( B ) Example of a chimeric MT generated from recombinant yeast tubulin containing tyrosinated ( red ) and detyrosinated ( blue ) sections . Most TMR - labeled DDB molecules ( green ) move processively through the annealed junction between the two types of MT lattice ( 65 % , n = 836 processive complexes , 65 MT - MT junctions , three independent experiments ) . Diffusive DDB molecules ( white arrow ) are primarily observed on the tyrosinated section of the MT and do not diffuse across the boundary . Right , video frames showing a processive DDB complex ( pink arrowhead ) moving across the junction ( yellow arrowhead ) of tyrosinated and detryosinated MT . The ( + ) and ( - ) signs denote MT polarity inferred from the directionality of DDB movement . Scale bars are 5 μm and 25 sec . See also Movie EV3 .

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(B) Example of a chimeric MT generated from recombinant yeast tubulin containing tyrosinated (red) and detyrosinated (blue) sections. Most TMR-labeled DDB molecules (green) move processively through the annealed junction between the two types of MT lattice (65%, n=836 processive complexes, 65 MT-MT junctions, three independent experiments). Diffusive DDB molecules (white arrow) are primarily observed on the tyrosinated section of the MT and do not diffuse across the boundary. Right, video frames showing a processive DDB complex (pink arrowhead) moving across the junction (yellow arrowhead) of tyrosinated and detryosinated MT. The (+) and (-) signs denote MT polarity inferred from the directionality of DDB movement. Scale bars are 5 μm and 25 sec. See also Movie EV3.


( C ) TIRF images of low ( 0 . 3 nM ) and high ( 10 nM ) concentrations of TMR - labeled p150 ( green ) bound to chimeric yeast MTs containing tyrosinated ( red ) and detyrosinated ( blue ) sections of MT . Representative kymographs below show diffusive behavior of p150 molecules on the MT . Note that p150 rarely diffuses into the detyrosinated section of MT , even at high concentrations . Scale bars are 5 μm and 10 sec .

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

(C) TIRF images of low (0.3 nM) and high (10 nM) concentrations of TMR-labeled p150 (green) bound to chimeric yeast MTs containing tyrosinated (red) and detyrosinated (blue) sections of MT. Representative kymographs below show diffusive behavior of p150 molecules on the MT. Note that p150 rarely diffuses into the detyrosinated section of MT, even at high concentrations. Scale bars are 5 μm and 10 sec.


( D - E ) TIRFimages and kymographs of annealed WT and CPA - treated porcineMTs ( D ) , or annealed WT MTs ( E ) . The majority of DDB complexes ( 78 % , n = 960 processive complexes , 45 MT - MT junctions , three independent experiments ) move processively across the boundary between WT and CPA - MTs ( D ) . Note TMR - labeled DDB complexes ( green ) in ( E ) traverse the annealed boundary without pausing ( 93 % , n = 677 processive complexes , 45 MT - MT junctions , three independent experiments ) , suggesting that the annealed junction does not affect DDB motility .

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

(D-E) TIRFimages and kymographs of annealed WT and CPA-treated porcineMTs(D), or annealed WT MTs(E). The majority of DDB complexes (78%, n=960 processive complexes, 45 MT-MT junctions, three independent experiments) move processively across the boundary between WT and CPA-MTs(D). Note TMR-labeled DDB complexes (green) in (E) traverse the annealed boundary without pausing (93%, n=677 processive complexes, 45 MT-MT junctions, three independent experiments), suggesting that the annealed junction does not affect DDB motility.


( F ) TIRF image and kymograph from a chimeric MT composed of WT ( red ) and subtilisin - treated porcine MTs ( blue ) . Note some DDB complexes ( pink arrows ) traverse the boundary and continue moving along the subtilisin - treated section of the MT ( 41 % , n = 709 processive complexes , 56 MT - MT junctions , 3 independent experiments ) . All scale bars in D - F are 5 μm and 25 sec .

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 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

(F) TIRF image and kymograph from a chimeric MT composed of WT (red) and subtilisin-treated porcine MTs (blue). Note some DDB complexes (pink arrows) traverse the boundary and continue moving along the subtilisin-treated section of the MT (41%, n=709 processive complexes, 56 MT-MT junctions, 3 independent experiments). All scale bars in D-F are 5 μm and 25 sec.


( A ) UASp - GFP - mCherry - DrAtg8a / + ; nosGAL4 / + flies conditioned on yeast paste had diffuse GFP - mCherry - DrAtg8a staining in midstage egg chambers . ( B ) Nondegenerating midstage egg chambers from starved flies contained autophagosomes ( yellow ) and autolysosomes ( red ) . ( C and D ) Egg chambers early in the degeneration process showed follicle cells that take up portions of the nurse cell cytoplasm ( C ) followed by condensation and fragmentation of the nurse cell nuclei and further uptake of the nurse cell cytoplasm into follicle cells ( D ) . ( E ) Late stage degenerating egg chambers lose all GFP staining and fluoresce red .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) UASp-GFP-mCherry-DrAtg8a/+;nosGAL4/+ flies conditioned on yeast paste had diffuse GFP-mCherry-DrAtg8a staining in midstage egg chambers. (B) Nondegenerating midstage egg chambers from starved flies contained autophagosomes (yellow) and autolysosomes (red). (C and D) Egg chambers early in the degeneration process showed follicle cells that take up portions of the nurse cell cytoplasm (C) followed by condensation and fragmentation of the nurse cell nuclei and further uptake of the nurse cell cytoplasm into follicle cells (D). (E) Late stage degenerating egg chambers lose all GFP staining and fluoresce red.


( F ) Fed Dcp - 1Prev1 / Dcp - 1Prev1 ; UASp - GFP - mCherry - DrAtg8a / nosGAL4 flies showed diffuse GFP - mCherry - DrAtg8a staining in the germline . ( G ) Starved Dcp - 1Prev1 / Dcp - 1Prev1 ; UASp - GFP - mCherry - DrAtg8a / nosGAL4 flies showed reduced autolysosomes in degenerating midstage egg chambers . ( H and I ) Fed flies overexpressing truncated Dcp - 1 ( tDcp - 1 ) in the germline showed increased autophagosomes and autolysosomes in nondegenerating midstage egg chambers ( H ) and also contained degenerating midstage egg chambers that lose all GFP fluorescence and fluoresce red ( I ) . Bars : ( main images ) 25 µm ; ( insets ) 12 . 5 µm . Insets in A - I show diffuse cytoplasmic staining of Atg8a or autophagosomes and autolysosomes . ( J and K ) The percentages of autolysosomes ( autolysosomes / total autophagic structures ) were manually calculated in at least eight egg chambers for each genotype as indicated . Error bars represent the means ± SD . Statistical testing was performed using one - way ANOVA with a Dunnet post test ( * * * * , P < 0 . 0001 ) or a two - tailed Student ' s t test ( * * , P < 0 . 005 ) .

O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR 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 B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Fed Dcp-1Prev1/Dcp-1Prev1;UASp-GFP-mCherry-DrAtg8a/nosGAL4 flies showed diffuse GFP-mCherry-DrAtg8a staining in the germline. (G) Starved Dcp-1Prev1/Dcp-1Prev1;UASp-GFP-mCherry-DrAtg8a/nosGAL4 flies showed reduced autolysosomes in degenerating midstage egg chambers. (H and I) Fed flies overexpressing truncated Dcp-1 (tDcp-1) in the germline showed increased autophagosomes and autolysosomes in nondegenerating midstage egg chambers (H) and also contained degenerating midstage egg chambers that lose all GFP fluorescence and fluoresce red (I). Bars: (main images) 25 µm; (insets) 12.5 µm. Insets in A-I show diffuse cytoplasmic staining of Atg8a or autophagosomes and autolysosomes. (J and K) The percentages of autolysosomes (autolysosomes/total autophagic structures) were manually calculated in at least eight egg chambers for each genotype as indicated. Error bars represent the means ± SD. Statistical testing was performed using one-way ANOVA with a Dunnet post test (****, P < 0.0001) or a two-tailed Student's t test (**, P < 0.005).


( A ) Drosophila S2 cells stably expressing GFP - RFP - Atg8a showed an increase in the percentage of cells containing two or more autolysosomes after starvation , which was blocked after Bafilomycin A1 ( BafA1 ) treatment . At least 50 cells were manually quantitated in three independent experiments ( n = 3 ) . Statistical significance was determined using one - way ANOVA with a Dunnett post test ( * * , P < 0 . 01 ) . ( B ) Representative images of S2 - GFP - RFP - Atg8a cells after the indicated treatments .

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

(A) Drosophila S2 cells stably expressing GFP-RFP-Atg8a showed an increase in the percentage of cells containing two or more autolysosomes after starvation, which was blocked after Bafilomycin A1 (BafA1) treatment. At least 50 cells were manually quantitated in three independent experiments (n = 3). Statistical significance was determined using one-way ANOVA with a Dunnett post test (**, P < 0.01). (B) Representative images of S2-GFP-RFP-Atg8a cells after the indicated treatments.


( C ) S2 - GFP - RFP - Atg8a cells were treated with dsRNAs and subjected to fed or starvation conditions as indicated . At least 50 cells were manually quantitated in three independent experiments ( n = 3 ) . Statistical significance was determined using one - way ANOVA with a Dunnett post test ( * * , P < 0 . 01 ; * * * , P < 0 . 001 ; * * * * , P < 0 . 0001 ) . ( D ) Representative images of S2 - GFP - RFP - Atg8a cells after the indicated RNAi treatments . Error bars represent the means ± SD . Bars , 10 µm .

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(C) S2-GFP-RFP-Atg8a cells were treated with dsRNAs and subjected to fed or starvation conditions as indicated. At least 50 cells were manually quantitated in three independent experiments (n = 3). Statistical significance was determined using one-way ANOVA with a Dunnett post test (**, P < 0.01; ***, P < 0.001; ****, P < 0.0001). (D) Representative images of S2-GFP-RFP-Atg8a cells after the indicated RNAi treatments. Error bars represent the means ± SD. Bars, 10 µm.


