Figure 4B ) Processing of endogenous Syntaxin 5 ( Stx5 ) and Syntaxin 6 ( Stx6 ) by SPPL2c wt by Western Blot . Non - transfected cells ( - ) , stably transfected but non - induced SPPL2c wt cells ( n / i ) and induced cells stably expressing SPPL2c D / A served as controls . Results are derived from one experiment , thus expression ( SPPL2c ) and loading ( Calnexin ) control are the same . Note , that Syntaxin 5 is cleaved by SPPL2c while Syntaxin 6 is not . C ) Syntaxin 5 protein levels were down regulated and processing and maturation of ectopically expressed GnT - V , as well maturation of the endogenous glycoproteins Nicastrin ( NCT ) and SPPL2a was analyzed in Western Blot . Calnexin served as loading control and ectopic expression of SPPL2c WT was confirmed using a SPPL2c specific antibody . While GnT - V and Nicastrin maturation were similarly affected by Syntaxin 5 knock down and SPPL2c overexpression , SPPL2a maturation was hardly impaired in Syntaxin 5 knock down cells . D ) Syntaxin 5 localization is impaired by overexpression of SPPL2c . Endogenous Syntaxin 5 was visualized in control cells without SPPL2c expression ( Ctr ) and in cells overexpressing SPPL2c wt or SPPL2c D / A using immunofluorescence . BiP was used as a marker protein of the ER and TGN46 as Golgi marker . White arrow indicating cells with disturbed Golgi and Syntaxin 5 staining . Scale bar 5 µm . E ) Syntaxin 5 knock down induces mislocalization of GnT - V similar to SPPL2c overexpression Cells ectopically expressing GnT - V were treated either with non - specific siRNA ( siCtr ) or with siRNA targeting Syntaxin 5 ( siStx5 ) . GnT - V was visualized in immunofluorescence using anti - V5 antibody targeting its C - terminal tag and either BIP was co - stained as ER marker protein or TGN46 as Golgi marker protein . Scale bar 5 µm .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4B) Processing of endogenous Syntaxin 5 (Stx5) and Syntaxin 6 (Stx6) by SPPL2c wt by Western Blot. Non-transfected cells (-), stably transfected but non-induced SPPL2c wt cells (n/i) and induced cells stably expressing SPPL2c D/A served as controls. Results are derived from one experiment, thus expression (SPPL2c) and loading (Calnexin) control are the same. Note, that Syntaxin 5 is cleaved by SPPL2c while Syntaxin 6 is not.C) Syntaxin 5 protein levels were down regulated and processing and maturation of ectopically expressed GnT-V, as well maturation of the endogenous glycoproteins Nicastrin (NCT) and SPPL2a was analyzed in Western Blot. Calnexin served as loading control and ectopic expression of SPPL2c WT was confirmed using a SPPL2c specific antibody. While GnT-V and Nicastrin maturation were similarly affected by Syntaxin 5 knock down and SPPL2c overexpression, SPPL2a maturation was hardly impaired in Syntaxin 5 knock down cells.D) Syntaxin 5 localization is impaired by overexpression of SPPL2c. Endogenous Syntaxin 5 was visualized in control cells without SPPL2c expression (Ctr) and in cells overexpressing SPPL2c wt or SPPL2c D/A using immunofluorescence. BiP was used as a marker protein of the ER and TGN46 as Golgi marker. White arrow indicating cells with disturbed Golgi and Syntaxin 5 staining. Scale bar 5 µm.E) Syntaxin 5 knock down induces mislocalization of GnT-V similar to SPPL2c overexpression Cells ectopically expressing GnT-V were treated either with non-specific siRNA (siCtr) or with siRNA targeting Syntaxin 5 (siStx5). GnT-V was visualized in immunofluorescence using anti-V5 antibody targeting its C-terminal tag and either BIP was co-stained as ER marker protein or TGN46 as Golgi marker protein. Scale bar 5 µm.


Figure 5After seeding cells were cultured for 72h and expression of catalytically active ( wt ) or non - active ( D / A ) SPPL2c was induced by addition of doxycycline for either 24h or 48h as indicated . Non - transfected cells ( Ctr ) or non - induced SPPL2c cells ( n / i ) served as controls . The ER was stained with the anti - BiP antibody , the cis / medial - Golgi with the anti - Giantin , the trans - Golgi Network with the anti - TGN46 antibody and all Golgi subcompartments with the CAB45 - specific antibody in immunofluorescence . Note that only upon expression of catalytically active SPPL2c the morphologies of ER and cis - Golgi change compared to controls . Scale bar 5 µm .

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Figure 5After seeding cells were cultured for 72h and expression of catalytically active (wt) or non-active (D/A) SPPL2c was induced by addition of doxycycline for either 24h or 48h as indicated. Non-transfected cells (Ctr) or non-induced SPPL2c cells (n/i) served as controls. The ER was stained with the anti-BiP antibody, the cis/medial-Golgi with the anti-Giantin, the trans-Golgi Network with the anti-TGN46 antibody and all Golgi subcompartments with the CAB45-specific antibody in immunofluorescence. Note that only upon expression of catalytically active SPPL2c the morphologies of ER and cis-Golgi change compared to controls. Scale bar 5 µm.


Figure 6A ) Western Blot analysis of SPPL2c in membrane preparations from HEK293 inducibly overexpressing SPPL2c , human testis and mouse testis tissue . For human and mouse tissue , 5 μg protein of each total testis preparation is loaded . The membrane fraction obtained from a confluent 6 cm dish of SPPL2c expressing HEK293 cells is diluted 1 : 50 ( 0 . 1 μg total protein ) . B ) Western Blot analysis of selected glycoproteins in mouse tissues from two wildtype C57BL / 6J ( Black6 ) mice . Endogenous Nicastrin ( NCT ) and SPPL2a were analysed in membrane preparations from testis , kidney , brain , spleen and liver in littermate male mice . Note that immature proteins ( im ) accumulate in testis compared to other tissues . SPPL2c protein is only detected in testis tissue . C ) Analysis of Syntaxin 8 ( Stx8 ) in SPPL2c - / - mice compared to wildtype ( wt ) littermates . Membrane isolates from whole testis were analyzed for Syntaxin 8 ( Stx8 ) and SPPL2c expression . TMEM192 serves as loading control . D ) Syntaxin 8 protein levels were quantified from the respective Western blots by densitometric analysis and normalized to those of TMEM192 . Bars represent mean values of n = 3 animals ± SD normalized to mean Syntaxin 8 levels of wildtype mice . Statistical significance was calculated applying an unpaired , two - sided Student ' s t - test . * p ≤ 0 . 05 . Note that Syntaxin 8 is accumulating in SPPL2c - / - mice . E ) Real - time qPCR analysis of Syntaxin 8 ( Stx8 ) mRNA levels in testis of SPPL2c - / - and the wildtype mice normalized to that of Tuba1a . The diagram depicts mean values of Stx8 mRNA levels normalized to that of wildtype animals of n = 6 individuals from two independent experiments ± SD . For statistical analysis an unpaired , two - sided Student ' s t - test was performed . ns = not significant . F ) Immunohistochemical stainings of cross - sectioned seminiferous tubules of SPPL2c + / + and SPPL2c - / - mice using anti - Cab45 antibody . Stages VII / VIII are depicted . Higher magnifications are indicated with dashed lines and black arrows indicate pre - acrosomal structures . Scale bar 50 µm . G ) Volcano plot depicting the binding affinities of lectins on sperm lysates from SPPL2c - / - mice compared to SPPL2c + / + ( wt ) . Each circle represents one lectin . The lectins above the dashed line ( light blue ) have a significant p - value < 0 . 05 . Lectins on the left side of the blot demonstrate reduced binding while UEA - I on the right side has increased binding . n = 4 animals per genotype , two - sided , unpaired Student ' s t - Test .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6A) Western Blot analysis of SPPL2c in membrane preparations from HEK293 inducibly overexpressing SPPL2c, human testis and mouse testis tissue. For human and mouse tissue, 5 μg protein of each total testis preparation is loaded. The membrane fraction obtained from a confluent 6 cm dish of SPPL2c expressing HEK293 cells is diluted 1:50 (0.1 μg total protein).B) Western Blot analysis of selected glycoproteins in mouse tissues from two wildtype C57BL/6J (Black6) mice. Endogenous Nicastrin (NCT) and SPPL2a were analysed in membrane preparations from testis, kidney, brain, spleen and liver in littermate male mice. Note that immature proteins (im) accumulate in testis compared to other tissues. SPPL2c protein is only detected in testis tissue.C) Analysis of Syntaxin 8 (Stx8) in SPPL2c-/- mice compared to wildtype (wt) littermates. Membrane isolates from whole testis were analyzed for Syntaxin 8 (Stx8) and SPPL2c expression. TMEM192 serves as loading control.D) Syntaxin 8 protein levels were quantified from the respective Western blots by densitometric analysis and normalized to those of TMEM192. Bars represent mean values of n=3 animals ± SD normalized to mean Syntaxin 8 levels of wildtype mice. Statistical significance was calculated applying an unpaired, two-sided Student's t-test. * p≤0.05. Note that Syntaxin 8 is accumulating in SPPL2c-/- mice.E) Real-time qPCR analysis of Syntaxin 8 (Stx8) mRNA levels in testis of SPPL2c-/- and the wildtype mice normalized to that of Tuba1a. The diagram depicts mean values of Stx8 mRNA levels normalized to that of wildtype animals of n=6 individuals from two independent experiments ± SD. For statistical analysis an unpaired, two-sided Student's t-test was performed. ns = not significant.F) Immunohistochemical stainings of cross-sectioned seminiferous tubules of SPPL2c+/+ and SPPL2c-/- mice using anti-Cab45 antibody. Stages VII/VIII are depicted. Higher magnifications are indicated with dashed lines and black arrows indicate pre-acrosomal structures. Scale bar 50 µm.G) Volcano plot depicting the binding affinities of lectins on sperm lysates from SPPL2c-/- mice compared to SPPL2c+/+ (wt). Each circle represents one lectin. The lectins above the dashed line (light blue) have a significant p-value<0.05. Lectins on the left side of the blot demonstrate reduced binding while UEA-I on the right side has increased binding. n=4 animals per genotype, two-sided, unpaired Student's t-Test.


