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

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

(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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 .

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

(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 .

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

(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 O O O O O O O O O O O B-CONTROLLED_VAR 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 O O O O O O O O O O O O O O O O O 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) 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 .

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 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

(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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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). (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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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- 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 .

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

(C) The 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 ) .

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

(B, C) 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 .

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

Quantitative 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 .

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 .

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

% 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 .

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 .

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

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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O 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± 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 .

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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

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 .

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

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 .

% 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 .

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

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 .

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

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 .

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 .

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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 .

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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 .

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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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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.


Examples 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 .

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Examples 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 .

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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 .

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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 .

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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 .

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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 .

% of fibrotic area evaluated by Sirius Red staining.


Quantification 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 .

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Quantification 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 .

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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 .

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(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 .

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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 .

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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 .

% 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 .

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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 .

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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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% 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.


Cloning 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 .

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Cloning 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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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.


Apg9p 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 .

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Apg9p 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 .

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. 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

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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 fraction


D , 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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D, 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.


Apg9p 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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Apg9p 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.


Apg9p does not comigrate with prAPI in a strain that accumulates Cvt / autophagic vesicles . Strain TK415 ( ypt7Δ ) was grown in YPD to log - phase , and osmotically lysed . Lysates were precleared , centrifuged at 100 , 000 g for 1 h , and the pellet fraction resuspended and centrifuged a second time . The resulting pellet fraction was subsequently loaded on a sucrose density gradient ranging from 18 - 55 % ( wt / wt ) , and centrifuged for 16 h at 174 , 000 g as described in Materials and Methods . 16 fractions were collected starting from the top and subjected to SDS - PAGE and immunoblot with antisera against Apg9p and API . The graph represents an average of two experiments .

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Apg9p does not comigrate with prAPI in a strain that accumulates Cvt/autophagic vesicles. Strain TK415 (ypt7Δ) was grown in YPD to log-phase, and osmotically lysed. Lysates were precleared, centrifuged at 100,000 g for 1 h, and the pellet fraction resuspended and centrifuged a second time. The resulting pellet fraction was subsequently loaded on a sucrose density gradient ranging from 18-55% (wt/wt), and centrifuged for 16 h at 174,000 g as described in Materials and Methods. 16 fractions were collected starting from the top and subjected to SDS-PAGE and immunoblot with antisera against Apg9p and API. The graph represents an average of two experiments.


Apg9p localizes to large perivacuolar punctate structures . Immunofluorescence microscopy of strains CTD1 ( apg9Δ ) , KSL12C ( vps4Δ ) , and SKD6 - 1D ( apg6Δ ) transformed with the multicopy plasmid 3 × HA APG9 . Cells were grown in YPD to log phase ( vegetative ) , and incubated further in SD ( − N ) medium with 1 mM PMSF for 3 h ( starvation ) . The cells were fixed with formaldehyde and examined by immunofluorescence microscopy as described in Materials and Methods . Anti - HA and FITC - conjugated antibodies mark Apg9p ( left ) , and the vacuole can be visualized by Nomarski optics ( middle ) . An overlay is shown in the right panels .

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Apg9p localizes to large perivacuolar punctate structures. Immunofluorescence microscopy of strains CTD1 (apg9Δ), KSL12C (vps4Δ), and SKD6-1D (apg6Δ) transformed with the multicopy plasmid 3×HA APG9. Cells were grown in YPD to log phase (vegetative), and incubated further in SD(−N) medium with 1 mM PMSF for 3 h (starvation). The cells were fixed with formaldehyde and examined by immunofluorescence microscopy as described in Materials and Methods. Anti-HA and FITC-conjugated antibodies mark Apg9p (left), and the vacuole can be visualized by Nomarski optics (middle). An overlay is shown in the right panels.


In vivo examination of the Apg9pGFP fusion protein . Wild - type ( SEY6210 ) cells expressing Apg9pGFP from a multicopy plasmid were grown in SMD to midlog phase and labeled with FM 4 - 64 to stain the vacuoles as described in Materials and Methods . The labeled cells were viewed directly with a Leica DM IRB confocal microscope . Large , punctate Apg9pGFP structures appear juxtaposed to the FM 4 - 64 - labeled vacuole membranes . In addition , smaller punctate structures appear throughout the cytoplasm .

