Figure 3 ( A ) Atg8WT - HA , Atg8F77A - HA , Atg8F79A - HA or Atg8F77 / 79A - HA was transformed into Δatg8 cells . Cell extracts were prepared and subjected to 13 . 5 % SDS - polyacrylamide gel electrophoresis ( SDS - PAGE ) containing 6 M urea , followed by immunoblot analysis with anti - Atg8 antibodies . ( B ) Prediction of protein - protein interaction sites in Atg8 ( Neuvirth et al , 2004 ) . A three ‐ dimensional model of Atg8 is coloured according to ProMate prediction ( see supplementary information online ) with red representing the predicted protein - protein binding sites , and Phe 77 and Phe 79 are marked in green within the prediction site . ( C ) Pull ‐ down assay of Atg8 mutants with Atg4 . His - Atg4 was attached to cobalt beads ( Talon metal ‐ affinity resin ) , washed and incubated with total cell extracts of Atg8 mutants . Elution was subjected to SDS - PAGE followed by western blot analysis using anti ‐ Atg8 and anti ‐ Atg4 antibodies . ( D ) Localization of green fluorescent protein ( GFP ) - Atg8wt and GFP - Atg877 / 79 cells carrying Atg4 plasmid under normal ( control ) and starvation conditions . Arrows point to the pre ‐ autophagosomal structure , and arrowheads point to GFP within the vacuoles . ( E ) Aminopeptidase I ( API ) maturation was tested in cells incubated in control or starvation medium by western blot analysis using anti ‐ API antibodies . mAPI , mature aminopeptidase I ; pAPI , premature aminopeptidase I ; WT , wild type .

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

Figure 3(A) Atg8WT-HA, Atg8F77A-HA, Atg8F79A-HA or Atg8F77/79A-HA was transformed into Δatg8 cells. Cell extracts were prepared and subjected to 13.5% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) containing 6 M urea, followed by immunoblot analysis with anti-Atg8 antibodies.(B) Prediction of protein-protein interaction sites in Atg8 ( Neuvirth et al, 2004). A three‐dimensional model of Atg8 is coloured according to ProMate prediction ( see supplementary information online) with red representing the predicted protein-protein binding sites, and Phe 77 and Phe 79 are marked in green within the prediction site.(C) Pull‐down assay of Atg8 mutants with Atg4. His-Atg4 was attached to cobalt beads (Talon metal‐affinity resin), washed and incubated with total cell extracts of Atg8 mutants. Elution was subjected to SDS-PAGE followed by western blot analysis using anti‐Atg8 and anti‐Atg4 antibodies.(D) Localization of green fluorescent protein (GFP)-Atg8wt and GFP-Atg877/79 cells carrying Atg4 plasmid under normal (control) and starvation conditions. Arrows point to the pre‐autophagosomal structure, and arrowheads point to GFP within the vacuoles.(E) Aminopeptidase I (API) maturation was tested in cells incubated in control or starvation medium by western blot analysis using anti‐API antibodies. mAPI, mature aminopeptidase I; pAPI, premature aminopeptidase I; WT, wild type.


Figure 4 ( A ) Cell extracts were subjected to 13 . 5 % SDS - polyacrylamide gel electrophoresis containing 6 M urea , followed by immunoblot analysis using anti ‐ Atg8 ‐ 15N peptide antibodies . ( B ) Cells were grown in control or in starvation medium and visualized using fluorescence microscopy . Arrows point to the pre ‐ autophagosomal structure , whereas arrowheads point to GFP within the vacuoles . ( C ) Aminopeptidase I ( API ) maturation and GFP cleavage were tested as described above . Asterisk represents nonspecific band . GFP , green fluorescent protein ; mAPI , mature aminopeptidase I ; pAPI , premature aminopeptidase I ; WT , wild type .

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

Figure 4(A) Cell extracts were subjected to 13.5% SDS-polyacrylamide gel electrophoresis containing 6 M urea, followed by immunoblot analysis using anti‐Atg8‐15N peptide antibodies.(B) Cells were grown in control or in starvation medium and visualized using fluorescence microscopy. Arrows point to the pre‐autophagosomal structure, whereas arrowheads point to GFP within the vacuoles.(C) Aminopeptidase I (API) maturation and GFP cleavage were tested as described above. Asterisk represents nonspecific band. GFP, green fluorescent protein; mAPI, mature aminopeptidase I; pAPI, premature aminopeptidase I; WT, wild type.


Figure 5 ( A ) Δatg8 cells transformed with GFP - Atg8WT , GFP - Atg8L50A or GFP - Atg8Y49A constructs were grown to mid ‐ exponential phase and then incubated in the presence of 3 . 5 % galactose to induce expression of GFP - Atg8 . API maturation was tested using anti ‐ Atg8 ( right bottom panel ) , anti ‐ GFP ( left bottom panel ) or anti ‐ API antibodies . ( B ) Starvation ‐ induced protein degradation of Δatg8 harbouring the different mutants was tested as described above . API , aminopeptidase I ; GFP , green fluorescent protein ; mAPI , mature aminopeptidase I ; pAPI , premature aminopeptidase I ; WT , wild type .

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

Figure 5(A) Δatg8 cells transformed with GFP-Atg8WT, GFP-Atg8L50A or GFP-Atg8Y49A constructs were grown to mid‐exponential phase and then incubated in the presence of 3.5% galactose to induce expression of GFP-Atg8. API maturation was tested using anti‐Atg8 (right bottom panel), anti‐GFP (left bottom panel) or anti‐API antibodies.(B) Starvation‐induced protein degradation of Δatg8 harbouring the different mutants was tested as described above. API, aminopeptidase I; GFP, green fluorescent protein; mAPI, mature aminopeptidase I; pAPI, premature aminopeptidase I; WT, wild type.


Figure 6 ( A ) GFP - Atg8 localization was determined using fluorescence microscopy ( arrows depict pre ‐ autophagosomal structure , and arrowheads point to GFP within vacuoles ) . Cells were also tested for the appearance of autophagic bodies by using light microscopy . ( B ) Immunoblot analysis of different cell extracts was carried out using anti ‐ GFP antibodies ( asterisk represents nonspecific band ) . ( C ) Atg8WT - HA , Atg8L50A - HA or Atg8Y49A - HA was transformed into Δatg8 and Δatg8Δatg4 double knockout strains and cell extracts were subjected to SDS - polyacrylamide gel electrophoresis ( SDS - PAGE ) followed by immunoblot analysis using anti ‐ HA antibodies ( lower panel ; asterisk represents nonspecific band ) . ( D ) Atg8WT , Atg8L50A or Atg8Y49A was transformed into Δatg8 and Δatg8Δatg4 double knockout strains and grown under control or starvation conditions for 4 h . Cell extracts were subjected to 13 . 5 % SDS - PAGE containing 6 M urea , followed by immunoblot analysis using anti ‐ Atg8 ‐ N15 antibodies . GFP , green fluorescent protein ; WT , wild type .

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

Figure 6(A) GFP-Atg8 localization was determined using fluorescence microscopy (arrows depict pre‐autophagosomal structure, and arrowheads point to GFP within vacuoles). Cells were also tested for the appearance of autophagic bodies by using light microscopy.(B) Immunoblot analysis of different cell extracts was carried out using anti‐GFP antibodies (asterisk represents nonspecific band).(C) Atg8WT-HA, Atg8L50A-HA or Atg8Y49A-HA was transformed into Δatg8 and Δatg8Δatg4 double knockout strains and cell extracts were subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE) followed by immunoblot analysis using anti‐HA antibodies (lower panel; asterisk represents nonspecific band).(D) Atg8WT, Atg8L50A or Atg8Y49A was transformed into Δatg8 and Δatg8Δatg4 double knockout strains and grown under control or starvation conditions for 4 h. Cell extracts were subjected to 13.5% SDS-PAGE containing 6 M urea, followed by immunoblot analysis using anti‐Atg8‐N15 antibodies. GFP, green fluorescent protein; WT, wild type.


Figure 1 A Western blot analysis of LC3 in SH - SY5Y cells after IU1 treatment ( 100 μM , 24h ) . Graphs represent mean ± SEM . N = 8 B - D Electron microscope images were taken to quantify the number of autophagosomes and autolysosome present in control ( DMSO ) or IU1 treated cells . At least 55 cells were analysed from 3 different experiments . Graphs represent mean ± SEM E Western blot analysis of mitochondrial marker proteins ATP5a , HSP60 and normalised by actin after 24h of DMSO ( C ) or IU1 ( 100 μM ) treatment . N = 3 . Graphs represent mean ± SEM F - I Confocal images of cells transfected with mito - YFP and treated with DMSO ( F , control ) or IU1 ( G ) for 24h . Fragmentation index ( H ) and total mitochondrial volume / cell were measured and represented in ( I ) , mean ± SEM . At least 35 cells were analysed from 3 different experimental conditions . Scale bar 10 μm J - L Mito - YFP transfected cells were treated with IU1 ( 100 μM , 24h ) , fixed and immuno labelled for LC3 . Number of LC3 dots / cell ( K ) and LC3 dot overlapping mitochondria ( L ) were counted . At least 35 cells from 3 independent experiments were analysed . Scale bar : 10 μm M - N Cells were transfected with mito - keima and treated with DMSO or IU1 for 24h and subjected to FACS analysis , counting 10 , 000 cells per experiment . ( M ) represents the shift of the signal intensity in IU1 treated cells when excited at 560 nm ( false colour ) . ( N ) represents the average intensity of the mito - keima protein when excited at 560 nm and the ratio between 560 : 405 nm . Graphs represent mean ± SEM from 3 different sets of

