Figure 5A Western blot analysis of Hwa in HEK293T cells co - transfected with Hwa - Flag , and ZNRF3 - HA ( 5 μg ) or ZNRF3ΔRING ( 2 . 5 μg ) . Lysate was immunoblotted with Flag , HA , and GAPDH antibodies . Blots are representative of 3 independent experiments . B Densitometry analysis of Hwa bands from the western blot in ( A ) for corresponding lanes normalized to GAPDH . n = 3 independent experiments , mean ± SEM , ns p > 0 . 05 , * * p < 0 . 01 by unpaired t test . C Western blot analysis of Hwa in HEK293T cells co - transfected with Hwa - Flag and ZNRF3 - HA . Cells were treated with or without Baf - A1 ( 50 nM ) or MG132 ( 20 μM ) for about 12 hours . Lysates were immunoblotted with Flag , HA , and α - tub ( α - tubulin ) antibodies . Blots are representative of 3 independent experiments . D Densitometry analysis of Hwa bands from the western blot in ( C ) for corresponding lanes normalized to α - tubulin . n = 3 independent experiments , mean ± SEM , ns p > 0 . 05 , * * p < 0 . 01 , * * * * p < 0 . 0001 by unpaired t test . F Colocalization by immunofluorescence of Hwa ( green signals ) with lysosome ( LAMP1 , red signals ) in Hela cells . Cells were co - transfected with Hwa - Flag , and ZNRF3 - HA or ZNRF3ΔRING - HA . Magnification , 100 × ; scale bars : 10 μm . I Surface biotinylation and streptavidin pulldown of Hwa - Myc - Flag . HEK293T cells were co - transfected with Hwa - MF ( Hwa - Myc - Flag ) , and ZNRF3 - Myc or ZNRF3ΔRING - Myc . Lysate was immunoblotted with Myc , Flag , and GAPDH antibodies . Lysates immunoprecipitated with anti - Flag ( 2nd elution ) were immunoblotted with Flag and HA antibodies .

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 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 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 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

Figure 5A Western blot analysis of Hwa in HEK293T cells co-transfected with Hwa-Flag, and ZNRF3-HA (5 μg) or ZNRF3ΔRING (2.5 μg). Lysate was immunoblotted with Flag, HA, and GAPDH antibodies. Blots are representative of 3 independent experiments. B Densitometry analysis of Hwa bands from the western blot in (A) for corresponding lanes normalized to GAPDH. n = 3 independent experiments, mean ± SEM, ns p > 0.05, **p < 0.01 by unpaired t test. C Western blot analysis of Hwa in HEK293T cells co-transfected with Hwa-Flag and ZNRF3-HA. Cells were treated with or without Baf-A1 (50 nM) or MG132 (20 μM) for about 12 hours. Lysates were immunoblotted with Flag, HA, and α-tub (α-tubulin) antibodies. Blots are representative of 3 independent experiments. D Densitometry analysis of Hwa bands from the western blot in (C) for corresponding lanes normalized to α-tubulin. n = 3 independent experiments, mean ± SEM, ns p > 0.05, **p < 0.01, ****p < 0.0001 by unpaired t test. F Colocalization by immunofluorescence of Hwa (green signals) with lysosome (LAMP1, red signals) in Hela cells. Cells were co-transfected with Hwa-Flag, and ZNRF3-HA or ZNRF3ΔRING-HA. Magnification, 100 ×; scale bars: 10 μm.I Surface biotinylation and streptavidin pulldown of Hwa-Myc-Flag. HEK293T cells were co-transfected with Hwa-MF (Hwa-Myc-Flag), and ZNRF3-Myc or ZNRF3ΔRING-Myc. Lysate was immunoblotted with Myc, Flag, and GAPDH antibodies. Lysates immunoprecipitated with anti-Flag (2nd elution) were immunoblotted with Flag and HA antibodies.


Figure 6C Whole mount in situ hybridization of Spemann organizer markers chrd and gsc in gastrula embryos . Embryos were obtained as descripted in ( B ) . Scale bars : 1mm . E Western blot analysis of Hwa or Hwa10KR in embryos at blastula stage . 1 - cell stage embryos were co - injected with hwa ( 100 pg ) or hwa10KR ( 100 pg ) and znrf3 ( 1 ng ) or znrf3ΔRING ( 100 pg ) as indicated . Lysates were immunoblotted with HA , Flag , and α - tub ( α - tubulin ) .

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Figure 6C Whole mount in situ hybridization of Spemann organizer markers chrd and gsc in gastrula embryos. Embryos were obtained as descripted in (B). Scale bars: 1mm.E Western blot analysis of Hwa or Hwa10KR in embryos at blastula stage. 1-cell stage embryos were co-injected with hwa (100 pg) or hwa10KR (100 pg) and znrf3 (1 ng) or znrf3ΔRING (100 pg) as indicated. Lysates were immunoblotted with HA, Flag, and α-tub (α-tubulin).


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 ) .

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 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

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 .

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 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

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 .

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

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 .

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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.


Figure 1 ( E ) Peptide competition assay with a WT LxxIxE peptide or a mutated variant that does not bind B56 ( LxxAxA ) . Binding of YFP - B56α to indicated proteins was determined and quantified by LiCor . ( G ) YFP - B56α pull down from cells stably expressing the indicated LxxIxE binding pocket variants of B56α and subsequent immunoblotting of indicated proteins . Representative of 4 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 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

Figure 1(E) Peptide competition assay with a WT LxxIxE peptide or a mutated variant that does not bind B56 (LxxAxA). Binding of YFP-B56α to indicated proteins was determined and quantified by LiCor.(G) YFP-B56α pull down from cells stably expressing the indicated LxxIxE binding pocket variants of B56α and subsequent immunoblotting of indicated proteins. Representative of 4 independent experiments.


Figure 2 ( B ) IP of YFP - B56α from cells stably expressing the B56α WT , R222E , and 5A followed by immunoblotting of indicated proteins . Representative blots are shown ( top ) . hSgo1 signals were normalized to YFP and plotted ( bottom ) . Error bars represent SD ( n = 4 ) . ( C ) Reciprocal IP of ( B ) . YFP - hSgo1 expression construct was transfected into cells stably expressing FLAG - B56α WT , R222E and 5A , followed by YFP IP and immunoblotting of indicated proteins . Representative blots are shown ( top ) . B56α signals were normalized to YFP and plotted ( bottom ) . Error bars represent SD ( n = 4 ) . ( D ) Representative images of chromosome spreads from the indicated conditions . Scale bar , 5μm . ( E ) Quantification of ( D ) . The distance between the two peak intensities of CREST was measured for 5 kinetochore pairs and averaged for a single cell and plotted . The data are from 4 independent experiments and the mean and SD are indicated . ( F ) hSgo1 RNAi and rescue with the indicated B56α variants fused to YFP and the Cenp B centromere - targeting domain ( CB ) . Representative images of chromosome spreads are shown . CB targets all the rescue constructs ( green ) to the centromere . Scale bar , 5μm . ( G ) Quantification of ( F ) . The distance between the two peak intensities of YFP was measured for 5 kinetochore pairs and averaged for a single cell and plotted . The data are from 3 independent experiments and the mean and SD are indicated .

