERES per cell in HeLa cells treated with different doses of CytoD and LatA for 30 min are displayed as % of control ± standard deviation (90 -100 cells from 3 experiments) (B).


( C ) Distribution of ManII - RUSH in HeLa . were pre - treated with DMSO ( Ctrl ) or 1 µM LatA for 15 min prior to addition of biotin to initiate ManII - trafficking . Golgi arrival for ManII - RUSH was monitored for 30 min .

O O O O O B-GENEPROD O B-EXP_ASSAY O B-CELL_LINE O O O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O B-GENEPROD O O O B-SUBCELLULAR O O B-GENEPROD O B-EXP_ASSAY O O O O O O

(C) Distribution of ManII-RUSH in HeLa cells. Cells were pre-treated with DMSO (Ctrl) or 1 µM LatA for 15 min prior to addition of biotin to initiate ManII-trafficking. Golgi arrival for ManII-RUSH was monitored for 30 min.


( D - E ) Graph showing of ManII within Golgi to outside Golgi . Values were derived from HeLa treated with LatA or CytoD as described in C . At least 30 per condition were used for in each experiment ( n = 3 ) .

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(D-E) Graph showing ratio of ManII within Golgi to outside Golgi. Values were derived from HeLa cells treated with LatA or CytoD as described in C. At least 30 cells per condition were used for quantification in each experiment (n = 3).


( F ) HeLa cultured on PDMS membranes were treated with DMSO or 1 µM cytochalasin D ( CytoD ) ERES were labelled with anti - Sec31A antibody before ( no ) or after 15 % equibiaxial ( 15 % ) . Anti - phalloidin was performed to visualize actin in . Representative are shown . ( G ) of experiment described in ( F ) . graph shows average ERES / . Data were derived from a total of 90 - 115 from 3 experiments . Each dot represents several error bars show standard deviation .

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(F) HeLa cells cultured on PDMS membranes were treated with DMSO or 1 µM cytochalasin D (CytoD) and ERES were labelled with anti-Sec31A antibody before (no strain) or after 15% equibiaxial strain (15% strain). Anti-phalloidin staining was performed to visualize actin in cells. Representative images are shown. (G) Quantification of experiment described in (F). The graph shows average ERES/Cell. Data were derived from a total of 90 - 115 cells from 3 experiments. Each dot represents several cells and error bars show standard deviation.


( H I ) siRNA mediated depletion of INF2 does not phenocopy Rac1 effect . HeLa were transfected with non - targeting control siRNA ( siCtrl ) or siRNA against INF2 ( siINF2 ) for 72 h before processing immunostaining with Sec31A to quantify ERES . Representative showing Sec31A - labelled ERES in HeLa ( H ) , of ERES per presented as % of control ( I ) . In total 137 ( siCtrl ) 113 ( siINF2 ) were used for ERES from three independent experiments .

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(H and I) siRNA mediated depletion of INF2 does not phenocopy Rac1 effect. HeLa cells were transfected with non-targeting control siRNA (siCtrl) or siRNA against INF2 (siINF2) for 72 h before processing cells immunostaining with Sec31A to quantify ERES. Representative images showing Sec31A-labelled ERES in HeLa cells (H), and quantification of ERES per cell presented as % of control (I). In total 137 cells (siCtrl) and 113 cells (siINF2) were used for ERES quantification from three independent experiments.


( J K ) Knockdown of INF2 does not alter kinetics of ER - to - Golgi transport . HeLa stably expressing ManII - RUSH were transfected with siRNAs as described in E , ER - to - Golgi transport of ManII - RUSH was followed by confocal microscopy ( J ) . Representative for indicated time points are shown . Scale bars , 5 µm . of ManII - RUSH within Golgi to outside Golgi was calculated for indicated time points ( K ) . Three independent experiments were performed a total of 82 ( siCtrl , 0 min ) , 72 ( siINF2 , 0 min ) 93 ( siCtrl siINF2 , 20 min ) were used for .

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(J and K) Knockdown of INF2 does not alter the kinetics of ER-to-Golgi transport. HeLa cells stably expressing ManII-RUSH were transfected with siRNAs as described in E, and the ER-to-Golgi transport of ManII-RUSH was followed by confocal microscopy (J). Representative images for indicated time points are shown. Scale bars, 5 µm. The ratio of ManII-RUSH within the Golgi to outside the Golgi was calculated for the indicated time points (K). Three independent experiments were performed and a total of 82 (siCtrl, 0 min), 72 (siINF2, 0 min) and 93 (siCtrl and siINF2, 20 min) were used for quantification.


( A ) Co - localization of mCherry - Rac1 GFP - Sec16A in HeLa as monitored by SRRF live imaging . See also movieS4 .

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(A) Co-localization of mCherry-Rac1 and GFP-Sec16A in HeLa cells as monitored by SRRF live cell imaging. See also movieS4.


( B ) Representative immunofluorescence of HeLa co - transfected with YFP ( N ) - Rac1 Sar1 - YFP ( C ) immunostained with Sec31A . Rac1 Sar1 form complexes at ERES in HeLa , which partially overlap with Sec31A . ( C ) Box plot showing fraction of ERES that overlap with Rac1 - Sar1 ( number of = 24 , number of experiments = 4 ) . data points shown in graph represent number of . Whiskers indicate min - max range central band indicates median of data .

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(B) Representative immunofluorescence images of HeLa cells co-transfected with YFP(N)-Rac1 and Sar1-YFP(C) and immunostained with Sec31A. Rac1 and Sar1 form complexes at ERES in HeLa cells, which partially overlap with Sec31A. (C) Box plot showing the fraction of ERES that overlap with the Rac1-Sar1 complex (number of cells = 24, number of experiments = 4). Individual data points shown in the graph represent number of images. Whiskers indicate min-max range and central band indicates the median of all data.


( D ) GFP - tagged Rac1 was immunoprecipitated by GFP - trap fraction was immunoblotted for endogenous Sar1 . Representative immunoblot showing co - immunoprecipitation of Sar1 by Rac1 .

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(D) GFP-tagged Rac1 was immunoprecipitated by GFP-trap and the fraction was immunoblotted for endogenous Sar1. Representative immunoblot showing co-immunoprecipitation of Sar1 by Rac1.


( E ) Representative immunofluorescence of HeLa co - transfected with YFP ( N ) - Rac1 Sar1 - YFP ( C ) cultured on crossbow micropatterns of indicated . were co - transfected with indicated constructs incubated overnight prior to trypsinization seeding on micropatterned coverslip . were allowed to grow for 3h , fixed processed for microscopy . ( F ) of Rac1 - Sar1 described in ( E ) ( number of experiments = 3 , number of = 28 ( small ) 27 ( large ) ) . In graph , error bars represent standard deviation asterisk ( * ) denotes statistical significance at p - value < 0 . 05 .

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(E) Representative immunofluorescence images of HeLa cells co-transfected with YFP(N)-Rac1 and Sar1-YFP(C) and cultured on crossbow micropatterns of indicated size. Cells were co-transfected with the indicated DNA constructs and incubated overnight prior to trypsinization and seeding on the micropatterned coverslip. Cells were allowed to grow for 3h, fixed and processed for microscopy. (F) Quantification of Rac1-Sar1 puncta described in (E) (number of experiments = 3, number of cells = 28 (small) and 27 (large)). In the quantification graph, error bars represent standard deviation and asterisk (*) denotes statistical significance at p-value < 0.05.


( E ) expressing GFP - Rac1 were lysed followed by immunoprecipitation against GFP immunoblotting against endogenous Sar1 Sec12 . Blotting against GFP was performed to determine efficiency of IP .

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(E) Cells expressing GFP-Rac1 were lysed followed by immunoprecipitation against GFP and immunoblotting against endogenous Sar1 and Sec12. Blotting against GFP was performed to determine efficiency of the IP.


( F ) HeLa were co - transfected with indicated plasmids were incubated overnight prior to lysis immunoprecipitation against GFP . Immunoblotting against anti - mCherry was performed to detect Sar1A - mCherry . IP efficiency was determined using anti - GFP . Different exposure times are shown for anti - mCherry blot .

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(F) HeLa cells were co-transfected with the indicated plasmids and cells were incubated overnight prior to cell lysis and immunoprecipitation against GFP. Immunoblotting against anti-mCherry was performed to detect Sar1A-mCherry. The IP efficiency was determined using anti-GFP. Different exposure times are shown for anti-mCherry blot.


( A ) Microsomes isolated from HeLa were incubated as indicated . Active Sar1 pulldown was then carried out , followed by immunoblotting against Sar1 .

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(A) Microsomes isolated from HeLa cells were incubated as indicated. Active Sar1 pulldown was then carried out, followed by immunoblotting against Sar1.


( B ) Microsomes ( M ) from HeLa stably expressing mCherry - ManII - RUSH were incubated with ATP an ATP - regenerating system , GTP , biotin cytosol in presence or absence of recombinant Rac1 for 30 min at 25 ° C . COPII vesicles ( V ) were pelleted by ultracentrifugation ( 100 , 000 xg ) immunoblotted for anti - mCherry to detect ManII - RUSH anti - calnexin ( ER marker ) . ( C ) data of two independent vesicle budding assays . values were normalized to 4 ℃ Ctrl condition .

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

(B) Microsomes (M) from HeLa cells stably expressing mCherry-ManII-RUSH were incubated with ATP and an ATP-regenerating system, GTP, biotin and cytosol in the presence or absence of recombinant Rac1 for 30 min at 25°C. COPII vesicles (V) were pelleted by ultracentrifugation (100,000 xg) and immunoblotted for anti-mCherry to detect ManII-RUSH and anti-calnexin (ER marker). (C) Quantification data of two independent vesicle budding assays. All values were normalized to 4 ℃ Ctrl condition.


