Equatorial region of eye imaginal discs where E ( spl ) mδ0 . 5 expression ( green ) becomes restricted to a single photoreceptor in each cluster ( magenta ) , as detected in control and Dl ∆ β1 - 2 / + discs ( top panels ) . In Df ( 3R ) DlFx3 / + and Dl ∆ β1 - 2 / Df ( 3R ) DlFx3 discs ( bottom panels ) E ( spl ) mδ0 . 5 expression is reduced ( Df ( 3R ) DlFx3 / + ) or absent from several clusters ( Dl ∆ β1 - 2 / Df ( 3R ) DlFx3 ) indicative of reduced Notch signalling . Scale bars correspond to 10 μm .

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

Equatorial region of eye imaginal discs where E(spl)mδ0.5 expression (green) becomes restricted to a single photoreceptor in each cluster (magenta), as detected in control and Dl∆β1-2/+ discs (top panels). In Df(3R)DlFx3 /+ and Dl∆β1-2/Df(3R)DlFx3 discs (bottom panels) E(spl)mδ0.5 expression is reduced (Df(3R)DlFx3 /+) or absent from several clusters (Dl∆β1-2/Df(3R)DlFx3) indicative of reduced Notch signalling. Scale bars correspond to 10 μm.


Proportion of photoreceptors clusters that fail to express the E ( spl ) mδ0 . 5 reporter in the indicated genotypes . ns , no significant difference , * * * P < 0 . 0001 ( one - way ANOVA ) . On the violin plot , dashed line represents the median and the dotted lines show the quartiles .

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

Proportion of photoreceptors clusters that fail to express the E(spl)mδ0.5 reporter in the indicated genotypes. ns, no significant difference, *** P<0.0001 (one-way ANOVA). On the violin plot, dashed line represents the median and the dotted lines show the quartiles.


Apical view of wing imaginal disc with homozygous Dl ∆ β1 - 2 clones ( GFP negative ) stained for Dl ( red ) and Cadherin ( blue ) . ( A ' ) Z - projection of apical layers spanning a Dl ∆ β1 - 2 clone ( GFP negative ) located at the DV boundary . No change in apical localisation of Dl ( red ) and Cadherin ( blue ) is detected . Panels on the right show the Dl ( grayscale ) apical localisation on the wing imaginal disc .

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

Apical view of wing imaginal disc with homozygous Dl∆β1-2 clones (GFP negative) stained for Dl (red) and Cadherin (blue). (A') Z-projection of apical layers spanning a Dl∆β1-2 clone (GFP negative) located at the DV boundary. No change in apical localisation of Dl (red) and Cadherin (blue) is detected. Panels on the right show the Dl (grayscale) apical localisation on the wing imaginal disc.


Uptake assay at t = 0 . After exposure to extracellular anti - Dl antibody , Dl protein ( red ) is detected at similar levels apical to Cadherin ( blue ) in wild - type ( GFP ) and Dl ∆ β1 - 2 mutant tissue ( GFP negative ) . ( B ' ) Cross - sectional view of B , Dl protein ( red ) is present apically relative to Cadherin ( Blue ) which marks adherens junctions . Middle and lower panels show the crosssection in grayscale of the apical marker Cadherin ( Cad ) and Delta ( Dl ) , respectively . Uptake assay after 30 minutes , internalised anti - Dl ( red ) enters the endocytic route and in the cross sectional view ( C ' ) can similarly be detected as puncta along the cell axis in wild - type ( GFP ) and homozygous Dl ∆ β1 - 2 ( GFP negative ) tissue . Middle and lower panels show the crosssection in grayscale of the apical marker Cadherin ( Cad ) and Delta ( Dl ) , respectively .

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

Uptake assay at t=0. After exposure to extracellular anti-Dl antibody, Dl protein (red) is detected at similar levels apical to Cadherin (blue) in wild-type (GFP) and Dl∆β1-2 mutant tissue (GFP negative). (B') Cross-sectional view of B, Dl protein (red) is present apically relative to Cadherin (Blue) which marks adherens junctions. Middle and lower panels show the crosssection in grayscale of the apical marker Cadherin (Cad) and Delta (Dl), respectively. Uptake assay after 30 minutes, internalised anti-Dl (red) enters the endocytic route and in the cross sectional view (C') can similarly be detected as puncta along the cell axis in wild-type (GFP) and homozygous Dl∆β1-2 (GFP negative) tissue. Middle and lower panels show the crosssection in grayscale of the apical marker Cadherin (Cad) and Delta (Dl), respectively.


( a ) Western blotting was performed to determine FoxO1 expression , p62 degradation and LC3 - II accumulation in H1299 , HCT116 and HeLa cells in response to serum starvation ( SS ) for 24 h or 0 . 5 mM H2O2 for 6 h in the presence or absence of E64 and pepstatin A ( PEPS A ) .

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

(a) Western blotting was performed to determine FoxO1 expression, p62 degradation and LC3-II accumulation in H1299, HCT116 and HeLa cells in response to serum starvation (SS) for 24 h or 0.5 mM H2O2 for 6 h in the presence or absence of E64 and pepstatin A (PEPS A).


( b , c ) Stable FoxO1 knockdown HCT116 cells , or cells with an empty RNAi plasmid , were incubated in a serum - free medium for up to 36 h ( b ) or treated with 0 . 5 mM H2O2 for up to 12 h ( c ) ; western blotting was performed to detect changes in FoxO1 , p62 , LC3 - II or β - actin .

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

(b, c) Stable FoxO1 knockdown HCT116 cells, or cells with an empty RNAi plasmid, were incubated in a serum-free medium for up to 36 h (b) or treated with 0.5 mM H2O2 for up to 12 h (c); western blotting was performed to detect changes in FoxO1, p62, LC3-II or β-actin.


( d ) HCT116 cells were labelled with [ 3H ] leucine for 48 h , washed and incubated for 24 h , before being subjected to serum starvation for 24 h or 0 . 5 mM H2O2 for 12 h in the presence or absence of 3 - MA . At 12 or 24 h after treatment , the relative degradation of long - lived proteins was measured by counting radioactivity . Data in d are means ± s . d . ( n = 3 ) . Two asterisks , P 0 . 01 ; asterisk , P 0 . 05 ; NS , not significant , P > 0 . 05 ; Student ' s t - test .

O O O O O O O O O O O O O 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 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 O O O O O O O O O O O O O O O O O O O O O O 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) HCT116 cells were labelled with [3H]leucine for 48 h, washed and incubated for 24 h, before being subjected to serum starvation for 24 h or 0.5 mM H2O2 for 12 h in the presence or absence of 3-MA. At 12 or 24 h after treatment, the relative degradation of long-lived proteins was measured by counting radioactivity. Data in d are means ± s.d. (n = 3). Two asterisks, P 0.01; asterisk, P 0.05; NS, not significant, P > 0.05; Student's t-test.


( e ) Autophagic vesicles or autophagosomes in HCT116 cells or stable FoxO1 - RNAi HCT116 cells , were observed by electron microscopy after 24 h of serum starvation . The bottom picture enlargement showing clearer autophagic vesicles indicated by an arrow . The graph shows a statistical analysis of autophagic vesicles in HCT116 cells with or without knockdown of FoxO1 in response to serum starvation . Data in e are means ± s . d . ( n = 3 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Autophagic vesicles or autophagosomes in HCT116 cells or stable FoxO1-RNAi HCT116 cells, were observed by electron microscopy after 24 h of serum starvation. The bottom picture enlargement showing clearer autophagic vesicles indicated by an arrow. The graph shows a statistical analysis of autophagic vesicles in HCT116 cells with or without knockdown of FoxO1 in response to serum starvation. Data in e are means ± s.d. (n = 3).


( f ) LC3 - immunopositive punctate signals were observed in HCT116 or in stable FoxO1 - RNAi HCT116 cells in response to serum starvation for 24 h or 0 . 5 mM H2O2 treatment . ( g ) Quantification of LC3 - positive punctate cells in f . Data in g are means ± s . d . ( n = 3 ) .

O O O O O O O O O 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

(f) LC3-immunopositive punctate signals were observed in HCT116 or in stable FoxO1-RNAi HCT116 cells in response to serum starvation for 24 h or 0.5 mM H2O2 treatment. (g) Quantification of LC3-positive punctate cells in f. Data in g are means ± s. d. (n = 3).


( h ) A rescue experiment using a specific mutated FoxO1 plasmid to validate the activity of FoxO1 in the induction of autophagy in HCT116 cells in the presence or absence of serum , with or without E64 and pepstatin A .

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

(h) A rescue experiment using a specific mutated FoxO1 plasmid to validate the activity of FoxO1 in the induction of autophagy in HCT116 cells in the presence or absence of serum, with or without E64 and pepstatin A.


( a ) A Flag - tagged FoxO1 ( WT ) , FoxO1 ( ΔDB ) , FoxO1 ( ΔDB3A ) or an empty plasmid was individually transfected into GFP - LC3 pre - transfected H1299 cells , and the cellular localization of FoxO1 ( upper panels ) or GFP - LC3 punctate signals ( lower panels ) was observed with a confocal microscope . Scale bars , 10 μm . ( b ) The numbers of punctate GFP - LC3 cells are represented by the ratio of GFP - LC3 - labelled punctate cells to overall survived H1299 cells ( at least 200 cells were counted ) . Data in b are means ± s . d . ( n = 3 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) A Flag-tagged FoxO1(WT), FoxO1(ΔDB), FoxO1(ΔDB3A) or an empty plasmid was individually transfected into GFP-LC3 pre-transfected H1299 cells, and the cellular localization of FoxO1 (upper panels) or GFP-LC3 punctate signals (lower panels) was observed with a confocal microscope. Scale bars, 10 μm. (b) The numbers of punctate GFP-LC3 cells are represented by the ratio of GFP-LC3-labelled punctate cells to overall survived H1299 cells (at least 200 cells were counted). Data in b are means ± s.d. (n = 3).


( c ) p62 degradation and LC3 - II accumulation were evaluated by western blotting in H1299 cells transfected with exogenous FoxO1 - expressing plasmids mentioned above .

O O O O 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) p62 degradation and LC3-II accumulation were evaluated by western blotting in H1299 cells transfected with exogenous FoxO1-expressing plasmids mentioned above.


( d ) H1299 cells were labelled with [ 3H ] leucine for 48 h , then transfected with a control plasmid or various Flag - FoxO1 plasmids , in the presence or absence of 3 - MA or E64 and pepstatin A . Degradation of long - lived proteins was measured at the indicated time , 24 h after transfection . Data in d are means ± s . d . ( n = 3 ) .