( A ) Nondegenerating midstage egg chambers from fed w1118flies showed Ref ( 2 ) P staining in follicle cells and nurse cells . ( B ) Starved w1118flies contained degenerating midstage egg chambers with reduced Ref ( 2 ) P . ( D ) Starved Atg7flies showed an accumulation of Ref ( 2 ) P in the follicle cells and nurse cells of degenerating midstage egg chambers . ( E ) Starved Dcp - 1Prev1flies contained increased levels of Ref ( 2 ) P in degenerating midstage egg chambers .

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

(A) Nondegenerating midstage egg chambers from fed w1118flies showed Ref(2)P staining in follicle cells and nurse cells. (B) Starved w1118flies contained degenerating midstage egg chambers with reduced Ref(2)P.(D) Starved Atg7flies showed an accumulation of Ref(2)P in the follicle cells and nurse cells of degenerating midstage egg chambers. (E) Starved Dcp-1Prev1flies contained increased levels of Ref(2)P in degenerating midstage egg chambers.


( C ) A representative Western blot of ovaries from w1118 and Atg7d77 / d14 flies subjected to fed or starvation conditions for 4 d . Ref ( 2 ) P was detected by immunoblotting . Actin served as a loading control . Densitometry was performed to quantitate Ref ( 2 ) P protein levels relative to actin . Graph represents ± SD from five independent experiments ( n = 5 ) . Statistical significance was determined using a two - tailed Student ' s t test . * , P = 0 . 004 for fed samples ; * , P = 0 . 008 for starved samples .

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 B-MEASURED_VAR I-MEASURED_VAR I-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 B-MEASURED_VAR I-MEASURED_VAR I-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 O O O O O O O O O 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) A representative Western blot of ovaries from w1118 and Atg7d77/d14 flies subjected to fed or starvation conditions for 4 d. Ref(2)P was detected by immunoblotting. Actin served as a loading control. Densitometry was performed to quantitate Ref(2)P protein levels relative to actin. Graph represents ± SD from five independent experiments (n = 5). Statistical significance was determined using a two-tailed Student's t test. *, P = 0.004 for fed samples; *, P = 0.008 for starved samples.


( F ) A representative Western blot of ovaries from w1118 and Dcp - 1Prev1 flies that were subjected to fed or starvation conditions for 4 d . Ref ( 2 ) P was detected by immunoblotting . Actin served as a loading control . Densitometry was performed to quantitate Ref ( 2 ) P protein levels relative to actin . Graph represents ± SD from five independent experiments ( n = 5 )

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

(F) A representative Western blot of ovaries from w1118 and Dcp-1Prev1 flies that were subjected to fed or starvation conditions for 4 d. Ref(2)P was detected by immunoblotting. Actin served as a loading control. Densitometry was performed to quantitate Ref(2)P protein levels relative to actin. Graph represents ± SD from five independent experiments (n = 5)


( A and B ) l ( 2 ) mbn cells were labeled with antibodies to Dcp - 1 and ATPsyn - α ( A ) or MitoTracker red ( MTR ; B ) . Merged images show colocalization between Dcp - 1 and the mitochondria . Boxes represent zoomed images . Bars : ( main images ) 5 µm ; ( zoomed images ) 1 . 25 µm .

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 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 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 O O O

(A and B) l(2)mbn cells were labeled with antibodies to Dcp-1 and ATPsyn-α (A) or MitoTracker red (MTR; B). Merged images show colocalization between Dcp-1 and the mitochondria. Boxes represent zoomed images. Bars: (main images) 5 µm; (zoomed images) 1.25 µm.


( C ) Western blot from l ( 2 ) mbn cells subjected to nutrient - rich or starvation conditions for 6 h . Cells were separated into cytosolic ( C ) and mitochondrial enriched ( M ) fractions .

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(C) Western blot from l(2)mbn cells subjected to nutrient-rich or starvation conditions for 6 h. Cells were separated into cytosolic (C) and mitochondrial enriched (M) fractions.


( D ) Ovaries from fed w1118 flies were separated into cytosolic and membrane - enriched fractions and probed with antibodies to VDAC , Tubulin , ATPsyn - α , and Dcp - 1 .

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(D) Ovaries from fed w1118 flies were separated into cytosolic and membrane-enriched fractions and probed with antibodies to VDAC, Tubulin, ATPsyn-α, and Dcp-1.


( E and F ) Intact and lysed mitochondria ( E ) or intact mitochondria isolated from l ( 2 ) mbn cells ( F ) were treated with proteinase K ( PK ) . The effects of proteinase K treatment were assessed by antibodies to VDAC , ATPsyn - α , Pink1 , and Dcp - 1 .

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(E and F) Intact and lysed mitochondria (E) or intact mitochondria isolated from l(2)mbn cells (F) were treated with proteinase K (PK). The effects of proteinase K treatment were assessed by antibodies to VDAC, ATPsyn-α, Pink1, and Dcp-1.


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


( G ) Control and Dcp - 1 RNAi - treated cells were subjected to nutrient - rich or starvation conditions and stained with NAO . Mean fluorescence was measured by flow cytometry . Graph represents ± SEM of three independent experiments ( n = 3 ) .

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(G) Control and Dcp-1 RNAi-treated cells were subjected to nutrient-rich or starvation conditions and stained with NAO. Mean fluorescence was measured by flow cytometry. Graph represents ± SEM of three independent experiments (n = 3).


( A ) l ( 2 ) mbn cells were labeled with the ATPsyn - α , and mitochondrial morphology was scored as fragmented , normal , or elongated . ( B ) Cells were treated with control or Dcp - 1 dsRNA and subjected to nutrient - rich media or 1 h of starvation . Quantifications represent the percentage of cells with elongated mitochondria divided by the total number of cells examined . At least 100 cells were examined manually in three independent experiments ( n = 3 ) . Error bars represent the mean ± SD . Statistical significance was determined using one - way ANOVA with a Bonferroni post test ( * , P < 0 . 05 ; * * , P < 0 . 01 ) .

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

(A) l(2)mbn cells were labeled with the ATPsyn-α, and mitochondrial morphology was scored as fragmented, normal, or elongated. (B) Cells were treated with control or Dcp-1 dsRNA and subjected to nutrient-rich media or 1 h of starvation. Quantifications represent the percentage of cells with elongated mitochondria divided by the total number of cells examined. At least 100 cells were examined manually in three independent experiments (n = 3). Error bars represent the mean ± SD. Statistical significance was determined using one-way ANOVA with a Bonferroni post test (*, P < 0.05; **, P < 0.01).


( C ) Mitochondrial targeted GFP ( mitoGFP ) was expressed in the germline using the nosGAL4 driver . Staining shows mitoGFP , Armadillo , and DNA . ( D ) Mitochondria were scored as healthy ( H ) , clustered ( C ) , or elongated and overly connected ( E ) . All of mitochondria from fed UASp - mitoGFP / + ; nosGAL4 / + flies were scored as healthy . n = 15 egg chambers manually scored . ( E ) mitoGFP was expressed in Dcp - 1Prev1 flies using the nosGAL4 driver . ( F ) 54 % of egg chambers from UASp - mitoGFP / + ; Dcp - 1Prev1 / Dcp - 1Prev1 ; nosGAL4 / + flies contained elongated mitochondria , 39 % contained mitochondria that were scored as healthy , and 7 % contained clustered mitochondria . n = 28 egg chambers manually scored .

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 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 B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR 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

(C) Mitochondrial targeted GFP (mitoGFP) was expressed in the germline using the nosGAL4 driver. Staining shows mitoGFP, Armadillo, and DNA. (D) Mitochondria were scored as healthy (H), clustered (C), or elongated and overly connected (E). All of mitochondria from fed UASp-mitoGFP/+;nosGAL4/+ flies were scored as healthy. n = 15 egg chambers manually scored. (E) mitoGFP was expressed in Dcp-1Prev1 flies using the nosGAL4 driver. (F) 54% of egg chambers from UASp-mitoGFP/+;Dcp-1Prev1/Dcp-1Prev1;nosGAL4/+ flies contained elongated mitochondria, 39% contained mitochondria that were scored as healthy, and 7% contained clustered mitochondria. n = 28 egg chambers manually scored.


( A ) Total cellular ATP levels were measured in l ( 2 ) mbn cells treated with control or Dcp - 1 dsRNA . Data represent ± SEM of three independent experiments ( n = 3 ) . Statistical significance was determined using a two - tailed Students t test ( * , P = 0 . 02 ) .

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(A) Total cellular ATP levels were measured in l(2)mbn cells treated with control or Dcp-1 dsRNA. Data represent ± SEM of three independent experiments (n = 3). Statistical significance was determined using a two-tailed Students t test (*, P = 0.02).


( B ) Total cellular ATP levels were measured in ovaries from fed or starved w1118 and Dcp - 1Prev1 flies . Data represent ± SEM of five independent experiments ( n = 5 ) . Statistical significance was determined using a two - tailed Student ' s t test ( * * , P = 0 . 014 ; * * * , P < 0 . 001 ) .

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(B) Total cellular ATP levels were measured in ovaries from fed or starved w1118 and Dcp-1Prev1 flies. Data represent ± SEM of five independent experiments (n = 5). Statistical significance was determined using a two-tailed Student's t test (**, P = 0.014; ***, P < 0.001).


( C ) Fed or starved w1118 and Dcp - 1Prev1 flies were treated with DMSO or 25 µg / ml oligomycin A , and Ref ( 2 ) P levels were assessed by immunoblot analysis . Actin served as a loading control . Densitometry was performed to quantitate protein levels relative to actin . Graph represents ± SD from three independent experiments ( n = 3 ) . Statistical significance was determined using a two - tailed Student ' s t test ( * , P = 0 . 02 ) .

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 B-MEASURED_VAR I-MEASURED_VAR I-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 O O O O O O O O O O O O O O O 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) Fed or starved w1118 and Dcp-1Prev1 flies were treated with DMSO or 25 µg/ml oligomycin A, and Ref(2)P levels were assessed by immunoblot analysis. Actin served as a loading control. Densitometry was performed to quantitate protein levels relative to actin. Graph represents ± SD from three independent experiments (n = 3). Statistical significance was determined using a two-tailed Student's t test (*, P = 0.02).