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In vivo examination of the Apg9pGFP fusion protein. Wild-type (SEY6210) cells expressing Apg9pGFP from a multicopy plasmid were grown in SMD to midlog phase and labeled with FM 4-64 to stain the vacuoles as described in Materials and Methods. The labeled cells were viewed directly with a Leica DM IRB confocal microscope. Large, punctate Apg9pGFP structures appear juxtaposed to the FM 4-64-labeled vacuole membranes. In addition, smaller punctate structures appear throughout the cytoplasm.


Ultrastructural localization of Apg9p for immunoelectron microscopy . Strain STY1 ( pep4Δ ) expressing 3 × HA Apg9p was grown in YPD to log phase and transferred to SD ( − N ) medium for 2 h . The cells were fixed and stained with anti - HA antibody and 5 - nm colloidal gold - conjugated goat anti - mouse IgG as described in Materials and Methods . A , Section showing autophagic bodies in the vacuole lumen . B , Section showing a cytosolic autophagosome . Apg9p is seen in patches near the vacuole , but not on autophagic bodies or autophagosomes . AB , autophagic body ; AP , autophagosome ; N , nucleus ; V , vacuole ; VM , vacuolar membrane . Arrows point to areas of concentrated Apg9p .

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Ultrastructural localization of Apg9p for immunoelectron microscopy. Strain STY1 (pep4Δ) expressing 3×HA Apg9p was grown in YPD to log phase and transferred to SD(−N) medium for 2 h. The cells were fixed and stained with anti-HA antibody and 5-nm colloidal gold-conjugated goat anti-mouse IgG as described in Materials and Methods. A, Section showing autophagic bodies in the vacuole lumen. B, Section showing a cytosolic autophagosome. Apg9p is seen in patches near the vacuole, but not on autophagic bodies or autophagosomes. AB, autophagic body; AP, autophagosome; N, nucleus; V, vacuole; VM, vacuolar membrane. Arrows point to areas of concentrated Apg9p.


Vesicle accumulation test and kinetic analysis of prAPI import in the temperature conditional Apg9p mutant . A , Morphological analysis of apg9Δ indicates an import defect at an early step in the pathway . Wild - type ( YW5 - 1B ) and apg9Δ ( CTD1 ) cells were grown in YPD ( vegetative ) or transferred to SD ( − N ) ( starvation ) for 3 h in the presence of PMSF ( starvation ) and examined by DIC ( Nomarski ) microscopy ( Zeiss Axioplan ) . PMSF inhibits the degradation of autophagic bodies . Under starvation conditions , wild - type cells accumulate autophagic bodies when vesicle breakdown is blocked . However , under the same conditions , apg9Δ cells do not accumulate autophagic bodies , indicating that Apg9p is required for a step before vesicle fusion and release of the autophagic body into the vacuolar lumen . The apg9Δ strain transformed with the APG9 plasmid shows the same result as wild - type ( data not shown ) . B , The apg9ts strain is tightly blocked for prAPI import at nonpermissive temperature . Wild - type ( SEY6210 ) and apg9Δ ( JKY007 ) cells transformed with the apg9ts centromeric plasmid were incubated at 24 and 38 ° C for 5 min , pulse - labeled for 10 min , and then subjected to nonradioactive chase reactions for the indicated times . Samples at each time point were immunoprecipitated with antiserum to API and resolved by SDS - PAGE as described in Materials and Methods . API - immunoprecipitated bands were quantified by a Molecular Dynamics STORM PhosphorImager and the results are presented in the graph . The percent mature API was determined by dividing the mature API value over the sum of the precursor and mature API values for each time point .

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

Vesicle accumulation test and kinetic analysis of prAPI import in the temperature conditional Apg9p mutant. A, Morphological analysis of apg9Δ indicates an import defect at an early step in the pathway. Wild-type (YW5-1B) and apg9Δ (CTD1) cells were grown in YPD (vegetative) or transferred to SD(−N) (starvation) for 3 h in the presence of PMSF (starvation) and examined by DIC (Nomarski) microscopy (Zeiss Axioplan). PMSF inhibits the degradation of autophagic bodies. Under starvation conditions, wild-type cells accumulate autophagic bodies when vesicle breakdown is blocked. However, under the same conditions, apg9Δ cells do not accumulate autophagic bodies, indicating that Apg9p is required for a step before vesicle fusion and release of the autophagic body into the vacuolar lumen. The apg9Δ strain transformed with the APG9 plasmid shows the same result as wild-type (data not shown). B, The apg9ts strain is tightly blocked for prAPI import at nonpermissive temperature. Wild-type (SEY6210) and apg9Δ (JKY007) cells transformed with the apg9ts centromeric plasmid were incubated at 24 and 38°C for 5 min, pulse-labeled for 10 min, and then subjected to nonradioactive chase reactions for the indicated times. Samples at each time point were immunoprecipitated with antiserum to API and resolved by SDS-PAGE as described in Materials and Methods. API-immunoprecipitated bands were quantified by a Molecular Dynamics STORM PhosphorImager and the results are presented in the graph. The percent mature API was determined by dividing the mature API value over the sum of the precursor and mature API values for each time point.


