( B ) Fluorescent composite images ( top ) and tracings ( bottom ) of mnr - 1 ( dz213 ) in an aman - 2 ( gk248486 ) and an aman - 2 ( gk248486 ) ; MGAT - 1 ( null ) background . An MGAT null mutant lacks the three C . elegans paralogs : gly - 12 , gly - 13 , and gly - 14 . PVD is visualized by the wyIs581 transgene . The cell body is denoted with an asterisk . Anterior is to the left and dorsal is up in all panels . Scale bars represent 20μm . ( C ) Quantification of full tertiaries of denoted genotypes . Data are represented as mean ± SEM . Statistical comparisons were performed using the Kruskal - Wallis test . Statistical significance is indicated ( * * * p ≤ 0 . 001 , * * * * p ≤ 0 . 0001 , ns = not significant ) . n = 15 for all genotypes and are biological replicates . ( D ) Quantification of the number of quaternary branches in wild type control , aman - 2 ( gk248486 ) null , and MGAT1 ( null ) animals . Loss of MGAT1 suppresses the decrease in quaternary branch number in the aman - 2 ( null ) background . Data are represented as mean ± SEM . Statistical significance was calculated using the Kruskal - Wallis test and is indicated ( * * p ≤ 0 . 01 , * * * * p ≤ 0 . 0001 ) . n = 15 for all genotypes and are biological replicates .

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(B) Fluorescent composite images (top) and tracings (bottom) of mnr-1(dz213) in an aman-2(gk248486) and an aman-2(gk248486); MGAT-1(null) background. An MGAT null mutant lacks the three C. elegans paralogs: gly-12, gly-13, and gly-14. PVD is visualized by the wyIs581 transgene. The cell body is denoted with an asterisk. Anterior is to the left and dorsal is up in all panels. Scale bars represent 20μm. (C) Quantification of full tertiaries of denoted genotypes. Data are represented as mean ± SEM. Statistical comparisons were performed using the Kruskal-Wallis test. Statistical significance is indicated (***p ≤ 0.001, ****p ≤ 0.0001, ns=not significant). n=15 for all genotypes and are biological replicates. (D) Quantification of the number of quaternary branches in wild type control, aman-2(gk248486) null ,and MGAT1(null) animals. Loss of MGAT1 suppresses the decrease in quaternary branch number in the aman-2(null) background. Data are represented as mean ± SEM. Statistical significance was calculated using the Kruskal-Wallis test and is indicated (**p ≤ 0.01, ****p ≤ 0.0001). n = 15 for all genotypes and are biological replicates.


Western blot against GFP in C . elegans lysate expressing no transgenes ( N2 ) and expressing DMA - 1 : : GFP ( qyIs369 ) , after precipitating with anti - GFP antibody . The red boxed plus sign indicates that the lysate is treated with the PNGase F glycosidase . The downwards size shift reveals that N - glycan structures are present on DMA - 1 . Ladder is marked in kilodaltons ( kDa ) . The GFP tag contains no N - glycosylation sites . The experiment was repeated four times with biological replicates .

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Western blot against GFP in C. elegans lysate expressing no transgenes (N2) and expressing DMA-1::GFP (qyIs369), after precipitating with anti-GFP antibody. The red boxed plus sign indicates that the lysate is treated with the PNGase F glycosidase. The downwards size shift reveals that N-glycan structures are present on DMA-1. Ladder is marked in kilodaltons (kDa). The GFP tag contains no N-glycosylation sites. The experiment was repeated four times with biological replicates.


Western blot against GFP in C . elegans lysate DMA - 1 : : GFP ( qyIs369 ) , after precipitating with anti - GFP antibody . Control indicates an otherwise wild type background as opposed to an aman - 2 ( gk248486 ) null background . The upward size shift in the mutant reveals that the loss of aman - 2 alters the identity of N - glycan structures on DMA - 1 . The experiment was repeated four times with biological replicates .

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Western blot against GFP in C. elegans lysate DMA-1::GFP (qyIs369), after precipitating with anti-GFP antibody. Control indicates an otherwise wild type background as opposed to an aman-2(gk248486) null background. The upward size shift in the mutant reveals that the loss of aman-2 alters the identity of N-glycan structures on DMA-1. The experiment was repeated four times with biological replicates.


Western blot against GFP in C . elegans lysate DMA - 1 : : GFP ( qyIs369 ) , after precipitating with anti - GFP antibody . Control indicates an otherwise wild type background as opposed to an aman - 2 ( gk248486 ) null background . The red boxed + F indicates that the lysate is treated with the PNGase F glycosidase , while the green boxed + H corresponds to the Endo H glycosidase , which cleaves high - mannose and hybrid type N - glycans . For complementary experiments using the Endo D glycosidase , which cleaves paucimannose type N - glycans , Size shifts indicate that some hybrid / high - mannose structures are present on DMA - 1 ( left ) , and that the aman - 2 mutant results in only hybrid / high - mannose structures on DMA - 1 ( right ) . The experiment was repeated four times with biological replicates .

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Western blot against GFP in C. elegans lysate DMA-1::GFP (qyIs369), after precipitating with anti-GFP antibody. Control indicates an otherwise wild type background as opposed to an aman-2(gk248486) null background. The red boxed +F indicates that the lysate is treated with the PNGase F glycosidase, while the green boxed +H corresponds to the Endo H glycosidase, which cleaves high-mannose and hybrid type N-glycans. For complementary experiments using the Endo D glycosidase, which cleaves paucimannose type N-glycans, Size shifts indicate that some hybrid/high-mannose structures are present on DMA-1 (left), and that the aman-2 mutant results in only hybrid/high-mannose structures on DMA-1 (right). The experiment was repeated four times with biological replicates.


Quantification of the number of quaternary branches in DMA - 1 : : 2xFLAG control animals and animals with combinations of DMA - 1 : : 2xFLAG N - glycan attachment sites mutated . Note that DMA - 1 : : 2xFLAG control animals display a slightly reduced number of quaternary dendrites compared to wild type animals Data are represented as mean ± SEM . Statistical significance was calculated using the Kruskal - Wallis test and Mann - Whitney between individual comparisons , and is indicated ( * * p ≤ 0 . 01 , * * * * p ≤ 0 . 0001 , ns = not significant ) . n = 15 for all genotypes and are biological replicates .

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Quantification of the number of quaternary branches in DMA-1::2xFLAG control animals and animals with combinations of DMA-1::2xFLAG N-glycan attachment sites mutated. Note that DMA-1::2xFLAG control animals display a slightly reduced number of quaternary dendrites compared to wild type animals Data are represented as mean ± SEM. Statistical significance was calculated using the Kruskal-Wallis test and Mann-Whitney between individual comparisons, and is indicated (**p ≤ 0.01, ****p ≤ 0.0001, ns=not significant). n = 15 for all genotypes and are biological replicates.


Quantification of the number of quaternary branches in DMA - 1 : : 2xFLAG control animals alone and in combination with an aman - 2 ( gk248486 ) null mutant ( shaded in red ) . Control data is identical as in ( B ) and shown for comparison only . Data are represented as mean ± SEM . Statistical significance was calculated using the Kruskal - Wallis test and Mann - Whitney between individual comparisons , and is indicated ( * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 , * * * * p ≤ 0 . 0001 , ns = not significant ) . n = 15 for all genotypes and are biological replicates .

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Quantification of the number of quaternary branches in DMA-1::2xFLAG control animals alone and in combination with an aman-2(gk248486) null mutant (shaded in red). Control data is identical as in (B) and shown for comparison only. Data are represented as mean ± SEM. Statistical significance was calculated using the Kruskal-Wallis test and Mann-Whitney between individual comparisons, and is indicated (**p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001, ns=not significant). n = 15 for all genotypes and are biological replicates.


Fluorescent images ( left ) and tracings ( center ) of PVD in animals of denoted backgrounds ( aman - 2 ( null ) is gk248486 ) . S4 corresponds to the endogenous alteration of N - glycan attachment site N386 of DMA - 1 . PVD is visualized by the dzIs117 transgene . The cell body is marked with an asterisk . Scale bars represent 20μm .

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Fluorescent images (left) and tracings (center) of PVD in animals of denoted backgrounds (aman-2(null) is gk248486). S4 corresponds to the endogenous alteration of N-glycan attachment site N386 of DMA-1. PVD is visualized by the dzIs117 transgene. The cell body is marked with an asterisk. Scale bars represent 20μm.


A . Histograms of spindle angle of GFP - H2B U2OS cells transfected with either GAPDH or Spindly siRNA and filmed on rectangular micropatterns . Cells were filmed 48 hours post - transfection .

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A. Histograms of spindle angle of GFP-H2B U2OS cells transfected with either GAPDH or Spindly siRNA and filmed on rectangular micropatterns. Cells were filmed 48 hours post-transfection.


B . Mitotic timing of control - depleted U2OS cells or CLIP - 170 - depleted cells . CLIP - 170 - depeleted cells are split in two categories ; ' aligned ' and ' misaligned ' , relating to the observed chromosome misalignment

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B. Mitotic timing of control-depleted U2OS cells or CLIP-170-depleted cells. CLIP-170-depeleted cells are split in two categories; 'aligned' and 'misaligned', relating to the observed chromosome misalignment phenotype


C . The histograms of spindle angles for the two different categories of CLIP - 170 - depleted U2OS cells ( Left graph with alignment defect , right graph without alignment defect ) .

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C. The histograms of spindle angles for the two different categories of CLIP-170-depleted U2OS cells (Left graph with alignment defect, right graph without alignment defect).


D and E . Mitotic timing and spindle angle histograms of control ( GAPDH ) - depleted U2OS cells and CENP - E - depleted cells .

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D and E. Mitotic timing and spindle angle histograms of control (GAPDH)-depleted U2OS cells and CENP-E-depleted cells.


A . HeLa cell stably expressing GFP - LGN and transiently expressing RFP - H2B was treated with noscapine to induce chromosome misalignments . Cells were imaged every 5 minutes . The time is relative to NEB . The red arrows indicate cortical regions where the chromosomes are in close proximity to the cell boundaries , accompanied by the displacement of LGN from the cortex .

