Bar graphs show time - course analyses of U87 ( E ) transduced with lentiviral LRG vector encoding sgRosa control or ATRX - targeted sgA # 10 or sgA # 19 . graph plots of GFP + over time following transduction with indicated sgRNAs . Data are expressed as means ± s . d . , * denotes ns ( not significant ) ; * * , p < 0 . 01 ; * * * , p < 0 . 001 . N = 3 , unpaired t .

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

Bar graphs show time-course analyses of U87 (E) transduced with lentiviral LRG vector encoding sgRosa control or ATRX-targeted sgA#10 or sgA#19. The graph plots the percentage of GFP+ cells over time following transduction with indicated sgRNAs. Data are expressed as means ± s.d., * denotes ns (not significant); **, p < 0.01; ***, p < 0.001. N = 3, unpaired t test.


Bar graphs show time - course analyses of U2OS ( F ) transduced with lentiviral LRG vector encoding sgRosa control or ATRX - targeted sgA # 10 or sgA # 19 . graph plots of GFP + over time following transduction with indicated sgRNAs . Data are expressed as means ± s . d . , * denotes ns ( not significant ) ; * * , p < 0 . 01 ; * * * , p < 0 . 001 . N = 3 , unpaired t .

Bar graphs show time-course analyses of U2OS cells (F) transduced with lentiviral LRG vector encoding sgRosa control or ATRX-targeted sgA#10 or sgA#19. The graph plots the percentage of GFP+ cells over time following transduction with indicated sgRNAs. Data are expressed as means ± s.d., * denotes ns (not significant); **, p < 0.01; ***, p < 0.001. N = 3, unpaired t test.


G , H 53BP1 / TRF2 co - immunofluorescence ( G ) revealed strongly increased TIF formation in lentiviral sgA # 10 or sgA # 19 transduced U87 assayed at day 45 but not day 15 post infections ( H ) . Scale bar , 15 µm . ( H ) Percentages of containing > 4 TIFs are expressed as means ± s . d . , N = 3 , unpaired t .

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

G, H 53BP1/TRF2 co-immunofluorescence (G) revealed strongly increased TIF formation in lentiviral sgA#10 or sgA#19 transduced U87 cells assayed at day 45 but not day 15 post infections (H). Scale bar, 15 µm. (H) Percentages of cells containing > 4 TIFs are expressed as means ± s.d., N = 3, unpaired t test.


I , J 53BP1 / TelG immuno - FISH ( I ) revealed strongly enhanced TIF formation in lentiviral sgA # 10 or sgA # 19 transduced IMR90 assayed at day 35 but not day 10 post infections ( J ) . Scale bar , 10 µm . ( J ) Percentages of containing > 4 TIFs are expressed as means ± s . d . , N = 3 , unpaired t .

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

I, J 53BP1/TelG immuno-FISH (I) revealed strongly enhanced TIF formation in lentiviral sgA#10 or sgA#19 transduced IMR90 cells assayed at day 35 but not day 10 post infections (J). Scale bar, 10 µm. (J) Percentages of cells containing > 4 TIFs are expressed as means ± s.d., N = 3, unpaired t test.


K Bar graphs show time - course analyses of IMR90 transduced with lentiviral sgRosa , sgA # 10 or sgA # 19 . Data are expressed as means ± s . d . , * denotes ns ( not significant ) ; * * , p < 0 . 01 ; * * * , p < 0 . 001 . N = 3 , unpaired t .

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

K Bar graphs show time-course analyses of IMR90 cells transduced with lentiviral sgRosa, sgA#10 or sgA#19. Data are expressed as means ± s.d., * denotes ns (not significant); **, p < 0.01; ***, p < 0.001. N = 3, unpaired t test.


A , B γH2AX / TelG immuno - FISH ( A ) revealed increased TIF formation in IMR90 transduced with DAXX - targeted lentiviral sgRNAs ( sgD # 1 # 2 ) ( B ) . Scale bar , 10 µm . Arrows denote telomeres that are positive for γH2AX signals . ( B ) of containing > 4 TIFs are expressed as means ± s . d . , N = 3 , unpaired t .

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

A, B γH2AX/TelG immuno-FISH (A) revealed increased TIF formation in IMR90 cells transduced with DAXX-targeted lentiviral sgRNAs (sgD#1 and #2) (B). Scale bar, 10 µm. Arrows denote telomeres that are positive for γH2AX signals. (B) Percentage of cells containing > 4 TIFs are expressed as means ± s.d., N = 3, unpaired t test.


C Western blots show depleted DAXX expression in Ad - Flp - infected cd - DAXX .

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C Western blots show depleted DAXX protein expression in Ad-Flp-infected cd-DAXX cells.


D , E PML / TelG immune - FISH ( D ) show enhanced APB formation in Ad - Flp infected cd - DAXX ( cd - D # 1 cd - D # 2 ) ( E ) . were analyzed at day 18 post Ad - Flp infection . are maximum - projections of ~ 10 stacks captured with a 63x lens with 2x zoom . Scale bar , 10 µm . ( E ) Percentages of APB + are expressed as means ± s . d . , N = 3 , unpaired t .

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D, E PML/TelG immune-FISH (D) show enhanced APB formation in Ad-Flp infected cd-DAXX cells (cd-D#1 and cd-D#2) (E). The cells were analyzed at day 18 post Ad-Flp infection. Images are maximum-intensity projections of ~10 stacks captured with a 63x lens with 2x zoom. Scale bar, 10 µm. (E) Percentages of APB+ cells are expressed as means ± s.d., N = 3, unpaired t test.


Depletion of DAXX induced delayed onset of growth arrest ( F ) ( F ) 2 , 000 were plated at day 4 post Ad - Flp infection . Data are expressed as means ± s . d . , N = 3 .

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Depletion of DAXX induced delayed onset of growth arrest (F) (F) 2,000 cells were plated at day 4 post Ad-Flp infection. Data are expressed as means ± s.d., N = 3.


Depletion of DAXX induced C - circle formation ( G ) .

Depletion of DAXX induced C-circle formation (G).


IMR90 at day 25 post infection of lentiviral control or sgA # 10 were synchronized by serum starvation switched to 15 % serum - containing medium assayed by 53BP1 / TelG immuno - FISH ( A ) at indicated time . Arrows denote telomeres that are positive for 53BP1 signals . Scale bar , 15 µm . ( B ) of containing ; 4 TIFs are expressed as means ± s . d . , N = 3 , unpaired t .

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

IMR90 cells at day 25 post infection of lentiviral control or sgA#10 were synchronized by serum starvation and switched to 15% serum-containing medium and assayed by 53BP1/TelG immuno-FISH (A) at indicated time. Arrows denote telomeres that are positive for 53BP1 signals. Scale bar, 15 µm. (B) Percentage of cells containing ; 4 TIFs are expressed as means ± s.d., N = 3, unpaired t test.


C , D RPA / TRF2 co - immunofluorescence ( C ) revealed strongly increased telomeric replication stress in U87 at day 30 but not day 10 post lentiviral sgA # 10 infection ( D ) . Lentiviral control sgA # 10 infected U87 at indicated time post - infection were released following thymidine ( Thy ) synchronization processed for RPA TRF2 co - . Scale bar , 10 µm . ( D ) Percentages of with > 4 RPA / TRF2 co - localized foci are expressed as means ± s . d . , N = 3 , unpaired t .

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

C, D RPA/TRF2 co-immunofluorescence (C) revealed strongly increased telomeric replication stress in U87 cells at day 30 but not day 10 post lentiviral sgA#10 infection (D). Lentiviral control and sgA#10 infected U87 cells at indicated time post-infection were released following thymidine (Thy) synchronization and processed for RPA and TRF2 co-staining. Scale bar, 10 µm. (D) Percentages of cells with > 4 RPA/TRF2 co-localized foci are expressed as means ± s.d., N = 3, unpaired t test.


A , B ATAC - seq analyses revealed progressive increased telomeric accessibilities in sgA # 10 transduced IMR90 . ( A ) graph plots show cumulative of total reads that contained at least indicated number of TTAGGG ( or CCCTAA ) tandem repeats . ( B ) of total reads that contains > 3 tandem telomeric repeats are expressed as means ± s . d . , N = 2 , unpaired t .

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

A, B ATAC-seq analyses revealed progressive increased telomeric DNA accessibilities in sgA#10 transduced IMR90 cells. (A) The graph plots show the cumulative percentage of total reads that contained at least indicated number of TTAGGG (or CCCTAA) tandem repeats. (B) The percentage of total reads that contains > 3 tandem telomeric repeats are expressed as means ± s.d., N = 2, unpaired t test.


C , D ChIP assays ( C ) show reduced nucleosome densities in sgA # 10 transduced IMR90 ( D ) . Input ChIP DNAs processed against indicated antibodies were assayed by dot - blotting hybridized with 32P - labeled probes specific for TelG . ( D ) relative enrichment was calculated after normalization of ChIP signals to respective input signals . Data are expressed as means ± s . d . , N = 3 , paired t .

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C, D ChIP assays (C) show reduced nucleosome densities in sgA#10 transduced IMR90 cells (D). Input and ChIP DNAs processed against indicated antibodies were assayed by dot-blotting and hybridized with 32P-labeled probes specific for TelG. (D) The relative enrichment was calculated after normalization of ChIP DNA signals to the respective input DNA signals. Data are expressed as means ± s.d., N = 3, paired t test.


A Growth curves show immortalization processes of lentiviral Rosa control ( sgRosa # 1 or # 2 ) or sgATRX ( sgA # 10 or # 19 ) transduced IMR90 . TERT transduced IMR90 culture was included as a positive control for immortalization .

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A Growth curves show the immortalization processes of lentiviral Rosa control (sgRosa#1 or #2) or sgATRX (sgA#10 or #19) transduced IMR90 cells. TERT transduced IMR90 culture was included as a positive control for immortalization.


B Quantitative RT - PCR of endogenous TERT gene expression revealed that sgATRX but not sgRosa transduced IMR90 adopted ALT during immortalization . Data are expressed as means ± s . d . , N = 3 , unpaired t .

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B Quantitative RT-PCR of endogenous TERT gene expression revealed that sgATRX but not sgRosa transduced IMR90 cells adopted ALT during immortalization. Data are expressed as means ± s.d., N = 3, unpaired t test.


C Western blots show depletion of ATRX expression in immortalized ALT IMR90 .

C Western blots show depletion of ATRX protein expression in the immortalized ALT IMR90 cells.


D C - circle shows increased C - circle formation in immortalized ALT . U2OS was used as a positive control , reactions lacking or ɸ29 serve as negative controls .

O O O O O O O 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 C-circle analysis shows increased C-circle formation in the immortalized ALT cells. U2OS was used as a positive control, and reactions lacking DNA or ɸ29 serve as negative controls.