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 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 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 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

Figure 2(B) IP of YFP-B56α from cells stably expressing the B56α WT, R222E, and 5A followed by immunoblotting of indicated proteins. Representative blots are shown (top). hSgo1 signals were normalized to YFP and plotted (bottom). Error bars represent SD (n=4).(C) Reciprocal IP of (B). YFP-hSgo1 expression construct was transfected into cells stably expressing FLAG-B56α WT, R222E and 5A, followed by YFP IP and immunoblotting of indicated proteins. Representative blots are shown (top). B56α signals were normalized to YFP and plotted (bottom). Error bars represent SD (n=4).(D) Representative images of chromosome spreads from the indicated conditions. Scale bar, 5μm. (E) Quantification of (D). The distance between the two peak intensities of CREST was measured for 5 kinetochore pairs and averaged for a single cell and plotted. The data are from 4 independent experiments and the mean and SD are indicated.(F) hSgo1 RNAi and rescue with the indicated B56α variants fused to YFP and the Cenp B centromere-targeting domain (CB). Representative images of chromosome spreads are shown. CB targets all the rescue constructs (green) to the centromere. Scale bar, 5μm. (G) Quantification of (F). The distance between the two peak intensities of YFP was measured for 5 kinetochore pairs and averaged for a single cell and plotted. The data are from 3 independent experiments and the mean and SD are indicated.


Figure 3 ( D ) Localization of B56α in cells depleted of hSgo1 and expressing the indicated YFP - hSgo1 variants . Representative immunofluorescent images are shown . Scale bar , 5μm . ( E ) B56α signal intensity was quantified . B56α signal from each cell was determined from 5 kinetochore pairs and normalized to YFP - hSgo1 signal . Each circle represents an individual cell , and the average and SD are indicated . ( F ) Mitotic U2OS LacO Haspin CM cells expressing hSgo1 - LacI - GFP variants or LacI - GFP ( control ) were stained for PP2A - C . Scale bar , 5μm . ( G ) PP2A - C signal intensity was quantified , normalized to GFP , and then plotted . Each circle represents an individual cell , and the mean fluorescent intensity is indicated . Representative of at least 3 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 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

Figure 3(D) Localization of B56α in cells depleted of hSgo1 and expressing the indicated YFP-hSgo1 variants. Representative immunofluorescent images are shown. Scale bar, 5μm. (E) B56α signal intensity was quantified. B56α signal from each cell was determined from 5 kinetochore pairs and normalized to YFP-hSgo1 signal. Each circle represents an individual cell, and the average and SD are indicated.(F) Mitotic U2OS LacO Haspin CM cells expressing hSgo1-LacI-GFP variants or LacI-GFP (control) were stained for PP2A-C. Scale bar, 5μm. (G) PP2A-C signal intensity was quantified, normalized to GFP, and then plotted. Each circle represents an individual cell, and the mean fluorescent intensity is indicated. Representative of at least 3 independent experiments.


Figure 4 ( A ) Representative images of chromosome spreads from hSgo1 RNAi treated cells stably expressing the indicated YFP - hSgo1 variants . All the YFP - hSgo1 rescue constructs ( green ) localize to the centromeres ( CREST , red ) . Scale bar , 5μm . ( B ) The distance between the two peak intensities of CREST was measured for 5 kinetochore pairs from the chromosome spreads in ( A ) and averaged for a single cell . The data are from 3 independent experiments and the mean and SD are indicated . ( C ) IP of YFP - hSgo1 from cells stably expressing hSgo1 WT , 3A , and 4A followed by immunoblotting of cohesin components , Smc1 and Smc3 . Representative blots are shown ( top ) . Signals were quantification by LiCor , normalized to YFP and plotted ( bottom ) . Error bars represent SD ( n = 4 for Smc1 and n = 3 for Smc3 ) . ( F ) hSgo1 RNAi and rescue with the indicated hSgo1 RNAi - resistant constructs was performed . Representative still images captured during the live cell imaging showing DIC and YFP - hSgo1 WT , 3A , and 4A localization during mitosis . Time ( min ) from nuclear envelop breakdown ( NEBD ) is indicated . Scale bar , 15μm .

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 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

Figure 4(A) Representative images of chromosome spreads from hSgo1 RNAi treated cells stably expressing the indicated YFP-hSgo1 variants. All the YFP-hSgo1 rescue constructs (green) localize to the centromeres (CREST, red). Scale bar, 5μm. (B) The distance between the two peak intensities of CREST was measured for 5 kinetochore pairs from the chromosome spreads in (A) and averaged for a single cell. The data are from 3 independent experiments and the mean and SD are indicated.(C) IP of YFP-hSgo1 from cells stably expressing hSgo1 WT, 3A, and 4A followed by immunoblotting of cohesin components, Smc1 and Smc3. Representative blots are shown (top). Signals were quantification by LiCor, normalized to YFP and plotted (bottom). Error bars represent SD (n=4 for Smc1 and n=3 for Smc3).(F) hSgo1 RNAi and rescue with the indicated hSgo1 RNAi-resistant constructs was performed. Representative still images captured during the live cell imaging showing DIC and YFP-hSgo1 WT, 3A, and 4A localization during mitosis. Time (min) from nuclear envelop breakdown (NEBD) is indicated. Scale bar, 15μm.


