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

O O O O O B-GENEPROD B-EXP_ASSAY O B-EXP_ASSAY O B-CELL_LINE B-CELL_TYPE O O O O O O O O O B-SUBCELLULAR O B-EXP_ASSAY O O O B-GENEPROD O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O B-SUBCELLULAR O B-CELL_LINE B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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 of H9C2 rat cardiomyoblasts expressing AcGFP - 53BP1 from 3 days after 10Gy irradiation at indicated times ( mins ) . are maximum 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 3 days after 10Gy irradiation at 10 minute intervals for 24 hours . > 500 foci from 10 were tracked per condition .

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


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

O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O 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-SUBCELLULAR O B-GENEPROD I-GENEPROD I-GENEPROD O B-CELL_TYPE O O O O O O O O O B-ORGANISM O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-CELL_TYPE O O O O O

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

O O B-ORGANISM B-CELL_TYPE I-CELL_TYPE O O O O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O B-EXP_ASSAY O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-SUBCELLULAR B-GENEPROD I-GENEPROD I-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY B-SUBCELLULAR O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O

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 - TRF1 - FokI - expressing CMs . Data are mean ± of n = 4 independent experiments . > 50 were analysed per condition . Statistical was performed by two - t - * * * P < 0 . 001 .

O O B-GENEPROD I-GENEPROD I-GENEPROD B-SUBCELLULAR B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-SUBCELLULAR O O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O B-CELL_TYPE O O O O O O O O O O O O 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 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 - TRF1 - FokI - expressing CMs . Data are mean ± of n = 4 independent experiments . > 50 were analysed per condition . Statistical was performed by two - t - * * * P < 0 . 001 .

O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O B-SUBCELLULAR O O O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O B-CELL_TYPE O O O O O O O O O O O O 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 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 for telomeres co - localising or not co - localising with γH2AX foci . Red dotted lines represent median . Mann - Whitney show no significant difference in telomere between TAF non - TAF .

O O B-SUBCELLULAR O B-SUBCELLULAR B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD B-SUBCELLULAR O O O O O O O O O O O O O O O B-SUBCELLULAR O B-SUBCELLULAR O O B-SUBCELLULAR 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 ± of n = 3 independent experiments . > 100 were quantified per condition . Statistical performed using two t ; * * P < 0 . 01 .

O O O B-GENEPROD O O B-CELL_TYPE O O O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O O O O O O O O O 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 Gapdh ) by real - time PCR in TRF1 - FokI - D450A TRF1 - FokI expressing CMs . Data are mean ± of n = 6 independent experiments . Statistical performed using two t ; * * * P < 0 . 001 .

O O B-GENEPROD O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 ± surface ( μm2 ) of FLAG - labelled CMs expressing TRF1 - FokI - D450A TRF1 - FokI . Statistical performed using two t ; * * * P < 0 . 001 . Scale bar represents 50μm .

O O B-EXP_ASSAY O O O O B-GENEPROD O O B-CELL_TYPE O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD 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 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 , 30 month old C57BL / 6 mice by immunohistochemistry ( ) . Data are mean ± of n = 4 per group . > 100 CMs were quantified per group .

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


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

O O O O O O O B-ORGANISM B-CELL_TYPE O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD I-GENEPROD O O O B-GENEPROD O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O O B-GENEPROD I-GENEPROD B-EXP_ASSAY B-CELL_TYPE 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-CELL_TYPE O B-ORGANISM O O B-ORGANISM O O O O O O O

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 30 month old mouse CMs positive for SADS in vivo , as detected by centromere - FISH . Data are mean ± of n = 4 per group . > 200 CMs per mouse were quantified .

O O O O O O B-CELL_TYPE B-SUBCELLULAR O O O O O B-ORGANISM B-CELL_TYPE O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O B-CELL_TYPE O B-ORGANISM 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 : Pearson correlation clustered showing a curated list of known SASP genes ( top panel ) or a selection of secreted SASP ( bottom panel ) in young ( 3 months ) old ( 20 months ) mouse CMs ( n = 5 per group ) . colour represents column Z - score , where red indicates highly blue lowly expressed .

O O O O O O O O O O O O O O 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-ORGANISM B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O

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

O O O B-EXP_ASSAY I-EXP_ASSAY O O B-CELL_TYPE O O B-ORGANISM O O O O O O B-EXP_ASSAY O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O B-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O B-CELL_TYPE O O O O O O O O O O O O O O B-ORGANISM O O O

(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 of immunofluorescent against α - SMA EdU in neonatal fibroblasts cultured in presence of CM from young old CMs .

O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD B-SMALL_MOLECULE O B-CELL_TYPE I-CELL_TYPE O O O O B-CELL_TYPE O O O B-CELL_TYPE O

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


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

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-CELL_TYPE I-CELL_TYPE O O O O O B-CELL_TYPE O O O B-CELL_TYPE O O O O O O O O O O O O O O O O B-ORGANISM 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 ( E ) in neonatal fibroblasts after treatment for 48h with CM from young old CM . Data are mean ± S . E . M of n = 3 - 4 mice per group ; > 200 were quantified per condition .

O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O B-CELL_TYPE O O O B-CELL_TYPE O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O

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

O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD B-SMALL_MOLECULE O B-CELL_TYPE O B-GENEPROD I-GENEPROD I-GENEPROD O B-CELL_TYPE O O

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).


