Figure 4 . ( 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 . ( 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 . ( 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 . ( 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 ) .

O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4.(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.(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.(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.(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).


Figure 5 . ( 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 .

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Figure 5.(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.


Figure 6 . ( 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 ) . ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6.(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).(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.


Figure 7 . ( 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 ) . ( 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 . ( 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 .

O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7.(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).(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.(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.


Figure 8 . ( 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 ) . ( 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 ) . ( 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 .

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Figure 8.(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).(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).(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.


Figure 1 ( 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 ) . ( 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 ) . ( 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 ) . ( 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 B-PANEL_START O O O O O O O O O O O O O O O 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-PANEL_START O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(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).(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).(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).(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.


Figure 2Protein 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 . 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 . 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 . ( 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 . 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2Protein 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.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.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. (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.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.


Figure 3 ( 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 . ( 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 . ( 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 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(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.(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.(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.


Figure 4 ( 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 . ( 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 . ( 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 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(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.(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.(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.


Figure 5Seven - 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 ) . ( 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 . 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 . 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5Seven-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). (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.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.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.


Figure 2 ( 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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Figure 2(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.


Figure 3 ( 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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Figure 3(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.


Figure 4 ( 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 ) .

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Figure 4(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).


Figure 5 ( 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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Figure 5(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.


Figure 6 ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6(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).


Figure 1Representative 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 . 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 . % 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 . 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 . % 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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Figure 1Representative 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.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.% 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.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.% 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.


Figure 2Representative 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 . 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 . 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 . 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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Figure 2Representative 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.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.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.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.


Figure 3Representative 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 . 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 . 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 . 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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Figure 3Representative 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.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.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.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.


Figure 4Real - 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 . 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 . 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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Figure 4Real-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.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.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.


Figure 5 ( 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 . 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 . 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 . 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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Figure 5(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.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.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.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.


Figure 6Mito 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 . 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 . 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 . Mean % of TAF - positive nuclei ( left graphs ) or mean % of TAF ( right graphs ) in WT vs . POLG double mutant mice . Data are mean ± S . E . M of n = 3 - 4 per group . > 100 CMs were quantified per age group . Schematic depicting isolated mouse adult CMs isolated from 4 animals were treated with or without 100nM rotenone either in the presence of 5mM NAC or vehicle control ( pre - treated for 30 minutes before rotenone treatment ) , for 24 h before fixation . Mean number of TAF ( top graph ) and mean % of TAF - positive nuclei ( bottom graph ) . Data are mean ± SEM from 4 separate CM cultures isolated from 3 month - old mice . 50 CMs were quantified per condition .

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Figure 6Mito 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.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.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.Mean % of TAF-positive nuclei (left graphs) or mean % of TAF (right graphs) in WT vs. POLG double mutant mice. Data are mean ±S.E.M of n=3-4 per group. >100 CMs were quantified per age group.Schematic depicting isolated mouse adult CMs isolated from 4 animals were treated with or without 100nM rotenone either in the presence of 5mM NAC or vehicle control (pre-treated for 30 minutes before rotenone treatment), for 24 h before fixation. Mean number of TAF (top graph) and mean % of TAF-positive nuclei (bottom graph). Data are mean ± SEM from 4 separate CM cultures isolated from 3 month-old mice. 50 CMs were quantified per condition.


Figure 7Examples of individual short axis cine - MR images of 3 or 20 month old mouse hearts . Ejection fraction and LV thickness was calculated based on manual measurements of Left ventricle epicardial and endocardial borders . % change in wall thickening calculation based on wall thickness at the 4 points indicated . Measurements were made in all cine slices at end diastole and end systole . Graphs representing data obtained from MRI analysis of > 7 animals per age group . Data are mean ± S . E . M . Asterisks denote a statistical significance at P < 0 . 05 using Mann Whitney U - test . Scale bars represent 5mm . Comparison between mean number of TAF per CM and CM area in 22 month old animals . Data are mean ± S . E . M . of n = 4 . > 100 CMs were quantified per mouse . Comparison between the % of p16 - or eGFP - positive CMs by RNA - in situ hybridization per plane in INK - ATTAC mice ( 28 - 29 month old ) treated with vehicle or AP20187 . Data are mean ± SEM of n = 5 per age group . 100 CMs were analysed per mouse . Mean number of TAF ( left graph ) and mean % of TAF - positive nuclei ( right graph ) in CMs . Data are mean ± S . E . M . of n = 6 per age group . 100 CMs were analysed per mouse . Mean CM area μm2 . Data are mean ± S . E . M . of n = 6 per age group , > 150 CMs analysed per mouse . % of fibrotic area evaluated by Sirius Red staining .