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

Figure 1 A Western blot analysis of LC3 in SH-SY5Y cells after IU1 treatment (100 μM, 24h). Graphs represent mean± SEM. N= 8  B-D Electron microscope images were taken to quantify the number of autophagosomes and autolysosome present in control (DMSO) or IU1 treated cells. At least 55 cells were analysed from 3 different experiments. Graphs represent mean± SEM  E Western blot analysis of mitochondrial marker proteins ATP5a, HSP60 and normalised by actin after 24h of DMSO (C) or IU1 (100 μM) treatment. N=3. Graphs represent mean± SEM  F-I Confocal images of cells transfected with mito-YFP and treated with DMSO (F, control) or IU1 (G) for 24h. Fragmentation index (H) and total mitochondrial volume / cell were measured and represented in (I), mean ±SEM. At least 35 cells were analysed from 3 different experimental conditions. Scale bar 10 μm  J-L Mito-YFP transfected cells were treated with IU1 (100 μM, 24h), fixed and immuno labelled for LC3. Number of LC3 dots / cell (K) and LC3 dot overlapping mitochondria (L) were counted. At least 35 cells from 3 independent experiments were analysed. Scale bar: 10 μm  M-N Cells were transfected with mito-keima and treated with DMSO or IU1 for 24h and subjected to FACS analysis, counting 10,000 cells per experiment. (M) represents the shift of the signal intensity in IU1 treated cells when excited at 560 nm (false colour). (N) represents the average intensity of the mito-keima protein when excited at 560 nm and the ratio between 560: 405 nm. Graphs represent mean ± SEM from 3 different sets of experiments


Figure 2 A - B Western blot analysis of the indicated proteins in cell lysates from SH - SY5Y cells , treated with different concentrations of IU1 ( 1 - 100 μM ) for 24h . Bar graphs represents mean ± SEM . Blots are representative of three independent experiments . ANOVA followed by Dunnets test C - D Representative images of MEF cells knocked out for the indicated mitochondrial dynamics protein and transfected with mito - YFP ( C ) with / without IU1 treatment ( 100 μM , 48h ) . Scale bar 10 μm . ( D ) Mitochondrial volume / cell was measured as described and represented as mean ± SEM . At least 35 cells were evaluated E - F Western blot analysis of HSP60 and ATP5a , as mitochondrial marker in the indicated knock out MEF cells ( with / without the indicated DRP1 variant ) after 48h of IU1 treatment . Bar graphs represent mean ± SEM , n = at least 3 . Student ' s t test ;

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

Figure 2 A-B Western blot analysis of the indicated proteins in cell lysates from SH-SY5Y cells, treated with different concentrations of IU1 (1-100 μM) for 24h. Bar graphs represents mean ± SEM. Blots are representative of three independent experiments. ANOVA followed by Dunnets test  C-D Representative images of MEF cells knocked out for the indicated mitochondrial dynamics protein and transfected with mito-YFP (C) with / without IU1 treatment (100 μM, 48h). Scale bar 10 μm. (D) Mitochondrial volume / cell was measured as described and represented as mean ±SEM. At least 35 cells were evaluated E-F Western blot analysis of HSP60 and ATP5a, as mitochondrial marker in the indicated knock out MEF cells (with / without the indicated DRP1 variant) after 48h of IU1 treatment. Bar graphs represent mean ± SEM, n= at least 3. Student's t test;


Figure 3 A - D Wild type MEF ( A ) , Hela ( B ) and PINK1 KO MEF ( C ) cells were treated with IU1 ( 48h , 100 μM ) and electron microscope images were evaluated for autophagic vesicle formation . ( D ) bar graphs represents mean ± SEM number of autophagosomes and autolysosome / cell from 3 different set of experiments ; at least 40 cells were analysed from each group E Western blot analysis of HSP60 protein with or without IU1 treatment in the indicated cell type . Bar graphs represent mean ± SEM . N = at least 3 independent experiments . Student ' s t test . * P ≤ 0 . 05 , * * P ≤ 0 . 01

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

Figure 3 A-D Wild type MEF (A), Hela (B) and PINK1 KO MEF (C) cells were treated with IU1 (48h, 100 μM) and electron microscope images were evaluated for autophagic vesicle formation. (D) bar graphs represents mean± SEM number of autophagosomes and autolysosome / cell from 3 different set of experiments; at least 40 cells were analysed from each group E Western blot analysis of HSP60 protein with or without IU1 treatment in the indicated cell type. Bar graphs represent mean ± SEM. N= at least 3 independent experiments. Student's t test. *P ≤ 0.05, **P ≤ 0.01


Figure 4 A - B SH - SY5Y cells were immunostained for ATP5a ( green ) and 20S proteasome core ( red ) after IU1 treatment ( 6h - 24h ) . Scale bar 10 μm . Colour mapping of the co - localisation of HSP60 and 20S proteasome from the selected regions were generated by image j and represented in the bottom panel . Intensity of the co - localisation is represented in the colour map scale at the bottom of the panel . ( B ) 20S proteasome subunit and mitochondria co - localisation was quantified by measuring Mander ' s coefficient . Bar graphs represent mean fold change ( mean ± SEM ) from 3 different experiments . At least 30 cells were evaluated for the calculations . ANOVA followed by Dunnet ' s test C Electron microscopy images showing subcellular localization of immuno - gold labelled 20S subunit of proteasome complex after 12h or 24h of IU1 treatment . Experiments were repeated twice in two biological replicates with similar results . The contrast in the magnified region is enhanced from the original image to highlight the immunogold labelled signals D - E Protein proximity ligation assay for Prohibitin2 ( PHB2 ) and LC3 after 24h IU1 treatment . Images showing signal are merged with bright field images to count the signal dots ( cyan false colour ) as depicted in the figure . In the inset only fluorescence signal is shown , without merging with the bright field from the selected regions . Scale bar 10 μm . ( E ) Bar graphs represent mean ± SEM of the number of signal dots per cell from 3 different experiments . Student ' s t test ; * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001

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

Figure 4 A-B SH-SY5Y cells were immunostained for ATP5a (green) and 20S proteasome core (red) after IU1 treatment (6h-24h). Scale bar 10 μm. Colour mapping of the co-localisation of HSP60 and 20S proteasome from the selected regions were generated by image j and represented in the bottom panel. Intensity of the co-localisation is represented in the colour map scale at the bottom of the panel. (B) 20S proteasome subunit and mitochondria co-localisation was quantified by measuring Mander's coefficient. Bar graphs represent mean fold change (mean ± SEM) from 3 different experiments. At least 30 cells were evaluated for the calculations. ANOVA followed by Dunnet's test  C Electron microscopy images showing subcellular localization of immuno- gold labelled 20S subunit of proteasome complex after 12h or 24h of IU1 treatment. Experiments were repeated twice in two biological replicates with similar results. The contrast in the magnified region is enhanced from the original image to highlight the immunogold labelled signals  D-E Protein proximity ligation assay for Prohibitin2 (PHB2) and LC3 after 24h IU1 treatment. Images showing signal are merged with bright field images to count the signal dots (cyan false colour) as depicted in the figure. In the inset only fluorescence signal is shown, without merging with the bright field from the selected regions. Scale bar 10 μm. (E) Bar graphs represent mean ±SEM of the number of signal dots per cell from 3 different experiments. Student's t test; *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001


Figure 5Electron microscope images of mitochondria from SH - SY5Y e , as indicated in the figure , with / without USP14 inhibition / knock down . Arrow heads in the IU1 treated groups indicate the mitochondrial rupture point . Bar graphs represent mean ( ± SEM ) of percentage ruptured mitochondria from at least 3 different experiments . For cells , total 150 - 300 mitochondria for the control group and 150 - 190 mitochondria from the IU1 treated groups were countedRepresentative immunoblot of isolated mitochondria from the indicated groups treated with / without trypsin and probed for HSP60 , ATP5a and TOM20 . Bar graphs represent mean ± SEM of the ratio of densitometric levels of the indicated proteins ( with : without trypsin ) from at least 3 independent experimentsElectron microscope images of mitochondria fro MEF WT , MEF PINK1 K as indicated in the figure , with / without USP14 inhibition / knock down . Arrow heads in the IU1 treated groups indicate the mitochondrial rupture point . Bar graphs represent mean ( ± SEM ) of percentage ruptured mitochondria from at least 3 different experiments . For cells , total 150 - 300 mitochondria for the control group and 150 - 190 mitochondria from the IU1 treated groups were countedRepresentative immunoblot of isolated mitochondria from the indicated groups treated with / without trypsin and probed for HSP60 , ATP5a and TOM20 . Bar graphs represent mean ± SEM of the ratio of densitometric levels of the indicated proteins ( with : without trypsin ) from at least 3 independent experimentsElectron microscope images of mitochondria fro Hela an e , as indicated in the figure , with / without USP14 inhibition / knock down . Arrow heads in the IU1 treated groups indicate the mitochondrial rupture point . Bar graphs represent mean ( ± SEM ) of percentage ruptured mitochondria from at least 3 different experiments . For cells , total 150 - 300 mitochondria for the control group and 150 - 190 mitochondria from the IU1 treated groups were countedRepresentative immunoblot of isolated mitochondria from the indicated groups treated with / without trypsin and probed for HSP60 , ATP5a and TOM20 . Bar graphs represent mean ± SEM of the ratio of densitometric levels of the indicated proteins ( with : without trypsin ) from at least 3 independent experimentsElectron microscope images of mitochondria fro Drosophila thoracic muscle , as indicated in the figure , with / without USP14 inhibition / knock down . Arrow heads in the IU1 treated groups indicate the mitochondrial rupture point . Bar graphs represent mean ( ± SEM ) of percentage ruptured mitochondria from at least 3 different experiments For flies , more than 200 mitochondria from each group were counted from 5 different experimentsRepresentative immunoblot of isolated mitochondria from the indicated groups treated with / without trypsin and probed for HSP60 , ATP5a and TOM20 . Bar graphs represent mean ± SEM of the ratio of densitometric levels of the indicated proteins ( with : without trypsin ) from at least 3 independent