Figure 1 ( A ) GFP - LC3 CHO cells were preincubated with 1 mM dybutiryl cAMP ( + dbcAMP , panels d , e , and f ) for 30 min and then were treated for 4 h with 10 µg / ml of α - hemolysin ( Hla ) or subjected to starvation conditions ( Stv ) in the continuous presence of dbcAMP . Cells without any treatment were used as control ( − dbcAMP , panels a , b , and c ) . Cells were immediately analyzed by confocal microscopy . A quantification of the percentage of cells presenting LC3 puncta ( i . e . , stimulated cells ) upon incubation with the different conditions is shown in the right panel ( n = 100 cells / condition , * p < 0 . 05 paired Student ' s t - test ) . These data are representative of three independent experiments . ( B ) GFP - LC3 CHO cells were incubated with complete medium in the absence ( − dbcAMP ) or presence of dbcAMP ( + dbcAMP ) and treated for 4 h with 10 µg / ml of α - hemolysin ( Hla ) or subjected to starvation conditions ( Stv ) with ( lower panel ) or without ( upper panel ) bafilomycin A1 to block lysosomal degradation . Afterwards , cells were lysed with sample buffer and the samples were subjected to Western blot analysis using a rabbit anti - LC3 and the corresponding HRP - labeled secondary antibody , and subsequently developed with an enhanced chemiluminescence detection kit . These data are representative of three independent experiments . ( C ) The band intensities of two independent experiments were quantificated with the Adobe Photoshop program , and normalized against tubulin . * p < 0 . 05 ( paired Student ' s t - test ) .

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Figure 1(A) GFP-LC3 CHO cells were preincubated with 1 mM dybutiryl cAMP (+dbcAMP, panels d, e, and f) for 30 min and then were treated for 4 h with 10 µg/ml of α-hemolysin (Hla) or subjected to starvation conditions (Stv) in the continuous presence of dbcAMP. Cells without any treatment were used as control (−dbcAMP, panels a, b, and c). Cells were immediately analyzed by confocal microscopy. A quantification of the percentage of cells presenting LC3 puncta (i.e., stimulated cells) upon incubation with the different conditions is shown in the right panel (n = 100 cells/condition, * p<0.05 paired Student's t-test). These data are representative of three independent experiments.(B) GFP-LC3 CHO cells were incubated with complete medium in the absence (−dbcAMP) or presence of dbcAMP (+dbcAMP) and treated for 4 h with 10 µg/ml of α-hemolysin (Hla) or subjected to starvation conditions (Stv) with (lower panel) or without (upper panel) bafilomycin A1 to block lysosomal degradation. Afterwards, cells were lysed with sample buffer and the samples were subjected to Western blot analysis using a rabbit anti-LC3 and the corresponding HRP-labeled secondary antibody, and subsequently developed with an enhanced chemiluminescence detection kit. These data are representative of three independent experiments. (C) The band intensities of two independent experiments were quantificated with the Adobe Photoshop program, and normalized against tubulin. * p<0.05 (paired Student's t-test).


Figure 2 ( A ) GFP - LC3 CHO cells were preincubated for 30 min with 10 µM H89 , a PKA inhibitor , in the presence ( panels a , b , and c ) or absence ( panels d , e , and f ) of 1 mM dbcAMP . Subsequently , they were incubated for 2 h in starvation medium ( Stv , panels b and e ) or treated for 4 h with 10 µg / ml of α - hemolysin ( Hla , panels c and f ) in full nutrient media . Cells without any treatment were used as control ( Ctr , panels a and d ) . Cells were finally analyzed by confocal microscopy . Images are representative of three independent experiments . ( B ) Quantification of the percentage of cells presenting LC3 puncta ( i . e . , stimulated cells ) upon incubation with the different conditions . * * p < 0 . 01 ( paired Student ' s t - test , n = 100 cells / condition ) . These data are representative of two independent experiments . ( C ) GFP - LC3 CHO cells were preincubated for 30 min in the presence or absence of 10 µM H89 , and then they were incubated for 2 h with 1 mM dbcAMP to verify the activity of H89 . Afterwards , cells were lysed with sample buffer and the samples were subjected to Western blot analysis using a rabbit anti - phosphoCREB and the corresponding HRP - labeled secondary antibody , and subsequently developed with an enhanced chemiluminescence detection kit . The band intensities were quantified with the Adobe Photoshop program ( lower panel ) . These data are representative of two independent experiments .

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Figure 2(A) GFP-LC3 CHO cells were preincubated for 30 min with 10 µM H89, a PKA inhibitor, in the presence (panels a, b, and c) or absence (panels d, e, and f) of 1 mM dbcAMP. Subsequently, they were incubated for 2 h in starvation medium (Stv, panels b and e) or treated for 4 h with 10 µg/ml of α-hemolysin (Hla, panels c and f) in full nutrient media. Cells without any treatment were used as control (Ctr, panels a and d). Cells were finally analyzed by confocal microscopy. Images are representative of three independent experiments.(B) Quantification of the percentage of cells presenting LC3 puncta (i.e., stimulated cells) upon incubation with the different conditions. ** p<0.01 (paired Student's t-test, n = 100 cells/condition). These data are representative of two independent experiments.(C) GFP-LC3 CHO cells were preincubated for 30 min in the presence or absence of 10 µM H89, and then they were incubated for 2 h with 1 mM dbcAMP to verify the activity of H89. Afterwards, cells were lysed with sample buffer and the samples were subjected to Western blot analysis using a rabbit anti-phosphoCREB and the corresponding HRP-labeled secondary antibody, and subsequently developed with an enhanced chemiluminescence detection kit. The band intensities were quantified with the Adobe Photoshop program (lower panel). These data are representative of two independent experiments.


Figure 3 ( A ) GFP - LC3 CHO cells were preincubated with 10 µM 8 - pCPT - 2 ′ - O - Me - cAMP ( 8 - pCPT ) for 30 min and then they were treated for 4 h with 10 µg / ml of α - hemolysin ( Hla ) or subjected to starvation conditions ( Stv ) . Cells without any treatment were used as control . Cells were immediately analyzed by confocal microscopy . Quantification of the percentage of cells presenting LC3 puncta upon incubation under the different conditions is shown in the right panel ( n = 50 cells / condition ) . These data are representative of two independent experiments . ( B ) CHO cells were cotransfected with GFP - LC3 and myc - EPAC wt ( left panel ) or myc - ΔEPAC ( right panel ) . Twenty - four hours after transfection they were incubated for 2 h in starvation medium ( Stv ) or treated for 4 h with 10 µg / ml of α - hemolysin ( Hla ) . Cells without any treatment were used as control ( Ctr ) . Cells were analyzed by confocal microscopy . Images are representative of two independent experiments . ( C ) Quantification of the percentage of cells presenting LC3 puncta upon incubation under the different conditions ( n = 20 cells / condition ) . These data are representative of two independent

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(A) GFP-LC3 CHO cells were preincubated with 10 µM 8-pCPT-2′-O-Me-cAMP (8-pCPT) for 30 min and then they were treated for 4 h with 10 µg/ml of α-hemolysin (Hla) or subjected to starvation conditions (Stv). Cells without any treatment were used as control. Cells were immediately analyzed by confocal microscopy. Quantification of the percentage of cells presenting LC3 puncta upon incubation under the different conditions is shown in the right panel (n = 50 cells/condition). These data are representative of two independent experiments.(B) CHO cells were cotransfected with GFP-LC3 and myc-EPAC wt (left panel) or myc-ΔEPAC (right panel). Twenty-four hours after transfection they were incubated for 2 h in starvation medium (Stv) or treated for 4 h with 10 µg/ml of α-hemolysin (Hla). Cells without any treatment were used as control (Ctr). Cells were analyzed by confocal microscopy. Images are representative of two independent experiments. (C) Quantification of the percentage of cells presenting LC3 puncta upon incubation under the different conditions (n = 20 cells/condition). These data are representative of two independent experiments


Figure 4 ( A ) CHO cells were cotransfected with RFP - LC3 and GFP - Rap2b wt ( left panel ) or GFP - Rap2b ΔAAX ( right panel ) . Twenty - four hours after transfection , they were incubated for 2 h in starvation medium ( Stv ) , with 50 ng / µl of rapamycin ( Rapa ) in full nutrient media , or treated for 4 h with 10 µg / ml of α - hemolysin ( Hla ) . Cells without any treatment were used as control ( Ctr ) . Cells were analyzed by confocal microscopy . Images are representative of three independent experiments . ( B ) Quantification of the percentage of cells presenting LC3 puncta ( i . e . , stimulated cells ) upon incubation with the different conditions is shown . * p < 0 . 05 , * * p < 0 . 01 ( paired Student ' s t - test , n = 50 cells / condition ) . These data are representative of three independent experiments . ( C ) CHO cells were transfected with GFP - Rap2b wt or GFP - Rap2b ΔAAX and incubated with complete medium without ( Ctr ) or with 10 µg / ml of α - hemolysin ( Hla ) for 4 h , with 50 ng / µl of rapamycin ( Rapa ) or subjected to starvation conditions ( Stv ) for 2 h in the absence or presence of bafilomycin A1 . Afterwards , cells were lysed with sample buffer and the samples were subjected to Western blot analysis using a rabbit anti - LC3 and the corresponding HRP - labeled secondary antibody , and subsequently developed with an enhanced chemiluminescence detection kit . These data are representative of two independent experiments . ( D ) Quantification of the Western blot bands intensities of two independent experiments with the Adobe Photoshop program .

