E - G ) Relative expression of the cell surface marker ( E ) CD36 ( biological replicates with n = 5 for APP / PS1 and n = 4 for APP / PS1 PD - 1 - / - mice , ANOVA ( DF = 1 , F = 0 . 002 , p = 0 . 94 ) , Tukey ' s HSD , * p < 0 . 05 , * * * p < 0 . 001 ) , ( F ) CD11b ( biological replicates with n = 5 for APP / PS1 and n = 4 for APP / PS1 PD - 1 - / - mice ) and ( G ) CD45 on methoxy - XO4 + versus methoxy - XO4 - microglia in APP / PS1 and APP / PS1 PD - 1 - / - mice normalized to methoxy - X04 - negative cells from APP / PS1 mice ( biological replicates with n = 5 for APP / PS1 and n = 4 for APP / PS1 PD - 1 - / - mice ) Data information : central bands represent median , boxes show interquartile range ( IQR ) and whiskers define the + / - 1 . 5xIQR .

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E-G) Relative expression of the cell surface marker (E) CD36 (biological replicates with n=5 for APP/PS1 and n=4 for APP/PS1 PD-1-/- mice, ANOVA (DF=1, F= 0.002, p=0.94), Tukey's HSD, * p<0.05, *** p<0.001), (F) CD11b (biological replicates with n=5 for APP/PS1 and n=4 for APP/PS1 PD-1-/- mice) and (G) CD45 on methoxy-XO4+ versus methoxy-XO4- microglia in APP/PS1 and APP/PS1 PD-1-/- mice normalized to methoxy-X04-negative cells from APP/PS1 mice (biological replicates with n=5 for APP/PS1 and n=4 for APP/PS1 PD-1-/- mice) Data information: central bands represent median, boxes show interquartile range (IQR) and whiskers define the +/-1.5xIQR.


B ) Different expression of inflammasome - related genes ( abs ( fold change ) > 1 . 5 , p < 0 . 05 , Benjamini - Hochberg adjusted ) in PD - 1 - / - vs . wild type microglia . C ) Different expression of complement system - related genes ( abs ( fold change ) > 1 . 5 , p < 0 . 05 , Benjamini - Hochberg adjusted ) in PD - 1 - / - vs . wild type microglia .

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B) Different expression of inflammasome-related genes (abs(fold change) > 1.5, p < 0.05, Benjamini- Hochberg adjusted) in PD-1-/- vs. wild type microglia. C) Different expression of complement system-related genes (abs(fold change) > 1.5, p < 0.05, Benjamini-Hochberg adjusted) in PD-1-/- vs. wild type microglia.


( a - d ) ELISA for IL - 1β ( a - c ) , IL - 18 ( d ) and IL - 6 ( e ) in supernatants of resting or LPS - primed bone marrow - derived macrophages ( BMMs ) stimulated with palmitate conjugated to BSA ( PA - BSA ) or BSA control .

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(a-d) ELISA for IL-1β (a-c), IL-18 (d) and IL-6 (e) in supernatants of resting or LPS-primed bone marrow-derived macrophages (BMMs) stimulated with palmitate conjugated to BSA (PA-BSA) or BSA control.


( e , f ) ELISA for IL - 1β ( e ) and IL - 18 ( f ) in supernatants . Resting or LPS - primed BMMs generated from wild - type ( WT ) , Nlrp3 − / − , Pycard − / − or Nlrc4 − / − mice were stimulated with PA - BSA .

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(e,f) ELISA for IL-1β (e) and IL-18 (f) in supernatants. Resting or LPS-primed BMMs generated from wild-type (WT), Nlrp3−/−, Pycard−/− or Nlrc4−/− mice were stimulated with PA-BSA.


( g ) IL - 1β ELISA of supernatants from LPS - primed BMMs stimulated with PA - BSA in the absence or presence of the pan - caspase inhibitor zVAD or caspase - 1 inhibitor zYVAD .

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(g) IL-1β ELISA of supernatants from LPS-primed BMMs stimulated with PA-BSA in the absence or presence of the pan-caspase inhibitor zVAD or caspase-1 inhibitor zYVAD.


( h ) ELISA for IL - 1β in supernatants . Resting or LPS - primed BMMs generated from WT or Casp1 - / - mice were stimulated with PA - BSA , BSA control , ATP or nigericin .

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(h) ELISA for IL-1β in supernatants. Resting or LPS-primed BMMs generated from WT or Casp1-/- mice were stimulated with PA-BSA, BSA control, ATP or nigericin.


( i ) ELISA for TNF in supernatants . Resting or LPS - primed BMMs generated from WT , Nlrp3 - / - , Pycard - / - or Casp1 - / - mice were stimulated with PA - BSA as indicated . Values are expressed as mean ± s . d . , and the results are representative of three independent experiments .

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(i) ELISA for TNF in supernatants. Resting or LPS-primed BMMs generated from WT, Nlrp3-/-, Pycard-/- or Casp1-/- mice were stimulated with PA-BSA as indicated. Values are expressed as mean ± s.d., and the results are representative of three independent experiments.


Immunoblotting for procaspase - 1 and activated caspase - 1 ( p10 ) , pro - IL - 1β and cleaved IL - 1β ( p17 ) in supernatants ( Sup ) and cell lysates ( Lys ) from resting or LPS - primed BMMs generated from WT , Nlrp3 − / − , Pycard − / − ( a ) or Nlrc4 − / − ( b ) mice stimulated with PA - BSA ( 0 . 2 or 0 . 5 mM ) as indicated at top . Data are representative of two independent experiments .

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Immunoblotting for procaspase-1 and activated caspase-1 (p10), pro-IL-1β and cleaved IL-1β (p17) in supernatants (Sup) and cell lysates (Lys) from resting or LPS-primed BMMs generated from WT, Nlrp3−/−, Pycard−/− (a) or Nlrc4−/− (b) mice stimulated with PA-BSA (0.2 or 0.5 mM) as indicated at top. Data are representative of two independent experiments.


( a ) ROS production determined by flow cytometry using the fluoroprobe dihydrorhodamine 123 ( DHR ) in LPS - primed BMMs stimulated with PA - BSA ( 0 . 5 mM ) in the absence or presence of ROS inhibitor APDC ( 50 μM ) . MFI , mean fluorescence intensity .

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(a) ROS production determined by flow cytometry using the fluoroprobe dihydrorhodamine 123 (DHR) in LPS-primed BMMs stimulated with PA-BSA (0.5 mM) in the absence or presence of ROS inhibitor APDC (50 μM). MFI, mean fluorescence intensity.


( b ) ELISA for IL - 1β on LPS - primed BMMs stimulated with PA - BSA in the absence of or presence of increasing concentrations of APDC .

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(b) ELISA for IL-1β on LPS-primed BMMs stimulated with PA-BSA in the absence of or presence of increasing concentrations of APDC.


( c ) immunoblotting for caspase - 1 p10 and IL - 1β p17 on LPS - primed BMMs stimulated with PA - BSA in the absence of or presence of increasing concentrations of APDC .

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(c) immunoblotting for caspase-1 p10 and IL-1β p17 on LPS-primed BMMs stimulated with PA-BSA in the absence of or presence of increasing concentrations of APDC.


( d ) ELISA for IL - 1β . Resting or LPS - primed BMMs were stimulated with PA - BSA ( 0 . 5 mM ) in the absence or presence of the ROS inhibitors APDC ( 10 or 50 μM ) and N - acetyl - cysteine ( NAC , 5 or 25 mM ) or the NAPDH oxidase inhibitor diphenyleneiodonium ( DPI , 5 or 25 μM ) . Values are expressed as mean ± s . d . In a , one of two independent experiments is shown . In each of b - d , the results are representative of three independent experiments .

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

(d) ELISA for IL-1β. Resting or LPS-primed BMMs were stimulated with PA-BSA (0.5 mM) in the absence or presence of the ROS inhibitors APDC (10 or 50 μM) and N-acetyl-cysteine (NAC, 5 or 25 mM) or the NAPDH oxidase inhibitor diphenyleneiodonium (DPI, 5 or 25 μM). Values are expressed as mean ± s.d. In a, one of two independent experiments is shown. In each of b-d, the results are representative of three independent experiments.


( a ) Thr172 - phosphorylated ( p - ) and total AMPK - α subunit determined by immunoblotting . Resting or LPS - primed BMMs were untreated or stimulated with PA - BSA ( 0 . 5 mM ) for 24 h .

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(a) Thr172-phosphorylated (p-) and total AMPK-α subunit determined by immunoblotting. Resting or LPS-primed BMMs were untreated or stimulated with PA-BSA (0.5 mM) for 24 h.


( b - e ) Analysis of LPS - primed BMMs stimulated with PA - BSA ( 0 . 5 mM ) in the absence or presence of AMPK agonist AICAR . ( b ) ROS production determined using the fluoroprobe DHR .

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(b-e) Analysis of LPS-primed BMMs stimulated with PA-BSA (0.5 mM) in the absence or presence of AMPK agonist AICAR. (b) ROS production determined using the fluoroprobe DHR.


( b - e ) Analysis of LPS - primed BMMs stimulated with PA - BSA ( 0 . 5 mM ) in the absence or presence of AMPK agonist AICAR . ( c , d ) IL - 1β ( c ) and IL - 6 ( d ) in supernatants , measured by ELISA .

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(b-e) Analysis of LPS-primed BMMs stimulated with PA-BSA (0.5 mM) in the absence or presence of AMPK agonist AICAR. (c,d) IL-1β (c) and IL-6 (d) in supernatants, measured by ELISA.


( b - e ) Analysis of LPS - primed BMMs stimulated with PA - BSA ( 0 . 5 mM ) in the absence or presence of AMPK agonist AICAR . ( e ) Immunoblotting for caspase - 1 , IL - 1β and AMPK signaling molecules .