Purified NK were activated with IL - 2 or IL - 15 in presence or absence of PGE2 ( 1µM ) tested for ( A ) proliferation by flow cytometry for Ki - 67 after four days of culture ( n = 6 , biological replicates ) ,

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Purified NK cells were activated with IL-2 or IL-15 in the presence or absence of PGE2 (1µM) and tested for (A) proliferation by flow cytometry for Ki-67 staining after four days of culture (n=6, biological replicates),


Purified NK were activated with IL - 2 or IL - 15 in presence or absence of PGE2 ( 1µM ) tested for , ( B ) ability to kill K562 after two days of culture in a 4 - hour chromium - at an E : T of 5 : 1 ( n = 4 , biological replicates ) .

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Purified NK cells were activated with IL-2 or IL-15 in the presence or absence of PGE2 (1µM) and tested for , (B) ability to kill K562 cells after two days of culture in a 4-hour chromium-assay at an E:T ratio of 5:1 (n=4, biological replicates).


Purified NK were activated with IL - 2 or IL - 15 in presence or absence of PGE2 ( 1µM ) tested for ( C ) Viability by flow cytometry using fixable live / dead after two days of culture ( n = 7 , biological replicates ) .

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Purified NK cells were activated with IL-2 or IL-15 in the presence or absence of PGE2 (1µM) and tested for (C) Viability by flow cytometry using fixable live/dead staining after two days of culture (n=7, biological replicates).


D ) Intracellular cAMP detected in two - day IL - 2 or IL - 15 activated NK before 5 minutes after addition of PGE2 ( 1 µM ) ( n = 3 , biological replicates )

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D) Intracellular cAMP detected in two-day IL-2 or IL-15 activated NK cells before and 5 minutes after the addition of PGE2 (1 µM) (n=3, biological replicates)


E ) mRNA of selected PGE2 response elements as measured by qPCR in NK ( n = 3 - 4 , biological replicates ) .

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E) mRNA levels of selected PGE2 response elements as measured by qPCR in NK cells (n=3-4, biological replicates).


F ) Fold change comparing flow cytometry mean ( MFI ) values of PDE4A expression between IL - 2 IL - 15 activated NK after two days activation with cytokines ( n = 5 , biological replicates ) .

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F) Fold change comparing flow cytometry mean fluorescence intensity (MFI) values of PDE4A expression between IL-2 and IL-15 activated NK cells after two days activation with cytokines (n=5, biological replicates).


G ) Representative showing PDE4A expression in NK activated by IL - 2 or IL - 15 for two days . Scale bar : 10 μm .

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G) Representative images showing PDE4A expression in NK cells activated by IL-2 or IL-15 for two days. Scale bar: 10 μm.


A - B ) NK were activated with IL - 2 or IL - 15 for two days in presence or absence of PGE2 ( 1µM ) analyzed for A ) of phosphorylated S6 ( pS235 / pS236 ) , STAT3 ( S727 ) , STAT5 ( Y694 ) , Akt ( S473 ) ( n = 4 , biological replicates ) B ) 16 categories of phosphorylation array showed . Red box indicates category 1 ( pS6 + , pSTAT3 + , pSTAT5 + , pAKT + ) .

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A-B) NK cells were activated with IL-2 or IL-15 for two days in the presence or absence of PGE2 (1µM) and analyzed for A) the levels of phosphorylated S6 (pS235/pS236), STAT3 (S727), STAT5 (Y694), Akt (S473) (n=4, biological replicates) and B)16 categories of phosphorylation array showed. Red box indicates category 1 (pS6+, pSTAT3+, pSTAT5+, pAKT+).


C ) Relative changes in frequency of NK expressing pS6 in presence absence of PGE2 ( n = 4 , biological replicates ) .

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C) Relative changes in frequency of NK cells expressing pS6 in the presence and absence of PGE2 (n=4, biological replicates).


D ) Flow cytometry of PDE4A in presence or absence of Torin - 1 ( 1µM ) ( left , n = 7 , biological replicates ) with representative histograms ( right ) .

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D) Flow cytometry analysis of PDE4A in the presence or absence of Torin-1 (1µM) (left, n=7, biological replicates) with representative histograms (right).


E ) NK - mediated cytotoxicity of K562 by 4 - hour chromium - release of two - day cytokine activated NK in presence or absence of Torin - 1 ( 1µM ) PGE2 ( 1µM ) ( n = 6 , biological replicates ) . E : T = 5 : 1 .

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E) NK cell-mediated cytotoxicity of K562 cells by 4-hour chromium-release assay of two-day cytokine activated NK cells in the presence or absence of Torin-1 (1µM) and PGE2 (1µM) (n=6, biological replicates). E:T ratio=5:1.


F , G ) ( F ) Flow cytometry of pS6 ( G ) 4 - hour chromium - release of PGE2 - treated NK against K562 measured after 48 hours treatment with Roflumilast ( PDE4i , 1µM ) ( n = 3 , biological replicates ) E : T = 5 : 1 .

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F, G) (F) Flow cytometry analysis of pS6 and (G) 4-hour chromium-release assay of PGE2-treated NK cells against K562 measured after 48 hours treatment with Roflumilast (PDE4i, 1µM) (n=3, biological replicates) E:T ratio=5:1.


B ) Volcano plot showing genes compared after two - days cytokine activated NK . Genes with significant changes ( p value < 0 . 05 ) > 1 . 5 log2 fold change between IL - 2 IL - 15 activated NK are labeled in red . Genes belonging to surface ( GO : 0009986 ) are annotated with gene name .

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B) Volcano plot showing genes compared after two-days cytokine activated NK cells. Genes with significant changes (p value<0.05) and >1.5 log2 fold change between IL-2 and IL-15 activated NK cells are labeled in red. Genes belonging to cell surface (GO:0009986) are annotated with gene name.


C ) of CD25 + CD54 + population changes after two days treatment ( n = 4 , biological replicates ) .

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C) Quantification of CD25+ CD54+ population changes after two days treatment (n=4, biological replicates).


D ) Representative flow cytometry graphs for CD25 CD54 in IL - 2 IL - 15 NK in presence or absence of PGE2 .

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D) Representative flow cytometry graphs for CD25 and CD54 staining in IL-2 and IL-15 NK cells in the presence or absence of PGE2.


E ) of number of clusters . NK clusters were defined as occupying an at least 2000 μm2 an eccentricity of < 0 . 8 ) . ( n = 4 , biological replicates ) .

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E) Quantification of the number of cell clusters. NK cell clusters were defined as cell aggregates occupying an area at least 2000 μm2 and an eccentricity of <0.8). (n=4, biological replicates).


F ) Representative phase contrast image of IL - 2 IL - 15 activated NK in presence or absence of PGE2 . Scale bar 50 µm .

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

F) Representative phase contrast image of IL-2 and IL-15 activated NK cells in the presence or absence of PGE2. Scale bar 50 µm.


G ) Chromium - release cytotoxicity by two - day cytokine activated NK in presence or absence of PGE2 ( 1µM ) anti - CD54 ( 20 µg / ml ) ( n = 4 , biological replicates ) , E : T = 5 : 1 .

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

G) Chromium-release cytotoxicity assay by two-day cytokine activated NK cells in the presence or absence of PGE2 (1µM) and anti-CD54 (20 µg/ml) (n=4, biological replicates), E:T ratio=5:1.


A ) NK were stimulated with either IL - 2 or IL - 15 in presence or absence of PGE2 for two days thereafter purified as CD25 + / CD54 + CD25 - / CD54 - tested in a 51Cr - release against K562 . ( E : T = 5 : 1 , n = 4 , biological replicates ) . Stars indicate significant p - values comparing CD25 + / CD54 + vs . CD25 - / CD54 - NK for each condition .

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

A) NK cells were stimulated with either IL-2 or IL-15 in the presence or absence of PGE2 for two days and thereafter purified as CD25+/CD54+ and CD25-/CD54- and tested in a 51Cr-release assay against K562. (E: T=5:1, n=4, biological replicates). Stars indicate significant p-values comparing CD25+/CD54+ vs. CD25-/CD54- NK cells for each condition.


B - F ) NK were stimulated with IL - 15 purified as CD25 + / CD54 + CD25 - / CD54 - co - cultures with K562 ( 5 : 1 ) for six hours analyzed for expression of B ) CD107a , C ) IFNγ , D ) Perforin , E ) TRAIL F ) Granzyme B were by flow cytometry ( n = 4 , biological replicates ) .

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

B-F) NK cells were stimulated with IL-15 and purified as CD25+/CD54+ and CD25-/CD54- and co-cultures with K562 cells (5:1 ratio) for six hours and analyzed for the expression of B) CD107a, C) IFNγ, D) Perforin, E) TRAIL and F) Granzyme B were by flow cytometry (n=4, biological replicates).


G ) Flow cytometry of ( Left = % frequency , right = fold change of mean ) of phosphorylated S6 ( S235 / 236 ) in CD25 + / CD54 + CD25 - / CD54 - IL - 15 activated NK ( n = 4 , biological replicates ) . H ) Flow cytometry of PDE4A expression in CD25 + / CD54 + CD25 - / CD54 - IL - 15 activated NK ( n = 4 , biological replicates ) .

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

G) Flow cytometry analysis of levels (Left=% frequency, right=fold change of mean fluorescence intensity) of phosphorylated S6 (S235/236) in CD25+/CD54+ and CD25-/CD54- IL-15 activated NK cells (n=4, biological replicates). H) Flow cytometry analysis of PDE4A expression in CD25+/CD54+ and CD25-/CD54- IL-15 activated NK cells (n=4, biological replicates).