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

O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-CELL_TYPE I-CELL_TYPE 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 indicated recombinant . Data are mean ± S . E . M of n = 3 - 4 independent experiments .

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


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

B-EXP_ASSAY O O O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O

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

B-SUBCELLULAR O O O B-EXP_ASSAY O O O O O O O B-SUBCELLULAR O O O O O O O B-ORGANISM B-CELL_TYPE O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY O

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 of MAO - A , MnSOD catalase in isolated mouse CMs from young ( 3 months ) old ( 20 months ) mice .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD O O B-ORGANISM B-CELL_TYPE O O O O O O O O O O O B-ORGANISM O

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 ± of n = 4 per group . 100 CMs were quantified per group .

O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O B-CELL_TYPE O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O B-CELL_TYPE O O O O O

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 of 4 to 24 Data are mean ± S . E . M of n = 3 - 4 per group . > 100 CMs were quantified per group .

O O O B-SUBCELLULAR O O B-SUBCELLULAR O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-ORGANISM O O O O O O O O O O O O O O B-SMALL_MOLECULE O 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-CELL_TYPE O O O O O

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 group .

O O O B-SUBCELLULAR O O B-SUBCELLULAR O O O O O O O O B-SUBCELLULAR O O O O O B-GENEPROD O O O O B-GENEPROD 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-CELL_TYPE O O O O O

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


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

O O O B-SUBCELLULAR O O B-SUBCELLULAR O O O O O O O O B-SUBCELLULAR O O O O O B-GENEPROD O O O O O B-GENEPROD 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-CELL_TYPE O O O O O

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


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 group .

O O O B-SUBCELLULAR O O B-SUBCELLULAR O O O O O O O O B-SUBCELLULAR O O O O O O O O B-GENEPROD O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O B-CELL_TYPE O O O O O

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 were treated with or without 100nM rotenone either in 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 ) mean % of TAF - positive nuclei ( bottom graph ) . Data are mean ± from 4 separate CM cultures isolated from 3 month - old mice . 50 CMs were quantified per condition .

O O O B-ORGANISM O B-CELL_TYPE O O O O O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O B-SUBCELLULAR O O B-SUBCELLULAR O O O O O O O O O O O O B-CELL_TYPE O O O O O O O B-ORGANISM O O B-CELL_TYPE O O O O O

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 short axis cine - MR of 3 or 20 month old mouse hearts . Ejection fraction LV thickness was calculated on manual measurements of Left ventricle epicardial endocardial borders . % change in wall thickening calculation on wall thickness at 4 points indicated . Measurements were made in cine slices at end diastole end systole . Graphs representing data obtained from MRI of > 7 per group . Data are mean ± S . E . M . Asterisks denote a statistical significance at P < 0 . 05 using Mann Whitney U - . 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 CM in 22 month old . 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 % of p16 - or eGFP - positive CMs by - in situ hybridization per plane in INK - ATTAC mice ( 28 - 29 month old ) treated with vehicle or AP20187 . Data are mean ± of n = 5 per 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 ) mean % of TAF - positive nuclei ( right graph ) in CMs . Data are mean ± S . E . M . of n = 6 per 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 μm2 . Data are mean ± S . E . M . of n = 6 per 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.


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% of fibrotic area evaluated by Sirius Red staining.


of mean number of TAF % of TAF - positive CMs in 24 months old wild - type mice treated with vehicle or navitoclax ( 50mg / kg / day ) . Data are mean ± of n = 5 - 8 mice per group . More than 100 CMs were quantified per .

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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 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 of Sirius red ( right ) % of fibrosis 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 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 .

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% 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 of CMs positive for CM marker troponin C ( TropC ) , EdU , Ki67 Aurora B from navitoclax treated . ( Upper Right panel ) arrows identify two nuclei in same CM that have incorporated EdU . ( Lower Right panel ) 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.


of EdU positive CMs ( mono - or multi - nucleated ) in vehicle navitoclax treated . 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 characterization of APG9 . A , Wild - type ( WT ; SEY6210 ) , apg9 ( THY154 ) , apg9Δ ( JKY007 ) , apg9Δ transformed with single copy ( CEN ; pAPG9 ( 414 ) ) or multicopy ( 2μ ; pAPG9 ( 424 ) ) plasmids encoding APG9 were grown to log phase in SMD . extracts were prepared analyzed by immunoblot using antiserum to API as described in Materials Methods . positions of precursor mature API are indicated . APG9 gene complements prAPI accumulation phenotype of 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 ) , apg9Δ transformed with pAPG9 ( 414 ) centromeric plasmid were grown in SMD transferred to SD ( − N ) as described in Materials Methods . Aliquots were removed at indicated times spread onto YPD plates in triplicate . Numbers of viable colonies were determined after two to three days . APG9 gene complements starvation - sensitive phenotype of 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 . A , Wild - type ( WT ; SEY6210 ) , apg9Δ ( JKY007 ) , apg9Δ transformed with single copy ( CEN ) or multicopy ( 2μ ) plasmids encoding APG9 were grown to log phase in SMD . extracts were prepared analyzed by immunoblot using antiserum to Apg9p . Apg9p is detected as an ∼ 125 - kD .