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

Figure 5Electron microscope images of mitochondria from SH-SY5Y e, as indicated in the figure, with / without USP14 inhibition/knock down. Arrow heads in the IU1 treated groups indicate the mitochondrial rupture point. Bar graphs represent mean (± SEM) of percentage ruptured mitochondria from at least 3 different experiments. For cells, total 150-300 mitochondria for the control group and 150-190 mitochondria from the IU1 treated groups were countedRepresentative immunoblot of isolated mitochondria from the indicated groups treated with / without trypsin and probed for HSP60, ATP5a and TOM20. Bar graphs represent mean ± SEM of the ratio of densitometric levels of the indicated proteins (with : without trypsin) from at least 3 independent experimentsElectron microscope images of mitochondria fro MEF WT, MEF PINK1 K as indicated in the figure, with / without USP14 inhibition/knock down. Arrow heads in the IU1 treated groups indicate the mitochondrial rupture point. Bar graphs represent mean (± SEM) of percentage ruptured mitochondria from at least 3 different experiments. For cells, total 150-300 mitochondria for the control group and 150-190 mitochondria from the IU1 treated groups were countedRepresentative immunoblot of isolated mitochondria from the indicated groups treated with / without trypsin and probed for HSP60, ATP5a and TOM20. Bar graphs represent mean ± SEM of the ratio of densitometric levels of the indicated proteins (with : without trypsin) from at least 3 independent experimentsElectron microscope images of mitochondria fro Hela an e, as indicated in the figure, with / without USP14 inhibition/knock down. Arrow heads in the IU1 treated groups indicate the mitochondrial rupture point. Bar graphs represent mean (± SEM) of percentage ruptured mitochondria from at least 3 different experiments. For cells, total 150-300 mitochondria for the control group and 150-190 mitochondria from the IU1 treated groups were countedRepresentative immunoblot of isolated mitochondria from the indicated groups treated with / without trypsin and probed for HSP60, ATP5a and TOM20. Bar graphs represent mean ± SEM of the ratio of densitometric levels of the indicated proteins (with : without trypsin) from at least 3 independent experimentsElectron microscope images of mitochondria fro Drosophila thoracic muscle, as indicated in the figure, with / without USP14 inhibition/knock down. Arrow heads in the IU1 treated groups indicate the mitochondrial rupture point. Bar graphs represent mean (± SEM) of percentage ruptured mitochondria from at least 3 different experiments For flies, more than 200 mitochondria from each group were counted from 5 different experimentsRepresentative immunoblot of isolated mitochondria from the indicated groups treated with / without trypsin and probed for HSP60, ATP5a and TOM20. Bar graphs represent mean ± SEM of the ratio of densitometric levels of the indicated proteins (with : without trypsin) from at least 3 independent experiments


Figure 6 A Life span analysis of flies of the indicated genetic background . At least 50 flies were monitored from each group . Log - rank test ( Mantel - Cox test , Mantel - Haenszel test , p < 0 . 0001 ) B Graph bar shows mean ± SEM of the climbing performance of flies of the indicated genotypes . n = 4 - 7 . ANOVA followed by Newman Keuls test C Quantitative analysis of respiratory fitness of isolated mitochondria extracted from flies of the indicated genotypes . Graph shows respiratory control rate ( RCR ) calculated as described in materials and methods . Graphs represent mean ± SEM . N = 4 - 5 , ANOVA followed by Newman Keuls test D Dopamine content in the fly heads ( 15 day old ) was measured by HPLC and the individual fold change values are represented in the scatter graph . Graphs represent mean ± SEM . N = 14 , 11 and 11 fly heads for control , PINK1 and PINK1 + USP14 KD fly respectively . ANOVA followed by Newman Keuls test E Enlarged TEM representative images of flight muscle mitochondria of the indicated genotypes . The experiment was repeated 3 times in replicates . Bar graphs represent quantification of electron dense mitochondria ( represented as grey bar ) and depolarised mitochondrial with ruptured cristae ( represented as white bars ) from the indicated genotypes

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

Figure 6 A Life span analysis of flies of the indicated genetic background. At least 50 flies were monitored from each group. Log-rank test (Mantel-Cox test, Mantel-Haenszel test, p < 0.0001)  B Graph bar shows mean± SEM of the climbing performance of flies of the indicated genotypes. n=4-7. ANOVA followed by Newman Keuls test  C Quantitative analysis of respiratory fitness of isolated mitochondria extracted from flies of the indicated genotypes. Graph shows respiratory control rate (RCR) calculated as described in materials and methods. Graphs represent mean± SEM. N=4-5, ANOVA followed by Newman Keuls test  D Dopamine content in the fly heads (15 day old) was measured by HPLC and the individual fold change values are represented in the scatter graph. Graphs represent mean± SEM. N= 14, 11 and 11 fly heads for control, PINK1 and PINK1+USP14 KD fly respectively. ANOVA followed by Newman Keuls test  E Enlarged TEM representative images of flight muscle mitochondria of the indicated genotypes. The experiment was repeated 3 times in replicates. Bar graphs represent quantification of electron dense mitochondria (represented as grey bar) and depolarised mitochondrial with ruptured cristae (represented as white bars) from the indicated genotypes


Figure 7 ( A ) Life span analysis of flies of the indicated genetic background . At least 50 flies were analysed for each group . Log - rank test ( Mantel - Cox test , Mantel - Haenszel test , p < 0 . 0001 ) ( B ) Graph bar shows mean ± SEM of the climbing performance of flies of the indicated genotypes . One way ANOVA , Newman Keuls test , n = 3 ( C ) Quantitative analysis of respiratory fitness of isolated mitochondria extracted from flies of the indicated genotypes . Graph shows ( mean ± SEM , n = 3 independent experiments ) respiratory control rate ( RCR ) calculated as described . One way ANOVA , Newman Keuls test ( D ) Enlarged TEM representative images of flight muscle mitochondria of the indicated genotypes . The experiment was repeated three times in replicates . Bar graphs represent quantification of electron dense mitochondria ( represented as grey bar ) and mitochondrial with ruptured cristae ( represented as white bars ) from the indicated genotypes

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Figure 7 (A) Life span analysis of flies of the indicated genetic background. At least 50 flies were analysed for each group. Log-rank test (Mantel-Cox test, Mantel-Haenszel test, p < 0.0001)  (B) Graph bar shows mean± SEM of the climbing performance of flies of the indicated genotypes. One way ANOVA, Newman Keuls test, n=3  (C) Quantitative analysis of respiratory fitness of isolated mitochondria extracted from flies of the indicated genotypes. Graph shows (mean± SEM, n=3 independent experiments) respiratory control rate (RCR) calculated as described. One way ANOVA, Newman Keuls test  (D) Enlarged TEM representative images of flight muscle mitochondria of the indicated genotypes. The experiment was repeated three times in replicates. Bar graphs represent quantification of electron dense mitochondria (represented as grey bar) and mitochondrial with ruptured cristae (represented as white bars) from the indicated genotypes


Figure 1 , B . survival of Ssrp1fl / fl ; CreERT2 + / + mice of different ages ( weeks ) after the start of tamoxifen ( TMX ) treatment . B . Kaplan - Meier survival curves . Log - rank test p = 0 . 032 for the oldest male age group versus all other male groups ; p = 0 . 013 for all females versus all males ; for all other comparisons p > 0 . 05 . n = 6 - 10 mice . C . Normalized blood cell counts in Ssrp1fl / fl ; CreERT2 + / + mice treated with vehicle or tamoxifen on day 7 after start of treatment . Mean ± SD , * indicates p < 0 . 05 ( unpaired t - test ) . n = 3 mice .

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Figure 1, B. survival of Ssrp1fl/fl; CreERT2+/+ mice of different ages (weeks) after the start of tamoxifen (TMX) treatment. B. Kaplan-Meier survival curves. Log-rank test p = 0.032 for the oldest male age group versus all other male groups; p = 0.013 for all females versus all males; for all other comparisons p > 0.05. n = 6-10 mice.C. Normalized blood cell counts in Ssrp1fl/fl; CreERT2+/+ mice treated with vehicle or tamoxifen on day 7 after start of treatment. Mean ± SD, * indicates p < 0.05 (unpaired t-test). n = 3 mice.


Figure 2A . H & E staining of mouse organ sections . Scale bars : spleen , colon 100 μM , bone marrow - 50 μM .

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Figure 2A. H&E staining of mouse organ sections. Scale bars: spleen, colon 100 μM, bone marrow - 50 μM.


Figure 3A , B . Effect of tamoxifen on replication in the spleen ( A ) and intestine ( B ) of Ssrp1fl / fl ; CreERT2 + / + mice . EdU was given to mice three days after the end of tamoxifen treatment ( 1 hour before tissue collection ) . Organ sections were stained for total DNA ( Hoechst ) , EdU , and SSRP1 . Scale bars - 100 μM . C . Quantitation of EdU staining . Mean number of EdU positive cells per field of view + / - SD . n > 5 fields of view per mouse . 3 mice were analyzed . D . Quantitation of cleaved caspase - 3 staining . Mean number of positive cells per fields of view + / - SD , n > 5 fields of view per mouse Two male and female mice were analyzed . Data information : P - values for C and D were calculated using the unpaired t - test .

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Figure 3A, B. Effect of tamoxifen on replication in the spleen (A) and intestine (B) of Ssrp1fl/fl; CreERT2+/+ mice. EdU was given to mice three days after the end of tamoxifen treatment (1 hour before tissue collection). Organ sections were stained for total DNA (Hoechst), EdU, and SSRP1. Scale bars - 100 μM. C. Quantitation of EdU staining. Mean number of EdU positive cells per field of view +/- SD. n > 5 fields of view per mouse. 3 mice were analyzed. D. Quantitation of cleaved caspase-3 staining. Mean number of positive cells per fields of view +/- SD, n > 5 fields of view per mouse Two male and female mice were analyzed. Data information: P-values for C and D were calculated using the unpaired t-test.


Figure 4D . Fold - change in the proportion of cells in each cluster shown in panel A in samples from tamoxifen - treated versus vehicle - treated mice . Cell types are identified by color as in panel B .

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Figure 4D. Fold-change in the proportion of cells in each cluster shown in panel A in samples from tamoxifen-treated versus vehicle-treated mice. Cell types are identified by color as in panel B.