Figure 1 . Mutations in epg - 7 cause defective degradation of SQST - 1 aggregates . ( A and B ) In wild - type embryos , SQST - 1 : : GFP is weakly expressed and diffusely localized in the cytoplasm . ( A ) DIC image of the embryo shown in B . ( C ) Elevated expression level and accumulation of many SQST - 1 : : GFP aggregates in epg - 7 mutant embryos . ( D ) Compared with wild type and epg - 2 mutants , levels of SQST - 1 are dramatically elevated in atg - 3 and epg - 7 mutant embryos in an immunoblotting assay . ( E - G ) Compared with wild - type embryos ( E and F ) , endogenous SQST - 1 shows dramatically elevated levels and accumulates into a large number of aggregates in epg - 7 mutant embryos ( G ) . ∼ 200 - cell stage embryos are shown in E - G . ( E ) DAPI image of the embryo shown in F . ( H ) Number of SQST - 1 aggregates per focal plane in wild type , atg - 3 mutants , and epg - 7 mutants . ( I - L ) At the ∼ 100 - cell stage , far fewer SQST - 1 aggregates are formed in epg - 7 mutants than in atg - 3 mutants . ( I and K ) DAPI images of the embryos shown in J and L , respectively . ( M ) SQST - 1 directly interacts with ZK1053 . 4 in a GST pull - down assay . ( N ) Compared with wild - type animals , levels of ZK1053 . 4 : : GFP are dramatically elevated in atg - 3 and epg - 7 mutants . ( O - R ) ZK1053 . 4 : : GFP is weakly expressed in wild - type embryos ( O - P ) and sqst - 1 mutants ( R ) , but forms a large number of aggregates in epg - 7 mutants ( Q ) . ( O ) DIC image of the embryo shown in P . ( S ) Survival curve of L1 larvae under food depletion conditions in wild type , epg - 1 mutants , and epg - 7 mutants . This experiment was completed once ( wild type : n = 2 , 964 ; epg - 7 : n = 602 ; epg - 1 : n = 215 ) . Statistical analysis of L1 survival shows that epg - 7 mutants exhibit significant difference to wild type and also to epg - 1 mutants ( log - rank test , P = 0 . 000 ) . ( T ) Loss of function of epg - 7 does not ameliorate the degeneration of mec - 4 : : gfp - labeled touch neurons caused by mec - 4 ( u231 ) . ( U ) Survival curves of wild type and various mutants . sqst - 1 ; epg - 7 mutants have a shortened mean and maximum life span . ( P = 0 . 000 for epg - 7 or wild type vs . sqst - 1 ; epg - 7 in all three repeats ; log - rank test ) . ( V ) Survival of hydrogen peroxide - treated animals . ( In all three repeats , P = 0 . 000 for epg - 7 vs . sqst - 1 ; epg - 7 ; P > 0 . 2 for wild type vs . sqst - 1 ; epg - 7 ; log - rank test ) . ( W ) Survival of animals at the indicated time after heat shock treatment . ( P < 0 . 05 for wild type vs . epg - 7 or epg - 7 vs . sqst - 1 ; epg - 7 at 5 , 6 , and 7 h ) . ( X ) Dauer formation of daf - 2 ( e1370 ) mutants in different backgrounds .

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

Figure 1.Mutations in epg-7 cause defective degradation of SQST-1 aggregates. (A and B) In wild-type embryos, SQST-1::GFP is weakly expressed and diffusely localized in the cytoplasm. (A) DIC image of the embryo shown in B. (C) Elevated expression level and accumulation of many SQST-1::GFP aggregates in epg-7 mutant embryos.(D) Compared with wild type and epg-2 mutants, levels of SQST-1 are dramatically elevated in atg-3 and epg-7 mutant embryos in an immunoblotting assay.(E-G) Compared with wild-type embryos (E and F), endogenous SQST-1 shows dramatically elevated levels and accumulates into a large number of aggregates in epg-7 mutant embryos (G). ∼200-cell stage embryos are shown in E-G. (E) DAPI image of the embryo shown in F.(H) Number of SQST-1 aggregates per focal plane in wild type, atg-3 mutants, and epg-7 mutants.(I-L) At the ∼100-cell stage, far fewer SQST-1 aggregates are formed in epg-7 mutants than in atg-3 mutants. (I and K) DAPI images of the embryos shown in J and L, respectively.(M) SQST-1 directly interacts with ZK1053.4 in a GST pull-down assay.(N) Compared with wild-type animals, levels of ZK1053.4::GFP are dramatically elevated in atg-3 and epg-7 mutants.(O-R) ZK1053.4::GFP is weakly expressed in wild-type embryos (O-P) and sqst-1 mutants (R), but forms a large number of aggregates in epg-7 mutants (Q). (O) DIC image of the embryo shown in P.(S) Survival curve of L1 larvae under food depletion conditions in wild type, epg-1 mutants, and epg-7 mutants. This experiment was completed once (wild type: n = 2,964; epg-7: n = 602; epg-1: n = 215). Statistical analysis of L1 survival shows that epg-7 mutants exhibit significant difference to wild type and also to epg-1 mutants (log-rank test, P = 0.000).(T) Loss of function of epg-7 does not ameliorate the degeneration of mec-4::gfp-labeled touch neurons caused by mec-4(u231).(U) Survival curves of wild type and various mutants. sqst-1;epg-7 mutants have a shortened mean and maximum life span. (P = 0.000 for epg-7 or wild type vs. sqst-1;epg-7 in all three repeats; log-rank test).(V) Survival of hydrogen peroxide-treated animals. (In all three repeats, P = 0.000 for epg-7 vs. sqst-1;epg-7; P > 0.2 for wild type vs. sqst-1;epg-7; log-rank test).(W) Survival of animals at the indicated time after heat shock treatment. (P < 0.05 for wild type vs. epg-7 or epg-7 vs. sqst-1;epg-7 at 5, 6, and 7 h).(X) Dauer formation of daf-2(e1370) mutants in different backgrounds.


Figure 2 . ( D ) A large number of SQST - 1 : : GFP aggregates accumulate in epg - 7 mutant embryos carrying an epg - 7 ( del Atg11 ) transgene . ( E and F ) No SQST - 1 aggregates are detected in epg - 7 mutants carrying an epg - 7 : : gfp reporter . ( E ) DAPI image of the embryo shown in F . ( G ) EPG - 7 : : GFP is weakly expressed and diffusely localized in the cytoplasm in wild - type embryos . ( H - I ) EPG - 7 : : GFP is expressed in pharyngeal muscles ( H , arrow ) and cells in the tail region ( I ) at larval stages . ( J ) Compared with wild - type animals , levels of EPG - 7 : : GFP are dramatically elevated in epg - 4 mutants , but remain unchanged in sqst - 1 mutants . ( K ) The expression level of EPG - 7 : : GFP is dramatically elevated and many EPG - 7 : : GFP aggregates are formed in lgg - 1 mutant embryos . ( L and M ) No EPG - 7 aggregates , detected by anti - EPG - 7 antibody , are formed in wild - type embryos . ( L ) DAPI image of the embryo shown in M . ( N ) Levels of EPG - 7 are dramatically elevated and a large number of EPG - 7 aggregates are formed in atg - 3 mutants . ( O ) No EPG - 7 aggregates are formed in sqst - 1 mutant embryos .