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Figure 4(A) CHO cells were cotransfected with RFP-LC3 and GFP-Rap2b wt (left panel) or GFP-Rap2b ΔAAX (right panel). Twenty-four hours after transfection, they were incubated for 2 h in starvation medium (Stv), with 50 ng/µl of rapamycin (Rapa) in full nutrient media, or treated for 4 h with 10 µg/ml of α-hemolysin (Hla). Cells without any treatment were used as control (Ctr). Cells were analyzed by confocal microscopy. Images are representative of three independent experiments.(B) Quantification of the percentage of cells presenting LC3 puncta (i.e., stimulated cells) upon incubation with the different conditions is shown. * p<0.05, ** p<0.01 (paired Student's t-test, n = 50 cells/condition). These data are representative of three independent experiments.(C) CHO cells were transfected with GFP-Rap2b wt or GFP-Rap2b ΔAAX and incubated with complete medium without (Ctr) or with 10 µg/ml of α-hemolysin (Hla) for 4 h, with 50 ng/µl of rapamycin (Rapa) or subjected to starvation conditions (Stv) for 2 h in the absence or presence of bafilomycin A1. Afterwards, cells were lysed with sample buffer and the samples were subjected to Western blot analysis using a rabbit anti-LC3 and the corresponding HRP-labeled secondary antibody, and subsequently developed with an enhanced chemiluminescence detection kit. These data are representative of two independent experiments.(D) Quantification of the Western blot bands intensities of two independent experiments with the Adobe Photoshop program.


Figure 5 ( A ) HeLa cells were cotransfected with Rap2b siRNA and GFP - LC3 ( right panel ) or irrelevant siRNA and GFP - LC3 ( left panel ) . Forty - eight hours after transfection , they were incubated for 2 h in starvation medium ( Stv ) , with 50 ng / µl of rapamycin ( Rapa ) or treated for 4 h with 10 µg / ml of α - hemolysin ( Hla ) . Cells without any treatment were used as control ( Ctr ) . Cells were analyzed by confocal microscopy . Images are representative of two independent experiments . ( B ) Quantification of the percentage of cells presenting LC3 puncta upon incubation under the different conditions . Data correspond to two independent experiments ( n = 50 cells / condition ) . ( C ) Upper panel : The knockdown of Rap2b was determined by Western blot as indicated in Materials and Methods . Lower panel : The band intensities of two independent experiments were quantified with the Adobe Photoshop program , and normalized against tubulin . * p < 0 . 05 ( paired Student ' s t - test ) . ( D ) HeLa cells were cotransfected with GFP - LC3 and Rap2b siRNA or an irrelevant siRNA and incubated for 4 h with complete medium in the absence ( Ctr ) or presence of 10 µg / ml of α - hemolysin ( Hla ) , with 50 ng / µl of rapamycin ( Rapa ) or subjected to starvation conditions ( Stv ) for 2 h . Afterwards , cells were lysed with sample buffer and the samples were subjected to Western blot analysis using a rabbit anti - LC3 and the corresponding HRP - labeled secondary antibody , and subsequently developed with an enhanced chemiluminescence detection kit . These data are representative of two independent experiments . ( E ) The band intensities of two independent experiments were quantified with the Adobe Photoshop program , and normalized against tubulin . * p < 0 . 05 ( paired Student ' s t - test ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(A) HeLa cells were cotransfected with Rap2b siRNA and GFP-LC3 (right panel) or irrelevant siRNA and GFP-LC3 (left panel). Forty-eight hours after transfection, they were incubated for 2 h in starvation medium (Stv), with 50 ng/µl of rapamycin (Rapa) or treated for 4 h with 10 µg/ml of α-hemolysin (Hla). Cells without any treatment were used as control (Ctr). Cells were analyzed by confocal microscopy. Images are representative of two independent experiments.(B) Quantification of the percentage of cells presenting LC3 puncta upon incubation under the different conditions. Data correspond to two independent experiments (n = 50 cells/condition).(C) Upper panel: The knockdown of Rap2b was determined by Western blot as indicated in Materials and Methods. Lower panel: The band intensities of two independent experiments were quantified with the Adobe Photoshop program, and normalized against tubulin. * p<0.05 (paired Student's t-test).(D) HeLa cells were cotransfected with GFP-LC3 and Rap2b siRNA or an irrelevant siRNA and incubated for 4 h with complete medium in the absence (Ctr) or presence of 10 µg/ml of α-hemolysin (Hla), with 50 ng/µl of rapamycin (Rapa) or subjected to starvation conditions (Stv) for 2 h. Afterwards, cells were lysed with sample buffer and the samples were subjected to Western blot analysis using a rabbit anti-LC3 and the corresponding HRP-labeled secondary antibody, and subsequently developed with an enhanced chemiluminescence detection kit. These data are representative of two independent experiments.(E) The band intensities of two independent experiments were quantified with the Adobe Photoshop program, and normalized against tubulin. * p<0.05 (paired Student's t-test).


Figure 6cAMP cannot inhibit the autophagy induced by the toxin in cells overexpressing the Rap2b negative mutant . ( A ) CHO cells were cotransfected with RFP - LC3 and GFP - Rap2b ΔAAX . Twenty - four hours after transfection they were incubated for 2 h in starvation medium ( Stv ) or treated for 4 h with 10 µg / ml of α - hemolysin ( Hla ) in the presence ( right panels ) or absence of 1 mM dbcAMP ( left panels ) . Cells without any treatment were used as control ( Ctr ) . Cells were analyzed by confocal microscopy . Images are representative of two independent experiments . ( B ) Quantification of the percentage of cells presenting LC3 puncta ( i . e . , stimulated cells ) upon incubation with the different conditions . These data are representative of two independent experiments .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6cAMP cannot inhibit the autophagy induced by the toxin in cells overexpressing the Rap2b negative mutant.(A) CHO cells were cotransfected with RFP-LC3 and GFP-Rap2b ΔAAX. Twenty-four hours after transfection they were incubated for 2 h in starvation medium (Stv) or treated for 4 h with 10 µg/ml of α-hemolysin (Hla) in the presence (right panels) or absence of 1 mM dbcAMP (left panels). Cells without any treatment were used as control (Ctr). Cells were analyzed by confocal microscopy. Images are representative of two independent experiments. (B) Quantification of the percentage of cells presenting LC3 puncta (i.e., stimulated cells) upon incubation with the different conditions. These data are representative of two independent experiments.


Figure 7 ( A ) GFP - LC3 CHO cells were preincubated for 30 min with 10 µM of calpeptin ( a calpain inhibitor , panels b , f , and j ) , with 1 mM dbcAMP ( panels d , h and l ) , or with Calpeptin + dbcAMP ( panels c , g , and k ) . Afterwards , they were incubated for 2 h in starvation medium ( Stv , panels e , f , g , and h ) or treated for 4 h with 10 µg / ml of α - hemolysin ( Hla , panels i , j , k , and l ) . Cells without any treatment were used as control ( Ctr , panels a , b , c and d ) . Cells were analyzed by confocal microscopy . Images are representative of two independent experiments . ( B ) Quantification of the percentage of cells presenting LC3 puncta ( i . e . , stimulated cells ) upon incubation with the different conditions . * p < 0 . 05 ( paired Student ' s t - test , n = 100 cells / condition ) . These data are representative of two independent experiments . ( C ) Upper panel : CHO cells were preincubated for 30 min with 10 µM of calpeptin , with 1 mM dbcAMP , or with calpeptin + dbcAMP , and then they were incubated for 4 h in complete medium in the presence ( Hla ) or absence ( Ctr ) of α - hemolysin . Afterwards , cells were lysed with sample buffer and the samples were subjected to Western blot analysis using a rabbit anti - LC3 and the corresponding HRP - labeled secondary antibody . The bands were subsequently developed with an enhanced chemiluminescence detection kit . Lower panel : Quantification of the band intensities with the Adobe Photoshop program . * p < 0 . 05 ( paired Student ' s t - test ) . These data are representative of three independent experiments .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7(A) GFP-LC3 CHO cells were preincubated for 30 min with 10 µM of calpeptin (a calpain inhibitor, panels b, f, and j), with 1 mM dbcAMP (panels d, h and l), or with Calpeptin+dbcAMP (panels c, g, and k). Afterwards, they were incubated for 2 h in starvation medium (Stv, panels e, f, g, and h) or treated for 4 h with 10 µg/ml of α-hemolysin (Hla, panels i, j, k, and l). Cells without any treatment were used as control (Ctr, panels a, b, c and d). Cells were analyzed by confocal microscopy. Images are representative of two independent experiments. (B) Quantification of the percentage of cells presenting LC3 puncta (i.e., stimulated cells) upon incubation with the different conditions. * p<0.05 (paired Student's t-test, n = 100 cells/condition). These data are representative of two independent experiments.(C) Upper panel: CHO cells were preincubated for 30 min with 10 µM of calpeptin, with 1 mM dbcAMP, or with calpeptin+dbcAMP, and then they were incubated for 4 h in complete medium in the presence (Hla) or absence (Ctr) of α-hemolysin. Afterwards, cells were lysed with sample buffer and the samples were subjected to Western blot analysis using a rabbit anti-LC3 and the corresponding HRP-labeled secondary antibody. The bands were subsequently developed with an enhanced chemiluminescence detection kit. Lower panel: Quantification of the band intensities with the Adobe Photoshop program. * p<0.05 (paired Student's t-test). These data are representative of three independent experiments.