I ) Representative image of PDE4A expression in CD25 + / CD54 + CD25 - / CD54 - IL - 15 activated NK . Scale bar : 10 µm .

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

I) Representative image of PDE4A expression in CD25+/CD54+ and CD25-/CD54- IL-15 activated NK cells. Scale bar: 10 µm.


J ) NK were first stimulated with IL - 15 for two days in absence of PGE2 , then purified as CD25 + / CD54 + or CD25 - / CD54 - NK tested in a 51Cr - release cytotoxicity against K562 at an E : T of 5 : 1 in presence ( filled bars ) or absence ( open bars ) of PGE2 ( 1 µM ) ( n = 3 , biological replicates ) .

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

J) NK cells were first stimulated with IL-15 for two days in the absence of PGE2, then purified as CD25+/CD54+ or CD25-/CD54- NK cells and tested in a 51Cr-release cytotoxicity assay against K562 cells at an E:T ratio of 5:1 in the presence (filled bars) or absence (open bars) of PGE2 (1 µM) (n=3, biological replicates).


K ) Mitochondria mass from purified CD25 + / CD54 + CD25 - / CD54 - NK measured by flow cytometry ( n = 5 , biological replicates ) .

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

K) Mitochondria mass from purified CD25+/CD54+ and CD25-/CD54- NK cells measured by flow cytometry (n=5, biological replicates).


L ) Representative image of Mitotracker Red FM in CD25 + / CD54 + NK CD25 - / CD54 - NK . Scale bar : 20 μm .

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

L) Representative image of Mitotracker Red FM in CD25+/CD54+ NK cells and CD25-/CD54- NK cells. Scale bar: 20 μm.


A ) Expression of PTGES in normal tissue .

A) Expression of PTGES in normal and tumor tissue.


B ) Kaplan - Meier of overall ( left ) progression - free ( right ) survival in with lung adenocarcinoma with high PTGES ( top 10 % ) low PTGES ( bottom 90 % ) expression .

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

B) Kaplan-Meier analysis of overall (left) and progression-free (right) survival in patients with lung adenocarcinoma with high PTGES (top 10%) and low PTGES (bottom 90%) expression.


C ) NK gene signature in normal tissues on a TCGA lung adenocarcinoma cohort of NK gene signature in with different stages of lung adenocarcinoma .

O O B-CELL_TYPE O O O O O O O O B-DISEASE I-DISEASE O O B-CELL_TYPE O O O O O O O B-DISEASE I-DISEASE O

C) NK cell gene signature in normal and tumor tissues based on a TCGA lung adenocarcinoma cohort and quantification of NK cell gene signature in patients with different stages of lung adenocarcinoma.


D ) IL15 gene expression in high low NK gene signature ( NK GS ) .

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

D) IL15 gene expression analysis in high and low NK gene signature (NK GS).


E ) Overall survival in with stage I ( n = 269 ) lung adenocarcinoma with high ( top 50 % ) low ( bottom 50 % ) NK gene expression signature expression .

O O O B-EXP_ASSAY O O O O O O O O O B-DISEASE I-DISEASE O O O O O O O O O O O O O B-CELL_TYPE B-EXP_ASSAY I-EXP_ASSAY O O O

E) Overall survival in patients with stage I (n=269) lung adenocarcinoma with high (top 50%) and low (bottom 50%) NK cell gene expression signature expression.


F ) Overall survival in with high ( top 50 % ) PTGES expression in relation to high ( top 50 % ) low ( bottom 50 % ) NK gene expression signature .

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

F) Overall survival in patients with high (top 50%) PTGES expression in relation to high (top 50%) and low (bottom 50%) NK cell gene expression signature.


A ) Real time image - killing of A549 by NK isolated from two healthy donors at an E : T of 10 : 1 .

O O O O B-EXP_ASSAY O O O B-CELL_LINE O B-CELL_TYPE O O O O O O O O O O O O O O O

A) Real time image-based killing of A549 cells by NK cells isolated from two individual healthy donors at an E:T ratio of 10:1.


B ) NK were stimulated with IL - 2 or IL - 15 in presence or absence of PGE2 then purified as CD25 + / CD54 + or CD25 - / CD54 - tested for cytotoxicity against A549 in a 51Cr - release . E : T = 5 : 1 ( n = 4 , biological replicates ) . Stars indicate significant p - values comparing CD25 + / CD54 + vs . CD25 - / CD54 - .

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

B) NK cells were stimulated with IL-2 or IL-15 in the presence or absence of PGE2 and then purified as CD25+/CD54+ or CD25-/CD54- cells and tested for cytotoxicity against A549 tumor cell in a 51Cr-release assay. E:T ratio = 5:1 (n=4, biological replicates). Stars indicate significant p-values comparing CD25+/CD54+ vs. CD25-/CD54-.


C ) Representative image of NK infiltration ( red ) into A549 spheroids at time of addition of NK at 20 hours after addition of NK , scale bar : 400 μm .

O O O B-EXP_ASSAY O B-CELL_TYPE O O O O O B-CELL_LINE O O O O O O B-CELL_TYPE O O O O O O B-CELL_TYPE O O O O O O O

C) Representative image of NK cell infiltration (red) into A549 tumor spheroids at the time of addition of NK cells and at 20 hours after the addition of NK cells, scale bar: 400 μm.


D , E ) Incucyte of D ) total E ) - infiltrating CD25 + / CD54 + CD25 - / CD54 - NK . RCU , Red Calibrated Unit , ( n = 4 , biological replicates ) .

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

D, E) Incucyte quantification of D) total and E) tumor-infiltrating CD25+/CD54+ and CD25-/CD54- NK cells. RCU, Red Calibrated Unit, (n=4, biological replicates).


F , G ) Flow cytometry of ( F ) - infiltrating ( G ) non - infiltrating NK . Results are depicted as total NK among CD45 positive live . ( n = 4 , biological replicates ) .

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

F, G) Flow cytometry quantification of (F) tumor-infiltrating and (G) non-infiltrating NK cells. Results are depicted as total NK cells among CD45 positive live cells. (n=4, biological replicates).


H ) 48 hours dpi zebrafish injected with A549 , A549 + CD25 + / CD54 + , or A549 + CD25 - / CD54 - NK after 24 hours . image was taken by Leica epi - stereomicroscope . Scale bar : 500 µm . injection ( perivitelline space ) is depicted with a dashed .

O O O O O B-ORGANISM O O B-CELL_LINE O B-CELL_LINE O B-GENEPROD O O B-GENEPROD O O O B-CELL_LINE O B-GENEPROD O O B-GENEPROD O B-CELL_TYPE O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O

H) 48 hours dpi zebrafish injected with A549, A549+ CD25+/CD54+, or A549+ CD25-/CD54- NK cells after 24 hours. Fluorescent image was taken by Leica epi-fluorescent stereomicroscope. Scale bar: 500 µm. The tumor injection area (perivitelline space) is depicted with a dashed line.


I ) 3D view of injection 24 hours posts inoculation ( left ) . Scale bar = 50 µm . injection ( perivitelline space ) is depicted with a dashed . Magnified view for ( right ) . A549 - td are annotated with a purple translucent mask NK are color - coded on to ( near - far : blue - red ) . Scale bar = 10 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 B-CELL_LINE O B-GENEPROD O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O

I) 3D view of tumor injection area 24 hours posts tumor inoculation (left). Scale bar=50 µm. The tumor injection area (perivitelline space) is depicted with a dashed line. Magnified view for tumor area (right). The A549-td tumor cells are annotated with a purple translucent mask and NK cells are color-coded based on the distance to the tumor area (near-far: blue-red). Scale bar=10 µm.


J ) between NK in CD25 - / CD54 - CD25 + / CD54 + NK treated fish ( n = 5 , biological replicates ) . K ) Frequency of infiltrating NK in in CD25 - / CD54 - CD25 + / CD54 + NK treated fish ( n = 5 , biological replicates ) .

O O O B-CELL_TYPE O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-CELL_TYPE O B-ORGANISM O O O O O O O O O O O O O O B-CELL_TYPE O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-CELL_TYPE O B-ORGANISM O O O O O O O O O

J) The distance between NK cells and tumor area in CD25-/CD54- and CD25+/CD54+ NK cell treated fish (n=5, biological replicates). K) Frequency of tumor infiltrating NK cells in in CD25-/CD54- and CD25+/CD54+ NK cell treated fish (n=5, biological replicates).


L , M ) of ( L ) ( M ) NK 24 hours after injection of NK by Imaris ( n = 5 , biological replicates ) .

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

L, M) Quantification of (L) tumor cell and (M) NK cell counts 24 hours after injection of NK cells by Imaris (n=5, biological replicates).


N ) Frequency of tissue - infiltrating total NK ( CD56 + / CD3 - ) ( left ) CD54 positive NK ( right ) among CD45 positive live ( n = 10 , biological replicates ) .

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

N) Frequency of tissue-infiltrating total NK cells (CD56+/CD3-) (left) and CD54 positive NK cells (right) among CD45 positive live cells (n=10, biological replicates).


( A ) . growth assessment of ΔIndLon ( upper plot ) ΔIndFtsH ( lower plot ) mutants grown under inducing ( blue ) or depleting conditions ( red ) . Growth was monitored by measuring biomass over time . average from two independent biological replicates is shown .

O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O

(A). Cell growth assessment of ΔIndLon (upper plot) and ΔIndFtsH (lower plot) mutants grown under inducing (blue) or depleting conditions (red). Growth was monitored by measuring DNA and protein biomass over time. The average from two independent biological replicates is shown.