( D and E ) Control UASp - GFP - mCherry - DrAtg8a / + ; nosGAL4 / + flies ( D ) and Dcp - 1Prev1 / Dcp - 1Prev1 ; UASp - GFP - mCherry - DrAtg8a / nosGAL4 flies ( E ) were subjected to starvation conditions supplemented with DMSO or 25 µg / ml oligomycin A . Bars , 25 µm . Quantifications show percentage of autolysosomes ( autolysosomes / total autophagic structures ) . At least eight egg chambers were manually quantitated per genotype per condition ( n = 8 ) . Statistical testing was determined using a two - tailed Student ' s t test . * * * , P = 0 . 0002 .

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

(D and E) Control UASp-GFP-mCherry-DrAtg8a/+;nosGAL4/+ flies (D) and Dcp-1Prev1/Dcp-1Prev1;UASp-GFP-mCherry-DrAtg8a/nosGAL4 flies (E) were subjected to starvation conditions supplemented with DMSO or 25 µg/ml oligomycin A. Bars, 25 µm. Quantifications show percentage of autolysosomes (autolysosomes/total autophagic structures). At least eight egg chambers were manually quantitated per genotype per condition (n = 8). Statistical testing was determined using a two-tailed Student's t test. ***, P = 0.0002.


( A ) A representative Western blot showing SesB and ATPsyn - α levels from fed or starved l ( 2 ) mbn cells treated with control or Dcp - 1 RNAi . Actin served as a loading control . Densitometry was performed to quantitate protein levels relative to actin . Graphs represent ± SD from three independent experiments ( n = 3 ) . Statistical significance was determined using a two - tailed Student ' s t test ( * , P < 0 . 05 ) .

O O O O O O O O B-MEASURED_VAR 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 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

(A) A representative Western blot showing SesB and ATPsyn-α levels from fed or starved l(2)mbn cells treated with control or Dcp-1 RNAi. Actin served as a loading control. Densitometry was performed to quantitate protein levels relative to actin. Graphs represent ± SD from three independent experiments (n = 3). Statistical significance was determined using a two-tailed Student's t test (*, P < 0.05).


( B ) A representative Western blot of ovaries from fed or starved w1118 or Dcp - 1Prev1 flies . SesB and ATPsyn - α were detected by immunoblotting . Actin served as a loading control . Densitometry was performed to quantitate protein levels relative to actin . Graphs represent ± SD from three independent experiments ( n = 3 ) . Statistical significance was determined using a two - tailed Student ' s t test ( * , P < 0 . 05 ) .

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 B-MEASURED_VAR 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 O O O O O O O O O O O O O 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) A representative Western blot of ovaries from fed or starved w1118 or Dcp-1Prev1 flies. SesB and ATPsyn-α were detected by immunoblotting. Actin served as a loading control. Densitometry was performed to quantitate protein levels relative to actin. Graphs represent ± SD from three independent experiments (n = 3). Statistical significance was determined using a two-tailed Student's t test (*, P < 0.05).


( C ) Purified catalytically active Dcp - 1FL , but not catalytically inactive Dcp - 1C < A , cleaved in vitro translated Drice . Cleavage of in vitro translated SesB was not detected .

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(C) Purified catalytically active Dcp-1FL, but not catalytically inactive Dcp-1C<A, cleaved in vitro translated Drice. Cleavage of in vitro translated SesB was not detected.


( D ) V5 - tagged Dcp - 1C < A or V5 vector only control was expressed in l ( 2 ) mbn cells and subjected to nutrient - full or starvation conditions for 2 h . After IP with anti - V5 antibodies , lysates were separated by SDS - PAGE . Proteins were visualized with colloidal Coomassie stain .

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(D) V5-tagged Dcp-1C<A or V5 vector only control was expressed in l(2)mbn cells and subjected to nutrient-full or starvation conditions for 2 h. After IP with anti-V5 antibodies, lysates were separated by SDS-PAGE. Proteins were visualized with colloidal Coomassie stain.


( E ) FLAG - SesB or a vector only control was expressed in l ( 2 ) mbn cells and was immunoprecipitated with FLAG - agarose . Immunoblots show the interaction between FLAG - SesB and endogenous pro - Dcp - 1 . The asterisk represents a nonspecific band .

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(E) FLAG-SesB or a vector only control was expressed in l(2)mbn cells and was immunoprecipitated with FLAG-agarose. Immunoblots show the interaction between FLAG-SesB and endogenous pro-Dcp-1. The asterisk represents a nonspecific band.


( A ) Ovaries from fed SesBOrg flies show reduced Ref ( 2 ) P staining in degenerating midstage egg chamber ( arrow ) .

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(A) Ovaries from fed SesBOrg flies show reduced Ref(2)P staining in degenerating midstage egg chamber (arrow).


( B ) Ovaries from fed w1118 and SesBOrg were analyzed by Western blotting using Ref ( 2 ) P . Actin served as a loading control . Results are representative of three independent experiments ( n = 3 ) .

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(B) Ovaries from fed w1118 and SesBOrg were analyzed by Western blotting using Ref(2)P. Actin served as a loading control. Results are representative of three independent experiments (n = 3).


( C ) SesBOrg flies expressing GFP - mCherry - DrAtg8a in the germline showed increased autophagosomes and autolysosomes in nondegenerating ( left ) and degenerating ( right ) midstage egg chambers . Insets show autophagosomes and autolysosomes ( left ) and autolysosomes ( right ) in SesBOrg midstage egg chambers .

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(C) SesBOrg flies expressing GFP-mCherry-DrAtg8a in the germline showed increased autophagosomes and autolysosomes in nondegenerating (left) and degenerating (right) midstage egg chambers. Insets show autophagosomes and autolysosomes (left) and autolysosomes (right) in SesBOrg midstage egg chambers.


( D ) Quantitation of percentages of autolysosomes ( autolysosomes / total autophagic structures ) in nondegenerating ovaries from fed w1118 and SesBOrg flies . 10 egg chambers were manually examined per genotype . Error bars represent the means ± SD . Statistical significance was determined using a two - tailed Student ' s t test ( * * * , P < 0 . 0001 ) .

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(D) Quantitation of percentages of autolysosomes (autolysosomes/total autophagic structures) in nondegenerating ovaries from fed w1118 and SesBOrg flies. 10 egg chambers were manually examined per genotype. Error bars represent the means ± SD. Statistical significance was determined using a two-tailed Student's t test (***, P < 0.0001).


( E ) Degenerating midstage egg chambers ( arrows ) from fed SesBOrg flies are TUNEL positive .

(E) Degenerating midstage egg chambers (arrows) from fed SesBOrg flies are TUNEL positive.


( F ) Degenerating midstage egg chambers from starved control SesBOrg / FM7a ; Dcp - 1Prev1 / Dcp - 1Prev1flies contained persisting nurse cellnuclei and increased Ref ( 2 ) P . n = 117 ovarioles examined . Box represents zoomed image . ( G ) Degenerating midstage egg chambers from fed SesBOrg ; Dcp - 1Prev1flies contained condensed and fragmented nurse cellnuclei and low levels of Ref ( 2 ) P . Box represents zoomed image . n = 156 ovarioles examined .

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

(F) Degenerating midstage egg chambers from starved control SesBOrg/FM7a;Dcp-1Prev1/Dcp-1Prev1flies contained persisting nurse cellnuclei and increased Ref(2)P. n = 117 ovarioles examined. Box represents zoomed image. (G) Degenerating midstage egg chambers from fed SesBOrg;Dcp-1Prev1flies contained condensed and fragmented nurse cellnuclei and low levels of Ref(2)P. Box represents zoomed image. n = 156 ovarioles examined.


( B ) Immunoblotting and quantification of brown preadipocytes ( WT - 1 ) stimulated with insulin at different doses for 5 min ( n = 3 ) . The molecular weight corresponded to the I - Afadin isoform .

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(B) Immunoblotting and quantification of brown preadipocytes (WT-1) stimulated with insulin at different doses for 5 min (n=3). The molecular weight corresponded to the I-Afadin isoform.


( C ) Immunoblotting of mature brown adipocytes ( WT - 1 ) stimulated with insulin ( 100nM ) for 5 min ( n = 4 ) .

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(C) Immunoblotting of mature brown adipocytes (WT-1) stimulated with insulin (100nM) for 5 min (n=4).


( D ) Immunoblotting of immortalized human mature white and brown adipocytes stimulated with insulin for 5 min ( n = 3 ) .

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(D) Immunoblotting of immortalized human mature white and brown adipocytes stimulated with insulin for 5 min (n=3).


( E ) Immunoblotting and quantification of primary human mature white adipocytes stimulated with insulin for 5 min ( n = 3 ) .

(E) Immunoblotting and quantification of primary human mature white adipocytes stimulated with insulin for 5 min (n=3).


( F ) Immunoblotting and quantification of isolated brown adipose tissue ( BAT ) from male C57BL / 6J mice injected with 5U insulin for 5 min ( n = 4 - 6 ) .

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(F) Immunoblotting and quantification of isolated brown adipose tissue (BAT) from male C57BL/6J mice injected with 5U insulin for 5 min (n=4-6).


( G ) Quantification of immunoblotting of isolated BAT , subcutaneous white adipose tissue ( sWAT ) and visceral white adipose tissue ( vWAT ) from male C57BL / 6J mice after fasting or re - feeding for 2 / 6 hrs ( n = 6 ) .

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(G) Quantification of immunoblotting of isolated BAT, subcutaneous white adipose tissue (sWAT) and visceral white adipose tissue (vWAT) from male C57BL/6J mice after fasting or re-feeding for 2/6 hrs (n=6).


( H ) Immunoblotting and quantification of total and Afadin S1795 phosphorylation in adipose tissue depots from 12 - week old C57BL / 6 male mice fed with either chow ( CD ) or high - fat diet ( HFD ) for 6 - weeks injected with vehicle or insulin ( n = 6 ) .

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(H) Immunoblotting and quantification of total and Afadin S1795 phosphorylation in adipose tissue depots from 12-week old C57BL/6 male mice fed with either chow (CD) or high-fat diet (HFD) for 6-weeks injected with vehicle or insulin (n=6).


( I ) Immunoblotting and quantification of Afadin S1718 phosphorylation in human Stromal vascular fractions ( SVF ) from lean , obese or Type 2 Diabetic ( T2DM ) subjects stimulated or not with insulin ( n = 6 ) . Representative Western blots are shown . Data are presented as means + SEM ; Student ' s unpaired t - test and ANOVA with Tukey or Dunnet ' s post hoc test : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , NS = no significance .

