B . Similar as described in supplementary figure 2D was carried out in presence of a small molecule inhibitor against Aurora - B ( ZM447439 ) in combination with proteasome inhibitor MG - 132 to keep arrested in mitosis in B . with an inhibitor against Plk1 ( BI2536 ) . Drugs were added at 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 quantifications of KT - localized Plk1 in U2OS after PBIP1 depletion . were either Mock transfected or with PBIP1 siRNA for 72 hours . were treated with nocodazole for 1 hour fixed . were stained with centromere marker ACA , Plk1 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 histograms showing effect of chromosome position on cortical LGN enrichment after 72 hours of Mock 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 histograms of GFP - H2B U2OS Mock transfected or with siPBIP1 filmed on rectangular micropatterns in presence of a CENP - E inhibitor . 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 . total spindle angle displacement within first hour of mitosis was determined by measuring spindle angle at each time point calculating sum of 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 of autophagosome marker LC3B - II were evaluated in wild - type ( WT ) 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 Ctns − / − fibroblasts were stained using LC3B antibodies analyzed by microscopy . number of LC3B - positive was quantified by ImagePro software normalized per arbitrary units . Results are mean ± ( n = 27 WT 24 Ctns − / − ) . * * * P < 0 . 001 ( unpaired t - ) .

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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 of liver ( C ) kidney ( D ) tissues from WT Ctns − / − GFP - LC3 transgenic mice using anti - GFP antibodies . of LC3 was obtained by counting number of GFP - positive structures relative to number of nuclei in same field , using ImagePro software . GFP - LC3 were counted by analyzing 6 to 10 fields per tissue section ( 200 - 400 nuclei per field ) , in a total of 3 WT 3 Ctns − / − mice expressing GFP - LC3 . Results are mean ± . In ( C ) , * * P = 0 . 0029 ; in ( D ) , * * P = 0 . 0025 ( unpaired t - ) . 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 of wild - type ( WT ) Ctns − / − mouse fibroblasts transfected with ptfLC3 vector under resting conditions ( fed ) , or after serum starvation ( Serum Starv . ) in presence or absence of alkalinizing drug chloroquine ( CQ ) . GFP RFP was analyzed by confocal microscopy . Examples of red - only ( mature autophagosomes ) are indicated with arrows . Scale bar : 5 μm . of mature autophagosomes ( red - only vesicles ) was calculated on between number of red - only total number of autophagosomes ( number of green red + red - only ) . graph is representative of three different experiments with similar results . Results are mean ± ( n = 7 per condition ) . In addition to experimental conditions shown in ( A ) , 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 Ctns − / − fibroblasts under resting conditions were treated with 100 nM bafilomycin A ( BafA ) for 2 h LC3B - II 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 of mTOR kinase 1 substrate S6K LC3B - II in WT Ctns − / − fibroblasts were measured by WB under resting conditions ( − ) , withdrawal of both serum ( Aa / Ser . Starv . ) subsequent recovery by replacement of starvation medium with normal growth medium ( Rec ) , in presence or absence of 100 nM BafA for 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 showing phosphorylation of mTOR substrate S6K expression of LC3B - II in resting or serum - starved WT , Ctns − / − 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.


of 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 lysates were resolved by SDS - PAGE analyzed by Western blot ( WB ; A ) . Equal loading was established by silver ( B ) . ( mean ± ) 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 using metabolic labeling was performed as described in Supplementary Materials Methods . data represent mean ± of nine independent samples analyzed in three independent experiments . * P = 0 . 009 for WT * P = 0 . 0013 for Ctns − / − , respectively . NS , not significant ( unpaired t - ) .

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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).


expression of endogenous LAMP2A LAMP1 were analyzed in wild - type ( WT ) Ctns − / − mousefibroblasts ( A ) , kidney ( B ) liverlysosomes ( C ) by Western blot . Quantitative densitometry of immunoblots obtained from different experiments was performed by calculating between LAMP2A actin signals in each lane . Results are mean ± of four independent experiments with mousefibroblasts , 5 WT 6 Ctns − / − mousekidneys , 3 WT 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 of amount of LAMP2A in wild - type or Ctns − / − liverlysosomes was performed using two different anti - LAMP2A antibodies . 15 μg of lysosomal were loaded in each lane . Ab1 , anti - LAMP2A antibody raised against twelve of 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 .