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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 of Apg9p on Kyte - Doolittle algorithm ( Kyte 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 , of wild - type YW5 - 1B were grown in YPD , converted to spheroplasts , osmotically lysed as described in Materials Methods . After a preclearing step to remove debris , 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 lysate into supernatant ( S ) pellet ( P ) fractions , which were analyzed by immunoblots with antiserum to Apg9p . Apg9p is found in low - speed pellet in absence of Triton X - 100 . Detergent extraction completely solubilizes Apg9p causes it to appear in 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 , CTD1 harboring a multicopy 3 × HA APG9 plasmid was grown in YPD converted to spheroplasts as described in Materials Methods . A crude lysate was prepared treated with or without endo H as indicated overnight at 37 ° C analyzed by immunoblots with anti - HA antibodies or anti - CPY serum . Endo H treatment removes N - linked glycosyl residues of CPY , causing a shift in its electrophoretic mobility . In contrast , Apg9p is not glycosylated remains unchanged in 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 B , STY1 ( pep4Δ ) was grown in YPD to log - phase , osmotically lysed . Lysates were precleared , loaded on a sucrose density gradient ranging from 18 - 55 % ( wt / wt ) , centrifuged for 2 . 5 h at 174 , 000 g as described in Materials Methods . Fractions were collected from top ( fraction number 1 ) to bottom ( fraction number 16 ) . Fractions were subjected to SDS - PAGE immunoblot with antiserum against Apg9p : in A , Kex2p ( trans Golgi network ) , Pho8p ( vacuole ) , Pma1p ( plasma ) ; in B , Sec12p ( ER ) Pep12p ( endosome ) . graphs in panels A B are from 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 that accumulates Cvt / autophagic vesicles . TK415 ( ypt7Δ ) was grown in YPD to log - phase , osmotically lysed . Lysates were precleared , centrifuged at 100 , 000 g for 1 h , pellet fraction resuspended centrifuged a second time . resulting pellet fraction was subsequently loaded on a sucrose density gradient ranging from 18 - 55 % ( wt / wt ) , centrifuged for 16 h at 174 , 000 g as described in Materials Methods . 16 fractions were collected starting from top subjected to SDS - PAGE immunoblot with antisera against Apg9p API . 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 ) , SKD6 - 1D ( apg6Δ ) transformed with multicopy plasmid 3 × HA APG9 . were grown in YPD to log phase ( vegetative ) , incubated further in SD ( − N ) medium with 1 mM PMSF for 3 h ( starvation ) . were fixed with formaldehyde examined by immunofluorescence microscopy as described in Materials Methods . Anti - HA FITC - conjugated antibodies mark Apg9p ( left ) , vacuole can be visualized by Nomarski optics ( middle ) . An overlay is shown in 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 Apg9pGFP fusion . Wild - type ( SEY6210 ) expressing Apg9pGFP from a multicopy plasmid were grown in SMD to midlog phase labeled with FM 4 - 64 to stain vacuoles as described in Materials Methods . labeled were viewed directly with a Leica DM IRB confocal microscope . Large , punctate Apg9pGFP structures appear juxtaposed to FM 4 - 64 - labeled vacuole membranes . In addition , smaller punctate structures appear throughout 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 . STY1 ( pep4Δ ) expressing 3 × HA Apg9p was grown in YPD to log phase transferred to SD ( − N ) medium for 2 h . were fixed stained with anti - HA antibody 5 - nm colloidal gold - conjugated goat anti - mouse IgG as described in Materials Methods . A , Section showing autophagic bodies in vacuole lumen . B , Section showing a autophagosome . Apg9p is seen in patches near vacuole , but not on autophagic bodies or autophagosomes . AB , autophagic ; AP , autophagosome ; N , nucleus ; V , vacuole ; VM , vacuolar . 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 kinetic of prAPI import in conditional Apg9p mutant . A , Morphological of apg9Δ indicates an import defect at an early step in pathway . Wild - type ( YW5 - 1B ) apg9Δ ( CTD1 ) were grown in YPD ( vegetative ) or transferred to SD ( − N ) ( starvation ) for 3 h in presence of PMSF ( starvation ) examined by DIC ( Nomarski ) microscopy ( Zeiss Axioplan ) . PMSF inhibits degradation of autophagic bodies . Under starvation conditions , wild - type accumulate autophagic bodies when vesicle breakdown is blocked . However , under same conditions , apg9Δ do not accumulate autophagic bodies , indicating that Apg9p is required for a step before vesicle fusion release of autophagic into vacuolar lumen . apg9Δ transformed with APG9 plasmid shows same result as wild - type ( data not shown ) . B , apg9ts is tightly blocked for prAPI import at nonpermissive . Wild - type ( SEY6210 ) apg9Δ ( JKY007 ) transformed with apg9ts centromeric plasmid were incubated at 24 38 ° C for 5 min , - labeled for 10 min , then subjected to nonradioactive chase reactions for indicated times . Samples at each time point were immunoprecipitated with antiserum to API resolved by SDS - PAGE as described in Materials Methods . API - immunoprecipitated bands were quantified by a Molecular Dynamics STORM PhosphorImager results are presented in graph . mature API was determined by dividing mature API value over sum of precursor mature API values for each time point .

O O O O B-GENEPROD O O O B-GENEPROD O O O O B-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O B-GENEPROD O B-ORGANISM O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE O O O O O B-EXP_ASSAY O B-EXP_ASSAY O O O O O O O B-SMALL_MOLECULE O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O B-GENEPROD O O O B-SUBCELLULAR I-SUBCELLULAR O O O B-GENEPROD O O O O O O O O O O B-SUBCELLULAR O B-SUBCELLULAR I-SUBCELLULAR O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O B-ORGANISM O B-GENEPROD O B-ORGANISM O O O B-GENEPROD O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O B-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O B-GENEPROD O B-EXP_ASSAY O O O O O O O O O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O B-GENEPROD 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.