E Telomere restriction fragment ( TRF ) show enhanced telomeric heterogeneities in immortalized ALT .

E Telomere restriction fragment (TRF) analysis show enhanced telomeric length heterogeneities in the immortalized ALT cells.


F , G PML / TRF2 co - immunofluorescence ( F ) shows increased APB formation in immortalized ALT ( G ) . Scale bar , 10 µm . ( G ) Percentages of APB positive are expressed as means ± s . d . , N = 3 , unpaired t .

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

F, G PML/TRF2 co-immunofluorescence (F) shows increased APB formation in the immortalized ALT cells (G). Scale bar, 10 µm. (G) Percentages of APB positive cells are expressed as means ± s.d., N = 3, unpaired t test.


( A ) Kaplan Meier plot for survival in NeuT ; Apln + / + ( n = 11 ) NeuT ; Apln - / - ( n = 10 ) mice with mammary cancer after onset . * P = 0 . 0185 ; Log rank .

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(A) Kaplan Meier plot for survival in NeuT;Apln+/+ (n=11) and NeuT;Apln-/- (n=10) mice with mammary cancer after tumor onset. *P=0.0185; Log rank test.


( B ) volumes , followed over time , of control mammary E0771 ( shRenilla ) Apelin - depleted ( shApln ) E0771 orthotopically - injected into both syngeneic C57BL / 6J Apln + / + Apln - / - mice ( 5x105 / mouse ) , respectively . volumes were determined using calipers are shown as mean volumes ± S . E . M . Data shown is pooled from 2 independent experiments . Apln + / + ; shRenilla ( n = 18 ) , Apln + / + ; shApln ( n = 17 ) , Apln - / - ; shRenilla ( n = 14 ) , Apln - / - ; shApln ( n = 15 ) ; * * P < 0 . 01 , * * * P < 0 . 001 ; two - way ANOVA .

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(B) Tumor volumes, followed over time, of control mammary tumor E0771 cells (shRenilla) and Apelin-depleted (shApln) E0771 cells orthotopically-injected into both syngeneic C57BL/6J Apln+/+ and Apln-/- mice (5x105 cells/mouse), respectively. Tumor volumes were determined using calipers and are shown as mean tumor volumes ± S.E.M. Data shown is pooled from 2 independent experiments. Apln+/+;shRenilla (n=18), Apln+/+;shApln (n=17), Apln-/-;shRenilla (n=14), Apln-/-;shApln (n=15); ** P<0.01, *** P<0.001; two-way ANOVA.


( C ) Mean percentages ( ± S . E . M . ) of CD31 + in E0771 shRenilla ( n = 3 ) or shApln ( n = 3 ) mammary tumors , assessed on day 23 post - orthotopic injection into C57BL / 6J Apln + / + or Apln - / - mice , respectively . * * P < 0 . 01 ; t . ( D ) Mean percentages ( ± S . E . M . ) of extravasated Dextran in E0771 shRenilla ( n = 9 ) or shApln ( n = 12 ) mammary tumors , assessed on day 19 post - orthotopic injection into C57BL / 6J Apln + / + or Apln - / - mice , respectively . * * P < 0 . 01 ; t . Right panel shows representative immunofluorescence of Dextran ( red ) , CD31 + vessels ( green ) , DAPI ( blue ) . arrows indicate regions of Dextran extravasation . Scale bars = 100

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

(C) Mean percentages (± S.E.M.) of CD31+ area in E0771 shRenilla (n=3) or shApln (n=3) mammary tumors, assessed on day 23 post-orthotopic injection into C57BL/6J Apln+/+ or Apln-/- mice, respectively. **P<0.01; t test. (D) Mean percentages (± S.E.M.) of extravasated Dextran in E0771 shRenilla (n=9) or shApln (n=12) mammary tumors, assessed on day 19 post-orthotopic injection into C57BL/6J Apln+/+ or Apln-/- mice, respectively. **P<0.01; t test. Right panel shows representative immunofluorescence of Dextran (red), CD31+ vessels (green), and DAPI (blue). The white arrows indicate regions of Dextran extravasation. Scale bars = 100 μm


( E ) Mean ( ± S . E . M . ) of pimonidazole positive foci , assessed on day 26 post - orthotopic injection of E0771 shRenilla ( n = 6 ) or shApln ( n = 4 ) into C57BL / 6J wild - type mice ( 5x105 / mouse ) . * P < 0 . 05 ; t . Right panels show representative immunohistochemical pimonidazole at two different magnifications ; Scale bars = 200 μm ( upper panels ) 50 μm ( lower panels ) .

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

(E) Mean counts (± S.E.M.) of pimonidazole positive foci, assessed on day 26 post-orthotopic injection of E0771 shRenilla (n=6) or shApln (n=4) into C57BL/6J wild-type mice (5x105 cells/mouse). *P<0.05; t test. Right panels show representative immunohistochemical pimonidazole staining at two different magnifications; Scale bars = 200 μm (upper panels) and 50 μm (lower panels).


( F ) Mean ( ± S . E . M . ) of - infiltrating immune normalized to CD45 + . E0771 shRenilla ( n = 8 ) or shApln ( n = 6 ) were orthotopically injected into C57BL / 6J Apln + / + or Apln - / - mice ( 5x105 / mouse ) , respectively tumors were harvested day 25 post - injection . * P < 0 . 05 , * * P < 0 . 01 ; t . immune populations were gated for viable CD45 + then further defined as : CD8 T ( Thy1 . 2 + , CD8 + ) , CD4 T , ( Thy1 . 2 + , CD4 + ) , Inflammatory Monocytes ( Ly6C + , Cd11b + , Ly6G - ) , PMN - MDSCs ( Ly6G + , Cd11b + ) , natural killer ( Thy1 . 2 - , Ly6G - , NK1 . 1 + ) , natural killer T ( Thy1 . 2 + , CD4 - , CD8 - , NK1 . 1 + ) peripheral dendritic ( Ly6G - , Ly6C + , Cd11b - , PDAC1 + , B220 + ) .

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(F) Mean percentage (± S.E.M.) of tumor-infiltrating immune cells normalized to CD45+. E0771 shRenilla (n=8) or shApln (n=6) were orthotopically injected into C57BL/6J Apln+/+ or Apln-/- mice (5x105 cells/mouse), respectively and tumors were harvested day 25 post-injection. *P<0.05, **P<0.01; t test. All immune cell populations were gated for viable CD45+ cells and then further defined as: CD8 T cells (Thy1.2+, CD8+), CD4 T cells, (Thy1.2+, CD4+), Inflammatory Monocytes (Ly6C+, Cd11b+, Ly6G-), PMN-MDSCs (Ly6G+, Cd11b+), natural killer cells (Thy1.2-, Ly6G-, NK1.1+), natural killer T cells (Thy1.2+, CD4-, CD8-, NK1.1+) and peripheral dendritic cells (Ly6G- ,Ly6C+, Cd11b- , PDAC1+, B220+).


( A ) Left - of - Seq transcriptome of CD31 + / CD105 + endothelial sorted from tumors established by E0771 shRenilla ( n = 6 ) or shApln ( n = 3 ) orthotopically injected into C57BL / 6J Apln + / + or Apln - / - mice , respectively . Tumors were harvested day 25 post - injection genes displayed are significantly deregulated at adjusted p - value cut - off of 0 . 05 . Right - Ingenuity Pathway for biological processes predicted to be decreased downstream of differentially expressed genes .

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(A) Left - Heatmap of RNA-Seq transcriptome analysis of CD31+/CD105+ endothelial cells sorted from tumors established by E0771 shRenilla (n=6) or shApln (n=3) cells orthotopically injected into C57BL/6J Apln+/+ or Apln-/- mice, respectively. Tumors were harvested day 25 post-injection and genes displayed are significantly deregulated at the adjusted p-value cut-off of 0.05. Right - Ingenuity Pathway Analysis for biological processes predicted to be decreased downstream of the differentially expressed genes.


( B ) of vessel sprouts ( mean values ± S . E . M . ) upon VEGF treatment ( 30ng / mL ) of embryoid bodies ( EB ) derived from murine ES ( mESCs ) with sense integrations in Apelin gene ( Apln STOP ) of splice acceptor described in Appendix Figure S1B or Cre - reverted antisense ( Apln GO ) sister . Apln GO ( Day 2 n = 6 ; Day 4 n = 40 , Day 6 n = 54 , Day 8 n = 32 , Day 10 n = 35 , Day 11 n = 31 ) , Apln STOP ( Day 2 n = 4 ; Day 4 n = 15 , Day 6 n = 31 , Day 8 n = 28 , Day 10 n = 32 , Day 11 n = 37 ) ; * * P < 0 . 01 , * * * P < 0 . 001 , two - way ANOVA . Representative brightfield automated of vessel sprouts by Definiens software are shown in bottom panels . Scale bars = 200

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

(B) Quantification of vessel sprouts (mean values ± S.E.M.) upon VEGF treatment (30ng/mL) of embryoid bodies (EB) derived from murine ES cells (mESCs) with sense integrations in the Apelin gene (Apln STOP) of the splice acceptor described in Appendix Figure S1B or Cre-reverted antisense (Apln GO) sister cells. Apln GO (Day 2 n=6; Day 4 n=40, Day 6 n=54, Day 8 n= 32, Day 10 n=35, Day 11 n=31), Apln STOP (Day 2 n=4; Day 4 n=15, Day 6 n=31, Day 8 n= 28, Day 10 n=32, Day 11 n=37); ** P<0.01, *** P<0.001, two-way ANOVA. Representative brightfield images and automated analysis of vessel sprouts by Definiens software are shown in the bottom panels. Scale bars = 200 μm


( C ) Differentially expressed genes using RNAseq transcriptome of CD31 + endothelial ( ECs ) sorted from sprouting EBs from Apelin STOP stimulated with VEGF ( 30ng / mL ) DMSO ( - Apln ) repaired Apelin GO sister stimulated with VEGF Apelin ( 1000nM ) ( + Apln ) . VEGF target genes angiogenesis - related genes , predicted by Ingenuity Pathway ( IPA ) software , are indicated by bars on upper axis of . GO terms were analyzed by DAVID on - software .

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(C) Differentially expressed genes using RNAseq transcriptome analysis of CD31+ endothelial cells (ECs) sorted from sprouting EBs from Apelin STOP cells stimulated with VEGF (30ng/mL) and DMSO (-Apln) and repaired Apelin GO sister cells stimulated with VEGF and Apelin (1000nM) (+Apln). VEGF target genes and angiogenesis-related genes, predicted by Ingenuity Pathway Analysis (IPA) software, are indicated by bars on the upper axis of the heatmap. GO terms were analyzed by DAVID on-line software.