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

(I) Immunoblotting and quantification of Afadin S1718 phosphorylation in human Stromal vascular fractions (SVF) from lean, obese or Type 2 Diabetic (T2DM) subjects stimulated or not with insulin (n=6). Representative Western blots are shown. Data are presented as means + SEM; Student's unpaired t-test and ANOVA with Tukey or Dunnet's post hoc test: *p<0.05, **p<0.01, ***p<0.001, NS= no significance.


Protein lysates from 10 - 13 - week old CTRL and FAKO ( Mllt4 - / - ) mice following retro - orbital injection of insulin ( 1U ) were immunoblotted and quantified . BAT ; Immunoblotting of Afadin S1795 phosphorylation ( n = 4 - 5 ) .

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Protein lysates from 10-13-week old CTRL and FAKO (Mllt4-/-) mice following retro-orbital injection of insulin (1U) were immunoblotted and quantified. BAT; Immunoblotting of Afadin S1795 phosphorylation (n=4-5).


Protein lysates from 10 - 13 - week old CTRL and FAKO ( Mllt4 - / - ) mice following retro - orbital injection of insulin ( 1U ) were immunoblotted and quantified . BAT quantification of Afadin S1795 phosphorylation ( n = 4 - 5 ) . Data are presented as means + SEM ; ANOVA with Tukey or Dunnet ' s post hoc test : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , NS = no significance .

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-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

Protein lysates from 10-13-week old CTRL and FAKO (Mllt4-/-) mice following retro-orbital injection of insulin (1U) were immunoblotted and quantified. BAT quantification of Afadin S1795 phosphorylation (n=4-5). Data are presented as means + SEM; ANOVA with Tukey or Dunnet's post hoc test: *p<0.05, **p<0.01, ***p<0.001, NS= no significance.


Protein lysates from 10 - 13 - week old CTRL and FAKO ( Mllt4 - / - ) mice following retro - orbital injection of insulin ( 1U ) were immunoblotted and quantified . sWAT ; Immunoblotting of Afadin S1795 phosphorylation ( n = 4 - 5 ) .

Protein lysates from 10-13-week old CTRL and FAKO (Mllt4-/-) mice following retro-orbital injection of insulin (1U) were immunoblotted and quantified. sWAT; Immunoblotting of Afadin S1795 phosphorylation (n=4-5).


Protein lysates from 10 - 13 - week old CTRL and FAKO ( Mllt4 - / - ) mice following retro - orbital injection of insulin ( 1U ) were immunoblotted and quantified . sWAT ; quantification of Afadin S1795 phosphorylation ( n = 4 - 5 ) . Data are presented as means + SEM ; ANOVA with Tukey or Dunnet ' s post hoc test : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , NS = no significance .

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 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

Protein lysates from 10-13-week old CTRL and FAKO (Mllt4-/-) mice following retro-orbital injection of insulin (1U) were immunoblotted and quantified. sWAT; quantification of Afadin S1795 phosphorylation (n=4-5). Data are presented as means + SEM; ANOVA with Tukey or Dunnet's post hoc test: *p<0.05, **p<0.01, ***p<0.001, NS= no significance.


Protein lysates from 10 - 13 - week old CTRL and FAKO ( Mllt4 - / - ) mice following retro - orbital injection of insulin ( 1U ) were immunoblotted and quantified . Liver ; Immunoblotting of Afadin S1795 phosphorylation ( n = 4 - 5 ) .

Protein lysates from 10-13-week old CTRL and FAKO (Mllt4-/-) mice following retro-orbital injection of insulin (1U) were immunoblotted and quantified. Liver; Immunoblotting of Afadin S1795 phosphorylation (n=4-5).


Protein lysates from 10 - 13 - week old CTRL and FAKO ( Mllt4 - / - ) mice following retro - orbital injection of insulin ( 1U ) were immunoblotted and quantified . Liver quantification of Afadin S1795 phosphorylation ( n = 4 - 5 ) . Data are presented as means + SEM ; ANOVA with Tukey or Dunnet ' s post hoc test : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , NS = no significance .

Protein lysates from 10-13-week old CTRL and FAKO (Mllt4-/-) mice following retro-orbital injection of insulin (1U) were immunoblotted and quantified. Liver quantification of Afadin S1795 phosphorylation (n=4-5). Data are presented as means + SEM; ANOVA with Tukey or Dunnet's post hoc test: *p<0.05, **p<0.01, ***p<0.001, NS= no significance.


Protein lysates from 10 - 13 - week old CTRL and FAKO ( Mllt4 - / - ) mice following retro - orbital injection of insulin ( 1U ) were immunoblotted and quantified . ( G ) Quantifications of phosphorylated Insulin Receptor β and phosphorylated Akt across the three tissues ( n = 4 - 5 ) . Data are presented as means + SEM ; ANOVA with Tukey or Dunnet ' s post hoc test : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , NS = no significance .

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 I-MEASURED_VAR I-MEASURED_VAR 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

Protein lysates from 10-13-week old CTRL and FAKO (Mllt4-/-) mice following retro-orbital injection of insulin (1U) were immunoblotted and quantified. (G) Quantifications of phosphorylated Insulin Receptor β and phosphorylated Akt across the three tissues (n=4-5). Data are presented as means + SEM; ANOVA with Tukey or Dunnet's post hoc test: *p<0.05, **p<0.01, ***p<0.001, NS= no significance.


Protein lysates from 10 - 13 - week old CTRL and FAKO ( Mllt4 - / - ) mice following retro - orbital injection of insulin ( 1U ) were immunoblotted and quantified . ( H ) Quantifications of Total IRβ / tubulin in BAT , sWAT and Liver ( n = 5 - 6 ) . Data are presented as means + SEM ; ANOVA with Tukey or Dunnet ' s post hoc test : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , NS = no significance .

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

Protein lysates from 10-13-week old CTRL and FAKO (Mllt4-/-) mice following retro-orbital injection of insulin (1U) were immunoblotted and quantified. (H) Quantifications of Total IRβ/tubulin in BAT, sWAT and Liver (n=5-6). Data are presented as means + SEM; ANOVA with Tukey or Dunnet's post hoc test: *p<0.05, **p<0.01, ***p<0.001, NS= no significance.


Protein lysates from 10 - 13 - week old CTRL and FAKO ( Mllt4 - / - ) mice following retro - orbital injection of insulin ( 1U ) were immunoblotted and quantified . ( I ) Insulin - induced lipogenesis in isolated mature adipocytes from CTRL and FAKO ( Mllt4 - / - ) mice ( n = 5 ) . Data are presented as means + SEM ; ANOVA with Tukey or Dunnet ' s post hoc test : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , NS = no significance .

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

Protein lysates from 10-13-week old CTRL and FAKO (Mllt4-/-) mice following retro-orbital injection of insulin (1U) were immunoblotted and quantified. (I) Insulin-induced lipogenesis in isolated mature adipocytes from CTRL and FAKO (Mllt4-/-) mice (n=5). Data are presented as means + SEM; ANOVA with Tukey or Dunnet's post hoc test: *p<0.05, **p<0.01, ***p<0.001, NS= no significance.


( A ) CRISPR / nCas9 - mediated ablation of Afadin protein levels in a clonal cell population termed Afdn - KO .

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(A) CRISPR/nCas9-mediated ablation of Afadin protein levels in a clonal cell population termed Afdn-KO.


( B ) Oil red O staining and gene expression of selected adipogenic genes in WT - 1 and Afdn - KO differentiated adipocytes ( n = 3 ) . Data are presented as means + SEM ; Student ' s unpaired t - test and ANOVA with Tukey or Dunnet ' s post hoc test : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , NS = no significance .

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(B) Oil red O staining and gene expression of selected adipogenic genes in WT-1 and Afdn-KO differentiated adipocytes (n=3). Data are presented as means + SEM; Student's unpaired t-test and ANOVA with Tukey or Dunnet's post hoc test: *p<0.05, **p<0.01, ***p<0.001, NS= no significance.


( C ) Wild - type , Afdn - KO and Afdn - KO with re - expression FLAG - tagged WT I - Afadin or a non - phosphorylable form ( S1795A ) stimulated with vehicle or insulin ( n = 4 ) for Immunoblotting .

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(C) Wild-type, Afdn-KO and Afdn-KO with re-expression FLAG-tagged WT I-Afadin or a non-phosphorylable form (S1795A) stimulated with vehicle or insulin (n=4) for Immunoblotting.


( D ) Quantification of insulin - induced Akt phosphorylation from immunoblots . Data are presented as means + SEM ; Student ' s unpaired t - test and ANOVA with Tukey or Dunnet ' s post hoc test : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , NS = no significance .

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(D) Quantification of insulin-induced Akt phosphorylation from immunoblots. Data are presented as means + SEM; Student's unpaired t-test and ANOVA with Tukey or Dunnet's post hoc test: *p<0.05, **p<0.01, ***p<0.001, NS= no significance.


( E ) Real - time measurement and ( F ) quantification of insulin - induced fatty - acid uptake ( n = 4 ) . Representative Western blots are shown . Data are presented as means + SEM ; Student ' s unpaired t - test and ANOVA with Tukey or Dunnet ' s post hoc test : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , NS = no significance .

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(E) Real-time measurement and (F) quantification of insulin-induced fatty-acid uptake (n=4). Representative Western blots are shown. Data are presented as means + SEM; Student's unpaired t-test and ANOVA with Tukey or Dunnet's post hoc test: *p<0.05, **p<0.01, ***p<0.001, NS= no significance.


( C ) Acetylated α - tubulin levels in cell lines ( n = 3 ) . Data are presented as means + SEM ; Student ' s unpaired t - test and ANOVA with Tukey or Dunnet ' s post hoc test : * p < 0 . 05 , * * p < 0 . 01 , NS = no significance .

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(C) Acetylated α-tubulin levels in cell lines (n=3). Data are presented as means + SEM; Student's unpaired t-test and ANOVA with Tukey or Dunnet's post hoc test: *p<0.05, **p<0.01, NS= no significance.