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

(d) H1299 cells were labelled with [3H]leucine for 48 h, then transfected with a control plasmid or various Flag-FoxO1 plasmids, in the presence or absence of 3-MA or E64 and pepstatin A. Degradation of long-lived proteins was measured at the indicated time, 24 h after transfection. Data in d are means ± s.d. (n = 3).


( e ) A GFP - tagged FoxO1 ( 3A ) or an empty plasmid was co - transfected with a FoxO1 - promoter luciferase plasmid into H1299 cells . After 24 h , luciferase reporter activity of FoxO1 was measured . RLU , relative luciferase units . Data are means ± s . d . ( n = 3 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) A GFP-tagged FoxO1(3A) or an empty plasmid was co-transfected with a FoxO1-promoter luciferase plasmid into H1299 cells. After 24 h, luciferase reporter activity of FoxO1 was measured. RLU, relative luciferase units. Data are means ± s.d. (n = 3).


( f ) A set of RNAi - resistant rescue forms of FoxO1 plasmids were transfected into stable FoxO1 - RNAi HCT116 cells in the presence or absence of E64 and pepstatin A . Western blotting was performed to detect p62 degradation and LC3 - II accumulation .

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

(f) A set of RNAi-resistant rescue forms of FoxO1 plasmids were transfected into stable FoxO1-RNAi HCT116 cells in the presence or absence of E64 and pepstatin A. Western blotting was performed to detect p62 degradation and LC3-II accumulation.


( g ) Flag - tagged FoxO1 ( WT ) r , FoxO1 ( 3A ) r , FoxO1 ( 3A - NLSm ) r , FoxO1 ( NESm ) r or an empty plasmid were individually transfected into GFP - LC3 pre - transfected HCT116 cells ( FoxO1 - RNAi treated ) , and the cellular localization of FoxO1 ( upper panels ) or GFP - LC3 punctate signals ( lower panels ) were observed with a confocal microscope . ( h ) The numbers of punctate GFP - LC3 cells are represented by the ratio of GFP - LC3 - labelled punctate cells to overall survived HCT116 cells . Data in h are means ± s . d . ( n = 3 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Flag-tagged FoxO1(WT)r, FoxO1(3A)r, FoxO1(3A-NLSm)r, FoxO1(NESm)r or an empty plasmid were individually transfected into GFP-LC3 pre-transfected HCT116 cells (FoxO1-RNAi treated), and the cellular localization of FoxO1 (upper panels) or GFP-LC3 punctate signals (lower panels) were observed with a confocal microscope. (h) The numbers of punctate GFP-LC3 cells are represented by the ratio of GFP-LC3-labelled punctate cells to overall survived HCT116 cells. Data in h are means ± s.d. (n = 3).


( a ) Different FoxO1 - expressing plasmids , namely FoxO1 ( WT ) , FoxO1 ( 3A ) , FoxO1 ( ΔDB ) , FoxO1 ( ΔDB3A ) , FoxO1 ( NESm ) and FoxO1 ( 3A - NLSm ) , were transfected into H1299 cells and cell fractionation was performed to analyse cellular localization ( N , nucleus ; C , cytoplasm ) of these FoxO1 mutants . PCAF is a loading control for nuclear protein , and actin is the loading control for cytoplasmic protein .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Different FoxO1-expressing plasmids, namely FoxO1(WT), FoxO1(3A), FoxO1(ΔDB), FoxO1(ΔDB3A), FoxO1(NESm) and FoxO1(3A-NLSm), were transfected into H1299 cells and cell fractionation was performed to analyse cellular localization (N, nucleus; C, cytoplasm) of these FoxO1 mutants. PCAF is a loading control for nuclear protein, and actin is the loading control for cytoplasmic protein.


( b ) Quantitative PCR was performed to measure the transactivity of autophagy - related genes in transfected H1299 cells with different FoxO1 - expressing plasmids ( upper panel ) or in stable FoxO1 - RNAi or non - RNAi - treated HCT116 cells in response to serum starvation for 24 h or 0 . 5 mM H2O2 for 12 h ( lower panel ) . All data are means ± s . d . ( n = 3 ) .

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

(b) Quantitative PCR was performed to measure the transactivity of autophagy-related genes in transfected H1299 cells with different FoxO1-expressing plasmids (upper panel) or in stable FoxO1-RNAi or non-RNAi-treated HCT116 cells in response to serum starvation for 24 h or 0.5 mM H2O2 for 12 h (lower panel). All data are means ± s.d. (n = 3).


( c ) Different FoxO1 - mutant - expressing plasmids were transfected into H1299 cells and a ChIP assay was performed to measure the binding activity of these mutated FoxO1 to the p27 promoter .

O O O O O O O O O O O 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) Different FoxO1-mutant-expressing plasmids were transfected into H1299 cells and a ChIP assay was performed to measure the binding activity of these mutated FoxO1 to the p27 promoter.


( a ) HCT116 cells were treated with serum starvation for 24 h ( upper panels ) or 0 . 5 mM H2O2 for 12 h ( lower panels ) ; cytosolic proteins were extracted for co - immunoprecipitation with anti - FoxO1 and probed with anti - acetylated lysine ( Ace - lyn ) . Both bottom panels display cytosolic FoxO1 . IP , immunoprecipitation .

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

(a) HCT116 cells were treated with serum starvation for 24 h (upper panels) or 0.5 mM H2O2 for 12 h (lower panels); cytosolic proteins were extracted for co-immunoprecipitation with anti-FoxO1 and probed with anti-acetylated lysine (Ace-lyn). Both bottom panels display cytosolic FoxO1. IP, immunoprecipitation.


( b ) HCT116 cells were serum - starved for 24 h and the cytosolic proteins were extracted for co - immunoprecipitation with anti - FoxO1 or anti - SIRT2 , followed by probing with anti - SIRT2 or anti - FoxO1 , respectively ( top and middle panels ) . FoxO1 ( WT ) was also transfected into H1299 cells and the cytosolic protein was extracted , followed by co - immunoprecipitation with anti - FoxO1 and probing with anti - SIRT2 ( bottom panels ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) HCT116 cells were serum-starved for 24 h and the cytosolic proteins were extracted for co-immunoprecipitation with anti-FoxO1 or anti-SIRT2, followed by probing with anti-SIRT2 or anti-FoxO1, respectively (top and middle panels). FoxO1(WT) was also transfected into H1299 cells and the cytosolic protein was extracted, followed by co-immunoprecipitation with anti-FoxO1 and probing with anti-SIRT2 (bottom panels).


( c ) HCT116 cells were transfected with SIRT2 RNAi plasmid or an empty control plasmid . At 48 h after transfection , the cytosolic lysate was extracted for probing with SIRT2 or FoxO1 ( upper panels ) , or for co - immunoprecipitation with anti - FoxO1 , followed by probing with anti - acetylated lysine ( lower panels ) . IB , immunoblot .

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

(c) HCT116 cells were transfected with SIRT2 RNAi plasmid or an empty control plasmid. At 48 h after transfection, the cytosolic lysate was extracted for probing with SIRT2 or FoxO1 (upper panels), or for co-immunoprecipitation with anti-FoxO1, followed by probing with anti-acetylated lysine (lower panels). IB, immunoblot.


( d ) HCT116 cells were treated with AGK2 for 24 h at different doses , as indicated . The cytosolic lysate was extracted for co - immunoprecipitation with anti - FoxO1 , followed by probing with anti - acetylated lysine .

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 B-MEASURED_VAR O

(d) HCT116 cells were treated with AGK2 for 24 h at different doses, as indicated. The cytosolic lysate was extracted for co-immunoprecipitation with anti-FoxO1, followed by probing with anti-acetylated lysine.


( a , b ) H1299 cells were transfected with an empty control plasmid or with the Flag - FoxO1 ( WT ) plasmid . At 24 h after transfection , the cytosolic lysate was extracted for co - immunoprecipitation with anti - FoxO1 , followed by probing with anti - mTOR , Beclin 1 , Vps34 or Atg1 ( a ) and with Atg3 , Atg4 , Atg10 , Atg7 or LC3 ( b ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) H1299 cells were transfected with an empty control plasmid or with the Flag-FoxO1(WT) plasmid. At 24 h after transfection, the cytosolic lysate was extracted for co-immunoprecipitation with anti-FoxO1, followed by probing with anti-mTOR, Beclin 1, Vps34 or Atg1 (a) and with Atg3, Atg4, Atg10, Atg7 or LC3 (b).


( c ) H1299 cells were co - transfected with Myc - Atg7 , with Flag - FoxO1 ( WT ) or with an empty control plasmid . At 24 h after transfection , the cytosolic lysate was extracted for Flag with anti - Flag , followed by probing with anti - Myc .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) H1299 cells were co-transfected with Myc-Atg7, with Flag-FoxO1(WT) or with an empty control plasmid. At 24 h after transfection, the cytosolic lysate was extracted for Flag with anti-Flag, followed by probing with anti-Myc.


HCT116 cells were serum - starved for 24 h ( d ) and the cytosolic lysate was extracted for co - immunoprecipitation with anti - FoxO1 , followed by probing with anti -

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

HCT116 cells were serum-starved for 24 h (d)and the cytosolic lysate was extracted for co-immunoprecipitation with anti-FoxO1, followed by probing with anti-Atg7


( e ) Different Myc - tagged FoxO1 fragments and Flag - tagged Atg7 were co - transfected into H1299 cells , and the cytosolic lysate was extracted for co - immunoprecipitation with anti - Myc , followed by probing with anti - Flag .

O O O O O O O O O O O O O O O O O O O O O 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) Different Myc-tagged FoxO1 fragments and Flag-tagged Atg7 were co-transfected into H1299 cells, and the cytosolic lysate was extracted for co-immunoprecipitation with anti-Myc, followed by probing with anti-Flag.


( f ) HCT116 cells were transfected with an empty control plasmid , an SIRT2 ( WT ) - expressing plasmid or an SIRT2 ( 168A ) - expressing plasmid . At 24 h after transfection , cells were serum - starved for 24 h , and the cytosolic lysate was extracted for co - immunoprecipitation with anti - FoxO1 , followed by probing with anti - Atg7 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) HCT116 cells were transfected with an empty control plasmid, an SIRT2(WT)-expressing plasmid or an SIRT2(168A)-expressing plasmid. At 24 h after transfection, cells were serum-starved for 24 h, and the cytosolic lysate was extracted for co-immunoprecipitation with anti-FoxO1, followed by probing with anti-Atg7.