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

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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 ) down ( blue ) regulated genes ( compared to healthy control samples ) in S . Typhi S . Paratyphi A positive individuals ( Nepal Oxford ) . Black numbers indicate up - 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 ) 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 DC BTMs of individuals with blood - culture confirmed enteric fever in Nepal 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 ) of 500 most variably expressed genes in samples of Nepali cohort . Bar graph on top of shows of each at time of sampling . Three samples labelled as ' 03NP - CONT ' are samples that grew contaminants were thus excluded from entire .

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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 on 5 - gene signature for samples in discovery cohort ( top ) 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 SPT samples are derived from 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 expression score calculated on bases of 5 - gene signature ( 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 5 - gene signature ( ∆ ∆ CT over PPIA ) for CTRLs , 03NP - sEF , 03NP - SPT 03NP - ST samples from Nepal . Yellow diamonds in 03NP - sEF category represent 9 classified as EF on random forest algorithm .

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(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 5 - gene signature in control samples ( Oxford 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 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 5 - gene signature . Black arrows indicate outlier samples .

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


( f ) CRP for samples of which data was available ( CRP was only measured in 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 5 - gene combined expression score ( left ) ( only nD7 TD samples from Oxford CHIM - Vi - TCV SPT ST cases from Nepal at presentation to hospital were included ) ( right ) CRP ( CRP was only available for Oxford CHIM - Vi - TCV samples we excluded D0 baseline measures ) .

O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O 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-ORGANISM O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O B-ORGANISM O B-ORGANISM I-ORGANISM I-ORGANISM O O O O B-DISEASE O B-DISEASE O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O B-DISEASE O O O O O O O O B-ORGANISM I-ORGANISM O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O

(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 . 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 lysates of HEK293T transfected with each plasmid subjected to in vitro kinase with bacterially purified GST - YAP in presence of [ 32P - ATP ] . Phosphorylation of GST - YAP autophosphorylation of NLK were examined by autoradiography ( top panel ) , Coomassie Blue showed similar of GST - YAP 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 endogenous level . Immunoprecipitates from HEK293T 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 . lysates from HEK293 transfected with empty vector ( - ) , Flag - NLK - WT , or Flag - NLK - KM were immunoblotted with antibodies shown in figure ( left panel ) . of p - YAP ( S127 ) or p - YAP ( S397 ) / total - YAP of three independent western blots was quantified ( right panel ) . Data are presented as mean ± . * * P < 0 . 01 . Student t - was used for statistical .

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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 two independent NLK knockout HEK293 were immunoblotted with antibodies indicated in figure ( left panel ) . of p - YAP ( S127 ) or p - YAP ( S397 ) / total - YAP of three independent western blots was quantified ( right panel ) . Data are presented as mean ± . * P < 0 . 05 * * P < 0 . 01 . Student t - was used for statistical .

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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 . lysates from HEK293 transfected with empty vector ( - ) , Flag - NLK - WT , or Flag - NLK - KM were immunoblotted with antibodies shown in 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 , vice versa . lysates from HEK293T transfected with empty vector ( - ) , EGFP - YAP , EGFP - YAP - S127A , or EGFP - YAP - S128A were immunoblotted with antibodies shown in 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.


A . Ectopic expression of NLK attenuates interaction between YAP 14 - 3 - 3 . Lysates from HEK293T transfected with plasmids indicated in figure were immunoprecipitated with anti - HA antibody immunoblotted with anti - GFP antibody ( top two panels ) . Whole lysates ( WCL ) were immunoblotted with antibodies shown in figure ( bottom three panels ) .

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A. Ectopic expression of NLK attenuates interaction between YAP and 14-3-3. Lysates from HEK293T cells transfected with plasmids indicated in the figure were immunoprecipitated with anti-HA antibody and immunoblotted with anti-GFP antibody (top two panels). Whole cell lysates (WCL) were immunoblotted with antibodies shown in the figure (bottom three panels).


B . Deficiency of NLK leads to increased interaction between YAP 14 - 3 - 3 . Lysates from NLK knockout HEK293 were immunoprecipitated with anti - YAP antibody immunoblotted with anti - 14 - 3 - 3 antibody ( top two panels ) . WCL were immunoblotted with antibodies indicated in figure ( bottom three panels ) .

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B. Deficiency of NLK leads to increased interaction between YAP and 14-3-3. Lysates from NLK knockout HEK293 cells were immunoprecipitated with anti-YAP antibody and immunoblotted with anti-14-3-3 antibody (top two panels). WCL were immunoblotted with antibodies indicated in the figure (bottom three panels).


C . Phosphorylation status of Ser127 or Ser128 determines interaction between 14 - 3 - 3 YAP . Lysates from HEK293T transfected with plasmids indicated in figure were immunoprecipitated with anti - Flag antibody immunoblotted with anti - HA antibodies ( top two panels ) . Lysates were immunoblotted with anti - HA or anti - Flag antibodies ( bottom two panels ) . " n . s . " arrow indicates a non - specific band also visible in non - transfected lane .

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C. Phosphorylation status of Ser127 or Ser128 determines interaction between 14-3-3 and YAP. Lysates from HEK293T cells transfected with plasmids indicated in the figure were immunoprecipitated with anti-Flag antibody and immunoblotted with anti-HA antibodies (top two panels). Lysates were immunoblotted with anti-HA or anti-Flag antibodies (bottom two panels). "n.s." arrow indicates a non-specific band also visible in the non-transfected lane.