( A ) Experimental set up ( right ) Kaplan Meier survival plot of NeuT ; Apln + / + NeuT ; Apln - / - mice with mammary cancer , left untreated ( control ) or treated with indicate dose of broad VEGFR blocker sunitinib . NeuT ; Apln + / + Control ( n = 8 ) , NeuT ; Apln - / - Control ( n = 11 ) , NeuT ; Apln + / + Sunitinib ( n = 11 ) , NeuT ; Apln - / - Sunitinib ( n = 12 ) ; * P < 0 . 05 ; Log rank . Mice were sacrificed when reached 1cm3 , following ethical guidelines . dotted indicates 50 % of survival .

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(A) Experimental set up and (right) Kaplan Meier survival plot of NeuT;Apln+/+ and NeuT;Apln-/- mice with mammary cancer, left untreated (control) or treated with the indicate dose of the broad VEGFR blocker sunitinib. NeuT;Apln+/+ Control (n=8), NeuT;Apln-/- Control (n=11), NeuT;Apln+/+ Sunitinib (n=11), NeuT;Apln-/- Sunitinib (n=12); *P<0.05; Log rank test. Mice were sacrificed when the tumor size reached 1cm3, following ethical guidelines. The dotted line indicates 50% of survival.


( B ) Percentages ( mean ± S . E . M . ) of burden in mammary of untreated ( control ) sunitinib - treated NeuT ; Apln + / + NeuT ; Apln - / - mice , assessed 4 after onset . NeuT ; Apln + / + Control ( n = 6 ) , NeuT ; Apln - / - Control ( n = 8 ) , NeuT ; Apln + / + Sunitinib ( n = 5 ) , NeuT ; Apln - / - Sunitinib ( n = 5 ) ; * P < 0 . 05 ; * * * P < 0 . 001 ; one - way ANOVA . Representative H & E are shown for each genotype in right panels . Scale bars = 1000 μm ( large panels ) 50 μm ( insets ) .

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

(B) Percentages (mean ± S.E.M.) of tumor burden in mammary glands of untreated (control) and sunitinib-treated NeuT;Apln+/+ and NeuT;Apln-/- mice, assessed 4 weeks after tumor onset. NeuT;Apln+/+ Control (n=6), NeuT;Apln-/- Control (n=8), NeuT;Apln+/+ Sunitinib (n=5), NeuT;Apln-/- Sunitinib (n=5); *P<0.05; ***P<0.001; one-way ANOVA. Representative H&E images are shown for each genotype in the right panels. Scale bars = 1000 μm (large panels) and 50 μm (insets).


( C ) volumes of untreated ( control ) sunitinib - treated NeuT ; Apln + / + NeuT ; Apln - / - mammary tumors , - matched at 20 - 70mm3 followed over time by MRI ; NeuT ; Apln + / + Control ( n = 7 ) , NeuT ; Apln - / - Control ( n = 5 ) , NeuT ; Apln + / + Sunitinib ( n = 5 ) , NeuT ; Apln - / - Sunitinib ( n = 6 ) mice per group ; Lines represent nonlinear fit of growth . Box arrow indicate time point used for in panel D . ( D ) Mitotic ( mean ± S . E . M . ) representative H & E of mammary tumors in untreated ( control ) sunitinib - treated NeuT ; Apln + / + NeuT ; Apln - / - mice , assessed 6 after onset . ; NeuT ; Apln + / + Control ( n = 7 ) , NeuT ; Apln - / - Control ( n = 6 ) , NeuT ; Apln + / + Sunitinib ( n = 9 ) , NeuT ; Apln - / - Sunitinib ( n = 4 ) tumors per group ; * P < 0 . 05 ; one - way ANOVA to sunitinib - treated NeuT ; Apln - / - . arrows indicate mitotic figures . Scale bars = 50

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

(C) Tumor volumes of untreated (control) and sunitinib-treated NeuT;Apln+/+ and NeuT;Apln-/- mammary tumors, size-matched at 20-70mm3 and followed over time by MRI analysis; NeuT;Apln+/+ Control (n=7), NeuT;Apln-/- Control (n=5), NeuT;Apln+/+ Sunitinib (n=5), NeuT;Apln-/- Sunitinib (n=6) mice per group; Lines represent nonlinear fit of tumor growth. Box and arrow indicate the time point used for analysis in panel D. (D) Mitotic counts (mean ± S.E.M.) and representative H&E images of mammary tumors in untreated (control) and sunitinib-treated NeuT;Apln+/+ and NeuT;Apln-/- mice, assessed 6 weeks after tumor onset.; NeuT;Apln+/+ Control (n=7), NeuT;Apln-/- Control (n=6), NeuT;Apln+/+ Sunitinib (n=9), NeuT;Apln-/- Sunitinib (n=4) tumors per group; *P<0.05; one-way ANOVA to sunitinib-treated NeuT;Apln-/-. White arrows indicate mitotic figures. Scale bars = 50 μm


( E ) volumes , followed over indicated time , of orthotopically injected E0771 shRenilla left untreated ( control ) or treated three times a week from day 8 after injection with an Apelin antagonist alone ( MM54 , 0 . 4μg / g ) , sunitinib alone ( 60mg / kg ) or a combination of both . volumes were measured using calipers are shown as mean volumes ± S . E . M . E0771 shRenilla Control ( n = 6 ) , MM54 ( n = 5 ) , Sunitinib ( n = 5 ) , Sunitinib + MM54 ( n = 5 ) ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , two - way ANOVA .

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(E) Tumor volumes, followed over the indicated time, of orthotopically injected E0771 shRenilla cells left untreated (control) or treated three times a week from day 8 after tumor injection with an Apelin antagonist alone (MM54, 0.4μg/g), sunitinib alone (60mg/kg) or a combination of both. Tumor volumes were measured using calipers and are shown as mean tumor volumes ± S.E.M. E0771 shRenilla Control (n=6), MM54 (n=5), Sunitinib (n=5), Sunitinib+MM54 (n=5); *P<0.05, **P<0.01, ***P<0.001, two-way ANOVA.


( B ) Representative MRI ; Scale bar = 5mm ( C ) ( mean ± S . E . M . ) of relative blood volume ( rTBV ) over time after NeuT + mammary tumors were - matched at 20 - 70mm3 ( 0 ) . Treatments genotypes are indicated . NeuT ; Apln + / + Control ( n = 4 ) , NeuT ; Apln - / - Control ( n = 4 ) , NeuT ; Apln + / + Sunitinib ( n = 5 ) , NeuT ; Apln - / - Sunitinib ( n = 5 ) ; * * P < 0 . 01 , * * * P < 0 . 001 compared to untreated NeuT ; Apln + / + mice # P < 0 . 05 compared to untreated control NeuT ; Apln - / - mice ; two - way ANOVA . Of note , in NeuT ; Apln + / + mice , only 2 tumors could be analyzed after 6 as some mice had to be sacrificed due to large sizes following ethical guidelines . Thus , we did not perform any statistical on 6 timepoints .

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

(B) Representative MRI images; Scale bar = 5mm and (C) quantification (mean ± S.E.M.) of relative tumor blood volume (rTBV) over time after NeuT+ mammary tumors were size-matched at 20-70mm3 (0 weeks). Treatments and genotypes are indicated. NeuT;Apln+/+ Control (n=4), NeuT;Apln-/- Control (n=4), NeuT;Apln+/+ Sunitinib (n=5), NeuT;Apln-/- Sunitinib (n=5); **P<0.01, and ***P<0.001 compared to untreated NeuT;Apln+/+ mice and #P<0.05 compared to untreated control NeuT;Apln-/- mice; two-way ANOVA. Of note, in NeuT;Apln+/+ mice, only 2 tumors could be analyzed after 6 weeks as some mice had to be sacrificed due to the large tumor sizes following ethical guidelines. Thus, we did not perform any statistical analysis on the 6 weeks timepoints.


( D ) ( mean values ± S . E . M . ) of CD31 + ( x104 μm2 ) / field , number of dilated vessels of alphaSMA + per CD31 + blood vessels in mammary tumors of untreated ( control ) sunitinib - treated NeuT ; Apln + / + NeuT ; Apln - / - mice , assessed 6 after mammary tumors were - matched . NeuT ; Apln + / + Control ( n = 4 ) , NeuT ; Apln - / - Control ( n = 4 ) , NeuT ; Apln + / + Sunitinib ( n = 5 ) , NeuT ; Apln - / - Sunitinib ( n = 4 ) ; * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; one - way ANOVA Kruskal - Wallis . ( E ) Representative immunofluorescence immunohistochemistry from Fig . 5D . Dilated blood vessels are marked by a red asterisk . DAPI ( blue ) is shown as a counterstain to visualize nuclei . Scale bars = 100 μm ( upper panels ) , 50 μm ( middel panels ) 20 μm ( lower panels ) .

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(D) Analysis (mean values ± S.E.M.) of CD31+ area (x104 μm2)/field, number of dilated tumor vessels and percentage of alphaSMA+ area per CD31+ blood vessels in mammary tumors of untreated (control) and sunitinib-treated NeuT;Apln+/+ and NeuT;Apln-/- mice, assessed 6 weeks after mammary tumors were size-matched. NeuT;Apln+/+ Control (n=4), NeuT;Apln-/- Control (n=4), NeuT;Apln+/+ Sunitinib (n=5), NeuT;Apln-/- Sunitinib (n=4); *P<0.05; **P<0.01; ***P<0.001; one-way ANOVA and Kruskal-Wallis test. (E) Representative immunofluorescence and immunohistochemistry images from Fig. 5D quantification. Dilated blood vessels are marked by a red asterisk. DAPI (blue) is shown as a counterstain to visualize nuclei. Scale bars = 100 μm (upper panels), 50 μm (middel panels) and 20 μm (lower panels).


( A ) Percentages of CA9 + adjacent to CD31 + blood vessels in untreated ( control ) sunitinib - treated NeuT ; Apln + / + NeuT ; Apln - / - mice , 6 after mammary tumors were - matched . NeuT ; Apln + / + Control ( n = 4 ) , NeuT ; Apln - / - Control ( n = 4 ) , NeuT ; Apln + / + Sunitinib ( n = 4 ) , NeuT ; Apln - / - Sunitinib ( n = 4 ) ; 100 - 200 peri - vascular intra - tumoral regions per group were counted . * * * P < 0 . 001 ; one - way ANOVA . Right panels show representative immunofluorescent . Areas limited by dotted lines indicate CA9 + areas . Scale bars = 50 μm .