( D ) Acetylated α - tubulin levels in isolated mature adipocytes from CTRL and FAKO ( Mllt4 - / - ) mice ( n = 4 ) . Data are presented as means + SEM ; Student ' s unpaired t - test and ANOVA with Tukey or Dunnet ' s post hoc test : * p < 0 . 05 , * * p < 0 . 01 , NS = no significance .

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(D) Acetylated α-tubulin levels in isolated mature adipocytes from CTRL and FAKO (Mllt4-/-) mice (n=4). Data are presented as means + SEM; Student's unpaired t-test and ANOVA with Tukey or Dunnet's post hoc test: *p<0.05, **p<0.01, NS= no significance.


( E ) IR abundance measured by immunoblotting following 4 hours pre - incubation with the HDAC6 inhibitor ( Tubastatin A ) in differentiated adipocytes exposed to insulin ( 100nM ) stimulation for 15 min ( n = 4 ) . Data are presented as means + SEM ; Student ' s unpaired t - test and ANOVA with Tukey or Dunnet ' s post hoc test : * p < 0 . 05 , * * p < 0 . 01 , NS = no significance .

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

(E) IR abundance measured by immunoblotting following 4 hours pre-incubation with the HDAC6 inhibitor (Tubastatin A) in differentiated adipocytes exposed to insulin (100nM) stimulation for 15 min (n=4). Data are presented as means + SEM; Student's unpaired t-test and ANOVA with Tukey or Dunnet's post hoc test: *p<0.05, **p<0.01, NS= no significance.


( F ) Immunolocalization of IR ( green ) and HDAC6 ( red ) in Afdn - KO and Afdn - KO with re - expression FLAG - tagged WT Afadin or S1795A Afadin in mature adipocytes stimulated with insulin . Nuclei were stained with DAPI ( blue ) . Co - localization was detected by the appearance of white pixels where the red and green overlap . Scale bars : 25 µm . Representative images are shown . The ratio of IR co - localized with HDAC6 ( Mandler ' s co - localization coefficient ) was quantified ( 98 - 112 individual cells from two independent experiments ) . Data are presented as means + SEM ; Student ' s unpaired t - test and ANOVA with Tukey or Dunnet ' s post hoc test : * p < 0 . 05 , * * p < 0 . 01 , NS = no significance .

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

(F) Immunolocalization of IR (green) and HDAC6 (red) in Afdn-KO and Afdn-KO with re-expression FLAG-tagged WT Afadin or S1795A Afadin in mature adipocytes stimulated with insulin. Nuclei were stained with DAPI (blue). Co-localization was detected by the appearance of white pixels where the red and green overlap. Scale bars: 25 µm. Representative images are shown. The ratio of IR co-localized with HDAC6 (Mandler's co-localization coefficient) was quantified (98-112 individual cells from two independent experiments). Data are presented as means + SEM; Student's unpaired t-test and ANOVA with Tukey or Dunnet's post hoc test: *p<0.05, **p<0.01, NS= no significance.


( G ) Immunoprecipitation of IR followed by immunoblotting using an anti - acetyl - Lys antibody in adipocytes lacking Afadin or re - expressing Afadin , with or without insulin stimulation ( n = 1 ) . Representative Western blots are shown . Data are presented as means + SEM ; Student ' s unpaired t - test and ANOVA with Tukey or Dunnet ' s post hoc test : * p < 0 . 05 , * * p < 0 . 01 , NS = no significance .

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(G) Immunoprecipitation of IR followed by immunoblotting using an anti-acetyl-Lys antibody in adipocytes lacking Afadin or re-expressing Afadin, with or without insulin stimulation (n=1). Representative Western blots are shown. Data are presented as means + SEM; Student's unpaired t-test and ANOVA with Tukey or Dunnet's post hoc test: *p<0.05, **p<0.01, NS= no significance.


Seven - week old CTRL and FAKO ( Mllt4 - / - ) mice were given control diet ( CD ) or high - fat diet ( HFD ) for 16 weeks ( n = 5 - 6 ) . ( A ) Body weight . Data are presented as means + SEM ; ANOVA with Tukey or Dunnet ' s post hoc test : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , NS = no significance .

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

Seven-week old CTRL and FAKO (Mllt4-/-) mice were given control diet (CD) or high-fat diet (HFD) for 16 weeks (n=5-6). (A) Body weight. Data are presented as means + SEM; ANOVA with Tukey or Dunnet's post hoc test: *p<0.05, **p<0.01, ***p<0.001, NS= no significance.


Seven - week old CTRL and FAKO ( Mllt4 - / - ) mice were given control diet ( CD ) or high - fat diet ( HFD ) for 16 weeks ( n = 5 - 6 ) . ( B ) Body composition . Data are presented as means + SEM ; ANOVA with Tukey or Dunnet ' s post hoc test : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , NS = no significance .

Seven-week old CTRL and FAKO (Mllt4-/-) mice were given control diet (CD) or high-fat diet (HFD) for 16 weeks (n=5-6). (B) Body composition. Data are presented as means + SEM; ANOVA with Tukey or Dunnet's post hoc test: *p<0.05, **p<0.01, ***p<0.001, NS= no significance.


Seven - week old CTRL and FAKO ( Mllt4 - / - ) mice were given control diet ( CD ) or high - fat diet ( HFD ) for 16 weeks ( n = 5 - 6 ) . ( C ) Sirius Red staining of representative embedded BAT , sWAT and vWAT from mice on HFD and quantification of adipocyte areas ( ~ 1000 adipocytes / genotype ) . Data are presented as means + SEM ; ANOVA with Tukey or Dunnet ' s post hoc test : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , NS = no significance .

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

Seven-week old CTRL and FAKO (Mllt4-/-) mice were given control diet (CD) or high-fat diet (HFD) for 16 weeks (n=5-6). (C) Sirius Red staining of representative embedded BAT, sWAT and vWAT from mice on HFD and quantification of adipocyte areas (~1000 adipocytes/genotype). Data are presented as means + SEM; ANOVA with Tukey or Dunnet's post hoc test: *p<0.05, **p<0.01, ***p<0.001, NS= no significance.


Seven - week old CTRL and FAKO ( Mllt4 - / - ) mice were given control diet ( CD ) or high - fat diet ( HFD ) for 16 weeks ( n = 5 - 6 ) . ( D ) IPGTT after 12 weeks of diet and AUC . Data are presented as means + SEM ; ANOVA with Tukey or Dunnet ' s post hoc test : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , NS = no significance .

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Seven-week old CTRL and FAKO (Mllt4-/-) mice were given control diet (CD) or high-fat diet (HFD) for 16 weeks (n=5-6). (D) IPGTT after 12 weeks of diet and AUC. Data are presented as means + SEM; ANOVA with Tukey or Dunnet's post hoc test: *p<0.05, **p<0.01, ***p<0.001, NS= no significance.


Seven - week old CTRL and FAKO ( Mllt4 - / - ) mice were given control diet ( CD ) or high - fat diet ( HFD ) for 16 weeks ( n = 5 - 6 ) . ( E ) ITT after 13 weeks of diet and AUC Data are presented as means + SEM ; ANOVA with Tukey or Dunnet ' s post hoc test : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , NS = no significance .

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

Seven-week old CTRL and FAKO (Mllt4-/-) mice were given control diet (CD) or high-fat diet (HFD) for 16 weeks (n=5-6). (E) ITT after 13 weeks of diet and AUC Data are presented as means + SEM; ANOVA with Tukey or Dunnet's post hoc test: *p<0.05, **p<0.01, ***p<0.001, NS= no significance.


Seven - week old CTRL and FAKO ( Mllt4 - / - ) mice were given high - fat diet ( HFD ) for 16 weeks ( n = 5 - 6 ) . ( F ) normalized to basal insulin . Data are presented as means + SEM ; ANOVA with Tukey or Dunnet ' s post hoc test : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , NS = no significance .

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

Seven-week old CTRL and FAKO (Mllt4-/-) mice were given high-fat diet (HFD) for 16 weeks (n=5-6). (F) normalized to basal insulin. Data are presented as means + SEM; ANOVA with Tukey or Dunnet's post hoc test: *p<0.05, **p<0.01, ***p<0.001, NS= no significance.


Seven - week old CTRL and FAKO ( Mllt4 - / - ) mice were given control diet ( CD ) or high - fat diet ( HFD ) for 16 weeks ( n = 5 - 6 ) . ( G ) Plasma insulin levels 5 min after an IPGTT performed after 18 weeks of diet . Data are presented as means + SEM ; ANOVA with Tukey or Dunnet ' s post hoc test : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , NS = no significance .

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 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

Seven-week old CTRL and FAKO (Mllt4-/-) mice were given control diet (CD) or high-fat diet (HFD) for 16 weeks (n=5-6). (G) Plasma insulin levels 5 min after an IPGTT performed after 18 weeks of diet. Data are presented as means + SEM; ANOVA with Tukey or Dunnet's post hoc test: *p<0.05, **p<0.01, ***p<0.001, NS= no significance.


( C ) Temporal analysis of antigen - specific CD8 + T cell responses . Peripheral blood was obtained on days 4 , 7 , 14 , 21 , 42 and 88 post - immunisation with WT ( triangles ; n = 3 - 9 ) , CSPSIINFEKL ( orange squares ; n = 4 - 8 ) or UIS4SIINFEKL ( blue circles ; n = 4 - 10 ) sporozoites , or no parasites ( diamonds ; n = 2 - 5 ) and stained for Kb - SIINFEKL + CD11ahi CD8 + T cells . Line graph shows mean values ( ± SEM ) from representative experiments . ( * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; Welch ' s t - test comparing CSPSIINFEKL and UIS4SIINFEKL ) . See Appendix Table S1 for the number of mice used per timepoint and per group . See Appendix Table S4 for exact p - values .

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(C) Temporal analysis of antigen-specific CD8+ T cell responses. Peripheral blood was obtained on days 4, 7, 14, 21, 42 and 88 post-immunisation with WT (triangles; n=3-9), CSPSIINFEKL (orange squares; n=4-8) or UIS4SIINFEKL (blue circles; n=4-10) sporozoites, or no parasites (diamonds; n=2-5) and stained for Kb-SIINFEKL+ CD11ahi CD8+ T cells. Line graph shows mean values (±SEM) from representative experiments. (*, p<0.05; **, p<0.01; ***, p<0.001; Welch's t-test comparing CSPSIINFEKL and UIS4SIINFEKL). See Appendix Table S1 for the number of mice used per timepoint and per group. See Appendix Table S4 for exact p-values.