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A. HeLa cell stably expressing GFP-LGN and transiently expressing RFP-H2B was treated with noscapine to induce chromosome misalignments. Cells were imaged every 5 minutes. The time is relative to NEB. The red arrows indicate cortical regions where the chromosomes are in close proximity to the cell boundaries, accompanied by the displacement of LGN from the cortex.


B . Similar assay as described in supplementary figure 2D was carried out in the presence of a small molecule inhibitor against Aurora - B ( ZM447439 ) in combination with the proteasome inhibitor MG - 132 to keep cells arrested in mitosis and in B . with an inhibitor against Plk1 ( BI2536 ) . Drugs were added at the same timing as Hoechst , 30 minutes prior to imaging .

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B. Similar assay as described in supplementary figure 2D was carried out in the presence of a small molecule inhibitor against Aurora-B (ZM447439) in combination with the proteasome inhibitor MG-132 to keep cells arrested in mitosis and in B. with an inhibitor against Plk1 (BI2536). Drugs were added at the same timing as Hoechst, 30 minutes prior to imaging.


A . Immunofluorescence images and quantifications of KT - localized Plk1 in U2OS cells after PBIP1 depletion . Cells were either Mock transfected or with PBIP1 siRNA for 72 hours . Cells were treated with nocodazole for 1 hour and fixed . Cells were stained with the centromere marker ACA , Plk1 and DAPI .

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A. Immunofluorescence images and quantifications of KT-localized Plk1 in U2OS cells after PBIP1 depletion. Cells were either Mock transfected or with PBIP1 siRNA for 72 hours. Cells were treated with nocodazole for 1 hour and fixed. Cells were stained with the centromere marker ACA, Plk1 and DAPI.


B . Representative images and histograms showing the effect of chromosome position on cortical LGN enrichment after 72 hours of Mock and PBIP1 depletion .

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B. Representative images and histograms showing the effect of chromosome position on cortical LGN enrichment after 72 hours of Mock and PBIP1 depletion.


C . Live cellimages and histograms of GFP - H2B U2OS cells Mock transfected or with siPBIP1 filmed on rectangular micropatterns in the presence of a CENP - E inhibitor . The spindle angles for both conditions were scored at 32 minutes after NEB .

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C. Live cellimages and histograms of GFP-H2B U2OS cells Mock transfected or with siPBIP1 filmed on rectangular micropatterns in the presence of a CENP-E inhibitor. The spindle angles for both conditions were scored at 32 minutes after NEB.


. D . The total spindle angle displacement within the first hour of mitosis was determined by measuring the spindle angle at each time point and calculating the sum of the Δ spindle angle between each time points .

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. D. The total spindle angle displacement within the first hour of mitosis was determined by measuring the spindle angle at each time point and calculating the sum of the Δ spindle angle between each time points.


Expression levels of the autophagosome marker LC3B - II were evaluated in wild - type ( WT ) and Ctns − / − fibroblasts by Western blot ( WB ) . Data are representative of five different experiments with similar results .

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Expression levels of the autophagosome marker LC3B-II were evaluated in wild-type (WT) and Ctns−/− fibroblasts by Western blot (WB). Data are representative of five different experiments with similar results.


WT and Ctns − / − fibroblasts were stained using LC3B antibodies and analyzed by fluorescence microscopy . The number of LC3B - positive puncta was quantified by the ImagePro software and normalized per area arbitrary units . Results are mean ± SEM ( n = 27 WT and 24 Ctns − / − cells ) . * * * P < 0 . 001 ( unpaired t - test ) .

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WT and Ctns−/− fibroblasts were stained using LC3B antibodies and analyzed by fluorescence microscopy. The number of LC3B-positive puncta was quantified by the ImagePro software and normalized per area arbitrary units. Results are mean ± SEM (n = 27 WT and 24 Ctns−/− cells). ***P < 0.001 (unpaired t-test).


Immunofluorescence analysis of liver ( C ) and kidney ( D ) tissues from WT and Ctns − / − GFP - LC3 transgenic mice using anti - GFP antibodies . Quantification of LC3 puncta was obtained by counting the number of GFP - positive structures relative to the number of nuclei in the same field , using the ImagePro software . GFP - LC3 puncta were counted by analyzing 6 to 10 fields per tissue section ( 200 - 400 nuclei per field ) , in a total of 3 WT and 3 Ctns − / − mice expressing GFP - LC3 . Results are mean ± SEM . In ( C ) , * * P = 0 . 0029 ; in ( D ) , * * P = 0 . 0025 ( unpaired t - test ) . Scale bar : 5 μm .

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Immunofluorescence analysis of liver (C) and kidney (D) tissues from WT and Ctns−/−GFP-LC3 transgenic mice using anti-GFP antibodies. Quantification of LC3 puncta was obtained by counting the number of GFP-positive structures relative to the number of nuclei in the same field, using the ImagePro software. GFP-LC3 puncta were counted by analyzing 6 to 10 fields per tissue section (200-400 nuclei per field), in a total of 3 WT and 3 Ctns−/−mice expressing GFP-LC3. Results are mean ± SEM. In (C), **P = 0.0029; in (D), **P = 0.0025 (unpaired t-test). Scale bar: 5 μm.


Representative images of wild - type ( WT ) and Ctns − / − mouse fibroblasts transfected with the ptfLC3 vector under resting conditions ( fed ) , or after serum starvation ( Serum Starv . ) in the presence or absence of the alkalinizing drug chloroquine ( CQ ) . GFP and RFP staining was analyzed by confocal microscopy . Examples of red - only puncta ( mature autophagosomes ) are indicated with arrows . Scale bar : 5 μm . The percentage of mature autophagosomes ( red - only vesicles ) was calculated based on the ratio between the number of red - only puncta and the total number of autophagosomes ( number of green and red + red - only puncta ) . The graph is representative of three different experiments with similar results . Results are mean ± SEM ( n = 7 cells per condition ) . In addition to the experimental conditions shown in ( A ) , the Aa / serum starvation condition is included .

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Representative images of wild-type (WT) and Ctns−/− mouse fibroblasts transfected with the ptfLC3 vector under resting conditions (fed), or after serum starvation (Serum Starv.) in the presence or absence of the alkalinizing drug chloroquine (CQ). GFP and RFP staining was analyzed by confocal microscopy. Examples of red-only puncta (mature autophagosomes) are indicated with arrows. Scale bar: 5 μm.The percentage of mature autophagosomes (red-only vesicles) was calculated based on the ratio between the number of red-only puncta and the total number of autophagosomes (number of green and red + red-only puncta). The graph is representative of three different experiments with similar results. Results are mean ± SEM (n = 7 cells per condition). In addition to the experimental conditions shown in (A), the Aa/serum starvation condition is included.


WT and Ctns − / − fibroblasts under resting conditions were treated with 100 nM bafilomycin A ( BafA ) for 2 h and LC3B - II levels were analyzed by Western blot ( WB ) .

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WT and Ctns−/− fibroblasts under resting conditions were treated with 100 nM bafilomycin A (BafA) for 2 h and LC3B-II levels were analyzed by Western blot (WB).


Phosphorylation levels of the mTOR complex kinase 1 substrate S6K and LC3B - II levels in WT and Ctns − / − fibroblasts were measured by WB under resting conditions ( − ) , withdrawal of both amino acids and serum ( Aa / Ser . Starv . ) and subsequent recovery by replacement of starvation medium with normal cell growth medium ( Rec ) , in the presence or absence of 100 nM BafA for the indicated time .

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Phosphorylation levels of the mTOR complex kinase 1 substrate S6K and LC3B-II levels in WT and Ctns−/− fibroblasts were measured by WB under resting conditions (−), withdrawal of both amino acids and serum (Aa/Ser. Starv.) and subsequent recovery by replacement of starvation medium with normal cell growth medium (Rec), in the presence or absence of 100 nM BafA for the indicated time.


WB analysis showing phosphorylation levels of the mTOR substrate S6K and expression of LC3B - II in resting or serum - starved WT , Ctns − / − and Ctns − / − fibroblasts expressing GFP - CTNS .

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WB analysis showing phosphorylation levels of the mTOR substrate S6K and expression of LC3B-II in resting or serum-starved WT,Ctns−/− and Ctns−/− fibroblasts expressing GFP-CTNS.


Analysis of the expression of endogenous SQSTM1 in wild - type or Ctns − / − mouse fibroblasts under fed ( serum ) or starved ( 15 h serum starvation ) conditions . In these assays , 20 μg of total protein lysates were resolved by SDS - PAGE and analyzed by Western blot ( WB ; A ) . Equal loading was established by silver staining ( B ) . Quantification ( mean ± SEM ) of five different areas of each silver - stained lane are shown in ( C ) .

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Analysis of the expression of endogenous SQSTM1 in wild-type or Ctns−/− mouse fibroblasts under fed (serum) or starved (15 h serum starvation) conditions. In these assays, 20 μg of total protein lysates were resolved by SDS-PAGE and analyzed by Western blot (WB; A). Equal loading was established by silver staining (B). Quantification (mean ± SEM) of five different areas of each silver-stained lane are shown in (C).


Degradation of long - lived proteins using metabolic labeling was performed as described in Supplementary Materials and Methods . The data represent the mean ± SEM of nine independent samples analyzed in three independent experiments . * P = 0 . 009 for WT and * P = 0 . 0013 for Ctns − / − , respectively . NS , not significant ( unpaired t - test ) .

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Degradation of long-lived proteins using metabolic labeling was performed as described in Supplementary Materials and Methods. The data represent the mean ± SEM of nine independent samples analyzed in three independent experiments. *P = 0.009 for WT and *P = 0.0013 for Ctns−/−, respectively. NS, not significant (unpaired t-test).