Figure 8 ( A ) GFP - LC3 CHO cells were preincubated with 1 mM dybutiryl cAMP ( + dbcAMP ) for 30 min and then were infected for 4 h in the presence of db cAMP with the wt strain of S . aureus ( wt ) , the mutant deficient for α - hemolysin ( Hla − ) , or the Hla ( − ) mutant expressing an α - hemolysin plasmid ( Hla ( − ) + pHla ) . Cells without any treatment were used as control ( − dbcAMP ) . The nucleus and the bacteria were labeled with TOPRO as indicated in Materials and Methods , and immediately visualized by confocal microscopy . Images are representative of two independent experiments . . ( B ) Quantification of the percentage of cells presenting LC3 puncta ( i . e . , stimulated cells ) upon incubation in the different conditions . * p < 0 . 05 , * * p < 0 . 01 ( paired Student ' s t - test , n = 50 cells / condition ) . These data are representative of two independent experiments . ( C ) Quantification of the number of CFU / ml of CHO cells infected for 3 h with the wt strain of S . aureus , the mutant deficient for α - hemolysin ( Hla − ) , or the Hla ( − ) mutant expressing an α - hemolysin plasmid in the absence or presence of cAMP . * p < 0 . 05 ( paired Student ' s t - test ) . These data are representative of two independent experiments .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 8(A) GFP-LC3 CHO cells were preincubated with 1 mM dybutiryl cAMP (+dbcAMP) for 30 min and then were infected for 4 h in the presence of db cAMP with the wt strain of S. aureus (wt), the mutant deficient for α-hemolysin (Hla−), or the Hla(−) mutant expressing an α-hemolysin plasmid (Hla(−) +pHla). Cells without any treatment were used as control (−dbcAMP). The nucleus and the bacteria were labeled with TOPRO as indicated in Materials and Methods, and immediately visualized by confocal microscopy. Images are representative of two independent experiments.. (B) Quantification of the percentage of cells presenting LC3 puncta (i.e., stimulated cells) upon incubation in the different conditions. * p<0.05, ** p<0.01 (paired Student's t-test, n = 50 cells/condition). These data are representative of two independent experiments.(C) Quantification of the number of CFU/ml of CHO cells infected for 3 h with the wt strain of S. aureus, the mutant deficient for α-hemolysin (Hla−), or the Hla(−) mutant expressing an α-hemolysin plasmid in the absence or presence of cAMP. * p<0.05 (paired Student's t-test). These data are representative of two independent experiments.


Figure 9CHO cells were cotransfected with GFP - LC3 and myc - EPAC wt ( upper panel ) or myc - ΔEPAC ( lower panel ) . Twenty - four hours after transfection , they were infected for 4 h with the wt strain of S . aureus ( wt ) , the mutant deficient for α - hemolysin ( Hla − ) , or the Hla ( − ) mutant expressing an α - hemolysin plasmid ( Hla ( − ) + pHla ) . The nucleus and the bacteria were labeled with TOPRO as indicated in Materials and Methods and immediately visualized by confocal microscopy . Images are representative of three independent experiments . B ) Quantification of the percentage of cells presenting LC3 puncta upon the infection . These data are representative of three independent experiments . ( C ) Quantification of the percentage of bacteria decorated with EPAC in cells infected with S . aureus . * * p < 0 . 01 ( paired Student ' s t - test , n = 20 cells / condition ) . These data are representative of three independent experiments .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 9CHO cells were cotransfected with GFP-LC3 and myc-EPAC wt (upper panel) or myc-ΔEPAC (lower panel). Twenty-four hours after transfection, they were infected for 4 h with the wt strain of S. aureus (wt), the mutant deficient for α-hemolysin (Hla−), or the Hla(−) mutant expressing an α-hemolysin plasmid (Hla(−)+pHla). The nucleus and the bacteria were labeled with TOPRO as indicated in Materials and Methods and immediately visualized by confocal microscopy. Images are representative of three independent experiments.B) Quantification of the percentage of cells presenting LC3 puncta upon the infection. These data are representative of three independent experiments.(C) Quantification of the percentage of bacteria decorated with EPAC in cells infected with S. aureus. ** p<0.01 (paired Student's t-test, n = 20 cells/condition). These data are representative of three independent experiments.


Figure 10 ( A ) CHO cells were cotransfected with RFP - LC3 and GFP - Rap2b wt ( upper panel ) or GFP - Rap2b ΔAAX ( lower panel ) . Twenty - four hours after transfection , they were infected for 4 h with the wt strain of S . aureus ( wt ) , the mutant deficient for α - hemolysin ( Hla − ) , or the Hla ( − ) mutant expressing an α - hemolysin plasmid ( Hla ( − ) + pHla ) . The nucleus and the bacteria were labeled with TOPRO as indicated in Materials and Methods and immediately visualized by confocal microscopy . These data are representative of three independent experiments . ( B ) Quantification of the percentage of infected cells presenting LC3 puncta upon the infection . * p < 0 . 05 , * * * p < 0 . 001 ( paired Student ' s t - test , n = 50 cells / condition ) . These data are representative of three independent experiments . ( C ) Quantification of the percentage of bacteria decorated with Rap2b wt upon the infection . * * p < 0 . 01 ( paired Student ' s t - test , n = 30 cells / condition ) . These data are representative of three independent

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 10(A) CHO cells were cotransfected with RFP-LC3 and GFP-Rap2b wt (upper panel) or GFP-Rap2b ΔAAX (lower panel). Twenty-four hours after transfection, they were infected for 4 h with the wt strain of S. aureus (wt), the mutant deficient for α-hemolysin (Hla−), or the Hla(−) mutant expressing an α-hemolysin plasmid (Hla(−)+pHla). The nucleus and the bacteria were labeled with TOPRO as indicated in Materials and Methods and immediately visualized by confocal microscopy. These data are representative of three independent experiments.(B) Quantification of the percentage of infected cells presenting LC3 puncta upon the infection. * p<0.05, *** p<0.001 (paired Student's t-test, n = 50 cells/condition). These data are representative of three independent experiments.(C) Quantification of the percentage of bacteria decorated with Rap2b wt upon the infection. ** p<0.01 (paired Student's t-test, n = 30 cells/condition). These data are representative of three independent experim


Figure 11 ( A ) HeLa cells were incubated for 4 h with complete medium in the presence ( Hla ) or absence ( Ctr ) of 10 µg / ml α - hemolysin or they were infected for 4 h with the wt strain of S . aureus ( wt ) or the α - hemolysin deficient mutant ( Hla − ) . Afterwards , cells were lysed with sample buffer and the samples were subjected to Western blot analysis using a rabbit anti - Rap2b and the corresponding HRP - labeled secondary antibody . The bands were subsequently developed with an enhanced chemiluminescence detection kit . A quantification of the bands intensities with the Adobe Photoshop program is shown in the lower panel . These data are representative of two independent experiments . B ) HeLa cells were incubated for 4 h with complete medium in the presence ( Hla ) or absence ( Ctr ) of 10 µg / ml α - hemolysin or they were infected for 4 h with the wt strain of S . aureus ( wt ) or the α - hemolysin deficient mutant ( Hla − ) . Cells were disrupted and whole cell lysates were subjected to pull - down assays using GST - Ral - GDS - RBD - sepharose . The levels of GTP - bound Rap2b were determined as was described in Materials and Methods by Western blot analysis using a rabbit anti - Rap2b and the corresponding HRP - labeled secondary antibody . The bands were subsequently developed with an enhanced chemiluminescence detection kit . The band intensities were quantificated with the Adobe Photoshop program is shown in the lower panel . * p < 0 . 05 ( paired Student ' s t - test ) . These data are representative of two independent experiments .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 11(A) HeLa cells were incubated for 4 h with complete medium in the presence (Hla) or absence (Ctr) of 10 µg/ml α-hemolysin or they were infected for 4 h with the wt strain of S. aureus (wt) or the α-hemolysin deficient mutant (Hla−). Afterwards, cells were lysed with sample buffer and the samples were subjected to Western blot analysis using a rabbit anti-Rap2b and the corresponding HRP-labeled secondary antibody. The bands were subsequently developed with an enhanced chemiluminescence detection kit. A quantification of the bands intensities with the Adobe Photoshop program is shown in the lower panel. These data are representative of two independent experiments.B) HeLa cells were incubated for 4 h with complete medium in the presence (Hla) or absence (Ctr) of 10 µg/ml α-hemolysin or they were infected for 4 h with the wt strain of S. aureus (wt) or the α-hemolysin deficient mutant (Hla−). Cells were disrupted and whole cell lysates were subjected to pull-down assays using GST-Ral-GDS-RBD-sepharose. The levels of GTP-bound Rap2b were determined as was described in Materials and Methods by Western blot analysis using a rabbit anti-Rap2b and the corresponding HRP-labeled secondary antibody. The bands were subsequently developed with an enhanced chemiluminescence detection kit. The band intensities were quantificated with the Adobe Photoshop program is shown in the lower panel. * p<0.05 (paired Student's t-test). These data are representative of two independent experiments.


f1 ( a ) Confocal analysis of the subcellular localization of HA - PLD1 ( green ) and endogenous LC3 ( red ) in HeLa cells in normal medium and under nutrient deprivation conditions . Left panels : normal medium condition ( Nm ) ; middle panels : 1 h with nutrient - deprived medium ( 1 h St ) ; right panels : 2 h with nutrient - deprived medium ( 2 h St ) . Insets denote high magnification of the selected area . Scale bar , 5 μm . ( b ) Immuno - gold EM micrographs of HeLa cells transfected with GFP - PLD1 and subjected to 1 h of nutrient deprivation in the presence ( St + B ) or absence ( St ) of bafilomycin ( 50 nM ) . Insets denote high magnification of the selected area . Scale bar , 500 nm . Right panel : Histogram showing morphometric analysis of the relative distribution of GFP - PLD1 - positive gold particles on the outer ( arrows ) and inner ( arrowheads ) membranes of the autophagosomal - like structures . Values denote means ± s . e . m . ( n = 14 ) . * * P0 . 01 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

f1(a) Confocal analysis of the subcellular localization of HA-PLD1 (green) and endogenous LC3 (red) in HeLa cells in normal medium and under nutrient deprivation conditions. Left panels: normal medium condition (Nm); middle panels: 1 h with nutrient-deprived medium (1 h St); right panels: 2 h with nutrient-deprived medium (2 h St). Insets denote high magnification of the selected area. Scale bar, 5 μm.(b) Immuno-gold EM micrographs of HeLa cells transfected with GFP-PLD1 and subjected to 1 h of nutrient deprivation in the presence (St+B) or absence (St) of bafilomycin (50 nM). Insets denote high magnification of the selected area. Scale bar, 500 nm. Right panel: Histogram showing morphometric analysis of the relative distribution of GFP-PLD1-positive gold particles on the outer (arrows) and inner (arrowheads) membranes of the autophagosomal-like structures. Values denote means±s.e.m. (n=14). **P0.01.


f2Confocal analysis of the subcellular localization of HA - PLD1 and endogenous LC3 using various organelle markers in cells deprived of nutrients for 2 h . Organelle markers used were as follows ( from top to bottom panels ) : Lamp1 ( late endosomes / lysosomes ) , giantin ( Golgi - TGN ) , Vps35 ( retromer - positive endosomes ) , GFP - Rab5 ( early endosomes ) , GFP - UVRAG ( early and late endosomes ) . Insets show high magnification of the selected areas . The triple insets at the bottom of each column of pictures indicate high magnification of the selected areas for two channels in various combinations . Scale bar , 5 μm .