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 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 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 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 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 2.(D) A large number of SQST-1::GFP aggregates accumulate in epg-7 mutant embryos carrying an epg-7(del Atg11) transgene.(E and F) No SQST-1 aggregates are detected in epg-7 mutants carrying an epg-7::gfp reporter. (E) DAPI image of the embryo shown in F.(G) EPG-7::GFP is weakly expressed and diffusely localized in the cytoplasm in wild-type embryos.(H-I) EPG-7::GFP is expressed in pharyngeal muscles (H, arrow) and cells in the tail region (I) at larval stages.(J) Compared with wild-type animals, levels of EPG-7::GFP are dramatically elevated in epg-4 mutants, but remain unchanged in sqst-1 mutants.(K) The expression level of EPG-7::GFP is dramatically elevated and many EPG-7::GFP aggregates are formed in lgg-1 mutant embryos.(L and M) No EPG-7 aggregates, detected by anti-EPG-7 antibody, are formed in wild-type embryos. (L) DAPI image of the embryo shown in M. (N) Levels of EPG-7 are dramatically elevated and a large number of EPG-7 aggregates are formed in atg-3 mutants. (O) No EPG-7 aggregates are formed in sqst-1 mutant embryos.


Figure 3 . ( A ) EPG - 7 colocalizes with SQST - 1 aggregates in epg - 8 mutant embryos . Colocalization was defined as two aggregates showing > 70 % overlap in their fluorescence signals . Insets show a magnified view . ( B ) Percentage of EPG - 7 aggregates colocalized with SQST - 1 in indicated autophagy mutants . ( C ) A coimmunoprecipitation assay reveals that EPG - 7 associates with SQST - 1 . ( D ) EPG - 7 interacts with SQST - 1 in a pull - down assay . ( E ) EPG - 7 self - associates in a pull - down assay . ( F ) SDS - PAGE and blue - native PAGE of purified EPG - 7 ( 520 - 714 ) proteins . Lane M shows molecular weight marker . Lanes 1 , 2 , 3 , and 4 contain 10 , 5 , 2 . 5 , and 1 . 25 µg of proteins , respectively . ( G ) An EPG - 7 fragment containing amino acids 531 - 631 self - interacts . ( H and I ) SQST - 1 aggregates accumulate in epg - 7 mutant embryos carrying an epg - 7 ( del 531 - 631 ) : : gfp transgene . ( H ) DAPI image of the embryo shown in I . ( J and K ) EPG - 7 ( del 531 - 631 ) : : GFP is weakly expressed and diffusely localized in epg - 7 ( tm2508 ) embryos . Its expression is not elevated in lgg - 1 ( RNAi ) animals . H - K show embryos carrying the same transgene . Images in J and K were taken using the same exposure time . ( L and M ) SQST - 1 self - interacts ( L ) and the self - interaction is mediated by a fragment containing amino acids 26 - 181 ( M ) . ( N ) Levels of SQST - 1 ( del 26 - 181 ) : : GFP are dramatically elevated in epg - 7 and atg - 3 mutants . ( O and P ) SQST - 1 ( del 26 - 181 ) : : GFP is weakly expressed and diffusely localized in sqst - 1 ( ok2892 ) mutant embryos . ( O ) DIC image of the embryo shown in P . ( Q and R ) SQST - 1 ( del 26 - 181 ) : : GFP is expressed at dramatically elevated levels and accumulates into a large number of aggregates in lgg - 1 ( Q ) and epg - 7 ( R ) mutants .

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 B-PANEL_START 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 B-PANEL_START 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 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 B-PANEL_START 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 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 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 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 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

Figure 3.(A) EPG-7 colocalizes with SQST-1 aggregates in epg-8 mutant embryos. Colocalization was defined as two aggregates showing >70% overlap in their fluorescence signals. Insets show a magnified view.(B) Percentage of EPG-7 aggregates colocalized with SQST-1 in indicated autophagy mutants.(C) A coimmunoprecipitation assay reveals that EPG-7 associates with SQST-1.(D) EPG-7 interacts with SQST-1 in a pull-down assay.(E) EPG-7 self-associates in a pull-down assay.(F) SDS-PAGE and blue-native PAGE of purified EPG-7(520-714) proteins. Lane M shows molecular weight marker. Lanes 1, 2, 3, and 4 contain 10, 5, 2.5, and 1.25 µg of proteins, respectively.(G) An EPG-7 fragment containing amino acids 531-631 self-interacts.(H and I) SQST-1 aggregates accumulate in epg-7 mutant embryos carrying an epg-7(del 531-631)::gfp transgene. (H) DAPI image of the embryo shown in I.(J and K) EPG-7(del 531-631)::GFP is weakly expressed and diffusely localized in epg-7(tm2508) embryos. Its expression is not elevated in lgg-1(RNAi) animals. H-K show embryos carrying the same transgene. Images in J and K were taken using the same exposure time.(L and M) SQST-1 self-interacts (L) and the self-interaction is mediated by a fragment containing amino acids 26-181 (M).(N) Levels of SQST-1(del 26-181)::GFP are dramatically elevated in epg-7 and atg-3 mutants.(O and P) SQST-1(del 26-181)::GFP is weakly expressed and diffusely localized in sqst-1(ok2892) mutant embryos. (O) DIC image of the embryo shown in P.(Q and R) SQST-1(del 26-181)::GFP is expressed at dramatically elevated levels and accumulates into a large number of aggregates in lgg-1 (Q) and epg-7 (R) mutants.


Figure 4 . ( A - D ) Expression of LGG - 1 in wild - type embryos at the ∼ 100 - cell ( A and B ) , the ∼ 200 - cell ( C ) , and the fourfold ( D ) stages . ( A ) DAPI image of the embryo shown in B . ( E ) Loss of function of epg - 7 does not increase levels of LGG - 1 and also does not suppress accumulation of LGG - 1 in epg - 3 mutant embryos . ( F - H ) Expression of LGG - 1 in epg - 7 mutant embryos before the ∼ 100 - cell stage ( F ) , at the ∼ 200 - cell stage ( G ) , and at the fourfold stage ( H ) . Images in B - D and F - H were taken using the same exposure time . ( I ) SQST - 1 aggregates are separable from LGG - 1 puncta in epg - 7 mutant embryos . Insets show a magnified view . ( J ) In epg - 6 mutant embryos , LGG - 1 puncta and SQST - 1 aggregates are enlarged and colocalized . ( K ) LGG - 1 and SQST - 1 aggregates are smaller in size and largely separable in epg - 6 ; epg - 7 double mutants . ( I - K ) DAPI images of the embryos shown in the same row . ( L - O ) Enlarged LGG - 1 and SQST - 1 aggregates are colocalized in epg - 4 mutants ( L and M ) . Simultaneously depleting epg - 7 reduces the size of LGG - 1 puncta and SQST - 1 aggregates , which become largely separable ( N and O ) . ( L and N ) DAPI images of the embryos shown in M and O , respectively . ( P ) In atg - 9 mutant embryos , LGG - 1 puncta largely colocalize with SQST - 1 aggregates at the comma stage . Notably , there are more SQST - 1 aggregates than LGG - 1 puncta . ( Q ) SQST - 1 aggregates are separable from LGG - 1 puncta in atg - 9 ; epg - 7 mutants at the comma stage . ( R ) Percentage of LGG - 1 puncta colocalized with SQST - 1 aggregates in indicated autophagy mutants . Comma - stage embryos were examined for atg - 9 and atg - 9 ; epg - 7 mutants . ∼ 200 - cell stage embryos were examined for other autophagy mutants .