The expression levels of the endogenous LAMP2A and LAMP1 proteins were analyzed in wild - type ( WT ) and Ctns − / − mousefibroblasts ( A ) , kidney ( B ) and liverlysosomes ( C ) by Western blot . Quantitative densitometry analysis of immunoblots obtained from different experiments was performed by calculating the ratio between LAMP2A and actin signals in each lane . Results are mean ± SEM of four independent experiments with mousefibroblasts , 5 WT and 6 Ctns − / − mousekidneys , 3 WT and 3 Ctns − / − mouselivers ( lysosomal extracts ) .

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The expression levels of the endogenous LAMP2A and LAMP1 proteins were analyzed in wild-type (WT) and Ctns−/−mousefibroblasts (A), kidney (B) and liverlysosomes (C) by Western blot. Quantitative densitometry analysis of immunoblots obtained from different experiments was performed by calculating the ratio between LAMP2A and actin signals in each lane. Results are mean ± SEM of four independent experiments with mousefibroblasts, 5 WT and 6 Ctns−/−mousekidneys, 3 WT and 3 Ctns−/−mouselivers (lysosomal extracts).


Comparative analysis of the amount of LAMP2A in wild - type or Ctns − / − liverlysosomes was performed using two different anti - LAMP2A antibodies . 15 μg of lysosomal proteins were loaded in each lane . Ab1 , anti - LAMP2A antibody raised against the twelve amino acids of the cytosolic region of ratLAMP2A largely validated to recognize mouseLAMP2A but not other LAMP2 isoforms ( Cuervo Dice , 1996 ) . Ab2 , anti - LAMP2A Abcam Ab18528 . Decreased lysosomalLAMP2A was evident using either antibody . LAMP1 was used as a loading control . Representative of two independent experiments with similar results .

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

Comparative analysis of the amount of LAMP2A in wild-type or Ctns−/−liverlysosomes was performed using two different anti-LAMP2A antibodies. 15 μg of lysosomal proteins were loaded in each lane. Ab1, anti-LAMP2A antibody raised against the twelve amino acids of the cytosolic region of ratLAMP2A largely validated to recognize mouseLAMP2A but not other LAMP2 isoforms (Cuervo Dice, 1996). Ab2, anti-LAMP2A Abcam Ab18528. Decreased lysosomalLAMP2A was evident using either antibody. LAMP1 was used as a loading control. Representative of two independent experiments with similar results.


Immunofluorescence analyses of endogenous LAMP1 , LAMP2A ( A , B ) , LAMP2B ( C ) and total LAMP2 ( D ) localization in wild - type ( WT ) and Ctns − / − mouse fibroblasts . ( A ) Confocal microscopy analysis . White arrows represent LAMP1 - positive circular structures ( lysosomes ) which were visible in both WT and Ctns − / − cells . Colocalization of LAMP1 and LAMP2A was observed in WT fibroblasts ( white arrows , merged panel ) but rarely in Ctns − / − cells ( red arrow ) . Most LAMP2A showed a punctate pattern and lack of colocalization with LAMP1 in Ctns − / − cells ( arrowheads , lower panels ) . Scale bars : 5 μm . Inset scale bars : 2 μm .

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Immunofluorescence analyses of endogenous LAMP1, LAMP2A (A, B), LAMP2B (C) and total LAMP2 (D) localization in wild-type (WT) and Ctns−/− mouse fibroblasts. (A) Confocal microscopy analysis. White arrows represent LAMP1-positive circular structures (lysosomes) which were visible in both WT and Ctns−/− cells. Colocalization of LAMP1 and LAMP2A was observed in WT fibroblasts (white arrows, merged panel) but rarely in Ctns−/− cells (red arrow). Most LAMP2A showed a punctate pattern and lack of colocalization with LAMP1 in Ctns−/− cells (arrowheads, lower panels). Scale bars: 5 μm. Inset scale bars: 2 μm.


Immunofluorescence analyses of endogenous LAMP1 , LAMP2A ( A , B ) , LAMP2B ( C ) and total LAMP2 ( D ) localization in wild - type ( WT ) and Ctns − / − mouse fibroblasts . ( B ) High - resolution stochastic optical reconstruction microscopy ( STORM ) analysis of the localization of LAMP1 and LAMP2A in wild - type and Ctns − / − cells . In Ctns − / − cells , LAMP2A was detected near ( arrowheads , estimated distance > 50 nm ) but not always adjacent ( 10 - 50 nm ) to LAMP1 or at LAMP1 - negative structures ( arrows ) . Scale bar : 0 . 5 μm .

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Immunofluorescence analyses of endogenous LAMP1, LAMP2A (A, B), LAMP2B (C) and total LAMP2 (D) localization in wild-type (WT) and Ctns−/− mouse fibroblasts.(B) High-resolution stochastic optical reconstruction microscopy (STORM) analysis of the localization of LAMP1 and LAMP2A in wild-type and Ctns−/− cells. In Ctns−/− cells, LAMP2A was detected near (arrowheads, estimated distance > 50 nm) but not always adjacent (10-50 nm) to LAMP1 or at LAMP1-negative structures (arrows). Scale bar: 0.5 μm.


Immunofluorescence analyses of endogenous LAMP1 , LAMP2A ( A , B ) , LAMP2B ( C ) and total LAMP2 ( D ) localization in wild - type ( WT ) and Ctns − / − mouse fibroblasts . ( C ) Immunofluorescence showing colocalization of endogenous LAMP1 and LAMP2B in both wild - type and CTNS - deficient fibroblasts ( arrows ) . Scale bar : 20 μm .

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Immunofluorescence analyses of endogenous LAMP1, LAMP2A (A, B), LAMP2B (C) and total LAMP2 (D) localization in wild-type (WT) and Ctns−/− mouse fibroblasts. (C) Immunofluorescence showing colocalization of endogenous LAMP1 and LAMP2B in both wild-type and CTNS-deficient fibroblasts (arrows). Scale bar: 20 μm.


Immunofluorescence analyses of endogenous LAMP1 , LAMP2A ( A , B ) , LAMP2B ( C ) and total LAMP2 ( D ) localization in wild - type ( WT ) and Ctns − / − mouse fibroblasts . ( D ) Immunofluorescence analysis of LAMP1 and total LAMP2 ( antibody ABL93 DSHB ) . Arrows show colocalization . Arrowheads show LAMP2 - positive LAMP1 - negative vesicles .

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Immunofluorescence analyses of endogenous LAMP1, LAMP2A (A, B), LAMP2B (C) and total LAMP2 (D) localization in wild-type (WT) and Ctns−/− mouse fibroblasts. (D) Immunofluorescence analysis of LAMP1 and total LAMP2 (antibody ABL93 DSHB). Arrows show colocalization. Arrowheads show LAMP2-positive LAMP1-negative vesicles.


Immunofluorescence analyses of endogenous LAMP2A , ER ( Grp78 / 94 ) ( A ) , which is expanded in cystinotic cells ( white arrow ) , VAMP7 - positive vesicles ( B ) and Rab11a - positive structures ( C ) was performed using wild - type or Ctns − / − fibroblasts as described in Materials and Methods . At least 2 independent experiments were performed for each vesicular trafficking or ER marker . ( B , C ) Results are expressed as the percentage of LAMP2A that colocalizes with VAMP7 or Rab11 . For Rab11 / LAMP2A colocalization , 29 wild - type and 15 Ctns − / − cells were quantified . For VAMP7 / LAMP2A colocalization ( white arrows ) , 49 WT and 57 Ctns − / − cells were analyzed . Data are expressed as mean ± SEM . * P < 0 . 001 ( unpaired t - test ) . Scale bars : 20 μm .

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

Immunofluorescence analyses of endogenous LAMP2A, ER (Grp78/94) (A), which is expanded in cystinotic cells (white arrow), VAMP7-positive vesicles (B) and Rab11a-positive structures (C) was performed using wild-type or Ctns−/− fibroblasts as described in Materials and Methods. At least 2 independent experiments were performed for each vesicular trafficking or ER marker. (B, C) Results are expressed as the percentage of LAMP2A that colocalizes with VAMP7 or Rab11. For Rab11/LAMP2A colocalization, 29 wild-type and 15 Ctns−/− cells were quantified. For VAMP7/LAMP2A colocalization (white arrows), 49 WT and 57 Ctns−/− cells were analyzed. Data are expressed as mean ± SEM. *P < 0.001 (unpaired t-test). Scale bars: 20 μm.


WT , Ctns − / − or Ctns − / − mouse fibroblasts treated for 20 h with either a combination of both leupeptin and chloroquine ( CQ / Leu ) or bafilomycin A ( BafA ) alone were fixed and immunostained with antibodies recognizing endogenous LAMP1 and LAMP2A proteins and samples were analyzed by confocal microscopy as described in Material and Methods . Lysosomal colocalization of LAMP1 and LAMP2A was evident in treated ( middle lower and bottom panels ) but not in untreated ( middle upper panels ) Ctns − / − cells . Scale bars : 5 μm . Inset scale bars : 2 μm .

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WT,Ctns−/− or Ctns−/− mouse fibroblasts treated for 20 h with either a combination of both leupeptin and chloroquine (CQ/Leu) or bafilomycin A (BafA) alone were fixed and immunostained with antibodies recognizing endogenous LAMP1 and LAMP2A proteins and samples were analyzed by confocal microscopy as described in Material and Methods. Lysosomal colocalization of LAMP1 and LAMP2A was evident in treated (middle lower and bottom panels) but not in untreated (middle upper panels) Ctns−/− cells. Scale bars: 5 μm. Inset scale bars: 2 μm.


Lysosomes were isolated from livers of starved wild - type ( WT ) and Ctns − / − mice as described in Supplementary Materials and Methods and incubated at 37 ° C for 30 min with the CMA substrate GAPDH , in the presence or absence of ATP ( necessary for CMA ) and protease inhibitors ( Prot . Inhib . ) . A fraction of the CMA reactions was then mixed with sample buffer and boiled at 95 ° C for 5 min , followed by SDS - PAGE and GAPDH and LAMP1 immunoblotting . Quantitative densitometry analysis of CMA activity performed in independent experiments using lysosomes isolated from a total of 11 WT and 10 Ctns − / − mice . Results are mean ± SEM . * * * P = 0 . 0005 ; * * P = 0 . 0041 ; N . S . , not significant ( unpaired t - test ) .