( D ) . Role of Lon FtsH under heat shock stress conditions . ΔIndLon ΔIndFtsH mutants were grown under inducing ( Lon + or FtsH + ) or depleting conditions ( Lon + or FtsH + , 60h of depletion for both , Lon FtsH ) , then exposed at 45 ° C during 0 , 5 , 10 , 15 or 20 min . Then , growth after heat treatment was monitored over time under inducing conditions by 430 / 560 absorbance rate index that shows pH changes in medium . average from two independent biological replicates is shown for each condition .

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

(D). Role of Lon and FtsH under heat shock stress conditions. ΔIndLon and ΔIndFtsH mutants were grown under inducing (Lon+ or FtsH+) or depleting conditions (Lon+ or FtsH+, 60h of depletion for both, Lon and FtsH), and then exposed at 45°C during 0, 5, 10, 15 or 20 min. Then, growth after the heat treatment was monitored over time under inducing conditions by the 430/560 absorbance rate index that shows pH changes in the medium. The average from two independent biological replicates is shown for each condition.


( C ) In vivo degradation assays of FtsA , FtsZ DnaB . ΔIndLon mutants expressing different N - terminal FLAG - tagged derivatives were grown in depleting conditions for 36h . Then , Lon expression was transiently induced for 3h before translation was blocked with gentamicin ( Gm ) . Samples were taken at indicated time points after gentamicin treatment , processed for immunoblot using anti - FLAG antibodies . LC , loading control . As controls , non - induced were also treated with gentamicin processed at same time points . Immunoblots are representative of two independent experiments .

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

(C) In vivo degradation assays of FtsA, FtsZ and DnaB. ΔIndLon mutants expressing the different N-terminal FLAG-tagged derivatives were grown in depleting conditions for 36h. Then, Lon expression was transiently induced for 3h before translation was blocked with gentamicin (Gm). Samples were taken at the indicated time points after gentamicin treatment, and processed for immunoblot analysis using anti-FLAG antibodies. LC, loading control. As controls, non-induced cells were also treated with gentamicin and processed at the same time points. Immunoblots are representative of two independent experiments.


( E F ) Similar to panel B ( E ) C ( F ) but for a C - terminal FLAG - tagged derivative of MPN201 as a representative of an unstable HsdS subunit .

O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O B-GENEPROD O O

(E and F) Similar to panel B (E) and C (F) but for a C-terminal FLAG-tagged derivative of MPN201 as a representative of an unstable HsdS subunit.


( H ) Similar to panel B but for C - terminal FLAG - tagged derivatives of FtsH candidate substrates . of FtsH candidate substrates were assessed by immunoblot using anti - FtsH anti - FLAG antibodies . LC , loading control .

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

(H) Similar to panel B but for C-terminal FLAG-tagged derivatives of FtsH candidate substrates. Protein levels of FtsH and candidate substrates were assessed by immunoblot analysis using anti-FtsH and anti-FLAG antibodies. LC, loading control.


( I ) Similar to panel C , but for FtsH candidate substrates . In this case , C - terminal FLAG - tagged derivatives were grown in depleting conditions for 60h before transient expression of FtsH for 3h gentamicin treatment .

O O O O O O O O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O B-GENEPROD O O B-SMALL_MOLECULE O O

(I) Similar to panel C, but for FtsH candidate substrates. In this case, C-terminal FLAG-tagged derivatives were grown in depleting conditions for 60h before transient expression of FtsH for 3h and gentamicin treatment.


( B ) stability assessment of Lon substrates containing multiple or single mutations in putative Lon degrons . N - terminal FLAG - tagged derivatives with mutations shown in panel A were expressed in ΔIndLon mutant . were then determined by immunoblot using anti - Lon anti - FLAG antibodies comparing inducing ( + ) or depleting ( - ) conditions ( 48h of depletion ) . LC , loading control .

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

(B) Protein stability assessment of Lon substrates containing multiple or single mutations in putative Lon degrons. N-terminal FLAG-tagged derivatives with mutations shown in panel A were expressed in the ΔIndLon mutant. Protein levels were then determined by immunoblot using anti-Lon and anti-FLAG antibodies comparing inducing (+) or depleting (-) conditions (48h of depletion). LC, loading control.


( B ) Lon surveillance of truncated variants . As an example , an N - terminal FLAG - tagged derivative of MPN304 , which encodes a truncated variant of ArcA ( ArcA - Nt ) , was expressed in ΔIndLon mutant , its accumulation monitored over time under non - inducing conditions using anti - FLAG antibodies . Anti - Lon antibodies were used to monitor Lon depletion along time course experiment ( western blot figure on left ) . MPN304 stability was also assessed by in vivo degradation experiments in Lon depleted mutants ( 36h of depletion ) , in which expression was blocked with gentamicin ( Gm ) during indicated time points after transient induction of Lon for 3h . As control , samples after Gm treatment were also taken from non - induced samples . LC , loading control ( western blot figure on right ) . Immunoblots are representative of two independent experiments . A schematic representation of arginine deiminase pathway , which is inactive in M . pneumoniae , estimated copy number of its components are also shown for ΔIndLon mutant grown in inducing ( + ) or depleting ( - ) conditions .

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

(B) Lon surveillance of truncated protein variants. As an example, an N-terminal FLAG-tagged derivative of MPN304, which encodes a truncated variant of ArcA (ArcA-Nt), was expressed in the ΔIndLon mutant, and its accumulation monitored over time under non-inducing conditions using anti-FLAG antibodies. Anti-Lon antibodies were used to monitor Lon depletion along the time course experiment (western blot figure on left). MPN304 stability was also assessed by in vivo degradation experiments in Lon depleted mutants (36h of depletion), in which protein expression was blocked with gentamicin (Gm) during the indicated time points after transient induction of Lon for 3h. As control, samples after Gm treatment were also taken from non-induced samples. LC, loading control (western blot figure on right). Immunoblots are representative of two independent experiments. A schematic representation of the arginine deiminase pathway, which is inactive in M. pneumoniae, and the estimated protein copy number of its components are also shown for the ΔIndLon mutant grown in inducing (+) or depleting (-) conditions.


( E ) quality control of terminal organelle assembly mediated by Lon . Top of panel E , a schematic representation of genetic organization at P65 operon after deleting N - terminal region of hmw2 gene in ΔIndLon mutant . expected PCR products for deletion are also indicated . Below , an agarose electrophoresis gel showing PCR validation of intended deletion , SDS - PAGE immunoblot analyses monitoring expression of specific terminal organelle , whose stability depend on HMW2 , for ΔIndLon or ΔIndLon _ Δhmw2Nt lysates grown in inducing ( + ) or depleting ( - ) conditions ( 48h of depletion ) . LC , loading control .

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

(E) Protein quality control of terminal organelle assembly mediated by Lon. Top of panel E, a schematic representation of the genetic organization at the P65 operon after deleting the N-terminal region of the hmw2 gene in the ΔIndLon mutant. The expected PCR products for the deletion are also indicated. Below, an agarose electrophoresis gel showing the PCR validation of the intended deletion, and SDS-PAGE and immunoblot analyses monitoring the expression of specific terminal organelle proteins, whose stability depend on HMW2, for ΔIndLon or ΔIndLon_Δhmw2Nt cell lysates grown in inducing (+) or depleting (-) conditions (48h of depletion). LC, loading control.


A , B , C . Immunostaining of Tom20 ( mitochondria ) in positive for β3 - tubulin in DIV6 Ifnb + / + Ifnb - / - CNs with or without rIFN - β for 24 h . A . Projection of 3D image of Tom20 . Scale bars equal 20 microns . B . of total mitochondrial mass per neuron . C . Mean mitochondria volume in Ifnb + / + Ifnb - / - CNs .

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

A, B, and C. Immunostaining of Tom20 (mitochondria) in cells positive for β3-tubulin in DIV6 Ifnb+/+ and Ifnb-/- CNs with or without rIFN-β for 24 h. A. Projection of 3D image of Tom20. Scale bars equal 20 microns. B. Quantification of total mitochondrial mass per neuron. C. Mean individual mitochondria volume in Ifnb+/+ and Ifnb-/- CNs.


D , E , F , G . 3D electron microscopy of thalamus in 12 - month - old Ifnb + / + Ifnb - / - mice . D . Volume rendering of mitochondria ( left panel ) corresponding mitochondria in 2D electron microscopy image ( right panel ) in bodies . Each mitochondrion is colored differently . Scale bars equal 2 microns . Voxel equals 0 . 00878908x0 . 00878908x0 . 03 microns for Ifnb + / + neuron 0 . 00439453x0 . 00438453x0 . 04 microns for Ifnb - / - . Nuclei are in grey marked with ' N . ' E . Reconstituted axonal mitochondria . extracted from Movies EV1 2 . F . of mitochondria in bodies non - myelinated myelinated axons . N > 30 ; error bars are SD . G . Number of branches per mitochondrion . N > 30 ; error bars are SD .

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

D, E, F, and G. 3D electron microscopy of thalamus in 12-month-old Ifnb+/+ and Ifnb-/- mice. D. Volume rendering of mitochondria (left panel) and corresponding mitochondria in 2D electron microscopy image (right panel) in cell bodies. Each individual mitochondrion is colored differently. Scale bars equal 2 microns. Voxel size equals 0.00878908x0.00878908x0.03 microns for the Ifnb+/+ neuron and 0.00439453x0.00438453x0.04 microns for Ifnb-/-. Nuclei are in grey and marked with 'N.' E. Reconstituted axonal mitochondria. Images extracted from Movies EV1 and 2. F. Size of individual mitochondria in cell bodies and non-myelinated and myelinated axons. N>30; error bars are SD. G. Number of branches per mitochondrion. N>30; error bars are SD.