Figure 5D . UMAP plots with color - coded intestinal stem cells identified by transcriptional signatures of intestinal stem cells or by expression of Lgr5 or Gkn3 genes .

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Figure 5D. UMAP plots with color-coded intestinal stem cells identified by transcriptional signatures of intestinal stem cells or by expression of Lgr5 or Gkn3 genes.


Figure 6A , B . Effect of 4 - OHT administration on colony formation by bone marrow cells of different genotypes . A . Photographs of colonies from bone marrow cells kept in culture for seven days in the presence or absence of 4 - OHT . B . Quantitation of bone marrow colony assay . Mean colony number per plate ± SD , n = 3 plates per mouse . P values < 0 . 05 are shown ( unpaired t - test ) . 2 mice of each genotype were analyzed . C , D . Growth of intestinal organoids in the presence or absence of 4 - OHT . C . Photographs of small intestine organoids on different days after plating . D . Number of organoids in the presence and absence of 4 - OHT . Mean fold change between number of organoids in control and 4 - OHT treated wells ± SD , n = 4 wells per mouse . P < 0 . 05 are shown ( unpaired t - test ) . Organoids were isolated from one mouse of each genotype in two independent experiments . F , G . Effect of 4 - OHT administration on intestinal organoids of different genotypes kept in the presence ( undifferentiated ) or absence ( differentiated ) of R - spondin - 1 . F . The viability of organoids assessed by the resorufin assay . Mean ± SD , n = 3 wells per mouse . P < 0 . 05 are shown ( unpaired t - test ) . Organoids were isolated from two mice of each genotype . G . IF images of intestinal organoids from Ssrp1fl / fl ; CreERT2 + / + mice kept in the presence or absence of R - Spondin - 1 . The organoids were stained with antibodies to LGR5 and SSRP1 and counterstained with Hoechst ( DNA ) . Effect of 4 - OHT administration on MSCs and their differentiated counterparts . H . Methylene blue viability assay for undifferentiated MSCs or MSCs differentiated into osteocytes or adipocytes for 21 days and then treated with vehicle or 4 - OHT . Mean ± SD , n = 3 plates per mouse . P < 0 . 05 are shown ( unpaired t - test ) . MSCs were isolated from two mice of each genotype .

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Figure 6A, B. Effect of 4-OHT administration on colony formation by bone marrow cells of different genotypes. A. Photographs of colonies from bone marrow cells kept in culture for seven days in the presence or absence of 4-OHT. B. Quantitation of bone marrow colony assay. Mean colony number per plate ± SD, n = 3 plates per mouse. P values < 0.05 are shown (unpaired t-test). 2 mice of each genotype were analyzed. C, D. Growth of intestinal organoids in the presence or absence of 4-OHT. C. Photographs of small intestine organoids on different days after plating. D. Number of organoids in the presence and absence of 4-OHT. Mean fold change between number of organoids in control and 4-OHT treated wells ± SD, n = 4 wells per mouse. P < 0.05 are shown (unpaired t-test). Organoids were isolated from one mouse of each genotype in two independent experiments.F, G. Effect of 4-OHT administration on intestinal organoids of different genotypes kept in the presence (undifferentiated) or absence (differentiated) of R-spondin-1. F. The viability of organoids assessed by the resorufin assay. Mean ± SD, n = 3 wells per mouse. P < 0.05 are shown (unpaired t-test). Organoids were isolated from two mice of each genotype. G. IF images of intestinal organoids from Ssrp1fl/fl; CreERT2+/+ mice kept in the presence or absence of R-Spondin-1. The organoids were stained with antibodies to LGR5 and SSRP1 and counterstained with Hoechst (DNA).Effect of 4-OHT administration on MSCs and their differentiated counterparts. H. Methylene blue viability assay for undifferentiated MSCs or MSCs differentiated into osteocytes or adipocytes for 21 days and then treated with vehicle or 4-OHT. Mean ± SD, n = 3 plates per mouse. P < 0.05 are shown (unpaired t-test). MSCs were isolated from two mice of each genotype.


Figure 7A . EU incorporation measured by flow cytometry . Histograms of EU fluorescent signals from two biological replicates for each condition ( except no EU ) . B . SSRP1 staining of the cells in A .

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Figure 7A. EU incorporation measured by flow cytometry. Histograms of EU fluorescent signals from two biological replicates for each condition (except no EU). B. SSRP1 staining of the cells in A.


Figure 8G . Box plots of mean read density ( y - axis ) at the gene body ( bins 21 - 100 out of 100 between TSS - TES ) for all genes and genes expressed at different levels . * * * - p < 1e - 10 ( paired t - test ) . n = 2 biological replicates . Central band is mean , box is interquartile range , whiskers are minimum and maximum . H . Comparison of Hoechst 33342 binding to live control and 4 - OHT - treated MSCs from different genotypes . Data are presented as the median fluorescence intensity ± SD , n = 4 biological replicates . P < 0 . 05 is shown ( unpaired t - test ) .

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Figure 8G. Box plots of mean read density (y-axis) at the gene body (bins 21-100 out of 100 between TSS - TES) for all genes and genes expressed at different levels. *** - p < 1e-10 (paired t-test). n = 2 biological replicates. Central band is mean, box is interquartile range, whiskers are minimum and maximum.H. Comparison of Hoechst 33342 binding to live control and 4-OHT-treated MSCs from different genotypes. Data are presented as the median fluorescence intensity ± SD, n = 4 biological replicates. P < 0.05 is shown (unpaired t-test).


Figure 1SUD binds to Paip1 and Paip1M with the same binding intensity . n . s . : not significant . The Mac2 subdomain is crucial for interacting with Paip1M .

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Figure 1SUD binds to Paip1 and Paip1M with the same binding intensity. n. s.: not significant.The Mac2 subdomain is crucial for interacting with Paip1M.


Figure 3Δ16 - Mac2 cannot bind Paip1M , Mac1 − 2 binds to Paip1M in the split - YFP assay . Scale bars represent 50 µM .

Figure 3Δ16-Mac2 cannot bind Paip1M, Mac1−2 binds to Paip1M in the split-YFP assay. Scale bars represent 50 µM.


Figure 4SUD and PABP do not compete with each other for interacting with Paip1 . ( A ) Excess expression of SUD - RFP does not impair the split - YFP signal between PABP and Paip1 ( lower images ) , compared to the control ( upper images ) . ( B ) Co - expression of excess RFP - PABP does not result in an impaired interaction between Paip1 and SUD either . RFP : red fluorescent protein . Scale bars represent 50 µM . GFP - trap - based co - immunoprecipitation ( CoIP ) assay demonstrating a complex formed by PABP , Paip1 , and SUD . GFP : green fluorescent protein .

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Figure 4SUD and PABP do not compete with each other for interacting with Paip1. (A) Excess expression of SUD-RFP does not impair the split-YFP signal between PABP and Paip1 (lower images), compared to the control (upper images). (B) Co-expression of excess RFP-PABP does not result in an impaired interaction between Paip1 and SUD either. RFP: red fluorescent protein. Scale bars represent 50 µM.GFP-trap-based co-immunoprecipitation (CoIP) assay demonstrating a complex formed by PABP, Paip1, and SUD. GFP: green fluorescent protein.


Figure 5Left image : SUD generally stimulates protein translation level detected by the luciferase - pcDNA3 reporter ( n = 6 ) . Middle image : SUD does not increase the amounts of total protein synthesis ( host and viral proteins ) in the replicon - transfected cells . Right image : SUD augments viral protein synthesis ( n = 8 ) . HEK - 293 cells growing in 12 - well plates were transfected with the indicated plasmids and replicon DNA . Twenty - four hours post - transfection , cells were harvested for Renilla luciferase activity measurement ( left and right image ) . For the ribopuromycylation assay , 24 hours post - transfection cells were pulsed with 3 µM puromycin for 1 hour at 37 ° C before harvesting for western blot analysis ( middle image ) .

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Figure 5Left image: SUD generally stimulates protein translation level detected by the luciferase-pcDNA3 reporter (n=6). Middle image: SUD does not increase the amounts of total protein synthesis (host and viral proteins) in the replicon-transfected cells. Right image: SUD augments viral protein synthesis (n=8). HEK-293 cells growing in 12-well plates were transfected with the indicated plasmids and replicon DNA. Twenty-four hours post-transfection, cells were harvested for Renilla luciferase activity measurement (left and right image). For the ribopuromycylation assay, 24 hours post-transfection cells were pulsed with 3 µM puromycin for 1 hour at 37°C before harvesting for western blot analysis (middle image).


Figure 6SARS - CoV - 2 SUD ( SARS2 - SUD ) interacts with Paip1 in HEK - 293 cells as demonstrated by the split - YFP assay . SARS2 - Mac2 binds to Paip1 . SARS2 - Mac2 but not Δ16 - SARS2 - Mac2 interacts with Paip1M . MERS - CoV SUD - MC ( Mac3 − DPUP ) or XMC ( Mac1 − Mac3 − DPUP ) do not bind to Paip1 . Scale bars represent 50 µM .

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Figure 6SARS-CoV-2 SUD (SARS2-SUD) interacts with Paip1 in HEK-293 cells as demonstrated by the split-YFP assay.SARS2-Mac2 binds to Paip1. SARS2-Mac2 but not Δ16-SARS2-Mac2 interacts with Paip1M.MERS-CoV SUD-MC (Mac3−DPUP) or XMC (Mac1−Mac3−DPUP) do not bind to Paip1. Scale bars represent 50 µM.


Figure 1Median CSF protein abundance distribution as calculated from MS - intensities of quantified peptides of each protein . The top ten most abundant proteins and hemoglobin proteins are highlighted .

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Figure 1Median CSF protein abundance distribution as calculated from MS-intensities of quantified peptides of each protein. The top ten most abundant proteins and hemoglobin proteins are highlighted.