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(A) Percentages of CA9+ cells adjacent to CD31+ tumor blood vessels in untreated (control) and sunitinib-treated NeuT;Apln+/+ and NeuT;Apln-/- mice, 6 weeks after mammary tumors were size-matched. NeuT;Apln+/+ Control (n=4), NeuT;Apln-/- Control (n=4), NeuT;Apln+/+ Sunitinib (n=4), NeuT;Apln-/- Sunitinib (n=4); 100-200 peri-vascular intra-tumoral regions per group were counted. ***P<0.001; one-way ANOVA. Right panels show representative immunofluorescent images. Areas limited by dotted white lines indicate CA9+ areas. Scale bars = 50 μm.


( B ) Representative MRI ; Scale bar = 5mm ( C ) ( mean ± S . E . M . ) of vessel permeability ( K2 ) in NeuT + mammary tumors followed over time . Treatments genotypes are indicated . NeuT ; Apln + / + Control ( n = 4 ) , NeuT ; Apln - / - Control ( n = 5 ) , NeuT ; Apln + / + Sunitinib ( n = 4 ) , NeuT ; Apln - / - Sunitinib ( n = 5 ) ; * * * P < 0 . 001 , compared to untreated control NeuT ; Apln + / + mice § P < 0 . 05 compared to sunitinib - treated NeuT ; Apln + / + mice ; two - way ANOVA . Of note , in NeuT ; Apln + / + mice , only 2 tumors could be analysed after 6 as some mice had to be sacrificed due to large sizes following ethical guidelines . Thus we did not perform any statistical on 6 timepoints .

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

(B) Representative MRI images; Scale bar = 5mm and (C) quantification (mean ± S.E.M.) of vessel permeability (K2) in NeuT+ mammary tumors followed over time. Treatments and genotypes are indicated. NeuT;Apln+/+ Control (n=4), NeuT;Apln-/- Control (n=5), NeuT;Apln+/+ Sunitinib (n=4), NeuT;Apln-/- Sunitinib (n=5); ***P<0.001, compared to untreated control NeuT;Apln+/+ mice and §P<0.05 compared to sunitinib-treated NeuT;Apln+/+ mice; two-way ANOVA. Of note, in NeuT;Apln+/+ mice, only 2 tumors could be analysed after 6 weeks as some mice had to be sacrificed due to the large tumor sizes following ethical guidelines. Thus we did not perform any statistical analysis on the 6 weeks timepoints.


( A ) Number of metastatic lung foci in untreated ( control ) sunitinib - treated ( 60mg / kg , three times a week from initiation ) NeuT ; Apln + / + or NeuT ; Apln - / - mice , assessed 6 after mammary tumors were - matched . Data of lung sections means ( black bars ) are shown . Right panels show representative H & E , where black arrows insets indicate metastatic foci . Scale bars = 1000 μm ( large panels ) 50 μm ( insets ) . * P < 0 . 05 , * * P < 0 . 01 ; * * * P < 0 . 001 ; Kruskal - Wallis ; n = 3 mice per cohort three sections per lung were analysed .

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(A) Number of metastatic lung foci in untreated (control) and sunitinib-treated (60mg/kg, three times a week from tumor initiation) NeuT;Apln+/+ or NeuT;Apln-/- mice, assessed 6 weeks after mammary tumors were size-matched. Data of individual lung sections and means (black bars) are shown. Right panels show representative H&E images, where black arrows and insets indicate metastatic foci. Scale bars = 1000 μm (large panels) and 50 μm (insets). *P<0.05, **P<0.01; ***P<0.001; Kruskal-Wallis test; n=3 mice per cohort and three sections per lung were analysed.


( B ) Kaplan Meier survival plot for distant metastasis free survival from KM - plotter database ( Data Ref : Győrffy et al , 2013 ) for high low Apelin ( APLN ) - expressing groups in human breast cancer . were split by median . Affymetrix Apelin ID 222856 _ at .

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(B) Kaplan Meier survival plot for distant metastasis free survival from the KM-plotter database (Data Ref: Győrffy et al, 2013) for high and low Apelin (APLN)-expressing groups in human breast cancer. Patients were split by the median. Affymetrix Apelin ID 222856_at.


( C ) Kaplan - Meier plot for progression - free survival stratifying RCC with high low APELIN serum 3 - 5 months after start date of sunitinib treatment . * P = 0 . 0367 ; Log rank .

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(C) Kaplan-Meier plot for progression-free survival stratifying RCC patients with high and low APELIN serum levels 3-5 months after the start date of sunitinib treatment. *P=0.0367; Log rank test.


( D ) Kaplan Meier plots for progression - free survival in RCC stratified into groups of high or low of APELIN VEGF . Cut - off were set by median . Serum was analysed 3 - 5 months after start date of sunitinib treatment . * P < 0 . 05 , * * P < 0 . 01 ; Log rank .

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(D) Kaplan Meier plots for progression-free survival in RCC patients stratified into groups of high or low levels of APELIN and VEGF. Cut-off levels were set by the median. Serum was analysed 3-5 months after the start date of sunitinib treatment. *P<0.05, **P<0.01; Log rank test.


A . Cytosolicnon - heme iron in wild - type mice subjected to sham or I / R procedure two days after surgery . Two - unpaired T - was performed . N = 4 mice for each group . B . Mitochondrialnon - heme iron in wild - type mice subjected to sham or I / R procedure two days after surgery . * P = 0 . 024 with two - unpaired T - . N = 4 mice for each group .

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A. Cytosolicnon-heme iron levels in wild-type mice subjected to sham or I/R procedure two days after surgery. Two-tailed unpaired T-test was performed. N=4 mice for each group.B. Mitochondrialnon-heme iron levels in wild-type mice subjected to sham or I/R procedure two days after surgery. * P=0.024 with two-tailed unpaired T-test. N=4 mice for each group.


C . Mitochondrial labile iron in H9c2 with or without H2O2 treatment measured using RPA . * P < 0 . 0001 with two - unpaired T - . N = 8 independent samples for PBS group N = 10 independent samples for H2O2 group . D . Cytosoliclabile iron in H9c2 with or without H2O2 treatment measured using calcein . * P = 0 . 0042 with two - unpaired T - . N = 11 independent samples in each group .

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C. Mitochondrial labile iron in H9c2 cells with or without H2O2 treatment measured using RPA fluorescence. * P<0.0001 with two-tailed unpaired T-test. N=8 independent samples for PBS group and N=10 independent samples for H2O2 group.D. Cytosoliclabile iron in H9c2 cells with or without H2O2 treatment measured using calcein fluorescence. * P=0.0042 with two-tailed unpaired T-test. N=11 independent samples in each group.


E . Mitochondrialiron in humancardiac tissue sample from non - failing hearts from hearts with ischemic cardiomyopathy ( ISCM ) . * P = 0 . 041 with two - unpaired T - . N = 4 independent samples in each group . F . Cytosoliciron in humancardiac tissue sample from non - failing hearts from hearts with ischemic cardiomyopathy ( ISCM ) . Two - unpaired T - was performed . N = 4 independent samples in each group . data are expressed as mean ± .

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E. Mitochondrialiron in humancardiac tissue sample from non-failing hearts and from hearts with ischemic cardiomyopathy (ISCM). * P=0.041 with two-tailed unpaired T-test. N=4 independent samples in each group.F. Cytosoliciron in humancardiac tissue sample from non-failing hearts and from hearts with ischemic cardiomyopathy (ISCM). Two-tailed unpaired T-test was performed. N=4 independent samples in each group. All data are expressed as mean ± SEM.


A . iron in H9c2 preloaded with radioactive 55Fe treated with indicated iron chelators . ANOVA followed by post - hoc Tukey was performed . * P = 7 . 9E - 8 PBS vs . DFO . * P = 9 . 4E - 6 BPD vs . DFO . * P = 0 . 003 PBS vs . BPD . N = 4 independent samples for PBS group N = 6 independent samples for other groups . B . iron in H9c2 preloaded with radioactive 55Fe treated with indicated iron chelators . ANOVA followed by post - hoc Tukey was performed . * P = 0 . 0003 PBS vs . BPD . * P = 5 . 58E - 5 PBS vs . DFO . N = 4 independent samples for PBS group N = 6 independent samples for other groups .

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A. Cytosolic iron in H9c2 cells preloaded with radioactive 55Fe and treated with indicated iron chelators. ANOVA followed by post-hoc Tukey test was performed. * P=7.9E-8 PBS vs. DFO. * P=9.4E-6 BPD vs. DFO. * P=0.003 PBS vs. BPD. N=4 independent samples for PBS group and N=6 independent samples for other groups.B. Nuclear iron in H9c2 cells preloaded with radioactive 55Fe and treated with indicated iron chelators. ANOVA followed by post-hoc Tukey test was performed. * P=0.0003 PBS vs. BPD. * P=5.58E-5 PBS vs. DFO. N=4 independent samples for PBS group and N=6 independent samples for other groups.


C . Representative RPA for labile mitochondrial iron in H9c2 with indicated iron chelator treatment . Scale bar = 100μm . D . Labile mitochondrial iron measured by RPA in H9c2 with indicated iron chelator treatment . ANOVA followed by post - hoc Tukey was performed . * P = 0 . 016 PBS vs . BPD . * P = 0 . 04 DFO vs . BPD . N = 8 independent samples for each group .

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C. Representative RPA fluorescence staining for labile mitochondrial iron in H9c2 cells with indicated iron chelator treatment. Scale bar = 100μm.D. Labile mitochondrial iron measured by RPA fluorescence in H9c2 cells with indicated iron chelator treatment. ANOVA followed by post-hoc Tukey test was performed.* P=0.016 PBS vs. BPD. * P=0.04 DFO vs. BPD. N=8 independent samples for each group.


E . H2O2 - induced in H9c2 with indicated treatments . ANOVA followed by post - hoc Tukey was performed . * P = 5E - 8 PBS - PBS vs . PBS - H2O2 . * P = 4E - 6 PBS - H2O2 vs . DFO - H2O2 . * P = 5E - 8 PBS - H2O2 vs . BPD - H2O2 . * P = 1E - 7 BPD - H2O2 vs . DFO - H2O2 . N = 6 independent samples for each group .

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E. H2O2-induced cell death in H9c2 cells with indicated treatments. ANOVA followed by post-hoc Tukey test was performed.* P=5E-8 PBS-PBS vs. PBS-H2O2. * P=4E-6 PBS-H2O2 vs. DFO-H2O2. * P=5E-8 PBS-H2O2 vs. BPD-H2O2. * P=1E-7 BPD-H2O2 vs. DFO-H2O2. N=6 independent samples for each group.