D - E . Respiration of isolated mitochondria from Ifnb + / + Ifnb - / - whole brains . OCR measurements under 1 substrates ( pyruvate / malate ) were obtained at baseline ( B ) on addition of ADP , oligomycin , FCCP rotenone / antimycin A to capture S3 , S4o , S3u , nonmitochondrial respiration . N = 3 - 4 mice per group ; bar graphs show mean + SE .

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

D-E. Respiration of isolated mitochondria from Ifnb+/+ and Ifnb-/- whole brains. OCR measurements under Complex 1 substrates (pyruvate/malate) were obtained at baseline (B) and on addition of ADP, oligomycin, FCCP and rotenone/antimycin A to capture S3, S4o, S3u, and nonmitochondrial respiration. N=3-4 mice per group; bar graphs show mean + SE.


K . Immunofluorescence of 8OHdG in CN cultures . Neurons were labeled with Nissl . Scale bars equal 50 microns . L . of 8OHdG by ELISA in brain tissues in young ( aged 1 . 5 - 3 months ) old ( aged 6 - 12 months ) Ifnb + / + Ifnb - / - mice .

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

K. Immunofluorescence of 8OHdG in CN cultures. Neurons were labeled with Nissl. Scale bars equal 50 microns. L. Quantification of 8OHdG by ELISA in brain tissues in young (aged 1.5-3 months) and old (aged 6-12 months) Ifnb+/+ and Ifnb-/- mice.


M . Immunoblot for optineurin in 6 - week - old midbrains of Ifnb + / + Ifnb - / - mice . N . Immunoblot for optineurin in DIV6 Ifnb + / + Ifnb - / - CNs with or without 30 U / mL rIFN - β .

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M. Immunoblot for optineurin in 6-week-old midbrains of Ifnb+/+ and Ifnb-/- mice. N. Immunoblot for optineurin in DIV6 Ifnb+/+ and Ifnb-/- CNs with or without 30 U/mL rIFN-β.


H . Kinetics of Drp1 dephosphorylation at S643 on rIFN - β treatment in wild - type CN cultures . PD - L1 was used as a positive control of rIFN - β activity .

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

H. Kinetics of Drp1 dephosphorylation at S643 on rIFN-β treatment in wild-type CN cultures. PD-L1 was used as a positive control of rIFN-β activity.


I . MFN2 OPA1 immunoblots in Ifnb + / + Ifnb - / - CN cultures . Vinculin was used as a loading control . Error bars are from 3 independent experiments .

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

I. MFN2 and OPA1 immunoblots and quantification in Ifnb+/+ and Ifnb-/- CN cultures and quantification. Vinculin was used as a loading control. Error bars are SEM from 3 independent experiments.


J . Optineurin Tom20 immunofluorescence in Ifnb + / + Ifnb - / - MEFs transfected with Drp1 or phosphomimetic Drp1 .

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

J. Optineurin and Tom20 immunofluorescence in Ifnb+/+ and Ifnb-/- MEFs transfected with Drp1 or phosphomimetic Drp1.


E . Volume rendering from 3D of Ifnb + / + Ifnb - / - thalami . Mitochondria are blue , ER is yellow , nuclei are grey . Mitochondria undergoing fission are cyan indicated with a arrow . 2D pictures are shown in Fig 2 .

O O O O O B-EXP_ASSAY O B-GENEPROD O O O B-GENEPROD O O O B-TISSUE O B-SUBCELLULAR O O O B-SUBCELLULAR O O O B-SUBCELLULAR O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O

E. Volume rendering from 3D TEM images of Ifnb+/+ and Ifnb-/- thalami. Mitochondria are blue, the ER is yellow, and nuclei are grey. Mitochondria undergoing fission are cyan and indicated with a white arrow. 2D pictures are shown in Fig 2.


H . Projections of 3D from immunostaining of Drp1 ( blue ) , Hsp60 ( mitochondria , green ) , INF2 ( yellow ) , calnexin ( ER , magenta ) in Ifnb - / - MEFs with or without rIFN - β treatment for 6 h . Scale bar equal 2 microns . Arrows indicate fission foci . I . Quantifications of triple - positive MT - DR + Drp1 + Cnx + in MEFs . are shown in Fig EV3 .

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

H. Projections of 3D images from immunostaining of Drp1 (blue), Hsp60 (mitochondria, green), INF2 (yellow), and calnexin (ER, magenta) in Ifnb-/- MEFs with or without rIFN-β treatment for 6 h. Scale bar equal 2 microns. Arrows indicate fission foci. I. Quantifications of triple-positive MT-DR+Drp1+Cnx+ in MEFs. Images are shown in Fig EV3.


D . Projections of 3D from immunostaining for phospho - S622 Drp1 ( yellow ) , Hsp60 ( mitochondria , green ) , calnexin ( ER , magenta ) in N2A depleted of STAT5 A B ( δStat5 ) or IFN - β ( δIfnb ) or with nontargeting control ( NTC ) . with or without rIFN - β treatment for 6 h . Scale bar equals 5 microns . Arrows indicate triple - positive foci . E . of D .

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

D. Projections of 3D images from immunostaining for phospho-S622 Drp1 (yellow), Hsp60 (mitochondria, green), and calnexin (ER, magenta) in N2A cells depleted of STAT5 A and B (δStat5) or IFN-β (δIfnb) or with nontargeting control (NTC). Cells with or without rIFN-β treatment for 6 h. Scale bar equals 5 microns. Arrows indicate triple-positive foci. E. Quantification of D.


L . Immunostaining of PGAM5 Hsp60 in NTC δSTAT5 N2As transfected with PGAM5 or empty vector control of mitochondrial section . Scale bars equal 5 microns . Error bars mean from 10 .

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

L. Immunostaining of PGAM5 and Hsp60 in NTC and δSTAT5 N2As transfected with PGAM5 or empty vector control and quantification of mitochondrial section area. Scale bars equal 5 microns. Error bars mean SEM from 10 individual cells.


M . Optineurin immunostaining in Ifnb + / + Ifnb - / - MEFs overexpressing PGAM5 . Empty vector was used as control . Additional controls are shown in Fig EV5L . Error bars are SD from N = 30 .

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

M. Optineurin immunostaining in Ifnb+/+ and Ifnb-/- MEFs overexpressing PGAM5 and quantification. Empty vector was used as control. Additional controls are shown in Fig EV5L. Error bars are SD from N=30 cells.


E - K . Immunofluorescence in striatal region in mice treated with 6OH - DA rIFN - β for optineurin ( E , quantified in F ) Tom20 ( E ) , with of mitochondrial by mitochondrial section ( G ) . 8OHdG - ( H - I ) , OxDJ1 - ( J - K ) , DAPI - labeled nuclei . Scale bars equal 10 microns .

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

E-K. Immunofluorescence in the striatal region in mice treated with 6OH-DA and rIFN-β for optineurin (E, quantified in F) and Tom20 (E), with quantification of individual mitochondrial size by mitochondrial section area (G). 8OHdG- (H-I), OxDJ1- (J-K), and DAPI-labeled nuclei. Scale bars equal 10 microns.


, Total of Munc18 - 1 . HEK293T transfected with WT or mutant Munc18 - 1 variants either GFP , syntaxin - 11 - 264 or syntaxin - 11 - 180 were lysed lysates were analyzed by quantitative immunoblotting to indicated , normalized to β - actin ( Synt - 1A = syntaxin - 1A ) . Data are means ± ( * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 by Kruskal - Wallis , followed by Dunn ' s multiple comparison ; n = 11 independent experiments ; exact p values are shown in Appendix Table S1 ) .

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

, Total protein levels of Munc18-1. HEK293T cells transfected with WT or mutant Munc18-1 variants and either GFP, syntaxin-11-264 or syntaxin-11-180 were lysed and lysates were analyzed by quantitative immunoblotting to indicated proteins, normalized to β-actin (Synt-1A = syntaxin-1A). Data are means ± SEM (*p < 0.05, **p < 0.01, ***p < 0.001, ****p<0.0001 by Kruskal-Wallis test, followed by Dunn's multiple comparison test; n = 11 independent experiments; exact p values are shown in Appendix Table S1).


Solubility of Munc18 - 1 . HEK293T transfected as in ( C ) were solubilized in 0 . 1 % Triton X - 100 ( TX ) equal volumes of soluble insoluble fractions were analyzed by quantitative immunoblotting . TX - soluble Munc18 - 1 was measured as of total Munc18 - 1 by quantitative immunoblotting . Data are means ± ( * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 by Kruskal - Wallis followed by Dunn ' s multiple comparison , or by one - way ANOVA followed by Bonferroni post - hoc ; n = 5 - 9 independent experiments ; exact n p values are shown in Appendix Table S1 ) .

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

Solubility of Munc18-1. HEK293T cells transfected as in (C) were solubilized in 0.1% Triton X-100 (TX) and equal volumes of soluble and insoluble fractions were analyzed by quantitative immunoblotting. TX-soluble Munc18-1 was measured as percent of total Munc18-1 by quantitative immunoblotting. Data are means ± SEM (*p<0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by Kruskal-Wallis test followed by Dunn's multiple comparison test, or by one-way ANOVA followed by Bonferroni post-hoc test; n = 5-9 independent experiments; exact n and p values are shown in Appendix Table S1).