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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 ) total LAMP2 ( D ) localization in wild - type ( WT ) Ctns − / − mouse fibroblasts . ( A ) Confocal microscopy . arrows represent LAMP1 - positive circular structures ( lysosomes ) which were visible in both WT Ctns − / − . Colocalization of LAMP1 LAMP2A was observed in WT fibroblasts ( arrows , merged panel ) but rarely in Ctns − / − ( red arrow ) . Most LAMP2A showed a punctate pattern lack of colocalization with LAMP1 in Ctns − / − ( 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 ) total LAMP2 ( D ) localization in wild - type ( WT ) Ctns − / − mouse fibroblasts . ( B ) High - resolution stochastic optical reconstruction microscopy ( STORM ) of localization of LAMP1 LAMP2A in wild - type Ctns − / − . In Ctns − / − , LAMP2A was detected near ( arrowheads , estimated > 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 ) total LAMP2 ( D ) localization in wild - type ( WT ) Ctns − / − mouse fibroblasts . ( C ) Immunofluorescence showing colocalization of endogenous LAMP1 LAMP2B in both wild - type 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 ) total LAMP2 ( D ) localization in wild - type ( WT ) Ctns − / − mouse fibroblasts . ( D ) Immunofluorescence of LAMP1 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 ( arrow ) , VAMP7 - positive vesicles ( B ) Rab11a - positive structures ( C ) was performed using wild - type or Ctns − / − fibroblasts as described in Materials Methods . At least 2 independent experiments were performed for each vesicular trafficking or ER marker . ( B , C ) Results are expressed as of LAMP2A that colocalizes with VAMP7 or Rab11 . For Rab11 / LAMP2A colocalization , 29 wild - type 15 Ctns − / − were quantified . For VAMP7 / LAMP2A colocalization ( arrows ) , 49 WT 57 Ctns − / − were analyzed . Data are expressed as mean ± . * P < 0 . 001 ( unpaired t - ) . Scale bars : 20 μm .

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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 chloroquine ( CQ / Leu ) or bafilomycin A ( BafA ) alone were fixed immunostained with antibodies recognizing endogenous LAMP1 LAMP2A samples were analyzed by confocal microscopy as described in Material Methods . Lysosomal colocalization of LAMP1 LAMP2A was evident in treated ( middle lower bottom panels ) but not in untreated ( middle upper panels ) Ctns − / − . 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 ) Ctns − / − mice as described in Supplementary Materials Methods incubated at 37 ° C for 30 min with CMA substrate GAPDH , in presence or absence of ATP ( necessary for CMA ) protease inhibitors ( Prot . Inhib . ) . A fraction of CMA reactions was then mixed with sample buffer boiled at 95 ° C for 5 min , followed by SDS - PAGE GAPDH LAMP1 immunoblotting . Quantitative densitometry of CMA activity performed in independent experiments using lysosomes isolated from a total of 11 WT 10 Ctns − / − mice . Results are mean ± . * * * P = 0 . 0005 ; * * P = 0 . 0041 ; N . S . , not significant ( unpaired t - ) .

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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).


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

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

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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 Ctns − / − were treated as described above samples analyzed by Western blot using antibodies recognizing ( 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 stained with anti - LAMP1 LAMP2A antibodies as indicated . For better visualization of LAMP1 / LAMP2A colocalization , GFP - CTNS was pseudocolored as magenta 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 − / − in cysteamine - treated Ctns − / − ) . Rescue of LAMP2A localization to lysosomes was observed in Ctns − / − expressing GFP - CTNS . Scale bar : 2 μm . of colocalization described in ( B ) . Calculation of Pearson ' s colocalization coefficient was done by analyzing 113 WT , 251 Ctns − / − , 88 cysteamine - treated 164 GFP - CTNS - expressing Ctns − / − , by using ZEN 2010 software . Results are mean ± . * * * P < 0 . 001 ; NS , not significant ( one - way ANOVA , Bonferroni ' s multiple comparisons ) .

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

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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 of LAMP2A expression in lysosomes isolated from WT , Ctns − / − 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 CMA substrate GAPDH , CMA activity was evaluated as in Fig8 as described in Materials Methods . Quantitative of substrate degradation is expressed as of residual amount of GAPDH in full reaction relative to its amount in presence of protease inhibitors . Results are mean ± ( n = 4 ) . * P = 0 . 0126 for WT versus Ctns − / − * P = 0 . 0436 for WT versus Ctns − / − + cysteamine ; NS , not significant ; unpaired t - .

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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 ) absolute ( right ) amount of 13C - Lys - labeled 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 . 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 mass spectra of doubly charged ions of unlabeled Aβ17 - 28 ( 663 . 3 m / z ) 13C - Lys Aβ17 - 28 ( 666 . 3 m / z ) in an APPtg brain after labeling from 75 - 175 d . % - ion , m / z - mass - to - charge . Data were acquired in data - independent mode ( MSE ) . D Representative chromatograms of unlabeled Aβ17 - 28 ( left ) 13C - Lys Aβ17 - 28 ( right ) in an APPtg brain after labeling from 50 - 175 d . intensities of 4 transitions ( y10 , y9 , y8 , 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 ) 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 ) . Aβ17 - 28 [ MH ] + fragment was detected with highest . Measurements in reflectron positive mode . of Aβ1 - 42 with tryptic cleavage sites is indicated at