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f2Confocal analysis of the subcellular localization of HA-PLD1 and endogenous LC3 using various organelle markers in cells deprived of nutrients for 2 h. Organelle markers used were as follows (from top to bottom panels): Lamp1 (late endosomes/lysosomes), giantin (Golgi-TGN), Vps35 (retromer-positive endosomes), GFP-Rab5 (early endosomes), GFP-UVRAG (early and late endosomes). Insets show high magnification of the selected areas. The triple insets at the bottom of each column of pictures indicate high magnification of the selected areas for two channels in various combinations. Scale bar, 5 μm.


f3 ( a ) Confocal analysis of HeLa cells subjected to nutrient deprivation for 2 h . Cells were transfected with HA - PLD1 and GFP - ATG14L ( upper panels ) or with HA - PLD1 alone ( lower panels ) . Upper panels : HA - PLD1 ( red ) , GFP - ATG14L ( green ) , endogenous LC3 ( blue ) . Lower panels : HA - PLD1 ( green ) , endogenous ATG16L ( red ) and LC3 ( blue ) . Insets show high magnification of the selected areas . The triple insets on the right indicate high magnification of the selected areas for two channels in various combinations . Scale bar , 5 μm . ( b ) Confocal analysis showing lack of co - localization of HA - PLD1 ( green ) with the isolation membrane marker ATG16L ( blue ) in HeLa cells expressing the strawberry - ATG4 - C74A mutant ( red ) and subjected to 1 h of nutrient deprivation . Insets denote high magnification of the selected area . Scale bar , 5 μm . ( c ) EM analysis showing that GFP - PLD1 does not localize to the isolation membrane ( arrows ) in cells expressing the strawberry - ATG4 - C74A mutant . Immuno - gold EM micrographs of HeLa cells transfected with GFP - PLD1 and pStrawberry - ATG4 - C74A and subjected to 1 h of nutrient deprivation . Inset denotes high magnification of the selected area . Scale bar , 250 nm .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

f3(a) Confocal analysis of HeLa cells subjected to nutrient deprivation for 2 h. Cells were transfected with HA-PLD1 and GFP-ATG14L (upper panels) or with HA-PLD1 alone (lower panels). Upper panels: HA-PLD1 (red), GFP-ATG14L (green), endogenous LC3 (blue). Lower panels: HA-PLD1 (green), endogenous ATG16L (red) and LC3 (blue). Insets show high magnification of the selected areas. The triple insets on the right indicate high magnification of the selected areas for two channels in various combinations. Scale bar, 5 μm.(b) Confocal analysis showing lack of co-localization of HA-PLD1 (green) with the isolation membrane marker ATG16L (blue) in HeLa cells expressing the strawberry-ATG4-C74A mutant (red) and subjected to 1 h of nutrient deprivation. Insets denote high magnification of the selected area. Scale bar, 5 μm.(c) EM analysis showing that GFP-PLD1 does not localize to the isolation membrane (arrows) in cells expressing the strawberry-ATG4-C74A mutant. Immuno-gold EM micrographs of HeLa cells transfected with GFP-PLD1 and pStrawberry-ATG4-C74A and subjected to 1 h of nutrient deprivation. Inset denotes high magnification of the selected area. Scale bar, 250 nm.


f4 ( a ) Histogram showing relative phospholipid levels in CHO cells treated with 0 . 3 % ethanol for 30 min before cell harvesting . Total PLD activity is measured as phosphatidylethanol ( PEtOH ) levels under the following conditions : normal medium ( Nm ) , serum - free medium ( SFM ) , serum - free medium with 100 nM wortmannin ( SFM + W ) , nutrient deprivation ( 0 . 5 h St ; 1 . 5 h St ; 3 . 0 h St ) , 30 min of nutrient deprivation in presence of 100nM wortmannin ( 0 . 5h St + W ) . Other phospholipids were used as controls : phosphatidylserine ( PS ) , phosphatidylethanolamine ( PE ) , phosphatidylcholine ( PC ) . Values denote means ± s . e . m . ( n = 9 , except for SFM + W and 0 . 5 h St + W , where n = 3 and 5 , respectively ) . * * P0 . 01 ; * , P0 . 05 . ( b ) Western blot analysis of HA - PLD1 and endogenous LC3 levels in post - nuclear supernatant ( PNS , upper panel ) , as well as in cytosolic / soluble ( S ) and particulate ( P ) fractions prepared from PNS after high - speed centrifugation ( middle panel ) . GAPDH was used as loading control in PNS fractions . HeLa cells were transfected with expression vectors encoding HA - PLD1 for 24 h and subjected to 2 h of nutrient deprivation in the presence or absence of the PI3K inhibitor wortmannin ( 100 nM ) . Lower panel : quantitative analysis of HA - PLD1 immunoreactivity expressed as P / S ratio . Conditions were as follows : normal medium ( Nm ) ; normal medium with wortmannin ( Nm + W ) ; starvation condition ( St ) ; starvation condition + wortmannin ( St + W ) . Values denote means ± s . e . m . ( n = 3 ) . ( c ) Confocal analysis of the co - localization between HA - PLD1 and Lamp1 in HeLa cells subjected to nutrient deprivation for 2 h in the presence ( ST + W ) or absence ( St ) of wortmannin 100 nM . Insets show high magnification of the selected areas . Scale bar , 5 μm .

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f4(a) Histogram showing relative phospholipid levels in CHO cells treated with 0.3% ethanol for 30 min before cell harvesting. Total PLD activity is measured as phosphatidylethanol (PEtOH) levels under the following conditions: normal medium (Nm), serum-free medium (SFM), serum-free medium with 100 nM wortmannin (SFM+W), nutrient deprivation (0.5 h St; 1.5 h St; 3.0 h St), 30 min of nutrient deprivation in presence of 100nM wortmannin (0.5h St+W). Other phospholipids were used as controls: phosphatidylserine (PS), phosphatidylethanolamine (PE), phosphatidylcholine (PC). Values denote means±s.e.m. (n=9, except for SFM+W and 0.5 h St+W, where n=3 and 5, respectively). **P0.01; *, P0.05.(b) Western blot analysis of HA-PLD1 and endogenous LC3 levels in post-nuclear supernatant (PNS, upper panel), as well as in cytosolic/soluble (S) and particulate (P) fractions prepared from PNS after high-speed centrifugation (middle panel). GAPDH was used as loading control in PNS fractions. HeLa cells were transfected with expression vectors encoding HA-PLD1 for 24 h and subjected to 2 h of nutrient deprivation in the presence or absence of the PI3K inhibitor wortmannin (100 nM). Lower panel: quantitative analysis of HA-PLD1 immunoreactivity expressed as P/S ratio. Conditions were as follows: normal medium (Nm); normal medium with wortmannin (Nm+W); starvation condition (St); starvation condition+wortmannin (St+W). Values denote means±s.e.m. (n=3).(c) Confocal analysis of the co-localization between HA-PLD1 and Lamp1 in HeLa cells subjected to nutrient deprivation for 2 h in the presence (ST+W) or absence (St) of wortmannin 100 nM. Insets show high magnification of the selected areas. Scale bar, 5 μm.


f5 ( a ) Western blot analysis showing the effects of siRNA - mediated Vps34 ( siVps34 ) downregulation on the expression levels of HA - PLD1 and Vps34 compared with control cells ( siCtrl ) . Cells were incubated in normal medium ( Nm ) or subjected to 2 h of nutrient deprivation in the presence of 50 nM bafilomycin ( St + B ) . HeLa cells were transfected for 48 h with siRNA directed to Vps34 ( siVps34 ) or a control sequence ( siCtrl ) and then transfected for 24 h with a plasmid encoding HA - PLD1 . ( b ) Confocal imaging showing the effects of Vps34 ( siVps34 ) downregulation on HA - PLD1 ( green ) , endogenous LC3 ( blue ) and Lamp1 ( red ) on 2 h of nutrient deprivation compared with control treatment ( siCtrl ) . Scale bar , 5 μm . ( c ) Confocal analysis showing the subcellular localization of HA - PLD1 mutated in its PX domain ( F120A / R179Q ) , so as to abolish phosphoinositide binding ( HA - PXmut ) . HeLa cells were transiently transfected with a plasmid encoding HA - PXmut and fixed after 2 h of nutrient deprivation . HA - PXmut ( green ) , endogenous LC3 ( blue ) and Lamp1 ( red ) . Scale bar , 10 μm . In b and c , insets show high magnification of the selected areas . The triple insets on the right indicate high magnification of the selected areas for two channels in various combinations .