Analysis of the apg9ts strain indicates Apg9p is directly required for the vesicle formation step . A , The nature of prAPI binding and pelleting in the apg9ts strain . Spheroplasts of apg9ts were labeled for 10 min and chased for 30 min at 38 ° C . The spheroplasts were osmotically lysed in a buffer containing no salt ( 20 mM Pipes , pH 6 . 8 ) or a physiological concentration of salt ( 100 mM KOAc , 50 mM KCl , 5 mM MgCl2 , 20 mM Pipes , pH 6 . 8 ) and pelleted at 5 , 000 g . All samples were immunoprecipitated with antiserum to API and the cytosolic marker PGK . apg9ts retains the salt dependent binding and pelleting of prAPI . B , prAPI in the apg9ts strain accumulates in both a membrane - associated state and a large pelletable complex . Spheroplasts of apg9ts and ypt7Δ were labeled for 10 min and chased for 30 min at 38 ° C . The labeled spheroplasts were then osmotically lysed and separated into supernatant ( S ) and pellet ( P ) fractions by centrifugation at 5 , 000 g for 5 min . An aliquot was removed for the total lysate control ( T ) . The pellet fraction ( P ) was resuspended in 15 % Ficoll - 400 in gradient buffer ( 20 mM Pipes , 5 mM MgCl2 , complete EDTA - free protease inhibitor cocktail ) in the presence or absence of Triton X - 100 and overlaid with 13 and 2 % Ficoll - 400 in gradient buffer . The step gradients were centrifuged at 13 , 000 g for 10 min . Membrane - containing float ( F ) , nonfloat ( NF ) , and pellet ( P2 ) fractions were immunoprecipitated with antiserum to API as described in Materials and Methods . The position of prAPI is indicated . C , prAPI in the apg9ts strain is protease accessible . Spheroplasts isolated from apg9ts and ypt7Δ cells were pulse - labeled for 10 min and chased for 30 min at 38 ° C . The labeled spheroplasts were then osmotically lysed and separated into low - speed supernatant ( S ) and pellet ( P ) fractions after a 5 , 000 g centrifugation step . The pellet fractions were subjected to protease treatment in the absence or presence of 0 . 2 % Triton X - 100 as described in Materials and Methods . The resulting samples were immunoprecipitated with antiserum to API and PGK . The immunoprecipitated bands were quantified by a Molecular Dynamics STORM PhosphorImager . The percent protease - protected prAPI was determined by dividing the value for prAPI in the protease - treated sample by the value for total prAPI before protease treatment . The percent PGK was determined by dividing the supernatant ( S ) or pellet ( P ) value over the sum of the S and P values .