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 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 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 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 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 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

Figure 4.(A-D) Expression of LGG-1 in wild-type embryos at the ∼100-cell (A and B), the ∼200-cell (C), and the fourfold (D) stages. (A) DAPI image of the embryo shown in B.(E) Loss of function of epg-7 does not increase levels of LGG-1 and also does not suppress accumulation of LGG-1 in epg-3 mutant embryos.(F-H) Expression of LGG-1 in epg-7 mutant embryos before the ∼100-cell stage (F), at the ∼200-cell stage (G), and at the fourfold stage (H). Images in B-D and F-H were taken using the same exposure time.(I) SQST-1 aggregates are separable from LGG-1 puncta in epg-7 mutant embryos. Insets show a magnified view. (J) In epg-6 mutant embryos, LGG-1 puncta and SQST-1 aggregates are enlarged and colocalized. (K) LGG-1 and SQST-1 aggregates are smaller in size and largely separable in epg-6;epg-7 double mutants. (I-K) DAPI images of the embryos shown in the same row.(L-O) Enlarged LGG-1 and SQST-1 aggregates are colocalized in epg-4 mutants (L and M). Simultaneously depleting epg-7 reduces the size of LGG-1 puncta and SQST-1 aggregates, which become largely separable (N and O). (L and N) DAPI images of the embryos shown in M and O, respectively.(P) In atg-9 mutant embryos, LGG-1 puncta largely colocalize with SQST-1 aggregates at the comma stage. Notably, there are more SQST-1 aggregates than LGG-1 puncta. (Q) SQST-1 aggregates are separable from LGG-1 puncta in atg-9;epg-7 mutants at the comma stage.(R) Percentage of LGG-1 puncta colocalized with SQST-1 aggregates in indicated autophagy mutants. Comma-stage embryos were examined for atg-9 and atg-9;epg-7 mutants. ∼200-cell stage embryos were examined for other autophagy mutants.


Figure 5 . ( A ) EPG - 7 coimmunoprecipitates with ATG - 9 : : HA . ( B ) EPG - 7 directly interacts with ATG - 9 in a GST pull - down assay . ( C and D ) Coimmunoprecipitation reveals that EPG - 7 associates with LGG - 3 ( C ) ( C and D ) Coimmunoprecipitation reveals that EPG - 7 associates with LGG - 1 ( D ) . ( E ) EPG - 7 directly interacts with LGG - 3 . ( F ) EPG - 7 fragments containing amino acids 852 - 1003 or 1004 - 1142 , but not 762 - 851 , interact with LGG - 1 . ( G ) Fragments of EPG - 7 containing amino acids 762 - 851 or 852 - 1003 directly interact with ATG - 18 . ( H ) Fragments of EPG - 7 containing amino acids 1 - 704 or 1143 - 1330 directly interact with UNC - 51 . ( I ) SQST - 1 associates with LGG - 1 in vivo . Embryonic extracts from epg - 7 mutants show that interaction of SQST - 1 with LGG - 1 is EPG - 7 independent . ( J ) Fragments of SQST - 1 containing amino acids 351 - 499 and 604 - 693 , but not 500 - 603 , interact with LGG - 1 . ( K ) Fragments of SQST - 1 - containing amino acids 418 - 499 and 604 - 630 bind to LGG - 1 . Mutating the putative LIR in the 604 - 630 fragment ( 605YPDL608 to APDA ) does not affect the binding . ( L and M ) SQST - 1 ( del LGG - 1 - 1 + 2 ) : : GFP , in which the two LGG - 1 - binding fragments ( amino acids 418 - 499 and 604 - 630 ) are deleted , is weakly expressed and diffusely localized in the cytoplasm in sqst - 1 ( ok2892 ) mutants . ( N and O ) Loss of function of lgg - 1 dramatically elevates the expression level of SQST - 1 ( del LGG - 1 - 1 + 2 ) : : GFP , which forms a large number of aggregates . ( L and N ) DIC images of the embryos shown in M and O , respectively ( P ) SQST - 1 ( del LGG - 1 - 1 + 2 ) : : GFP aggregates colocalize with EPG - 7 in lgg - 1 ; sqst - 1 mutants . Insets show a magnified view .

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

Figure 5.(A) EPG-7 coimmunoprecipitates with ATG-9::HA.(B) EPG-7 directly interacts with ATG-9 in a GST pull-down assay.(C and D) Coimmunoprecipitation reveals that EPG-7 associates with LGG-3 (C)(C and D) Coimmunoprecipitation reveals that EPG-7 associates with LGG-1 (D).(E) EPG-7 directly interacts with LGG-3.(F) EPG-7 fragments containing amino acids 852-1003 or 1004-1142, but not 762-851, interact with LGG-1.(G) Fragments of EPG-7 containing amino acids 762-851 or 852-1003 directly interact with ATG-18.(H) Fragments of EPG-7 containing amino acids 1-704 or 1143-1330 directly interact with UNC-51.(I) SQST-1 associates with LGG-1 in vivo. Embryonic extracts from epg-7 mutants show that interaction of SQST-1 with LGG-1 is EPG-7 independent.(J) Fragments of SQST-1 containing amino acids 351-499 and 604-693, but not 500-603, interact with LGG-1.(K) Fragments of SQST-1-containing amino acids 418-499 and 604-630 bind to LGG-1. Mutating the putative LIR in the 604-630 fragment (605YPDL608 to APDA) does not affect the binding.(L and M) SQST-1(del LGG-1-1+2)::GFP, in which the two LGG-1-binding fragments (amino acids 418-499 and 604-630) are deleted, is weakly expressed and diffusely localized in the cytoplasm in sqst-1(ok2892) mutants. (N and O) Loss of function of lgg-1 dramatically elevates the expression level of SQST-1(del LGG-1-1+2)::GFP, which forms a large number of aggregates. (L and N) DIC images of the embryos shown in M and O, respectively(P) SQST-1(del LGG-1-1+2)::GFP aggregates colocalize with EPG-7 in lgg-1;sqst-1 mutants. Insets show a magnified view.