D . Phosphorylation status of Ser127 or Ser128 determines subcellular localization of YAP . Immunofluorescence was performed in HeLa transfected with EGFP - YAP , EGFP - YAP - S127A , EGFP - YAP - S128A , or EGFP - YAP - S128D at high density . Figure shows a representative of multiple areas . Nuclei were stained with Hoechst 33342 . Scale bars : 20 μm .

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D. Phosphorylation status of Ser127 or Ser128 determines subcellular localization of YAP. Immunofluorescence analysis was performed in HeLa cells transfected with EGFP-YAP, EGFP-YAP-S127A, EGFP-YAP-S128A, or EGFP-YAP-S128D at high cell density. Figure shows a representative of multiple areas. Nuclei were stained with Hoechst 33342. Scale bars: 20 μm.


E . NLK knockout leads to increased localization of endogenous YAP . Immunoblot shows significant reduction of NLK level in NLK knockout HEK293 pools ( top panel ) .

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E. NLK knockout leads to increased cytoplasmic localization of endogenous YAP. Immunoblot analysis shows significant reduction of NLK level in NLK knockout HEK293 cell pools (top panel).


E . NLK knockout leads to increased localization of endogenous YAP . To determine subcellular localization of YAP indirect immunofluorescence was performed in NLK knockout HEK293 pools ( bottom panel ) . Figure shows a representative of multiple areas . Nuclei were stained with DAPI . Scale bars : 20 μm .

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E. NLK knockout leads to increased cytoplasmic localization of endogenous YAP. To determine subcellular localization of YAP indirect immunofluorescence analysis was performed in NLK knockout HEK293 cell pools (bottom panel). Figure shows a representative of multiple areas. Nuclei were stained with DAPI. Scale bars: 20 μm.


A . Localization of endogenous YAP NLK in low ( LD ) or high density ( HD ) MCF10A ( upper panel ) or NIH3T3 ( bottom panel ) was examined by indirect immunofluorescence . Nuclei were stained with DAPI . Scale bars : 20 μm .

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A. Localization of endogenous YAP and NLK in low (LD) or high density (HD) MCF10A (upper panel) or NIH3T3 cells (bottom panel) was examined by indirect immunofluorescence. Nuclei were stained with DAPI. Scale bars: 20 μm.


B . Level of NLK decreases in a density - dependent manner . Lysates from low ( LD ) high density ( HD ) of MCF10A , HEK293T , NIH3T3 were immunoblotted with antibodies indicated in figure .

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B. Level of NLK decreases in a cell density-dependent manner. Lysates from low (LD) and high density (HD) of MCF10A, HEK293T, and NIH3T3 cells were immunoblotted with antibodies indicated in the figure.


C . Level of endogenous NLK mRNA is unaffected , whereas expression of YAP target genes was reduced , at high density . Quantitative real - time PCR analyses for expression of AMOTL2 , CTGF , CYR61 , NLK in low ( LD ) high density ( HD ) NIH3T3 were performed . of AMOTL2 , CTGF , CYR61 , NLK mRNA was normalized with level of Gapdh . Data represent average values from a representative of multiple experiments performed in triplicate . Error bars indicate standard deviations of triplicate measurements . Data are presented as mean ± . * P < 0 . 05 * * * P < 0 . 005 . Student t - was used for statistical .

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C. Level of endogenous NLK mRNA is unaffected, whereas the expression of YAP target genes was reduced, at high cell density. Quantitative real-time PCR analyses for expression of AMOTL2, CTGF, CYR61, and NLK in low (LD) and high density (HD) NIH3T3 cells were performed. Quantification of AMOTL2, CTGF, CYR61, and NLK mRNA was normalized with the level of Gapdh. Data represent average values from a representative of multiple experiments performed in triplicate. Error bars indicate standard deviations of triplicate measurements. Data are presented as mean ± sem. *P < 0.05 and ***P < 0.005. Student t-test was used for statistical analysis.


A . Knockout of NLK reduces interaction between endogenous YAP TEAD . Lysates from NLK knockout HEK293 were immunoprecipitated with anti - TEAD1 antibody immunoblotted with anti - TEAD1 antibody or anti - YAP antibody ( top panel ) . Whole lysates ( WCL ) were immunoblotted with antibodies indicated in figure .

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A. Knockout of NLK reduces the interaction between endogenous YAP and TEAD. Lysates from NLK knockout HEK293 cells were immunoprecipitated with anti-TEAD1 antibody and immunoblotted with anti-TEAD1 antibody or anti-YAP antibody (top panel). Whole cell lysates (WCL) were immunoblotted with antibodies indicated in the figure.


B . Phosphorylation status of Ser127 or Ser128 determines interaction between TEAD1 YAP . Lysates from HEK293T transfected with plasmids indicated in figure were immunoprecipitated with anti - Flag antibody immunoblotted with indicated antibodies in figure .

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B. Phosphorylation status of Ser127 or Ser128 determines interaction between TEAD1 and YAP. Lysates from HEK293T cells transfected with plasmids indicated in the figure were immunoprecipitated with anti-Flag antibody and immunoblotted with indicated antibodies in the figure.