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(b-e) Analysis of LPS-primed BMMs stimulated with PA-BSA (0.5 mM) in the absence or presence of AMPK agonist AICAR. (e) Immunoblotting for caspase-1, IL-1β and AMPK signaling molecules.


( f ) Transfection of BMMs with empty vector ( EV ) or constitutively active ( CA ) AMPK - α1 subunit . N , non - transfected . Efficiency was determined 16 h later by flow cytometric analysis of GFP expression in macrophages cotransfected with CA AMPK - α1 and pmaxGFP . Values are percentages of GFP - positive cells in the ranges indicated by the bars . ( g ) ELISA for IL - 1β in supernatants of cells transfected as in f and stimulated with PA - BSA ( 0 . 5 mM ) for 24 h . In each of a , b and f , one of two independent experiments is shown . In each of c - e and g , the results are representative of three independent experiments . Values are expressed as mean ± s . d . * P 0 . 05 versus controls .

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

(f) Transfection of BMMs with empty vector (EV) or constitutively active (CA) AMPK-α1 subunit. N, non-transfected. Efficiency was determined 16 h later by flow cytometric analysis of GFP expression in macrophages cotransfected with CA AMPK-α1 and pmaxGFP. Values are percentages of GFP-positive cells in the ranges indicated by the bars. (g) ELISA for IL-1β in supernatants of cells transfected as in f and stimulated with PA-BSA (0.5 mM) for 24 h. In each of a, b and f, one of two independent experiments is shown. In each of c-e and g, the results are representative of three independent experiments. Values are expressed as mean ± s.d. *P 0.05 versus controls.


( a ) Immunoblotting for AMPK activation ( Thr172 p - AMPK - α ) and LC3B ( top ) , and densitometric analysis to quantify ratio of LC3B - II to actin ( bottom ) . BMMs were pretreated with LPS ( 200 ng / ml ) for 3 h , followed by PA - BSA ( 0 . 5 mM ) treatment for 24 h in the absence or presence of chloroquine ( 50 μM ) . Med , medium control .

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(a) Immunoblotting for AMPK activation (Thr172 p-AMPK-α) and LC3B (top), and densitometric analysis to quantify ratio of LC3B-II to actin (bottom). BMMs were pretreated with LPS (200 ng/ml) for 3 h, followed by PA-BSA (0.5 mM) treatment for 24 h in the absence or presence of chloroquine (50 μM). Med, medium control.


( b , c ) LC3B and autophagosomes in BMMs pretreated with LPS for 3 h , followed by PA - BSA treatment for 24 h in the absence or presence of AICAR . Cells were fixed and stained for LC3B ( b , left ) . Quantitation of autophagosomes was performed by counting LC3B puncta in 100 cells ( b , right ) .

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(b,c) LC3B and autophagosomes in BMMs pretreated with LPS for 3 h, followed by PA-BSA treatment for 24 h in the absence or presence of AICAR. Cells were fixed and stained for LC3B (b, left). Quantitation of autophagosomes was performed by counting LC3B puncta in 100 cells (b, right).


( b , c ) LC3B and autophagosomes in BMMs pretreated with LPS for 3 h , followed by PA - BSA treatment for 24 h in the absence or presence of AICAR . Cells were fixed and stained for LC3B ( b , left ) . Quantitation of autophagosomes was performed by counting LC3B puncta in 100 cells ( b , right ) . Cells were fixed and examined by transmission electron microscopy for autophagosomes ( c ) . Quantitation ( c , right ) is based on counting autophagosomes in ten cells per treatment .

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

(b,c) LC3B and autophagosomes in BMMs pretreated with LPS for 3 h, followed by PA-BSA treatment for 24 h in the absence or presence of AICAR. Cells were fixed and stained for LC3B (b, left). Quantitation of autophagosomes was performed by counting LC3B puncta in 100 cells (b, right). Cells were fixed and examined by transmission electron microscopy for autophagosomes (c). Quantitation (c, right) is based on counting autophagosomes in ten cells per treatment.


( d ) Immunoblotting ( left ) and densitometric analysis to quantify ratio of LC3B - II to actin and ULK1 species ( right ) in treated BMMs .

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(d) Immunoblotting (left) and densitometric analysis to quantify ratio of LC3B-II to actin and ULK1 species (right) in treated BMMs.


( e ) Mitochondrial ROS production determined using the MitoSOX fluorescence indicator . In each of a and e , one of three independent experiments is shown . In each of b and d , one of two independent experiments is shown . Values are expressed as mean ± s . d . * P 0 . 05 versus controls .

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(e) Mitochondrial ROS production determined using the MitoSOX fluorescence indicator. In each of a and e, one of three independent experiments is shown. In each of b and d, one of two independent experiments is shown. Values are expressed as mean ± s.d. *P 0.05 versus controls.


( a , b ) Analysis of FL83B mouse liver cells pretreated with mouse recombinant IL - 1β ( 2 ng / ml ) ( a ) or TNF ( 2 ng / ml ) ( b ) for 24 h , then stimulated with insulin ( 200 nM ) for 10 min . p - Akt ( Ser473 ) was determined by flow cytometry ( right ) with quantification shown in the graph at left ( a ) . p - Akt ( Ser473 ) was determined by immunoblotting ( b ) .

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(a,b) Analysis of FL83B mouse liver cells pretreated with mouse recombinant IL-1β (2 ng/ml) (a) or TNF (2 ng/ml) (b) for 24 h, then stimulated with insulin (200 nM) for 10 min. p-Akt (Ser473) was determined by flow cytometry (right) with quantification shown in the graph at left (a). p-Akt (Ser473) was determined by immunoblotting (b).


( c ) p - IRS1 ( Ser307 ) , determined by immunoblotting , in FL83B mouse liver cells treated with either IL - 1β or TNF for 24 h .

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(c) p-IRS1 (Ser307), determined by immunoblotting, in FL83B mouse liver cells treated with either IL-1β or TNF for 24 h.


( d - f ) Akt Ser473 phosphorylation measured by flow cytometry ( d , e ) or immunoblotting ( f ) . FL83B cells ( d , e ) or WT primary hepatocytes ( f ) were pretreated with conditioned medium ( CM ) generated from WT , Pycard − / − ( d ) , Nlrp3 − / − or Casp1 − / − ( e ) macrophages for 24 h , then stimulated with insulin . Control medium was obtained from wild - type BMM cultures without stimulation .

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(d-f) Akt Ser473 phosphorylation measured by flow cytometry (d,e) or immunoblotting (f). FL83B cells (d,e) or WT primary hepatocytes (f) were pretreated with conditioned medium (CM) generated from WT, Pycard−/− (d), Nlrp3−/− or Casp1−/− (e) macrophages for 24 h, then stimulated with insulin. Control medium was obtained from wild-type BMM cultures without stimulation.


( d - f ) Akt Ser473 phosphorylation measured by flow cytometry ( d , e ) or immunoblotting ( f ) . FL83B cells ( d , e ) or WT primary hepatocytes ( f ) were pretreated with conditioned medium ( CM ) generated from WT , Pycard − / − ( d ) , Nlrp3 − / − or Casp1 − / − ( e ) macrophages for 24 h , then stimulated with insulin . Control medium was obtained from wild - type BMM cultures without

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

(d-f) Akt Ser473 phosphorylation measured by flow cytometry (d,e) or immunoblotting (f). FL83B cells (d,e) or WT primary hepatocytes (f) were pretreated with conditioned medium (CM) generated from WT, Pycard−/− (d), Nlrp3−/− or Casp1−/− (e) macrophages for 24 h, then stimulated with insulin. Control medium was obtained from wild-type BMM cultures without stimulation


( g ) Akt serine 473 phosphorylation measured by flow cytometry . FL83B cells were pretreated with WT or Pycard − / − CM in the absence or presence of the IL - 1 receptor antagonist anakinra ( 1 μg / ml ) for 24 h , then stimulated with insulin .

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(g) Akt serine 473 phosphorylation measured by flow cytometry. FL83B cells were pretreated with WT or Pycard−/− CM in the absence or presence of the IL-1 receptor antagonist anakinra (1 μg/ml) for 24 h, then stimulated with insulin.


( h ) p - Akt ( Ser473 ) analyzed by immunoblotting and quantified by densitometric analysis in FL83B cells pretreated with WT CM in the absence or presence of either anakinra or neutralizing antibody to TNF . The results shown are representative of three independent experiments and are expressed as mean ± s . d .

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(h) p-Akt (Ser473) analyzed by immunoblotting and quantified by densitometric analysis in FL83B cells pretreated with WT CM in the absence or presence of either anakinra or neutralizing antibody to TNF. The results shown are representative of three independent experiments and are expressed as mean ± s.d.


( a ) ITT performed 2 h after IL - 1β injection . Eight - week old male C57BL / 6 mice were intraperitoneally injected with saline or mouse recombinant IL - 1β ( 1 μg / kg ) .

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(a) ITT performed 2 h after IL-1β injection. Eight-week old male C57BL/6 mice were intraperitoneally injected with saline or mouse recombinant IL-1β (1 μg/kg).


( b ) ITT in WT mice on regular diet ( RD ) or HFD for 12 weeks , or Il1b - / - mice on HFD for 12 weeks .

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(b) ITT in WT mice on regular diet (RD) or HFD for 12 weeks, or Il1b-/- mice on HFD for 12 weeks.


( c , d ) ITT ( c ) and serum IL - 1β ( d , left ) and TNF ( d , right ) determined by ELISA , 2 h after IL - 1β injection . Recombinant mouse IL - 1β was administered to WT and Tnfa − / − mice ( n = 5 for each group ) . One of two independent experiments is shown ( mean ± s . d . ) . * P 0 . 05 versus controls .