Figure 2 A - C Protein AD / non - AD fold changes plotted vs statistical significance for Sweden ( A ) , Magdeburg / Kiel ( B ) , and Berlin ( C ) cohort . Proteins associated with the GO annotation neuron projection labelled in orange . Proteins above the dashed green line are statistically significant ( p < 0 . 05 ) and those above the black curves have a q - value below 0 . 05

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Figure 2 A-C Protein AD/non-AD fold changes plotted vs statistical significance for Sweden (A), Magdeburg/Kiel (B), and Berlin (C) cohort. Proteins associated with the GO annotation neuron projection labelled in orange. Proteins above the dashed green line are statistically significant (p < 0.05) and those above the black curves have a q-value below 0.05


Figure 3Correlation of protein AD / non - AD fold changes in pairwise combinations of two cohorts each . Combinations are Sweden vs Magdeburg / Kiel Proteins included differ significantly ( p < 0 . 05 ) and consistently in abundance by AD status in both cohorts each . Correlation of protein AD / non - AD fold changes in pairwise combinations of two cohorts each . Combinations are Sweden vs Berlin ( D ) , and Magdeburg / Kiel vs Berlin ( E ) . Proteins included differ significantly ( p < 0 . 05 ) and consistently in abundance by AD status in both cohorts each . Proteins that differ significantly ( p - value < 0 . 05 in E ; q - value < 0 . 05 in F ) in abundance by AD status across all three cohorts . Z - scored abundances of proteins in the AD and non - AD groups of all cohorts shown by the heat map Hierarchical clustering separates AD from non - AD groups . Pyruvat kinase PKM ( PKM ) was quantified in two isoforms and UniProt IDs are given in parenthesis . Black frames highlight proteins with consistent AD / non - AD fold changes across cohorts . Proteins that differ significantly ( p - value < 0 . 05 in E ; q - value < 0 . 05 in F ) in abundance by AD status across all three cohorts . Z - scored abundances of proteins in the AD and non - AD groups of all cohorts shown by the heat map Hierarchical clustering separates AD from non - AD groups . Pyruvat kinase PKM ( PKM ) was quantified in two isoforms and UniProt IDs are given in parenthesis . Black frames highlight proteins with consistent AD / non - AD fold changes across cohorts .

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Figure 3Correlation of protein AD/non-AD fold changes in pairwise combinations of two cohorts each. Combinations are Sweden vs Magdeburg/Kiel Proteins included differ significantly (p < 0.05) and consistently in abundance by AD status in both cohorts each. Correlation of protein AD/non-AD fold changes in pairwise combinations of two cohorts each. Combinations are Sweden vs Berlin (D), and Magdeburg/Kiel vs Berlin (E). Proteins included differ significantly (p < 0.05) and consistently in abundance by AD status in both cohorts each. Proteins that differ significantly (p-value < 0.05 in E; q-value < 0.05 in F) in abundance by AD status across all three cohorts. Z-scored abundances of proteins in the AD and non-AD groups of all cohorts shown by the heat map Hierarchical clustering separates AD from non-AD groups. Pyruvat kinase PKM (PKM) was quantified in two isoforms and UniProt IDs are given in parenthesis. Black frames highlight proteins with consistent AD/non-AD fold changes across cohorts.Proteins that differ significantly (p-value < 0.05 in E; q-value < 0.05 in F) in abundance by AD status across all three cohorts. Z-scored abundances of proteins in the AD and non-AD groups of all cohorts shown by the heat map Hierarchical clustering separates AD from non-AD groups. Pyruvat kinase PKM (PKM) was quantified in two isoforms and UniProt IDs are given in parenthesis. Black frames highlight proteins with consistent AD/non-AD fold changes across cohorts.


Figure 4 A - C ) Correlation of proteins with ELISA - measured t - tau concentration across samples within the Sweden ( A ) , Magdeburg ( B ) and Berlin ( C ) cohorts . Proteins with a q - value below 0 . 05 are labeled in yellow . Proteins of the 40 protein signature are colored in red for those with higher abundance in AD CSF and in blue for those with higher abundance in non - AD CSF . D ) Three cohort summary of proteins significantly correlating with ELISA - measured t - tau . Protein names given for the 29 proteins out of the 40 protein signature with significant ( p < 0 . 05 ) correlation in each of the three cohorts . Pyruvat kinase PKM ( PKM ) was quantified in two isoforms and UniProt IDs are given in parenthesis . Protein abundance distribution of CSF showing the abundances of AD - modulated CSF proteins . Proteins of our 40 protein signature are highlighted in red ( elevated abundance in AD ) and blue ( elevated abundance in non - AD ) . Proteins linked to glucose metabolism are highlighted in purple and labelled . Protein abundance distribution of brain showing the abundances of AD - modulated CSF proteins . Proteins of our 40 protein signature are highlighted in red ( elevated abundance in AD ) and blue ( elevated abundance in non - AD ) . Proteins linked to glucose metabolism are highlighted in purple and labelled .

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

Figure 4 A-C) Correlation of proteins with ELISA-measured t-tau concentration across samples within the Sweden (A), Magdeburg (B) and Berlin (C) cohorts. Proteins with a q-value below 0.05 are labeled in yellow. Proteins of the 40 protein signature are colored in red for those with higher abundance in AD CSF and in blue for those with higher abundance in non-AD CSF.  D) Three cohort summary of proteins significantly correlating with ELISA-measured t-tau. Protein names given for the 29 proteins out of the 40 protein signature with significant (p < 0.05) correlation in each of the three cohorts. Pyruvat kinase PKM (PKM) was quantified in two isoforms and UniProt IDs are given in parenthesis.  Protein abundance distribution of CSF showing the abundances of AD-modulated CSF proteins. Proteins of our 40 protein signature are highlighted in red (elevated abundance in AD) and blue (elevated abundance in non-AD). Proteins linked to glucose metabolism are highlighted in purple and labelled.  Protein abundance distribution of brain showing the abundances of AD-modulated CSF proteins. Proteins of our 40 protein signature are highlighted in red (elevated abundance in AD) and blue (elevated abundance in non-AD). Proteins linked to glucose metabolism are highlighted in purple and labelled.


Figure 1Volcano plot highlighting proteins significantly enriched ( open circles , FDR < 0 . 05 ) in eNOS - FLAG immunoprecipitates from growth factor - stimulated human endothelial cells ; n = 3 independent cell batches . Nitrite in the cell supernatant of HEK293 cells expressing wild - type ( WT ) , Y657F ( YF ) or Y657D ( YD ) eNOS and treated with solvent ( Sol ) or ionomycin ( Io , 1 µmol / L , 15 min ) ; n = 6 independent experiments ( 2 - way ANOVA and Bonferroni ) . The FMN / FAD ratio measured in eNOS immunoprecipitates from cells expressing WT , YF or YD eNOS ; n = 6 independent experiments ( 1 - way ANOVA and Newman - Keuls ) . Consequence of mutating Y657 on the growth factor - induced formation of eNOS complexes with Hsp90 and calmodulin ( CaM ) ; n = 6 - 19 independent cell batches ( 1 - way ANOVA and Newman - Keuls ) . Effect of mutating Y657 on the formation of complexes between eNOS , Hsp90 and PKM2 in growth factor - stimulated human endothelial cells . Complex formation was assessed in eNOS - FLAG immunoprecipitates ( E ) , or PKM2 immunoprecipitates ( F ) ; n = 4 - 8 independent cell batches ( 1 - way ANOVA and Newman - Keuls ) .

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

Figure 1Volcano plot highlighting proteins significantly enriched (open circles, FDR<0.05) in eNOS-FLAG immunoprecipitates from growth factor-stimulated human endothelial cells; n=3 independent cell batches.Nitrite in the cell supernatant of HEK293 cells expressing wild-type (WT), Y657F (YF) or Y657D (YD) eNOS and treated with solvent (Sol) or ionomycin (Io, 1 µmol/L, 15 min); n=6 independent experiments (2-way ANOVA and Bonferroni).The FMN/FAD ratio measured in eNOS immunoprecipitates from cells expressing WT, YF or YD eNOS; n=6 independent experiments (1-way ANOVA and Newman-Keuls).Consequence of mutating Y657 on the growth factor-induced formation of eNOS complexes with Hsp90 and calmodulin (CaM); n=6-19 independent cell batches (1-way ANOVA and Newman-Keuls).Effect of mutating Y657 on the formation of complexes between eNOS, Hsp90 and PKM2 in growth factor-stimulated human endothelial cells. Complex formation was assessed in eNOS-FLAG immunoprecipitates (E), or PKM2 immunoprecipitates (F); n=4-8 independent cell batches (1-way ANOVA and Newman-Keuls).


Figure 2Pyruvate kinase ( PK ) activity in FLAG immunoprecipitates from human endothelial cells expressing FLAG - tagged wild - type ( WT ) , Y657F ( YF ) or Y657D ( YD ) eNOS ; n = 6 independent cell batches ( 1 - way ANOVA and Tukey ) . PK activity in eNOS immunoprecipitates from HEK293 cells expressing WT , YF , YD or S633D ( SD ) eNOS ; n = 5 - 9 independent experiments ( 1 - way ANOVA and Bonferroni ) . S - nitrosation of PKM2 in human endothelial cells expressing FLAG - tagged WT or YF eNOS and treated with H2O2 ( 30 µmol / L ) for 15 min ; n = 8 independent cell batches ( Unpaired Student ' s t test ) . Dithiothreitol ( DTT ) was included to demonstrate specificity of the S - NO signal . PKM2 S - nitrosation in HEK293 cells expressing YF or YD eNOS and either FLAG - tagged WT PKM2 or a C358S ( CS ) PKM2 mutant . SNO - FLAG - PKM2 was detected with an anti - FLAG antibody after biotin switch technique ; n = 4 independent experiments ( 2 - way ANOVA & Bonferroni ) . DTT treatment was included to demonstrate specificity of the SNO signal . PK activity in eNOS immunoprecipitates from HEK293 cells co - expressing YF or YD eNOS and either WT PKM2 or the CS PKM2 mutant ; n = 6 independent experiments ( 2 - way ANOVA & Bonferroni ) . PKM2 enzyme kinetics measured with increasing concentrations of phosphoenolpyruvate ( PEP ) in eNOS immunoprecipitates from HEK293 cells expressing YF or YD eNOS . The data were fit with the Michaelis - Menten equation to determine Vmax and Km ; n = 4 independent experiments ( 2 - way ANOVA & Bonferroni ) . Quantification of pentose phosphate pathway ( PPP ) intermediates ; gluconate - 6 - P ( G6P ) , ribulose - 5 - P ( Rl5P ) , ribose - 5 - P ( R5P ) , sedoheptulose - 7 - P ( S7P ) , fructose - 6 - P ( F6P ) and erythrose - 4 - P ( E4P ) in human endothelial cells expressing YF or YD eNOS ; n = 6 - 8 independent cell batches ( Unpaired Student ' s t test ) . NADPH / NADP + and GSH / GSSG ratios in human endothelial cells expressing YF or YD eNOS ; n = 6 - 8 independent cell batches ( Unpaired Student ' s t test ) . NADPH / NADP + and GSH / GSSG ratios in human endothelial cells treated with solvent ( Sol ) or the PKM2 inhibitor shikonin ( SKN , 1 µmol / L ) for 45 minutes ; n = 4 independent cell batches ( Unpaired Student ' s t test ) .