HCT116 cells weretreated with AGK2 for 24 h ( g ) , and the cytosolic lysate was extracted for co - immunoprecipitation with anti - FoxO1 , followed by probing with anti -

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

HCT116 cells weretreated with AGK2 for 24 h (g), and the cytosolic lysate was extracted for co-immunoprecipitation with anti-FoxO1, followed by probing with anti-Atg7


( a ) HCT116 cells or stable FoxO1 - RNAi HCT116 cells were treated with a non - specific RNAi control or SIRT2 - RNAi , and LC3 - II accumulation or p62 degradation was detected in the presence or absence of E64 and pepstatin A .

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

(a) HCT116 cells or stable FoxO1-RNAi HCT116 cells were treated with a non-specific RNAi control or SIRT2-RNAi, and LC3-II accumulation or p62 degradation was detected in the presence or absence of E64 and pepstatin A.


( b ) HCT116 cells or stable FoxO1 - RNAi HCT116 cells were treated with 10 μM AGK2 for 24 h , in the presence or absence of E64 and pepstatin A . Changes in FoxO1 , p62 and LC3 - II were determined by western blotting ( upper panels ) , or the formation of LC3 punctate signals was observed ( lower panels ) . HCT116 cells were treated with 10 μM AGK2 for 24 h and the formation of LC3 punctate signals was observed . Quantification of the ratio of LC3 punctate cells to overall HCT116 cells is shown in the lower right panel . Data are means ± s . d . ( n = 3 ) .

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(b) HCT116 cells or stable FoxO1-RNAi HCT116 cells were treated with 10 μM AGK2 for 24 h, in the presence or absence of E64 and pepstatin A. Changes in FoxO1, p62 and LC3-II were determined by western blotting (upper panels), or the formation of LC3 punctate signals was observed (lower panels). HCT116 cells were treated with 10 μM AGK2 for 24 h and the formation of LC3 punctate signals was observed. Quantification of the ratio of LC3 punctate cells to overall HCT116 cells is shown in the lower right panel. Data are means ± s.d. (n = 3).


( c ) H1299 cells were transfected with Flag - FoxO1 ( WT ) , SIRT2 ( WT ) or SIRT2 ( 168A ) . At 24 h after transfection , the cell lysate was extracted and immunoblotted with anti - Flag , anti - SIRT2 , anti - LC3 , anti - p62 or anti - β - actin . .

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(c) H1299 cells were transfected with Flag-FoxO1(WT), SIRT2(WT) or SIRT2(168A). At 24 h after transfection, the cell lysate was extracted and immunoblotted with anti-Flag, anti-SIRT2, anti-LC3, anti-p62 or anti-β-actin..


( d ) HCT116 cells were transfected with a control plasmid , a SIRT2 ( WT ) - expressing plasmid or a SIRT2 ( 168A ) - expressing plasmid . At 24 h after transfection , the cells were subjected to serum starvation for 24 h . The cell lysate was extracted and immunoblotted with anti - SIRT2 , anti - LC3 , anti - p62 or anti - β - actin .

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(d) HCT116 cells were transfected with a control plasmid, a SIRT2(WT)-expressing plasmid or a SIRT2(168A)-expressing plasmid. At 24 h after transfection, the cells were subjected to serum starvation for 24 h. The cell lysate was extracted and immunoblotted with anti-SIRT2, anti-LC3, anti-p62 or anti-β-actin.


( e ) H1299 cells were transfected with Myc - Atg7 , Flag - FoxO1 ( WT ) or mutated Flag - FoxO1 ( KR ) . At 24 h after transfection , the cytosolic lysate was extracted for co - immunoprecipitation with anti - Myc , followed by probing with anti - Flag .

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(e) H1299 cells were transfected with Myc-Atg7, Flag-FoxO1(WT) or mutated Flag-FoxO1(KR). At 24 h after transfection, the cytosolic lysate was extracted for co-immunoprecipitation with anti-Myc, followed by probing with anti-Flag.


( f ) H1299 cells were transfected with a control plasmid , Flag - FoxO1 ( WT ) , Flag - FoxO1 ( K262R ) , Flag - FoxO1 ( K265R ) , Flag - FoxO1 ( K274R ) or Flag - FoxO1 ( KR ) . At 24h after transfection in the presence or absence of E64 and pepstatin A and immunoblotted , the cell lysate was extracted and immunoblotted with anti - Flag , anti - LC3 , anti - p62 or anti - β - actin .

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(f) H1299 cells were transfected with a control plasmid, Flag-FoxO1(WT), Flag-FoxO1(K262R), Flag-FoxO1(K265R), Flag-FoxO1(K274R) or Flag-FoxO1(KR). At 24h after transfection in the presence or absence of E64 and pepstatin A and immunoblotted, the cell lysate was extracted and immunoblotted with anti-Flag, anti-LC3, anti-p62 or anti-β-actin.


( g ) Left : H1299 cells were co - transfected with GFP - LC3 , Flag - FoxO1 ( WT ) or Flag - FoxO1 ( KR ) . The formation of GFP - LC3 - labelled punctate signals was observed 24 h after transfection . Right : quantification of the ratio of GFP - LC3 - labelled punctate cells to overall survived H1299 cells . Data in g are means ± s . d . ( n = 3 ) . Scale bars , 25 μm ( b ) , 10 μm ( g ) .

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(g) Left: H1299 cells were co-transfected with GFP-LC3, Flag-FoxO1(WT) or Flag-FoxO1(KR). The formation of GFP-LC3-labelled punctate signals was observed 24 h after transfection. Right: quantification of the ratio of GFP-LC3-labelled punctate cells to overall survived H1299 cells. Data in g are means ± s.d. (n = 3). Scale bars, 25 μm (b), 10 μm (g).


( a ) H1299 cells were transfected with a control plasmid , Flag - FoxO1 ( WT ) , Flag - FoxO1 ( 3A ) , Flag - FoxO1 ( ΔDB ) or Flag - FoxO1 ( ΔDB3A ) , in the presence or absence of 10 mM 3 - MA ( upper panel ) or in the presence or absence of 50 μM Z - VAD - fmk ( lower panel ) ; cells were stained with propidium iodide ( PI ) 48 h after transfection . PI - stained ( dead ) cells were counted by flow cytometry and the percentage cell loss was calculated .

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(a) H1299 cells were transfected with a control plasmid, Flag-FoxO1(WT), Flag-FoxO1(3A), Flag-FoxO1(ΔDB) or Flag-FoxO1(ΔDB3A), in the presence or absence of 10 mM 3-MA (upper panel) or in the presence or absence of 50 μM Z-VAD-fmk (lower panel); cells were stained with propidium iodide (PI) 48 h after transfection. PI-stained (dead) cells were counted by flow cytometry and the percentage cell loss was calculated.


( b ) FoxO1 ( WT ) or Flag - FoxO1 ( ΔDB ) was transfected into H1299 cells ; 48 h later the autophagic vesicles were observed under an electron microscope . Enlarged images ( right ) show clearer autophagic vesicles indicated by arrows . Scale bars , 2 μm .

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(b) FoxO1(WT) or Flag-FoxO1(ΔDB) was transfected into H1299 cells; 48 h later the autophagic vesicles were observed under an electron microscope. Enlarged images (right) show clearer autophagic vesicles indicated by arrows. Scale bars, 2 μm.


( c ) 48 h cells with or without Atg5 RNAi were transfected with FoxO1 ( WT ) , and cell death was determined by staining with PI at 48 h after transfection . Data in c are means ± s . d . ( n = 3 ) .

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(c) 48 h cells with or without Atg5 RNAi were transfected with FoxO1(WT), and cell death was determined by staining with PI at 48 h after transfection. Data in c are means ± s.d. (n = 3).


( d ) The stable Atg5 knockdown H1299 cells was further transfected with a control plasmid , Flag - FoxO1 ( WT ) , or Flag - FoxO1 ( ΔDB ) , and the percentage cell loss was quantified by flow cytometry at 48 h after transfection . Data in d are means ± s . d . ( n = 3 ) .

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(d) The stable Atg5 knockdown H1299 cells was further transfected with a control plasmid, Flag-FoxO1(WT), or Flag-FoxO1(ΔDB), and the percentage cell loss was quantified by flow cytometry at 48 h after transfection. Data in d are means ± s.d. (n = 3).


( a ) Stable H1299 cells with or without Atg7 knockdown were transfected with a control plasmid ( group 1 ) , Flag - FoxO1 ( ΔDB ) ( group 2 ) or Flag - FoxO1 ( ΔDB - 3A ) ( group 3 ) ; 107 cells were then injected into 18 nude mice , with 6 mice per group . Seven weeks later the mice were killed and the tumours were weighed and analysed ( bottom graph ) . Group 1 : tumour weights were 1 . 22 ± 0 . 78 g ( without Atg7 RNAi ) and 1 . 31 ± 0 . 43 g ( with Atg7 RNAi ) , P = 0 . 426 . Group 2 : tumour weights were 0 . 49 ± 0 . 18 g ( without Atg7 RNAi ) and 0 . 80 ± 0 . 20 g ( with Atg7 RNAi ) , P = 0 . 017 . Group 3 : tumour weights were 1 . 00 ± 0 . 40 g ( without Atg7 RNAi ) and 1 . 10 ± 0 . 36 g ( with Atg7 RNAi ) , P = 0 . 500 . Data are means ± s . d . ( n = 6 ) .

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(a) Stable H1299 cells with or without Atg7 knockdown were transfected with a control plasmid (group 1), Flag-FoxO1(ΔDB) (group 2) or Flag-FoxO1(ΔDB-3A) (group 3); 107 cells were then injected into 18 nude mice, with 6 mice per group. Seven weeks later the mice were killed and the tumours were weighed and analysed (bottom graph). Group 1: tumour weights were 1.22 ± 0.78 g (without Atg7 RNAi) and 1.31 ± 0.43 g (with Atg7 RNAi), P = 0.426. Group 2: tumour weights were 0.49 ± 0.18 g (without Atg7 RNAi) and 0.80 ± 0.20 g (with Atg7 RNAi), P = 0.017. Group 3: tumour weights were 1.00 ± 0.40 g (without Atg7 RNAi) and 1.10 ± 0.36 g (with Atg7 RNAi), P = 0.500. Data are means ± s.d. (n = 6).


( b ) Expression levels of FoxO1 and Atg7 conjugate in mice tumours were determined by western blotting .

(b) Expression levels of FoxO1 and Atg7 conjugate in mice tumours were determined by western blotting.