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(c,d) ITT (c) and serum IL-1β (d, left) and TNF (d, right) determined by ELISA, 2 h after IL-1β injection. Recombinant mouse IL-1β was administered to WT and Tnfa−/− mice (n = 5 for each group). One of two independent experiments is shown (mean ± s.d.). *P 0.05 versus controls.


( a ) Blood glucose and insulin levels measured in WT ( n = 6 ) , Nlrp3 − / − ( n = 5 ) or Pycard − / − ( n = 6 ) mice under fasting or refed conditions after 12 weeks of HFD .

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(a) Blood glucose and insulin levels measured in WT (n = 6), Nlrp3−/− (n = 5) or Pycard−/− (n = 6) mice under fasting or refed conditions after 12 weeks of HFD.


( b - e ) Glucose tolerance test ( GTT ) ( b , d ) and insulin tolerance test ( ITT ) ( c , e ) performed for WT , Nlrp3 − / − ( b , c ) and Pycard − / − ( d , e ) mice on HFD for 12 weeks .

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(b-e) Glucose tolerance test (GTT) (b,d) and insulin tolerance test (ITT) (c,e) performed for WT, Nlrp3−/− (b,c) and Pycard−/− (d,e) mice on HFD for 12 weeks.


( f ) GTT conducted on the indicated bone marrow chimeric mice on HFD for 12 weeks .

(f) GTT conducted on the indicated bone marrow chimeric mice on HFD for 12 weeks.


( g , h ) Insulin - stimulated phosphorylation of IRβ , IRS1 and Akt ( Ser473 ) in liver tissues of individual WT and Pycard − / − mice ( g ) , and p - Akt ( Ser473 ) in liver , white adipose ( WAT ) and muscle tissues of individual WT and Nlrp3 − / − mice ( h ) on HFD for 12 weeks , after insulin ( 2 IU / kg ) infusion . Graphs at right of blots show the quantitation of each molecule . pY , phosphotyrosine .

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(g,h) Insulin-stimulated phosphorylation of IRβ, IRS1 and Akt (Ser473) in liver tissues of individual WT and Pycard−/− mice (g), and p-Akt (Ser473) in liver, white adipose (WAT) and muscle tissues of individual WT and Nlrp3−/− mice (h) on HFD for 12 weeks, after insulin (2 IU/kg) infusion. Graphs at right of blots show the quantitation of each molecule. pY, phosphotyrosine.


( i , j ) Expression of Tnfa and Mcp1 mRNA relative to Actb in liver tissues of Nlrp3 − / − mice ( i ) and Pycard − / − mice ( j ) on regular diet ( RD ) or HFD for 12 weeks . One of two independent experiments is shown ( mean ± s . d . ) . * P 0 . 05 versus controls .

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(i,j) Expression of Tnfa and Mcp1 mRNA relative to Actb in liver tissues of Nlrp3−/− mice (i) and Pycard−/− mice (j) on regular diet (RD) or HFD for 12 weeks. One of two independent experiments is shown (mean ± s.d.). *P 0.05 versus controls.


B . Phenotype of the patients as indicated . ( a ) Sparse woolly hair . ( b ) Protrusion of upper lip due to deformity of teeth . ( c ) Ichthyosis - like fine scale and erythema .

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B. Phenotype of the patients as indicated. (a) Sparse woolly hair. (b) Protrusion of upper lip due to deformity of teeth. (c) Ichthyosis-like fine scale and erythema.


C . Images of the heart of the patients as indicated . ( a ) M - mode echocardiography taken from the parasternal long axis view of the left ventricle ( LV ) illustrating poor LV function with severe involvement of the interventricular septum ( IVS ) and reduced motion of the left ventricular posterior wall ( LVPW ) . ( b ) bi - dimensional apical four chamber view with swirling of SMOG heart chambers as a result of poor myocardial function . The SMOG is enhanced in the right atrium . ( c ) bi - dimensional apical four chamber view with free tricuspid valve regurgitation ( TR ) due to lack of cooptation of the tricuspid valve leaflets during systole ( marked by a red bar ) as a result of critical reduction of right ventricular function and right ventricular enlargement . ( d ) Right and left ventricular thrombi seen in the short axis apical parasternal view . T1 - large right ventricular thrombus , T2 & amp ; amp ; T3 - left ventricular thrombi . ( e ) The color - flow Doppler picture : TR . Free regurgitation of the tricuspid valve shown in modified four chambers apical view . RA - right atrium , RV - right ventricle , LA - left atrium , LV - left ventricle , TR - tricuspid regurgitation color - flow Doppler .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Images of the heart of the patients as indicated. (a) M-mode echocardiography taken from the parasternal long axis view of the left ventricle (LV) illustrating poor LV function with severe involvement of the interventricular septum (IVS) and reduced motion of the left ventricular posterior wall (LVPW). (b) bi-dimensional apical four chamber view with swirling of SMOG heart chambers as a result of poor myocardial function. The SMOG is enhanced in the right atrium. (c) bi-dimensional apical four chamber view with free tricuspid valve regurgitation (TR) due to lack of cooptation of the tricuspid valve leaflets during systole (marked by a red bar) as a result of critical reduction of right ventricular function and right ventricular enlargement. (d) Right and left ventricular thrombi seen in the short axis apical parasternal view. T1-large right ventricular thrombus, T2&amp;T3-left ventricular thrombi. (e) The color-flow Doppler picture: TR. Free regurgitation of the tricuspid valve shown in modified four chambers apical view. RA-right atrium, RV-right ventricle, LA-left atrium, LV-left ventricle, TR-tricuspid regurgitation color-flow Doppler.


D . Photomicrografh demonstrating fibrosis and interstitial inflammation in the heart of patient VI10 . a c - post - mortem heart sections ( 2 . 7Y ) . ( a ) Prominent fibrosis and lymphocytic interstitial inflammation ( Hematoxylin Eosin x50 ) . ( c ) Sub - epicardial inflammation and mild interstitial " myocarditis " ( Hematoxylin Eosin x100 ) . ( b d ) - Fetal heart ( VI7 ) homozygous for p . Tyr747Ter . No inflammatory infiltrate or other histological abnormalities are seen . ( b ) Normal arrayed myocardium ( Hematoxylin Eosin x50 ) . ( d ) Sub - epicardial region with no histopathological changes . ( Hematoxylin Eosin x100 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Photomicrografh demonstrating fibrosis and interstitial inflammation in the heart of patient VI10. a c- post-mortem heart sections (2.7Y). (a) Prominent fibrosis and lymphocytic interstitial inflammation (Hematoxylin Eosin x50). (c) Sub-epicardial inflammation and mild interstitial "myocarditis" (Hematoxylin Eosin x100). (b d)- Fetal heart (VI7) homozygous for p.Tyr747Ter. No inflammatory infiltrate or other histological abnormalities are seen. (b) Normal arrayed myocardium (Hematoxylin Eosin x50). (d) Sub-epicardial region with no histopathological changes. (Hematoxylin Eosin x100).


F . Segregation of c . 2241C > G , p . Y747X in PPP1R13L in the studied family : The affected girl VI10 , is homozygous for the causative SV p . Y747X , as are the four fetuses that were aborted . The parents are obligate carriers as shown and , of the two healthy sisters , one carries the wild allele only , and the other is heterozygous for the causative SV .

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F. Segregation of c.2241C> G, p.Y747X in PPP1R13L in the studied family: The affected girl VI10, is homozygous for the causative SV p.Y747X, as are the four fetuses that were aborted. The parents are obligate carriers as shown and, of the two healthy sisters, one carries the wild allele only, and the other is heterozygous for the causative SV.


A . PPP1R13L mRNA expression level is lower in VI10 PDFs ( set as 1 ) than in CFs .

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A. PPP1R13L mRNA expression level is lower in VI10 PDFs (set as 1) than in CFs.


B . and C . Western blots ( WBs ) analyses demonstrated the absence of iASPP in VI10 , VI4 and VII1 PDFs .

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B. and C. Western blots (WBs) analyses demonstrated the absence of iASPP in VI10, VI4 and VII1 PDFs.


D . WB analysis demonstrated that PPP1R13L - shRNA downregulated the putative iASPP band .

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

D. WB analysis demonstrated that PPP1R13L-shRNA downregulated the putative iASPP band.


A . Higher induction of inflammatory cytokine mRNA levels in LPS - stimulated VI10 PDFs than in CFs . The expression level in resting conditions of the PDFs was set as 1 . Differences between knockdown and control with p values ≤ 0 . 05 ( Student ' s t test ) are indicated with an asterisk . B . as in A . for patient VII1 .

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A. Higher induction of inflammatory cytokine mRNA levels in LPS-stimulated VI10 PDFs than in CFs. The expression level in resting conditions of the PDFs was set as 1. Differences between knockdown and control with p values ≤ 0.05 (Student's t test) are indicated with an asterisk. B. as in A. for patient VII1.


C . knockdown of PPP1R13L in CFs resulted in increased mRNA expression levels of inflammatory mediators . Differences between knockdown and control with p values ( Student ' s t test ) are indicated with asterisks in the graph .

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C. knockdown of PPP1R13L in CFs resulted in increased mRNA expression levels of inflammatory mediators. Differences between knockdown and control with p values (Student's t test) are indicated with asterisks in the graph.


A . WB analysis of nuclear and cytosolic fractions demonstrated that the duration of p65 in the nucleus is longer in the VI10 PDFs than in CFs .

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A. WB analysis of nuclear and cytosolic fractions demonstrated that the duration of p65 in the nucleus is longer in the VI10 PDFs than in CFs.


B . ChIP assay shows that p65 is associated more strongly with inflammatory cytokine gene promoters in LPS - stimulated PDFs than in LPS - stimulated - CFs . The binding activity at the IL1B promoter was set as 1 . Differences between knockdown and control with p value = 0 . 00436 ( Student ' s t test ) are indicated with an asterisk .