Figure 6 . Formation of ATG - 9 puncta in various autophagy mutants . ( A and B ) In wild - type embryos , ATG - 9 : : GFP is diffusely localized in the cytoplasm . ( A ) DIC image of the embryo shown in B . Insets show a magnified view . ( C ) ATG - 9 : : GFP forms a large number of small , intense puncta in epg - 1 mutants . ( D and E ) Accumulation of ATG - 9 : : GFP puncta in unc - 51 ( D ) and epg - 1 ( E ) mutants is independent of lgg - 1 . ( F ) The ATG - 9 : : GFP puncta largely disappear in epg - 1 ; epg - 7 mutants . ( G ) ATG - 9 : : GFP accumulates into large punctate structures in epg - 6 mutants . ( H ) Loss of function of lgg - 1 suppresses the accumulation of ATG - 9 : : GFP puncta in epg - 6 mutants . ( I ) Loss of function of epg - 7 has no effect on the formation of ATG - 9 : : GFP puncta in epg - 6 mutants . The ATG - 9 : : GFP puncta are smaller in epg - 6 ; epg - 7 mutants than those in epg - 6 single mutants . ( J - L ) ATG - 9 : : GFP accumulates into large punctate structures in epg - 4 mutants ( J ) and the accumulation is suppressed by simultaneously depleting the activity of lgg - 1 ( K ) or epg - 7 ( L ) . ( M and N ) In epg - 8 mutants , ATG - 9 accumulates into aggregates ( M ) that are suppressed by loss of lgg - 1 activity ( N ) . ( O ) epg - 7 mutants exhibit the same distribution pattern of ATG - 9 : : GFP as wild - type embryos . ( P ) Percentage of ATG - 9 : : GFP puncta colocalized with EPG - 7 and SQST - 1 aggregates in indicated autophagy mutants . ( Q - T ) The ATG - 9 : : GFP puncta in epg - 6 ( Q ) , epg - 8 ( R ) , unc - 51 ( S ) , and atg - 18 ( T ) mutants colocalize with EPG - 7 aggregates . ( U and V ) ATG - 9 : : GFP puncta in epg - 6 ( U ) and epg - 8 ( V ) mutants colocalize with SQST - 1 aggregates . ( W and X ) In unc - 51 ( W ) and atg - 18 ( X ) mutants , ATG - 9 : : GFP partially colocalizes with SQST - 1 aggregates .

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

Figure 6.Formation of ATG-9 puncta in various autophagy mutants. (A and B) In wild-type embryos, ATG-9::GFP is diffusely localized in the cytoplasm. (A) DIC image of the embryo shown in B. Insets show a magnified view. (C) ATG-9::GFP forms a large number of small, intense puncta in epg-1 mutants. (D and E) Accumulation of ATG-9::GFP puncta in unc-51(D) and epg-1 (E) mutants is independent of lgg-1. (F) The ATG-9::GFP puncta largely disappear in epg-1;epg-7 mutants. (G) ATG-9::GFP accumulates into large punctate structures in epg-6 mutants. (H) Loss of function of lgg-1 suppresses the accumulation of ATG-9::GFP puncta in epg-6 mutants. (I) Loss of function of epg-7 has no effect on the formation of ATG-9::GFP puncta in epg-6 mutants. The ATG-9::GFP puncta are smaller in epg-6;epg-7 mutants than those in epg-6 single mutants. (J-L) ATG-9::GFP accumulates into large punctate structures in epg-4 mutants (J) and the accumulation is suppressed by simultaneously depleting the activity of lgg-1 (K) or epg-7 (L). (M and N) In epg-8 mutants, ATG-9 accumulates into aggregates (M) that are suppressed by loss of lgg-1 activity (N). (O) epg-7 mutants exhibit the same distribution pattern of ATG-9::GFP as wild-type embryos. (P) Percentage of ATG-9::GFP puncta colocalized with EPG-7 and SQST-1 aggregates in indicated autophagy mutants.(Q-T) The ATG-9::GFP puncta in epg-6 (Q), epg-8 (R), unc-51 (S), and atg-18 (T) mutants colocalize with EPG-7 aggregates.(U and V) ATG-9::GFP puncta in epg-6 (U) and epg-8 (V) mutants colocalize with SQST-1 aggregates. (W and X) In unc-51 (W) and atg-18 (X) mutants, ATG-9::GFP partially colocalizes with SQST-1 aggregates.


Figure 7 . ( A2 ) Levels of W07G4 . 5 : : GFP in wild type , epg - 7 mutants , and atg - 3 L1 larvae before and after 6 h starvation treatment . ( B2 ) Model for the role of epg - 7 in degradation of SQST - 1 aggregates . SQST - 1 directly interacts with EPG - 7 and is recruited into EPG - 7 aggregates . EPG - 7 also associates with multiple Atg proteins , which may be recruited to the SQST - 1 - EPG - 7 complex in a hierarchical order . IM , isolation membrane . ( C2 ) Hierarchical relationship of the scaffold proteins EPG - 2 and EPG - 7 and other essential ATG and EPG proteins in the aggrephagy pathway ( A and B ) C17E4 . 2 : : GFP is weakly expressed and diffusely localized in a wild - type embryo . ( A ) DIC image of the embryo shown in B . ( C and D ) C17E4 . 2 : : GFP is expressed at dramatically elevated levels and accumulates into many aggregates in lgg - 1 ( C ) and epg - 7 ( D ) mutant embryos . ( E and F ) W07G4 . 5 : : GFP is expressed in the intestine and a few aggregates are formed in a wild - type embryo at the fourfold stage . ( E ) DIC image of the embryo shown in F . ( G and H ) W07G4 . 5 : : GFP is expressed at dramatically elevated levels and accumulates into a large number of aggregates in atg - 3 ( G ) and epg - 7 ( H ) mutants . ( I ) Levels of C33D9 . 6 : : GFP and W07G4 . 5 : : GFP are dramatically elevated in atg - 3 and epg - 7 mutant embryos . ( J and K ) C17E4 . 2 aggregates in atg - 3 mutants overlap with EPG - 7 aggregates ( J ) , but are separable from SQST - 1 aggregates ( K ) . Insets show a magnified view . ( L ) Colocalization frequency of C17E4 . 2 , C33D9 . 6 , and W07G4 . 5 aggregates with EPG - 7 and SQST - 1 aggregates in atg - 3 mutants . ( M ) SQST - 1 : : GFP accumulates in hypodermal cells in epg - 7 mutants at the larval stage . ( N ) SQST - 1 : : GFP aggregates disappear in rheb - 1 ( RNAi ) ; epg - 7 mutant larvae . ( O ) Dramatic accumulation of SQST - 1 : : GFP aggregates in hypodermal cells in atg - 3 mutant larvae . ( P ) SQST - 1 : : GFP aggregates persist in rheb - 1 ( RNAi ) ; atg - 3 mutant larvae . ( Q - T ) At larval stages , W07G4 . 5 : : GFP forms aggregates in intestinal cells ( Q and R ) . These aggregates disappear upon RNAi knockdown of let - 363 ( S and T ) . ( U - X ) W07G4 . 5 : : GFP aggregates accumulate in intestinal cells in epg - 7 mutant larvae ( U and V ) . These aggregates disappear when let - 363 is simultaneously depleted ( W and X ) . ( Y and Z ) In atg - 3 mutants , W07G4 . 5 : : GFP aggregates accumulate in intestinal cells ( Y ) and persist after RNAi knockdown of let - 363 ( Z ) .