( c ) HCT116 cells ( 107 ) or stable FoxO1 - knockdown HCT116 cells ( 107 ) were injected into the left or right hind legs of three nude mice . Six weeks later the mice were killed and the tumours were weighed and analysed ( bottom graph ) . Tumour weights were 0 . 08 ± 0 . 10 g ( without FoxO1 RNAi ) and 0 . 48 ± 0 . 06 g ( with FoxO1 RNAi ) , P = 0 . 020 . Data are means ± sd . n = 3 .

(c) HCT116 cells (107) or stable FoxO1-knockdown HCT116 cells (107) were injected into the left or right hind legs of three nude mice. Six weeks later the mice were killed and the tumours were weighed and analysed (bottom graph). Tumour weights were 0.08 ± 0.10 g (without FoxO1 RNAi) and 0.48 ± 0.06 g (with FoxO1 RNAi), P = 0.020. Data are means ± sd. n = 3.


( d ) Protein was extracted from one paired colon tumour ( T ) or adjacent normal tissue ( N ) from one patient , and western blotting was performed to detect changes in FoxO1 or p62 .

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(d) Protein was extracted from one paired colon tumour (T) or adjacent normal tissue (N) from one patient, and western blotting was performed to detect changes in FoxO1 or p62.


( e ) Proteins were extracted from 16 paired samples from patients for western blotting with anti - FoxO1 or with anti - p62 antibody . Protein bands were scanned with a phosphorimager and relative band intensity was normalized to the β - actin band . FoxO1 of normal tissue , 0 . 37 ; FoxO1 of tumour tissue , 0 . 10 ; P = 0 . 001 , two - sided t - test . p62 of normal tissue , 0 . 32 ; p62 of tumour tissue , 1 . 00 ; P = 0 . 009 , two - sided t - test . Data in e are means ± s . d . ( n = 16 ) .

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(e) Proteins were extracted from 16 paired samples from patients for western blotting with anti-FoxO1 or with anti-p62 antibody. Protein bands were scanned with a phosphorimager and relative band intensity was normalized to the β-actin band. FoxO1 of normal tissue, 0.37; FoxO1 of tumour tissue, 0.10; P = 0.001, two-sided t-test. p62 of normal tissue, 0.32; p62 of tumour tissue, 1.00; P = 0.009, two-sided t-test. Data in e are means ± s.d. (n = 16).


Representative dual - channel kymographs showing that Rheb is recruited to mitochondria in axons and co - migrates with mitochondria in a predominant retrograde direction upon mitochondrial membrane potential ( ∆ ψm ) dissipation . The motility of mitochondria ( Mito ) and Rheb - associated mitochondria ( Rheb - Mito ) in axons were quantified , respectively . Vertical lines represent stationary organelles . Slanted lines or curves to the right ( negative slope ) represent anterograde movement ; those to the left ( positive slope ) indicate retrograde movement . An organelle was considered stationary if it remained immotile ( displacement ≤ 5 μm ) .

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Representative dual-channel kymographs showing that Rheb is recruited to mitochondria in axons and co-migrates with mitochondria in a predominant retrograde direction upon mitochondrial membrane potential (∆ψm) dissipation. The motility of mitochondria (Mito) and Rheb-associated mitochondria (Rheb-Mito) in axons were quantified, respectively. Vertical lines represent stationary organelles. Slanted lines or curves to the right (negative slope) represent anterograde movement; those to the left (positive slope) indicate retrograde movement. An organelle was considered stationary if it remained immotile (displacement ≤ 5 μm).


Time - lapse images ( B ) showing colocalization and comigration ( white arrows ) of Rheb with mitochondria in a retrograde direction along axons during a 148 - s recording period . Cortical neurons were transfected with GFP - Rheb and DsRed - Mito at 5 - 7 DIV and incubated with DMSO or 10 μM CCCP for 24 hr prior to imaging at 10 - 12 DIV .

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Time-lapse images (B) showing colocalization and comigration (white arrows) of Rheb with mitochondria in a retrograde direction along axons during a 148-s recording period. Cortical neurons were transfected with GFP-Rheb and DsRed-Mito at 5-7 DIV and incubated with DMSO or 10 μM CCCP for 24 hr prior to imaging at 10-12 DIV.


quantitative analysis showing that Rheb is recruited to mitochondria in axons and co - migrates with mitochondria in a predominant retrograde direction upon mitochondrial membrane potential ( ∆ ψm ) dissipation . The motility of mitochondria ( Mito ) and Rheb - associated mitochondria ( Rheb - Mito ) in axons were quantified , respectively . Vertical lines represent stationary organelles . Slanted lines or curves to the right ( negative slope ) represent anterograde movement ; those to the left ( positive slope ) indicate retrograde movement . An organelle was considered stationary if it remained immotile ( displacement ≤ 5 μm ) .

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

quantitative analysis showing that Rheb is recruited to mitochondria in axons and co-migrates with mitochondria in a predominant retrograde direction upon mitochondrial membrane potential (∆ψm) dissipation. The motility of mitochondria (Mito) and Rheb-associated mitochondria (Rheb-Mito) in axons were quantified, respectively. Vertical lines represent stationary organelles. Slanted lines or curves to the right (negative slope) represent anterograde movement; those to the left (positive slope) indicate retrograde movement. An organelle was considered stationary if it remained immotile (displacement ≤ 5 μm).


Quantitative analysis showing robust association of Rheb with depolarized mitochondria within axons treated with CCCP . The percentages of mitochondria targeted by Rheb or Rheb - SSVM in the presence or absence of CCCP were quantified ,

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Quantitative analysis showing robust association of Rheb with depolarized mitochondria within axons treated with CCCP. The percentages of mitochondria targeted by Rheb or Rheb-SSVM in the presence or absence of CCCP were quantified, respectively


representative images showing robust association of Rheb with depolarized mitochondria within axons treated with CCCP . Rheb - SSVM , a farnesylation deficient mutant of Rheb , fails to be recruited to mitochondria upon dissipating ∆ ψm . Mitochondria associated with Rheb were marked by white arrows .

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representative images showing robust association of Rheb with depolarized mitochondria within axons treated with CCCP. Rheb-SSVM, a farnesylation deficient mutant of Rheb, fails to be recruited to mitochondria upon dissipating ∆ψm. Mitochondria associated with Rheb were marked by white arrows.


F - H Representative dual - channel kymographs ( F ) and quantitative analysis ( G , H ) showing that Rheb - mediated mitophagy activation depends on Rheb recruitment to mitochondria through Rheb farnesylation . Note that Rheb , but not Rheb - SSVM , is associated with autophagic vacuoles ( AVs ) within axons upon ∆ ψm dissipation . Rheb - associated AVs move in a predominant retrograde direction along axons . The number of Rheb - associated AVs ( Rheb - AVs ) with or without CCCP treatment and the relative motilities of AVs and Rheb - AVs in CCCP - treated axons were quantified ,

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F-H Representative dual-channel kymographs (F) and quantitative analysis (G, H) showing that Rheb-mediated mitophagy activation depends on Rheb recruitment to mitochondria through Rheb farnesylation. Note that Rheb, but not Rheb-SSVM, is associated with autophagic vacuoles (AVs) within axons upon ∆ψm dissipation. Rheb-associated AVs move in a predominant retrograde direction along axons. The number of Rheb-associated AVs (Rheb-AVs) with or without CCCP treatment and the relative motilities of AVs and Rheb-AVs in CCCP-treated axons were quantified, respectively


Representative dual - channel kymographs showing that Rheb enhances mitophagy in CCCP - treated axons .

Representative dual-channel kymographs showing that Rheb enhances mitophagy in CCCP-treated axons.


quantitative analysis showing that Rheb enhances mitophagy in CCCP - treated axons . The numbers of mitochondria ( Mito ) , AVs , and Mito - AVs / mitophagosomes , the percentage of Mito within AVs , were

quantitative analysis showing that Rheb enhances mitophagy in CCCP-treated axons. The numbers of mitochondria (Mito), AVs, and Mito-AVs/mitophagosomes, the percentage of Mito within AVs, were quantified


quantitative analysis the percentage of Mito within AVs , and the motilities of AVs and Mito - AVs along axons with and without elevated Rheb expression were

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quantitative analysis the percentage of Mito within AVs, and the motilities of AVs and Mito-AVs along axons with and without elevated Rheb expression were quantified


D , E Representative images ( D ) and quantitative analysis ( E ) showing that mitophagy in axons requires Rheb and Nix , but not Parkin . The number of mitophagosomes indicated by white arrows and the percentage of Mito within AVs in CCCP - treated axons expressing scrambled shRNA , Rheb shRNA , Nix shRNA , or Parkin shRNA were quantified , respectively ( E ) .

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D,E Representative images (D) and quantitative analysis (E) showing that mitophagy in axons requires Rheb and Nix, but not Parkin. The number of mitophagosomes indicated by white arrows and the percentage of Mito within AVs in CCCP-treated axons expressing scrambled shRNA, Rheb shRNA, Nix shRNA, or Parkin shRNA were quantified, respectively (E).


F , G Quantitative analysis ( F ) and representative blots ( G ) showing increased mitochondrial association with Rheb and LC3 - II in mature cortical neurons upon ∆ ψm depolarization . Cortical neurons at 14 DIV were incubated with DMSO or 10 μM CCCP for 24 hr and then subjected to fractionation into post - nuclear supernatant ( P ) , mitochondria - enriched membrane fraction ( M ) , and cytosolic supernatant ( S ) . Equal amounts of protein ( 20 μg ) were sequentially immunoblotted with antibodies against mitophagic / autophagic proteins Rheb , LC3 , and Rab7 , mitochondrial protein VDAC , and cytosolic protein GAPDH on the same membranes after stripping between each antibody application . The purity of mitochondrial fractions was confirmed by enrichment of VDAC and less abundance of GAPDH , compared to post - nuclear supernatant . Protein levels in mitochondrial fractions of CCCP - treated neurons were normalized to those in neurons incubated with DMSO . Data were quantified from four independent repeats .

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F,G Quantitative analysis (F) and representative blots (G) showing increased mitochondrial association with Rheb and LC3-II in mature cortical neurons upon ∆ψm depolarization. Cortical neurons at 14 DIV were incubated with DMSO or 10 μM CCCP for 24 hr and then subjected to fractionation into post-nuclear supernatant (P), mitochondria-enriched membrane fraction (M), and cytosolic supernatant (S). Equal amounts of protein (20 μg) were sequentially immunoblotted with antibodies against mitophagic/autophagic proteins Rheb, LC3, and Rab7, mitochondrial protein VDAC, and cytosolic protein GAPDH on the same membranes after stripping between each antibody application. The purity of mitochondrial fractions was confirmed by enrichment of VDAC and less abundance of GAPDH, compared to post-nuclear supernatant. Protein levels in mitochondrial fractions of CCCP-treated neurons were normalized to those in neurons incubated with DMSO. Data were quantified from four independent repeats.