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B. ChIP assay shows that p65 is associated more strongly with inflammatory cytokine gene promoters in LPS-stimulated PDFs than in LPS-stimulated-CFs. The binding activity at the IL1B promoter was set as 1. Differences between knockdown and control with p value= 0.00436 (Student's t test) are indicated with an asterisk.


C . NF - kB inhibitor BOT - 64 blocked the inducible mRNA expression of the inflammatory cytokines . Differences between knockdown and control with p values ( Student ' s t test ) are indicated with an asterisks and exact values .

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C. NF-kB inhibitor BOT-64 blocked the inducible mRNA expression of the inflammatory cytokines. Differences between knockdown and control with p values (Student's t test) are indicated with an asterisks and exact values.


A . WB analysis confirmed the reduced expression of iASPP in ppp1r13l knocked down cardiomyocytes .

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A. WB analysis confirmed the reduced expression of iASPP in ppp1r13l knocked down cardiomyocytes.


B . qPCR showed that ppp1r13l - knocked down murine cardiomyocytes ( using two alternative shRNA sequences ) expressed higher level of pro - inflammatory cytokine mRNAs after 4 hrs of LPS stimulation . The expression in ppp1r13l - knocked down murine cardiomyocytes was set as 1 . Genome wide expression analysis is presented in Figure 1S . Differences between knockdown and control with p values ( Student ' s t test ) are indicated with asterisks and exact values .

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B. qPCR showed that ppp1r13l-knocked down murine cardiomyocytes (using two alternative shRNA sequences) expressed higher level of pro-inflammatory cytokine mRNAs after 4 hrs of LPS stimulation. The expression in ppp1r13l-knocked down murine cardiomyocytes was set as 1. Genome wide expression analysis is presented in Figure 1S. Differences between knockdown and control with p values (Student's t test) are indicated with asterisks and exact values.


A wa3 hearts express higher level of Il1 than littermate WT hearts without significant changes in selected structural and regulatory genes as myosin binding protein C ( Mybpc3 ) , phospholamban ( Pln ) , ryanodine receptor 2 ( Ryr2 ) , and sarcoglycan gamma ( Sgcg ) . Hearts removed from perfused mice and mRNA was subjected for q PCR . Difference between knockdown and control with p values = 0 . 03 ( Student ' s t test ) is indicated with an asterisk .

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

A wa3 hearts express higher level of Il1 than littermate WT hearts without significant changes in selected structural and regulatory genes as myosin binding protein C (Mybpc3), phospholamban (Pln), ryanodine receptor 2 (Ryr2), and sarcoglycan gamma (Sgcg). Hearts removed from perfused mice and mRNA was subjected for q PCR. Difference between knockdown and control with p values = 0.03 (Student's t test) is indicated with an asterisk.


B . A scatter plot showing the deviation from normal expression by highlighting the differentially expressed genes of newborn , 7 - week - and 12 - week - hearts ( p < 0 . 05 threshold , calculated by Deseq algorithm " R " ) . The expression level of s100a9 was higher in wa3 mice than in WT by 2 . 8 times . For the full list of genes see Tables S1 and S2 , and functional analyses in Figures S3 - S6 .

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B. A scatter plot showing the deviation from normal expression by highlighting the differentially expressed genes of newborn, 7-week- and 12-week- hearts (p<0.05 threshold, calculated by Deseq algorithm "R"). The expression level of s100a9 was higher in wa3 mice than in WT by 2.8 times. For the full list of genes see Tables S1 and S2, and functional analyses in Figures S3-S6.


C . Hierarchical clustering heat map of differentially expressed genes in the hearts of 7 - and 12 - week old wa3 mice in comparison to WT .

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C. Hierarchical clustering heat map of differentially expressed genes in the hearts of 7- and 12- week old wa3 mice in comparison to WT.


A . LPS ( 5mg / Kg for 2 hrs ) induced higher expression levels of cytokine genes in wa3 hearts than in littermate WT hearts . The results are presented as fold induction of untreated controls . The mRNA expression levels of Pln and Ryr2 was reduced in both wa3 and WT hearts , while of Mybpc3 and Sgcg was unchanged . For the full list see Dataset EV3 and for functional clustering in Appendix Figure S7 . Differences between knockdown and control with p values = 0 . 04998 ( Student ' s t test ) are indicated with an asterisk .

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A. LPS (5mg/Kg for 2 hrs) induced higher expression levels of cytokine genes in wa3 hearts than in littermate WT hearts. The results are presented as fold induction of untreated controls. The mRNA expression levels of Pln and Ryr2 was reduced in both wa3 and WT hearts, while of Mybpc3 and Sgcg was unchanged. For the full list see Dataset EV3 and for functional clustering in Appendix Figure S7. Differences between knockdown and control with p values =0.04998 (Student's t test) are indicated with an asterisk.


B shows that LPS induced a higher mortality rate of wa3 mice than of littermate controls following two weekly subsequent injections of LPS ( 1mg / Kg ) .

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B shows that LPS induced a higher mortality rate of wa3 mice than of littermate controls following two weekly subsequent injections of LPS (1mg/Kg).


A - D SQST ‐ 1 : : GFP is very weakly expressed and diffusely localized in the cytoplasm in wild ‐ type larval intestine , but is expressed at much higher levels and accumulates into numerous aggregates in the intestine in bp399 mutant larvae . ( A ) and ( C ) represent DIC images of the animals in ( B ) and ( D ) , respectively .

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A-D SQST‐1::GFP is very weakly expressed and diffusely localized in the cytoplasm in wild‐type larval intestine, but is expressed at much higher levels and accumulates into numerous aggregates in the intestine in bp399 mutant larvae. (A) and (C) represent DIC images of the animals in (B) and (D), respectively.


E Compared to wild ‐ type , SQST ‐ 1 : : GFP levels are much higher in bp399 and bp412 mutant larvae as shown by immunoblotting assay . SQST ‐ 1 : : GFP levels in bp399 mutants are greatly reduced after 12 h of starvation . 200 young adult animals for each genotype were collected for analysis .

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E Compared to wild‐type, SQST‐1::GFP levels are much higher in bp399 and bp412 mutant larvae as shown by immunoblotting assay. SQST‐1::GFP levels in bp399 mutants are greatly reduced after 12 h of starvation. 200 young adult animals for each genotype were collected for analysis.


H , I SQST ‐ 1 : : GFP aggregates in rpl ‐ 43 ( bp399 ) mutants are separable from LAAT ‐ 1 : : Cherry ‐ labeled lysosomes under normal conditions , but are delivered to lysosomes upon starvation . Some aggregates are enclosed by lysosomes ( inserts ) . Scale bar : 20 μm .

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H, I SQST‐1::GFP aggregates in rpl‐43(bp399) mutants are separable from LAAT‐1::Cherry‐labeled lysosomes under normal conditions, but are delivered to lysosomes upon starvation. Some aggregates are enclosed by lysosomes (inserts). Scale bar: 20 μm.


J - Q RNAi inactivation of let ‐ 363 , rpn ‐ 2 , cogc ‐ 1 , or hgrs ‐ 1 suppresses the accumulation of SQST ‐ 1 : : GFP aggregates in rpl ‐ 43 ( bp399 ) mutants . ( J ) , ( L ) , ( N ) , and ( P ) : DIC images of the animals in ( K ) , ( M ) , ( O ) , and ( Q ) . Scale bar : 20 μm .

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J-Q RNAi inactivation of let‐363, rpn‐2, cogc‐1, or hgrs‐1 suppresses the accumulation of SQST‐1::GFP aggregates in rpl‐43(bp399) mutants. (J), (L), (N), and (P): DIC images of the animals in (K), (M), (O), and (Q). Scale bar: 20 μm.


R SQST ‐ 1 : : GFP levels in rpl ‐ 43 ( bp399 ) mutants are greatly reduced by inactivation of rpt ‐ 3 and rpn ‐ 2 . 200 young adult animals for each genotype were collected for analysis .

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R SQST‐1::GFP levels in rpl‐43(bp399) mutants are greatly reduced by inactivation of rpt‐3 and rpn‐2. 200 young adult animals for each genotype were collected for analysis.


A , B SQST ‐ 1 : : GFP aggregates are absent in the rpl ‐ 43 ( bp399 ) ; sma ‐ 3 ( wk20 ) mutant intestine .

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A, B SQST‐1::GFP aggregates are absent in the rpl‐43(bp399); sma‐3(wk20) mutant intestine.


C Percentage of the indicated animals with different levels of SQST ‐ 1 : : GFP aggregates . S : many SQST ‐ 1 : : GFP aggregates in all intestinal cells , as in rpl ‐ 43 ( bp399 ) mutants ; M : fewer SQST ‐ 1 : : GFP aggregates in some but not all intestinal cells ; N : no or very few SQST ‐ 1 : : GFP aggregates in intestinal cells . For each genotype , > 30 animals were examined .

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C Percentage of the indicated animals with different levels of SQST‐1::GFP aggregates. S: many SQST‐1::GFP aggregates in all intestinal cells, as in rpl‐43(bp399) mutants; M: fewer SQST‐1::GFP aggregates in some but not all intestinal cells; N: no or very few SQST‐1::GFP aggregates in intestinal cells. For each genotype, > 30 animals were examined.


D SQST ‐ 1 : : GFP levels in rpl ‐ 43 ( bp399 ) mutants are dramatically decreased in a Western blot when sma ‐ 3 , lin ‐ 35 , or daf ‐ 2 is simultaneously depleted . Loss of function of daf ‐ 16 partially restores SQST ‐ 1 levels in rpl ‐ 43 ( bp399 ) ; daf ‐ 2 ( RNAi ) mutants .

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D SQST‐1::GFP levels in rpl‐43(bp399) mutants are dramatically decreased in a Western blot when sma‐3, lin‐35, or daf‐2 is simultaneously depleted. Loss of function of daf‐16 partially restores SQST‐1 levels in rpl‐43(bp399); daf‐2(RNAi) mutants.