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f5(a) Western blot analysis showing the effects of siRNA-mediated Vps34 (siVps34) downregulation on the expression levels of HA-PLD1 and Vps34 compared with control cells (siCtrl). Cells were incubated in normal medium (Nm) or subjected to 2 h of nutrient deprivation in the presence of 50 nM bafilomycin (St+B). HeLa cells were transfected for 48 h with siRNA directed to Vps34 (siVps34) or a control sequence (siCtrl) and then transfected for 24 h with a plasmid encoding HA-PLD1.(b) Confocal imaging showing the effects of Vps34 (siVps34) downregulation on HA-PLD1 (green), endogenous LC3 (blue) and Lamp1 (red) on 2 h of nutrient deprivation compared with control treatment (siCtrl). Scale bar, 5 μm. (c) Confocal analysis showing the subcellular localization of HA-PLD1 mutated in its PX domain (F120A/R179Q), so as to abolish phosphoinositide binding (HA-PXmut). HeLa cells were transiently transfected with a plasmid encoding HA-PXmut and fixed after 2 h of nutrient deprivation. HA-PXmut (green), endogenous LC3 (blue) and Lamp1 (red). Scale bar, 10 μm. In b and c, insets show high magnification of the selected areas. The triple insets on the right indicate high magnification of the selected areas for two channels in various combinations.


f6 ( a ) Confocal analysis showing the effects of PLD inhibition ( FIPI , 750 nM ) on the levels of endogenous LC3 . CHO cells were treated with PLD inhibitor FIPI or DMSO for 3 h in the presence of serum ( normal medium , Nm ) or in the absence of nutrients ( starvation , St ) . Insets denote high magnification of the selected areas . Scale bar , 10 μm . ( b ) Quantification of the average number , size and surface area of LC3 - positive compartments per cell after FIPI treatment . Black bars , DMSO ( vehicle ) ; grey bars , FIPI treatment . Values denote means ± s . e . m . ( n = 180 - 285 cells for Nm ; n = 449 - 519 cells for St ) . * * P0 . 01 . ( c ) Western blot analysis showing levels of LC3 - I and LC3 - II in control - and FIPI - treated CHO cells grown in normal medium ( Nm ) , after nutrient starvation ( St ) or after nutrient starvation in the presence of 50 nM bafilomycin ( St + B ) . Treatments were for 3 h . Right panel : quantification of protein signal intensities showing LC3 - II protein levels in extracts from nutrient - starved and bafilomycin - treated CHO cells following FIPI treatment . Black bars , DMSO ( vehicle ) ; grey bars , FIPI treatment . Values denote means ± s . e . m . ( n = 4 ) . ( d ) Western blot analysis of the effects of PLD inhibition on the levels of total Tau , phosphorylated Tau ( p - Tau ) and detergent - insoluble Tau . Organotypic brain slices from hTau mice were treated with either DMSO ( D ) or 750nM FIPI ( F ) and harvested after 5 days of treatment . Left panel : western blot analysis showing immunoreactivities for total human Tau ( CP27 ) , hyperphosphorylated tau ( p - Tau , ser202 / ser205 , CP13 ) , p62 and tubulin from the supernatant fraction and aggregated tau ( CP27 ) and p62 from the sarkosyl - insoluble fraction . Two representative pairs are displayed . Right panel , quantification of total proteins ( Tau , p - Tau , p62 and tubulin ) and sarkosyl - insoluble aggregates of tau ( insoluble Tau ) and p62 ( insoluble p62 ) . Black bars , DMSO ( vehicle ) ; grey bars , FIPI treatment . Values denote means ± s . e . m . ( n = 7 ) . * * P0 . 01 ; * , P0 . 05 . For the statistical analysis of FIPI - treated samples , tau and p62 protein levels were compared with tubulin .

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f6(a) Confocal analysis showing the effects of PLD inhibition (FIPI, 750 nM) on the levels of endogenous LC3. CHO cells were treated with PLD inhibitor FIPI or DMSO for 3 h in the presence of serum (normal medium, Nm) or in the absence of nutrients (starvation, St). Insets denote high magnification of the selected areas. Scale bar, 10 μm. (b) Quantification of the average number, size and surface area of LC3-positive compartments per cell after FIPI treatment. Black bars, DMSO (vehicle); grey bars, FIPI treatment. Values denote means±s.e.m. (n=180-285 cells for Nm; n=449-519 cells for St). **P0.01.(c) Western blot analysis showing levels of LC3-I and LC3-II in control- and FIPI-treated CHO cells grown in normal medium (Nm), after nutrient starvation (St) or after nutrient starvation in the presence of 50 nM bafilomycin (St+B). Treatments were for 3 h. Right panel: quantification of protein signal intensities showing LC3-II protein levels in extracts from nutrient-starved and bafilomycin-treated CHO cells following FIPI treatment. Black bars, DMSO (vehicle); grey bars, FIPI treatment. Values denote means±s.e.m. (n=4).(d) Western blot analysis of the effects of PLD inhibition on the levels of total Tau, phosphorylated Tau (p-Tau) and detergent-insoluble Tau. Organotypic brain slices from hTau mice were treated with either DMSO (D) or 750nM FIPI (F) and harvested after 5 days of treatment. Left panel: western blot analysis showing immunoreactivities for total human Tau (CP27), hyperphosphorylated tau (p-Tau, ser202/ser205, CP13), p62 and tubulin from the supernatant fraction and aggregated tau (CP27) and p62 from the sarkosyl-insoluble fraction. Two representative pairs are displayed. Right panel, quantification of total proteins (Tau, p-Tau, p62 and tubulin) and sarkosyl-insoluble aggregates of tau (insoluble Tau) and p62 (insoluble p62). Black bars, DMSO (vehicle); grey bars, FIPI treatment. Values denote means±s.e.m. (n=7). **P0.01; *, P0.05. For the statistical analysis of FIPI-treated samples, tau and p62 protein levels were compared with tubulin.


f7a ) Western blot analysis showing reduced levels of LC3 - II in KO MEFs . ( b ) Quantification of protein signal intensities from western blots showing LC3 - II levels in MEFs extracts after normalization to the control protein GAPDH . In a and b , MEFs were grown in normal medium ( Nm ) , treated for 2 h with 50 nM bafilomycin in normal medium ( B ) , or grown in nutrient - deprived medium in the absence ( St ) or presence of 50 nM bafilomycin ( St + B ) . Values denote means ± s . e . m . ( n = 4 ) . ( c ) Immunostaining of endogenous LC3 in Pld1 WT and KO MEFs showing a reduced number and size of the LC3 - positive compartment in KO MEFs . The DAPI staining is shown in blue . Cells were deprived of nutrients for 2 h in the absence ( St , upper panels ) or presence of bafilomycin ( St + B , lower panels ) . Scale bar , 5 μm . ( d ) Quantification of the average size ( top panel ) and number of LC3 - positive compartments per cell ( bottom panel ) in Pld1 WT and KO MEFs starved for 2 h . Values denote means ± s . e . m . ( n = 6 , number of cells = 26 - 45 for all the conditions ) . * * P0 . 01 ; * P0 . 05 .

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f7a) Western blot analysis showing reduced levels of LC3-II in KO MEFs. (b) Quantification of protein signal intensities from western blots showing LC3-II levels in MEFs extracts after normalization to the control protein GAPDH. In a and b, MEFs were grown in normal medium (Nm), treated for 2 h with 50 nM bafilomycin in normal medium (B), or grown in nutrient-deprived medium in the absence (St) or presence of 50 nM bafilomycin (St+B). Values denote means±s.e.m. (n=4).(c) Immunostaining of endogenous LC3 in Pld1 WT and KO MEFs showing a reduced number and size of the LC3-positive compartment in KO MEFs. The DAPI staining is shown in blue. Cells were deprived of nutrients for 2 h in the absence (St, upper panels) or presence of bafilomycin (St+B, lower panels). Scale bar, 5 μm. (d) Quantification of the average size (top panel) and number of LC3-positive compartments per cell (bottom panel) in Pld1 WT and KO MEFs starved for 2 h. Values denote means±s.e.m. (n=6, number of cells=26-45 for all the conditions). **P0.01; *P0.05.


f8 ( a ) Immunofluorescent detection of endogenous LC3 ( red ) in liver sections from fed or starved Pld1 WT and KO mice . Sections were counterstained with DAPI . Scale bar , 5 μm . ( b ) Quantitative analysis of endogenous LC3 immunofluorescence in liver sections derived from fed or starved Pld1 WT and KO mice . Histograms show the average number , size and surface area of LC3 - positive compartments . Values denote means ± s . e . m . ( ( n = 25 - 28 ) , 4 mice per genotype ) . * * P0 . 01 . ( c ) Electron microscopic analysis showing a reduction in the size and number of autophagosome / amphisome - like structures ( AP ) in PLD1 - deficient mice after food restriction . Representative electron micrographs from animals analysed in a showing APs ( arrows ) and autophagolysosomes / lysosomes ( AL , arrowheads ) in liver sections from starved WT and KO mice . Insets show high magnification of the selected areas . Scale bar , 1 μm . ( d ) Morphometric analysis of the number ( per 100 μm2 cytoplasmic area ) and relative surface area ( % cytoplasmic area ) of APs and ALs . Values denote means ± s . e . m . ( n = 36 electron micrograms , from a total of 3 , 600 μm2 cytoplasmic area per genotype ) . * * P0 . 01 . ( c ) Morphometric analysis of the diameter of APs and ALs in the liver of PLD1 - deficient mice after food restriction . Values denote means ± s . e . m . ( n = 60 ) . * * P0 . 01 .

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f8(a) Immunofluorescent detection of endogenous LC3 (red) in liver sections from fed or starved Pld1 WT and KO mice. Sections were counterstained with DAPI. Scale bar, 5 μm. (b) Quantitative analysis of endogenous LC3 immunofluorescence in liver sections derived from fed or starved Pld1 WT and KO mice. Histograms show the average number, size and surface area of LC3-positive compartments. Values denote means±s.e.m. ((n=25-28), 4 mice per genotype). **P0.01.(c) Electron microscopic analysis showing a reduction in the size and number of autophagosome/amphisome-like structures (AP) in PLD1-deficient mice after food restriction. Representative electron micrographs from animals analysed in a showing APs (arrows) and autophagolysosomes/lysosomes (AL, arrowheads) in liver sections from starved WT and KO mice. Insets show high magnification of the selected areas. Scale bar, 1 μm. (d) Morphometric analysis of the number (per 100 μm2 cytoplasmic area) and relative surface area (% cytoplasmic area) of APs and ALs. Values denote means±s.e.m. (n=36 electron micrograms, from a total of 3,600 μm2 cytoplasmic area per genotype). **P0.01. (c) Morphometric analysis of the diameter of APs and ALs in the liver of PLD1-deficient mice after food restriction. Values denote means±s.e.m. (n=60). **P0.01.