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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 13C15N - Lys Aβ17 - 28 synthetic peptides in 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 + ) their derived fragments are indicated as a series of y ions ( y4 - y10 ) . 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 analyzed by MRM . intensities of 4 transitions ( y10 , y9 , y8 , 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 insoluble fraction of APPtg brains , immunoprecipitated , trypsin - digested , analyzed by MRM . Assessment of intensities of y10 , y9 , y8 y7 showed a detection limit level of 50 amol for 13C15N - Lys Aβ17 - 28 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 of 13C - Lys Aβ17 - 28 unlabeled Aβ17 - 28 in liver , spleen , mesenteric lymph nodes , peritoneal after 13C - Lys - APPtg brain extract i . p . injection ( n = 3 mice per time point tissue at 1 - 10 d , except for n = 2 mice for peritoneal at 3 d ; n = 4 mice per time point 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 as a spiked internal standard additionally to organ ( 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 of Aβ42 in liver , spleen , mesenteric lymph nodes , peritoneal 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 3 d , n = 3 mice at 1 d , n = 1 mouse at 5 7 d , except for peritoneal where n = 1 mouse at 5 h 3 d , n = 2 mice at 1 d ) . Error bars show median ± interquartile range . D Normalization of APPtg extract group data presented in ( C ) to organ ( mg ) 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 of unlabeled Aβ17 - 28 13C - Lys Aβ17 - 28 in 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 as a spiked internal standard brain ( mg ) . 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 of 13C - Lys Aβ17 - 28 ( B ) in insoluble brain fraction after 13C - Lys - APPtg brain extract ( donors labeled for 50 - 125 d ) 13C - Lys - non - tg brain extract ( donors labeled for 10 d ) i . p . injection ( n = 3 mice per group time point at 1 - 10 d , n = 4 mice per group time point at 25 , 50 , 100 d , n = 3 mice in APPtg extract group n = 4 in non - tg extract group at 75 d ) , right side zoom for time points 1 - 10 d . Unpaired two - t - * 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 as a spiked internal standard brain ( mg ) . 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 of unlabeled Aβ17 - 28 ( C ) in insoluble brain fraction after 13C - Lys - APPtg brain extract ( donors labeled for 50 - 125 d ) 13C - Lys - non - tg brain extract ( donors labeled for 10 d ) i . p . injection ( n = 3 mice per group time point at 1 - 10 d , n = 4 mice per group time point at 25 , 50 , 100 d , n = 3 mice in APPtg extract group n = 4 in non - tg extract group at 75 d ) , right side zoom for time points 1 - 10 d . Unpaired two - t - * 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 as a spiked internal standard brain ( mg ) . 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 of Aβ42 in insoluble brain fraction after 13C - Lys APPtg or 13C - Lys non - tg brain extract i . p . injection ( n = 3 mice per group time point at 1 - 10 d , n = 4 mice per group time point at 25 - 100 d ) , right side zoom for time points 1 - 10 d . Normalization to .

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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 ) 13C - Lys Aβ17 - 28 ( right ) in brain of an unlabeled 50 - day - old APPtg mouse not injected with labeled brain extract . Note 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 of unlabeled 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 as a spiked internal standard brain ( mg ) . 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 of 13C - Lys Aβ17 - 28 in insoluble brain fraction after 13C - Lys APPtg 13C - Lys non - tg brain extract ( 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 as a spiked internal standard brain ( mg ) . 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 of unlabeled 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 as a spiked internal standard brain ( mg ) . 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 was transfected with increasing concentrations of MDM2 , RB + 5 ´ UTR exogenous ( left panel ) endogenous ( right panel ) were evaluated . B ) Evaluation of RB expression as in ( A ) but inducing 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 RB in an MDM2 dose - dependent fashion under normal genotoxic stress induced by doxorubicin 16 h in HEK 293 . D ) Evaluation of RB in a MDM2 dose - dependent fashion under normal genotoxic stress induced by doxorubicin 16 h in C33 .

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

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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 , normalised by GAPDH , in an MDM2 dose - dependent manner . H1299 were treated with doxorubicin during 16 h to induce genotoxic stress . Endogenous ( upper panel ) ; exogenous ( middle panel ) ; endogenous of RB but using 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 endogenous RB expression in H1299 from 0 to 48h . were treated with cichlohexamide ( CHX ) was added to stop translation , under normal conditions ( upper panel ) treated with doxorubicin ( lower panel ) . 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 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 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 ; RB constructs of expression were evaluated using HA antibody . bar diagrams show of western blots data . A ) Evaluation of small pocket expression in an MDM2 dose - dependent manner . B ) were transfected with 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 ; RB constructs of expression were evaluated using HA antibody . bar diagrams show of western blots data . C ) were transfected with RB full without 5 ´ URT ( named RB - HA ) . D ) were transfected with RB full - 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 ) binding of MDM2 to RB mRNA in H1299 as estimated using CoIP . 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 GAPDH of MDM2 - immunoprecipitation from 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 using anti - RB anti - MDM2 antibodies , H1299 were treated or not with etoposide to induce stress , endogenous were visualised . nuclei were visualised with DAPI blue . red dots show interaction between two . 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 ) - PLA using rabbit anti - MDM2 a mouse biotin - antibody . H1299 - RBKO was stable transfected with RB - HA or RB + 5 ´ UTR . nuclei were visualised with DAPI blue . red dots show interaction between mRNA . 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 ) cycle ( FACS ) of C33 after treatment with etoposide . contents of were evaluated by propidium iodide , ( PE - A ; Phycoerythrin ) . inset shows % of in each 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 ) cycle of synchronised C33 during time .