E Endogenous LGG ‐ 1 is elevated in a Western blot in dbl ‐ 1 ( wk70 ) , sma ‐ 2 ( e502 ) , and sma ‐ 3 ( wk20 ) mutants .

E Endogenous LGG‐1 is elevated in a Western blot in dbl‐1(wk70), sma‐2(e502), and sma‐3(wk20) mutants.


F mRNA levels of bec ‐ 1 , epg ‐ 8 , lgg ‐ 1 , and atg ‐ 7 are upregulated in dbl ‐ 1 ( wk70 ) , sma ‐ 2 ( e502 ) , and sma ‐ 3 ( wk20 ) mutants . * * P 0 . 01 . 500 young adult animals were collected for analysis . Error bars indicate s . d . from three experiments .

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F mRNA levels of bec‐1, epg‐8, lgg‐1, and atg‐7 are upregulated in dbl‐1(wk70), sma‐2(e502), and sma‐3(wk20) mutants. **P 0.01. 500 young adult animals were collected for analysis. Error bars indicate s.d. from three experiments.


G , H No SQST ‐ 1 : : GFP aggregates are observed in the lin ‐ 35 ( n745 ) ; rpl ‐ 43 ( bp399 ) mutant intestine .

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G, H No SQST‐1::GFP aggregates are observed in the lin‐35(n745); rpl‐43(bp399) mutant intestine.


I , J Numerous SQST ‐ 1 : : GFP aggregates are formed in rpl ‐ 43 ( bp399 ) ; lin ‐ 15A ( n767 ) mutants .

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I, J Numerous SQST‐1::GFP aggregates are formed in rpl‐43(bp399); lin‐15A(n767) mutants.


K Endogenous LGG ‐ 1 levels are increased in lin ‐ 9 ( n112 ) , lin ‐ 15B ( n744 ) , and lin ‐ 35 ( n745 ) mutants .

K Endogenous LGG‐1 levels are increased in lin‐9(n112), lin‐15B(n744), and lin‐35(n745) mutants.


L mRNA levels of bec ‐ 1 , epg ‐ 8 , lgg ‐ 1 , and atg ‐ 7 are upregulated in lin ‐ 9 ( n112 ) , lin ‐ 15B ( n744 ) , and lin ‐ 35 ( n745 ) mutants . * * P 0 . 01 . 500 young adult animals were collected for analysis . Error bars indicate s . d . from three experiments .

L mRNA levels of bec‐1, epg‐8, lgg‐1, and atg‐7 are upregulated in lin‐9(n112), lin‐15B(n744), and lin‐35(n745) mutants. **P 0.01. 500 young adult animals were collected for analysis. Error bars indicate s.d. from three experiments.


M , N rpl ‐ 43 ( bp399 ) ; daf ‐ 2 ( RNAi ) mutant larvae contain no intestinal SQST ‐ 1 : : GFP aggregates .

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M, N rpl‐43(bp399); daf‐2(RNAi) mutant larvae contain no intestinal SQST‐1::GFP aggregates.


O , P SQST ‐ 1 : : GFP aggregates are partially restored in daf ‐ 16 ( mu86 ) ; rpl ‐ 43 ( bp399 ) ; daf ‐ 2 ( RNAi ) animals .

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O, P SQST‐1::GFP aggregates are partially restored in daf‐16(mu86); rpl‐43(bp399); daf‐2(RNAi) animals.


A , B hsp ‐ 4 : : GFP is very weakly expressed in wild ‐ type animals .

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A, B hsp‐4::GFP is very weakly expressed in wild‐type animals.


C Percentage of indicated animals with different levels of SQST ‐ 1 : : GFP aggregates . S : strong . M : medium . N : none . > 30 animals were examined for each genotype .

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C Percentage of indicated animals with different levels of SQST‐1::GFP aggregates. S: strong. M: medium. N: none. >30 animals were examined for each genotype.


D , E Phsp ‐ 4 : : GFP expression is much higher in rpt ‐ 3 ( RNAi ) animals .

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D, E Phsp‐4::GFP expression is much higher in rpt‐3(RNAi) animals.


F , G Upregulation of Phsp ‐ 4 : : GFP expression in rpt ‐ 3 ( RNAi ) animals is suppressed by the xbp ‐ 1 ( zc12 ) mutation .

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F, G Upregulation of Phsp‐4::GFP expression in rpt‐3(RNAi) animals is suppressed by the xbp‐1(zc12) mutation.


H , I SQST ‐ 1 : : GFP aggregates are absent in rpl ‐ 43 ( bp399 ) ; rpt ‐ 3 ( RNAi ) animals .

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H, I SQST‐1::GFP aggregates are absent in rpl‐43(bp399); rpt‐3(RNAi) animals.


J , K Loss of function of xbp ‐ 1 restores SQST ‐ 1 : : GFP aggregates in rpl ‐ 43 ( bp399 ) ; rpt ‐ 3 ( RNAi ) animals .

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J, K Loss of function of xbp‐1 restores SQST‐1::GFP aggregates in rpl‐43(bp399); rpt‐3(RNAi) animals.


M The increase in bec ‐ 1 , epg ‐ 8 , lgg ‐ 1 , and atg ‐ 7 mRNA levels in npl ‐ 4 . 1 , rpt ‐ 3 , and pep ‐ 2 RNAi animals is dependent on xbp ‐ 1 . * P 0 . 05 , * * P 0 . 01 . 500 young adult animals were collected for analysis . Error bars indicate s . d . from three experiments .

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M The increase in bec‐1, epg‐8, lgg‐1, and atg‐7 mRNA levels in npl‐4.1, rpt‐3, and pep‐2 RNAi animals is dependent on xbp‐1. *P 0.05, **P 0.01. 500 young adult animals were collected for analysis. Error bars indicate s.d. from three experiments.


A Phsp ‐ 60 : : GFP is weakly expressed in wild ‐ type animals .

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A Phsp‐60::GFP is weakly expressed in wild‐type animals.


B egl ‐ 46 ( RNAi ) causes upregulation of Phsp ‐ 60 : : GFP .

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B egl‐46(RNAi) causes upregulation of Phsp‐60::GFP.


C Percentage of indicated mutant animals with different levels of SQST ‐ 1 : : GFP aggregates . S : strong . M : medium . N : none . > 30 animals were examined in each group .

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C Percentage of indicated mutant animals with different levels of SQST‐1::GFP aggregates. S: strong. M: medium. N: none. >30 animals were examined in each group.


D Levels of SQST ‐ 1 : : GFP in various genetic mutants . 200 young adult animals for each genotype were collected for analysis .

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D Levels of SQST‐1::GFP in various genetic mutants. 200 young adult animals for each genotype were collected for analysis.


E , F Loss of function of atfs ‐ 1 suppresses the upregulation of Phsp ‐ 60 : : GFP in egl ‐ 46 ( n1126 ) mutants . ( E ) : DIC image of ( F ) .

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E, F Loss of function of atfs‐1 suppresses the upregulation of Phsp‐60::GFP in egl‐46(n1126) mutants. (E): DIC image of (F).


G , H The dramatic decrease in the number of SQST ‐ 1 : : GFP aggregates in the intestine in rpl ‐ 43 ( bp399 ) ; egl ‐ 46 ( RNAi ) mutants ( G ) is restored by simultaneous loss of function of atfs ‐ 1 ( gk3094 ) ( H ) .

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G, H The dramatic decrease in the number of SQST‐1::GFP aggregates in the intestine in rpl‐43(bp399); egl‐46(RNAi) mutants (G) is restored by simultaneous loss of function of atfs‐1(gk3094) (H).


I , J GFP : : LGG ‐ 1 forms numerous puncta in the intestine in egl ‐ 46 ( RNAi ) ( I ) , gfi ‐ 1 ( RNAi ) ( J ) animals .

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I, J GFP::LGG‐1 forms numerous puncta in the intestine in egl‐46(RNAi) (I), gfi‐1(RNAi) (J) animals.


( a ) Western blot analysis of myosin - VI - depleted HeLa cells untreated or treated with 100 nM bafilomycin A1 . ( b ) Quantification of western blot LC3 - II intensity ( ± s . d . ; n = 3 ) . * * P0 . 01 , * * * P0 .

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(a) Western blot analysis of myosin-VI-depleted HeLa cells untreated or treated with 100 nM bafilomycin A1. (b) Quantification of western blot LC3-II intensity (±s.d.; n = 3). **P0.01, ***P0.001


( c ) Confocal immunofluorescence microscopy of LC3 punctae ( green ) in HeLa cells following knockdown of myosin VI . Hoechst labels the nuclei ( blue ) . The insets show higher magnifications of the areas outlined in the main images ( d ) Quantification of LC3 - and p62 - positive punctae was performed and the results are represented as the average punctae fluorescence intensity per cell ( ± s . d . ; n = 3 ) from > 1 , 500 cells per experiment .

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(c) Confocal immunofluorescence microscopy of LC3 punctae (green) in HeLa cells following knockdown of myosin VI. Hoechst labels the nuclei (blue). The insets show higher magnifications of the areas outlined in the main images (d) Quantification of LC3- and p62-positive punctae was performed and the results are represented as the average punctae fluorescence intensity per cell (±s.d.; n = 3) from >1,500 cells per experiment.


( e ) Western blot analysis of parental HeLa cells or HeLa cells stably expressing siRNA - resistant GFP - myosin VI transiently transfected with a single myosin VI siRNA oligonucleotide following treatment with 1 μM MG132 .

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(e) Western blot analysis of parental HeLa cells or HeLa cells stably expressing siRNA-resistant GFP-myosin VI transiently transfected with a single myosin VI siRNA oligonucleotide following treatment with 1 μM MG132.