Quantitative analysis showing that Rheb RNAi augments axonal retention of oxidized mitochondria upon ∆ ψm depolarization . The fluorescence of MitoROGFP was emitted at 510 nm and excited at 405 nm or 488 nm , respectively . Mean fluorescence intensity ratios evoked by the two excitation wavelengths at individual mitochondria in the CCCP - treated axons expressing scrambled shRNA or Rheb shRNA were quantified and normalized to those of control neurons transfected with scrambled shRNA ( H ) .

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Quantitative analysis showing that Rheb RNAi augments axonal retention of oxidized mitochondria upon ∆ψm depolarization. The fluorescence of MitoROGFP was emitted at 510 nm and excited at 405 nm or 488 nm, respectively. Mean fluorescence intensity ratios evoked by the two excitation wavelengths at individual mitochondria in the CCCP-treated axons expressing scrambled shRNA or Rheb shRNA were quantified and normalized to those of control neurons transfected with scrambled shRNA (H).


representative images showing that Rheb RNAi augments axonal retention of oxidized mitochondria upon ∆ ψm depolarization . The fluorescence of MitoROGFP was emitted at 510 nm and excited at 405 nm or 488 nm , respectively . Ratiometric images were generated from fluorescence excited by 405 nm light relative to that excited by 488 nm light . The ratio has been false colored with the indicated heat map , with high intensity indicative of ROGFP fluorescence in a more oxidative

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representative images showing that Rheb RNAi augments axonal retention of oxidized mitochondria upon ∆ψm depolarization. The fluorescence of MitoROGFP was emitted at 510 nm and excited at 405 nm or 488 nm, respectively. Ratiometric images were generated from fluorescence excited by 405 nm light relative to that excited by 488 nm light. The ratio has been false colored with the indicated heat map, with high intensity indicative of ROGFP fluorescence in a more oxidative level


Aberrant accumulation of mitophagosomes co - labeled by antibodies against LC3 and cytochrome c ( Cyto c ) in the hippocampal mossy fibers of mutant hAPP Tg mouse brains . Note that mitophagosomes indicated by white arrows are not readily observed in WT mouse

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Aberrant accumulation of mitophagosomes co-labeled by antibodies against LC3 and cytochrome c (Cyto c) in the hippocampal mossy fibers of mutant hAPP Tg mouse brains. Note that mitophagosomes indicated by white arrows are not readily observed in WT mouse brains


The average number of mitophagosomes per section ( 320 μm × 320 μm ) was quantified from the total number of brain slice sections ( n ) indicated on the top of the

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The average number of mitophagosomes per section (320 μm × 320 μm) was quantified from the total number of brain slice sections (n) indicated on the top of the bars


C Clustering of mitophagosomes ( white arrows ) co - labeled by antibodies against LC3 and Cyto c or p62 and HSP60 within swollen / dystrophic presynaptic terminals indicated by synaptophysin ( SYP ) surrounding amyloid plaques in AD mouse brains .

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C Clustering of mitophagosomes (white arrows) co-labeled by antibodies against LC3 and Cyto c or p62 and HSP60 within swollen/dystrophic presynaptic terminals indicated by synaptophysin (SYP) surrounding amyloid plaques in AD mouse brains.


Quantitative analysis showing presynaptic retention of mitophagosome - like structures in the hippocampal regions of mutant hAPP mouse brains . Data were quantified from the total number of the imaging fields ( n ) indicated in

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Quantitative analysis showing presynaptic retention of mitophagosome-like structures in the hippocampal regions of mutant hAPP mouse brains. Data were quantified from the total number of the imaging fields (n) indicated in parentheses


representative TEM images showing presynaptic retention of mitophagosome - like structures in the hippocampal regions of mutant hAPP mouse brains . White arrow - labeled mitophagosomes are not readily detected in WT mouse brains . Black arrows marked amphisomes or degradative autophagic vacuoles ( AVd ) .

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representative TEM images showing presynaptic retention of mitophagosome-like structures in the hippocampal regions of mutant hAPP mouse brains. White arrow-labeled mitophagosomes are not readily detected in WT mouse brains. Black arrows marked amphisomes or degradative autophagic vacuoles (AVd).


F , G Mitophagic accumulation at Aβ - enriched synapses in AD mouse brains . Note that the levels of mitophagic / autophagic proteins ( red box ) along with Aβ ( green box ) are increased in synaptic fractions isolated from AD mouse brains . Equal amounts ( 10 μg ) of synaptosomal preparations ( Syn ) and post - nuclear supernatants ( PNS ) from WT and mutant hAPP mouse brains were sequentially immunoblotted with antibodies against hAPP / Aβ , Rheb , LC3 , p62 , and Rab7 , as well as mitochondrial proteins TOM20 and SOD2 on the same membrane after stripping between each antibody application . The purity of synaptosomal fractions was confirmed by relative enrichment of SYP and less abundance of p115 and GAPDH ( F ) . Relative protein levels in the synaptosomal preparations from mutant hAPP mice were compared to those in WT littermate controls . Data were quantified from five repeats ( G ) .

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 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 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

F,G Mitophagic accumulation at Aβ-enriched synapses in AD mouse brains. Note that the levels of mitophagic/autophagic proteins (red box) along with Aβ (green box) are increased in synaptic fractions isolated from AD mouse brains. Equal amounts (10 μg) of synaptosomal preparations (Syn) and post-nuclear supernatants (PNS) from WT and mutant hAPP mouse brains were sequentially immunoblotted with antibodies against hAPP/Aβ, Rheb, LC3, p62, and Rab7, as well as mitochondrial proteins TOM20 and SOD2 on the same membrane after stripping between each antibody application. The purity of synaptosomal fractions was confirmed by relative enrichment of SYP and less abundance of p115 and GAPDH (F). Relative protein levels in the synaptosomal preparations from mutant hAPP mice were compared to those in WT littermate controls. Data were quantified from five repeats (G).


H , I Representative blots ( H ) and quantitative analysis ( I ) showing increased levels of Rheb along with LC3 - II , p62 , and Rab7 ( red box ) in Mito fractions purified from AD mouse brains . The purity of Mito fractions was confirmed by enrichment of mitochondrial proteins VDAC and TOM20 and by the absence of GAPDH and p115 , relative to PNS fractions . Data were quantified from four repeats .

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H,I Representative blots (H) and quantitative analysis (I) showing increased levels of Rheb along with LC3-II, p62, and Rab7 (red box) in Mito fractions purified from AD mouse brains. The purity of Mito fractions was confirmed by enrichment of mitochondrial proteins VDAC and TOM20 and by the absence of GAPDH and p115, relative to PNS fractions. Data were quantified from four repeats.


Representative images showing increased mitochondrial association with Rheb in AD axons , relative to that of WT littermate controls . Note that CCCP treatment enhances Rheb localization to mitochondria . The percentages of white arrow - marked mitochondria that were targeted by Rheb in WT and mutant hAPP axons in the absence and presence of

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Representative images showing increased mitochondrial association with Rheb in AD axons, relative to that of WT littermate controls. Note that CCCP treatment enhances Rheb localization to mitochondria. The percentages of white arrow-marked mitochondria that were targeted by Rheb in WT and mutant hAPP axons in the absence and presence of CCCP


quantitative analysis The percentages of white arrow - marked mitochondria that were targeted by Rheb in WT and mutant hAPP axons in the absence and presence of CCCP were quantified ,

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quantitative analysis The percentages of white arrow-marked mitochondria that were targeted by Rheb in WT and mutant hAPP axons in the absence and presence of CCCP were quantified, respectively


C - Representative dual - channel kymographs showing defective retrograde transport and mitophagic accumulation within AD axons . mitochondria within AVs in WT or mutant hAPP axons with and without CCCP were

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C- Representative dual-channel kymographs showing defective retrograde transport and mitophagic accumulation within AD axons. mitochondria within AVs in WT or mutant hAPP axons with and without CCCP were quantified


quantitative analysis , the numbers of Mito , AVs , and Mito - AVs , and the percentage of mitochondria within AVs in WT or mutant hAPP axons with and without CCCP were quantified ,

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quantitative analysis , the numbers of Mito, AVs, and Mito-AVs, and the percentage of mitochondria within AVs in WT or mutant hAPP axons with and without CCCP were quantified, respectively


quantitative analysis Relative motility of AVs and Mito - AVs / mitophagosomes in WT or mutant hAPP axons with and without CCCP were

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quantitative analysis Relative motility of AVs and Mito-AVs/mitophagosomes in WT or mutant hAPP axons with and without CCCP were quantified


F , G Quantitative analysis ( F ) and representative blots ( G ) showing that ∆ ψm depolarization augments mitophagy activation in cortical neurons cultured from AD mouse brains . Note increased localization of Rheb along with LC3 - II and Rab7 to mitochondria in mutant hAPP Tg neurons after CCCP treatment ( red box ) . Data were quantified from three independent repeats . P : post - nuclear supernatants ; M : mitochondria - enriched membrane fractions ; S : cytosolic supernatants .

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F,G Quantitative analysis (F) and representative blots (G) showing that ∆ψm depolarization augments mitophagy activation in cortical neurons cultured from AD mouse brains. Note increased localization of Rheb along with LC3-II and Rab7 to mitochondria in mutant hAPP Tg neurons after CCCP treatment (red box). Data were quantified from three independent repeats. P: post-nuclear supernatants; M: mitochondria-enriched membrane fractions; S: cytosolic supernatants.


Quantitative analysis Mean fluorescence intensity ratios evoked by the two excitation wavelengths ( 405 nm and 488 nm ) at individual mitochondria in mutant hAPP axons with and without CCCP treatment were quantified and normalized to those of WT

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Quantitative analysis Mean fluorescence intensity ratios evoked by the two excitation wavelengths (405 nm and 488 nm) at individual mitochondria in mutant hAPP axons with and without CCCP treatment were quantified and normalized to those of WT controls


representative images showing abnormal accumulation of oxidatively damaged mitochondria in AD axons , which is exacerbated after dissipating ∆ ψm . Mean fluorescence intensity ratios evoked by the two excitation wavelengths ( 405 nm and 488 nm ) at individual mitochondria in mutant hAPP axons with and without CCCP treatment were quantified and normalized to those of WT

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representative images showing abnormal accumulation of oxidatively damaged mitochondria in AD axons, which is exacerbated after dissipating ∆ψm. Mean fluorescence intensity ratios evoked by the two excitation wavelengths (405 nm and 488 nm) at individual mitochondria in mutant hAPP axons with and without CCCP treatment were quantified and normalized to those of WT controls


A , B Representative images ( A ) and quantitative analysis ( B ) showing aberrant accumulation of mitophagosomes labeled by LC3 and Cyto c antibodies in the hippocampal mossy fibers of snapin - cKO mouse brains . Note that white arrow - labeled mitophagosomes are not readily observed in WT mouse brains . The average number of mitophagosomes per section ( 320 μm × 320 μm ) was quantified .