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Lysosomes were isolated from livers of starved wild-type (WT) and Ctns−/− mice as described in Supplementary Materials and Methods and incubated at 37°C for 30 min with the CMA substrate GAPDH, in the presence or absence of ATP (necessary for CMA) and protease inhibitors (Prot. Inhib.). A fraction of the CMA reactions was then mixed with sample buffer and boiled at 95°C for 5 min, followed by SDS-PAGE and GAPDH and LAMP1 immunoblotting.Quantitative densitometry analysis of CMA activity performed in independent experiments using lysosomes isolated from a total of 11 WT and 10 Ctns−/− mice. Results are mean ± SEM. ***P = 0.0005; **P = 0.0041; N.S., not significant (unpaired t-test).


The ability of lysosomal proteases to mediate GAPDH degradation was assessed by incubating Triton X - 100 - treated WT and Ctns − / − lysosomes with GAPDH for 30 min in acidic reaction buffer and compared with input ( reaction with no lysosomes ) . Quantitative densitometry analysis of independent reactions performed with lysosomes from a total of 9 WT and 9 Ctns − / − mice shows no significant difference in protease activity . Results are mean ± SEM .

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The ability of lysosomal proteases to mediate GAPDH degradation was assessed by incubating Triton X-100-treated WT and Ctns−/− lysosomes with GAPDH for 30 min in acidic reaction buffer and compared with input (reaction with no lysosomes). Quantitative densitometry analysis of independent reactions performed with lysosomes from a total of 9 WT and 9 Ctns−/− mice shows no significant difference in protease activity. Results are mean ± SEM.


WT and Ctns − / − mouse fibroblasts were treated with either 50 μM H2O2 or 1 mM paraquat ( PQ ) for 4 h and stained with ( A ) FITC - annexin V or ( B ) propidium iodide ( PI ) and analyzed by FACS . Results from 3 different experiments are shown as mean ± SEM . In ( A ) , * P = 0 . 05 ; * * * P = 0 . 0002 . In ( B ) , * P = 0 . 046 ; * * P = 0 . 0065 . Unpaired t - test .

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WT and Ctns−/− mouse fibroblasts were treated with either 50 μM H2O2 or 1 mM paraquat (PQ) for 4 h and stained with (A) FITC-annexin V or (B) propidium iodide (PI) and analyzed by FACS. Results from 3 different experiments are shown as mean ± SEM. In (A), *P = 0.05; ***P = 0.0002. In (B), *P = 0.046; **P = 0.0065. Unpaired t-test.


WT and Ctns − / − cells were treated as described above and samples analyzed by Western blot using antibodies recognizing cleaved ( active ) caspase 3 . Data are representative of 2 independent experiments with similar results .

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WT and Ctns−/− cells were treated as described above and samples analyzed by Western blot using antibodies recognizing cleaved (active) caspase 3. Data are representative of 2 independent experiments with similar results.


Cystine content in WT , Ctns − / − , cysteamine - treated Ctns − / − fibroblasts or Ctns − / − fibroblasts expressing GFP - CTNS was assessed by mass spectrometry .

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Cystine content in WT,Ctns−/−, cysteamine-treated Ctns−/− fibroblasts or Ctns−/− fibroblasts expressing GFP-CTNS was assessed by mass spectrometry.


WT , Ctns − / − , Ctns − / − fibroblasts treated with 1 mM cysteamine ( 48 h ) , or Ctns − / − fibroblasts expressing GFP - CTNS were fixed and stained with anti - LAMP1 and LAMP2A antibodies as indicated . For better visualization of LAMP1 / LAMP2A colocalization , GFP - CTNS was pseudocolored as magenta and LAMP1 was pseudocolored as green ( lower panels ) . Some lysosomes are indicated with arrows . Arrowheads indicate LAMP2A distribution to structures different from lysosomes ( only observed in Ctns − / − cells and in cysteamine - treated Ctns − / − cells ) . Rescue of the LAMP2A localization to lysosomes was observed in Ctns − / − cells expressing GFP - CTNS . Scale bar : 2 μm . Quantification of the colocalization analysis described in ( B ) . Calculation of the Pearson ' s colocalization coefficient was done by analyzing 113 WT , 251 Ctns − / − , 88 cysteamine - treated and 164 GFP - CTNS - expressing Ctns − / − cells , by using the ZEN 2010 software . Results are mean ± SEM . * * * P < 0 . 001 ; NS , not significant ( one - way ANOVA , Bonferroni ' s multiple comparisons test ) .

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WT, Ctns−/−, Ctns−/− fibroblasts treated with 1 mM cysteamine (48 h), or Ctns−/− fibroblasts expressing GFP-CTNS were fixed and stained with anti-LAMP1 and LAMP2A antibodies as indicated. For better visualization of LAMP1/LAMP2A colocalization, GFP-CTNS was pseudocolored as magenta and LAMP1 was pseudocolored as green (lower panels). Some lysosomes are indicated with arrows. Arrowheads indicate LAMP2A distribution to structures different from lysosomes (only observed in Ctns−/− cells and in cysteamine-treated Ctns−/− cells). Rescue of the LAMP2A localization to lysosomes was observed in Ctns−/− cells expressing GFP-CTNS. Scale bar: 2 μm.Quantification of the colocalization analysis described in (B). Calculation of the Pearson's colocalization coefficient was done by analyzing 113 WT, 251 Ctns−/−, 88 cysteamine-treated and 164 GFP-CTNS-expressing Ctns−/− cells, by using the ZEN 2010 software. Results are mean ± SEM. ***P < 0.001; NS, not significant (one-way ANOVA, Bonferroni's multiple comparisons test).


Cystine content in WT , Ctns − / − and cysteamine - treated Ctns − / − mouse livers was assessed by mass spectrometry . Results are mean ± SEM ( n = 4 ) . * * * P < 0 . 001 ; * * P = 0 . 0026 ( one - way ANOVA , Bonferroni ' s multiple comparisons test ) .

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Cystine content in WT,Ctns−/− and cysteamine-treated Ctns−/− mouse livers was assessed by mass spectrometry. Results are mean ± SEM (n = 4). ***P < 0.001; **P = 0.0026 (one-way ANOVA, Bonferroni's multiple comparisons test).


Western blot analysis of LAMP2A expression in lysosomes isolated from WT , Ctns − / − and cysteamine - treated Ctns − / − mouse livers .

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Western blot analysis of LAMP2A expression in lysosomes isolated from WT,Ctns−/− and cysteamine-treated Ctns−/− mouse livers.


Lysosomes , isolated as in ( B ) , were incubated with the CMA substrate GAPDH , and CMA activity was evaluated as in Fig8 and as described in Materials and Methods . Quantitative analysis of substrate degradation is expressed as percent of the residual amount of GAPDH in the full reaction relative to its amount in the presence of protease inhibitors . Results are mean ± SEM ( n = 4 ) . * P = 0 . 0126 for WT versus Ctns − / − and * P = 0 . 0436 for WT versus Ctns − / − + cysteamine ; NS , not significant ; unpaired t - test .

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Lysosomes, isolated as in (B), were incubated with the CMA substrate GAPDH, and CMA activity was evaluated as in Fig8 and as described in Materials and Methods.Quantitative analysis of substrate degradation is expressed as percent of the residual amount of GAPDH in the full reaction relative to its amount in the presence of protease inhibitors. Results are mean ± SEM (n = 4). *P = 0.0126 for WT versus Ctns−/− and *P = 0.0436 for WT versus Ctns−/− + cysteamine; NS, not significant; unpaired t-test.


B Relative ( left ) and absolute ( right ) amount of 13C - Lys - labeled and unlabeled Aβ in APPtg mouse brains after labeling from 75 - 125 d ( n = 1 ) , 75 - 175 d ( n = 4 ) , 50 - 100 d ( n = 5 ) , 50 - 125 d ( n = 2 ) , 50 - 150 ( n = 4 ) or 50 - 175 d ( n = 9 ) of age . Error bars show mean ± SD .

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B Relative (left) and absolute (right) amount of 13C-Lys-labeled and unlabeled Aβ in APPtg mouse brains after labeling from 75-125 d (n = 1), 75-175 d (n = 4), 50-100 d (n = 5), 50-125 d (n = 2), 50-150 (n = 4) or 50-175 d (n = 9) of age. Error bars show mean ± SD.


C Representative chromatogram and mass spectra of doubly charged ions of unlabeled Aβ17 - 28 ( 663 . 3 m / z ) and 13C - Lys Aβ17 - 28 ( 666 . 3 m / z ) in an APPtg brain after labeling from 75 - 175 d . % - ion intensity , m / z - mass - to - charge . Data were acquired in data - independent analysis mode ( MSE ) . D Representative chromatograms of unlabeled Aβ17 - 28 ( left ) and 13C - Lys Aβ17 - 28 ( right ) in an APPtg brain after labeling from 50 - 175 d . The intensities of 4 transitions ( y10 , y9 , y8 , and y7 ) were assessed . Data were acquired by MRM in nanoflow mode .

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C Representative chromatogram and mass spectra of doubly charged ions of unlabeled Aβ17-28 (663.3 m/z) and 13C-Lys Aβ17-28 (666.3 m/z) in an APPtg brain after labeling from 75-175 d. %- ion intensity, m/z- mass-to-charge. Data were acquired in data-independent analysis mode (MSE). D Representative chromatograms of unlabeled Aβ17-28 (left) and 13C-Lys Aβ17-28 (right) in an APPtg brain after labeling from 50-175 d. The intensities of 4 transitions (y10, y9, y8, and y7) were assessed. Data were acquired by MRM in nanoflow mode.