F . Labile mitochondrial iron in H9c2 with indicated treatment . PBS with without H2O2 data were copied from Fig 1C . ANOVA followed by post - hoc Tukey was performed . * P = 1E - 8 PBS - PBS vs . PBS - H2O2 . * P = 3E - 9 PBS - H2O2 vs . BPD - H2O2 . N = 8 independent samples for PBS - PBS , N = 12 for other groups . data are expressed as mean ± . N . S . = not signigicant .

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F. Labile mitochondrial iron in H9c2 cells with indicated treatment. PBS with and without H2O2 data were copied from Fig 1C. ANOVA followed by post-hoc Tukey test was performed. * P=1E-8 PBS-PBS vs. PBS-H2O2. * P=3E-9 PBS-H2O2 vs. BPD-H2O2. N=8 independent samples for PBS-PBS, and N=12 for the other groups. All data are expressed as mean ± SEM. N.S. = not signigicant.


A . Mitochondrial non - heme iron in ABCB8 NTG TG mice . N = 4 mice for NTG 6 mice for TG . * P = 0 . 021 with two - unpaired T - . B . non - heme iron in ABCB8 NTG TG mice . N = 4 mice for NTG 6 mice for TG . Two - unpaired T - was performed .

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A. Mitochondrial non-heme iron in ABCB8 NTG and TG mice. N=4 mice for NTG and 6 mice for TG. * P=0.021 with two-tailed unpaired T-test.B. Cytosolic non-heme iron in ABCB8 NTG and TG mice. N=4 mice for NTG and 6 mice for TG. Two-tailed unpaired T-test was performed.


C . Baseline ejection fraction of littermate ABCB8 NTG TG mice . N = 6 mice in each group . Two - unpaired T - was performed . D . Baseline fractional shortening of littermate ABCB8 NTG TG mice . N = 6 mice in each group . Two - unpaired T - was performed .

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C. Baseline ejection fraction of littermate ABCB8 NTG and TG mice. N=6 mice in each group. Two-tailed unpaired T-test was performed.D. Baseline fractional shortening of littermate ABCB8 NTG and TG mice. N=6 mice in each group. Two-tailed unpaired T-test was performed.


E . Lipid peroxidation products in hearts of TG NTG mice . N = 6 mice in each group . Two - unpaired T - was performed .

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E. Lipid peroxidation products in hearts of TG and NTG mice. N=6 mice in each group. Two-tailed unpaired T-test was performed.


F . Relative expression of antioxidant genes in NTG TG mice . N = 6 mice in each group . Two - unpaired T - was performed . data are expressed as mean ± . N . S . = not signigicant .

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F. Relative expression of antioxidant genes in NTG and TG mice. N=6 mice in each group. Two-tailed unpaired T-test was performed. All data are expressed as mean ± SEM. N.S. = not signigicant.


A . Cardiac function in ABCB8 NTG TG mice undergoing sham or I / R procedure . ANOVA followed by post - hoc Tukey was performed for each time point . * P < 0 . 05 compared with TG - sham at same time point . $ P < 0 . 05 compared with NTG - I / R at same time point . § P < 0 . 05 compared with NTG - sham at same time point . N = 5 mice for NTG - sham NTG - I / R , N = 6 mice for TG - sham TG - I / R . Exact P - values are included in Appendix Table S3 .

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A. Cardiac function in ABCB8 NTG and TG mice undergoing sham or I/R procedure. ANOVA followed by post-hoc Tukey test was performed for each time point. * P<0.05 compared with TG-sham at the same time point. $ P<0.05 compared with NTG-I/R at the same time point. § P<0.05 compared with NTG-sham at the same time point. N=5 mice for NTG-sham and NTG-I/R, and N=6 mice for TG-sham and TG-I/R. Exact P-values are included in Appendix Table S3.


B . Representative of TUNEL for mice with indicated genotype undergone indicated procedure . Scale bar = 100μm .

B. Representative images of TUNEL staining for mice with indicated genotype undergone indicated procedure. Scale bar = 100μm.


C . of apoptosis in ABCB8 TG NTG mice with indicated procedure . ANOVA followed by post - hoc Tukey was performed . * P = 0 . 035 . N = 4 mice for TG - I / R , N = 6 mice for other groups .

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C. Quantification of apoptosis in ABCB8 TG and NTG mice with indicated procedure. ANOVA followed by post-hoc Tukey test was performed. * P=0.035. N=4 mice for TG-I/R, and N=6 mice for all other groups.


D . Nppa expression in mice subjected to sham or I / R procedure . ANOVA followed by post - hoc Tukey was performed . * P = 3 . 24E - 4 NTG - sham vs . NTG - I / R . * P = 0 . 002 TG - sham vs . TG - I / R . * P = 8E - 6 NTG - I / R vs . TG - I / R . N = 5 mice for NTG - sham NTG - I / R , N = 6 mice for TG - sham TG - I / R . E . Nppb expression in mice subjected to sham or I / R procedure . ANOVA followed by post - hoc Tukey was performed . * P < 1E - 8 NTG - sham vs . NTG - I / R . * P = 0 . 032 NTG - I / R vs . TG - I / R . N = 5 mice for NTG - sham NTG - I / R , N = 6 mice for TG - sham TG - I / R . F . Myh7 expression in mice subjected to sham or I / R procedure . ANOVA followed by post - hoc Tukey was performed . * P = 0 . 04 NTG - sham vs . NTG - I / R . * P = 0 . 034 NTG - I / R vs . TG - I / R . N = 5 mice for NTG - sham NTG - I / R , N = 6 mice for TG - sham TG - I / R . data are expressed as mean ± . N . S . = not signigicant .

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D. Nppa expression in mice subjected to sham or I/R procedure. ANOVA followed by post-hoc Tukey test was performed. * P=3.24E-4 NTG-sham vs. NTG-I/R. * P=0.002 TG-sham vs. TG-I/R. * P=8E-6 NTG-I/R vs. TG-I/R. N=5 mice for NTG-sham and NTG-I/R, and N=6 mice for TG-sham and TG-I/R.E. Nppb expression in mice subjected to sham or I/R procedure. ANOVA followed by post-hoc Tukey test was performed. * P<1E-8 NTG-sham vs. NTG-I/R. * P=0.032 NTG-I/R vs. TG-I/R. N=5 mice for NTG-sham and NTG-I/R, and N=6 mice for TG-sham and TG-I/R.F. Myh7 expression in mice subjected to sham or I/R procedure. ANOVA followed by post-hoc Tukey test was performed. * P=0.04 NTG-sham vs. NTG-I/R. * P=0.034 NTG-I/R vs. TG-I/R. N=5 mice for NTG-sham and NTG-I/R, and N=6 mice for TG-sham and TG-I/R. All data are expressed as mean ± SEM. N.S. = not signigicant.


A . Representative H & amp ; amp ; E of peri - infarct demonstrated reduced injury in ABCB8 TG mice after I / R . Scale bar = 115μm . B . Representative H & amp ; amp ; E Masson Trichrome ( MT ) in mouseheart 28 days after indicated procedure . Scale bar = 1100μm . C . of tissue fibrosis in ABCB8 NTG TG mice subjected to I / R . * P = 0 . 047 with two - unpaired T - . 2 - 3 sections from each mice were quantified ; N = 4 mice for NTG N = 6 mice for TG .

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A. Representative H&amp;E staining of peri-infarct area demonstrated reduced cellular injury in ABCB8 TG mice after I/R. Scale bar = 115μm.B. Representative H&amp;E and Masson Trichrome (MT) staining in mouseheart 28 days after indicated procedure. Scale bar = 1100μm.C. Quantification of tissue fibrosis in ABCB8 NTG and TG mice subjected to I/R. * P=0.047 with two-tailed unpaired T-test. 2-3 sections from each mice were quantified; N=4 mice for NTG and N=6 mice for TG.


D . Nppa expression in mice subjected to sham or I / R procedure . ANOVA followed by post - hoc Tukey was performed . * P = 0 . 006 NTG - sham vs . NTG - I / R . * P = 0 . 015 NTG - I / R vs . TG - I / R . N = 5 mice for NTG - sham TG - I / R , N = 6 mice for TG - sham , N = 4 mice for NTG - I / R . E . Nppb expression in mice subjected to sham or I / R procedure . ANOVA followed by post - hoc Tukey was performed . * P = 0 . 001 NTG - sham vs . NTG - I / R . * P = 0 . 004 NTG - I / R vs . TG - I / R . N = 5 mice for NTG - sham TG - I / R , N = 6 mice for TG - sham , N = 4 mice for NTG - I / R . F . Myh7 expression in mice subjected to sham or I / R procedure . ANOVA followed by post - hoc Tukey was performed . * P = 0 . 002 NTG - sham vs . NTG - I / R . * P = 0 . 011 NTG - I / R vs . TG - I / R . N = 5 mice for NTG - sham TG - I / R , N = 6 mice for TG - sham , N = 4 mice for NTG - I / R . data are expressed as mean ± . N . S . = not signigicant .

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D. Nppa expression in mice subjected to sham or I/R procedure. ANOVA followed by post-hoc Tukey test was performed. * P=0.006 NTG-sham vs. NTG-I/R. * P=0.015 NTG-I/R vs. TG-I/R. N=5 mice for NTG-sham and TG-I/R, N=6 mice for TG-sham, and N=4 mice for NTG-I/R.E. Nppb expression in mice subjected to sham or I/R procedure. ANOVA followed by post-hoc Tukey test was performed. * P=0.001 NTG-sham vs. NTG-I/R. * P=0.004 NTG-I/R vs. TG-I/R. N=5 mice for NTG-sham and TG-I/R, N=6 mice for TG-sham, and N=4 mice for NTG-I/R.F. Myh7 expression in mice subjected to sham or I/R procedure. ANOVA followed by post-hoc Tukey test was performed. * P=0.002 NTG-sham vs. NTG-I/R. * P=0.011 NTG-I/R vs. TG-I/R. N=5 mice for NTG-sham and TG-I/R, N=6 mice for TG-sham, and N=4 mice for NTG-I/R. All data are expressed as mean ± SEM. N.S. = not signigicant.


A . Mitochondrial non - heme iron in wild - type mice treated with vehicle control or indicated iron chelator for 7 days . ANOVA followed by post - hoc Tukey was performed . * P = 0 . 03 . N = 4 mice for PBS DFO , N = 5 mice for BPD . B . non - heme iron in wild - type mice treated with vehicle control or indicated iron chelator for 7 days . ANOVA followed by post - hoc Tukey was performed . * P = 5E - 4 PBS vs . DFO . * P = 0 . 01 DFO vs . BPD . P = 0 . 06 PBS vs . DFO . N = 4 mice for PBS DFO , N = 5 mice for BPD . C . non - heme iron in wild - type mice treated with vehicle control or indicated iron chelator for 7 days . N = 4 mice for PBS DFO N = 5 mice for BPD .