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A,B Representative images (A) and quantitative analysis (B) showing aberrant accumulation of mitophagosomes labeled by LC3 and Cyto c antibodies in the hippocampal mossy fibers of snapin-cKO mouse brains. Note that white arrow-labeled mitophagosomes are not readily observed in WT mouse brains. The average number of mitophagosomes per section (320 μm × 320 μm) was quantified.


Representative blots showing aberrant mitophagic retention at synaptic terminals in snapin - mutant mouse brains . The levels of Rheb along with LC3 - II , p62 , and Rab7 are significantly elevated in the synaptosomal fractions ( Syn ) purified from snapin - deficient mouse brains , relative to those of WT littermate controls ( red box ) . Equal amounts ( 10 μg ) of Syn and post - nuclear supernatants (

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Representative blots showing aberrant mitophagic retention at synaptic terminals in snapin-mutant mouse brains. The levels of Rheb along with LC3-II, p62, and Rab7 are significantly elevated in the synaptosomal fractions (Syn) purified from snapin-deficient mouse brains, relative to those of WT littermate controls (red box). Equal amounts (10 μg) of Syn and post-nuclear supernatants (PNS


quantitative analysis The levels of Rheb along with LC3 - II , p62 , and Rab7 are significantly elevated in the synaptosomal fractions ( Syn ) purified from snapin - deficient mouse brains , relative to those of WT littermate controls WT and snapin - mutant mouse brains were sequentially immunoblotted with antibodies against TOM20 on the same membrane after stripping between each antibody application . The purity of synaptosomal fractions was confirmed by relative enrichment of SYP and PSD95 and less abundance of GAPDH . Data were quantified from five independent repeats .

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quantitative analysis The levels of Rheb along with LC3-II, p62, and Rab7 are significantly elevated in the synaptosomal fractions (Syn) purified from snapin-deficient mouse brains, relative to those of WT littermate controls WT and snapin-mutant mouse brains were sequentially immunoblotted with antibodies against TOM20 on the same membrane after stripping between each antibody application. The purity of synaptosomal fractions was confirmed by relative enrichment of SYP and PSD95 and less abundance of GAPDH. Data were quantified from five independent repeats.


E , F Representative blots ( E ) and quantitative analysis ( F ) showing mitophagic accumulation in the brains of snapin conditional KO ( cKO ) mice by crossing with Thy1 - cre Tg mice . Note increased levels of Rheb along with LC3 - II , p62 , and Rab7 in mitochondrial ( Mito ) fractions isolated from snapin - deficient mouse brains , relative to those of WT littermate controls ( red box ) . Data were quantified from five independent repeats . PNS : post - nuclear supernatants .

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E,F Representative blots (E) and quantitative analysis (F) showing mitophagic accumulation in the brains of snapin conditional KO (cKO) mice by crossing with Thy1-cre Tg mice. Note increased levels of Rheb along with LC3-II, p62, and Rab7 in mitochondrial (Mito) fractions isolated from snapin-deficient mouse brains, relative to those of WT littermate controls (red box). Data were quantified from five independent repeats. PNS: post-nuclear supernatants.


G , H Representative TEM images ( G ) and quantitative analysis ( H ) showing abnormal presynaptic retention of mitophagosome - like structures in the hippocampal regions of snapin - deficient mouse brains . Mitophagosomes , indicated by black arrows , are not readily detected in WT mouse brains . Data were quantified from the total number of the imaging fields ( n ) indicated in parentheses ( H ) .

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G,H Representative TEM images (G) and quantitative analysis (H) showing abnormal presynaptic retention of mitophagosome-like structures in the hippocampal regions of snapin-deficient mouse brains. Mitophagosomes, indicated by black arrows, are not readily detected in WT mouse brains. Data were quantified from the total number of the imaging fields (n) indicated in parentheses (H).


I , J Representative images ( I ) and quantitative analysis ( J ) showing that deletion of snapin in mouse brains induces synaptic damage . Note that the density of presynaptic terminals in the hippocampal mossy fibers revealed by SYP immunostaining is significantly reduced in snapin - mutant mice crossed with CamKII - cre Tg mice at the age of 8 months , relative to that of WT littermate controls .

I,J Representative images (I) and quantitative analysis (J) showing that deletion of snapin in mouse brains induces synaptic damage. Note that the density of presynaptic terminals in the hippocampal mossy fibers revealed by SYP immunostaining is significantly reduced in snapin-mutant mice crossed with CamKII-cre Tg mice at the age of 8 months, relative to that of WT littermate controls.


Representative dual - channel kymographs ( A ) and quantitative analysis ( B showing that overexpression of Snapin , but not Snapin - L99K , a Snapin mutant deficient in dynein motor binding , reduces mitophagic accumulation within AD axons incubated with CCCP .

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Representative dual-channel kymographs (A) and quantitative analysis (B showing that overexpression of Snapin, but not Snapin-L99K, a Snapin mutant deficient in dynein motor binding, reduces mitophagic accumulation within AD axons incubated with CCCP.


Representative quantitative analysis showing that overexpression of Snapin , but not Snapin - L99K , a

Representative quantitative analysis showing that overexpression of Snapin, but not Snapin-L99K, a Snapin


D Mean fluorescence intensity ratios evoked by 405 nm and 488 nm excitation wavelengths at individual mitochondria in mutant hAPP axons expressing Snapin or Snapin - L99K were quantified and normalized to those of mutant hAPP controls . Note that oxidatively stressed mitochondria are reduced within AD axons expressing Snapin after CCCP treatment .

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D Mean fluorescence intensity ratios evoked by 405 nm and 488 nm excitation wavelengths at individual mitochondria in mutant hAPP axons expressing Snapin or Snapin-L99K were quantified and normalized to those of mutant hAPP controls. Note that oxidatively stressed mitochondria are reduced within AD axons expressing Snapin after CCCP treatment.


A Representative images showing gene delivery into the hippocampus of AD mice injected with AAV - mCherry or AAV - mCherry - Snapin . mCherry fluorescence indicated by white arrows is present in the soma and processes of transduced neurons in the hippocampal CA3 and dentate gyrus ( DG ) of mutant hAPP mouse brains . NeuN , a neuronal nuclear marker , indicates neurons .

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A Representative images showing gene delivery into the hippocampus of AD mice injected with AAV-mCherry or AAV-mCherry-Snapin. mCherry fluorescence indicated by white arrows is present in the soma and processes of transduced neurons in the hippocampal CA3 and dentate gyrus (DG) of mutant hAPP mouse brains. NeuN, a neuronal nuclear marker, indicates neurons.


Attenuation of mitophagic accumulation in the hippocampal mossy fibers of AD mice transduced with AAV - mCherry - Snapin . Note a marked reduction of mitophagosomes ( white arrows ) in mutant hAPP Tg mouse brains with elevated Snapin expression Cyto c * : color is converted from blue to red for better

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Attenuation of mitophagic accumulation in the hippocampal mossy fibers of AD mice transduced with AAV-mCherry-Snapin. Note a marked reduction of mitophagosomes (white arrows) in mutant hAPP Tg mouse brains with elevated Snapin expression Cyto c*: color is converted from blue to red for better contrast


Attenuation of mitophagic accumulation in the hippocampal mossy fibers of AD mice transduced with AAV - mCherry - Snapin . Note a marked reduction of mitophagosomes ( white arrows ) in mutant hAPP Tg mouse brains with elevated Snapin

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Attenuation of mitophagic accumulation in the hippocampal mossy fibers of AD mice transduced with AAV-mCherry-Snapin. Note a marked reduction of mitophagosomes (white arrows) in mutant hAPP Tg mouse brains with elevated Snapin expression


D , E Quantitative analysis ( D ) and representative images ( E ) showing increased density of presynaptic terminals in the hippocampal mossy fibers of AD mice infected with AAV - mCherry - Snapin . Blue indicates the signal of DAPI staining . The percentage area of SYP - labeled presynaptic terminals was quantified and normalized to that of control AD mice injected with AAV - mCherry ( D ) . Data information : Data were quantified from a total number of 40 - 44 imaging slice sections of three pairs of mutant hAPP Tg mice with AAV injection .

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D,E Quantitative analysis (D) and representative images (E) showing increased density of presynaptic terminals in the hippocampal mossy fibers of AD mice infected with AAV-mCherry-Snapin. Blue indicates the signal of DAPI staining. The percentage area of SYP-labeled presynaptic terminals was quantified and normalized to that of control AD mice injected with AAV-mCherry (D). Data information: Data were quantified from a total number of 40-44 imaging slice sections of three pairs of mutant hAPP Tg mice with AAV injection.


B Plasmids encoding truncated mutants of the Smad6 MH2 domain were co - transfected into HEK293 cells with HA - tagged Pellino - 1N plasmid . Cell lysates were immunoprecipitated with anti - HA antibody and immunoblotted with anti - Myc antibody . Data are representative of at least three independent experiments . IP , immunoprecipitation ; IB , immunoblot ; TCL , total cell lysates .

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B Plasmids encoding truncated mutants of the Smad6 MH2 domain were co-transfected into HEK293 cells with HA-tagged Pellino-1N plasmid. Cell lysates were immunoprecipitated with anti-HA antibody and immunoblotted with anti-Myc antibody. Data are representative of at least three independent experiments. IP, immunoprecipitation; IB, immunoblot; TCL, total cell lysates.


C , D The SBE - Luc reporter plasmid was co - transfected with an empty vector , Myc - Smad6 ( 400 - 441 ) , or full - length Smad6 - expressing plasmids into CMT - 93 cells , respectively . After 24 h , cells were treated with TGF - β1 for 2 h and luciferase activities were measured and normalized . Data were statistically analyzed by a t - test and show the mean ± SD of three independent experiments . * * P < 0 . 05 , * * * P < 0 . 001 .

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C, D The SBE-Luc reporter plasmid was co-transfected with an empty vector, Myc-Smad6 (400-441), or full-length Smad6-expressing plasmids into CMT-93 cells, respectively. After 24 h, cells were treated with TGF-β1 for 2 h and luciferase activities were measured and normalized. Data were statistically analyzed by a t-test and show the mean ± SD of three independent experiments. **P < 0.05, ***P < 0.001.