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

Figure 2Pyruvate kinase (PK) activity in FLAG immunoprecipitates from human endothelial cells expressing FLAG-tagged wild-type (WT), Y657F (YF) or Y657D (YD) eNOS; n=6 independent cell batches (1-way ANOVA and Tukey).PK activity in eNOS immunoprecipitates from HEK293 cells expressing WT, YF, YD or S633D (SD) eNOS; n=5-9 independent experiments (1-way ANOVA and Bonferroni).S-nitrosation of PKM2 in human endothelial cells expressing FLAG-tagged WT or YF eNOS and treated with H2O2 (30 µmol/L) for 15 min; n=8 independent cell batches (Unpaired Student's t test). Dithiothreitol (DTT) was included to demonstrate specificity of the S-NO signal.PKM2 S-nitrosation in HEK293 cells expressing YF or YD eNOS and either FLAG-tagged WT PKM2 or a C358S (CS) PKM2 mutant. SNO-FLAG-PKM2 was detected with an anti-FLAG antibody after biotin switch technique; n=4 independent experiments (2-way ANOVA & Bonferroni). DTT treatment was included to demonstrate specificity of the SNO signal.PK activity in eNOS immunoprecipitates from HEK293 cells co-expressing YF or YD eNOS and either WT PKM2 or the CS PKM2 mutant; n=6 independent experiments (2-way ANOVA & Bonferroni).PKM2 enzyme kinetics measured with increasing concentrations of phosphoenolpyruvate (PEP) in eNOS immunoprecipitates from HEK293 cells expressing YF or YD eNOS. The data were fit with the Michaelis-Menten equation to determine Vmax and Km; n=4 independent experiments (2-way ANOVA & Bonferroni).Quantification of pentose phosphate pathway (PPP) intermediates; gluconate-6-P (G6P), ribulose-5-P (Rl5P), ribose-5-P (R5P), sedoheptulose-7-P (S7P), fructose-6-P (F6P) and erythrose-4-P (E4P) in human endothelial cells expressing YF or YD eNOS; n=6-8 independent cell batches (Unpaired Student's t test).NADPH/NADP+ and GSH/GSSG ratios in human endothelial cells expressing YF or YD eNOS; n=6-8 independent cell batches (Unpaired Student's t test).NADPH/NADP+ and GSH/GSSG ratios in human endothelial cells treated with solvent (Sol) or the PKM2 inhibitor shikonin (SKN, 1 µmol/L) for 45 minutes; n=4 independent cell batches (Unpaired Student's t test).


Figure 3Systolic blood pressure ( SBP ) in wild - type ( WT ) and YF - eNOS ( YF ) mice ; n = 9 animals per group ( Unpaired Student ' s t test ) . Phenylephrine ( PE ) - induced contraction of endothelium - intact aortic rings from WT and YF mice . Experiments were performed in the absence and presence of L - NAME ( LN , 300 µmol / L ) ; n = 6 - 10 animals per group ( 2 - way ANOVA and Bonferroni ) . acetylcholine ( Ach ) - induced relaxation ( C ) of endothelium - intact aortic rings from WT and YF mice . Experiments were performed in the absence and presence of L - NAME ( LN , 300 µmol / L ) ; n = 6 - 10 animals per group ( 2 - way ANOVA and Bonferroni ) . Acetylcholine ( ACh ) - induced vasodilatation of the buffer - perfused hindlimb in situ . Experiments were performed in the absence and presence of L - NAME ( LN , 300 µmol / L ) ; n = 12 - 13 animals per group ( 2 - way ANOVA and Bonferroni ) . PKM2 S - nitrosation in pulmonary endothelial cells from WT and YF mice ; n = 5 - 6 independent cell batches ( Unpaired Student ' s t test ) . Dithiothreitol ( DTT ) treatment was included to demonstrate specificity of the SNO signal . The blots show non adjacent bands cropped from the same membranes . PK activity in eNOS immunoprecipitates from WT and YF pulmonary endothelial cells ; n = 7 - 10 independent cell batches ( Unpaired Student ' s t test ) . PK activity in eNOS immunoprecipitates from hearts from WT and YF mice ; n = 6 mice per group ( Unpaired Student ' s t test ) . Representative images of eNOS - PKM2 interaction ( PLA foci , red ) in mouse pulmonary endothelial cells ; the Golgi apparatus and endosomes were stained with Golph4 ( green ) , the plasma membrane was stained with CD144 and nuclei were highlighted with DAPI ( gray ) . Only rare PLA foci were found in samples incubated with control mouse and rabbit IgGs , demonstrating the specificity of the reaction ; n = 3 independent cell batches . Scale bars : 20 µm .

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

Figure 3Systolic blood pressure (SBP) in wild-type (WT) and YF-eNOS (YF) mice; n=9 animals per group (Unpaired Student's t test).Phenylephrine (PE)-induced contraction of endothelium-intact aortic rings from WT and YF mice. Experiments were performed in the absence and presence of L-NAME (LN, 300 µmol/L); n=6-10 animals per group (2-way ANOVA and Bonferroni).acetylcholine (Ach)-induced relaxation (C) of endothelium-intact aortic rings from WT and YF mice. Experiments were performed in the absence and presence of L-NAME (LN, 300 µmol/L); n=6-10 animals per group (2-way ANOVA and Bonferroni).Acetylcholine (ACh)-induced vasodilatation of the buffer-perfused hindlimb in situ. Experiments were performed in the absence and presence of L-NAME (LN, 300 µmol/L); n=12-13 animals per group (2-way ANOVA and Bonferroni).PKM2 S-nitrosation in pulmonary endothelial cells from WT and YF mice; n=5-6 independent cell batches (Unpaired Student's t test). Dithiothreitol (DTT) treatment was included to demonstrate specificity of the SNO signal. The blots show non adjacent bands cropped from the same membranes.PK activity in eNOS immunoprecipitates from WT and YF pulmonary endothelial cells; n=7-10 independent cell batches (Unpaired Student's t test). PK activity in eNOS immunoprecipitates from hearts from WT and YF mice; n=6 mice per group (Unpaired Student's t test).Representative images of eNOS-PKM2 interaction (PLA foci, red) in mouse pulmonary endothelial cells; the Golgi apparatus and endosomes were stained with Golph4 (green), the plasma membrane was stained with CD144 and nuclei were highlighted with DAPI (gray). Only rare PLA foci were found in samples incubated with control mouse and rabbit IgGs, demonstrating the specificity of the reaction; n=3 independent cell batches. Scale bars: 20 µm.


Figure 4Wild - type ( WT ) and YF - eNOS ( YF ) mice were treated with vehicle ( Veh ) or angiotensin II ( AII , 1 . 44 mg / kg / day ) for up to 28 days . Phosphorylation of eNOS on Tyr656 in lung lysates from animals treated for 7 days , n = 6 - 7 mice per group ( Unpaired Student ' s t test ) . Wild - type ( WT ) and YF - eNOS ( YF ) mice were treated with vehicle ( Veh ) or angiotensin II ( AII , 1 . 44 mg / kg / day ) for up to 28 days . Systolic blood pressure ( SBP ) in WT and YF mice treated for 28 days , n = 10 mice per group ( Unpaired Student ' s t test ) . Wild - type ( WT ) and YF - eNOS ( YF ) mice were treated with vehicle ( Veh ) or angiotensin II ( AII , 1 . 44 mg / kg / day ) for up to 28 days . Acetylcholine ( ACh ) - induced relaxation of aortic rings from the same animals as in panel B , n = 8 mice per group ( 2 - way ANOVA and Bonferroni ) . Wild - type ( WT ) and YF - eNOS ( YF ) mice were treated with vehicle ( Veh ) or angiotensin II ( AII , 1 . 44 mg / kg / day ) for up to 28 days . PKM2 S - nitrosation in lungs from WT and YF mice administered AII for 28 days , n = 7 - 8 mice per group ( Unpaired Student ' s t test ) . DTT treatment was included to demonstrate specificity of the SNO signal . The blots show non adjacent lanes from the same membranes . Wild - type ( WT ) and YF - eNOS ( YF ) mice were treated with vehicle ( Veh ) or angiotensin II ( AII , 1 . 44 mg / kg / day ) for up to 28 days . NADPH / NADP + and GSH / GSSG ratios in lungs from WT and YF mice treated with AII for 28 days ; n = 8 - 9 mice per group ( Unpaired Student ' s t test ) . Wild - type ( WT ) and YF - eNOS ( YF ) mice were treated with vehicle ( Veh ) or angiotensin II ( AII , 1 . 44 mg / kg / day ) for up to 28 days . 3 - Nitrotyrosine ( 3 - NT ) levels in lungs from WT and YF mice treated with vehicle ( Veh ) or AII ( AII ) for 28 days ; n = 7 - 9 mice per group ( 2 - way ANOVA and Bonferroni ) . Wild - type ( WT ) and YF - eNOS ( YF ) mice were treated with vehicle ( Veh ) or angiotensin II ( AII , 1 . 44 mg / kg / day ) for up to 28 days . NADPH / NADP + and GSH / GSSG ratios in pulmonary endothelial cells from WT and YF mice treated with AII for 30 min ; n = 8 cell batches ( 2 - way ANOVA and Tukey ) . Wild - type ( WT ) and YF - eNOS ( YF ) mice were treated with vehicle ( Veh ) or angiotensin II ( AII , 1 . 44 mg / kg / day ) for up to 28 days . H2O2 levels in pulmonary endothelial cells from WT and YF mice after treatment with AII ( 1 µmol / L ) for 30 min ; n = 8 cell batches ( 2 - way ANOVA and Tukey ) .