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A. Mitochondrial non-heme iron in wild-type mice treated with vehicle control or indicated iron chelator for 7 days. ANOVA followed by post-hoc Tukey test was performed. * P=0.03. N=4 mice for PBS and DFO, and N=5 mice for BPD.B. Cytosolic non-heme iron in wild-type mice treated with vehicle control or indicated iron chelator for 7 days. ANOVA followed by post-hoc Tukey test was performed. * P=5E-4 PBS vs. DFO. * P=0.01 DFO vs. BPD. P=0.06 PBS vs. DFO. N=4 mice for PBS and DFO, and N=5 mice for BPD.C. Nuclear non-heme iron in wild-type mice treated with vehicle control or indicated iron chelator for 7 days. N=4 mice for PBS and DFO and N=5 mice for BPD.


D . Cardiac function in wild - type mice treated with vehicle control or indicated iron chelator for 7 days . N = 5 mice for each group .

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D. Cardiac function in wild-type mice treated with vehicle control or indicated iron chelator for 7 days. N=5 mice for each group.


E . Cardiac function of chelator - treated mice after I / R . ANOVA followed by post - hoc Tukey was performed for each time point . * P < 0 . 0001 compared to PBS - I / R group at same time point . # P < 0 . 0001 compared to PBS - sham group at same time point . Exact P - values are included in Appendix Table S3 . N = 5 micePBS - sham BPD - I / R , N = 6 mice for other groups .

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E. Cardiac function of chelator-treated mice after I/R. ANOVA followed by post-hoc Tukey test was performed for each time point. * P<0.0001 compared to PBS-I/R group at the same time point. # P<0.0001 compared to PBS-sham group at the same time point. Exact P-values are included in Appendix Table S3. N=5 micePBS-sham and BPD-I/R, N=6 mice for all other groups.


F . Representative Hematoxylin Eosin ( Hamp ; amp ; E ) Masson Trichrome ( MT ) of heart sections in mice with indicated chelator treatment undergone sham or I / R . Scale bar = 1100μm . Bar graph represent of tissue fibrosis . ANOVA followed by post - hoc Tukey was performed . * P = 0 . 048 . 2 - 3 sections from each mice were quantified , N = 4 mice for PBS , N = 5 mice for DFO N = 6 mice for BPD .

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F. Representative Hematoxylin Eosin (Hamp;amp;E) and Masson Trichrome (MT) staining of heart sections in mice with indicated chelator treatment undergone sham or I/R. Scale bar = 1100μm. Bar graph represent quantification of tissue fibrosis. ANOVA followed by post-hoc Tukey test was performed. * P=0.048. 2-3 sections from each mice were quantified, N=4 mice for PBS, N=5 mice for DFO and N=6 mice for BPD.


G . Nppa expression in mice subjected to sham or I / R procedure . ANOVA followed by post - hoc Tukey was performed . * P = 0 . 002 PBS - sham vs . PBS - I / R . * P = 0 . 02 DFO - sham vs . DFO I / R . * P = 0 . 007 PBS - I / R vs . BPD I / R . N = 6 mice for PBS - sham DFO - sham , N = 4 mice for other groups . H . Nppb expression in mice subjected to sham or I / R procedure . ANOVA followed by post - hoc Tukey was performed . * P = 0 . 0007 PBS - sham vs . PBS - I / R . * P = 0 . 014 DFO - sham vs . DFO I / R . * P = 0 . 003 PBS - I / R vs . BPD I / R . N = 6 mice for PBS - sham DFO - sham , N = 4 mice for other groups . I . Myh7 expression in mice subjected to sham or I / R procedure . ANOVA followed by post - hoc Tukey was performed . * P = 0 . 0001 PBS - sham vs . PBS - I / R . * P = 0 . 038 DFO - sham vs . DFO I / R . * P = 0 . 006 PBS - I / R vs . BPD I / R . N = 6 mice for PBS - sham DFO - sham , N = 4 mice for other groups . data are expressed as mean ± . N . S . = not signigicant .

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G. Nppa expression in mice subjected to sham or I/R procedure. ANOVA followed by post-hoc Tukey test was performed. * P=0.002 PBS-sham vs. PBS-I/R. * P=0.02 DFO-sham vs. DFO I/R. * P=0.007 PBS-I/R vs. BPD I/R. N=6 mice for PBS-sham and DFO-sham, N=4 mice for all other groups.H. Nppb expression in mice subjected to sham or I/R procedure. ANOVA followed by post-hoc Tukey test was performed. * P= 0.0007 PBS-sham vs. PBS-I/R. * P= 0.014 DFO-sham vs. DFO I/R. * P= 0.003 PBS-I/R vs. BPD I/R. N=6 mice for PBS-sham and DFO-sham, N=4 mice for all other groups.I. Myh7 expression in mice subjected to sham or I/R procedure. ANOVA followed by post-hoc Tukey test was performed. * P=0.0001 PBS-sham vs. PBS-I/R. * P=0.038 DFO-sham vs. DFO I/R. * P=0.006 PBS-I/R vs. BPD I/R. N=6 mice for PBS-sham and DFO-sham, N=4 mice for all other groups. All data are expressed as mean ± SEM. N.S. = not signigicant.


A . Cardiac mitochondrial iron in ABCB8 KO WT mice with indicated treatment harvested 4 after tamoxifen treatment . ANOVA followed by post - hoc Tukey was performed . * P = 0 . 02 WT - vehicle vs . WT - BPD . * P = 0 . 001 WT - vehicle vs . ABCB8 KO - vehicle . * P = 0 . 004 ABCB8 KO - vehicle vs . ABCB8 KO - BPD . N = 8 for WT - vehicle N = 7 for other groups .

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A. Cardiac mitochondrial iron in ABCB8 KO and WT mice with indicated treatment harvested 4 weeks after the tamoxifen treatment. ANOVA followed by post-hoc Tukey test was performed. * P=0.02 WT-vehicle vs. WT-BPD. * P= 0.001 WT-vehicle vs. ABCB8 KO-vehicle. * P=0.004 ABCB8 KO-vehicle vs. ABCB8 KO-BPD. N=8 for WT-vehicle and N=7 for all other groups.


B . Ejection fraction in ABCB8 KO WT mice with indicated treatment 4 after gene knockout . ANOVA followed by post - hoc Tukey was performed . * P < 1E - 8 WT - vehicle vs . ABCB8 KO - vehicle . * P < 1E - 8 ABCB8 - vehicle vs . ABCB8 KO - BPD . N = 8 for WT - vehicle N = 7 for other groups .

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B. Ejection fraction in ABCB8 KO and WT mice with indicated treatment 4 weeks after gene knockout. ANOVA followed by post-hoc Tukey test was performed. * P<1E-8 WT-vehicle vs. ABCB8 KO-vehicle. * P<1E-8 ABCB8-vehicle vs. ABCB8 KO-BPD. N=8 for WT-vehicle and N=7 for all other groups.


C . Fractional shortening in ABCB8 KO WT mice with indicated treatment 4 after gene knockout . ANOVA followed by post - hoc Tukey was performed . * P < 1E - 8 WT - vehicle vs . ABCB8 KO - vehicle . * P < 1E - 8 ABCB8 - vehicle vs . ABCB8 KO - BPD . N = 8 for WT - vehicle N = 7 for other groups .

C. Fractional shortening in ABCB8 KO and WT mice with indicated treatment 4 weeks after gene knockout. ANOVA followed by post-hoc Tukey test was performed. * P<1E-8 WT-vehicle vs. ABCB8 KO-vehicle. * P<1E-8 ABCB8-vehicle vs. ABCB8 KO-BPD. N=8 for WT-vehicle and N=7 for all other groups.


D . Nppa expression in mice subjected to sham or I / R procedure . ANOVA followed by post - hoc Tukey was performed . * P = 0 . 017 WT - vehicle vs . ABCB8 KO - vehicle . * P = 0 . 01 ABCB8 - vehicle vs . ABCB8 KO - BPD . N = 8 for WT - vehicle N = 7 for other groups . E . Nppb expression in mice subjected to sham or I / R procedure . ANOVA followed by post - hoc Tukey was performed . * P = 0 . 001 WT - vehicle vs . ABCB8 KO - vehicle . * P = 0 . 0003 ABCB8 - vehicle vs . ABCB8 KO - BPD . N = 8 for WT - vehicle N = 7 for other groups . F . Myh7 expression in mice subjected to sham or I / R procedure . ANOVA followed by post - hoc Tukey was performed . * P = 0 . 011 WT - vehicle vs . ABCB8 KO - vehicle . * P = 0 . 045 ABCB8 - vehicle vs . ABCB8 KO - BPD . N = 8 for WT - vehicle N = 7 for other groups . data are expressed as mean ± . N . S . = not signigicant .

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D. Nppa expression in mice subjected to sham or I/R procedure. ANOVA followed by post-hoc Tukey test was performed. * P=0.017 WT-vehicle vs. ABCB8 KO-vehicle. * P=0.01 ABCB8-vehicle vs. ABCB8 KO-BPD. N=8 for WT-vehicle and N=7 for all other groups.E. Nppb expression in mice subjected to sham or I/R procedure. ANOVA followed by post-hoc Tukey test was performed. * P=0.001 WT-vehicle vs. ABCB8 KO-vehicle. * P=0.0003 ABCB8-vehicle vs. ABCB8 KO-BPD. N=8 for WT-vehicle and N=7 for all other groups.F. Myh7 expression in mice subjected to sham or I/R procedure. ANOVA followed by post-hoc Tukey test was performed. * P=0.011 WT-vehicle vs. ABCB8 KO-vehicle. * P=0.045 ABCB8-vehicle vs. ABCB8 KO-BPD. N=8 for WT-vehicle and N=7 for all other groups. All data are expressed as mean ± SEM. N.S. = not signigicant.


A . Representative western blot demonstrating ABCB8 overexpression in H9c2 . EV = empty vector . N = 3 independent samples for each group .

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A. Representative western blot demonstrating ABCB8 overexpression in H9c2 cells. EV = empty vector. N=3 independent samples for each group.


B . I ROS production in mitochondria with ABCB8 downregulation with or without rotenone . ANOVA followed by post - hoc Tukey was performed . N = 6 independent samples for each group .

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B. Complex I ROS production in mitochondria with ABCB8 downregulation with or without rotenone. ANOVA followed by post-hoc Tukey test was performed. N= 6 independent samples for each group.


C . III ROS production in mitochondria with ABCB8 downregulation with or without antimycin A . ANOVA followed by post - hoc Tukey was performed . N = 6 independent samples for each group .

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C. Complex III ROS production in mitochondria with ABCB8 downregulation with or without antimycin A. ANOVA followed by post-hoc Tukey test was performed. N= 6 independent samples for each group.