F Representative chromatograms of unlabeled Aβ17 - 28 ( left ) and 13C - Lys Aβ17 - 28 ( right ) in a non - APPtg brain after labeling from 90 - 100 d . Data were acquired by MRM in microflow mode .

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F Representative chromatograms of unlabeled Aβ17-28 (left) and 13C-Lys Aβ17-28 (right) in a non-APPtg brain after labeling from 90-100 d. Data were acquired by MRM in microflow mode.


A MALDI - TOF mass spectrum of trypsin - digested , synthetic Aβ1 - 42 ( 10 pmol ) . The Aβ17 - 28 [ MH ] + fragment was detected with the highest intensity . Measurements in reflectron positive mode . The sequence of Aβ1 - 42 with the tryptic cleavage sites is indicated at the

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A MALDI-TOF mass spectrum of trypsin-digested, synthetic Aβ1-42 (10 pmol). The Aβ17-28 [MH]+ fragment was detected with the highest intensity. Measurements in reflectron positive mode. The sequence of Aβ1-42 with the tryptic cleavage sites is indicated at the top


B MS / MS spectrum of unlabeled Aβ17 - 28 and 13C15N - Lys Aβ17 - 28 synthetic peptides in the 50 fmol range . m / z of precursors ions ( m / z 663 . 34 for Aβ17 - 28 [ MH ] 2 + , m / z 667 . 36 for 13C15N - Lys Aβ17 - 28 [ MH ] 2 + ) and their derived fragments are indicated as a series of y ions ( y4 - y10 ) . The peptides were analyzed in data independent acquisition MSE

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B MS/MS spectrum of unlabeled Aβ17-28 and 13C15N-Lys Aβ17-28 synthetic peptides in the 50 fmol range. m/z of precursors ions (m/z 663.34 for Aβ17-28 [MH]2+, m/z 667.36 for 13C15N-Lys Aβ17-28 [MH]2+) and their derived fragments are indicated as a series of y ions (y4-y10). The peptides were analyzed in data independent acquisition MSE mode


C Decreasing quantities of synthetic 13C15N - Lys Aβ1 - 42 were trypsin - digested and analyzed by MRM . The intensities of 4 transitions ( y10 , y9 , y8 , and y7 ) were assessed for 13C15N - Lys Aβ17 - 28 , showing a detection limit level of 25 amol . Measurements in technical triplicates , normalization to total ion current . Error bars show mean ±

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C Decreasing quantities of synthetic 13C15N-Lys Aβ1-42 were trypsin-digested and analyzed by MRM. The intensities of 4 transitions (y10, y9, y8, and y7) were assessed for 13C15N-Lys Aβ17-28, showing a detection limit level of 25 amol. Measurements in technical triplicates, normalization to total ion current. Error bars show mean ± SD


D Decreasing quantities of synthetic 13C15N - Lys Aβ1 - 42 were spiked into the insoluble fraction of APPtg brains , immunoprecipitated , trypsin - digested , and analyzed by MRM . Assessment of the intensities of y10 , y9 , y8 and y7 showed a detection limit level of 50 amol for 13C15N - Lys Aβ17 - 28 and detected an excess of brain - derived unlabeled Aβ17 - 28 . Measurements in technical triplicates , normalization to total ion current . Error bars show mean ±

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D Decreasing quantities of synthetic 13C15N-Lys Aβ1-42 were spiked into the insoluble fraction of APPtg brains, immunoprecipitated, trypsin-digested, and analyzed by MRM. Assessment of the intensities of y10, y9, y8 and y7 showed a detection limit level of 50 amol for 13C15N-Lys Aβ17-28 and detected an excess of brain-derived unlabeled Aβ17-28. Measurements in technical triplicates, normalization to total ion current. Error bars show mean ± SD


B Temporal MRM analysis of 13C - Lys Aβ17 - 28 and unlabeled Aβ17 - 28 in the liver , spleen , mesenteric lymph nodes , and peritoneal cells after 13C - Lys - APPtg brain extract i . p . injection ( n = 3 mice per time point and tissue at 1 - 10 d , except for n = 2 mice for peritoneal cells at 3 d ; n = 4 mice per time point and tissue at 25 - 100 d , except for n = 3 mice for spleen at 100 d ) . Measurements in technical triplicates , normalization to 13C15N - Lys Aβ17 - 28 intensity as a spiked internal standard and additionally to organ weight ( mg ) for lymph nodes . Error bars show median ± interquartile range .

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B Temporal MRM analysis of 13C-Lys Aβ17-28 and unlabeled Aβ17-28 in the liver, spleen, mesenteric lymph nodes, and peritoneal cells after 13C-Lys-APPtg brain extract i.p. injection (n = 3 mice per time point and tissue at 1-10 d, except for n = 2 mice for peritoneal cells at 3 d; n = 4 mice per time point and tissue at 25-100 d, except for n = 3 mice for spleen at 100 d). Measurements in technical triplicates, normalization to 13C15N-Lys Aβ17-28 intensity as a spiked internal standard and additionally to organ weight (mg) for lymph nodes. Error bars show median ± interquartile range.


C Temporal 4G8 immunoassay quantification of Aβ42 in the liver , spleen , mesenteric lymph nodes , and peritoneal cells after unlabeled APPtg ( n = 8 mice at 5 h - 7 d , except for n = 5 mice for peritoneal lavage , n = 5 mice at 10 - 14 d , n = 4 mice at 21 d ) or non - tg brain extract injection ( n = 2 mice at 5 h and 3 d , n = 3 mice at 1 d , n = 1 mouse at 5 and 7 d , except for peritoneal cells where n = 1 mouse at 5 h and 3 d , and n = 2 mice at 1 d ) . Error bars show median ± interquartile range . D Normalization of APPtg extract group data presented in ( C ) to organ weight ( mg ) and injected Aβ amount ( range 1 , 027 - 1 , 426 ng ) . Error bars show median ± interquartile range .

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C Temporal 4G8 immunoassay quantification of Aβ42 in the liver, spleen, mesenteric lymph nodes, and peritoneal cells after unlabeled APPtg (n = 8 mice at 5 h - 7 d, except for n = 5 mice for peritoneal lavage, n = 5 mice at 10-14 d, n = 4 mice at 21 d) or non-tg brain extract injection (n = 2 mice at 5 h and 3 d, n = 3 mice at 1 d, n = 1 mouse at 5 and 7 d, except for peritoneal cells where n = 1 mouse at 5 h and 3 d, and n = 2 mice at 1 d). Error bars show median ± interquartile range. D Normalization of APPtg extract group data presented in (C) to organ weight (mg) and injected Aβ amount (range 1,027-1,426 ng). Error bars show median ± interquartile range.


A Temporal MRM analysis of unlabeled Aβ17 - 28 and 13C - Lys Aβ17 - 28 in the soluble brain fraction after 13C - Lys APPtg brain extract i . p . injection ( n = 3 mice per time point at 1 - 10 d , n = 4 mice per time point at 25 - 100 d ) . Data information : measurements in technical triplicates , normalization to 13C15N - Lys Aβ17 - 28 intensity as a spiked internal standard and brain weight ( mg ) . All error bars show mean ± SD .

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A Temporal MRM analysis of unlabeled Aβ17-28 and 13C-Lys Aβ17-28 in the soluble brain fraction after 13C-Lys APPtg brain extract i.p. injection (n = 3 mice per time point at 1-10 d, n = 4 mice per time point at 25-100 d). Data information: measurements in technical triplicates, normalization to 13C15N-Lys Aβ17-28 intensity as a spiked internal standard and brain weight (mg). All error bars show mean ± SD.


Comparative MRM analysis of 13C - Lys Aβ17 - 28 ( B ) in the insoluble brain fraction after 13C - Lys - APPtg brain extract ( donors labeled for 50 - 125 d ) and 13C - Lys - non - tg brain extract ( donors labeled for 10 d ) i . p . injection ( n = 3 mice per group and time point at 1 - 10 d , n = 4 mice per group and time point at 25 , 50 , 100 d , n = 3 mice in the APPtg extract group and n = 4 in the non - tg extract group at 75 d ) , right side zoom for time points 1 - 10 d . Unpaired two - tailed t - test * p < 0 . 05 ( P = 0 . 0129 at 3 d , P = 0 . 0156 at 10 d , P = 0 . 0109 at 25 d ) , * * p < 0 . 01 ( P = 0 . 0091 at 1 d , P = 0 . 0011 at 5 d ) , * * * p < 0 . 001 ( P = 0 . 0006 at 7 d ) , 13C - Lys APPtg extract - injected vs 13C - Lys non - tg extract - injected for each time point . Data information : measurements in technical triplicates , normalization to 13C15N - Lys Aβ17 - 28 intensity as a spiked internal standard and brain weight ( mg ) . All error bars show mean ± SD .

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Comparative MRM analysis of 13C-Lys Aβ17-28 (B) in the insoluble brain fraction after 13C-Lys-APPtg brain extract (donors labeled for 50-125 d) and 13C-Lys-non-tg brain extract (donors labeled for 10 d) i.p. injection (n = 3 mice per group and time point at 1-10 d, n = 4 mice per group and time point at 25, 50, 100 d, n = 3 mice in the APPtg extract group and n = 4 in the non-tg extract group at 75 d), right side zoom for time points 1-10 d. Unpaired two-tailed t-test * p<0.05 (P=0.0129 at 3 d, P=0.0156 at 10 d, P=0.0109 at 25 d), ** p<0.01 (P=0.0091 at 1 d, P=0.0011 at 5 d), *** p<0.001 (P=0.0006 at 7 d), 13C-Lys APPtg extract-injected vs 13C-Lys non-tg extract-injected for each time point. Data information: measurements in technical triplicates, normalization to 13C15N-Lys Aβ17-28 intensity as a spiked internal standard and brain weight (mg). All error bars show mean ± SD.