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


C ) Evaluation of increasing concentrations of MDM2 on cycle of C33 after genotoxic stress induced with etoposide , ( EV ; empty vector ) . D ) same as in ( C ) but under normal 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 of after 14 h of 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 G1 cycle phase after 14 h of release of nocodazole treatment . Data information : Data are means ± s . d . of three independent experiments , student t 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 synchronised in G1 or G2 / M 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 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 . Increasing concentrations of MDM2 were transfected in H1299 - RBKO stable transfected with RB - HA or RB + 5 ´ UTR . were treated with doxorubicin to induce genotoxic stress conditions . Data are means ± s . d . of three independent experiments , student t 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 in an MDM2 dose - dependent manner . H1299 were treated with doxorubicin during 16 h to induce genotoxic stress . Data are means ± s . d . of three independent experiments , student t 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 lysates were prepared , blotted with 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 . number of endogenous LC3 or GFP - Atg5 dots was counted ( c , d ) . 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 in MEFs . Wortmannin is an autophagy inhibitor . results shown are mean1s . d . Statistical significance ( P value ) was determined by Student ' s t - .

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 Student ' s t - . * 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 of caspase - 1 IL - 1b in macrophages . Lys . , 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 indicated phenotype .

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


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

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


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

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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 .

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


a , Macrophages were stimulated with LPS ( 100 ng ml - 1 ) for indicated time period . number of endogenous LC3 dots within each was counted . 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 of p62 LC3 in macrophages .

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b, Expression levels of p62 and LC3 in macrophages.


c , IL - 1b IL - 6 production by wild - type or Atg7 - deficient macrophages stimulated with LPS . Statistical significance was determined by Student ' s t - . * 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 then stimulated with indicated ligands .

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


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

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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 initiation of DSS administration . 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 of IL - 1b 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 anti - IL - 18 neutralizing antibodies ( squares ; n55 ) or isotype control IgG ( circles ; n55 ) at days 1 , 3 , 5 7 . survival ( e ) 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 in control , susceptible resilient mice following 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 resilient mice ( n = 20 , 20 , 12 mice . Treatment F2 , 49 = 16 . 05 , P < 0 . 0001 , one - way ANOVA with Fisher ' s LSD ) .

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

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

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 time in open arms ( F ) closed arms ( G ) in 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 ) .

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

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

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

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

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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 effects of apyrase ( 40 , 80 160 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 - ) .

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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 effects of apyrase ( 40 , 80 160 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 ) .

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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 effect of apyrase ( 40 U / ml ) infusion into hippocampal on 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 ) .

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

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

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

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 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 - ) .

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

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).


H sucrose preference in mice with ATP intra - hippocampal infusion after apyrase exposure ( n = 10 , 8 , 8 mice . Treatment F2 , 23 = 3 . 32 , P = 0 . 0540 ; Apyrase - ACSF vs Vehicle , P = 0 . 0466 , one - way ANOVA with Fisher ' s LSD ) .

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 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

H The sucrose preference in mice with ATP intra-hippocampal infusion after apyrase exposure (n = 10, 8, 8 mice. Treatment F2, 23 = 3.32, P = 0.0540; Apyrase-ACSF vs Vehicle, P =0.0466, one-way ANOVA with Fisher's LSD test).


B ATPase activity of mice exposure to ARL67156 ( n = 7 , 7 , 5 , 5 mice . Treatment F3 , 20 = 16 . 81 , P < 0 . 0001 ; CSDS - ACSF vs Ctrl - ACSF , P < 0 . 0001 ; CSDS - ARL67156 vs CSDS - ACSF , P = 0 . 0405 . One - way ANOVA with Fisher ' s LSD ) .

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

B The ATPase activity of mice exposure to ARL67156 (n = 7, 7, 5, 5 mice. Treatment F3, 20 = 16.81, P < 0.0001; CSDS - ACSF vs Ctrl - ACSF, P < 0.0001; CSDS - ARL67156 vs CSDS - ACSF, P = 0.0405. One-way ANOVA with Fisher's LSD test).


C social avoidance behavior of susceptible mice with intracerebroventricular infusion of ARL67156 ( n = 12 , 10 , 9 , 9 mice . Interaction F3 , 36 = 5 . 119 , P = 0 . 0047 ; Target F1 , 36 = 0 . 005 , P = 0 . 9437 ; Drug F3 , 36 = 3 . 626 , P = 0 . 022 . For Target , CSDS - ACSF vs Ctrl - ACSF , P = 0 . 0001 ; CSDS - ARL67156 vs CSDS - ACSF , P = 0 . 0054 . Two - way ANOVA with Tukey ' s post - ) .

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

C The social avoidance behavior of susceptible mice with intracerebroventricular infusion of ARL67156 (n = 12, 10, 9, 9 mice. Interaction F3, 36 = 5.119, P = 0.0047; Target F1, 36 = 0.005, P = 0.9437; Drug F3, 36 = 3.626, P = 0.022. For Target, CSDS - ACSF vs Ctrl -ACSF, P = 0.0001; CSDS - ARL67156 vs CSDS - ACSF, P = 0.0054. Two-way ANOVA with Tukey's post-test).