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Figure 7Examples of individual short axis cine-MR images of 3 or 20 month old mouse hearts. Ejection fraction and LV thickness was calculated based on manual measurements of Left ventricle epicardial and endocardial borders. % change in wall thickening calculation based on wall thickness at the 4 points indicated. Measurements were made in all cine slices at end diastole and end systole. Graphs representing data obtained from MRI analysis of >7 animals per age group. Data are mean ± S.E.M. Asterisks denote a statistical significance at P<0.05 using Mann Whitney U-test. Scale bars represent 5mm.Comparison between mean number of TAF per CM and CM area in 22 month old animals. Data are mean ± S.E.M. of n=4. >100 CMs were quantified per mouse.Comparison between the % of p16- or eGFP-positive CMs by RNA-in situ hybridization per plane in INK-ATTAC mice (28-29 month old) treated with vehicle or AP20187. Data are mean ± SEM of n=5 per age group. 100 CMs were analysed per mouse.Mean number of TAF (left graph) and mean % of TAF-positive nuclei (right graph) in CMs. Data are mean ± S.E.M. of n=6 per age group. 100 CMs were analysed per mouse.Mean CM area μm2. Data are mean ±S.E.M. of n=6 per age group, >150 CMs analysed per mouse.% of fibrotic area evaluated by Sirius Red staining.


Figure 8Quantification of mean number of TAF and % of TAF - positive CMs in 24 months old wild - type mice treated with vehicle or navitoclax ( 50mg / kg / day ) . Data are mean ± SEM of n = 5 - 8 mice per group . More than 100 CMs were quantified per animal . CM cross - sectional area in 24 months old wild - type mice treated with vehicle or navitoclax . ( Left ) Representative images of Sirius red staining and ( right ) % of fibrosis area in 24 month - old mice treated or not with navitoclax . MRI analysis of ejection fraction ( EF % ) in 24 month - old mice treated or not with navitoclax . Data are mean ± S . E . M of n = 6 mice per treatment group . left ventricle mass ( LVmass ) index in 24 month - old mice treated or not with navitoclax . Data are mean ± S . E . M of n = 6 mice per treatment group . % of ventricle wall thickness ( % WTC ) in 24 month - old mice treated or not with navitoclax . Data are mean ± S . E . M of n = 6 mice per treatment group . Examples of confocal microscopy images of CMs positive for CM marker troponin C ( TropC ) , EdU , Ki67 and Aurora B from navitoclax treated animals . ( Upper Right panel ) white arrows identify two nuclei in the same CM that have incorporated EdU . ( Lower Right panel ) white arrow identify EdU expressing Aurora B symmetrically between two nuclei . Scale bars represent 20µm . Quantification of EdU positive CMs ( mono - or multi - nucleated ) in vehicle and navitoclax treated animals . Data are mean ± S . E . M of n = 5 - 6 mice per group . % of Ki67 positive CMs in 29 month old INK - ATTAC mice treated with vehicle or AP20187 . Data are mean ± S . E . M of n = 4 - 6 mice per group .