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 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 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 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 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 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 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

Figure 7.(A2) Levels of W07G4.5::GFP in wild type, epg-7 mutants, and atg-3 L1 larvae before and after 6 h starvation treatment. (B2) Model for the role of epg-7 in degradation of SQST-1 aggregates. SQST-1 directly interacts with EPG-7 and is recruited into EPG-7 aggregates. EPG-7 also associates with multiple Atg proteins, which may be recruited to the SQST-1-EPG-7 complex in a hierarchical order. IM, isolation membrane. (C2) Hierarchical relationship of the scaffold proteins EPG-2 and EPG-7 and other essential ATG and EPG proteins in the aggrephagy pathway(A and B) C17E4.2::GFP is weakly expressed and diffusely localized in a wild-type embryo. (A) DIC image of the embryo shown in B. (C and D) C17E4.2::GFP is expressed at dramatically elevated levels and accumulates into many aggregates in lgg-1 (C) and epg-7 (D) mutant embryos.(E and F) W07G4.5::GFP is expressed in the intestine and a few aggregates are formed in a wild-type embryo at the fourfold stage. (E) DIC image of the embryo shown in F. (G and H) W07G4.5::GFP is expressed at dramatically elevated levels and accumulates into a large number of aggregates in atg-3 (G) and epg-7 (H) mutants.(I) Levels of C33D9.6::GFP and W07G4.5::GFP are dramatically elevated in atg-3 and epg-7 mutant embryos.(J and K) C17E4.2 aggregates in atg-3 mutants overlap with EPG-7 aggregates (J), but are separable from SQST-1 aggregates (K). Insets show a magnified view. (L) Colocalization frequency of C17E4.2, C33D9.6, and W07G4.5 aggregates with EPG-7 and SQST-1 aggregates in atg-3 mutants.(M) SQST-1::GFP accumulates in hypodermal cells in epg-7 mutants at the larval stage. (N) SQST-1::GFP aggregates disappear in rheb-1(RNAi);epg-7 mutant larvae. (O) Dramatic accumulation of SQST-1::GFP aggregates in hypodermal cells in atg-3 mutant larvae. (P) SQST-1::GFP aggregates persist in rheb-1(RNAi);atg-3 mutant larvae.(Q-T) At larval stages, W07G4.5::GFP forms aggregates in intestinal cells (Q and R). These aggregates disappear upon RNAi knockdown of let-363 (S and T). (U-X) W07G4.5::GFP aggregates accumulate in intestinal cells in epg-7 mutant larvae (U and V). These aggregates disappear when let-363 is simultaneously depleted (W and X). (Y and Z) In atg-3 mutants, W07G4.5::GFP aggregates accumulate in intestinal cells (Y) and persist after RNAi knockdown of let-363 (Z).


Figure 1A , B Regression plot showing the association between baseline AV45 PET ( x - axis ) and annual AV45 PET change ( y - axis ) across the entire sample ( N = 300 ) . The vertical dashed line represents the Aβ - positivity threshold of AV45 PET SUVR > 0 . 079 ( A ) . Regression plot for the association between baseline AV45 PET and annual AV45 PET change stratified by baseline CSF sTREM2 levels ( B ) .

O 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

Figure 1A, B Regression plot showing the association between baseline AV45 PET (x-axis) and annual AV45 PET change (y-axis) across the entire sample (N=300). The vertical dashed line represents the Aβ-positivity threshold of AV45 PET SUVR > 0.079 (A). Regression plot for the association between baseline AV45 PET and annual AV45 PET change stratified by baseline CSF sTREM2 levels (B).


Figure 2Boxplots ( central band = median , boxes = Quartiles , whiskers = 1 . 5 * interquartile range ) , illustrating the differences in GE180 PET ( A ) compared between wild type C57BL / 6 mice ( n = 43 ) and APPNL - G - G ( n = 15 ) . B Boxplots ( central band = median , boxes = Quartiles , whiskers = 1 . 5 * interquartile range ) , illustrating the differences in the rate of change in Florbetaben PET ( B ) compared between wild type C57BL / 6 mice ( n = 43 ) and APPNL - G - G ( n = 15 ) . C The regression plot shows lower rate of change in Florbetaben PET to be associated with lower GE180 PET at baseline , i . e . at 5 months of age ( C ) . The shaded area corresponds to the 95 % CI of the regression line .

O 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 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

Figure 2Boxplots (central band = median, boxes = Quartiles, whiskers = 1.5 * interquartile range), illustrating the differences in GE180 PET (A) compared between wild type C57BL/6 mice (n=43) and APPNL-G-G (n=15).B Boxplots (central band = median, boxes = Quartiles, whiskers = 1.5 * interquartile range), illustrating the differences in the rate of change in Florbetaben PET (B) compared between wild type C57BL/6 mice (n=43) and APPNL-G-G (n=15).C The regression plot shows lower rate of change in Florbetaben PET to be associated with lower GE180 PET at baseline, i.e. at 5 months of age (C). The shaded area corresponds to the 95% CI of the regression line.


Figure 3A - C Coronal ( top row ) and axial ( bottom row ) slices of average 18 - F GE - 180 TSPO ( microglia ) tracer and 18F - florbetaben amyloid - PET split up by group ( A - C ) : C57BL / 6 ( A ) , APPNL - G - F mice subgroup with a low ( < median ) GE - 180 PET ( B ) , and the APPNL - G - F mice subgroup with a high ( > median ) GE - 180 PET ( C ) . APPNL - G - F mice with low baseline GE180 PET levels showed faster increase in Florbetaben PET between 5 and 10 months ( B ) compared to those at high baseline level of GE - 180 PET ( C ) .

O 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 3A-C Coronal (top row) and axial (bottom row) slices of average 18-F GE-180 TSPO (microglia) tracer and 18F-florbetaben amyloid-PET split up by group (A - C): C57BL/6 (A), APPNL-G-F mice subgroup with a low (< median) GE-180 PET (B), and the APPNL-G-F mice subgroup with a high (> median) GE-180 PET (C). APPNL-G-F mice with low baseline GE180 PET levels showed faster increase in Florbetaben PET between 5 and 10 months (B) compared to those at high baseline level of GE-180 PET (C).