D sucrose preference of susceptible mice with intracerebroventricular infusion of ARL67156 ( n = 12 , 10 , 10 , 10 mice . Interaction F1 , 38 = 5 . 352 , P = 0 . 0262 ; Group F1 , 38 = 5 . 711 , P = 0 . 0219 ; Drug F1 , 38 = 9 . 703 , P = 0 . 0035 ; CSDS - ACSF vs Ctrl - ACSF , P = 0 . 0083 ; Ctrl - ARL67156 vs CSDS - ACSF , P = 0 . 0027 ; CSDS - ARL67156 vs CSDS - ACSF , P = 0 . 0031 . Two - way ANOVA with Tukey ' s post - ) .

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

D The sucrose preference of susceptible mice with intracerebroventricular infusion of ARL67156 (n = 12, 10, 10, 10 mice. Interaction F1, 38 = 5.352, P = 0.0262; Group F1, 38 = 5.711, P = 0.0219; Drug F1, 38 = 9.703, P = 0.0035; CSDS - ACSF vs Ctrl - ACSF, P = 0.0083; Ctrl - ARL67156 vs CSDS - ACSF, P = 0.0027; CSDS - ARL67156 vs CSDS - ACSF, P = 0.0031. Two-way ANOVA with Tukey's post-test).


social avoidance behavior ( E ) in mice with intra - hippocampal infusion of ARL67156 after CSDS ( n = 9 , 9 , 9 , 8 mice . For social behavior , Interaction F3 , 31 = 5 . 579 , P = 0 . 0035 ; Target F1 , 31 = 0 . 5121 , P = 0 . 4796 ; Drug F3 , 31 = 8 . 367 , P = 0 . 0003 ; CSDS - ACSF vs Ctrl - ACSF , P < 0 . 0001 ; CSDS - ARL67156 vs CSDS - ACSF , P = 0 . 0131 .

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

The social avoidance behavior (E) in mice with intra-hippocampal infusion of ARL67156 after CSDS (n = 9, 9, 9, 8 mice. For social behavior, Interaction F3, 31 = 5.579, P = 0.0035; Target F1, 31 = 0.5121, P = 0.4796; Drug F3, 31 = 8.367, P = 0.0003; CSDS - ACSF vs Ctrl - ACSF, P < 0.0001; CSDS - ARL67156 vs CSDS - ACSF, P = 0.0131.


sucrose preference ( F ) in mice with intra - hippocampal infusion of ARL67156 after CSDS ( n = 9 , 9 , 9 , 8 mice . Sucrose , Interaction F1 , 30 = 0 . 3342 , P = 0 . 5675 ; Group F1 , 30 = 17 . 22 , P = 0 . 0003 ; Drug F1 , 30 = 0 . 05903 , P = 0 . 8097 ; CSDS - ACSF vs Ctrl - ACSF , P = 0 . 0761 . Two - way ANOVA with Tukey ' s post - ) .

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

sucrose preference (F) in mice with intra-hippocampal infusion of ARL67156 after CSDS (n = 9, 9, 9, 8 mice. Sucrose, Interaction F1, 30 = 0.3342, P = 0.5675; Group F1, 30 = 17.22, P = 0.0003; Drug F1, 30 = 0.05903, P = 0.8097; CSDS- ACSF vs Ctrl - ACSF, P = 0.0761. Two-way ANOVA with Tukey's post-test).


G immobility time in TST of mice with intra - hippocampal infusion of ARL67156 after CSDS ( n = 16 , 12 , 21 , 19 mice . Treatment F 3 , 64 = 13 . 61 , P < 0 . 0001 ; CSDS - ACSF vs Ctrl - ACSF , P = 0 . 0059 ; Ctrl - ARL67156 vs Ctrl - ACSF , P = 0 . 0011 ; CSDS - ARL67156 vs CSDS - ACSF , P = 0 . 0002 . One - way ANOVA with Fisher ' s LSD ) .

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

G The immobility time in the TST of mice with intra-hippocampal infusion of ARL67156 after CSDS (n = 16, 12, 21, 19 mice. Treatment F 3, 64= 13.61, P < 0.0001; CSDS - ACSF vs Ctrl - ACSF, P = 0.0059; Ctrl - ARL67156 vs Ctrl - ACSF, P = 0.0011; CSDS - ARL67156 vs CSDS - ACSF, P = 0.0002. One-way ANOVA with Fisher's LSD test).


H immobility time in FST with intra - hippocampal infusion of ARL67156 after CSDS ( n = 21 , 12 , 23 , 19 mice . Treatment F 3 , 71 = 12 . 94 , P < 0 . 0001 ; CSDS - ACSF vs Ctrl - ACSF , P = 0 . 0036 ; Ctrl - ARL67156 vs Ctrl - ACSF , P = 0 . 0428 ; CSDS - ARL67156 vs CSDS - ACSF , P < 0 . 0001 . One - way ANOVA with Fisher ' s LSD ) . Data information : data are expressed as means ± . NS P > 0 . 05 , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . Ctrl , control .

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

H The immobility time in the FST with intra-hippocampal infusion of ARL67156 after CSDS (n = 21, 12, 23, 19 mice. Treatment F 3, 71 = 12.94, P < 0.0001; CSDS - ACSF vs Ctrl - ACSF, P = 0.0036; Ctrl - ARL67156 vs Ctrl - ACSF, P = 0.0428; CSDS - ARL67156 vs CSDS - ACSF, P < 0.0001. One-way ANOVA with Fisher's LSD test).Data information: data are expressed as the means ± SEM. NS P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001. Ctrl, control.