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

Analysis of the apg9ts strain indicates Apg9p is directly required for the vesicle formation step. A, The nature of prAPI binding and pelleting in the apg9ts strain. Spheroplasts of apg9ts were labeled for 10 min and chased for 30 min at 38°C. The spheroplasts were osmotically lysed in a buffer containing no salt (20 mM Pipes, pH 6.8) or a physiological concentration of salt (100 mM KOAc, 50 mM KCl, 5 mM MgCl2, 20 mM Pipes, pH 6.8) and pelleted at 5,000 g. All samples were immunoprecipitated with antiserum to API and the cytosolic marker PGK. apg9ts retains the salt dependent binding and pelleting of prAPI. B, prAPI in the apg9ts strain accumulates in both a membrane-associated state and a large pelletable complex. Spheroplasts of apg9ts and ypt7Δ were labeled for 10 min and chased for 30 min at 38°C. The labeled spheroplasts were then osmotically lysed and separated into supernatant (S) and pellet (P) fractions by centrifugation at 5,000 g for 5 min. An aliquot was removed for the total lysate control (T). The pellet fraction (P) was resuspended in 15% Ficoll-400 in gradient buffer (20 mM Pipes, 5 mM MgCl2, complete EDTA-free protease inhibitor cocktail) in the presence or absence of Triton X-100 and overlaid with 13 and 2% Ficoll-400 in gradient buffer. The step gradients were centrifuged at 13,000 g for 10 min. Membrane-containing float (F), nonfloat (NF), and pellet (P2) fractions were immunoprecipitated with antiserum to API as described in Materials and Methods. The position of prAPI is indicated. C, prAPI in the apg9ts strain is protease accessible. Spheroplasts isolated from apg9ts and ypt7Δ cells were pulse-labeled for 10 min and chased for 30 min at 38°C. The labeled spheroplasts were then osmotically lysed and separated into low-speed supernatant (S) and pellet (P) fractions after a 5,000 g centrifugation step. The pellet fractions were subjected to protease treatment in the absence or presence of 0.2% Triton X-100 as described in Materials and Methods. The resulting samples were immunoprecipitated with antiserum to API and PGK. The immunoprecipitated bands were quantified by a Molecular Dynamics STORM PhosphorImager. The percent protease-protected prAPI was determined by dividing the value for prAPI in the protease-treated sample by the value for total prAPI before protease treatment. The percent PGK was determined by dividing the supernatant (S) or pellet (P) value over the sum of the S and P values.


( b ) Volcano plots of up ( red ) and down ( blue ) regulated genes ( compared to healthy control samples ) in S . Typhi and S . Paratyphi A positive individuals ( Nepal and Oxford ) . Black numbers indicate the up - and down - regulated genes compared to healthy controls .

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(b) Volcano plots of up (red) and down (blue) regulated genes (compared to healthy control samples) in S. Typhi and S. Paratyphi A positive individuals (Nepal and Oxford). Black numbers indicate the up- and down-regulated genes compared to healthy controls.


( d - e ) Scatter plots of BTMs enriched ( p > 0 . 05 ) in blood - culture positive samples in Nepal ( y - axis ) versus Oxford ( x - axis ) for typhoid fever ( d ) and paratyphoid fever ( e ) . For further details on BTMs refer to reference ( Chaussabel et al . , 2014 ) .

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(d-e) Scatter plots of BTMs enriched (p>0.05) in blood-culture positive samples in Nepal (y-axis) versus Oxford (x-axis) for typhoid fever (d) and paratyphoid fever (e). For further details on BTMs refer to reference (Chaussabel et al., 2014).


( a ) Single - sample GSEA Normalised Enrichment Scores ( NES ) of IFN and DC BTMs of individuals with blood - culture confirmed enteric fever in Nepal and Oxford .

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(a) Single-sample GSEA Normalised Enrichment Scores (NES) of IFN and DC BTMs of individuals with blood-culture confirmed enteric fever in Nepal and Oxford.


( b ) Heatmap of the 500 most variably expressed genes in samples of the Nepali cohort . Bar graph on top of the heatmap shows temperature of each individual at the time of sampling . Three samples labelled as ' 03NP - CONT ' are samples that grew bacterial contaminants and were thus excluded from the entire analysis .

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(b) Heatmap of the 500 most variably expressed genes in samples of the Nepali cohort. Bar graph on top of the heatmap shows temperature of each individual at the time of sampling. Three samples labelled as '03NP-CONT' are samples that grew bacterial contaminants and were thus excluded from the entire analysis.


( d ) Combined expression score for samples based on the 5 - gene signature for samples in the discovery cohort ( top ) and validation cohort ( bottom ) . Ox . CTRL : Oxford controls ( D0 ) ; CTRL : Nepali control samples . PTB : pulmonary TB ; DENV : Dengue samples ; bsPf : blood - stage P . falciparum ; SPT : S . Paratyphi A ; ST : S . Typhi . ST and SPT samples are derived from the challenge models as well as from Nepal .

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(d) Combined expression score for samples based on the 5-gene signature for samples in the discovery cohort (top) and validation cohort (bottom). Ox.CTRL: Oxford controls (D0); CTRL: Nepali control samples. PTB: pulmonary TB; DENV: Dengue samples; bsPf: blood-stage P. falciparum; SPT: S. Paratyphi A; ST: S. Typhi. ST and SPT samples are derived from the challenge models as well as from Nepal.