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

Figure 4Wild-type (WT) and YF-eNOS (YF) mice were treated with vehicle (Veh) or angiotensin II (AII, 1.44 mg/kg/day) for up to 28 days. Phosphorylation of eNOS on Tyr656 in lung lysates from animals treated for 7 days, n=6-7 mice per group (Unpaired Student's t test).Wild-type (WT) and YF-eNOS (YF) mice were treated with vehicle (Veh) or angiotensin II (AII, 1.44 mg/kg/day) for up to 28 days. Systolic blood pressure (SBP) in WT and YF mice treated for 28 days, n=10 mice per group (Unpaired Student's t test).Wild-type (WT) and YF-eNOS (YF) mice were treated with vehicle (Veh) or angiotensin II (AII, 1.44 mg/kg/day) for up to 28 days. Acetylcholine (ACh)-induced relaxation of aortic rings from the same animals as in panel B, n=8 mice per group (2-way ANOVA and Bonferroni).Wild-type (WT) and YF-eNOS (YF) mice were treated with vehicle (Veh) or angiotensin II (AII, 1.44 mg/kg/day) for up to 28 days. PKM2 S-nitrosation in lungs from WT and YF mice administered AII for 28 days, n=7-8 mice per group (Unpaired Student's t test). DTT treatment was included to demonstrate specificity of the SNO signal. The blots show non adjacent lanes from the same membranes.Wild-type (WT) and YF-eNOS (YF) mice were treated with vehicle (Veh) or angiotensin II (AII, 1.44 mg/kg/day) for up to 28 days. NADPH/NADP+ and GSH/GSSG ratios in lungs from WT and YF mice treated with AII for 28 days; n=8-9 mice per group (Unpaired Student's t test).Wild-type (WT) and YF-eNOS (YF) mice were treated with vehicle (Veh) or angiotensin II (AII, 1.44 mg/kg/day) for up to 28 days. 3-Nitrotyrosine (3-NT) levels in lungs from WT and YF mice treated with vehicle (Veh) or AII (AII) for 28 days; n=7-9 mice per group (2-way ANOVA and Bonferroni).Wild-type (WT) and YF-eNOS (YF) mice were treated with vehicle (Veh) or angiotensin II (AII, 1.44 mg/kg/day) for up to 28 days. NADPH/NADP+ and GSH/GSSG ratios in pulmonary endothelial cells from WT and YF mice treated with AII for 30 min; n=8 cell batches (2-way ANOVA and Tukey).Wild-type (WT) and YF-eNOS (YF) mice were treated with vehicle (Veh) or angiotensin II (AII, 1.44 mg/kg/day) for up to 28 days. H2O2 levels in pulmonary endothelial cells from WT and YF mice after treatment with AII (1 µmol/L) for 30 min; n=8 cell batches (2-way ANOVA and Tukey).


Figure 5ApoE - / - ( - / - ) and ApoEYF ( YF ) mice were subjected to partial ligation of the left carotid artery ( LCA ) and given a cholesterol - rich diet . The right , non - ligated artery ( RCA ) was used as a control . Proximity ligation assays showing S - nitrosated PKM2 ( yellow ) in the RCA and LCA 2 days after ligation . Phalloidin staining ( blue ) was included to highlight the vessel wall ; n = 4 - 5 mice per group ( 2 - way ANOVA and Bonferroni ) . Scale bars : 50 µm . ApoE - / - ( - / - ) and ApoEYF ( YF ) mice were subjected to partial ligation of the left carotid artery ( LCA ) and given a cholesterol - rich diet . The right , non - ligated artery ( RCA ) was used as a control . GSH ( green ) levels in the RCA and LCA 2 days after ligation . CD31 ( red ) was included to label endothelial cells . n = 4 - 5 mice per group ( 2 - way ANOVA and Bonferroni ) . Scale bars : 50 µm . ApoE - / - ( - / - ) and ApoEYF ( YF ) mice were subjected to partial ligation of the left carotid artery ( LCA ) and given a cholesterol - rich diet . The right , non - ligated artery ( RCA ) was used as a control . 3 - nitrotyrosine ( 3NT , green ) levels in the RCA and LCA 2 days after ligation . CD31 ( red ) was included to label endothelial cells . n = 5 mice per group ( 2 - way ANOVA and Bonferroni ) . Scale bars : 50 µm . ApoE - / - ( - / - ) and ApoEYF ( YF ) mice were subjected to partial ligation of the left carotid artery ( LCA ) and given a cholesterol - rich diet . The right , non - ligated artery ( RCA ) was used as a control . Acetylcholine ( ACh ) - induced relaxation of the phenylephrine contracted LCA 7 days after ligation , n = 7 - 8 mice per group ( 2 - way ANOVA and Bonferroni ) . Acetylcholine ( ACh ) - induced relaxation of the phenylephrine contracted aorta 7 days after ligation , n = 7 - 8 mice per group ( 2 - way ANOVA and Bonferroni ) .

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

Figure 5ApoE-/- (-/-) and ApoEYF (YF) mice were subjected to partial ligation of the left carotid artery (LCA) and given a cholesterol-rich diet. The right, non-ligated artery (RCA) was used as a control. Proximity ligation assays showing S-nitrosated PKM2 (yellow) in the RCA and LCA 2 days after ligation. Phalloidin staining (blue) was included to highlight the vessel wall; n=4-5 mice per group (2-way ANOVA and Bonferroni). Scale bars: 50 µm.ApoE-/- (-/-) and ApoEYF (YF) mice were subjected to partial ligation of the left carotid artery (LCA) and given a cholesterol-rich diet. The right, non-ligated artery (RCA) was used as a control. GSH (green) levels in the RCA and LCA 2 days after ligation. CD31 (red) was included to label endothelial cells. n=4-5 mice per group (2-way ANOVA and Bonferroni). Scale bars: 50 µm.ApoE-/- (-/-) and ApoEYF (YF) mice were subjected to partial ligation of the left carotid artery (LCA) and given a cholesterol-rich diet. The right, non-ligated artery (RCA) was used as a control. 3-nitrotyrosine (3NT, green) levels in the RCA and LCA 2 days after ligation. CD31 (red) was included to label endothelial cells. n=5 mice per group (2-way ANOVA and Bonferroni). Scale bars: 50 µm.ApoE-/- (-/-) and ApoEYF (YF) mice were subjected to partial ligation of the left carotid artery (LCA) and given a cholesterol-rich diet. The right, non-ligated artery (RCA) was used as a control. Acetylcholine (ACh)-induced relaxation of the phenylephrine contracted LCA 7 days after ligation, n=7-8 mice per group (2-way ANOVA and Bonferroni). Acetylcholine (ACh)-induced relaxation of the phenylephrine contracted aorta 7 days after ligation, n=7-8 mice per group (2-way ANOVA and Bonferroni).


Figure 6ApoE - / - mice were treated with vehicle ( Veh ) or the PKM2 inhibitor shikonin ( SKN , 1 . 2 mg / kg ) , subjected to partial ligation of the left carotid artery ( LCA ) and given a cholesterol - rich diet . The right , non - ligated artery ( RCA ) was used as a control . GSH ( green ) levels in the RCA and LCA 3 days after ligation . CD31 ( red ) was included to label endothelial cells . n = 5 mice per group ( 2 - way ANOVA and Bonferroni ) . Scale bars : 50 µm . ApoE - / - mice were treated with vehicle ( Veh ) or the PKM2 inhibitor shikonin ( SKN , 1 . 2 mg / kg ) , subjected to partial ligation of the left carotid artery ( LCA ) and given a cholesterol - rich diet . The right , non - ligated artery ( RCA ) was used as a control . 3 - Nitrotyrosine ( 3NT , green ) levels in the RCA and LCA 3 days after ligation . CD31 ( red ) was included to label endothelial cells . n = 5 mice per group ( 2 - way ANOVA and Bonferroni ) . Scale bars : 50 µm .

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

Figure 6ApoE-/- mice were treated with vehicle (Veh) or the PKM2 inhibitor shikonin (SKN, 1.2 mg/kg), subjected to partial ligation of the left carotid artery (LCA) and given a cholesterol-rich diet. The right, non-ligated artery (RCA) was used as a control. GSH (green) levels in the RCA and LCA 3 days after ligation. CD31 (red) was included to label endothelial cells. n=5 mice per group (2-way ANOVA and Bonferroni). Scale bars: 50 µm.ApoE-/- mice were treated with vehicle (Veh) or the PKM2 inhibitor shikonin (SKN, 1.2 mg/kg), subjected to partial ligation of the left carotid artery (LCA) and given a cholesterol-rich diet. The right, non-ligated artery (RCA) was used as a control. 3-Nitrotyrosine (3NT, green) levels in the RCA and LCA 3 days after ligation. CD31 (red) was included to label endothelial cells. n=5 mice per group (2-way ANOVA and Bonferroni). Scale bars: 50 µm.