( f ) Western blot analysis of MEFs cultured from wild - type and Snell ' s waltzer ( SV ) mice treated with 1 μM MG132 or 100 nM bafilomycin A1 .

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(f) Western blot analysis of MEFs cultured from wild-type and Snell's waltzer (SV) mice treated with 1 μM MG132 or 100 nM bafilomycin A1.


( g ) Quantification of western blot LC3 - II intensity ( ± s . d . ; n = 3 ) . * * P0 . 01 . ( h ) To evaluate the effects on autophagosome biogenesis , the results are shown as the fold increase in normalized LC3 - II intensity with bafilomycin A1 compared with the untreated control . ( ± s . d . ; n = 3 ) .

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(g) Quantification of western blot LC3-II intensity (±s.d.; n = 3). **P0.01. (h) To evaluate the effects on autophagosome biogenesis, the results are shown as the fold increase in normalized LC3-II intensity with bafilomycin A1 compared with the untreated control. (±s.d.; n = 3).


( i ) Western blot analysis of cortical neurons from wild - type and SV mice treated with 1 μM MG132 in the absence or presence of 100 nM bafilomycin A1 . ( j ) Quantification of western blot LC3 - II intensity was performed ( n = 2 ) . ( k ) To evaluate the effects on autophagosome biogenesis , the results are shown as the fold increase in normalized LC3 - II intensity with bafilomycin A1 compared with the untreated control ( n = 2 ) .

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(i) Western blot analysis of cortical neurons from wild-type and SV mice treated with 1 μM MG132 in the absence or presence of 100 nM bafilomycin A1. (j) Quantification of western blot LC3-II intensity was performed (n = 2). (k) To evaluate the effects on autophagosome biogenesis, the results are shown as the fold increase in normalized LC3-II intensity with bafilomycin A1 compared with the untreated control (n = 2).


( l ) Western blot ( top ) and confocal immunofluorescence microscopy ( bottom ) of mock - or myosin - VI - siRNA - treated HeLa cells stably expressing the RFP - GFP - LC3 reporter . Hoechst labels the nuclei ( blue ) . ( m ) Quantitative data of RFP and GFP signal overlap from confocal images of mock - or myosin - VI - siRNA - treated HeLa cells expressing RFP - GFP - LC3 . The data are represented as the Pearson ' s coefficient of the RFP and GFP signal correlation from > 100cells per experiment ( ± s . d . ; n = 3 ) .

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(l) Western blot (top) and confocal immunofluorescence microscopy (bottom) of mock- or myosin-VI-siRNA-treated HeLa cells stably expressing the RFP-GFP-LC3 reporter. Hoechst labels the nuclei (blue). (m) Quantitative data of RFP and GFP signal overlap from confocal images of mock- or myosin-VI-siRNA-treated HeLa cells expressing RFP-GFP-LC3. The data are represented as the Pearson's coefficient of the RFP and GFP signal correlation from >100cells per experiment (±s.d.; n = 3).


n ) Confocal immunofluorescence microscopy of myosin - VI - depleted RPE cells stably expressing GFP - LC3 immunostained against GFP ( green ) and cathepsin D ( red ) . Hoechst labels the nuclei ( blue ) . The insets show higher magnifications of the areas outlined in the main images . Scale bars , 20 μm . Uncropped images of blots are shown in Supplementary Fig . S9 .

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n) Confocal immunofluorescence microscopy of myosin-VI-depleted RPE cells stably expressing GFP-LC3 immunostained against GFP (green) and cathepsin D (red). Hoechst labels the nuclei (blue). The insets show higher magnifications of the areas outlined in the main images. Scale bars, 20 μm. Uncropped images of blots are shown in Supplementary Fig. S9.


( a ) RPE cells transiently transfected with siRNA against myosin VI and Atg5 were treated with 1 μM MG132 for 16 h . Cells were processed for immunofluorescence microscopy following 16 h MG132 treatment ( zero time point ) and 8 h post - washout of inhibitor . Immunolabelling for p62 was performed to visualize aggregates ( red ) and Hoechst was used to identify nuclei ( blue ) .

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(a) RPE cells transiently transfected with siRNA against myosin VI and Atg5 were treated with 1 μM MG132 for 16 h. Cells were processed for immunofluorescence microscopy following 16 h MG132 treatment (zero time point) and 8 h post-washout of inhibitor. Immunolabelling for p62 was performed to visualize aggregates (red) and Hoechst was used to identify nuclei (blue).


( b ) Top , western blot analysis confirming siRNA depletion . Bottom , quantification of immunofluorescent p62 - positive aggregates was evaluated using an automated Cellomics VTi microscope . The results were calculated as the average p62 fluorescence intensity at 8 h post - MG132 washout normalized to the zero time point and are represented as the percentage of the control ( ± s . d . ; n = 3 ) . * P0 . 05 , * * * P0 . 001 . See Methods and Supplementary Table S1 for details of the SMARTpool ( SP ) and single ( 07 ) oligonucleotides .

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(b) Top, western blot analysis confirming siRNA depletion. Bottom, quantification of immunofluorescent p62-positive aggregates was evaluated using an automated Cellomics VTi microscope. The results were calculated as the average p62 fluorescence intensity at 8 h post-MG132 washout normalized to the zero time point and are represented as the percentage of the control (±s.d.; n = 3). *P0.05, ***P0.001. See Methods and Supplementary Table S1 for details of the SMARTpool (SP) and single (07) oligonucleotides.


( c ) Parental HeLa cells or HeLa cells stable expressing siRNA - resistant GFP - myosin VI were transiently transfected with a single siRNA oligonucleotide targeting myosin VI and were subsequently treated with 1 μM MG132 for 16 h . Cells were processed for immunofluorescence microscopy at 16 h post - MG132 treatment ( t = 0 ) or allowed to recover following washout of MG132 for 8 h ( t = 8 ) . Cells were processed for quantification with the automated Cellomics VTi microscope to evaluate the p62 fluorescence intensity . The results represent the fold increase in p62 fluorescence intensity of myosin VI siRNA compared with mock control cells following recovery from MG132 washout ( t = 8 ; ± s . d . ; n = 3 ) .

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(c) Parental HeLa cells or HeLa cells stable expressing siRNA-resistant GFP-myosin VI were transiently transfected with a single siRNA oligonucleotide targeting myosin VI and were subsequently treated with 1 μM MG132 for 16 h. Cells were processed for immunofluorescence microscopy at 16 h post-MG132 treatment (t = 0) or allowed to recover following washout of MG132 for 8 h (t = 8). Cells were processed for quantification with the automated Cellomics VTi microscope to evaluate the p62 fluorescence intensity. The results represent the fold increase in p62 fluorescence intensity of myosin VI siRNA compared with mock control cells following recovery from MG132 washout (t = 8; ±s.d.; n = 3).


( d ) HeLa cells with stable expression of HttQ72 - GFP were transiently transfected with siRNA against myosin VI followed by saponin extraction and processing for immunofluorescence microscopy . Immunolabelling was performed for GFP ( green ) and p62 ( red ) . Nuclei are labelled with Hoechst ( blue ) . ( e ) Quantification of HttQ72 - GFP aggregates was performed on myosin - VI - siRNA - transfected HeLa cells . The results were calculated as the percentage of GFP - expressing cells with greater than 15 GFP - positive spots per cell . The results represent the mean ( ± s . d . ) from n = 3independent experiments , * * * P0 . 001 . The insets show higher magnifications of the areas outlined in the main images . Scale bars , 20 μm . Uncropped images of blots are shown in Supplementary Fig . S9 .

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(d) HeLa cells with stable expression of HttQ72-GFP were transiently transfected with siRNA against myosin VI followed by saponin extraction and processing for immunofluorescence microscopy. Immunolabelling was performed for GFP (green) and p62 (red). Nuclei are labelled with Hoechst (blue). (e) Quantification of HttQ72-GFP aggregates was performed on myosin-VI-siRNA-transfected HeLa cells. The results were calculated as the percentage of GFP-expressing cells with greater than 15 GFP-positive spots per cell. The results represent the mean (±s.d.) from n = 3independent experiments, ***P0.001. The insets show higher magnifications of the areas outlined in the main images. Scale bars, 20 μm. Uncropped images of blots are shown in Supplementary Fig. S9.


( a ) RPE cells transiently co - transfected with GFP - myosin VI and cherry - LC3 and either left untreated ( top row ) or treated with 250 nM Torin1 for 2 h ( bottom row ) were processed for immunofluorescence microscopy . The arrows highlight areas of co - localization and the insets show higher magnifications of the areas outlined in the main images .

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(a) RPE cells transiently co-transfected with GFP-myosin VI and cherry-LC3 and either left untreated (top row) or treated with 250 nM Torin1 for 2 h (bottom row) were processed for immunofluorescence microscopy. The arrows highlight areas of co-localization and the insets show higher magnifications of the areas outlined in the main images.


( b ) RPE cells treated with 100 nM bafilomycin A1 were processed for immunofluorescence microscopy to evaluate endogenous myosin VI and endogenous LC3 co - localization . The arrows highlight areas of co - localization and the insets show higher magnifications of the areas outlined in the main images .

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(b) RPE cells treated with 100 nM bafilomycin A1 were processed for immunofluorescence microscopy to evaluate endogenous myosin VI and endogenous LC3 co-localization. The arrows highlight areas of co-localization and the insets show higher magnifications of the areas outlined in the main images.


( c ) Image stills acquired from spinning - disc time - lapse video microscopy of RPE cells transiently co - transfected with cherry - LC3 and GFP - myosin VI . The outlined regions highlight areas of interest within the cell that are shown to the right ( i - iii ) as video image stills captured at 1 - min intervals . The arrowheads highlight myosin - VI - positive vesicles ( green ) coming into contact and co - localizing with LC3 - positive vesicles ( red ) .