( b ) Dot plot of prediction probability of being class EF versus the expression score calculated on the bases of the 5 - gene signature ( based on gene array data ) .

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(b) Dot plot of prediction probability of being class EF versus the expression score calculated on the bases of the 5-gene signature (based on gene array data).


( c ) qPCR gene expression scores of the 5 - gene signature ( ∆ ∆ CT over PPIA ) for CTRLs , 03NP - sEF , 03NP - SPT and 03NP - ST samples from Nepal . Yellow diamonds in the 03NP - sEF category represent the 9 patients classified as EF based on the random forest algorithm .

(c) qPCR gene expression scores of the 5-gene signature (∆∆CT over PPIA) for CTRLs, 03NP-sEF, 03NP-SPT and 03NP-ST samples from Nepal. Yellow diamonds in the 03NP-sEF category represent the 9 patients classified as EF based on the random forest algorithm.


( d ) qPCR expression values ( ∆ ∆ Ct over PPIA ) of the 5 - gene signature in control samples ( Oxford and Nepal ) , S . Paratyphi A ( 03NP - SPT ) or S . Typhi ( 03NP - ST ) in Nepal , samples at day 7 after challenge of participants who stayed well following challenge with S . Typhi ( nD7 ) , or typhoid diagnosis after challenge ( TD ) in the Vi - TCV

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(d) qPCR expression values (∆∆Ct over PPIA) of the 5-gene signature in control samples (Oxford and Nepal), S. Paratyphi A (03NP-SPT) or S. Typhi (03NP-ST) in Nepal, samples at day 7 after challenge of participants who stayed well following challenge with S. Typhi (nD7), or typhoid diagnosis after challenge (TD) in the Vi-TCV study


( e ) Combined qPCR expression score of the 5 - gene signature . Black arrows indicate outlier samples .

(e) Combined qPCR expression score of the 5-gene signature. Black arrows indicate outlier samples.


( f ) Temperature and CRP for samples of which data was available ( CRP was only measured in the Oxford CHIM ) . D0 = pre - challenge baseline Vi - TCV study ; nD7 = day 7 samples of participants who stayed well following challenge ( Vi - TCV study ) ; SPT = S . Paratyphi A ( 03NP ) ; ST = S . Typhi ( 03NP ) ; TD = Typhoid diagnosis ( Vi - TCV study ) . ( g ) Spearman ' s rank correlation of the 5 - gene combined expression score and ( left ) temperature ( only nD7 and TD samples from the Oxford CHIM - Vi - TCV and SPT and ST cases from Nepal at presentation to hospital were included ) and ( right ) CRP ( CRP was only available for Oxford CHIM - Vi - TCV samples and we excluded D0 baseline measures ) .

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(f) Temperature and CRP for samples of which data was available (CRP was only measured in the Oxford CHIM). D0 = pre-challenge baseline Vi-TCV study; nD7 = day 7 samples of participants who stayed well following challenge (Vi-TCV study); SPT = S. Paratyphi A (03NP); ST = S. Typhi (03NP); TD = Typhoid diagnosis (Vi-TCV study). (g) Spearman's rank correlation of the 5-gene combined expression score and (left) temperature (only nD7 and TD samples from the Oxford CHIM - Vi-TCV and SPT and ST cases from Nepal at presentation to hospital were included) and (right) CRP (CRP was only available for Oxford CHIM - Vi-TCV samples and we excluded D0 baseline measures).


A . NLK but not kinase - negative NLK ( Flag - NLK - KM ; K155M ) caused mobility shift of EGFP - YAP . EGFP - YAP was transfected with empty vector , Flag - NLK - WT , or Flag - NLK - KM into HEK293T cells . Mobility shift was shown by immunoblotting .

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A. NLK but not kinase-negative NLK (Flag-NLK-KM; K155M) caused mobility shift of EGFP-YAP. EGFP-YAP was transfected with empty vector, Flag-NLK-WT, or Flag-NLK-KM into HEK293T cells. Mobility shift was shown by immunoblotting.