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Figure 8Quantification of mean number of TAF and % of TAF-positive CMs in 24 months old wild-type mice treated with vehicle or navitoclax (50mg/kg/day). Data are mean± SEM of n=5-8 mice per group. More than 100 CMs were quantified per animal.CM cross-sectional area in 24 months old wild-type mice treated with vehicle or navitoclax.(Left) Representative images of Sirius red staining and (right) % of fibrosis area in 24 month-old mice treated or not with navitoclax.MRI analysis of ejection fraction (EF%) in 24 month-old mice treated or not with navitoclax. Data are mean±S.E.M of n=6 mice per treatment group.left ventricle mass (LVmass) index in 24 month-old mice treated or not with navitoclax. Data are mean±S.E.M of n=6 mice per treatment group.% of ventricle wall thickness (%WTC) in 24 month-old mice treated or not with navitoclax. Data are mean±S.E.M of n=6 mice per treatment group.Examples of confocal microscopy images of CMs positive for CM marker troponin C (TropC), EdU, Ki67 and Aurora B from navitoclax treated animals. (Upper Right panel) white arrows identify two nuclei in the same CM that have incorporated EdU. (Lower Right panel) white arrow identify EdU expressing Aurora B symmetrically between two nuclei. Scale bars represent 20µm.Quantification of EdU positive CMs (mono- or multi-nucleated) in vehicle and navitoclax treated animals. Data are mean±S.E.M of n=5-6 mice per group.% of Ki67 positive CMs in 29 month old INK-ATTAC mice treated with vehicle or AP20187. Data are mean±S.E.M of n=4-6 mice per group.


Figure 2Cloning and characterization of APG9 . A , Wild - type ( WT ; SEY6210 ) , apg9 ( THY154 ) , apg9Δ ( JKY007 ) , and the apg9Δ strain transformed with single copy ( CEN ; pAPG9 ( 414 ) ) or multicopy ( 2μ ; pAPG9 ( 424 ) ) plasmids encoding APG9 were grown to log phase in SMD . Protein extracts were prepared and analyzed by immunoblot using antiserum to API as described in Materials and Methods . The positions of precursor and mature API are indicated . The APG9 gene complements the prAPI accumulation phenotype of the apg9Δ mutant . B , WT ( SEY6210 ) , apg9Δ ( JKY007 ) , and the apg9Δ strain transformed with pAPG9 ( 414 ) centromeric plasmid were grown in SMD and transferred to SD ( − N ) as described in Materials and Methods . Aliquots were removed at the indicated times and spread onto YPD plates in triplicate . Numbers of viable colonies were determined after two to three days . The APG9 gene complements the starvation - sensitive phenotype of the apg9Δ mutant .

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Figure 2Cloning and characterization of APG9. A, Wild-type (WT; SEY6210), apg9 (THY154), apg9Δ (JKY007), and the apg9Δ strain transformed with single copy (CEN; pAPG9(414)) or multicopy (2μ; pAPG9(424)) plasmids encoding APG9 were grown to log phase in SMD. Protein extracts were prepared and analyzed by immunoblot using antiserum to API as described in Materials and Methods. The positions of precursor and mature API are indicated. The APG9 gene complements the prAPI accumulation phenotype of the apg9Δ mutant.B, WT (SEY6210), apg9Δ (JKY007), and the apg9Δ strain transformed with pAPG9(414) centromeric plasmid were grown in SMD and transferred to SD(−N) as described in Materials and Methods. Aliquots were removed at the indicated times and spread onto YPD plates in triplicate. Numbers of viable colonies were determined after two to three days. The APG9 gene complements the starvation-sensitive phenotype of the apg9Δ mutant.


Figure 3Apg9p is an unglycosylated , integral membrane protein . A , Wild - type ( WT ; SEY6210 ) , apg9Δ ( JKY007 ) , and the apg9Δ strain transformed with single copy ( CEN ) or multicopy ( 2μ ) plasmids encoding APG9 were grown to log phase in SMD . Protein extracts were prepared and analyzed by immunoblot using antiserum to Apg9p . Apg9p is detected as an ∼ 125 - kD protein . . B , Hydrophobicity analysis of Apg9p based on the Kyte - Doolittle algorithm ( Kyte and Doolittle 1982 ) predicts six to eight transmembrane domains . C , Cells of wild - type strain YW5 - 1B were grown in YPD , converted to spheroplasts , and osmotically lysed as described in Materials and Methods . After a preclearing step to remove cell debris , the lysates were incubated with or without 1 % Triton X - 100 on ice for 30 min , as indicated . Centrifugation at 10 , 000 g for 1 h separated the lysate into supernatant ( S ) and pellet ( P ) fractions , which were analyzed by immunoblots with antiserum to Apg9p . Apg9p is found in the low - speed pellet in the absence of Triton X - 100 . Detergent extraction completely solubilizes Apg9p and causes it to appear in the supernatant fractionD , Strain CTD1 harboring a multicopy 3 × HA APG9 plasmid was grown in YPD and converted to spheroplasts as described in Materials and Methods . A crude lysate was prepared and treated with or without endo H as indicated overnight at 37 ° C and analyzed by immunoblots with anti - HA antibodies or anti - CPY serum . Endo H treatment removes the N - linked glycosyl residues of CPY , causing a shift in its electrophoretic mobility . In contrast , Apg9p is not glycosylated and remains unchanged in the presence or absence of endo H treatment .