Same as in ( A , B ) except that compounds 9 , 10 , 11 , 13 , 14 16 were added at 0 . 25 , 1 , 5 , 20 , 100 , or 250 µM . Note , that data for 250 µM were not plotted in ( D ) because of causing neuronal . Data are means ± ( * p < 0 . 05 , * * p < 0 . 01 by Kruskal - Wallis with Dunn ' s multiple comparison , or one - way ANOVA with Dunnett ' s post - hoc ; n = 6 - 15 independent experiments ; exact n p values are shown in Appendix Table S1 ) .

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

Same as in (A, B) except that compounds 9, 10, 11, 13, 14 and 16 were added at 0.25, 1, 5, 20, 100, or 250 µM. Note, that the data for 250 µM were not plotted in (D) because of causing neuronal death. Data are means ± SEM (*p < 0.05, **p < 0.01 by Kruskal-Wallis test with Dunn's multiple comparison test, or one-way ANOVA with Dunnett's post-hoc test; n = 6-15 independent experiments; exact n and p values are shown in Appendix Table S1).


Limited proteolysis . Recombinant purified were incubated with increasing concentrations of trypsin . Remaining were analyzed by quantitative immunoblotting . Data are means ± ( * p < 0 . 05 , * * p < 0 . 01 , * * * * p < 0 . 0001 , by 2 - way ANOVA Dunnett ' s multiple - comparison ; n = 4 independent experiments ; exact p values are shown in Appendix Table S1 ) .

O B-EXP_ASSAY O O O O O O O O O B-SMALL_MOLECULE O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Limited proteolysis. Recombinant purified proteins were incubated with increasing concentrations of trypsin. Remaining protein levels were analyzed by quantitative immunoblotting. Data are means ± SEM (* p < 0.05, **p < 0.01, ****p < 0.0001, by 2-way ANOVA and Dunnett's multiple-comparison test; n = 4 independent experiments; exact p values are shown in Appendix Table S1).


Munc18 - 1 knockout neurons expressing G544D Munc18 - 1 were subjected to of mean firing rate before addition of compounds ( 0 h ) or 48 hours after vehicle ( DMSO ) or compound addition . 16 electrodes per well were analyzed for neuronal firing ( A ; purple boxes indicate network activity ) . Data are means ± ( * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 by one - way ANOVA Dunnett ' s multiple comparison , or Kruskal - Wallis followed by Dunn ' s post - hoc ; n = 11 - 16 independent experiments ; exact n p values are shown in Appendix Table S1 ) .

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

Munc18-1 knockout neurons expressing G544D Munc18-1 were subjected to analysis of mean firing rate before addition of compounds (0 h) or 48 hours after vehicle (DMSO) or compound addition. 16 electrodes per well were analyzed for neuronal firing (A; purple boxes indicate network activity). Data are means ± SEM (*p < 0.05, **p < 0.01, ***p < 0.001 by one-way ANOVA and Dunnett's multiple comparison test, or Kruskal-Wallis test followed by Dunn's post-hoc test; n = 11-16 independent experiments; exact n and p values are shown in Appendix Table S1).


Uptake of synaptotagmin - 1 antibody during high K + stimulation . Neurons expressing cre recombinase / or WT , R406H or G544D Munc18 - 1b with or without compound were subjected 7 days after lentiviral infection to an antibody uptake . Endocytosed synaptotagmin - 1 antibody was quantified by immunostaining ( A - C ; scale bar = 30 µm ; 4 - PBA = 4 - phenylbutyrate ) , via counting number of pixels > of 15 ( D ) . Data are means ± ( * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 by one - way ANOVA Dunnett ' s multiple comparison ; n = 3 - 10 independent experiments ; exact n p values are shown in Appendix Table S1 ) .

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

Uptake of synaptotagmin-1 antibody during high K+ stimulation. Neurons expressing cre recombinase and/or WT, R406H or G544D Munc18-1b with or without compound were subjected 7 days after lentiviral infection to an antibody uptake assay. Endocytosed synaptotagmin-1 antibody was quantified by immunostaining (A-C; scale bar = 30 µm; 4-PBA = 4-phenylbutyrate), via counting the number of pixels > intensity of 15 (D). Data are means ± SEM (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by one-way ANOVA and Dunnett's multiple comparison test; n = 3-10 independent experiments; exact n and p values are shown in Appendix Table S1).


Plot of change in from baseline to peak for Munc18 - 1 G544D ( H ) Data are means ± ( * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 by one - way one - way ANOVA Dunnett ' s multiple comparison ; n = 7 - 23 ; exact n p values are shown in Appendix Table S1 ) . Note , that compound 10 is at a similar wavelength than pHluorin , so experiments at concentrations higher than 1 µM were not possible .

O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O B-GENEPROD O O O O O O O O O O O O O

Plot of change in fluorescence from baseline to peak fluorescence for Munc18-1 G544D (H) Data are means ± SEM (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by one-way one-way ANOVA and Dunnett's multiple comparison test; n = 7-23; exact n and p values are shown in Appendix Table S1). Note, that compound 10 is fluorescent at a similar wavelength than pHluorin, so experiments at concentrations higher than 1 µM were not possible.


Heat - induced paralysis . Indicated worm strains maintained in compounds at indicated concentrations were exposed to 37 ° C over a period of 120 min , paralysis was scored at indicated time points . Data are means ± ( n = 3 - 4 independent experiments on ten worms per experiment ; exact n p values are shown in Appendix Table S1 ) . ( B , D , F , H , J , L ) of paralysis at 20 minutes . Data are means ± ( * , # p < 0 . 05 , * * , # # p < 0 . 01 , * * * , # # # p < 0 . 001 , # as compared to WT , * as compared to mutant by two - way ANOVA Dunnett ' s multiple comparison ; n = 3 - 4 independent experiments on ten worms per experiment ; exact n p values are shown in Appendix Table S1 ) .

O O O B-DISEASE O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O B-DISEASE O 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-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-DISEASE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-ORGANISM O O O O O O O O O O O O O O O

Heat-induced paralysis. Indicated worm strains maintained in compounds at indicated concentrations were exposed to 37 °C over a period of 120 min, and paralysis was scored at indicated time points. Data are means ± SEM (n = 3-4 independent experiments on ten worms per experiment; exact n and p values are shown in Appendix Table S1). (B, D, F, H, J, L) Quantification of paralysis at 20 minutes. Data are means ± SEM (*,#p < 0.05, **,##p < 0.01, ***,###p < 0.001, # as compared to WT, * as compared to mutant by two-way ANOVA and Dunnett's multiple comparison test; n = 3-4 independent experiments on ten worms per experiment; exact n and p values are shown in Appendix Table S1).


Rescue of subcellular localization of UNC - 18 in worms expressing G544D UNC - 18 . C . elegans expressing WT : : GFP or G544D : : GFP were immobilized , ventral nerve cord was imaged . Solid arrowheads point to pairs of bigger , broken arrowheads to single , smaller ( N , O ) . Scale bar in ( N ) ( O ) = 10 µm . 4 - PBA = 4 - phenylbutyrate .

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

Rescue of the subcellular localization of UNC-18 in worms expressing G544D UNC-18. C. elegans expressing WT::GFP or G544D::GFP were immobilized, and the ventral nerve cord was imaged. Solid arrowheads point to pairs of bigger puncta, broken arrowheads to single, smaller puncta (N, O). Scale bar in (N) and (O) = 10 µm. 4-PBA = 4-phenylbutyrate.


Rescue of subcellular localization of UNC - 18 in worms expressing G544D UNC - 18 . C . elegans expressing WT : : GFP or G544D : : GFP at 1 , 5 , 20 or 100 µM compound were immobilized , ventral nerve cord was imaged . Solid arrowheads point to pairs of bigger , broken arrowheads to single , smaller

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

Rescue of the subcellular localization of UNC-18 in worms expressing G544D UNC-18. C. elegans expressing WT::GFP or G544D::GFP at 1, 5, 20 or 100 µM compound were immobilized, and the ventral nerve cord was imaged. Solid arrowheads point to pairs of bigger puncta, broken arrowheads to single, smaller puncta


of soluble aggregated Munc18 - 1 . Data are means ± ( * * p < 0 . 01 , * * * p < 0 . 001 by one - way ANOVA Dunnett ' s multiple comparison ; n = 3 - 4 worms ; exact n p values are shown in Appendix Table S1 ) .

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

Quantification of soluble and aggregated Munc18-1. Data are means ± SEM (**p < 0.01, ***p < 0.001 by one-way ANOVA and Dunnett's multiple comparison test; n = 3-4 worms; exact n and p values are shown in Appendix Table S1).


( a ) Ba / F3 expressing myrPI ( 3 ) K - ER , myrPKB - ER , FOXO3 ( A3 ) - ER or FOXO4 ( A3 ) - ER were stimulated with 4 - OHT for 4 h ( myrPI ( 3 ) K - ER , myrPKB - ER ) or 8 h ( FOXO3 ( A3 ) - ER FOXO4 ( A3 ) - ER ) microarray analyses were performed . Shown are fold changes relative to control for glutamine synthetase ( GS ) , Mxi1 Pink1 . Data are represented as mean values of one experiment performed in quadruplicate .

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

(a) Ba/F3 cells expressing myrPI(3)K-ER, myrPKB-ER, FOXO3(A3)-ER or FOXO4(A3)-ER were stimulated with 4-OHT for 4 h (myrPI(3)K-ER, myrPKB-ER) or 8 h (FOXO3(A3)-ER and FOXO4(A3)-ER) and microarray analyses were performed. Shown are the fold changes relative to control cells for glutamine synthetase (GS), Mxi1 and Pink1. Data are represented as mean values of one experiment performed in quadruplicate.