C , D The 5xNF - κB - Luc reporter plasmid was co - transfected with an empty vector , Myc - Smad6 ( 400 - 441 ) , or full - length Smad6 - expressing plasmids into CMT - 93 cells , respectively . After 24 h , cells were treated with LPS for 2 h and luciferase activities were measured and normalized . Data were statistically analyzed by a t - test and show the mean ± SD of three independent experiments . * * P < 0 . 05 , * * * P < 0 . 001 .

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C, D The 5xNF-κB-Luc reporter plasmid was co-transfected with an empty vector, Myc-Smad6 (400-441), or full-length Smad6-expressing plasmids into CMT-93 cells, respectively. After 24 h, cells were treated with LPS for 2 h and luciferase activities were measured and normalized. Data were statistically analyzed by a t-test and show the mean ± SD of three independent experiments. **P < 0.05, ***P < 0.001.


E CMT - 93 cell lines stably expressing Smad6 amino acids 400 - 441 were treated with LPS for the indicated time and expression of IκBα , IKKα , and phospho - IKKα / β was monitored by immunoblotting . As a positive control , CMT - 93 cells were pre - treated with TGF - β1 for 2 h . CMT - 93 cells stably expressing the empty vector pMSCV - puro were used as a negative control . β - actin was used as a loading control . All data are representative of three independent experiments .

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E CMT-93 cell lines stably expressing Smad6 amino acids 400-441 were treated with LPS for the indicated time and expression of IκBα, IKKα, and phospho-IKKα/β was monitored by immunoblotting. As a positive control, CMT-93 cells were pre-treated with TGF-β1 for 2 h. CMT-93 cells stably expressing the empty vector pMSCV-puro were used as a negative control. β-actin was used as a loading control. All data are representative of three independent experiments.


B A plasmid encoding the truncated mutant composed of Smad6 amino acids 422 - 441 ( Myc - Smad6 ( 422 - 441 ) ) or wild - type Smad6 MH2 domain ( Myc - Smad6 - MH2 ) was co - transfected with the HA - tagged full - length Pellino - 1 plasmid into HEK293 cells . Cell lysates were immunoprecipitated ( IP ) with anti - Myc or anti - HA antibody , and immunoblotted ( IB ) with anti - HA or anti - Myc antibody , respectively . The vector , CS3MTBXA - 6xMyc , was transfected as a negative control . IP , immunoprecipitation ; IB , immunoblot ; TCL , total cell lysates . Data are representative of at least three independent experiments .

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B A plasmid encoding the truncated mutant composed of Smad6 amino acids 422-441 (Myc-Smad6(422-441)) or wild-type Smad6 MH2 domain (Myc-Smad6-MH2) was co-transfected with the HA-tagged full-length Pellino-1 plasmid into HEK293 cells. Cell lysates were immunoprecipitated (IP) with anti-Myc or anti-HA antibody, and immunoblotted (IB) with anti-HA or anti-Myc antibody, respectively. The vector, CS3MTBXA-6xMyc, was transfected as a negative control. IP, immunoprecipitation; IB, immunoblot; TCL, total cell lysates. Data are representative of at least three independent experiments.


C The Myc - Smad6 ( 422 - 441 ) plasmid was co - transfected with full - length Flag - IRAK1 , Flag - TRAF6 , Flag - MyD88 , or HA - Pellino - 1 plasmid into HEK293 cells . Cell lysates were immunoprecipitated with the indicated antibodies and immunoblotted with anti - Myc antibody . IgG was added as a negative control for IP . Data are representative of at least three independent experiments .

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C The Myc-Smad6(422-441) plasmid was co-transfected with full-length Flag-IRAK1, Flag-TRAF6, Flag-MyD88, or HA-Pellino-1 plasmid into HEK293 cells. Cell lysates were immunoprecipitated with the indicated antibodies and immunoblotted with anti-Myc antibody. IgG was added as a negative control for IP. Data are representative of at least three independent experiments.


D , E The SBE - Luc or 5xNF - κB - Luc reporter plasmids were co - transfected with an empty vector or the Myc - Smad6 ( 422 - 441 ) plasmid into CMT - 93 cells , respectively . After 24 h , cells were treated with TGF - β1 for 6 h or LPS for 2 h , and luciferase activities were measured and normalized .

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D, E The SBE-Luc or 5xNF-κB-Luc reporter plasmids were co-transfected with an empty vector or the Myc-Smad6(422-441) plasmid into CMT-93 cells, respectively. After 24 h, cells were treated with TGF-β1 for 6 h or LPS for 2 h, and luciferase activities were measured and normalized.


F The BRE - Luc reporter plasmid was co - transfected with an empty vector or the Myc - Smad6 ( 422 - 441 ) plasmid or full - length Smad6 into RAW264 . 7 cells , respectively . After 24 h , cells were treated with BMP4 for 6 h and luciferase activity was measured and normalized .

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F The BRE-Luc reporter plasmid was co-transfected with an empty vector or the Myc-Smad6(422-441) plasmid or full-length Smad6 into RAW264.7 cells, respectively. After 24 h, cells were treated with BMP4 for 6 h and luciferase activity was measured and normalized.


G A plasmid encoding the truncated mutant composed of Smad6 amino acids 422 - 441 ( Myc - Smad6 ( 422 - 441 ) ) or wild - type Smad6 MH2 domain ( Myc - Smad6 - MH2 ) or full - length Smad6 ( Myc - Smad6 ) was co - transfected with HA - tagged full - length Smad4 or HA - tagged full - length Pellino - 1 plasmid into HEK293 cells , respectively . Cell lysates were immunoprecipitated ( IP ) with anti - Myc and immunoblotted ( IB ) with anti - HA or anti - Myc antibody , respectively . The vector , pCS3MTBXA - 6xMyc , was co - transfected with HA - Smad4 or HA - Pellino - 1 as a negative control . Data are representative of at least three independent experiments .

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G A plasmid encoding the truncated mutant composed of Smad6 amino acids 422-441 (Myc-Smad6(422-441)) or wild-type Smad6 MH2 domain (Myc-Smad6-MH2) or full-length Smad6 (Myc-Smad6) was co-transfected with HA-tagged full-length Smad4 or HA-tagged full-length Pellino-1 plasmid into HEK293 cells, respectively. Cell lysates were immunoprecipitated (IP) with anti-Myc and immunoblotted (IB) with anti-HA or anti-Myc antibody, respectively. The vector, pCS3MTBXA-6xMyc, was co-transfected with HA-Smad4 or HA-Pellino-1 as a negative control. Data are representative of at least three independent experiments.


A Pre - treatment of Smaducin - 6 reduces LPS - induced interleukin - 6 ( Il6 ) gene expression in a dose - dependent manner in RAW264 . 7 cells . Il6 gene expression was analyzed by qRT - PCR .

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A Pre-treatment of Smaducin-6 reduces LPS-induced interleukin-6 (Il6) gene expression in a dose-dependent manner in RAW264.7 cells. Il6 gene expression was analyzed by qRT-PCR.


B , C Pre - treatment of 100 nM Smaducin - 6 or scrambled peptide ( Pal - Scram # 1 ) for 30 min inhibits ( B ) NF - κB - mediated luciferase gene expression when RAW264 . 7 cells are treated with LPS for 2 h . Luciferase activity in ( B ) was normalized to β - galactosidase activity .

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B, C Pre-treatment of 100 nM Smaducin-6 or scrambled peptide (Pal-Scram #1) for 30 min inhibits (B) NF-κB-mediated luciferase gene expression when RAW264.7 cells are treated with LPS for 2 h. Luciferase activity in (B) was normalized to β-galactosidase activity.


B , C Pre - treatment of 100 nM Smaducin - 6 or scrambled peptide ( Pal - Scram # 1 ) for 30 min inhibits ( C ) IκBα degradation and IKKα / β phosphorylation when RAW264 . 7 cells are treated with LPS for 2 h .

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B, C Pre-treatment of 100 nM Smaducin-6 or scrambled peptide (Pal-Scram #1) for 30 min inhibits (C) IκBα degradation and IKKα/β phosphorylation when RAW264.7 cells are treated with LPS for 2 h.


D Peli1 knockdown or wild - type humanTHP1 cells were pre - treated with 100 nM Pal - Scram peptide and Smaducin - 6 for 30 min and subsequently treated with LPS for 2 h . Il6 and Peli1 gene expression were analyzed by qRT - PCR . Data show the mean ± SD of three independent experiments .

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D Peli1 knockdown or wild-type humanTHP1 cells were pre-treated with 100 nM Pal-Scram peptide and Smaducin-6 for 30 min and subsequently treated with LPS for 2 h. Il6 and Peli1 gene expression were analyzed by qRT-PCR. Data show the mean ± SD of three independent experiments.


E RAW264 . 7 cells were treated with 100 nM FITC - conjugated Smaducin - 6 and TAT - S6 ( 422 - 441 ) , and localization was observed by confocal microscopy . Scale bar , 10 μm ( magnification , 200 × ) . Nuclei were stained with DAPI .

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E RAW264.7 cells were treated with 100 nM FITC-conjugated Smaducin-6 and TAT-S6(422-441), and localization was observed by confocal microscopy. Scale bar, 10 μm (magnification, 200×). Nuclei were stained with DAPI.


F After pre - treating RAW264 . 7 cells with biotin - conjugated Smaducin - 6 ( 100 nM ) for 30 min , cells were treated with LPS for 2 h . Subsequent precipitation by streptavidin - agarose showed that endogenous Pellino - 1 binds to the Smaducin - 6 peptide . A biotin - conjugated scrambled peptide was used as a negative control .

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F After pre-treating RAW264.7 cells with biotin-conjugated Smaducin-6 (100 nM) for 30 min, cells were treated with LPS for 2 h. Subsequent precipitation by streptavidin-agarose showed that endogenous Pellino-1 binds to the Smaducin-6 peptide. A biotin-conjugated scrambled peptide was used as a negative control.


G - I Immunoprecipitation assays in primary peritoneal macrophages show that Smaducin - 6 inhibits the formation of endogenous ( G ) IRAK1 - , ( H ) RIP1 - , or ( I ) IKKε - mediated signaling complexes induced by 2 h LPS treatment , compared to a scrambled peptide . IB , immunoblot ; TCL , total cell lysates . Data shown are representative of three independent experiments .

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G-I Immunoprecipitation assays in primary peritoneal macrophages show that Smaducin-6 inhibits the formation of endogenous (G) IRAK1-, (H) RIP1-, or (I) IKKε-mediated signaling complexes induced by 2 h LPS treatment, compared to a scrambled peptide. IB, immunoblot; TCL, total cell lysates. Data shown are representative of three independent experiments.