( D - H ) C57BL / 6 mice ( n = 4 per group ) , which received 2x106 CFSE - labelled OT - I splenocytes , were immunised with 10 , 000 γ - radiation attenuated WT , CSPSIINFEKL or UIS4SIINFEKL sporozoites intravenously . 5 days later , mice were sacrificed , spleens harvested and splenocytes assessed for ( D ) CFSE dilution of CD8 + T cells , ( E ) CFSE dilution of antigen experienced Kb - SIINFEKL + CD11ahi CD8 + T cells and stained ex vivo ( F - H ) for effector CD8 + T cell surface markers . Shown are flow cytometry plots of Kb - SIINFEKL co - staining with markers of effector phenotypes : ( F ) CD11ahi , ( G ) CD62Llo and ( H ) CD49dhi .

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(D-H) C57BL/6 mice (n=4 per group), which received 2x106 CFSE-labelled OT-I splenocytes, were immunised with 10,000 γ-radiation attenuated WT, CSPSIINFEKL or UIS4SIINFEKL sporozoites intravenously. 5 days later, mice were sacrificed, spleens harvested and splenocytes assessed for (D) CFSE dilution of CD8+ T cells, (E) CFSE dilution of antigen experienced Kb-SIINFEKL+ CD11ahi CD8+ T cells and stained ex vivo (F-H) for effector CD8+ T cell surface markers. Shown are flow cytometry plots of Kb-SIINFEKL co-staining with markers of effector phenotypes: (F) CD11ahi, (G) CD62Llo and (H) CD49dhi.


( B Proportions and numbers of ( B - Kb - SIINFEKL + CD8 + T cells were enumerated , or ( E - IFN - γ - secreting CD8 + T cells following restimulation ex vivo with SIINFEKL peptide were quantified . Flow cytometry plots show representative percentages of CD8 + T cells co - stained with CD11a and ( B ) Kb - SIINFEKL or ( E ) IFN - γ .

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(B Proportions and numbers of (B- Kb-SIINFEKL+ CD8+ T cells were enumerated, or (E- IFN-γ-secreting CD8+ T cells following restimulation ex vivo with SIINFEKL peptide were quantified. Flow cytometry plots show representative percentages of CD8+ T cells co-stained with CD11a and (B) Kb-SIINFEKL or (E) IFN-γ.


( B - C57BL / 6 mice received 10 , 000 γ - radiation attenuated WT , CSPSIINFEKL or UIS4SIINFEKL sporozoites , either intravenously ( n = 6 - 8 mice per group ) or ( E - intradermally ( n = 4 mice per group ) . Additional control mice did not receive sporozoites ( n = 6 - 8 intravenously , n = 4 intradermally ) . Spleens and livers were harvested either at day 14 ( n = 5 livers and n = 6 spleens for intravenous immunisation ) or day 42 ( n = 8 for intravenous immunisation ) , and IFN - γ - secreting lymphocytes following restimulation ex vivo with SIINFEKL peptide were quantified . Flow cytometry plots show representative percentages of CD8 + T cells co - stained with IFN - γ and CD11a ( B , E ) .

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

(B- C57BL/6 mice received 10,000 γ-radiation attenuated WT, CSPSIINFEKL or UIS4SIINFEKL sporozoites, either intravenously (n=6-8 mice per group) or (E- intradermally (n=4 mice per group). Additional control mice did not receive sporozoites (n=6-8 intravenously, n=4 intradermally). Spleens and livers were harvested either at day 14 (n=5 livers and n=6 spleens for intravenous immunisation) or day 42 (n=8 for intravenous immunisation), and IFN-γ-secreting lymphocytes following restimulation ex vivo with SIINFEKL peptide were quantified. Flow cytometry plots show representative percentages of CD8+ T cells co-stained with IFN-γ and CD11a (B, E).


( C ) The upper panel of bar charts show the percentage of CD11ahi IFN - γ + CD8 + T cells and the lower panel the absolute cell counts . Bar charts show mean values ( ± SEM ) from representative experiments ( * * * , p < 0 . 001 ; one - way ANOVA with Tukey ' s multiple comparison test ) . See Appendix Table S4 for exact p - values .

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(C) The upper panel of bar charts show the percentage of CD11ahi IFN-γ+ CD8+ T cells and the lower panel the absolute cell counts. Bar charts show mean values (±SEM) from representative experiments (***, p<0.001; one-way ANOVA with Tukey's multiple comparison test). See Appendix Table S4 for exact p-values.


( B , C ) Flow cytometry ( B ) and scatter plots ( C ) represent CD8 + T cells derived from peripheral blood co - stained with IFN - γ and CD11a , following ex vivo restimulation with SIINFEKL . Data information : Data are representative of two experiments performed with scatter plots showing mean values ( ± SEM ) .

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(B, C) Flow cytometry (B) and scatter plots (C) represent CD8+ T cells derived from peripheral blood co-stained with IFN-γ and CD11a, following ex vivo restimulation with SIINFEKL. Data information: Data are representative of two experiments performed with scatter plots showing mean values (±SEM).


( D ) Protective efficacy as measured by quantitative real - time PCR . Groups of mice were vaccinated as described and challenged 19 days later with 10 , 000 WT ( n = 7 non - vaccinated , n = 9 vaccinated ) , CSPSIINFEKL ( n = 6 non - vaccinated , n = 4 vaccinated ) or UIS4SIINFEKL sporozoites ( n = 3 non - vaccinated , n = 11 vaccinated ) . 42 hours later livers were removed , and parasite load was assessed by qPCR . Plots show the relative parasite load of mice in each condition ( * * , p < 0 . 01 ; Mann - Whitney U test ) . See Appendix Table S4 for exact p - values . Data information : Data are representative of two experiments performed with scatter plots showing mean values ( ± SEM ) .

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

(D) Protective efficacy as measured by quantitative real-time PCR. Groups of mice were vaccinated as described and challenged 19 days later with 10,000 WT (n=7 non-vaccinated, n=9 vaccinated), CSPSIINFEKL (n=6 non-vaccinated, n=4 vaccinated) or UIS4SIINFEKL sporozoites (n=3 non-vaccinated, n=11 vaccinated). 42 hours later livers were removed, and parasite load was assessed by qPCR. Plots show the relative parasite load of mice in each condition (**, p<0.01; Mann-Whitney U test). See Appendix Table S4 for exact p-values. Data information: Data are representative of two experiments performed with scatter plots showing mean values (±SEM).


Representative images of γH2AX immuno - FISH in troponin - C / α - actinin ( top left and bottom left panels , respectively ) positive mouse CMs ( white - troponin - C / α - actinin ; red - telo - FISH ; green - γH2AX ) . Images are Z - projections of 0 . 1μm stacks taken with a 100x oil objective . Scale bar : 20μm . Right panels represent a single Z - plane where co - localisation between a γH2AX foci and telomere was observed , Scale bars 1μm . ( Below ) 3D reconstruction of Immuno - FISH using STED microscopy for γH2A . X and telomeres in a CM from 30 month old mice . Scale bars as indicated and scale the same for both individual TAF examples using STED .

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Representative images of γH2AX immuno-FISH in troponin-C/α-actinin (top left and bottom left panels, respectively) positive mouse CMs (white - troponin-C/α-actinin; red - telo-FISH; green - γH2AX). Images are Z-projections of 0.1μm stacks taken with a 100x oil objective. Scale bar: 20μm. Right panels represent a single Z-plane where co-localisation between a γH2AX foci and telomere was observed, Scale bars 1μm. (Below) 3D reconstruction of Immuno-FISH using STED microscopy for γH2A.X and telomeres in a CM from 30 month old mice. Scale bars as indicated and scale the same for both individual TAF examples using STED.


Mean number of TAF α - actinin positive CMs from C57BL / 6 mice . Data are mean ± SEM of n = 4 - 7 mice per age group .

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Mean number of TAF α-actinin positive CMs from C57BL/6 mice. Data are mean ± SEM of n=4-7 mice per age group.


α - actinin positive CMs from C57BL / 6 mice . Data are mean ± SEM of n = 4 - 7 mice per age group . 100 α - actinin positive CMs were quantified per mouse .

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α-actinin positive CMs from C57BL/6 mice. Data are mean ± SEM of n=4-7 mice per age group. 100 α-actinin positive CMs were quantified per mouse.


Mean number of total γH2A . X foci in α - actinin positive CMs from C57BL / 6 mice . Data are mean ± SEM of n = 4 - 7 mice per age group . 100 α - actinin positive CMs were quantified per mouse .

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Mean number of total γH2A.X foci in α-actinin positive CMs from C57BL/6 mice. Data are mean ± SEM of n=4-7 mice per age group. 100 α-actinin positive CMs were quantified per mouse.


% of γH2A . X foci - positive nuclei ( E ) in α - actinin positive CMs from C57BL / 6 mice . Data are mean ± SEM of n = 4 - 7 mice per age group . 100 α - actinin positive CMs were quantified per mouse .

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% of γH2A.X foci -positive nuclei (E) in α-actinin positive CMs from C57BL/6 mice. Data are mean ± SEM of n=4-7 mice per age group. 100 α-actinin positive CMs were quantified per mouse.


Fold enrichment of γH2AX at telomere repeats by real - time PCR . Graph represents fold enrichment of γH2AX at telomeric repeats between IgG control , 3 and 30 month old whole mouse hearts . Data are mean ± SEM of n = 3 mice per age group .

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Fold enrichment of γH2AX at telomere repeats by real-time PCR. Graph represents fold enrichment of γH2AX at telomeric repeats between IgG control, 3 and 30 month old whole mouse hearts. Data are mean ± SEM of n=3 mice per age group.


Quantitative PCR - ELISA TRAP assay comparing telomerase activity of 3 and 30 month old C57BL / 6 mice whole heart lysates . Data are mean ± SEM of n = 4 mice per age group .

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Quantitative PCR-ELISA TRAP assay comparing telomerase activity of 3 and 30 month old C57BL/6 mice whole heart lysates. Data are mean ± SEM of n=4 mice per age group.