( b ) Ba / F3 expressing either myrPI ( 3 ) K - ER or myrPKB - ER were cytokine starved overnight stimulated with 4 - OHT . Relative mRNA of glutamine synthetase were analysed using quantitative rtPCR . Data are represented as mean ± s . e . m . normalized for B2M ( n = 4 ) . * P0 . 05 , * * P0 . 01 .

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

(b) Ba/F3 cells expressing either myrPI(3)K-ER or myrPKB-ER were cytokine starved overnight and stimulated with 4-OHT. Relative mRNA levels of glutamine synthetase were analysed using quantitative rtPCR. Data are represented as mean ± s.e.m. normalized for B2M (n=4). *P0.05, **P0.01.


( c ) Ba / F3 expressing either myrPI ( 3 ) K - ER or myrPKB - ER were cytokine starved overnight stimulated with 4 - OHT . lysates were analysed for of phospho - FOXO3 ( T32 ) , glutamine synthetase , p27 actin . Shown are representative blots ( n = 4 ) .

O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O B-GENEPROD O O O O B-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD O O O O O O O O O O O

(c) Ba/F3 cells expressing either myrPI(3)K-ER or myrPKB-ER were cytokine starved overnight and stimulated with 4-OHT. Cell lysates were analysed for protein levels of phospho-FOXO3 (T32), glutamine synthetase, p27 and actin. Shown are representative blots (n=4).


( d ) Ba / F3 expressing either FOXO3 ( A3 ) - ER or FOXO4 ( A3 ) - ER were stimulated with 4 - OHT in presence of mIL - 3 . Relative mRNA of glutamine synthetase were analysed using quantitative rtPCR . Data are represented as mean ± s . e . m . values normalized for B2M ( n = 3 ) . * * P0 . 01 .

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

(d) Ba/F3 cells expressing either FOXO3(A3)-ER or FOXO4(A3)-ER were stimulated with 4-OHT in the presence of mIL-3. Relative mRNA levels of glutamine synthetase were analysed using quantitative rtPCR. Data are represented as mean ±  s.e.m. values normalized for B2M (n=3). **P0.01.


( e ) Ba / F3 expressing either FOXO3 ( A3 ) - ER or FOXO4 ( A3 ) - ER were stimulated with 4 - OHT in presence of mIL - 3 . lysates were analysed for of glutamine synthetase , p27 actin . Shown are representative blots ( n = 3 ) .

O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-GENEPROD I-GENEPROD O O O O O O B-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD O O O O O O O O O O O

(e) Ba/F3 cells expressing either FOXO3(A3)-ER or FOXO4(A3)-ER were stimulated with 4-OHT in the presence of mIL-3. Cell lysates were analysed for protein levels of glutamine synthetase, p27 and actin. Shown are representative blots (n=3).


( f ) Wild - type Ba / F3 were cytokine starved overnight stimulated with mIL - 3 . lysates were analysed for of phospho - FOXO3 ( T32 ) , glutamine synthetase , p27 actin . Shown are representative blots ( n = 4 ) .

O O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O B-SMALL_MOLECULE O O O O O B-GENEPROD I-GENEPROD O O O O O O O O B-GENEPROD O O O O B-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD O O O O O O O O O O O

(f) Wild-type Ba/F3 cells were cytokine starved overnight and stimulated with mIL-3. Cell lysates were analysed for protein levels of phospho-FOXO3 (T32), glutamine synthetase, p27 and actin. Shown are representative blots (n=4).


( g ) Wild - type Ba / F3 were incubated with LY294002 in presence of mIL - 3 . lysates were analysed for of glutamine synthetase , p27 actin . Shown are representative blots ( n = 3 ) .

O O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O B-SMALL_MOLECULE O O O O B-GENEPROD I-GENEPROD O O O O O O B-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD O O O O O O O O O O O

(g) Wild-type Ba/F3 cells were incubated with LY294002 in the presence of mIL-3. Cell lysates were analysed for protein levels of glutamine synthetase, p27 and actin. Shown are representative blots (n=3).


( h ) FOXO1 , 3 , 4 − / − MEFs wild - type MEFs were incubated with LY294002 ( 10 μM ) for indicated times . was isolated relative mRNA of glutamine synthetase were analysed using quantitative PCR . Data are represented as mean values normalized for B2M ( n = 2 ) . Uncropped of blots are shown in Supplementary Fig . S7 .

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

(h) FOXO1,3,4−/− MEFs and wild-type MEFs were incubated with LY294002 (10 μM) for the indicated times. Cell RNA was isolated and relative mRNA levels of glutamine synthetase were analysed using quantitative PCR. Data are represented as mean values normalized for B2M (n=2). Uncropped images of blots are shown in Supplementary Fig. S7.


( a ) Ba / F3 expressing FOXO3 ( A3 ) - ER were stimulated with 4 - OHT actinomycin D ( 1 μg ml − 1 ) or dimethylsulphoxide ( DMSO ) as a control in presence of mIL - 3 for 16 h . lysates were analysed for of glutamine synthetase ( GS ) , p27 actin . Shown are representative blots ( n = 3 ) .

O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O B-GENEPROD O O O O B-GENEPROD O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O B-GENEPROD I-GENEPROD O O O O O O O O O O B-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD O O O O O O O O O O O

(a) Ba/F3 cells expressing FOXO3(A3)-ER were stimulated with 4-OHT and actinomycin D (1 μg ml−1) or dimethylsulphoxide (DMSO) as a control in the presence of mIL-3 for 16 h. Cell lysates were analysed for protein levels of glutamine synthetase (GS), p27 and actin. Shown are representative blots (n=3).


( b ) Glutamine synthetase reporter plasmids carrying mutations in putative FOXO - binding sites were transfected in HEK293 together with Renilla FOXO3 ( A3 ) as indicated . Luciferase activity was measured 40 h after transfection . Data are depicted as relative luciferase units ( RLU ) compared with control . Shown are mean ± s . e . m . values ( n = 3 )

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

(b) Glutamine synthetase reporter plasmids carrying mutations in putative FOXO-binding sites were transfected in HEK293 cells together with Renilla and FOXO3(A3) as indicated. Luciferase activity was measured 40 h after transfection. Data are depicted as relative luciferase units (RLU) compared with the control. Shown are mean ± s.e.m. values (n=3)


( a ) DLD1 expressing FOXO3 ( A3 ) - ER were transiently transfected with GFP - ULK2 stimulated with 4 - OHT MSO for 24 h in glutamine - free DMEM containing 0 . 1 % FCS . were stained for LC3 analysed by confocal microscopy . Shown are representative pictures ( n = 3 ) . Scale bars , 20 μm .

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

(a) DLD1 cells expressing FOXO3(A3)-ER were transiently transfected with GFP-ULK2 and stimulated with 4-OHT and MSO for 24 h in glutamine-free DMEM containing 0.1% FCS. Cells were stained for LC3 and analysed by confocal microscopy. Shown are representative pictures (n=3). Scale bars, 20 μm.


( d ) DLD1 expressing FOXO3 ( A3 ) - ER were stimulated with 4 - OHT . lysates were analysed for of glutamine synthetase , p27 actin . Shown are representative blots ( n = 4 ) .

O O O B-CELL_LINE O B-GENEPROD O O O O B-GENEPROD O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O B-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD O O O O O O O O O O O

(d) DLD1 cells expressing FOXO3(A3)-ER were stimulated with 4-OHT. Cell lysates were analysed for protein levels of glutamine synthetase, p27 and actin. Shown are representative blots (n=4).


( e ) osteosarcoma U2OS expressing FOXO ( A3 ) - ER was stimulated with 4 - OHT , were lysed equal amounts of were analysed for of glutamine synthetase actin .

O O O O B-CELL_LINE O O O O O O B-GENEPROD O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O

(e) The osteosarcoma cell line U2OS expressing FOXO(A3)-ER was stimulated with 4-OHT, cells were lysed and equal amounts of proteins were analysed for levels of glutamine synthetase and actin.


( f ) MSCs expressing FOXO ( A3 ) - ER were stimulated with 4 - OHT , were lysed equal amounts of were analysed for of glutamine synthetase actin . Shown are representative blots ( n = 3 ) .

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

(f) MSCs expressing FOXO(A3)-ER were stimulated with 4-OHT, cells were lysed and equal amounts of proteins were analysed for levels of glutamine synthetase and actin. Shown are representative blots (n=3).


( g ) Wild - type , daf - 2 daf - 16 mutant worms were synchronized to L1 by hypochlorite treatment were placed on nematode growth medium agar plates . After five days worms were lysed in imidazole analysed for glutamine synthetase activity .

O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-ORGANISM O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O B-GENEPROD I-GENEPROD O O

(g) Wild-type, daf-2 and daf-16 mutant worms were synchronized to L1 by hypochlorite treatment and were placed on nematode growth medium agar plates. After five days worms were lysed in imidazole and analysed for glutamine synthetase activity.


( h ) Wild - type N2 worms or daf - 2 mutants were synchronized to L1 by hypochlorite treatment placed on nematode growth medium plates with or without expressing DAF - 16 double - stranded . After five days worms were lysed in imidazole analysed for glutamine synthetase activity . ( g , h ) Shown are means of five independent plates for each condition . Uncropped of blots are shown in Supplementary Fig . S7 .

O O O O O O O B-ORGANISM O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-ORGANISM O O O B-SMALL_MOLECULE O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(h) Wild-type N2 worms or daf-2 mutants were synchronized to L1 by hypochlorite treatment and placed on nematode growth medium plates with or without bacteria expressing DAF-16 double-stranded RNA. After five days worms were lysed in imidazole and analysed for glutamine synthetase activity. (g,h) Shown are the means of five independent plates for each condition. Uncropped images of blots are shown in Supplementary Fig. S7.