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(c) Image stills acquired from spinning-disc time-lapse video microscopy of RPE cells transiently co-transfected with cherry-LC3 and GFP-myosin VI. The outlined regions highlight areas of interest within the cell that are shown to the right (i-iii) as video image stills captured at 1-min intervals. The arrowheads highlight myosin-VI-positive vesicles (green) coming into contact and co-localizing with LC3-positive vesicles (red).


( d ) Confocal immunofluorescence microscopy of RPE cells stably expressing cherry - LC3 following transient transfection with GFP - myosin VI . Immunostaining for GFP ( green ) and cherry ( red ) was performed and actin was visualized with phalloidin ( white ) . Nuclei were labelled with Hoechst ( blue ) . The arrowheads indicate areas of actin - rich myosin - VI / LC3 - positive autophagosomes . Scale bars , 20 μm ( a , b , d ) ; 10 μm ( c , whole - cell image ) ; 2 μm ( c , cropped images ) .

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(d) Confocal immunofluorescence microscopy of RPE cells stably expressing cherry-LC3 following transient transfection with GFP-myosin VI. Immunostaining for GFP (green) and cherry (red) was performed and actin was visualized with phalloidin (white). Nuclei were labelled with Hoechst (blue). The arrowheads indicate areas of actin-rich myosin-VI/LC3-positive autophagosomes. Scale bars, 20 μm (a,b,d); 10 μm (c, whole-cell image); 2 μm (c, cropped images).


( b ) RPE cells with stable expression of cherry - LC3 were transiently transfected with the GFP - myosin VI cargo - binding tail domain containing various mutations in the protein - interaction ( ΔWWY and ΔRRL ) and ubiquitin - binding motifs ( A1013G ) , followed by treatment with 250 nM Torin1 for 3 h to induce autophagy . Immunofluorescence microscopy was performed either in the absence or presence of saponin extraction . The arrowheads indicate areas of co - localization . Scale bar , 20 μm . ( c ) A Pearson ' s coefficient was calculated on the basis of the degree of co - localization between the different GFP - myosin VI mutant tails and cherry - LC3 from confocal immunofluorescence micrographs . The graph represents data from more than 20 transfected cells from n = 2 independent experiments .

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(b) RPE cells with stable expression of cherry-LC3 were transiently transfected with the GFP-myosin VI cargo-binding tail domain containing various mutations in the protein-interaction (ΔWWY and ΔRRL) and ubiquitin-binding motifs (A1013G), followed by treatment with 250 nM Torin1 for 3 h to induce autophagy. Immunofluorescence microscopy was performed either in the absence or presence of saponin extraction. The arrowheads indicate areas of co-localization. Scale bar, 20 μm. (c) A Pearson's coefficient was calculated on the basis of the degree of co-localization between the different GFP-myosin VI mutant tails and cherry-LC3 from confocal immunofluorescence micrographs. The graph represents data from more than 20 transfected cells from n = 2 independent experiments.


( a ) Left , immunofluorescence microscopy was performed on RPE cells following treatment with 100 nM bafilomycin A1 for 2 h . Immunolabelling was performed against the indicated endogenous proteins optineurin , T6BP and NDP52 ( red ) , and LC3 ( green ) . The insets show higher magnifications of the areas outlined in the main images . Right , from the confocal images , a Pearson ' s coefficient was calculated to estimate the degree of co - localization of the different autophagy adaptors with LC3 . As a negative control , a Pearson ' s coefficient was calculated after rotation of one colour image by 90 ° . The results represent > 20 cells from n = 2 independent experiments .

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(a) Left, immunofluorescence microscopy was performed on RPE cells following treatment with 100 nM bafilomycin A1 for 2 h. Immunolabelling was performed against the indicated endogenous proteins optineurin, T6BP and NDP52 (red), and LC3 (green). The insets show higher magnifications of the areas outlined in the main images. Right, from the confocal images, a Pearson's coefficient was calculated to estimate the degree of co-localization of the different autophagy adaptors with LC3. As a negative control, a Pearson's coefficient was calculated after rotation of one colour image by 90°. The results represent >20 cells from n = 2 independent experiments.


( b ) Left , stable cherry - LC3 - expressing RPE cells were transiently transfected with GFP - myosin VI , treated with Torin1 for 3 h to induce autophagy , and subsequently processed for confocal immunofluorescence microscopy . Areas of co - localization are white in the merged three - colour images and the insets show higher magnifications of the areas outlined in the main images . Right , a Pearson ' s coefficient was calculated for the degree of co - localization between the three colours . As a negative control , a Pearson ' s coefficient was calculated after rotating one colour image by 90 ° . Results represent > 10 cells from n = 2 independent experiments .

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(b) Left, stable cherry-LC3-expressing RPE cells were transiently transfected with GFP-myosin VI, treated with Torin1 for 3 h to induce autophagy, and subsequently processed for confocal immunofluorescence microscopy. Areas of co-localization are white in the merged three-colour images and the insets show higher magnifications of the areas outlined in the main images. Right, a Pearson's coefficient was calculated for the degree of co-localization between the three colours. As a negative control, a Pearson's coefficient was calculated after rotating one colour image by 90°. Results represent >10 cells from n = 2 independent experiments.


( c ) HeLa cells were either mock transfected or co - transfected with siRNA targeted against TNO . Cells were either left untreated or treated with100 nM bafilomycin A1 ( BfnA1 ) for 4 h . Western blot analysis was performed against the indicated proteins . ( d ) Quantification of LC3 - II intensity ( ± s . d . ) was performed from western blot data of TNO - siRNA treated HeLa cells . The results represent n = 3independent experiments . * * P0 . 01 , * * * P0 .

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(c) HeLa cells were either mock transfected or co-transfected with siRNA targeted against TNO. Cells were either left untreated or treated with100 nM bafilomycin A1 (BfnA1) for 4 h. Western blot analysis was performed against the indicated proteins. (d) Quantification of LC3-II intensity (±s.d.) was performed from western blot data of TNO-siRNA treated HeLa cells. The results represent n = 3independent experiments. **P0.01, ***P0.001


( e ) Immunofluorescence microscopy of RFP - GFP - LC3 - expressing HeLa cells transfected with TNO siRNA was performed and confocal images were evaluated for the correlation in GFP and RFP punctae signal overlap using the Image JACoP plugin . The results are calculated from > 100 cells from n = 2 independent experiments represented as the Pearson ' s coefficient of GFP / RFP signal overlap . Scale bars , 20 μm . Uncropped images of blots are shown in Supplementary Fig . S9 .

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(e) Immunofluorescence microscopy of RFP-GFP-LC3-expressing HeLa cells transfected with TNO siRNA was performed and confocal images were evaluated for the correlation in GFP and RFP punctae signal overlap using the Image JACoP plugin. The results are calculated from >100 cells from n = 2 independent experiments represented as the Pearson's coefficient of GFP/RFP signal overlap. Scale bars, 20 μm. Uncropped images of blots are shown in Supplementary Fig. S9.


( a ) Myosin VI and Tom1 / Tom1L2 co - immunoprecipitate from LNCAP cell lysates . Immunoprecipitation of myosin VI with three separate affinity - purified rabbit polyclonal antibodies ( B4 , B7 , 2401 ) was performed alongside an IgG control immunoprecipitation . The immunoprecipitates were analysed by western blotting with antibodies against Tom1 / Tom1L2 .

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(a) Myosin VI and Tom1/Tom1L2 co-immunoprecipitate from LNCAP cell lysates. Immunoprecipitation of myosin VI with three separate affinity-purified rabbit polyclonal antibodies (B4, B7, 2401) was performed alongside an IgG control immunoprecipitation. The immunoprecipitates were analysed by western blotting with antibodies against Tom1/Tom1L2.


( b ) Binding of myosin VI to Tom1 requires the WWY motif . Bottom , the mammalian two - hybrid assay was used to test binding of full - length Tom1 and Tom1L2 or various truncated versions of Tom1 , annotated by amino acid numbers , against wild - type myosin VI tail or the myosin VI ΔWWY or ΔRRL tail . The graph shows the mean values from n = 2 independent experiments . Top , Tom1 domain structure and truncation mutants . VHS , Vps - 27 , Hrs and STAM ; GAT , GGA and Tom1 .

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(b) Binding of myosin VI to Tom1 requires the WWY motif. Bottom, the mammalian two-hybrid assay was used to test binding of full-length Tom1 and Tom1L2 or various truncated versions of Tom1, annotated by amino acid numbers, against wild-type myosin VI tail or the myosin VI ΔWWY or ΔRRL tail. The graph shows the mean values from n = 2 independent experiments. Top, Tom1 domain structure and truncation mutants. VHS, Vps-27, Hrs and STAM; GAT, GGA and Tom1.


( c ) RPE cells stably expressing GFP - myosin VI were processed for immunofluorescence microscopy to evaluate GFP - myosin VI and endogenous Tom1 / Tom1L2 or Rab5 co - localization . The insets show higher magnifications of the areas outlined in the main panels . The arrows indicate areas of co - localization .

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(c) RPE cells stably expressing GFP-myosin VI were processed for immunofluorescence microscopy to evaluate GFP-myosin VI and endogenous Tom1/Tom1L2 or Rab5 co-localization. The insets show higher magnifications of the areas outlined in the main panels. The arrows indicate areas of co-localization.


( d ) RPE cells with stable expression of GFP - myosin VI tail were subjected to mock or Tom1 siRNA transfection followed by western blot analysis ( left ) and immunofluorescence microscopy ( middle ) to evaluate GFP - myosin VI tail localization . Cells were either fixed directly or saponin - extracted before fixation . Quantification ( right ) was performed on RPE cells stably expressing GFP - myosin VI tail and transfected with mock , Tom1 or TNO siRNA . Cells were processed for immunocytochemistry , immunolabelled for GFP and nuclei labelled with Hoechst followed by quantification of GFP - myosin VI tail punctae per cell using an automated Cellomics VTi microscope . More than 600 cells per group from n = 3 independent experiments were analysed and are represented as the mean number of GFP - myosin VI tail punctae per cell ( ± s . d . ) . Scale bars , 20 μm . Uncropped images of blots are shown in Supplementary Fig . S9 .