B Imaging of GFP expression in hippocampus 3 after vector injection . Scale bar : 250 μm .

B Imaging of GFP expression in the hippocampus 3 weeks after virus vector injection. Scale bar: 250 μm.


C Expression of CD39 in hippocampus of mice injected with LV - siCD39 ( n = 6 mice / group . LV - siCD39 vs Control , P = 0 . 0006 , Student ' s t ) .

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 O O O O O O O

C Expression of CD39 in the hippocampus of mice injected with LV-siCD39 (n = 6 mice/group. LV-siCD39 vs Control, P = 0.0006, Student's t test).


D social avoidance behavior for stressed mice after knocking down CD39 ( n = 15 , 15 , 14 mice . Interaction F2 , 41 = 17 . 96 , P < 0 . 0001 ; Target F1 , 41 = 3 . 75e - 005 , P = 0 . 9951 ; Drug F2 , 41 = 6 . 099 , P = 0 . 0048 ; For Target CSDS - LV - GFP vs Ctrl - LV - GFP , P < 0 . 0001 ; CSDS - LV - siCD39 vs CSDS - LV - GFP , P < 0 . 0001 . Two - way ANOVA with Tukey ' s post - ) .

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

D The social avoidance behavior for the stressed mice after knocking down CD39 (n = 15, 15, 14 mice. Interaction F2, 41 = 17.96, P < 0.0001; Target F1, 41 = 3.75e-005, P = 0.9951; Drug F2, 41 = 6.099, P = 0.0048; For Target CSDS - LV-GFP vs Ctrl - LV-GFP, P < 0.0001; CSDS - LV-siCD39 vs CSDS - LV-GFP, P < 0.0001. Two-way ANOVA with Tukey's post-test).


E immobility time in TST of mice injected with LV - siCD39 ( n = 11 , 19 , 15 mice . Treatment F 2 , 42 = 5 . 807 , P = 0 . 0059 ; CSDS - LV - GFP vs Ctrl - LV - GFP , P = 0 . 0069 ; CSDS - LV - siCD39 vs CSDS - LV - GFP , P = 0 . 0065 . One - way ANOVA with Fisher ' s LSD ) .

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

E The immobility time in the TST of mice injected with LV- siCD39 (n = 11, 19, 15 mice. Treatment F 2, 42 = 5.807, P = 0.0059; CSDS - LV-GFP vs Ctrl - LV-GFP, P = 0.0069; CSDS - LV-siCD39 vs CSDS - LV-GFP, P = 0.0065. One-way ANOVA with Fisher's LSD test).


F immobility time spent in FST of mice treatment with LV - siCD39 ( n = 12 , 19 , 15 mice . Treatment F2 , 43 = 5 . 237 , P = 0 . 0092 ; CSDS - LV - GFP vs Ctrl - LV - GFP , P = 0 . 0095 ; CSDS - LV - siCD39 vs CSDS - LV - GFP , P = 0 . 0093 . One - way ANOVA with Fisher ' s LSD ) .

F The immobility time spent in the FST of mice treatment with LV-siCD39 (n = 12, 19, 15 mice. Treatment F2, 43 = 5.237, P = 0.0092; CSDS - LV-GFP vs Ctrl - LV-GFP, P = 0.0095; CSDS - LV-siCD39 vs CSDS - LV-GFP, P = 0.0093. One-way ANOVA with Fisher's LSD test).


G ATPase hyperactivity of mice after knocking down CD39 in hippocampal ( n = 5 , 6 , 5 mice . Treatment F2 , 13 = 8 . 45 , P = 0 . 0044 ; CSDS - LV - GFP vs Ctrl - LV - GFP , P = 0 . 0016 ; CSDS - LV - CD39 vs CSDS - LV - GFP , P = 0 . 0166 . One - way ANOVA with Fisher ' s LSD ) .

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

G The ATPase hyperactivity of mice after knocking down CD39 in the hippocampal (n = 5, 6, 5 mice. Treatment F2, 13 = 8.45, P = 0.0044; CSDS - LV-GFP vs Ctrl - LV-GFP, P = 0.0016; CSDS - LV-CD39 vs CSDS - LV-GFP, P = 0.0166. One-way ANOVA with Fisher's LSD test).


H ATP level of mice after knocking down CD39 in hippocampal ( n = 5 mice / group . Treatment F2 , 12 = 6 . 04 , P = 0 . 0153 ; CSDS - LV - GFP vs Ctrl - LV - GFP , P = 0 . 0444 ; CSDS - LV - CD39 vs CSDS - LV - GFP , P = 0 . 0051 . One - way ANOVA with Fisher ' s LSD ) .

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

H The ATP level of mice after knocking down CD39 in the hippocampal (n = 5 mice/group. Treatment F2, 12 = 6.04, P = 0.0153; CSDS - LV-GFP vs Ctrl - LV-GFP, P = 0.0444; CSDS - LV-CD39 vs CSDS - LV-GFP, P = 0.0051. One-way ANOVA with Fisher's LSD test).