Histograms displaying distributions of telomere intensity in CMs analysed by 3D Q - FISH in young ( 4 months ) and old ( 30 months ) wild - type mice . Data are from n = 3 mice . > 100 CMs were analysed per mouse .

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Histograms displaying distributions of telomere intensity in CMs analysed by 3D Q-FISH in young (4 months) and old (30 months) wild-type mice. Data are from n=3 mice. > 100 CMs were analysed per mouse.


% of telomere FISH signal loss in 30 months old wild - type mice and late generation Terc - / - mice ( 6 months old ) in comparison to 4 months old wild - type mice . Data are from n = 3 mice . > 100 CMs were analysed per mouse .

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% of telomere FISH signal loss in 30 months old wild-type mice and late generation Terc-/- mice (6 months old) in comparison to 4 months old wild-type mice. Data are from n=3 mice. >100 CMs were analysed per mouse.


Representative images of mouse embryonic cardiomyocytes at days 0 , 3 , 5 and 10 days following 10Gy X - irradiation . Left panels represent troponin C - positive embryonic cardiomyocytes ( troponin C - magenta ; DAPI - light blue ) . Middle panels display γH2AX foci ( green ) and telomeres ( red ) in Z projections of 0 . 1µm slices , with white arrows indicating co - localisation . Co - localising foci are amplified in the right - hand panels ( amplified images represent a single Z - plane where co - localisation was observed ) . Scale bars represent 10μm . Scale bars in single plane images 500nm .

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Representative images of mouse embryonic cardiomyocytes at days 0, 3, 5 and 10 days following 10Gy X-irradiation. Left panels represent troponin C-positive embryonic cardiomyocytes (troponin C - magenta; DAPI - light blue). Middle panels display γH2AX foci (green) and telomeres (red) in Z projections of 0.1µm slices, with white arrows indicating co-localisation. Co-localising foci are amplified in the right-hand panels (amplified images represent a single Z-plane where co-localisation was observed). Scale bars represent 10μm. Scale bars in single plane images 500nm.


( Left ) Mean number of both TAF and non - TAF in troponin I - positive mouse embryonic cardiomyocytes at days 0 , 3 , 5 and 10 following 10Gy X - irradiation . Data are mean ± S . E . M of n = 3 independent experiments ; 30 - 50 troponin positive cardiomyocytes were analysed per experiment . ( Right ) Mean percentage of γH2AX foci co - localising with telomeres ( % TAF ) in troponin C - positive mouse embryonic cardiomyocytes at days 0 , 3 , 5 and 10 following 10Gy X - irradiation . Statistical analysis performed using one - way ANOVA ( Holm - Sidak method ) ; * P < 0 . 05 . Significant differences were found for mean number of non - TAF , but not for mean number of TAF .

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(Left) Mean number of both TAF and non-TAF in troponin I-positive mouse embryonic cardiomyocytes at days 0, 3, 5 and 10 following 10Gy X-irradiation. Data are mean ±S.E.M of n=3 independent experiments; 30-50 troponin positive cardiomyocytes were analysed per experiment. (Right) Mean percentage of γH2AX foci co-localising with telomeres (% TAF) in troponin C-positive mouse embryonic cardiomyocytes at days 0, 3, 5 and 10 following 10Gy X-irradiation. Statistical analysis performed using one-way ANOVA (Holm-Sidak method); * P<0.05. Significant differences were found for mean number of non-TAF, but not for mean number of TAF.


Mean number of both TAF and non - TAF in neonatal rat cardiomyocytes at days 0 , 3 , 5 , 10 days following treatment for 24h with H2O2 . Data are mean ± S . E . M of n = 3 . > 50 cells were quantified per condition .

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Mean number of both TAF and non-TAF in neonatal rat cardiomyocytes at days 0, 3, 5, 10 days following treatment for 24h with H2O2. Data are mean ±S.E.M of n=3. >50 cells were quantified per condition.


( Left ) Representative images of γH2AX immuno - FISH in H9C2 myoblasts 3 days 10Gy X - irradiation ( red - telo - FISH ; green - γH2AX ) . White arrows identify areas shown in higher magnification panels . ( Right ) Mean number of both TAF and non - TAF in H9C2 myoblasts at days 3 and 5 following 10Gy X - irradiation . Data are mean ± S . E . M of n = 3 . > 50 cells were quantified per condition . Statistical analysis performed using one - way ANOVA ( Holm - Sidak method ) ; * * * P < 0 . 001 . Scale bars represent 1μm . Scale bars in single plane images 500nm .

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(Left) Representative images of γH2AX immuno-FISH in H9C2 myoblasts 3 days 10Gy X-irradiation (red - telo-FISH; green - γH2AX). White arrows identify areas shown in higher magnification panels. (Right) Mean number of both TAF and non-TAF in H9C2 myoblasts at days 3 and 5 following 10Gy X-irradiation. Data are mean ±S.E.M of n=3. >50 cells were quantified per condition. Statistical analysis performed using one-way ANOVA (Holm-Sidak method); ***P<0.001. Scale bars represent 1μm. Scale bars in single plane images 500nm.


Representative time - lapse images of H9C2 rat cardiomyoblasts expressing AcGFP - 53BP1 from 3 days after 10Gy irradiation at the indicated times ( mins ) . Images are maximum intensity projections with a 6 . 7µm focal depth .

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Representative time-lapse images of H9C2 rat cardiomyoblasts expressing AcGFP-53BP1 from 3 days after 10Gy irradiation at the indicated times (mins). Images are maximum intensity projections with a 6.7µm focal depth.


Kaplan - Meyer survival curves for AcGFP - 53BP1c DDR foci in H9C2 cells 3 days after 10Gy irradiation at 10 minute intervals for 24 hours . > 500 foci from 10 cells were tracked per condition .

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Kaplan-Meyer survival curves for AcGFP-53BP1c DDR foci in H9C2 cells 3 days after 10Gy irradiation at 10 minute intervals for 24 hours. >500 foci from 10 cells were tracked per condition.


Schematic illustration showing 1 month old C57BL / 6 mice treated with 2Gy whole body X - irradiation , followed by a recovery period of 11 months before culling at 12 months of age . Mean number of TAF in α - actinin positive cardiomyocytes . Data are mean ± SEM of n = 3 mice per group . 90 α - actinin positive cardiomyocytes were quantified per condition .

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Schematic illustration showing 1 month old C57BL/6 mice treated with 2Gy whole body X-irradiation, followed by a recovery period of 11 months before culling at 12 months of age. Mean number of TAF in α-actinin positive cardiomyocytes. Data are mean ± SEM of n=3 mice per group. 90 α-actinin positive cardiomyocytes were quantified per condition.


Representative images of rat neonatal CMs 4 days following transfection with a FLAG - tagged TRF1 - FokI - D450A ( top row ) or TRF1 - FokI ( middle and bottom row ) fusion protein ( cell treatments the same for all subsequent panels in Figure ) ( red - telo - FISH ; green - γH2A . X ) . Images are z - projections of 0 . 1µm stacks taken with 100X objective . White arrows indicate co - localisation between telomeres and γH2A . X , with co - localising foci amplified in the right panels ( taken from single z - planes where co - localisation was found ) . Scale bar represents 3 . 5μm . Scale bar in magnified images showing individual co - localisation 500nm .

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Representative images of rat neonatal CMs 4 days following transfection with a FLAG-tagged TRF1-FokI-D450A (top row) or TRF1-FokI (middle and bottom row) fusion protein (cell treatments the same for all subsequent panels in Figure) (red - telo-FISH; green - γH2A.X). Images are z-projections of 0.1µm stacks taken with 100X objective. White arrows indicate co-localisation between telomeres and γH2A.X, with co-localising foci amplified in the right panels (taken from single z-planes where co-localisation was found). Scale bar represents 3.5μm. Scale bar in magnified images showing individual co-localisation 500nm.


% of γH2A . X foci co - localising with telomeres ( B ) in FLAG - tagged TRF1 - FokI - D450A - and TRF1 - FokI - expressing CMs . Data are mean ± SEM of n = 4 independent experiments . > 50 cells were analysed per condition . Statistical analysis was performed by two - tailed t - test * * * P < 0 . 001 .

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% of γH2A.X foci co-localising with telomeres (B) in FLAG-tagged TRF1-FokI-D450A- and TRF1-FokI-expressing CMs. Data are mean ± SEM of n=4 independent experiments. >50 cells were analysed per condition. Statistical analysis was performed by two-tailed t-test ***P<0.001.


mean number of Telomere - associated Foci ( TAF ) ( C ) in FLAG - tagged TRF1 - FokI - D450A - and TRF1 - FokI - expressing CMs . Data are mean ± SEM of n = 4 independent experiments . > 50 cells were analysed per condition . Statistical analysis was performed by two - tailed t - test * * * P < 0 . 001 .

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mean number of Telomere-associated Foci (TAF) (C) in FLAG-tagged TRF1-FokI-D450A- and TRF1-FokI-expressing CMs. Data are mean ± SEM of n=4 independent experiments. >50 cells were analysed per condition. Statistical analysis was performed by two-tailed t-test ***P<0.001.


Histograms displaying telomere intensity for telomeres co - localising or not co - localising with γH2AX foci . Red dotted lines represent median . Mann - Whitney test show no significant difference in telomere intensity between TAF and non - TAF .

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Histograms displaying telomere intensity for telomeres co-localising or not co-localising with γH2AX foci. Red dotted lines represent median. Mann-Whitney test show no significant difference in telomere intensity between TAF and non-TAF.


Mean % of FLAG - labelled CMs positive for SA - β - Gal activity . Data are mean ± SEM of n = 3 independent experiments . > 100 cells were quantified per condition . Statistical analysis performed using two tailed t test ; * * P < 0 . 01 .

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Mean % of FLAG-labelled CMs positive for SA-β-Gal activity. Data are mean ± SEM of n=3 independent experiments. >100 cells were quantified per condition. Statistical analysis performed using two tailed t test; **P<0.01.


Expression of p21 mRNA ( as a function of β - actin and Gapdh ) by real - time PCR in TRF1 - FokI - D450A and TRF1 - FokI expressing CMs . Data are mean ± SEM of n = 6 independent experiments . Statistical analysis performed using two tailed t test ; * * * P < 0 . 001 .