C Comparative MRM analysis of unlabeled Aβ17 - 28 ( C ) in the insoluble brain fraction after 13C - Lys - APPtg brain extract ( donors labeled for 50 - 125 d ) and 13C - Lys - non - tg brain extract ( donors labeled for 10 d ) i . p . injection ( n = 3 mice per group and time point at 1 - 10 d , n = 4 mice per group and time point at 25 , 50 , 100 d , n = 3 mice in the APPtg extract group and n = 4 in the non - tg extract group at 75 d ) , right side zoom for time points 1 - 10 d . Unpaired two - tailed t - test * p < 0 . 05 ( P = 0 . 0129 at 3 d , P = 0 . 0156 at 10 d , P = 0 . 0109 at 25 d ) , * * p < 0 . 01 ( P = 0 . 0091 at 1 d , P = 0 . 0011 at 5 d ) , * * * p < 0 . 001 ( P = 0 . 0006 at 7 d ) , 13C - Lys APPtg extract - injected vs 13C - Lys non - tg extract - injected for each time point . Data information : measurements in technical triplicates , normalization to 13C15N - Lys Aβ17 - 28 intensity as a spiked internal standard and brain weight ( mg ) . All error bars show mean ± SD .

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C Comparative MRM analysis of unlabeled Aβ17-28 (C) in the insoluble brain fraction after 13C-Lys-APPtg brain extract (donors labeled for 50-125 d) and 13C-Lys-non-tg brain extract (donors labeled for 10 d) i.p. injection (n = 3 mice per group and time point at 1-10 d, n = 4 mice per group and time point at 25, 50, 100 d, n = 3 mice in the APPtg extract group and n = 4 in the non-tg extract group at 75 d), right side zoom for time points 1-10 d. Unpaired two-tailed t-test * p<0.05 (P=0.0129 at 3 d, P=0.0156 at 10 d, P=0.0109 at 25 d), ** p<0.01 (P=0.0091 at 1 d, P=0.0011 at 5 d), *** p<0.001 (P=0.0006 at 7 d), 13C-Lys APPtg extract-injected vs 13C-Lys non-tg extract-injected for each time point. Data information: measurements in technical triplicates, normalization to 13C15N-Lys Aβ17-28 intensity as a spiked internal standard and brain weight (mg). All error bars show mean ± SD.


D Comparative 4G8 immunoassay quantification of Aβ42 in the insoluble brain fraction after 13C - Lys APPtg or 13C - Lys non - tg brain extract i . p . injection ( n = 3 mice per group and time point at 1 - 10 d , n = 4 mice per group and time point at 25 - 100 d ) , right side zoom for time points 1 - 10 d . Normalization to protein concentration .

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D Comparative 4G8 immunoassay quantification of Aβ42 in the insoluble brain fraction after 13C-Lys APPtg or 13C-Lys non-tg brain extract i.p. injection (n = 3 mice per group and time point at 1-10 d, n = 4 mice per group and time point at 25-100 d), right side zoom for time points 1-10 d. Normalization to protein concentration.


E Representative chromatograms of unlabeled Aβ17 - 28 ( left ) and 13C - Lys Aβ17 - 28 ( right ) in the brain of an unlabeled 50 - day - old APPtg mouse not injected with labeled brain extract . Note the absence of 13C - Lys Aβ17 - 28 .

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E Representative chromatograms of unlabeled Aβ17-28 (left) and 13C-Lys Aβ17-28 (right) in the brain of an unlabeled 50-day-old APPtg mouse not injected with labeled brain extract. Note the absence of 13C-Lys Aβ17-28.


F MRM analysis of unlabeled and 13C - Lys - labeled Aβ17 - 28 in APPtg mouse brains after 13C - Lys - labeled diet for 1 or 3 d ( n = 3 mice per time point ) . Data information : measurements in technical triplicates , normalization to 13C15N - Lys Aβ17 - 28 intensity as a spiked internal standard and brain weight ( mg ) . All error bars show mean ± SD .

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F MRM analysis of unlabeled and 13C-Lys-labeled Aβ17-28 in APPtg mouse brains after 13C-Lys-labeled diet for 1 or 3 d (n = 3 mice per time point). Data information: measurements in technical triplicates, normalization to 13C15N-Lys Aβ17-28 intensity as a spiked internal standard and brain weight (mg). All error bars show mean ± SD.


G Comparative MRM analysis of 13C - Lys Aβ17 - 28 in the insoluble brain fraction after 13C - Lys APPtg and 13C - Lys non - tg brain extract ( all donors labeled for 100 d ) i . p . injection ( n = 6 mice per group ) . Data information : measurements in technical triplicates , normalization to 13C15N - Lys Aβ17 - 28 intensity as a spiked internal standard and brain weight ( mg ) . All error bars show mean ± SD .

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G Comparative MRM analysis of 13C-Lys Aβ17-28 in the insoluble brain fraction after 13C-Lys APPtg and 13C-Lys non-tg brain extract (all donors labeled for 100 d) i.p. injection (n = 6 mice per group). Data information: measurements in technical triplicates, normalization to 13C15N-Lys Aβ17-28 intensity as a spiked internal standard and brain weight (mg). All error bars show mean ± SD.


H MRM analysis of unlabeled and 13C - Lys - labeled Aβ17 - 28 in non - APPtg mouse brains after 13C - Lys - labeled diet for 1 d ( n = 3 mice ) or at 1 d after 13C - Lys - APPtg brain extract i . p . injection ( n = 3 mice ) . Data information : measurements in technical triplicates , normalization to 13C15N - Lys Aβ17 - 28 intensity as a spiked internal standard and brain weight ( mg ) . All error bars show mean ± SD .

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H MRM analysis of unlabeled and 13C-Lys-labeled Aβ17-28 in non-APPtg mouse brains after 13C-Lys-labeled diet for 1 d (n = 3 mice) or at 1 d after 13C-Lys-APPtg brain extract i.p. injection (n = 3 mice). Data information: measurements in technical triplicates, normalization to 13C15N-Lys Aβ17-28 intensity as a spiked internal standard and brain weight (mg). All error bars show mean ± SD.


A ) H1299 cell line was transfected with increasing concentrations of MDM2 , and the RB + 5 ´ UTR exogenous ( left panel ) and endogenous ( right panel ) levels were evaluated . B ) Evaluation of the RB expression as in ( A ) but inducing DNA damage with doxorubicin 1 µM during 16 h .

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A) H1299 cell line was transfected with increasing concentrations of MDM2, and the RB+5´UTR exogenous (left panel) and endogenous (right panel) levels were evaluated. B) Evaluation of the RB expression as in (A) but inducing DNA damage with doxorubicin 1 µM during 16 h.


C ) Evaluation of the RB levels in an MDM2 dose - dependent fashion under normal and genotoxic stress induced by doxorubicin 16 h in HEK 293 cell line . D ) Evaluation of the RB levels in a MDM2 dose - dependent fashion under normal and genotoxic stress induced by doxorubicin 16 h in C33 cell line .

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C) Evaluation of the RB levels in an MDM2 dose-dependent fashion under normal and genotoxic stress induced by doxorubicin 16 h in HEK 293 cell line. D) Evaluation of the RB levels in a MDM2 dose-dependent fashion under normal and genotoxic stress induced by doxorubicin 16 h in C33 cell line.


E ) Evaluation of the endogenous RB expression using H1299 cell line , under normal conditions , transfecting the cells with an increasing amount of the phosphomimetic mutant MDM2 ( S395D ) . F ) Evaluation of the endogenous RB as in ( E ) but using C33 cell line .

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E) Evaluation of the endogenous RB expression using H1299 cell line, under normal conditions, transfecting the cells with an increasing amount of the phosphomimetic mutant MDM2(S395D). F) Evaluation of the endogenous RB as in (E) but using C33 cell line.


A ) Evaluation of RB mRNA levels , normalised by GAPDH , in an MDM2 dose - dependent manner . The H1299 cells were treated with doxorubicin during 16 h to induce genotoxic stress . Endogenous levels ( upper panel ) ; exogenous levels ( middle panel ) ; endogenous levels of RB but using the MDM2 ( S395D ) under normal conditions ( lower panel ) .

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A) Evaluation of RB mRNA levels, normalised by GAPDH, in an MDM2 dose-dependent manner. The H1299 cells were treated with doxorubicin during 16 h to induce genotoxic stress. Endogenous levels (upper panel); exogenous levels (middle panel); endogenous levels of RB but using the MDM2(S395D) under normal conditions (lower panel).


B ) Kinetic of the endogenous RB expression in H1299 cell line from 0 to 48h . The cells were treated with cichlohexamide ( CHX ) was added to stop translation , under normal conditions ( upper panel ) and treated with doxorubicin ( lower panel ) . The cells were transfected with MDM2 .

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B) Kinetic of the endogenous RB expression in H1299 cell line from 0 to 48h. The cells were treated with cichlohexamide (CHX) was added to stop translation, under normal conditions (upper panel) and treated with doxorubicin (lower panel). The cells were transfected with MDM2.


C ) Kinetic of the endogenous RB expression in H1299 under genotoxic stress conditions in presence ( upper panel ) or in absence ( lower panel ) of CHX .

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C) Kinetic of the endogenous RB expression in H1299 under genotoxic stress conditions in presence (upper panel) or in absence (lower panel) of CHX.


D ) Evaluation of the levels of expression of RB treated with doxorubicin to generate stress in absence of CHX .

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D) Evaluation of the levels of expression of RB treated with doxorubicin to generate stress in absence of CHX.


Different constructs of RB were transfected in H1299 - RBKO treated with doxorubicin 16 h to induce genotoxic stress ; the RB constructs levels of expression were evaluated using HA antibody . The bar diagrams show the quantification of the western blots data . A ) Evaluation of the small pocket expression in an MDM2 dose - dependent manner . B ) Cells were transfected with the RB large pocket construct .

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Different constructs of RB were transfected in H1299-RBKO treated with doxorubicin 16 h to induce genotoxic stress; the RB constructs levels of expression were evaluated using HA antibody. The bar diagrams show the quantification of the western blots data. A) Evaluation of the small pocket expression in an MDM2 dose-dependent manner. B) Cells were transfected with the RB large pocket construct.