D . I ROS production in mitochondria with ABCB8 overexpression with or without rotenone . EV = empty vector . ANOVA followed by post - hoc Tukey was performed . N = 9 independent samples for EV - baseline N = 8 independent samples for other groups .

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D. Complex I ROS production in mitochondria with ABCB8 overexpression with or without rotenone. EV= empty vector. ANOVA followed by post-hoc Tukey test was performed. N=9 independent samples for EV-baseline and N=8 independent samples for the other groups.


E . IIIROS production in mitochondria with ABCB8 overexpression with or without antimycin A . EV = empty vector . ANOVA followed by post - hoc Tukey was performed . N = 9 independent samples for EV - baseline N = 8 independent samples for other groups .

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E. Complex IIIROS production in mitochondria with ABCB8 overexpression with or without antimycin A. EV= empty vector. ANOVA followed by post-hoc Tukey test was performed. N=9 independent samples for EV-baseline and N=8 independent samples for the other groups.


F . Mitochondrial ROS in H9c2 with various treatments with or without oxidative stress . N = 6 independent samples for each group . ANOVA followed by post - hoc Tukey was performed . * P = 2E - 8 PBS - PBS vs . PBS - H2O2 . * P = 2E - 8 DFO - H2O2 vs . BPD - H2O2 .

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F. Mitochondrial ROS in H9c2 cells with various treatments with or without oxidative stress. N=6 independent samples for each group. ANOVA followed by post-hoc Tukey test was performed. * P=2E-8 PBS-PBS vs. PBS-H2O2. * P=2E-8 DFO- H2O2 vs. BPD-H2O2.


G . Mitochondrial potential as measured by TMRE in H9c2 treated with indicated iron chelator with or without oxidative stress . N = 6 independent samples for each group . ANOVA followed by post - hoc Tukey was performed . * P = 1E - 8 PBS - PBS vs . PBS - H2O2 . * P = 1 . 8E - 8 DFO - PBS vs . DFO - H2O2 .

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G. Mitochondrial membrane potential as measured by TMRE intensity in H9c2 cells treated with indicated iron chelator with or without oxidative stress. N= 6 independent samples for each group. ANOVA followed by post-hoc Tukey test was performed. * P=1E-8 PBS-PBS vs. PBS-H2O2. * P=1.8E-8 DFO-PBS vs. DFO-H2O2.


H . of lipid peroxidation products in ABCB8 KO mice with or without chelator treatment . N = 4 mice for WT - vehicle , N = 6 mice for ABCB8 KO - vehicle , N = 5 mice for other groups . ANOVA followed by post - hoc Tukey was performed . * P = 0 . 004 WT - vehicle vs . ABCB8 KO - vehicle . * P = 1E - 6 ABCB8 KO - vehicle vs . ABCB8 KO - BPD .

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H. The levels of lipid peroxidation products in ABCB8 KO mice with or without chelator treatment. N=4 mice for WT-vehicle, N=6 mice for ABCB8 KO-vehicle, and N=5 mice for all other groups. ANOVA followed by post-hoc Tukey test was performed. * P=0.004 WT-vehicle vs. ABCB8 KO-vehicle. * P=1E-6 ABCB8 KO-vehicle vs. ABCB8 KO-BPD.


I . Lipid peroxidation products in ABCB8 TG NTG mice two days after I / R . N = 6 mice for NTG - sham , N = 4 mice for other groups . ANOVA followed by post - hoc Tukey was performed . * P = 0 . 024 NTG - sham vs . NTG - I / R . * P = 0 . 016 NTG - I / R vs . TG - I / R . data are expressed as mean ± . N . S . = not signigicant .

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I. Lipid peroxidation products in ABCB8 TG and NTG mice two days after I/R. N=6 mice for NTG-sham, and N=4 mice for the other groups. ANOVA followed by post-hoc Tukey test was performed. * P=0.024 NTG-sham vs. NTG-I/R. * P=0.016 NTG-I/R vs. TG-I/R. All data are expressed as mean ± SEM. N.S. = not signigicant.


A . Mitochondrial aconitase activity in H9c2 with or without ABCB8 overexpression treated with or without H2O2 . N = 4 independent samples for each group . ANOVA followed by post - hoc Tukey was performed . * P = 0 . 0004 EV - PBS vs . EV - H2O2 . * P = 0 . 0001 EV - H2O2 vs . ABCB8 - H2O2 .

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A. Mitochondrial aconitase activity in H9c2 cells with or without ABCB8 overexpression and treated with or without H2O2. N=4 independent samples for each group. ANOVA followed by post-hoc Tukey test was performed. * P=0.0004 EV-PBS vs. EV-H2O2. * P=0.0001 EV-H2O2 vs. ABCB8-H2O2.


B . I activity in H9c2 with or without ABCB8 overexpression treated with or without H2O2 . N = 6 independent samples for each group . ANOVA followed by post - hoc Tukey was performed . * P = 0 . 0005 EV - PBS vs . EV - H2O2 . * P = 0 . 00001 EV - H2O2 vs . ABCB8 - H2O2 . C . II activity in H9c2 with or without ABCB8 overexpression treated with or without H2O2 . N = 6 independent samples for each group . ANOVA followed by post - hoc Tukey was performed . * P = 0 . 001 EV - PBS vs . EV - H2O2 . * P = 0 . 001 EV - H2O2 vs . ABCB8 - H2O2 .

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B. Complex I activity in H9c2 cells with or without ABCB8 overexpression and treated with or without H2O2. N=6 independent samples for each group. ANOVA followed by post-hoc Tukey test was performed. * P=0.0005 EV-PBS vs. EV-H2O2. * P=0.00001 EV-H2O2 vs. ABCB8-H2O2.C. Complex II activity in H9c2 cells with or without ABCB8 overexpression and treated with or without H2O2. N=6 independent samples for each group. ANOVA followed by post-hoc Tukey test was performed. * P=0.001 EV-PBS vs. EV-H2O2. * P=0.001 EV-H2O2 vs. ABCB8-H2O2.


D . IV activity in H9c2 with or without ABCB8 overexpression treated with or without H2O2 . N = 6 independent samples for each group . ANOVA followed by post - hoc Tukey was performed . * P = 0 . 0008 EV - PBS vs . EV - H2O2 . * P = 0 . 0006 EV - H2O2 vs . ABCB8 - H2O2 .

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D. Complex IV activity in H9c2 cells with or without ABCB8 overexpression and treated with or without H2O2. N=6 independent samples for each group. ANOVA followed by post-hoc Tukey test was performed. * P=0.0008 EV-PBS vs. EV-H2O2. * P=0.0006 EV-H2O2 vs. ABCB8-H2O2.


E . Mitochondrial aconitase activity in H9c2 treated with indicated iron chelators with or without H2O2 . N = 4 independent samples in each group . ANOVA followed by post - hoc Tukey was performed . * P = 0 . 036 PBS - PBS vs . PBS - H2O2 . * P = 0 . 0047 DFO - PBS vs . DFO - H2O2 . * P = 0 . 035 PBS - H2O2 vs . BPD - H2O2 .

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E. Mitochondrial aconitase activity in H9c2 cells treated with indicated iron chelators with or without H2O2. N=4 independent samples in each group. ANOVA followed by post-hoc Tukey test was performed. * P=0.036 PBS-PBS vs. PBS-H2O2. * P=0.0047 DFO-PBS vs. DFO-H2O2. * P=0.035 PBS- H2O2 vs. BPD-H2O2.


F . I activity in H9c2 treated with indicated iron chelators with or without H2O2 . N = 6 independent samples for each group . ANOVA followed by post - hoc Tukey was performed . * P = 0 . 0004 PBS - PBS vs . PBS - H2O2 . * P = 0 . 0003 DFO - PBS vs . DFO - H2O2 . * P < 0 . 0001 PBS - H2O2 vs . BPD - H2O2 . G . II activity in H9c2 treated with indicated iron chelators with or without H2O2 . N = 6 independent samples for each group . ANOVA followed by post - hoc Tukey was performed . * P = 0 . 0001 PBS - PBS vs . PBS - H2O2 . * P < 0 . 0001 DFO - PBS vs . DFO - H2O2 . * P < 0 . 0001 PBS - H2O2 vs . BPD - H2O2 . H . IV activity in H9c2 treated with indicated iron chelators with or without H2O2 . N = 6 independent samples for each group . ANOVA followed by post - hoc Tukey was performed . * P = 0 . 02 PBS - PBS vs . PBS - H2O2 . * P = 0 . 0009 DFO - PBS vs . DFO - H2O2 . * P = 0 . 006 PBS - H2O2 vs . BPD - H2O2 . data are expressed as mean ± .

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F. Complex I activity in H9c2 cells treated with indicated iron chelators with or without H2O2. N=6 independent samples for each group. ANOVA followed by post-hoc Tukey test was performed. * P=0.0004 PBS-PBS vs. PBS-H2O2. * P=0.0003 DFO-PBS vs. DFO-H2O2. * P<0.0001 PBS- H2O2 vs. BPD-H2O2.G. Complex II activity in H9c2 cells treated with indicated iron chelators with or without H2O2. N=6 independent samples for each group. ANOVA followed by post-hoc Tukey test was performed. * P=0.0001 PBS-PBS vs. PBS-H2O2. * P<0.0001 DFO-PBS vs. DFO-H2O2. * P<0.0001 PBS- H2O2 vs. BPD-H2O2.H. Complex IV activity in H9c2 cells treated with indicated iron chelators with or without H2O2. N=6 independent samples for each group. ANOVA followed by post-hoc Tukey test was performed. * P=0.02 PBS-PBS vs. PBS-H2O2. * P=0.0009 DFO-PBS vs. DFO-H2O2. * P=0.006 PBS- H2O2 vs. BPD-H2O2. All data are expressed as mean ± SEM.


Adult Swiss mice ( n = 11 Veh ; 15 AβOs ) received a single i . c . v . injection of vehicle or 10 pmol AβOs were assessed in a glucose tolerance ( 2 g glucose / kg , i . p . ) 7 days after injection . Blood of glucose were measured at several time points following glucose administration . Bar graph represents areas under curves in time course plot . Data are representative of three independent experiments with similar results . Left panel : * * * P = 0 . 0006 , two - way ANOVA followed by Bonferroni post hoc ; right panel : * P = 0 . 0207 , Student ' s t - .

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Adult Swiss mice (n = 11 Veh; 15 AβOs) received a single i.c.v. injection of vehicle or 10 pmol AβOs and were assessed in a glucose tolerance test (2 g glucose/kg body weight, i.p.) 7 days after injection. Blood levels of glucose were measured at several time points following glucose administration. Bar graph represents areas under the curves in the time course plot. Data are representative of three independent experiments with similar results. Left panel: ***P = 0.0006, two-way ANOVA followed by Bonferroni post hoc test; right panel: *P = 0.0207, Student's t-test.