C . Downregulation of nemo with RNAi achieved in posterior compartment of wing imaginal discs ( co - expression of GFP serves to mark posterior compartment ) using hh - Gal4 driver results in decreased expression of Yorkie target gene DIAP1 . Two different RNAi constructs produce similar effect . Panels below show Z - stacks of magnified areas around A / P border ( left ) . of DIAP1 achieved upon posterior - downregulation of nemo , measured as normalized posterior - to - anterior compared to similar expression of GFP . Data are present as mean ± , n = 8 discs . * P < 0 . 05 . Student t - was used for statistical ( right ) . Scale bars : 50 µm .

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C. Downregulation of nemo with RNAi achieved in the posterior compartment of wing imaginal discs (co-expression of GFP serves to mark the posterior compartment) using the hh-Gal4 driver results in decreased expression of the Yorkie target gene DIAP1. Two different RNAi constructs produce similar effect. Panels below show Z-stacks of magnified areas around the A/P border (left). Quantification of DIAP1 levels achieved upon posterior-downregulation of nemo, measured as normalized posterior-to-anterior levels and compared to similar expression of GFP. Data are present as mean ± sem, n=8 discs. *P < 0.05. Student t-test was used for statistical analysis (right). Scale bars: 50 µm.


D . Yorkie is epistatic to Nemo : co - overexpression of constitutively active mutant yki [ S168A ] together with nmo - RNAi produces typical yki [ S168A ] : severe overgrowth of affected with concomitant increase in yki target gene expression ( DIAP1 ) . Due to lethality of hh - Gal4 - driven co - overexpression , another posterior driver en - Gal4 was used instead ; anti - Hh ( green ) was used to mark posterior compartment ( left panel ) . posterior DIAP1 in UAS - RNAi - nemo ; en - Gal4 UAS - yki [ S168A ] ; en - Gal4 , presented as in ( C ) ; n = 3 - 6 discs . Posterior DIAP1 in UAS - RNAi - nemo ; en - Gal4 are also significantly different from control UAS - GFP ; en - Gal4 discs ( right panel ) . Data are presented as mean ± . * * * P < 0 . 005 . Student t - was used for statistical ( right ) . Scale bars : 50 µm .

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D. Yorkie is epistatic to Nemo: co-overexpression of the constitutively active mutant yki[S168A] together with nmo-RNAi produces typical yki[S168A] phenotypes: severe overgrowth of the affected area with concomitant increase in yki target gene expression (DIAP1). Due to lethality of the hh-Gal4-driven co-overexpression, another posterior driver en-Gal4 was used instead; anti-Hh staining (green) was used to mark the posterior compartment (left panel). Quantification posterior DIAP1 levels in UAS-RNAi-nemo; en-Gal4 and UAS-yki[S168A]; en-Gal4, presented as in (C); n= 3-6 discs. Posterior DIAP1 levels in UAS-RNAi-nemo; en-Gal4 are also significantly different from control UAS-GFP; en-Gal4 discs (right panel). Data are presented as mean ± sem. ***P < 0.005. Student t-test was used for statistical analysis (right). Scale bars: 50 µm.


E . Mutant forms of YAP which have different phosphorylation status of Ser127 or S128 differentially rescue retarded wound healing caused by NLK knockout . HEK293 wild type or HEK293 NLK knockout were transfected with indicated plasmids ( " EV " stands for " empty vector " ) , wounded areas after 0 24 hr were measured by using TScratch program ( left panel ) . Graphs show of unfilled areas at 24 hr compared to 0 hr , average values from a representative of multiple experiments performed in triplicate ( right - top panel ) . Lysates were immunoblotted with antibodies indicated in figure ( right - bottom panel ) . Error bars indicate standard deviations of triplicate measurements . Data are presented as mean ± . * P < 0 . 05 . Student t - was used for statistical ( right ) . Scale bars : 200 μm .

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E. Mutant forms of YAP which have different phosphorylation status of Ser127 or S128 differentially rescue retarded wound healing caused by NLK knockout. HEK293 wild type or HEK293 NLK knockout cells were transfected with indicated plasmids ("EV" stands for "empty vector"), and the wounded areas after 0 and 24 hr were measured by using the TScratch program (left panel). Graphs show percentage of unfilled areas at 24 hr compared to 0 hr, and average values from a representative of multiple experiments performed in triplicate (right-top panel). Lysates were immunoblotted with antibodies indicated in the figure (right-bottom panel). Error bars indicate standard deviations of triplicate measurements. Data are presented as mean ± sem. *P < 0.05. Student t-test was used for statistical analysis (right). Scale bars: 200 μm.


E . Mutant forms of YAP which have different phosphorylation status of Ser127 or S128 differentially rescue retarded wound healing caused by NLK knockout . HEK293 wild type or HEK293 NLK knockout were transfected with indicated plasmids ( " EV " stands for " empty vector " ) , wounded areas after 0 24 hr were measured by using TScratch program ( left panel ) . Graphs show of unfilled areas at 24 hr compared to 0 hr , average values from a representative of multiple experiments performed in triplicate ( right - top panel ) .

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E. Mutant forms of YAP which have different phosphorylation status of Ser127 or S128 differentially rescue retarded wound healing caused by NLK knockout. HEK293 wild type or HEK293 NLK knockout cells were transfected with indicated plasmids ("EV" stands for "empty vector"), and the wounded areas after 0 and 24 hr were measured by using the TScratch program (left panel). Graphs show percentage of unfilled areas at 24 hr compared to 0 hr, and average values from a representative of multiple experiments performed in triplicate (right-top panel).