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Expression of p21 mRNA (as a function of β-actin and Gapdh) by real-time PCR in TRF1-FokI-D450A and TRF1-FokI expressing CMs. Data are mean ± SEM of n=6 independent experiments. Statistical analysis performed using two tailed t test; ***P<0.001.


Mean ± SEM cell surface area ( μm2 ) of FLAG - labelled CMs expressing TRF1 - FokI - D450A and TRF1 - FokI . Statistical analysis performed using two tailed t test ; * * * P < 0 . 001 . Scale bar represents 50μm .

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Mean± SEM cell surface area (μm2) of FLAG-labelled CMs expressing TRF1-FokI-D450A and TRF1-FokI. Statistical analysis performed using two tailed t test; ***P<0.001. Scale bar represents 50μm.


Real - time PCR gene expression analysis in isolated mouse CMs from C57BL / 6 mice .

Real-time PCR gene expression analysis in isolated mouse CMs from C57BL/6 mice.


Mean % of p21 - positive CM nuclei from 3 , 15 , and 30 month old C57BL / 6 mice by immunohistochemistry ( IHC ) . Data are mean ± SEM of n = 4 per age group . > 100 CMs were quantified per age group .

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Mean % of p21-positive CM nuclei from 3, 15, and 30 month old C57BL/6 mice by immunohistochemistry (IHC). Data are mean ± SEM of n=4 per age group. >100 CMs were quantified per age group.


Mean % of 3 and 24 month old mouse CMs staining positive for SA - β - Gal in vivo with representative images above ( blue - SA - β - Gal ; green - troponin C ; red - WGA ) . Black arrows indicate SA - β - Gal expression in a troponin C expressing CM . Statistical analysis performed using two - tailed t - test ; * P < 0 . 05 . Data are mean ± SEM from the analysis of > 500 CMs per mouse , 4 mice per age group . Scale bar 20µm .

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Mean % of 3 and 24 month old mouse CMs staining positive for SA-β-Gal in vivo with representative images above (blue - SA-β-Gal; green - troponin C; red - WGA). Black arrows indicate SA-β-Gal expression in a troponin C expressing CM. Statistical analysis performed using two-tailed t-test; *P<0.05. Data are mean ± SEM from the analysis of >500 CMs per mouse, 4 mice per age group. Scale bar 20µm.


Mean % of SADS - positive CM nuclei from 3 and 30 month old mouse CMs positive for SADS in vivo , as detected by centromere - FISH . Data are mean ± SEM of n = 4 per age group . > 200 CMs per mouse were quantified .

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Mean % of SADS-positive CM nuclei from 3 and 30 month old mouse CMs positive for SADS in vivo, as detected by centromere-FISH. Data are mean ± SEM of n=4 per age group. >200 CMs per mouse were quantified.


SASP heatmap : Pearson correlation clustered heatmap showing a curated list of known SASP genes ( top panel ) or a selection of secreted SASP proteins ( bottom panel ) in young ( 3 months ) and old ( 20 months ) mouse CMs ( n = 5 per age group ) . The colour intensity represents column Z - score , where red indicates highly and blue lowly expressed .

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SASP heatmap: Pearson correlation clustered heatmap showing a curated list of known SASP genes (top panel) or a selection of secreted SASP proteins (bottom panel) in young (3 months) and old (20 months) mouse CMs (n=5 per age group). The colour intensity represents column Z-score, where red indicates highly and blue lowly expressed.


( Above ) RNA - sequencing of purified CMs from 4 mice per age group reveal age - dependent increased expression of 3 secreted proteins Edn3 , Tgfb2 , and Gdf15 ; the colour intensity represents column Z - score , where red indicates highly and blue lowly expressed . ( Below ) mRNA expression of Edn3 , Tgfb2 , and Gdf15 was independently validated by RT - PCR in young and old isolated adult CMs . Data are mean ± S . E . M of n = 8 mice per group .

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(Above) RNA-sequencing of purified CMs from 4 mice per age group reveal age-dependent increased expression of 3 secreted proteins Edn3, Tgfb2, and Gdf15; the colour intensity represents column Z-score, where red indicates highly and blue lowly expressed. (Below) mRNA expression of Edn3, Tgfb2, and Gdf15 was independently validated by RT-PCR in young and old isolated adult CMs. Data are mean ±S.E.M of n=8 mice per group.


Representative images of immunofluorescent staining against α - SMA and EdU in neonatal fibroblasts cultured in the presence of CM from young and old CMs .

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Representative images of immunofluorescent staining against α-SMA and EdU in neonatal fibroblasts cultured in the presence of CM from young and old CMs.


Quantification of % of EdU incorporation ( D ) in neonatal fibroblasts after treatment for 48h with CM from young and old CM . Data are mean ± S . E . M of n = 3 - 4 mice per age group ; > 200 cells were quantified per condition .

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Quantification of % of EdU incorporation (D) in neonatal fibroblasts after treatment for 48h with CM from young and old CM. Data are mean ±S.E.M of n=3-4 mice per age group; >200 cells were quantified per condition.


% of a - SMA positive cells ( E ) in neonatal fibroblasts after treatment for 48h with CM from young and old CM . Data are mean ± S . E . M of n = 3 - 4 mice per age group ; > 200 cells were quantified per condition .

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% of a-SMA positive cells (E) in neonatal fibroblasts after treatment for 48h with CM from young and old CM. Data are mean ±S.E.M of n=3-4 mice per age group; >200 cells were quantified per condition.


Representative micrographs of neonatal fibroblasts and CMs treated with recombinant proteins : Tgfb2 , Edn3 , and Gdf15 for 48h and immunostained against α - SMA and EdU ( fibroblasts ) and α - actinin ( CMs ) .

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Representative micrographs of neonatal fibroblasts and CMs treated with recombinant proteins: Tgfb2, Edn3, and Gdf15 for 48h and immunostained against α-SMA and EdU (fibroblasts) and α-actinin (CMs).


Quantification of α - SMA ( G ) in neonatal CMs following treatment with the indicated recombinant proteins . Data are mean ± S . E . M of n = 3 - 4 independent experiments .

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Quantification of α-SMA (G) in neonatal CMs following treatment with the indicated recombinant proteins. Data are mean ±S.E.M of n=3-4 independent experiments.


EdU positive neonatal fibroblasts ( H ) in neonatal CMs following treatment with the indicated recombinant proteins . Data are mean ± S . E . M of n = 3 - 4 independent experiments .

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EdU positive neonatal fibroblasts (H) in neonatal CMs following treatment with the indicated recombinant proteins. Data are mean ±S.E.M of n=3-4 independent experiments.


surface area ( μm2 ) ( I ) in neonatal CMs following treatment with the indicated recombinant proteins . Data are mean ± S . E . M of n = 3 - 4 independent experiments .

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surface area (μm2) (I) in neonatal CMs following treatment with the indicated recombinant proteins. Data are mean ±S.E.M of n=3-4 independent experiments.


Mito and ETC genes with GSEA analysis : Clustered heatmap showing all genes associated with the " Mitochondrion " GO term in young and old , mouse CMs as observed by the GSEA pre - ranked list enrichment analysis ( normalised enrichment score : - 1 . 70 ; FDR q - value < 0 . 05 ) . Alongside this is a column clustered heatmap displaying a list of genes from the electron transport chain ( ETC ) GO ontology . In both instances , genes are by column and samples by row with the colour intensity representing column Z - score , where red indicates highly and blue lowly expressed .

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Mito and ETC genes with GSEA analysis: Clustered heatmap showing all genes associated with the "Mitochondrion" GO term in young and old, mouse CMs as observed by the GSEA pre-ranked list enrichment analysis (normalised enrichment score: -1.70; FDR q-value < 0.05). Alongside this is a column clustered heatmap displaying a list of genes from the electron transport chain (ETC) GO ontology. In both instances, genes are by column and samples by row with the colour intensity representing column Z-score, where red indicates highly and blue lowly expressed.


Real - time PCR gene expression analysis of MAO - A , MnSOD and catalase in isolated mouse CMs from young ( 3 months ) and old ( 20 months ) mice .

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Real-time PCR gene expression analysis of MAO-A, MnSOD and catalase in isolated mouse CMs from young (3 months) and old (20 months) mice.


Mean % of 4 - HNE - ( top ) or 8oxodG - ( bottom ) positive CMs from 3 month ( young ) or 30 month ( old ) aged mice . Data are mean ± SEM of n = 4 per age group . 100 CMs were quantified per age group .

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Mean % of 4-HNE- (top) or 8oxodG- (bottom) positive CMs from 3 month (young) or 30 month (old) aged mice. Data are mean ± SEM of n=4 per age group. 100 CMs were quantified per age group.


Mean % of TAF - positive nuclei ( left graphs ) or mean % of TAF ( right graphs ) in wild - type ( control ) compared to MAO - A transgenic mice with or without drinking water supplemented with 1 . 5g / kg / day NAC from the age of 4 to 24 weeks Data are mean ± S . E . M of n = 3 - 4 per group . > 100 CMs were quantified per age group .

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Mean % of TAF-positive nuclei (left graphs) or mean % of TAF (right graphs) in wild-type (control) compared to MAO-A transgenic mice with or without drinking water supplemented with 1.5g/kg/day NAC from the age of 4 to 24 weeks Data are mean ±S.E.M of n=3-4 per group. >100 CMs were quantified per age group.


Mean % of TAF - positive nuclei ( left graphs ) or mean % of TAF ( right graphs ) in MnSOD + / + vs MnSOD - / + , Data are mean ± S . E . M of n = 3 - 4 per group . > 100 CMs were quantified per age group .

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 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

Mean % of TAF-positive nuclei (left graphs) or mean % of TAF (right graphs) in MnSOD+/+ vs MnSOD-/+, Data are mean ±S.E.M of n=3-4 per group. >100 CMs were quantified per age group.


Mean % of TAF - positive nuclei ( left graphs ) or mean % of TAF ( right graphs ) in Catalase + / + vs . Catalase - / - , Data are mean ± S . E . M of n = 3 - 4 per group . > 100 CMs were quantified per age group .

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 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

Mean % of TAF-positive nuclei (left graphs) or mean % of TAF (right graphs) in Catalase+/+ vs. Catalase-/-, Data are mean ±S.E.M of n=3-4 per group. >100 CMs were quantified per age group.