Insulin tolerance ( 1 IU insulin / kg , i . p . ) ( n = 7 Veh ; 8 AβOs ) . Blood of glucose were measured at several time points following insulin administration . Bar graph represents kinetic constants for glucose disappearance ( Kitt ) calculated from time course plot . Data are representative of two independent experiments with similar results . Left panel : * P = 0 . 0456 * * * P = 0 . 0007 , two - way ANOVA followed by Bonferroni post hoc ; right panel : * * P = 0 . 0033 , Student ' s t - .

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Insulin tolerance test (1 IU insulin/kg body weight, i.p.) (n = 7 Veh; 8 AβOs). Blood levels of glucose were measured at several time points following insulin administration. Bar graph represents the kinetic constants for glucose disappearance (Kitt) calculated from the time course plot. Data are representative of two independent experiments with similar results. Left panel: *P = 0.0456 and ***P = 0.0007, two-way ANOVA followed by Bonferroni post hoc test; right panel: **P = 0.0033, Student's t-test.


Glucose tolerance ( 2 g glucose / kg , i . p . ) in mice 7 days after a single intracaudal ( C ; n = 8 / group ) or intraperitoneal ( D ; n = 13 / group ) injection of AβOs ( 10 pmol ) or vehicle .

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Glucose tolerance test (2 g glucose/kg body weight, i.p.) in mice 7 days after a single intracaudal (C; n = 8 animals/group) or intraperitoneal (D; n = 13 animals/group) injection of AβOs (10 pmol) or vehicle.


Glucose tolerance ( 2 g glucose / kg , i . p . ) in 8 - to 13 - month - old APP / PS1 mice ( E ; n = 9 / group ) or 6 - month - old 3xTg - AD mice ( F ; n = 10 WT ; 9 3xTg ) , or their corresponding wild - type . Bar graph represents areas under curves in time course plots . In ( E ) , left panel : * P = 0 . 0466 , two - way ANOVA followed by Bonferroni post hoc ; right panel : & amp ; amp ; amp ; P = 0 . 072 , Student ' s t - . In ( F ) , left panel : * P = 0 . 0171 # P = 0 . 0781 , two - way ANOVA followed by Bonferroni post hoc ; right panel : * P = 0 . 0101 , Student ' s t - .

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Glucose tolerance test (2 g glucose/kg body weight, i.p.) in 8- to 13-month-old APP/PS1 mice (E; n = 9 animals/group) or 6-month-old 3xTg-AD male mice (F; n = 10 WT; 9 3xTg), or their corresponding wild-type littermates. Bar graph represents areas under the curves in the time course plots. In (E), left panel: *P = 0.0466, two-way ANOVA followed by Bonferroni post hoc test; right panel: &amp;amp;P = 0.072, Student's t-test. In (F), left panel: *P = 0.0171 and #P = 0.0781, two-way ANOVA followed by Bonferroni post hoc test; right panel: *P = 0.0101, Student's t-test.


CD68 immunoreactivity in adipose tissue ( scale bar = 25 μm , representative of one each from a total of four per experimental group ) . Arrow points to a region stained with CD68 antibody . * P = 0 . 0109 , Student ' s t - .

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CD68 immunoreactivity in white adipose tissue (scale bar = 25 μm, images representative of one animal each from a total of four animals per experimental group). Arrow points to a region stained with CD68 antibody. *P = 0.0109, Student's t-test.


Epididymal fat mass was analyzed in mice ( n = 6 / group ) 7 days after i . c . v . injection of vehicle or AβOs . Data are representative of three independent experiments with similar results . * P = 0 . 0255 .

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Epididymal fat mass was analyzed in mice (n = 6 animals/group) 7 days after i.c.v. injection of vehicle or AβOs. Data are representative of three independent experiments with similar results. *P = 0.0255.


Relative expression of leptin ( C ) , TNF - α ( D ) IL - 6 ( E ) , respectively , in adipose tissue of mice ( n = 7 Veh ; 9 AβOs ) 7 days after i . c . v . injection of vehicle or AβOs . In ( C ) , * P = 0 . 0394 ; in ( D ) , * * P = 0 . 0038 ; in ( E ) , * P = 0 . 0305 ; Student ' s t - .

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Relative expression of leptin (C), TNF-α (D) and IL-6 (E), respectively, in white adipose tissue of mice (n = 7 Veh; 9 AβOs) 7 days after i.c.v. injection of vehicle or AβOs. In (C), *P = 0.0394; in (D), **P = 0.0038; in (E), *P = 0.0305; Student's t-test.


p - JNK ( F ; n = 5 / group ) IRS - 1pSer312 ( G ; n = 6 / group ) ( normalized by total JNK total IRS - 1 , respectively ) in skeletal muscle of mice 7 days after i . c . v . injection of vehicle or AβOs . In ( F ) , * P = 0 . 0464 ; in ( G ) , * P = 0 . 0081 ; Student ' s t - .

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p-JNK (F; n = 5 animals/group) and IRS-1pSer312 (G; n = 6 animals/group) levels (normalized by total JNK and total IRS-1, respectively) in skeletal muscle of mice 7 days after i.c.v. injection of vehicle or AβOs. In (F), *P = 0.0464; in (G), *P = 0.0081; Student's t-test.


Representative of GLUT - 4 immunofluorescence in insulin - stimulated skeletal muscle from mice that were i . c . v . - injected with vehicle ( Veh ) or 10 pmol AβOs . Bar graphs show of GLUT - 4 surface immunoreactivity in skeletal muscle of mice that received intraperitoneal injections of PBS or insulin ( 1 IU / kg ) 7 days after i . c . v . injection of vehicle or AβOs , as indicated ( n = 5 / group ) . Scale bar = 25 μm . * P = 0 . 0144 , one - way ANOVA followed by Bonferroni post hoc .

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Representative images of GLUT-4 immunofluorescence in insulin-stimulated skeletal muscle from mice that were i.c.v.-injected with vehicle (Veh) or 10 pmol AβOs. Bar graphs show quantification of GLUT-4 surface immunoreactivity in skeletal muscle of mice that received intraperitoneal injections of PBS or insulin (1 IU/kg body weight) 7 days after i.c.v. injection of vehicle or AβOs, as indicated (n = 5 animals/group). Scale bar = 25 μm. *P = 0.0144, one-way ANOVA followed by Bonferroni post hoc test.


GLUT - 4 mRNA ( n = 4 / group ) total ( normalized to actin ; n = 5 Veh ; 6 AβOs ) were unchanged in skeletal muscle of Swiss mice injected with vehicle ( Veh ) or 10 pmol AβOs .

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GLUT-4 mRNA (n = 4 animals/group) and total protein levels (normalized to actin levels; n = 5 Veh; 6 AβOs) were unchanged in skeletal muscle of Swiss mice injected with vehicle (Veh) or 10 pmol AβOs.


Plasma of insulin ( K ; n = 12 / group ) , leptin ( L ; n = 11 Veh ; 12 AβOs ) , cholesterol ( M ; n = 8 Veh ; 6 AβOs ) , triglycerides ( N ; n = 8 Veh ; 6 AβOs ) or noradrenaline ( O ; n = 7 Veh ; 8 AβOs ) measured 7 days after i . c . v . injection of vehicle ( Veh ) or 10 pmol AβOs . In ( O ) , * P = 0 . 0361 , Student ' s t - .

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Plasma levels of insulin (K; n = 12 animals/group), leptin (L; n = 11 Veh; 12 AβOs), cholesterol (M; n = 8 Veh; 6 AβOs), triglycerides (N; n = 8 Veh; 6 AβOs) or noradrenaline (O; n = 7 Veh; 8 AβOs) measured 7 days after i.c.v. injection of vehicle (Veh) or 10 pmol AβOs. In (O), *P = 0.0361, Student's t-test.


Representative immunocytochemistry of mature hypothalamic neurons in culture exposed to vehicle ( Veh ) or AβOs ( 500 nM ) for 3 h . Binding of AβOs was detected using anti - oligomer monoclonal antibody NU4 ( red ) . Neurons were double - labeled using MAP - 2 antibody ( green ) . represent typical results from experiments with three independent hypothalamic cultures ( three coverslips / experimental condition per independent experiment ) . Scale bar = 30 10 μm for main panels insets , respectively .

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Representative immunocytochemistry images of mature hypothalamic neurons in culture exposed to vehicle (Veh) or AβOs (500 nM) for 3 h. Binding of AβOs was detected using anti-oligomer monoclonal antibody NU4 (red). Neurons were double-labeled using MAP-2 antibody (green). Images represent typical results from experiments with three independent hypothalamic cultures (three coverslips/experimental condition per independent experiment). Scale bar = 30 and 10 μm for main panels and insets, respectively.


Representative immunocytochemistry image of mature hypothalamic culture exposed to AβOs ( 500 nM ) immunolabeled with anti - GFAP ( green ) NU4 ( red ) antibodies . Insets show AβOs binding to neuronal dendrites , whereas no binding was detected to GFAP - positive .

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Representative immunocytochemistry image of mature hypothalamic culture exposed to AβOs (500 nM) and immunolabeled with anti-GFAP (green) and NU4 (red) antibodies. Insets show AβOs binding to neuronal dendrites, whereas no binding was detected to GFAP-positive cells.


Representative DCFfluorescence from hypothalamic neuronalcultures exposed to vehicle or AβOs ( 500 nM , 4 h ) . Insets show optical zoom of indicated areas . Scale bars = 100 50 μm for main panels insets , respectively . Graph shows integrated DCFfluorescence intensities ( relative units ; see Materials Methods ) ( n = 3 independent hypothalamic cultures ; three wells / experimental condition per experiment ; three acquired per well ) . Bars represent means ± . # P = 0 . 0604 , one - sample t - compared with a fixed value of 100 RUs .

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Representative DCFfluorescence images from hypothalamic neuronalcultures exposed to vehicle or AβOs (500 nM, 4 h). Insets show optical zoom images of the indicated areas. Scale bars = 100 and 50 μm for main panels and insets, respectively. Graph shows integrated DCFfluorescence intensities (relative units; see Materials and Methods) (n = 3 independent hypothalamic cultures; three wells/experimental condition per experiment; three images acquired per well). Bars represent means ± SEM. #P = 0.0604, one-sample t-test compared with a fixed value of 100 RUs.


LDH activity ( IU / l ) in culture media of hypothalamic cultures exposed to vehicle or AβOs ( 500 nM , 3 h ) .

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LDH activity (IU/l) in culture media of hypothalamic cultures exposed to vehicle or AβOs (500 nM, 3 h).