Figure 7ApoE - / - ( - / - ) and ApoEYF ( YF ) mice were subjected to partial ligation of the left carotid artery ( LCA ) and given a cholesterol - rich diet . ICAM1 and VCAM1 ( green ) expression in the LCA 2 days after ligation . CD31 ( red ) was included to label endothelial cells and nuclei are highlighted with DAPI ( gray ) . Comparable images were obtained in 4 additional animals in each group . Scale bars : 50 µm . ApoE - / - ( - / - ) and ApoEYF ( YF ) mice were subjected to partial ligation of the left carotid artery ( LCA ) and given a cholesterol - rich diet . Percentage of neutrophils and monocytes infiltrated into the LCA 7 days after ligation and high cholesterol diet feeding ; n = 9 - 10 mice per group ( Unpaired Student ' s t test ) . Representative images and quantification of ICAM1 ( green ) and VCAM1 ( red ) staining in human endothelial cells pre - treated with the PKM2 inhibitor shikonin ( SKN , 1 µmol / L ) or solvent ( Sol ) for 45 minutes and then stimulated with interleukin - 1β ( IL - 1 , 20 ng / ml ) or solvent ( CTL ) for 90 min . Nuclei are highlighted with DAPI ( gray ) . n = 6 independent cell batches ( 2 - way ANOVA and Bonferroni ) . Scale bars : 20 µm . Percentage of neutrophils and macrophages infiltrated into the LCA from ApoE - / - mice treated with Veh or SKN ( 1 . 2 mg / kg ) 7 days after ligation and high cholesterol diet feeding ; n = 4 - 6 mice per group ( Unpaired Student ' s t test ) .

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

Figure 7ApoE-/- (-/-) and ApoEYF (YF) mice were subjected to partial ligation of the left carotid artery (LCA) and given a cholesterol-rich diet. ICAM1 and VCAM1 (green) expression in the LCA 2 days after ligation. CD31 (red) was included to label endothelial cells and nuclei are highlighted with DAPI (gray). Comparable images were obtained in 4 additional animals in each group. Scale bars: 50 µm.ApoE-/- (-/-) and ApoEYF (YF) mice were subjected to partial ligation of the left carotid artery (LCA) and given a cholesterol-rich diet. Percentage of neutrophils and monocytes infiltrated into the LCA 7 days after ligation and high cholesterol diet feeding; n=9-10 mice per group (Unpaired Student's t test).Representative images and quantification of ICAM1 (green) and VCAM1 (red) staining in human endothelial cells pre-treated with the PKM2 inhibitor shikonin (SKN, 1 µmol/L) or solvent (Sol) for 45 minutes and then stimulated with interleukin-1β (IL-1, 20 ng/ml) or solvent (CTL) for 90 min. Nuclei are highlighted with DAPI (gray). n=6 independent cell batches (2-way ANOVA and Bonferroni). Scale bars: 20 µm.Percentage of neutrophils and macrophages infiltrated into the LCA from ApoE-/- mice treated with Veh or SKN (1.2 mg/kg) 7 days after ligation and high cholesterol diet feeding; n=4-6 mice per group (Unpaired Student's t test).


Figure 8Plaque burden in ligated carotid arteries from ApoE - / - ( - / - ) and ApoEYF ( YF ) mice fed a high cholesterol diet for 21 days ; scale bars : 2 mm . Intimal and medial thickness was determined at four specific locations ( proximal , middle 1 , middle 2 , distal ) in cryo - sectioned arteries stained with Oil Red O ; scale bars : 200 µm . n = 7 mice per group ( Unpaired Student ' s t test ) . Oil Red O staining of plaques in aortae from mice fed a Western diet for four ( B ) or six months ( C ) ; scale bars : 3 mm . n = 6 ( B ) or n = 12 ( C ) mice per group ( Unpaired Student ' s t test ) .

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

Figure 8Plaque burden in ligated carotid arteries from ApoE-/- (-/-) and ApoEYF (YF) mice fed a high cholesterol diet for 21 days; scale bars: 2 mm. Intimal and medial thickness was determined at four specific locations (proximal, middle 1, middle 2, distal) in cryo-sectioned arteries stained with Oil Red O; scale bars: 200 µm. n=7 mice per group (Unpaired Student's t test).Oil Red O staining of plaques in aortae from mice fed a Western diet for four (B) or six months (C); scale bars: 3 mm. n=6 (B) or n=12 (C) mice per group (Unpaired Student's t test).


Figure 1A . Rab25 expression decreases nutrient withdrawal ( 24 h ) induced cell death in ovarian A2780 and IOSE29 cells measured using the Cell death ELISA plus assay ( left ) ; cells were cultured in complete media ( containing 5 % FBS ) , serum free media ( SF ) , complete media plus 2DG , serum and glucose free media ( SF - glu ) , or in amino acid , glucose and SF Earls buffer salt solution ( EBSS ) . ( a ) p < 0 . 01 versus control and ( b ) p < 0 . 05 versus pcDNA . ( Right ) Percentage of apoptotic cells ( sub - G0 population ) by flow cytometry in A2780 cells following glucose and serum withdrawal ( Glu / SF ) . ( a ) p < 0 . 001 versus A2780pcDNA control and ( b ) p < 0 . 05 A2780Rab25 versus A2780pcDNA control . B . Decreasing Rab25 expression increases sensitivity to nutrient stress induced cell death . Expression of Rab25 was down - regulated by siRNA or shRNA specific to Rab25 . C . Rab25 regulates autophagy activity in ovarian cancer cells under serum and glucose deprivation conditions . Western blot for the 16kD LC3 - II fragment , indicative of autophagy activity , in A2780 cells after 4 and 6 h of serum and glucose withdrawal . D . Electron microscopy of autophagy in ovarian A2780 cells after 4 and 24 h of serum and glucose withdrawal ( upper panel , low magnification ) . High magnification images of boxed areas with arrowheads depicting autophagic vacuoles ( lower panel , inset ; N : nucleus ) . Average number of autophagosomes per cell was calculated by counting the number of autophagosomes in 16 individual cells from two - independent experiments . E - H . Expression of Rab25 decreases glucose and serum deprivation induced signalling activation . Protein expression was measured by RPPA or WB analysis . E . RPPA detection of time - dependent activation of AMPK after nutrient withdrawal . E - H . Expression of Rab25 decreases glucose and serum deprivation induced signalling activation . Protein expression was measured by RPPA or WB analysis . F . WB analysis of AMPK and acetyl - CoA carboxylase ( ACC ) phosphorylation in HEY ovarian cancer cells ( upper panel ) . WB of phospho - ACC levels in A2780 , IOSE80ht and SKOV3 ovarian cells after 1 h of nutrient withdrawal ( lower panel ) . E - H . Expression of Rab25 decreases glucose and serum deprivation induced signalling activation . Protein expression was measured by RPPA or WB analysis . G . RPPA detection of phosphorylation of ACC after withdrawal of serum and glucose . E - H . Expression of Rab25 decreases glucose and serum deprivation induced signalling activation . Protein expression was measured by RPPA or WB analysis . H . Effect of Rab25 down - regulation on AMPK ( left panel ) and ACC ( right panel ) phosphorylation . Total cellular protein , isolated from A2780pcDNA and Rab25 expressing cells 72 h post - transfection with either non - target ( NT ) RNAi or Rab25 specific RNAi , was separated by polyacrylamide gel electrophoresis ( PAGE ) followed by WB analysis . ( a ) p < 0 . 001 versus NT RNAi control and ( b ) p < 0 . 001 , pcDNA versus Rab25 .

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

Figure 1A. Rab25 expression decreases nutrient withdrawal (24 h) induced cell death in ovarian A2780 and IOSE29 cells measured using the Cell death ELISA plus assay (left); cells were cultured in complete media (containing 5% FBS), serum free media (SF), complete media plus 2DG, serum and glucose free media (SF-glu), or in amino acid, glucose and SF Earls buffer salt solution (EBSS). (a) p < 0.01 versus control and (b) p < 0.05 versus pcDNA. (Right) Percentage of apoptotic cells (sub-G0 population) by flow cytometry in A2780 cells following glucose and serum withdrawal (Glu/SF). (a) p < 0.001 versus A2780pcDNA control and (b) p < 0.05 A2780Rab25 versus A2780pcDNA control.B. Decreasing Rab25 expression increases sensitivity to nutrient stress induced cell death. Expression of Rab25 was down-regulated by siRNA or shRNA specific to Rab25.C. Rab25 regulates autophagy activity in ovarian cancer cells under serum and glucose deprivation conditions. Western blot for the 16kD LC3-II fragment, indicative of autophagy activity, in A2780 cells after 4 and 6 h of serum and glucose withdrawal.D. Electron microscopy of autophagy in ovarian A2780 cells after 4 and 24 h of serum and glucose withdrawal (upper panel, low magnification). High magnification images of boxed areas with arrowheads depicting autophagic vacuoles (lower panel, inset; N: nucleus). Average number of autophagosomes per cell was calculated by counting the number of autophagosomes in 16 individual cells from two-independent experiments.E-H. Expression of Rab25 decreases glucose and serum deprivation induced signalling activation. Protein expression was measured by RPPA or WB analysis.E. RPPA detection of time-dependent activation of AMPK after nutrient withdrawal.E-H. Expression of Rab25 decreases glucose and serum deprivation induced signalling activation. Protein expression was measured by RPPA or WB analysis.F. WB analysis of AMPK and acetyl-CoA carboxylase (ACC) phosphorylation in HEY ovarian cancer cells (upper panel). WB of phospho-ACC levels in A2780, IOSE80ht and SKOV3 ovarian cells after 1 h of nutrient withdrawal (lower panel).E-H. Expression of Rab25 decreases glucose and serum deprivation induced signalling activation. Protein expression was measured by RPPA or WB analysis.G. RPPA detection of phosphorylation of ACC after withdrawal of serum and glucose.E-H. Expression of Rab25 decreases glucose and serum deprivation induced signalling activation. Protein expression was measured by RPPA or WB analysis.H. Effect of Rab25 down-regulation on AMPK (left panel) and ACC (right panel) phosphorylation. Total cellular protein, isolated from A2780pcDNA and Rab25 expressing cells 72 h post-transfection with either non-target (NT) RNAi or Rab25 specific RNAi, was separated by polyacrylamide gel electrophoresis (PAGE) followed by WB analysis. (a) p < 0.001 versus NT RNAi control and (b) p < 0.001, pcDNA versus Rab25.