Figure 1A . MS / MS analysis of peptides corresponding to amino acids 380 - 399 of STAT2 . 293T cells expressing flag - tagged STAT2 were used . B . Abundance of T387 phosphorylated STAT2 in 293T cells expressing flag - tagged STAT2 .

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Figure 1A. MS/MS analysis of peptides corresponding to amino acids 380-399 of STAT2. 293T cells expressing flag-tagged STAT2 were used.B. Abundance of T387 phosphorylated STAT2 in 293T cells expressing flag-tagged STAT2.


Figure 2A . U6A cells expressing wild - type or T387A STAT2 were treated with IFN - β ( 100 IU / ml ) . Cells were harvested after 4 h and total RNAs were analyzed by real - time PCR . Values are the means ± SD from three independent experiments . B . U6A cells expressing wild - type , T387A , or T387D STAT2 were treated with IFN - β ( 100 IU / ml ) . Cells were harvested after 4 h and total RNAs were analyzed by real - time PCR . Values are the means ± SD from three independent experiments . C , D . U6A cells expressing wild - type or T387A STAT2 were treated with IFN - β for 0 , 4 , 8 , or 24 h . Total RNAs were analyzed by using an Illumina HumanHT - 12 v4 Expression BeadChip array . The average signal for each probe was used to determine expression levels . Genes with average signals below 25 and detection P values greater than 0 . 01 in the untreated or treated cells were excluded from the analysis . Inductions of less than 2 - fold were not scored . The Venn diagram shows the numbers of genes induced by 1 . 2 fold or more in U6A cells expressing T387A STAT2 , relative to untreated control cells . The numbers in the table are fold changes calculated from the ratios T387A STAT2 treated / untreated and wild - type STAT2 treated / untreated .

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Figure 2A. U6A cells expressing wild-type or T387A STAT2 were treated with IFN-β (100 IU/ml). Cells were harvested after 4 h and total RNAs were analyzed by real-time PCR. Values are the means ± SD from three independent experiments.B. U6A cells expressing wild-type, T387A, or T387D STAT2 were treated with IFN-β (100 IU/ml). Cells were harvested after 4 h and total RNAs were analyzed by real-time PCR. Values are the means ± SD from three independent experiments.C, D. U6A cells expressing wild-type or T387A STAT2 were treated with IFN-β for 0, 4, 8, or 24 h. Total RNAs were analyzed by using an Illumina HumanHT-12 v4 Expression BeadChip array. The average signal for each probe was used to determine expression levels. Genes with average signals below 25 and detection P values greater than 0.01 in the untreated or treated cells were excluded from the analysis. Inductions of less than 2-fold were not scored. The Venn diagram shows the numbers of genes induced by 1.2 fold or more in U6A cells expressing T387A STAT2, relative to untreated control cells. The numbers in the table are fold changes calculated from the ratios T387A STAT2 treated / untreated and wild-type STAT2 treated / untreated.


Figure 3A . HME cells expressing wild - type , T387A , or T387D STAT2 were seeded in 96 - well plates at 8000 cells / well . The cells were exposed to vesicular stomatitis virus ( VSV ) for 2 h , with or without pre - treatment with IFN - β ( 100 IU / ml ) . After 20 h , the amounts of the VSV - M and STAT2 proteins were analyzed by the Western method . B , C . HME cells expressing wild - type , T387A , or T387D STAT2 were seeded in 96 - well plates at 8000 cells / well . On the second day , the cells were treated with IFN - β ( 10 IU / ml ) for 2 h . The cells were then exposed to different multiplicities of infection of VSV for 2 h , followed by fresh media containing 10 IU / ml of IFN - β . The cells were analyzed by staining with crystal violet ( B ) after 48 h . B , C . HME cells expressing wild - type , T387A , or T387D STAT2 were seeded in 96 - well plates at 8000 cells / well . On the second day , the cells were treated with IFN - β ( 10 IU / ml ) for 2 h . The cells were then exposed to different multiplicities of infection of VSV for 2 h , followed by fresh media containing 10 IU / ml of IFN - β . The cells were analyzed by the MTT assay ( C ) after 48 h . D . U6A cells expressing wild - type , T387A , or T387D STAT2 were placed into 96 well plates ( 2000 cells / well ) and the cells were treated with IFN - β ( 1000 IU / ml ) for 96 h . Cell survival was analyzed by the MTT assay . Each experiment was carried out two independent times , with results similar to the representative examples that are shown .

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Figure 3A. HME cells expressing wild-type, T387A, or T387D STAT2 were seeded in 96-well plates at 8000 cells/well. The cells were exposed to vesicular stomatitis virus (VSV) for 2 h, with or without pre-treatment with IFN-β (100 IU/ml). After 20 h, the amounts of the VSV-M and STAT2 proteins were analyzed by the Western method.B, C. HME cells expressing wild-type, T387A, or T387D STAT2 were seeded in 96-well plates at 8000 cells/well. On the second day, the cells were treated with IFN-β (10 IU/ml) for 2 h. The cells were then exposed to different multiplicities of infection of VSV for 2 h, followed by fresh media containing 10 IU/ml of IFN-β. The cells were analyzed by staining with crystal violet (B) after 48 h.B, C. HME cells expressing wild-type, T387A, or T387D STAT2 were seeded in 96-well plates at 8000 cells/well. On the second day, the cells were treated with IFN-β (10 IU/ml) for 2 h. The cells were then exposed to different multiplicities of infection of VSV for 2 h, followed by fresh media containing 10 IU/ml of IFN-β. The cells were analyzed by the MTT assay (C) after 48 h.D. U6A cells expressing wild-type, T387A, or T387D STAT2 were placed into 96 well plates (2000 cells/well) and the cells were treated with IFN-β (1000 IU/ml) for 96 h. Cell survival was analyzed by the MTT assay. Each experiment was carried out two independent times, with results similar to the representative examples that are shown.


Figure 4A . An immuno - precipitation was performed using whole cell lysates from cells expressing Flag - tagged wild - type STAT2 , and samples were analyzed by the Western method . The numbers in the chart are the proportions of T387 phosphorylated STAT2 in each cell type , calculated in comparison with 293T cells . B . HME cells expressing wild - type STAT2 were treated with IFN - β ( 100 IU / ml ) . Whole cell lysates harvested at the times indicated were analyzed by the Western method ; C . HME cells expressing wild - type STAT2 were seeded in complete medium . On the second day , the cells were re - fed with medium lacking supplements . On the third day , the cells were treated with each supplement for 4 h , and total lysates were analyzed by the Western method . 1 . EGF , 2 . Insulin , 3 . Hydrocortisone , 4 . GA - 1000 , 5 . BPE . D . HME cells were pretreated with hydrocortisone ( 0 . 04mg / ml and 0 . 4mg / ml ) for 3 h and then treated with IFN - β ( 100 IU / ml ) . Cells were harvested after 4 h and total RNAs were analyzed by real - time PCR . Values are the means ± SD from three independent experiments . F . HCC827 and LnCAP cells expressing wild - type STAT2 were treated with CDK inhibitors ( 1 µM ) for 4 h and whole - cell lysates were analyzed by the Western method . 1 . Flavopiridol , 2 . AT7519 , 3 . Dinaciclib .

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Figure 4A. An immuno-precipitation was performed using whole cell lysates from cells expressing Flag-tagged wild-type STAT2, and samples were analyzed by the Western method. The numbers in the chart are the proportions of T387 phosphorylated STAT2 in each cell type, calculated in comparison with 293T cells.B. HME cells expressing wild-type STAT2 were treated with IFN-β (100 IU/ml). Whole cell lysates harvested at the times indicated were analyzed by the Western method;C. HME cells expressing wild-type STAT2 were seeded in complete medium. On the second day, the cells were re-fed with medium lacking supplements. On the third day, the cells were treated with each supplement for 4 h, and total lysates were analyzed by the Western method. 1. EGF, 2. Insulin, 3. Hydrocortisone, 4. GA-1000, 5. BPE.D. HME cells were pretreated with hydrocortisone (0.04mg/ml and 0.4mg/ml) for 3 h and then treated with IFN-β (100 IU/ml). Cells were harvested after 4 h and total RNAs were analyzed by real-time PCR. Values are the means ± SD from three independent experiments.F. HCC827 and LnCAP cells expressing wild-type STAT2 were treated with CDK inhibitors (1 µM) for 4 h and whole-cell lysates were analyzed by the Western method. 1. Flavopiridol, 2. AT7519, 3. Dinaciclib.


Figure 5A . U6A cells expressing wild - type or T387A STAT2 were treated with IFN - β ( 100 IU / ml ) for 30 min or were untreated . The cells were washed with PBS and the media was replaced with fresh media containing staurosporine ( 500 nM ) . Whole - cell lysates were harvested and analyzed by the Western method ; B . U6A cells expressing wild - type or T387A STAT2 were treated with IFN - β ( 100 IU / ml ) for 4 h or were untreated . Whole - cell lysates were used for immunoprecipitations of IRF9 and Flag - STAT2 . C . U2A cells expressing wild - type or T387A STAT2 were treated with IFN - β ( 100 IU / ml ) for 4 h or were untreated . Whole - cell lysates were used for immunoprecipitation of Flag - STAT2 . D . U3A cells expressing wild - type or T387A STAT2 were treated with IFN - β ( 100 IU / ml ) for 4 h or were untreated . Whole - cell lysates were used for immunoprecipitation of Flag - tagged STAT2 . E . EMSAs using an ISRE probe . Extracts from U6A cells expressing wild - type or T387A STAT2 treated with IFN - β for 1 h were analyzed . The position of ISGF3 is labeled .