B Immunostaining for BrdU ( blue ) DCX ( green ) in DG region of control , susceptible ARL67156 treatment mice . Arrows indicate BrdU + DCX + . Scale bar : 50 μm .

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 B-MEASURED_VAR O O O O O O O O

B Immunostaining for BrdU (blue) and DCX (green) in the DG region of control, susceptible and ARL67156 treatment mice. Arrows indicate the BrdU+ DCX+ cells. Scale bar: 50 μm.


C , D of BrdU + ( C ) BrdU + DCX + ( D ) in DG of susceptible mice with ARL67156 compared with vehicle treatment ( n = 4 mice / group . For BrdU + Treatment F2 , 9 = 10 . 07 , P = 0 . 0051 ; CSDS - ACSF vs Ctrl - ACSF , P = 0 . 0147 ; CSDS - ARL67156 vs CSDS - ACSF , P = 0 . 0018 ; BrdU + DCX + Treatment F2 , 9 = 9 . 005 , P = 0 . 0071 ; CSDS - ACSF vs Ctrl - ACSF , P = 0 . 0211 ; CSDS - ARL67156 vs CSDS - ACSF , P = 0 . 0024 , one - way ANOVA with Fisher ' s LSD ) .

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

C, D Quantification of BrdU+ cells (C) and BrdU+ DCX+ cells (D) in the DG of susceptible mice with ARL67156 compared with vehicle treatment (n = 4 mice/group. For BrdU+ Treatment F2, 9 = 10.07, P =0.0051; CSDS - ACSF vs Ctrl - ACSF, P = 0.0147; CSDS - ARL67156 vs CSDS - ACSF, P = 0.0018; BrdU+ DCX+ Treatment F2, 9 = 9.005, P = 0.0071; CSDS - ACSF vs Ctrl - ACSF, P = 0.0211; CSDS - ARL67156 vs CSDS - ACSF, P = 0.0024, one-way ANOVA with Fisher's LSD test).


F Confocal of BrdU ( blue ) DCX ( red ) in mice treatment with LV - siCD39 in DG . Arrows indicate BrdU + DCX + . Scale bar : 50 μm .

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

F Confocal images of BrdU (blue) and DCX (red) in mice treatment with LV-siCD39 in the DG. Arrows indicate the BrdU+ DCX+ cells. Scale bar: 50 μm.


G , H Quantitative of BrdU + ( G ) BrdU + DCX + ( H ) in DG of susceptible mice with LV - siCD39 compared with LV - GFP treatment ( n = 3 , 6 , 6 mice . For BrdU + Treatment F2 , 12 = 15 . 79 , P = 0 . 0004 ; CSDS - LV - GFP vs Ctrl - LV - GFP , P = 0 . 0012 ; CSDS - LV - siCD39 vs CSDS - LV - GFP , P = 0 . 0003 ; BrdU + DCX + Treatment F2 , 12 = 12 . 79 , P = 0 . 0011 ; CSDS - LV - GFP vs Ctrl - LV - GFP , P = 0 . 05 ; CSDS - LV - siCD39 vs CSDS - LV - GFP , P = 0 . 0003 , one - way ANOVA with Fisher ' s LSD ) .

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

G, H Quantitative of BrdU+ (G) and BrdU+ DCX+ (H) cells in the DG of susceptible mice with LV-siCD39 compared with LV-GFP treatment (n = 3, 6, 6 mice. For BrdU+ Treatment F2, 12 = 15.79, P =0.0004; CSDS - LV-GFP vs Ctrl - LV-GFP, P = 0.0012; CSDS - LV-siCD39 vs CSDS - LV-GFP, P = 0.0003; BrdU+ DCX+ Treatment F2, 12 = 12.79, P = 0.0011; CSDS - LV-GFP vs Ctrl - LV-GFP, P = 0.05; CSDS - LV-siCD39 vs CSDS - LV-GFP, P = 0.0003, one-way ANOVA with Fisher's LSD test).


Representative confocal z - stack of LV - infected dendritic segments in DG granule neurons from control susceptible mice with different treatment . arrows indicate stubby spines , yellow arrows indicate mushroom spines , blue arrows indicate thin spines .

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

Representative confocal z-stack images of LV-infected dendritic segments in the DG granule neurons from control and susceptible mice with different treatment. White arrows indicate stubby spines, yellow arrows indicate mushroom spines, and blue arrows indicate thin spines.


B - E of total ( B ) , stubby ( C ) , mushroom ( D ) thin ( E ) spine density in DG from control susceptible mice infected with LV - GFP or LV - siCD39 ( n = 15 segments from 4 mice / group . For total density : Treatment F2 , 42 = 3 . 12 , P = 0 . 0544 ; Ctrl - LV - GFP vs CSDS - LV - siCD39 , P = 0 . 0312 ; CSDS - LV - siCD39 vs CSDS - LV - GFP , P = 0 . 0425 ; stubby density : Treatment F2 , 42 = 10 . 93 , P = 0 . 0002 ; P = 0 . 0026 ; CSDS - LV - CD39 vs CSDS - LV - GFP , P < 0 . 0001 ; mushroom density : Treatment F2 , 42 = 0 . 34 , P = 0 . 7161 ; thin density : F2 , 42 = 1 . 00 , P = 0 . 3758 . One - way ANOVA with Fisher ' s LSD ) .