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Figure 3Apg9p is an unglycosylated, integral membrane protein. A, Wild-type (WT; SEY6210), apg9Δ (JKY007), and the apg9Δ strain transformed with single copy (CEN) or multicopy (2μ) plasmids encoding APG9 were grown to log phase in SMD. Protein extracts were prepared and analyzed by immunoblot using antiserum to Apg9p. Apg9p is detected as an ∼125-kD protein.. B, Hydrophobicity analysis of Apg9p based on the Kyte-Doolittle algorithm (Kyte and Doolittle 1982) predicts six to eight transmembrane domains.C, Cells of wild-type strain YW5-1B were grown in YPD, converted to spheroplasts, and osmotically lysed as described in Materials and Methods. After a preclearing step to remove cell debris, the lysates were incubated with or without 1% Triton X-100 on ice for 30 min, as indicated. Centrifugation at 10,000 g for 1 h separated the lysate into supernatant (S) and pellet (P) fractions, which were analyzed by immunoblots with antiserum to Apg9p. Apg9p is found in the low-speed pellet in the absence of Triton X-100. Detergent extraction completely solubilizes Apg9p and causes it to appear in the supernatant fractionD, Strain CTD1 harboring a multicopy 3×HA APG9 plasmid was grown in YPD and converted to spheroplasts as described in Materials and Methods. A crude lysate was prepared and treated with or without endo H as indicated overnight at 37°C and analyzed by immunoblots with anti-HA antibodies or anti-CPY serum. Endo H treatment removes the N-linked glycosyl residues of CPY, causing a shift in its electrophoretic mobility. In contrast, Apg9p is not glycosylated and remains unchanged in the presence or absence of endo H treatment.


Figure 4Apg9p does not comigrate with typical endomembrane markers in sucrose density gradients . A and B , Strain STY1 ( pep4Δ ) was grown in YPD to log - phase , and osmotically lysed . Lysates were precleared , loaded on a sucrose density gradient ranging from 18 - 55 % ( wt / wt ) , and centrifuged for 2 . 5 h at 174 , 000 g as described in Materials and Methods . Fractions were collected from the top ( fraction number 1 ) to the bottom ( fraction number 16 ) . Fractions were subjected to SDS - PAGE and immunoblot with antiserum against Apg9p and : in A , Kex2p ( trans Golgi network ) , Pho8p ( vacuole ) , and Pma1p ( plasma membrane ) ; and in B , Sec12p ( ER ) and Pep12p ( endosome ) . The graphs in panels A and B are from the same gradient , but are presented separately for clarity .

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Figure 4Apg9p does not comigrate with typical endomembrane markers in sucrose density gradients. A and B, Strain STY1 (pep4Δ) was grown in YPD to log-phase, and osmotically lysed. Lysates were precleared, loaded on a sucrose density gradient ranging from 18-55% (wt/wt), and centrifuged for 2.5 h at 174,000 g as described in Materials and Methods. Fractions were collected from the top (fraction number 1) to the bottom (fraction number 16). Fractions were subjected to SDS-PAGE and immunoblot with antiserum against Apg9p and: in A, Kex2p (trans Golgi network), Pho8p (vacuole), and Pma1p (plasma membrane); and in B, Sec12p (ER) and Pep12p (endosome). The graphs in panels A and B are from the same gradient, but are presented separately for clarity.