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Figure 5A. U6A cells expressing wild-type or T387A STAT2 were treated with IFN-β (100 IU/ml) for 30 min or were untreated. The cells were washed with PBS and the media was replaced with fresh media containing staurosporine (500 nM). Whole-cell lysates were harvested and analyzed by the Western method;B. U6A cells expressing wild-type or T387A STAT2 were treated with IFN-β (100 IU/ml) for 4 h or were untreated. Whole-cell lysates were used for immunoprecipitations of IRF9 and Flag-STAT2.C. U2A cells expressing wild-type or T387A STAT2 were treated with IFN-β (100 IU/ml) for 4 h or were untreated. Whole-cell lysates were used for immunoprecipitation of Flag-STAT2.D. U3A cells expressing wild-type or T387A STAT2 were treated with IFN-β (100 IU/ml) for 4 h or were untreated. Whole-cell lysates were used for immunoprecipitation of Flag-tagged STAT2.E. EMSAs using an ISRE probe. Extracts from U6A cells expressing wild-type or T387A STAT2 treated with IFN-β for 1 h were analyzed. The position of ISGF3 is labeled.


Figure 1Activated GFAP + astrocytes in white matter MS lesions with inflammatory infiltrates and demyelination show an upregulation of OTUB1 ( arrows ) . Photomicrographs are obtained from case 9 ( A ) and case 5 ( B ) listed in Table EV1 . Data are representative for all 10 MS patients analyzed . Double immunofluorescence with rabbit anti - OTUB1 ( Cy3 ) and mouse anti - GFAP ( FITC ) ; original magnification × 400 ; scale bars correspond to 50 µm .

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Figure 1Activated GFAP+ astrocytes in white matter MS lesions with inflammatory infiltrates and demyelination show an upregulation of OTUB1 (arrows). Photomicrographs are obtained from case 9 (A) and case 5 (B) listed in Table EV1. Data are representative for all 10 MS patients analyzed. Double immunofluorescence with rabbit anti-OTUB1 (Cy3) and mouse anti-GFAP (FITC); original magnification ×400; scale bars correspond to 50 µm.


Figure 2WB analysis of OTUB1 expression in cultured primary astrocytes from OTUB1fl / fl and GFAP - Cre OTUB1fl / fl mice . Direct ex vivo WB analysis of OTUB1 expression in astrocytes isolated from adult OTUB1fl / fl and GFAP - Cre OTUB1fl / fl mice . Normal CNS architecture in OTUB1fl / fl and GFAP - Cre OTUB1fl / fl mice . Myelination is normal in the brain and spinal cord of an OTUB1fl / fl and a GFAP - Cre OTUB1fl / fl mouse . CV - LFB staining ; original magnification × 50 ; scale bars correspond to 100 µm . All photographs are representative of three mice per group . Absolute numbers of CD45 + cells in the spinal cord of OTUB1fl / fl and GFAP - Cre OTUB1fl / fl mice ( n = 5 for both groups , mean + SEM ) . Absolute numbers of different subpopulations of leukocytes infiltrating the spinal cord of OTUB1fl / fl and GFAP - Cre OTUB1fl / fl mice were analyzed by flow cytometry ( n = 5 for both groups , mean + SEM ) . In an OTUB1fl / fl and a GFAP - Cre OTUB1fl / fl mouse , OTUB1 is strongly expressed by NeuN + neurons in the brain and spinal cord . Double immunofluorescence with rabbit anti - OTUB1 ( Cy3 ) and mouse anti - NeuN ( FITC ) . Single NogoA + oligodendrocytes in the brain and spinal cord ( arrows ) of an OTUB1fl / fl and a GFAP - Cre OTUB1fl / fl mouse express OTUB1 . Double immunofluorescence with rabbit anti - OTUB1 ( Cy3 ) and mouse anti - NogoA ( Alexa Fluor 488 ) . GFAP + astrocytes are of normal morphology and distribution in the brain and spinal cord of a non - immunized OTUB1fl / fl mouse and a GFAP - Cre OTUB1fl / fl mouse . Note that GFAP + astrocytes of an OTUB1fl / fl mouse do not express OTUB1 . GFAP + astrocytes of a GFAP - Cre OTUB1fl / fl mouse are also OTUB1 - negative and represent a negative control for OTUB1 staining . Double immunofluorescence with rabbit anti - OTUB1 ( Cy3 ) and mouse anti - GFAP ( FITC ) .

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Figure 2WB analysis of OTUB1 expression in cultured primary astrocytes from OTUB1fl/fl and GFAP-Cre OTUB1fl/fl mice.Direct ex vivo WB analysis of OTUB1 expression in astrocytes isolated from adult OTUB1fl/fl and GFAP-Cre OTUB1fl/fl mice.Normal CNS architecture in OTUB1fl/fl and GFAP-Cre OTUB1fl/fl mice. Myelination is normal in the brain and spinal cord of an OTUB1fl/fl and a GFAP-Cre OTUB1fl/fl mouse. CV-LFB staining; original magnification ×50; scale bars correspond to 100 µm. All photographs are representative of three mice per group.Absolute numbers of CD45+ cells in the spinal cord of OTUB1fl/fl and GFAP-Cre OTUB1fl/fl mice (n = 5 for both groups, mean + SEM). Absolute numbers of different subpopulations of leukocytes infiltrating the spinal cord of OTUB1fl/fl and GFAP-Cre OTUB1fl/fl mice were analyzed by flow cytometry (n = 5 for both groups, mean + SEM).In an OTUB1fl/fl and a GFAP-Cre OTUB1fl/fl mouse, OTUB1 is strongly expressed by NeuN+ neurons in the brain and spinal cord. Double immunofluorescence with rabbit anti-OTUB1 (Cy3) and mouse anti-NeuN (FITC). Single NogoA+ oligodendrocytes in the brain and spinal cord (arrows) of an OTUB1fl/fl and a GFAP-Cre OTUB1fl/fl mouse express OTUB1. Double immunofluorescence with rabbit anti-OTUB1 (Cy3) and mouse anti-NogoA (Alexa Fluor 488). GFAP+ astrocytes are of normal morphology and distribution in the brain and spinal cord of a non-immunized OTUB1fl/fl mouse and a GFAP-Cre OTUB1fl/fl mouse. Note that GFAP+ astrocytes of an OTUB1fl/fl mouse do not express OTUB1. GFAP+ astrocytes of a GFAP-Cre OTUB1fl/fl mouse are also OTUB1-negative and represent a negative control for OTUB1 staining. Double immunofluorescence with rabbit anti-OTUB1 (Cy3) and mouse anti-GFAP (FITC).


Figure 3Activated GFAP + astrocytes in the spinal cord upregulate OTUB1 in EAE at maximal disease activity ( day 15 p . i . ) in an OTUB1fl / fl mouse ( arrowheads ) , but not in a GFAP - Cre OTUB1fl / fl mouse . Note the OTUB1 - expressing neuron in an OTUB1fl / fl mouse which is surrounded by GFAP - expressing processes of an activated astrocyte ( asterisk ) . At day 22 p . i . , astrocytes have downregulated OTUB1 expression while neurons express OTUB1 in an OTUB1fl / fl mouse ( asterisk ) . Astrocytes in a GFAP - Cre OTUB1fl / fl mouse are OTUB1 - negative while neurons express OTUB1 ( asterisk ) . At day 22 p . i . , demyelination in a GFAP - Cre OTUB1fl / fl mouse is much more severe and extended as compared to an OTUB1fl / fl mouse . Inflammation persists in the GFAP - Cre OTUB1fl / fl mouse , whereas it has resolved in an OTUB1fl / fl mouse . All photographs are representative of three mice per group ; original magnification × 400 ; scale bars correspond to 50 µm . ( A , C ) Double immunofluorescence with rabbit anti - OTUB1 ( Cy3 ) and mouse anti - GFAP ( FITC ) . ( B , D ) CV - LFB staining . EAE was induced in GFAP - Cre OTUB1fl / fl mice ( n = 29 ) and OTUB1fl / fl control littermates ( n = 29 ) by MOG35 - 55 peptide immunization with pertussis toxin . Graph represents data pooled from 4 experiments with 7 - 8 mice per group and shows the mean clinical scores + SEM . Statistical analysis performed using Mann - Whitney U test ; * p < 0 . 05 . EAE was induced in GFAP - Cre OTUB1fl / fl mice ( n = 12 ) and OTUB1fl / fl control littermates ( n = 12 ) by MOG35 - 55 peptide immunization without pertussis toxin . Graph represents the mean clinical scores + SEM . Statistical analysis performed using Mann - Whitney U test ; * p < 0 . 05 .

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Figure 3Activated GFAP+ astrocytes in the spinal cord upregulate OTUB1 in EAE at maximal disease activity (day 15 p.i.) in an OTUB1fl/fl mouse (arrowheads), but not in a GFAP-Cre OTUB1fl/fl mouse. Note the OTUB1-expressing neuron in an OTUB1fl/fl mouse which is surrounded by GFAP-expressing processes of an activated astrocyte (asterisk). At day 22 p.i., astrocytes have downregulated OTUB1 expression while neurons express OTUB1 in an OTUB1fl/fl mouse (asterisk). Astrocytes in a GFAP-Cre OTUB1fl/fl mouse are OTUB1-negative while neurons express OTUB1 (asterisk). At day 22 p.i., demyelination in a GFAP-Cre OTUB1fl/fl mouse is much more severe and extended as compared to an OTUB1fl/fl mouse. Inflammation persists in the GFAP-Cre OTUB1fl/fl mouse, whereas it has resolved in an OTUB1fl/fl mouse. All photographs are representative of three mice per group; original magnification ×400; scale bars correspond to 50 µm. (A, C) Double immunofluorescence with rabbit anti-OTUB1 (Cy3) and mouse anti-GFAP (FITC). (B, D) CV-LFB staining.EAE was induced in GFAP-Cre OTUB1fl/fl mice (n = 29) and OTUB1fl/fl control littermates (n = 29) by MOG35-55 peptide immunization with pertussis toxin. Graph represents data pooled from 4 experiments with 7-8 mice per group and shows the mean clinical scores + SEM. Statistical analysis performed using Mann-Whitney U test; * p < 0.05. EAE was induced in GFAP-Cre OTUB1fl/fl mice (n = 12) and OTUB1fl/fl control littermates (n = 12) by MOG35-55 peptide immunization without pertussis toxin. Graph represents the mean clinical scores + SEM. Statistical analysis performed using Mann-Whitney U test; * p < 0.05.