Different constructs of RB were transfected in H1299 - RBKO treated with doxorubicin 16 h to induce genotoxic stress ; the RB constructs levels of expression were evaluated using HA antibody . The bar diagrams show the quantification of the western blots data . C ) Cells were transfected with RB full length without the 5 ´ URT ( named RB - HA ) . D ) Cells were transfected with the RB full - length plus 260 nt of its own 5 ´ UTR ( named RB + 5 ´ UTR ) .

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Different constructs of RB were transfected in H1299-RBKO treated with doxorubicin 16 h to induce genotoxic stress; the RB constructs levels of expression were evaluated using HA antibody. The bar diagrams show the quantification of the western blots data. C) Cells were transfected with RB full length without the 5´URT (named RB-HA). D) Cells were transfected with the RB full-length plus 260 nt of its own 5´ UTR (named RB+5´UTR).


E ) The binding of MDM2 to the RB mRNA in H1299 cells as estimated using RNA CoIP . Cells were treated with doxorubicin ( 1 µM for 16 h ) or DMSO .

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E) The binding of MDM2 to the RB mRNA in H1299 cells as estimated using RNA CoIP. Cells were treated with doxorubicin (1 µM for 16 h) or DMSO.


F ) RT - qPCR of RB and GAPDH of the MDM2 - immunoprecipitation from the polysomal fractionation ( see EV2D ) . Data are representative of three similar independent experiments .

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F) RT-qPCR of RB and GAPDH of the MDM2-immunoprecipitation from the polysomal fractionation (see EV2D). Data are representative of three similar independent experiments.


G ) PLA assay using anti - RB and anti - MDM2 antibodies , H1299 cell were treated or not with etoposide to induce stress , the endogenous proteins were visualised . Cell nuclei were visualised with DAPI blue . The red dots show the interaction between the two proteins . The scale bar corresponds to 20 μm .

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G) PLA assay using anti-RB and anti-MDM2 antibodies, H1299 cell were treated or not with etoposide to induce stress, the endogenous proteins were visualised. Cell nuclei were visualised with DAPI blue. The red dots show the interaction between the two proteins. The scale bar corresponds to 20 μm.


H ) RNA - PLA assay using rabbit anti - MDM2 and a mouse biotin - antibody . H1299 - RBKO was stable transfected with RB - HA or RB + 5 ´ UTR . Cell nuclei were visualised with DAPI blue . The red dots show the interaction between the protein and the mRNA . The scale bar corresponds to 20 μm .

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H) RNA-PLA assay using rabbit anti-MDM2 and a mouse biotin-antibody. H1299- RBKO was stable transfected with RB-HA or RB+5´UTR. Cell nuclei were visualised with DAPI blue. The red dots show the interaction between the protein and the mRNA. The scale bar corresponds to 20 μm.


A ) Cell cycle analysis ( FACS ) of C33 cell line after treatment with etoposide . The DNA contents of the cells were evaluated by propidium iodide staining , ( PE - A ; Phycoerythrin ) . The inset shows the % of the cells in each cell cycle phase .

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A) Cell cycle analysis (FACS) of C33 cell line after treatment with etoposide. The DNA contents of the cells were evaluated by propidium iodide staining, (PE-A; Phycoerythrin). The inset shows the % of the cells in each cell cycle phase.


B ) The cell cycle analysis of synchronised C33 cell line during the time .

B) The cell cycle analysis of synchronised C33 cell line during the time.


C ) Evaluation of increasing concentrations of MDM2 on the cell cycle of C33 cell line after genotoxic stress induced with etoposide , ( EV ; empty vector ) . D ) The same as in ( C ) but under normal cellular conditions .

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C) Evaluation of increasing concentrations of MDM2 on the cell cycle of C33 cell line after genotoxic stress induced with etoposide, (EV; empty vector). D) The same as in (C) but under normal cellular conditions.


E ) RB levels of the cells after 14 h of the release of nocodazole treatment . Etoposide was used to induce genotoxic stress .

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E) RB levels of the cells after 14 h of the release of nocodazole treatment. Etoposide was used to induce genotoxic stress.


F ) Evaluation of the G1 cell cycle phase after 14 h of the release of nocodazole treatment . Data information : Data are means ± s . d . of three independent experiments , student t test was used to calculate statistical significance ( * p < 0 . 05 , * * p < 0 . 01 ) .

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F) Evaluation of the G1 cell cycle phase after 14 h of the release of nocodazole treatment. Data information: Data are means ± s.d. of three independent experiments, student t test was used to calculate statistical significance (*p <0.05, **p <0.01).


A ) Ubiquitination of endogenous RB in C33 cells synchronised in the G1 or G2 / M cell cycle phases under stress conditions .

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A) Ubiquitination of endogenous RB in C33 cells synchronised in the G1 or G2/M cell cycle phases under stress conditions.


B ) In vitro RB ubiquitination by MDM2 wild type and the phosphomimetic mutant MDM2 ( S395D ) .

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B) In vitro RB ubiquitination by MDM2 wild type and the phosphomimetic mutant MDM2(S395D).


C ) In vitro RB ubiquitination by MDM2 ( S395D ) in presence or absence of RB mRNA .

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C) In vitro RB ubiquitination by MDM2(S395D) in presence or absence of RB mRNA.


D ) A representative clonogenic assay . Increasing concentrations of MDM2 were transfected in H1299 - RBKO stable transfected with RB - HA or RB + 5 ´ UTR . The cells were treated with doxorubicin to induce genotoxic stress conditions . Data are means ± s . d . of three independent experiments , student t test was used to calculate statistical significance ( * p < 0 . 05 ) .

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D) A representative clonogenic assay. Increasing concentrations of MDM2 were transfected in H1299-RBKO stable transfected with RB-HA or RB+5´UTR. The cells were treated with doxorubicin to induce genotoxic stress conditions. Data are means ± s.d. of three independent experiments, student t test was used to calculate statistical significance (*p <0.05).


E ) Evaluation of cyclin E mRNA levels in an MDM2 dose - dependent manner . The H1299 cells were treated with doxorubicin during 16 h to induce genotoxic stress . Data are means ± s . d . of three independent experiments , student t test was used to calculate statistical significance ( * p < 0 . 05 , * * * p < 0 . 001 ) .

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E) Evaluation of cyclin E mRNA levels in an MDM2 dose-dependent manner. The H1299 cells were treated with doxorubicin during 16 h to induce genotoxic stress. Data are means ± s.d. of three independent experiments, student t test was used to calculate statistical significance (*p <0.05, ***p< 0.001).


b - d , MEFs were cultured in nutrient - rich medium ( fed ) or Hanks ' buffer ( starved ) for 1 h . Total cell lysates were prepared , and blotted with the indicated antibodies ( b ) .

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b-d, MEFs were cultured in nutrient-rich medium (fed) or Hanks' buffer (starved) for 1 h. Total cell lysates were prepared, and blotted with the indicated antibodies (b).


α , β , indicate Atg16L1 isoform α , β ; ∆ CCD indicates Atg16L1 ∆ CCD ; asterisk indicates a non - specific band . Atg16L1 ∆ / ∆ indicates mice expressing ∆ Atg16L1 . The number of endogenous LC3 or GFP - Atg5 dots was counted ( c , d ) . The results shown are mean + s . d . ( n > 20 ) .

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α, β, indicate Atg16L1 isoform α, β; ∆CCD indicates Atg16L1∆CCD; asterisk indicates a non-specific band. Atg16L1∆/∆ indicates mice expressing ∆Atg16L1. The number of endogenous LC3 or GFP-Atg5 dots was counted (c, d). The results shown are mean + s.d. (n > 20).


e , Electron micrograph of starved MEFs .

e, Electron micrograph of starved MEFs.


f , Degradation of long - lived proteins in MEFs . Wortmannin is an autophagy inhibitor . The results shown are mean1s . d . Statistical significance ( P value ) was determined by the Student ' s t - test .

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f, Degradation of long-lived proteins in MEFs. Wortmannin is an autophagy inhibitor. The results shown are mean1s.d. Statistical significance (P value) was determined by the Student's t-test.


a , Cytokine production from macrophages stimulated with LPS ( 100 ng ml - 1 ) for 24 h . Statistical significance was determined by the Student ' s t - test . * P 0 . 01 .

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a, Cytokine production from macrophages stimulated with LPS (100 ng ml-1) for 24 h. Statistical significance was determined by the Student's t-test. *P  0.01.


b , IL - 1b production from macrophages stimulated with indicated ligands .

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b, IL-1b production from macrophages stimulated with indicated ligands.


c , IL - 1b production from LPS - primed macrophages infected with S . typhimurium ( multiplicity of infection , m . o . i . , 1 ) , or stimulated with ATP or MSU for 1 h .

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c, IL-1b production from LPS-primed macrophages infected with S. typhimurium (multiplicity of infection, m.o.i., 1), or stimulated with ATP or MSU for 1 h.


d , Expression levels of caspase - 1 and IL - 1b in macrophages . Lys . , cell lysates ; Sup . , culture supernatants .

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d, Expression levels of caspase-1 and IL-1b in macrophages. Lys., cell lysates; Sup., culture supernatants.


e , LPS - induced production of IL - 1b from macrophages with the indicated phenotype .

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e, LPS-induced production of IL-1b from macrophages with the indicated phenotype.


f , Expression levels of caspase - 1 and IL - 1b in macrophages treated as in e .

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f, Expression levels of caspase-1 and IL-1b in macrophages treated as in e.


g , Effect of the ROS scavenger FeTPPS ( 25 mM ) , N - acetyl - L - cysteine ( NAC ; 25mM ) or P2X7 receptor antagonist oxidized ATP ( 250 mM ) on IL - 1b production .

g, Effect of the ROS scavenger FeTPPS (25 mM), N-acetyl-L-cysteine (NAC; 25mM) or P2X7 receptor antagonist oxidized ATP (250 mM) on IL-1b production.


h , ROS in LPS - stimulated macrophages were detected by CM - H2DCFDA staining .