( a ) Ba / F3 expressing FOXO3 ( A3 ) - ER were stimulated with 4 - OHT in presence of mIL - 3 . lysates were analysed for glutamine synthetase ( GS ) activity in an enzyme ( left ) . In addition , expression of glutamine synthetase , p27 actin was determined using western blot ( right ) . Shown are mean ± s . e . m . ( n = 3 ) representative blots of these experiments . * * P0 . 01 .

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

(a) Ba/F3 cells expressing FOXO3(A3)-ER were stimulated with 4-OHT in the presence of mIL-3. Cell lysates were analysed for glutamine synthetase (GS) activity in an enzyme assay (left). In addition, the expression of glutamine synthetase, p27 and actin was determined using western blot (right). Shown are the mean ± s.e.m. (n=3) and representative blots of these experiments. **P0.01.


( b ) Ba / F3 expressing FOXO3 ( A3 ) - ER were stimulated with 4 - OHT for 16 h in presence of mIL - 3 together with MSO , as indicated . lysates were analysed for glutamine synthetase activity in an enzyme ( left ) . Furthermore expression of glutamine synthetase , p27 actin were determined using western blot ( right ) . Shown are mean ± s . e . m . ( n = 3 ) representative blots of these experiments . * P0 . 05 , * * P0 . 01 .

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

(b) Ba/F3 cells expressing FOXO3(A3)-ER were stimulated with 4-OHT for 16 h in the presence of mIL-3 together with MSO, as indicated. Cell lysates were analysed for glutamine synthetase activity in an enzyme assay (left). Furthermore the expression levels of glutamine synthetase, p27 and actin were determined using western blot (right). Shown are the mean ±  s.e.m. (n=3) and representative blots of these experiments. *P0.05, **P0.01.


( c ) Ba / F3 expressing myrPI ( 3 ) K - ER were cytokine - starved overnight . next day , were washed in PBS put in a medium without serum , with or without 4 - OHT . At times indicated , medium samples were taken analysed for acid by high - performance liquid chromatography . were lysed equal amounts of were analysed by western blotting for of phospho - PKB ( S473 ) , phospho - FOXO3 ( T32 ) actin ( inset ) . Shown are mean of relative acid , compared with t = 0 ( n = 2 ) , representative blots of these experiments .

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

(c) Ba/F3 cells expressing myrPI(3)K-ER were cytokine-starved overnight. The next day, cells were washed in PBS and put in a medium without serum, with or without 4-OHT. At the times indicated, medium samples were taken and analysed for amino acid levels by high-performance liquid chromatography. Cells were lysed and equal amounts of protein were analysed by western blotting for levels of phospho-PKB (S473), phospho-FOXO3 (T32) and actin (inset). Shown are the mean of relative amino acid levels, compared with t=0 (n=2), and representative blots of these experiments.


( d ) Ba / F3 expressing FOXO3 ( A3 ) - ER were stimulated with 4 - OHT in serum - free medium containing mIL - 3 . medium was analysed for acid by high - performance liquid chromatography . were lysed analysed for of glutamine synthetase , p27 actin ( inset ) . Shown are mean ± s . e . m . of relative acid , compared with t = 0 ( n = 4 ) , representative blots of these experiments . * * P0 . 01 . Uncropped of blots are shown in Supplementary Fig . S7 .

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

(d) Ba/F3 cells expressing FOXO3(A3)-ER were stimulated with 4-OHT in serum-free medium containing mIL-3. The medium was analysed for amino acid levels by high-performance liquid chromatography. Cells were lysed and analysed for protein levels of glutamine synthetase, p27 and actin (inset). Shown are the mean ±  s.e.m. of relative amino acid levels, compared with t=0 (n=4), and representative blots of these experiments. **P0.01. Uncropped images of blots are shown in Supplementary Fig. S7.


( a ) DLD1 expressing FOXO3 ( A3 ) - ER were stimulated with 4 - OHT with or without MSO . After 24 h , were starved of serum , glucose in D - PBS containing indicated inhibitors stimulated with for 10 min . lysates were analysed for of phospho - S6K ( Thr 389 ) actin . Shown are representative blots of three independent experiments .

O O O B-CELL_LINE O B-GENEPROD O O O O B-GENEPROD O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O

(a) DLD1 cells expressing FOXO3(A3)-ER were stimulated with 4-OHT with or without MSO. After 24 h, the cells were starved of serum, amino acids and glucose in D-PBS containing the indicated inhibitors and stimulated with amino acids for 10 min. Cell lysates were analysed for protein levels of phospho-S6K (Thr 389) and actin. Shown are representative blots of three independent experiments.


( b , c ) DLD1 expressing FOXO3 ( A3 ) - ER were stimulated with 4 - OHT MSO ( 0 . 25 M ) for 24 h in glutamine - free DMEM containing 0 . 1 % FCS . were stained for mTOR LAMP2 analysed by confocal microscopy . ( b ) Shown are representative pictures ( n = 4 ) . Scale bars , 100 μm . ( c ) of co - localization between mTOR LAMP2 as seen in b . Depicted are mean ± s . e . m . of of co - localized pixels from four experiments . * P0 . 05 , * * P0 . 01 .

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

(b,c) DLD1 cells expressing FOXO3(A3)-ER were stimulated with 4-OHT and MSO (0.25 M) for 24 h in glutamine-free DMEM containing 0.1% FCS. Cells were stained for mTOR and LAMP2 and analysed by confocal microscopy. (b) Shown are representative pictures (n=4). Scale bars, 100 μm. (c) Quantification of co-localization between mTOR and LAMP2 as seen in b. Depicted are the mean ±  s.e.m. of the percentage of co-localized pixels from four experiments. *P0.05, **P0.01.


( d ) DLD1 expressing FOXO3 ( A3 ) - ER were treated with or without 4 - OHT , MSO BafA1 ( 200 nM ) . After 24 h , lysates were analysed for of LC3 tubulin . Shown are representative blots ( n = 4 ) .

O O O B-CELL_LINE O B-GENEPROD O O O O B-GENEPROD O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD O O O O O O O O O O O

(d) DLD1 cells expressing FOXO3(A3)-ER were treated with or without 4-OHT, MSO and BafA1 (200 nM). After 24 h, cell lysates were analysed for protein levels of LC3 and tubulin. Shown are representative blots (n=4).


( e ) DLD1 expressing FOXO3 ( A3 ) - ER were transfected with glutamine synthetase ( GS ) siRNA or a non - targeting siRNA stimulated with 4 - OHT in glutamine - free DMEM containing 0 . 1 % FBS . After 24 h , lysates were analysed for of glutamine synthetase , LC3 actin . Shown are representative blots ( n = 2 )

O O O B-CELL_LINE O B-GENEPROD O O O O B-GENEPROD O O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD O O O O O O O O O O

(e) DLD1 cells expressing FOXO3(A3)-ER were transfected with glutamine synthetase (GS) siRNA or a non-targeting siRNA and stimulated with 4-OHT in glutamine-free DMEM containing 0.1% FBS. After 24 h, cell lysates were analysed for protein levels of glutamine synthetase, LC3 and actin. Shown are representative blots (n=2)


( f ) DLD1 expressing FOXO3 ( A3 ) - ER were transfected with glutamine synthetasesiRNA or non - template ( NT ) control siRNA stimulated with 4 - OHT for 16 h . were lysed equal amounts of were analysed for of p62 or glutamine synthetase . Shown are representative blots ( n = 2 ) .

O O O B-CELL_LINE O B-GENEPROD O O O O B-GENEPROD O O O B-GENEPROD I-GENEPROD O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD O O O O O O O O O O O

(f) DLD1 cells expressing FOXO3(A3)-ER were transfected with glutamine synthetasesiRNA or non-template (NT) control siRNA and stimulated with 4-OHT for 16 h. Cells were lysed and equal amounts of proteins were analysed for levels of p62 or glutamine synthetase. Shown are representative blots (n=2).


( g ) DLD1 were stimulated with LY294002 or PKB inhibitor VIII for 24 h . lysates were analysed for of LC3 actin . Shown are representative blots ( n = 4 ) . Uncropped of blots are shown in Supplementary Figs S7 S8 .

O O O B-CELL_LINE O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O

(g) DLD1 cells were stimulated with LY294002 or PKB inhibitor VIII for 24 h. Cell lysates were analysed for protein levels of LC3 and actin. Shown are representative blots (n=4). Uncropped images of blots are shown in Supplementary Figs S7 and S8.


( a ) DLD1 expressing FOXO3 ( A3 ) - ER were stimulated with 4 - OHT MSO for 24 h in glutamine - free DMEM containing 0 . 1 % FCS . were stained for LC3 ER analysed by confocal microscopy . Shown are representative pictures ( n = 3 ) . Scale bars , 20 μm . ( b ) of LC3 - positive spots as seen in a using ImageJ software . Depicted are mean ± s . e . m . of number of LC3 - positive spots divided by number of DAPI - positive ( n = 4 ) . * P0 . 05 .

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

(a) DLD1 cells expressing FOXO3(A3)-ER were stimulated with 4-OHT and MSO for 24 h in glutamine-free DMEM containing 0.1% FCS. Cells were stained for LC3 and ER and analysed by confocal microscopy. Shown are representative pictures (n=3). Scale bars, 20 μm. (b) Quantification of LC3-positive spots as seen in a using ImageJ software. Depicted are mean ±  s.e.m. of the number of LC3-positive spots divided by the number of DAPI-positive cells (n=4). *P0.05.