Immunoblot of ADAR1 p110 , ADAR1 p150 ADAR2 expression in various organs isolated from wild - type ( WT ) mice . expression of GAPDH is shown as a reference .

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Immunoblot analysis of ADAR1 p110, ADAR1 p150 and ADAR2 expression in various organs isolated from wild-type (WT) mice. The expression of GAPDH is shown as a reference.


relative expression of ADAR1 p150 mRNA ( B ) total ADAR1 mRNA ( C ) in sorted DN , DP , 4SP 8SP thymocytes isolated from WT mice . relative expression of ADAR1 p150 mRNA ( B ) total ADAR1 mRNA ( C ) were normalized to level of expression in DN thymocytes ( DN values set to 1 ) . Values represent relative gene expression normalized to GAPDH mRNA are displayed as mean ± ( n = 5 ; Mann - Whitney U - , * p < 0 . 05 , * * p < 0 . 01 ) .

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The relative expression of ADAR1 p150 mRNA (B) and total ADAR1 mRNA (C) in sorted DN, DP, 4SP and 8SP thymocytes isolated from WT mice. The relative expression of ADAR1 p150 mRNA (B) and total ADAR1 mRNA (C) were normalized to the level of expression in DN thymocytes (DN values set to 1). Values represent the relative gene expression normalized to GAPDH mRNA and are displayed as the mean ± SEM (n = 5; Mann-Whitney U-test, *p < 0.05, **p < 0.01).


relative expression of type I interferon - stimulated genes ( ISGs ) in sorted DN , DP , 4SP 8SP thymocytes isolated from WT mice . Values represent relative gene expression normalized to GAPDH mRNA are displayed as mean ± ( n = 3 ; Mann - Whitney U - , * p < 0 . 05 ) . relative expression of ISGs was normalized against level of expression in DN thymocytes ( DN values set to 1 ) .

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The relative expression of type I interferon-stimulated genes (ISGs) in sorted DN, DP, 4SP and 8SP thymocytes isolated from WT mice. Values represent the relative gene expression normalized to GAPDH mRNA and are displayed as the mean ± SEM (n = 3; Mann-Whitney U-test, *p < 0.05). The relative expression of ISGs was normalized against the level of expression in DN thymocytes (DN values set to 1).


Total number of thymocytes in wild - type ( WT ) CD4 + - specific Adar1 knockout ( Cd4cre A1fl / fl ) mice .

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Total number of thymocytes in wild-type (WT) and CD4+ cell-specific Adar1 knockout (Cd4cre A1fl/fl) mice.


Thymocytes isolated from WT Cd4cre A1fl / fl mice were stained with anti - CD4 anti - CD8 antibodies analyzed by flow cytometry . Representative are shown ( B ) .

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Thymocytes isolated from WT and Cd4cre A1fl/fl mice were stained with anti-CD4 and anti-CD8 antibodies and analyzed by flow cytometry. Representative images are shown (B).


percentages of DN , DP , 4SP , 8SP thymocytes were compared between WT Cd4cre A1fl / fl mice ( C ) .

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The percentages of DN, DP, 4SP, and 8SP thymocytes were compared between WT and Cd4cre A1fl/fl mice (C).


Thymocytes isolated from WT Cd4cre A1fl / fl mice were stained with anti - CD4 , anti - CD8 anti - TCRβ antibodies were gated by high expression of TCRβ ( TCRβhigh ) . Representative are shown ( D ) .

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Thymocytes isolated from WT and Cd4cre A1fl/fl mice were stained with anti-CD4, anti-CD8 and anti-TCRβ antibodies and were gated by the high expression of TCRβ (TCRβhigh). Representative images are shown (D).


percentages of DN , DP , 4SP , 8SP thymocytes were compared between WT Cd4cre A1fl / fl mice ( E ) .

The percentages of DN, DP, 4SP, and 8SP thymocytes were compared between WT and Cd4cre A1fl/fl mice (E).


Thymocytes isolated from WT Cd4cre A1fl / fl mice were stained with anti - CD4 , anti - CD8 , anti - CD69 anti - MHC - I antibodies were gated on 4SP thymocytes . Representative are shown ( upper panel ) . percentages of CD69 + MHC - I - ( SM ) , CD69 + MHC - I + ( M1 ) , CD69 - MHC - I - ( M2 ) thymocytes were compared between WT Cd4cre A1fl / fl mice ( lower panel ) .

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Thymocytes isolated from WT and Cd4cre A1fl/fl mice were stained with anti-CD4, anti-CD8, anti-CD69 and anti-MHC-I antibodies and were gated on 4SP thymocytes. Representative images are shown (upper panel). The percentages of CD69+MHC-I- (SM), CD69+MHC-I+ (M1), and CD69-MHC-I- (M2) thymocytes were compared between WT and Cd4cre A1fl/fl mice (lower panel).


Thymocytes isolated from WT Cd4cre A1fl / fl mice were stained with antibodies against CD5 analyzed by flow cytometry . Representative flow cytometric for DP 4SP thymocytes are shown ( upper panel ) . percentages of CD5 - DP 4SP thymocytes were compared between WT Cd4cre A1fl / fl mice ( lower panel ) .

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Thymocytes isolated from WT and Cd4cre A1fl/fl mice were stained with antibodies against CD5 and analyzed by flow cytometry. Representative flow cytometric images for DP and 4SP thymocytes are shown (upper panel). The percentages of CD5- DP and 4SP thymocytes were compared between WT and Cd4cre A1fl/fl mice (lower panel).