Subcutaneous injection of Smaducin - 6 increases the survival rate of severe CLP - induced sepsis mice ( BALB / c mice ) . Different amounts of Smaducin - 6 were subcutaneously injected at 2 h post - CLP followed by three injections at 12 - h intervals . n = 10 mice per group per experiment .

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Subcutaneous injection of Smaducin-6 increases the survival rate of severe CLP-induced sepsis mice (BALB/c mice). Different amounts of Smaducin-6 were subcutaneously injected at 2 h post-CLP followed by three injections at 12-h intervals. n = 10 mice per group per experiment.


Smaducin - 6 treatment reduces abnormal nuclei and cell morphology observed in tissues of CLP - induced BALB / c mice , as observed by hematoxylin and eosin ( H / E ) staining . Data shown are representative of five independent experiments . Scale bar , 40 μm ( magnification , 400 × ) .

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Smaducin-6 treatment reduces abnormal nuclei and cell morphology observed in tissues of CLP-induced BALB/c mice, as observed by hematoxylin and eosin (H/E) staining. Data shown are representative of five independent experiments. Scale bar, 40 μm (magnification, 400×).


The survival rates of severe CLP - induced sepsis mice by subcutaneously injected Smaducin - 6 were compared with those by intraperitoneally injected antibiotics ( 8 mg / kg gentamycin plus 8 mg / kg cephalosporin ) . Antibiotics were intraperitoneally injected into CLP mice at 2 h post - CLP followed by an additional injection after 12 h . n = 10 mice per group per experiment .

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The survival rates of severe CLP-induced sepsis mice by subcutaneously injected Smaducin-6 were compared with those by intraperitoneally injected antibiotics (8 mg/kg gentamycin plus 8 mg/kg cephalosporin). Antibiotics were intraperitoneally injected into CLP mice at 2 h post-CLP followed by an additional injection after 12 h. n = 10 mice per group per experiment.


Therapeutic effects of Smaducin - 6 alone , or antibiotics ( 8 mg / kg gentamycin plus 8 mg / kg cephalosporin ) , or different amounts of Smaducin - 6 plus antibiotics were observed in CLP mice . Antibiotics were intraperitoneally injected into CLP mice at 2 h post - CLP followed by an additional injection after 12 h . Smaducin - 6 was subcutaneously injected at 2 h post - CLP followed by three injections at 12 - h intervals . n = 10 mice per group .

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Therapeutic effects of Smaducin-6 alone, or antibiotics (8 mg/kg gentamycin plus 8 mg/kg cephalosporin), or different amounts of Smaducin-6 plus antibiotics were observed in CLP mice. Antibiotics were intraperitoneally injected into CLP mice at 2 h post-CLP followed by an additional injection after 12 h. Smaducin-6 was subcutaneously injected at 2 h post-CLP followed by three injections at 12-h intervals. n = 10 mice per group.


Comparison of mortality of severe CLP - induced sepsis mice upon subcutaneous injections of a total of 16 mg / kg Smaducin - 6 or TAT - S6 ( 422 - 441 ) peptide . n = 10 mice per group .

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Comparison of mortality of severe CLP-induced sepsis mice upon subcutaneous injections of a total of 16 mg/kg Smaducin-6 or TAT-S6(422-441) peptide. n = 10 mice per group.


A - I Smaducin - 6 treatment decreased the concentration of IL - 6 , TNF - α , IFN - γ , and IL - 1β in the blood of CLP mice ( A - D ) , but not IL - 4 , IL - 10 , TGF - β1 , IL - 12 , or IL - 17A ( E - I ) . Cytokine concentrations were analyzed by ELISA . n = 10 mice per group per experiment . Data were statistically analyzed by the Dunnett ' s multiple comparison test ( one - way ANOVA ) . * * * P < 0 . 001 , * * P < 0 . 005 , * P < 0 . 05 compared to sham or vehicle control ( CLP + Pal - Scram # 1 ) .

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A-I Smaducin-6 treatment decreased the concentration of IL-6, TNF-α, IFN-γ, and IL-1β in the blood of CLP mice (A-D), but not IL-4, IL-10, TGF-β1, IL-12, or IL-17A (E-I). Cytokine concentrations were analyzed by ELISA. n = 10 mice per group per experiment. Data were statistically analyzed by the Dunnett's multiple comparison test (one-way ANOVA). ***P < 0.001, **P < 0.005, *P < 0.05 compared to sham or vehicle control (CLP + Pal-Scram #1).


J Expression of the IL - 6 protein is reduced in the spleen and liver of Smaducin - 6 - treated CLP mice . Each lane represents independent mice .

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J Expression of the IL-6 protein is reduced in the spleen and liver of Smaducin-6-treated CLP mice. Each lane represents independent mice.


Subcutaneous injection of Smaducin - 6 into CLP mice reduced bacterial loads in peritoneal fluid . n = 10 mice per group per experiment . Data were statistically analyzed by the Mann - Whitney U - test . * * P < 0 . 005 compared to sham or vehicle control ( CLP + Pal - Scram # 1 ) .

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Subcutaneous injection of Smaducin-6 into CLP mice reduced bacterial loads in peritoneal fluid. n = 10 mice per group per experiment. Data were statistically analyzed by the Mann-Whitney U-test. **P < 0.005 compared to sham or vehicle control (CLP + Pal-Scram #1).


Smaducin - 6 increases neutrophil numbers in peritoneal lavage fluids from LPS - induced endotoxemia BALB / c mice . n = 5 mice per group per experiment . Data were statistically analyzed by a t - test . * * P < 0 . 005 compared to sham or vehicle control ( CLP + Pal - Scram # 1 ) .

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Smaducin-6 increases neutrophil numbers in peritoneal lavage fluids from LPS-induced endotoxemia BALB/c mice. n = 5 mice per group per experiment. Data were statistically analyzed by a t-test. **P < 0.005 compared to sham or vehicle control (CLP + Pal-Scram #1).


CXCR2 expression in human neutrophils , measured by FACS , was decreased by LPS treatment and restored by Smaducin - 6 treatment .

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CXCR2 expression in human neutrophils, measured by FACS, was decreased by LPS treatment and restored by Smaducin-6 treatment.


Treatment with Smaducin - 6 downregulates LPS - induced GRK2 expression in RAW264 . 7 cells and human neutrophils .

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Treatment with Smaducin-6 downregulates LPS-induced GRK2 expression in RAW264.7 cells and human neutrophils.


CXCR2 - expressing neutrophils isolated from CLP mice in the presence of Smaducin - 6 or Pal - Scram peptide were analyzed by FACS . n = 5 mice per group were used . Data were statistically analyzed by a t - test and show the mean ± SD of at least three independent experiments . * * P < 0 . 005 compared to sham or vehicle controls ( CLP + PBS and CLP + Pal - Scram # 1 ) .

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CXCR2-expressing neutrophils isolated from CLP mice in the presence of Smaducin-6 or Pal-Scram peptide were analyzed by FACS. n = 5 mice per group were used. Data were statistically analyzed by a t-test and show the mean ± SD of at least three independent experiments. **P < 0.005 compared to sham or vehicle controls (CLP + PBS and CLP + Pal-Scram #1).


GRK2 expression was directly analyzed by immunoblotting in neutrophils isolated from CLP mice in the presence of Smaducin - 6 or Pal - Scram # 1 peptide .

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GRK2 expression was directly analyzed by immunoblotting in neutrophils isolated from CLP mice in the presence of Smaducin-6 or Pal-Scram #1 peptide.


A , B Quantitative analysis of ( A ) TUNEL - positive cells and ( B ) caspase - 3 - positive cells in IHC in the spleens of CLP + PBS , CLP + Pal - Scram # 1 , and CLP + Smaducin - 6 mice . Three independent experiments ( n = 3 mice per group per experiments ) were performed . At least five hot spots in a section of TUNEL and IHC per experiment were selected , and average count was determined . The data were expressed as a mean percentage of total cell numbers and statistically analyzed by a t - test and show the mean ± SD of three independent experiments . * * P < 0 . 005 , * * * P < 0 . 001 compared to sham or vehicle control ( CLP + Pal - Scram # 1 ) .

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A, B Quantitative analysis of (A) TUNEL-positive cells and (B) caspase-3-positive cells in IHC in the spleens of CLP + PBS, CLP + Pal-Scram #1, and CLP + Smaducin-6 mice. Three independent experiments (n = 3 mice per group per experiments) were performed. At least five hot spots in a section of TUNEL and IHC per experiment were selected, and average count was determined. The data were expressed as a mean percentage of total cell numbers and statistically analyzed by a t-test and show the mean ± SD of three independent experiments. **P < 0.005, ***P < 0.001 compared to sham or vehicle control (CLP + Pal-Scram #1).


C ELISA showing that Smaducin - 6 treatment reduced IFN - β1 levels in peritoneal lavage fluids compared to Pal - Scram # 1 - treated CLP mice . n = 10 mice per group per experiment . Data were statistically analyzed by the Dunnett ' s multiple comparison test ( one - way ANOVA ) . * * * P < 0 . 001 compared to sham or vehicle control ( CLP + Pal - Scram # 1 ) .

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C ELISA showing that Smaducin-6 treatment reduced IFN-β1 levels in peritoneal lavage fluids compared to Pal-Scram #1-treated CLP mice. n = 10 mice per group per experiment. Data were statistically analyzed by the Dunnett's multiple comparison test (one-way ANOVA). ***P < 0.001 compared to sham or vehicle control (CLP + Pal-Scram #1).


D , E IFN - β1 ( D ) and TRAIL ( E ) expression detected by FACS in splenocytes is decreased in Smaducin - 6 - treated CLP mice compared to scrambled peptide - treated CLP mice . Data are representative of three independent experiments .

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D, E IFN-β1 (D) and TRAIL (E) expression detected by FACS in splenocytes is decreased in Smaducin-6-treated CLP mice compared to scrambled peptide-treated CLP mice. Data are representative of three independent experiments.


F Decreased expression of TRAIL was detected by IHC in the spleen of CLP - induced mice treated with Smaducin - 6 compared to mice treated with Pal - Scram . Scale bar , 100 μm ( magnification , 200 × ) . Data are representative of three independent experiments .

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F Decreased expression of TRAIL was detected by IHC in the spleen of CLP-induced mice treated with Smaducin-6 compared to mice treated with Pal-Scram. Scale bar, 100 μm (magnification, 200×). Data are representative of three independent experiments.