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

B-E Quantification of total (B), stubby (C), mushroom (D) and thin (E) spine density in DG from control and susceptible mice infected with LV-GFP or LV-siCD39 (n = 15 segments from 4 mice/group. For total density: Treatment F2, 42 = 3.12, P = 0.0544; Ctrl - LV-GFP vs CSDS - LV-siCD39, P = 0.0312; CSDS - LV-siCD39 vs CSDS - LV-GFP, P = 0.0425; stubby density: Treatment F2, 42 = 10.93, P=0.0002; P = 0.0026; CSDS - LV-CD39 vs CSDS - LV-GFP, P < 0.0001; mushroom density: Treatment F2, 42 = 0.34, P = 0.7161; thin density: F2, 42 = 1.00, P = 0.3758. One-way ANOVA with Fisher's LSD test).


F Representative confocal z - stack of LV - infected dendritic segments in DG granule neurons from control susceptible mice with different treatment . arrows indicate stubby spines , yellow arrows indicate mushroom spines , blue arrows indicate thin spines .

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

F Representative confocal z-stack images of LV-infected dendritic segments in the DG granule neurons from control and susceptible mice with different treatment. White arrows indicate stubby spines, yellow arrows indicate mushroom spines, and blue arrows indicate thin spines.


G - J of total ( G ) , stubby ( H ) , mushroom ( I ) thin ( J ) spine density in DG from control susceptible mice treatment with ACSF or ARL67156 ( n = 11 , 13 , 10 segments from 2 , 4 , 2 mice , respectively . For total density : Treatment F2 , 31 = 4 . 474 , P = 0 . 0196 ; CSDS - ASCF vs Ctrl - ACSF , P = 0 . 0366 ; CSDS - ARL67156 vs CSDS - ACSF , P = 0 . 0086 ; stubby density : Treatment F2 , 31 = 3 . 327 , P = 0 . 0491 ; CSDS - ASCF vs Ctrl - ACSF , P = 0 . 1026 ; CSDS - ARL67156 vs CSDS - ACSF , P = 0 . 0179 ; mushroom density : Treatment F2 , 31 = 1 . 986 , P = 0 . 1543 ; thin density : F2 , 31 = 1 . 475 , P = 0 . 2445 . One - way ANOVA with Fisher ' s LSD ) .

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

G-J Quantification of total (G), stubby (H), mushroom (I) and thin (J) spine density in DG from control and susceptible mice treatment with ACSF or ARL67156 (n = 11, 13, 10 segments from 2, 4, 2 mice, respectively. For total density: Treatment F2, 31 = 4.474, P = 0.0196; CSDS - ASCF vs Ctrl - ACSF, P = 0.0366; CSDS - ARL67156 vs CSDS - ACSF, P = 0.0086; stubby density: Treatment F2, 31 = 3.327, P=0.0491; CSDS - ASCF vs Ctrl - ACSF, P = 0.1026; CSDS - ARL67156 vs CSDS - ACSF, P = 0.0179; mushroom density: Treatment F2, 31 = 1.986, P = 0.1543; thin density: F2, 31 = 1.475, P = 0.2445. One-way ANOVA with Fisher's LSD test).


a , b , Representative of GFP - LC3 ( autophagosomes ) in skeletal ( vastus lateralis ) ( a ) cardiac muscle ( b ) from GFP - LC3 transgenic mice at serial time points after exercise . Scale bar , 20 µm . c , of data ( mean ± s . e . m . of 10 tissue sections ) in a b . * * P 0 . 01 , * * * P 0 . 001 ( one - way ANOVA ) .

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

a, b, Representative images of GFP-LC3 puncta (autophagosomes) in skeletal (vastus lateralis) (a) and cardiac muscle (b) from GFP-LC3 transgenic mice at serial time points after exercise. Scale bar, 20 µm. c, Quantification of data (mean ± s.e.m. of 10 tissue sections) in a and b. **P  0.01, ***P  0.001 (one-way ANOVA).


d , Western blot of LC3 - I / II ( non - lipidated lipidated forms of MAP1LC3 , respectively ) p62 in indicated tissue from mice at ( - ) or after maximal exercise ( + ) . Skeletal cardiac indicate skeletal cardiac muscle , respectively .

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

d, Western blot analysis of LC3-I/II (non-lipidated and lipidated forms of MAP1LC3, respectively) and p62 levels in indicated tissue from mice at rest (-) or after maximal exercise (+). Skeletal and cardiac indicate skeletal and cardiac muscle, respectively.


e , Co - immunoprecipitation of beclin 1 with BCL2 in muscle tissue from mice at indicated time points after exercise . IP , immunoprecipitate ; WB , western blot ; WCL , whole lysates .

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

e, Co-immunoprecipitation of beclin 1 with BCL2 in muscle tissue from mice at indicated time points after exercise. IP, immunoprecipitate; WB, western blot; WCL, whole cell lysates.