Thymocytes isolated from WT Cd4cre A1fl / fl mice were stained with anti - CD4 , anti - CD8 , anti - CD25 anti - Foxp3 antibodies were gated on 4SP thymocytes . Representative are shown ( upper panel ) . percentages of CD25 + Foxp3 + 4SP thymocytes ( thymic Treg ) were compared between WT Cd4cre A1fl / fl mice ( lower panel ) .

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Thymocytes isolated from WT and Cd4cre A1fl/fl mice were stained with anti-CD4, anti-CD8, anti-CD25 and anti-Foxp3 antibodies and were gated on 4SP thymocytes. Representative images are shown (upper panel). The percentages of CD25+Foxp3+ 4SP thymocytes (thymic Treg cells) were compared between WT and Cd4cre A1fl/fl mice (lower panel).


CD4 + - specific Adar1 knockout ( Cd4cre A1fl / fl ) mice were crossed with HY - TCR + mice to generate Cd4cre A1fl / fl HY - TCR + mice . Thymocytes isolated from ( A ) ( B ) HY - TCR + or Cd4cre A1fl / fl HY - TCR + mice were stained with anti - CD4 anti - CD8 antibodies analyzed by flow cytometry . Representative are shown ( left panels ) . absolute number of DP 8SP thymocytes were compared between HY - TCR + Cd4cre A1fl / fl HY - TCR + mice ( right panels ) .

B-GENEPROD O O O B-GENEPROD O O B-GENEPROD I-GENEPROD O O O B-ORGANISM O O O O O B-GENEPROD O B-ORGANISM O O B-GENEPROD I-GENEPROD O O O O B-GENEPROD O B-ORGANISM O B-CELL_TYPE O O O O O O O O O O B-GENEPROD O O B-GENEPROD I-GENEPROD O O O O B-GENEPROD O B-ORGANISM O B-EXP_ASSAY O O O B-GENEPROD O O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O B-CELL_TYPE O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD O O O O B-GENEPROD O B-ORGANISM O O O O O

CD4+ cell-specific Adar1 knockout (Cd4cre A1fl/fl) mice were crossed with HY-TCR+ mice to generate Cd4cre A1fl/fl HY-TCR+ mice. Thymocytes isolated from male (A) and female (B) HY-TCR+ or Cd4cre A1fl/fl HY-TCR+ mice were stained with anti-CD4 and anti-CD8 antibodies and analyzed by flow cytometry. Representative images are shown (left panels). The absolute number of DP and 8SP thymocytes were compared between HY-TCR+ and Cd4cre A1fl/fl HY-TCR+ mice (right panels).


Thymocytes isolated from WT Cd4cre A1fl / fl mice were labeled with Proliferation Dye then stimulated with plate - bound anti - CD3 anti - CD28 antibodies for 4 days . Apoptotic ( Annexin V + ) proliferating thymocytes ( reduced of Proliferation Dye ) were determined by flow cytometry . Representative flow cytometric are shown ( left panel ) . percentages of either Annexin V + or Annexin V - proliferating thymocytes were compared between WT ( n = 5 ) Cd4cre A1fl / fl ( n = 4 ) mice ( right panel ) .

B-CELL_TYPE O O O B-GENEPROD I-GENEPROD O O B-ORGANISM O O O B-EXP_ASSAY O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD O O B-EXP_ASSAY B-CELL_TYPE O O O B-EXP_ASSAY O O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O B-GENEPROD I-GENEPROD O O B-GENEPROD I-GENEPROD O B-EXP_ASSAY B-CELL_TYPE O O O O O O O O O B-GENEPROD I-GENEPROD O O O O O O O B-ORGANISM O O O O O

Thymocytes isolated from WT and Cd4cre A1fl/fl mice were labeled with Cell Proliferation Dye and then stimulated with plate-bound anti-CD3 and anti-CD28 antibodies for 4 days. Apoptotic (Annexin V+) and proliferating thymocytes (reduced intensity of Proliferation Dye) were determined by flow cytometry. Representative flow cytometric images are shown (left panel). The percentages of either Annexin V+ or Annexin V- proliferating thymocytes were compared between WT (n = 5) and Cd4cre A1fl/fl (n = 4) mice (right panel).


relative expression of anti - apoptotic genes in sorted 4SP thymocytes isolated from WT Cd4cre A1fl / fl mice . Values represent relative gene expression normalized to Actinβ mRNA are displayed as mean ± ( n = 3 ; Mann - Whitney U - , * p < 0 . 05 , N . S . ; not significant ) . mean value for relative expression of each anti - apoptotic gene in WT 4SP thymocytes was set as one .

O B-EXP_ASSAY O O O B-EXP_ASSAY O O O O B-CELL_TYPE O O O B-GENEPROD I-GENEPROD O O B-ORGANISM O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O O O O O O O O O O O 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-EXP_ASSAY O O O O B-EXP_ASSAY O O O O B-CELL_TYPE O O O O O

The relative expression of anti-apoptotic genes in sorted 4SP thymocytes isolated from WT and Cd4cre A1fl/fl mice. Values represent the relative gene expression normalized to Actinβ mRNA and are displayed as the mean ± SEM (n = 3; Mann-Whitney U-test, *p < 0.05, N.S.; not significant). The mean value for the relative expression of each anti-apoptotic gene in WT 4SP thymocytes was set as one.