Figure 4Effects of sTREM2 on AV1451 - PET - assessed tau pathology stratified by Braak - stages shown as boxplots ( central band = median , boxes = Quartiles , whiskers = 1 . 5 * interquartile range ) .

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Figure 4Effects of sTREM2 on AV1451-PET-assessed tau pathology stratified by Braak-stages shown as boxplots (central band = median, boxes = Quartiles, whiskers = 1.5 * interquartile range).


Figure 1 ( a ) 30 and 150 days post fertilization ( dpf ) zebrafish ventricles were isolated and relative protein levels determined ( n = 3 ) . ( c ) qPCR analysis of mRNA levels of glycolytic enzyme genes in 5 dpci remote and wound border cardiac tissue ( n = 2 - 3 technical replicates using pooled cDNA from 10 ventricles for each condition ( n = 2 for pkmb and pdk4 , and n = 3 for the other genes ) ) . d , Immunostaining of heart sections for PCNA and MEF2 in 5 dpci animals treated with PBS , 2 - DG or DCA ; magnified view of area in white boxes shown below ; white dashed lines mark the wound border ; arrowheads point to PCNA + CMs ; percentage of PCNA + CMs in the border zone shown on the right ( n = 4 - 5 ventricles ) . e ) Immunostaining of heart sections for MEF2 N2 . 261 and MEF2 in 5 dpci animals treated with PBS , 2 - DG or DCA ; magnified view of area in white boxes shown below ; white dashed lines mark the wound border ; arrowheads point to N2 . 261 + CMs ; percentage of N2 . 261 + CMs in the border zone shown on the right ( n = 4 - 5 ventricles ) .

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 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 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

Figure 1(a) 30 and 150 days post fertilization (dpf) zebrafish ventricles were isolated and relative protein levels determined (n = 3).(c) qPCR analysis of mRNA levels of glycolytic enzyme genes in 5 dpci remote and wound border cardiac tissue (n = 2-3 technical replicates using pooled cDNA from 10 ventricles for each condition (n = 2 for pkmb and pdk4, and n = 3 for the other genes)).d, Immunostaining of heart sections for PCNA and MEF2 in 5 dpci animals treated with PBS, 2-DG or DCA; magnified view of area in white boxes shown below; white dashed lines mark the wound border; arrowheads point to PCNA+ CMs; percentage of PCNA+ CMs in the border zone shown on the right (n = 4-5 ventricles).e) Immunostaining of heart sections for MEF2 N2.261 and MEF2 in 5 dpci animals treated with PBS, 2-DG or DCA; magnified view of area in white boxes shown below; white dashed lines mark the wound border; arrowheads point to N2.261+ CMs; percentage of N2.261+ CMs in the border zone shown on the right (n = 4-5 ventricles).


Figure 2 ( a Immunostaining of heart sections for PCNA and MEF2 in 5 dpci pkma2 + / - ; pkmb + / - and pkma2 - / - ; pkmb + / - animals ; magnified view of area in white boxes shown below ; arrowheads point to PCNA + CMs ; white dashed lines mark the wound border ; percentage of PCNA + CMs in the border zone shown on the right ( n = 4 - 6 ventricles of each genotype ) . Immunostaining of heart sections for N2 . 261 and MEF2 in 5 dpci pkma2 + / - ; pkmb + / - and pkma2 - / - ; pkmb + / - animals ; magnified view of area in white boxes shown below ; arrowheads point to N2 . 261 + CMs ; white dashed lines mark the wound border ; percentage of N2 . 261 + in the border zone shown on the right ( n = 4 - 6 ventricles of each genotype ) . Immunostaining of heart sections for PCNA and MEF2 in 5 dpci ppargc1a + / + and ppargc1a - / - animals ; magnified view of area in white boxes shown below ; arrowheads point to PCNA + CMs ; white dashed lines mark the wound border ; percentage of PCNA + CMs in the border zone shown on the right ( n = 4 - 6 ventricles of each genotype ) . d ) Immunostaining of heart sections for N2 . 261 and MEF2 in 5 dpci ppargc1a + / + and ppargc1a - / - animals ; magnified view of area in white boxes shown below ; arrowheads point to N2 . 261 + CMs ; white dashed lines mark the wound border ; percentage of N2 . 261 + CMs in the border zone shown on the right ( n = 4 - 6 ventricles of each genotype ) . ( e AFOG staining of heart sections from 60 dpci pkma2 + / - ; pkmb + / - and pkma2 - / - ; pkmb + / - animals ; black dashed lines outline the scar area ; scar area measured on the right ( n = 3 - 5 ventricles of each genotype ) . f ) AFOG staining of heart sections from 45 dpci ppargc1a + / + and ppargc1a - / - animals ; black dashed lines outline the scar area ; scar area measured on the right ( n = 3 - 5 ventricles of each genotype ) .

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 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 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 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 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 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

Figure 2(a Immunostaining of heart sections for PCNA and MEF2 in 5 dpci pkma2+/-; pkmb+/- and pkma2-/-; pkmb+/- animals; magnified view of area in white boxes shown below; arrowheads point to PCNA+ CMs; white dashed lines mark the wound border; percentage of PCNA+ CMs in the border zone shown on the right (n = 4-6 ventricles of each genotype).Immunostaining of heart sections for N2.261 and MEF2 in 5 dpci pkma2+/-; pkmb+/- and pkma2-/-; pkmb+/- animals; magnified view of area in white boxes shown below; arrowheads point to N2.261+ CMs; white dashed lines mark the wound border; percentage of N2.261+ in the border zone shown on the right (n = 4-6 ventricles of each genotype).Immunostaining of heart sections for PCNA and MEF2 in 5 dpci ppargc1a+/+ and ppargc1a-/- animals; magnified view of area in white boxes shown below; arrowheads point to PCNA+ CMs; white dashed lines mark the wound border; percentage of PCNA+ CMs in the border zone shown on the right (n = 4-6 ventricles of each genotype).d) Immunostaining of heart sections for N2.261 and MEF2 in 5 dpci ppargc1a+/+ and ppargc1a-/- animals; magnified view of area in white boxes shown below; arrowheads point to N2.261+ CMs; white dashed lines mark the wound border; percentage of N2.261+ CMs in the border zone shown on the right (n = 4-6 ventricles of each genotype).(e AFOG staining of heart sections from 60 dpci pkma2+/-; pkmb+/- and pkma2-/-; pkmb+/- animals; black dashed lines outline the scar area; scar area measured on the right (n = 3-5 ventricles of each genotype).f) AFOG staining of heart sections from 45 dpci ppargc1a+/+ and ppargc1a-/- animals; black dashed lines outline the scar area; scar area measured on the right (n = 3-5 ventricles of each genotype).