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(d) RPE cells with stable expression of GFP-myosin VI tail were subjected to mock or Tom1 siRNA transfection followed by western blot analysis (left) and immunofluorescence microscopy (middle) to evaluate GFP-myosin VI tail localization. Cells were either fixed directly or saponin-extracted before fixation. Quantification (right) was performed on RPE cells stably expressing GFP-myosin VI tail and transfected with mock, Tom1 or TNO siRNA. Cells were processed for immunocytochemistry, immunolabelled for GFP and nuclei labelled with Hoechst followed by quantification of GFP-myosin VI tail punctae per cell using an automated Cellomics VTi microscope. More than 600 cells per group from n = 3 independent experiments were analysed and are represented as the mean number of GFP-myosin VI tail punctae per cell (±s.d.). Scale bars, 20 μm. Uncropped images of blots are shown in Supplementary Fig. S9.


( a ) HeLa cells transiently transfected with siRNA against Tom1 were left untreated or treated with 100 nM bafilomycin A1 for 4 h . Western blot analysis was performed on whole - cell lysates against the indicated proteins . ( b ) Quantification of LC3 - II intensity of western blots was performed by an infrared imaging system . ( ± s . d . ; n = 3 ) . * P0 . 05 , * * P0 . 01 .

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(a) HeLa cells transiently transfected with siRNA against Tom1 were left untreated or treated with 100 nM bafilomycin A1 for 4 h. Western blot analysis was performed on whole-cell lysates against the indicated proteins. (b) Quantification of LC3-II intensity of western blots was performed by an infrared imaging system. (±s.d.; n = 3). *P0.05, **P0.01.


( c , left ) Confocal immunofluorescence microscopy was performed on HeLa cells transfected with Tom1 siRNA to evaluate LC3 punctae formation ( green ) . The insets show higher magnifications of the areas outlined in the main images . Nuclei are labelled with Hoechst ( blue ) . ( c , right , d ) Quantification of LC3 - positive punctae ( c , right ) and p62 - positive punctae ( d ) was performed in a 96 - well format on an Arrayscan VTi HCS microscope . Cells were identified by Hoechst staining of nuclei ( blue ) . The results are represented as the average punctae fluorescence intensity per cell ( ± s . d . ) from n = 3 independent experiments each performed in triplicate wells , each with > 500 cells .

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(c, left) Confocal immunofluorescence microscopy was performed on HeLa cells transfected with Tom1 siRNA to evaluate LC3 punctae formation (green). The insets show higher magnifications of the areas outlined in the main images. Nuclei are labelled with Hoechst (blue). (c,right,d) Quantification of LC3-positive punctae (c, right) and p62-positive punctae (d) was performed in a 96-well format on an Arrayscan VTi HCS microscope. Cells were identified by Hoechst staining of nuclei (blue). The results are represented as the average punctae fluorescence intensity per cell (±s.d.) from n = 3 independent experiments each performed in triplicate wells, each with >500 cells.


( e ) HeLa cells with stable expression of the RFP - GFP - LC3 reporter construct were subjected to mock or Tom1 siRNA mediated knockdown ( left ) . Confocal immunofluorescence microscopy ( middle ) was performed and images were subsequently quantified for the correlation between the GFP and RFP signals using the ImageJ JACoP plugin ( right ) . The results are for at least 100 individual cells from n = 3 independent experiments and are represented as the Pearson ' s coefficient of GFP / RFP overlap ( ± s . d . ) . A higher Pearson ' s coefficient of GFP / RFP signal overlap represents a greater number of autophagosomes when compared with autolysosomes .

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(e) HeLa cells with stable expression of the RFP-GFP-LC3 reporter construct were subjected to mock or Tom1 siRNA mediated knockdown (left). Confocal immunofluorescence microscopy (middle) was performed and images were subsequently quantified for the correlation between the GFP and RFP signals using the ImageJ JACoP plugin (right). The results are for at least 100 individual cells from n = 3 independent experiments and are represented as the Pearson's coefficient of GFP/RFP overlap (±s.d.). A higher Pearson's coefficient of GFP/RFP signal overlap represents a greater number of autophagosomes when compared with autolysosomes.


( f ) Confocal immunofluorescence microscopy of Tom1 - depleted RPE cells stably expressing GFP - LC3 immunostained against GFP ( green ) and cathepsin D ( red ) . Hoechst labels the nuclei ( blue ) . The insets show higher magnifications of the areas outlined in the main images .

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(f) Confocal immunofluorescence microscopy of Tom1-depleted RPE cells stably expressing GFP-LC3 immunostained against GFP (green) and cathepsin D (red). Hoechst labels the nuclei (blue). The insets show higher magnifications of the areas outlined in the main images.


( g ) HeLa cells with stable expression of HttQ72 - GFP were transiently transfected with Tom1 siRNA ( top right ) , saponin - extracted , and processed for immunofluorescence microscopy ( left ) to quantify HttQ72 - GFP punctae ( bottom right ) . Immunolabelling against GFP ( green ) and p62 ( red ) was performed . Nuclei ( blue ) were labelled with Hoechst . The results are represented as the number of GFP - expressing cells with greater than 15 GFP - positive spots per cell ( ± s . d . ; n = 3 ) . Scale bars , 20 μm . Uncropped images of blots are shown in Supplementary Fig . S9 .

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(g) HeLa cells with stable expression of HttQ72-GFP were transiently transfected with Tom1 siRNA (top right), saponin-extracted, and processed for immunofluorescence microscopy (left) to quantify HttQ72-GFP punctae (bottom right). Immunolabelling against GFP (green) and p62 (red) was performed. Nuclei (blue) were labelled with Hoechst. The results are represented as the number of GFP-expressing cells with greater than 15 GFP-positive spots per cell (±s.d.; n = 3). Scale bars, 20 μm. Uncropped images of blots are shown in Supplementary Fig. S9.


( a ) Left , RPE cells with stable expression of GFP - LC3 following mock - or myosin - VI - siRNA transfection were processed for confocal immunofluorescence microscopy and immunolabelled for Tom1 / Tom1L2 ( red ) . Nuclei ( blue ) were labelled with Hoechst . The insets show higher magnifications of the areas outlined in the main panels and the arrows highlight areas of complete overlapping co - localization and arrowheads highlight adjacent localization of Tom1 relative to LC3 . Right , quantification was performed on > 75 cells for the relative position ( adjacent or complete overlap ) of Tom1 - positive vesicles in relation to LC3 - positive vesicles . The results are represented as the average number of Tom1 - positive vesicles per cell categorized by their relative position to LC3 ( ± s . d . ; n = 3 ) .

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(a) Left, RPE cells with stable expression of GFP-LC3 following mock- or myosin-VI-siRNA transfection were processed for confocal immunofluorescence microscopy and immunolabelled for Tom1/Tom1L2 (red). Nuclei (blue) were labelled with Hoechst. The insets show higher magnifications of the areas outlined in the main panels and the arrows highlight areas of complete overlapping co-localization and arrowheads highlight adjacent localization of Tom1 relative to LC3. Right, quantification was performed on >75 cells for the relative position (adjacent or complete overlap) of Tom1-positive vesicles in relation to LC3-positive vesicles. The results are represented as the average number of Tom1-positive vesicles per cell categorized by their relative position to LC3 (±s.d.; n = 3).


( b ) GFP - LC3 - expressing RPE cells were depleted of myosin VI by siRNA . Cells were pulse labelled with Texas - red - dextran ( red ) for 4 h , before chase into fresh medium containing 1 μM MG132 for 2 h . Cells were processed for immunofluorescence microscopy and immunostained for GFP ( green ) . Nuclei were labelled with Hoechst ( blue ) . The insets represent higher magnifications of the areas outlined in the main panels . ( c ) Mander ' s overlap coefficients for the degree of LC3 signal co - localizing with dextran were calculated using Volocity software . The results represent > 20 cells from n = 3 independent experiments and are illustrated as a box and whisker plot . The box represents the median , 25th and 75th percentiles and whiskers represent the maximum and minimum .

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(b) GFP-LC3-expressing RPE cells were depleted of myosin VI by siRNA. Cells were pulse labelled with Texas-red-dextran (red) for 4 h, before chase into fresh medium containing 1 μM MG132 for 2 h. Cells were processed for immunofluorescence microscopy and immunostained for GFP (green). Nuclei were labelled with Hoechst (blue). The insets represent higher magnifications of the areas outlined in the main panels. (c) Mander's overlap coefficients for the degree of LC3 signal co-localizing with dextran were calculated using Volocity software. The results represent >20 cells from n = 3 independent experiments and are illustrated as a box and whisker plot. The box represents the median, 25th and 75th percentiles and whiskers represent the maximum and minimum.


( d ) RPE cells stably expressing GFP - LC3 were depleted of myosin VI and Tom1 by siRNA . Cells were pulse - labelled with Texas - red ( TR ) - dextran for 16 h followed by a chase period of 4 h . Cells were processed for immunoelectron microscopy and labelled with 15 nm gold particles against GFP - LC3 and 5 nm gold particles against Texas - red - dextran . Scale bars , 20 μm ( a , b ) ; 200 nm ( d ) .

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(d) RPE cells stably expressing GFP-LC3 were depleted of myosin VI and Tom1 by siRNA. Cells were pulse-labelled with Texas-red (TR)-dextran for 16 h followed by a chase period of 4 h. Cells were processed for immunoelectron microscopy and labelled with 15 nm gold particles against GFP-LC3 and 5 nm gold particles against Texas-red-dextran. Scale bars, 20 μm (a,b); 200 nm (d).