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h, ROS in LPS-stimulated macrophages were detected by CM-H2DCFDA staining.


a , Macrophages were stimulated with LPS ( 100 ng ml - 1 ) for the indicated time period . The number of endogenous LC3 dots within each cell was counted . The results shown are mean + s . d . ( n > 100 ) .

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a, Macrophages were stimulated with LPS (100 ng ml-1) for the indicated time period. The number of endogenous LC3 dots within each cell was counted. The results shown are mean + s.d. (n > 100).


b , Expression levels of p62 and LC3 in macrophages .

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b, Expression levels of p62 and LC3 in macrophages.


c , IL - 1b and IL - 6 production by wild - type or Atg7 - deficient macrophages stimulated with LPS . Statistical significance was determined by the Student ' s t - test . * P , 0 . 01 .

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c, IL-1b and IL-6 production by wild-type or Atg7-deficient macrophages stimulated with LPS. Statistical significance was determined by the Student's t-test. *P,0.01.


d , Macrophages were pre - treated with or without 10 mM 3MA and then stimulated with the indicated ligands .

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d, Macrophages were pre-treated with or without 10 mM 3MA and then stimulated with the indicated ligands.


a , b , Fetal liver chimaeric mice were given 5 % DSS in drinking water for 7 days . The survival ( a ) and weight loss ( b ) of each mouse genotype were plotted . The results shown are mean ± s . d . Statistical significance was determined by the Students t - test .

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a, b, Fetal liver chimaeric mice were given 5% DSS in drinking water for 7 days. The survival (a) and weight loss (b) of each mouse genotype were plotted. The results shown are mean ± s.d. Statistical significance was determined by the Students t-test.


c , Typical distal colon appearance 6 days after the initiation of DSS administration . The results shown are mean ± s . d . ( n = 3 , each group ) .

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c, Typical distal colon appearance 6  days after the initiation of DSS administration. The results shown are mean ± s.d. (n = 3, each group).


d , Expression levels of IL - 1b and IL - 18 in serum ( n55 , each group ) .

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d, Expression levels of IL-1b and IL-18 in serum (n55, each group).


e , f , Atg16L1 - deficient chimaeric mice given 5 % DSS in drinking water were intraperitoneally injected with both anti - IL - 1b and anti - IL - 18 neutralizing antibodies ( squares ; n55 ) or isotype control IgG ( circles ; n55 ) at days 1 , 3 , 5 and 7 . The survival ( e ) and weight loss ( f ) of each mouse genotype were plotted .

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e, f, Atg16L1-deficient chimaeric mice given 5% DSS in drinking water were intraperitoneally injected with both anti-IL-1b and anti- IL-18 neutralizing antibodies (squares; n55) or isotype control IgG (circles;n55) at days 1, 3, 5 and 7. The survival (e) and weight loss (f) of each mouse genotype were plotted.


B Social interaction ratio in control , susceptible and resilient mice following the CSDS protocol ( n = 44 , 55 , 44 mice ) .

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B Social interaction ratio in control, susceptible and resilient mice following the CSDS protocol (n = 44, 55, 44 mice).


C Sucrose preference in control , susceptible and resilient mice ( n = 20 , 20 , 12 mice . Treatment F2 , 49 = 16 . 05 , P < 0 . 0001 , one - way ANOVA with Fisher ' s LSD test ) .

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C Sucrose preference in control, susceptible and resilient mice (n = 20, 20, 12 mice. Treatment F2, 49 = 16.05, P < 0.0001, one-way ANOVA with Fisher's LSD test).


D The immobility time in TST in susceptible mice , compared to control mice ( n = 16 , 32 mice . Susceptible vs control , P = 0 . 002 , Student ' s t test ) .

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D The immobility time in TST in susceptible mice, compared to control mice (n = 16, 32 mice. Susceptible vs control, P = 0.002, Student's t test).


E The immobility time in FST in susceptible mice , compared to control mice ( n = 17 , 35 mice . Susceptible vs control , P = 0 . 0003 , Student ' s t test ) .

E The immobility time in FST in susceptible mice, compared to control mice (n = 17, 35 mice. Susceptible vs control, P = 0.0003, Student's t test).


F , G The time in the open arms ( F ) and closed arms ( G ) in the EPM in susceptible mice , compared to control mice ( n = 19 , 33 mice . Susceptible vs control , for open arms , P < 0 . 0001 ; For closed time , P < 0 . 0001 , Student ' s t test ) .

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F, G The time in the open arms (F) and closed arms (G) in the EPM in susceptible mice, compared to control mice (n = 19, 33 mice. Susceptible vs control, for open arms, P < 0.0001; For closed time, P < 0.0001, Student's t test).


H The central distance in the OFT in susceptible mice , compared to control mice ( n = 13 , 28 mice . Susceptible vs control , P = 0 . 0089 , Student ' s t test ) .

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H The central distance in the OFT in susceptible mice, compared to control mice (n = 13, 28 mice. Susceptible vs control, P = 0.0089, Student's t test).


I The mRNA level of CD39 , ENTPD2 and ENTPD3 in the hippocampus of susceptible mice after CSDS ( n = 7 , 8 mice . CSDS vs control , CD39 P = 0 . 0002 , ENTPD2 P = 0 . 0025 , Student ' s t test ) .

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I The mRNA level of CD39, ENTPD2 and ENTPD3 in the hippocampus of susceptible mice after CSDS (n = 7, 8 mice. CSDS vs control, CD39 P = 0.0002, ENTPD2 P = 0.0025, Student's t test).


J The CD39 protein expression in the hippocampus of susceptible mice induced by CSDS ( n = 6 , 7 mice . CSDS vs control , P = 0 . 0003 , Student ' s t test ) .

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J The CD39 protein expression in the hippocampus of susceptible mice induced by CSDS (n = 6, 7 mice. CSDS vs control, P = 0.0003, Student's t test).


K The ATPase activity in the hippocampus of stressed animals ( n = 6 mice / group . CSDS vs control , P = 0 . 0041 , Student ' s t test ) .

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K The ATPase activity in the hippocampus of stressed animals (n = 6 mice/group. CSDS vs control, P =0.0041, Student's t test).


B The effects of apyrase ( 40 , 80 and 160 U / ml ) infusion into cerebral ventricle on social interaction time ( n = 8 , 10 , 10 , 10 mice . Interaction F3 , 34 = 3 . 917 , P = 0 . 0167 ; Target F1 , 34 = 0 . 0004 , P = 0 . 9834 ; Drug F3 , 34 = 2 . 295 , P = 0 . 0954 ; For target , Apyrase 80 U / ml vs control , P = 0 . 0008 , Two - way ANOVA with Tukey ' s post - test ) .

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B The effects of apyrase (40, 80 and 160 U/ml) infusion into cerebral ventricle on social interaction time (n = 8, 10, 10, 10 mice. Interaction F3, 34 = 3.917, P = 0.0167; Target F1, 34 = 0.0004, P = 0.9834; Drug F3, 34 = 2.295, P = 0.0954; For target, Apyrase 80 U/ml vs control, P = 0.0008, Two-way ANOVA with Tukey's post-test).


C The effects of apyrase ( 40 , 80 and 160 U / ml ) infusion into cerebral ventricle on sucrose preference ( n = 8 , 10 , 9 , 11 mice . Treatment F3 , 34 = 2 . 87 , P = 0 . 0505 ; Apyrase 80 U / ml vs control , P = 0 . 007 , one - way ANOVA with Fisher ' s LSD test ) .

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C The effects of apyrase (40, 80 and 160 U/ml) infusion into cerebral ventricle on sucrose preference (n = 8, 10, 9, 11 mice. Treatment F3, 34 = 2.87, P = 0.0505; Apyrase 80 U/ml vs control, P = 0.007, one-way ANOVA with Fisher's LSD test).


E The effect of apyrase ( 40 U / ml ) infusion into hippocampal on the social interaction time ( n = 14 , 20 mice . Interaction F1 , 32 = 7 . 0754 , P = 0 . 0121 ; Target F1 , 32 = 0 . 68447 , P = 0 . 4142 ; Drug F1 , 32 = 18 . 211 , P = 0 . 0002 ; Apyrase vs control , P < 0 . 0001 , Student ' s t test ) .

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E The effect of apyrase (40 U/ml) infusion into hippocampal on the social interaction time (n = 14, 20 mice. Interaction F1, 32 = 7.0754, P = 0.0121; Target F1, 32 = 0.68447, P = 0.4142; Drug F1, 32 = 18.211, P = 0.0002; Apyrase vs control, P < 0.0001, Student's t test).


F The effect of apyrase ( 40 U / ml ) infusion into hippocampal on the sucrose preference ( n = 10 , 16 mice . Apyrase vs control , P = 0 . 0003 , Student ' s t test ) .

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F The effect of apyrase (40 U/ml) infusion into hippocampal on the sucrose preference (n = 10, 16 mice. Apyrase vs control, P = 0.0003, Student's t test).


G The social avoidance behavior in mice with ATP intra - hippocampal infusion after apyrase exposure ( n = 14 , 10 , 10 mice . Interaction F2 , 31 = 2 . 051 , P = 0 . 1457 ; Target F1 , 31 = 2 . 666 , P = 0 . 1127 ; Drug F2 , 31 = 3 . 711 , P = 0 . 0359 ; For Target , Apyrase - ACSF vs Vehicle , P = 0 . 0387 ; Apyrase - ATP vs Apyrase - ACSF , P = 0 . 0099 , Two - way ANOVA with Tukey ' s post - test ) .

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G The social avoidance behavior in mice with ATP intra-hippocampal infusion after apyrase exposure (n = 14, 10, 10 mice. Interaction F2, 31 = 2.051, P = 0.1457; Target F1, 31 = 2.666, P = 0.1127; Drug F2, 31 = 3.711, P = 0.0359; For Target, Apyrase-ACSF vs Vehicle, P = 0.0387; Apyrase-ATP vs Apyrase-ACSF, P = 0.0099, Two-way ANOVA with Tukey's post-test).