B . NLK phosphorylates YAP in vitro . Flag - NLK - WT , or Flag - NLK - KM was immunoprecipitated with anti - Flag antibody from cell lysates of HEK293T cells transfected with each plasmid and subjected to in vitro kinase assay with bacterially purified GST - YAP in the presence of [ 32P - ATP ] . Phosphorylation of GST - YAP and autophosphorylation of NLK were examined by autoradiography ( top panel ) , and Coomassie Blue staining showed similar levels of GST - YAP and Flag - NLK ( bottom panel ) .

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B. NLK phosphorylates YAP in vitro. Flag-NLK-WT, or Flag-NLK-KM was immunoprecipitated with anti-Flag antibody from cell lysates of HEK293T cells transfected with each plasmid and subjected to in vitro kinase assay with bacterially purified GST-YAP in the presence of [32P-ATP]. Phosphorylation of GST-YAP and autophosphorylation of NLK were examined by autoradiography (top panel), and Coomassie Blue staining showed similar levels of GST-YAP and Flag-NLK (bottom panel).


C . NLK interacts with YAP at the endogenous level . Immunoprecipitates from HEK293T cell lysates with indicated antibody were blotted with anti - YAP or anti - NLK antibody .

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C. NLK interacts with YAP at the endogenous level. Immunoprecipitates from HEK293T cell lysates with indicated antibody were blotted with anti-YAP or anti-NLK antibody.


B . NLK - WT but not NLK - KM reduces Ser127 phosphorylation of endogenous YAP . Cell lysates from HEK293 cells transfected with empty vector ( - ) , Flag - NLK - WT , or Flag - NLK - KM were immunoblotted with antibodies shown in the figure ( left panel ) . The ratio of p - YAP ( S127 ) or p - YAP ( S397 ) / total - YAP of three independent western blots was quantified ( right panel ) . Data are presented as mean ± sem . * * P < 0 . 01 . Student t - test was used for statistical analysis .

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B. NLK-WT but not NLK-KM reduces Ser127 phosphorylation of endogenous YAP. Cell lysates from HEK293 cells transfected with empty vector (-), Flag-NLK-WT, or Flag-NLK-KM were immunoblotted with antibodies shown in the figure (left panel). The ratio of p-YAP(S127) or p-YAP(S397)/total-YAP of three independent western blots was quantified (right panel). Data are presented as mean ± sem. **P < 0.01. Student t-test was used for statistical analysis.


C . Deficiency of NLK leads to increased YAP phosphorylation at Ser127 . Lysates from wild type and two independent NLK knockout HEK293 cells were immunoblotted with antibodies indicated in the figure ( left panel ) . The ratio of p - YAP ( S127 ) or p - YAP ( S397 ) / total - YAP of three independent western blots was quantified ( right panel ) . Data are presented as mean ± sem . * P < 0 . 05 and * * P < 0 . 01 . Student t - test was used for statistical analysis .

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C. Deficiency of NLK leads to increased YAP phosphorylation at Ser127. Lysates from wild type and two independent NLK knockout HEK293 cells were immunoblotted with antibodies indicated in the figure (left panel). The ratio of p-YAP(S127) or p-YAP(S397)/total-YAP of three independent western blots was quantified (right panel). Data are presented as mean ± sem. *P < 0.05 and **P < 0.01. Student t-test was used for statistical analysis.


D . Ectopic expression of NLK attenuates YAP phosphorylation at Ser127 while increases phosphorylation at Ser128 . Cell lysates from HEK293 cells transfected with empty vector ( - ) , Flag - NLK - WT , or Flag - NLK - KM were immunoblotted with antibodies shown in the figure .

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D. Ectopic expression of NLK attenuates YAP phosphorylation at Ser127 while increases phosphorylation at Ser128. Cell lysates from HEK293 cells transfected with empty vector (-), Flag-NLK-WT, or Flag-NLK-KM were immunoblotted with antibodies shown in the figure.


E . Substitution of Ser127 with alanine enhances phosphorylation at Ser128 , and vice versa . Cell lysates from HEK293T cells transfected with empty vector ( - ) , EGFP - YAP , EGFP - YAP - S127A , or EGFP - YAP - S128A were immunoblotted with antibodies shown in the figure .

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E. Substitution of Ser127 with alanine enhances phosphorylation at Ser128, and vice versa. Cell lysates from HEK293T cells transfected with empty vector (-), EGFP-YAP, EGFP-YAP-S127A, or EGFP-YAP-S128A were immunoblotted with antibodies shown in the figure.