A , 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 .

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A, 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 ) .

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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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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.


A . 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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A. 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.


G . 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 .

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G. 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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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).


SUD binds to Paip1 and Paip1M with the same binding intensity . n . s . : not significant .

SUD binds to Paip1 and Paip1M with the same binding intensity. n. s.: not significant.


The Mac2 subdomain is crucial for interacting with Paip1M .

The Mac2 subdomain is crucial for interacting with Paip1M.


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

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


SUD 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 .

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SUD 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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GFP-trap-based co-immunoprecipitation (CoIP) assay demonstrating a complex formed by PABP, Paip1, and SUD. GFP: green fluorescent protein.


Left 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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Left 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).


SARS - CoV - 2 SUD ( SARS2 - SUD ) interacts with Paip1 in HEK - 293 cells as demonstrated by the split - YFP assay .

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SARS-CoV-2 SUD (SARS2-SUD) interacts with Paip1 in HEK-293 cells as demonstrated by the split-YFP assay.


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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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MERS-CoV SUD-MC (Mac3−DPUP) or XMC (Mac1−Mac3−DPUP) do not bind to Paip1. Scale bars represent 50 µM.


Median 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 .

Median 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.


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

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


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 .

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Correlation 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 .

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Correlation 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 .

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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 .

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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.


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 .

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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 .

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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 .

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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 .

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.


Volcano 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 .

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Volcano 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 ) .

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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 ) .

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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 ) .

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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 ) .

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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).


Pyruvate 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 ) .

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Pyruvate 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 ) .

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 .

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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 .

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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 ) .

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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 ) .

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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 ) .

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 ) .

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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 ) .

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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).


Systolic blood pressure ( SBP ) in wild - type ( WT ) and YF - eNOS ( YF ) mice ; n = 9 animals per group ( Unpaired Student ' s t test ) .

Systolic 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 ) .

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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 ) .

B-CONTROLLED_VAR 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-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR 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

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 ) .

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

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 .

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

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 ) .

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

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

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.


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 . 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 ) .

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 I-CONTROLLED_VAR 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

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. 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 ) .

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

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 ) .

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

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 .

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

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 ) .

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

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 ) .

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

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 ) .

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

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

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).


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 . 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 .

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

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. 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 .

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

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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 I-MEASURED_VAR I-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

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

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).


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 . 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 .

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

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

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.


ApoE - / - ( - / - ) 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 .

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

ApoE-/- (-/-) 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 ) .

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

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 .

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

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 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

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).


Plaque 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 ) .

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

Plaque 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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).


A . 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 .

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A. 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 .

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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 .

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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 .

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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 .

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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 ) .

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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 .

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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 .

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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.


Expression of Rab25 increases endogenous ATP level in ovarian cancer cells . * p < 0 . 001 Rab25 versus pcDNA . The mean cellular concentration of ATP per mg of protein ( based on the standard curve generated using a known amount of ATP ) is 2 . 36 − 10 moles / mg for IOSE29htpcDNA cells , 6 . 21 − 10 moles / mg for IOSE29htRab25 cells , 8 . 14 − 11 moles / mg for IOSE80htpcDNA cells , 1 . 05 − 10 moles / mg for IOSE80htRab25 cells , 2 . 85 − 10 moles / mg for A2780pcDNA cells , 3 . 81 − 10 moles / mg for A2780Rab25 cells , 7 . 23 − 10 moles / mg for HEYpcDNA cells and 9 . 82 − 10 moles / mg for HEYRab25 cells .

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Expression of Rab25 increases endogenous ATP level in ovarian cancer cells. *p < 0.001 Rab25 versus pcDNA. The mean cellular concentration of ATP per mg of protein (based on the standard curve generated using a known amount of ATP) is 2.36−10 moles/mg for IOSE29htpcDNA cells, 6.21−10 moles/mg for IOSE29htRab25 cells, 8.14−11 moles/mg for IOSE80htpcDNA cells, 1.05−10 moles/mg for IOSE80htRab25 cells, 2.85−10 moles/mg for A2780pcDNA cells, 3.81−10 moles/mg for A2780Rab25 cells, 7.23−10 moles/mg for HEYpcDNA cells and 9.82−10 moles/mg for HEYRab25 cells.


Rab25 expression increases ATP synthesis and delays fall in total cellular ATP levels after glucose and serum withdrawal , p < 0 . 001 Rab25 versus control pcDNA .

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Rab25 expression increases ATP synthesis and delays fall in total cellular ATP levels after glucose and serum withdrawal, p < 0.001 Rab25 versus control pcDNA.


Down - regulation of Rab25 expression decreases ability to maintain ATP after glucose and serum withdrawal ( a ) p < 0 . 05 SF - Glu versus FBS and ( b ) p < 0 . 05 versus control .

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Down-regulation of Rab25 expression decreases ability to maintain ATP after glucose and serum withdrawal (a) p < 0.05 SF-Glu versus FBS and (b) p < 0.05 versus control.


Expression of Rab25 increases total cellular glycogen content while down - regulation of Rab25 expression decreases total cellular glycogen content ( µg of glycogen / mg protein ) . Cells were cultured in complete media for 24 h before glycogen measurement . ( a ) p < 0 . 01 Rab25 versus pcDNA , ( b ) p < 0 . 05 Rab25 RNAi versus NT RNAi and ( c ) p < 0 . 05 shRAb25 versus shRNA control .

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Expression of Rab25 increases total cellular glycogen content while down-regulation of Rab25 expression decreases total cellular glycogen content (µg of glycogen/mg protein). Cells were cultured in complete media for 24 h before glycogen measurement. (a) p < 0.01 Rab25 versus pcDNA, (b) p < 0.05 Rab25 RNAi versus NT RNAi and (c) p < 0.05 shRAb25 versus shRNA control.


Decrease in cellular glycogen content after glucose and serum withdrawal * p < 0 . 001 Rab25 versus pcDNA .

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Decrease in cellular glycogen content after glucose and serum withdrawal *p < 0.001 Rab25 versus pcDNA.


Effect of GPi ( Bay U6571 ) and oligomycin on ATP production . A2780pcDNA transfected and Rab25 expressing cells were pretreated with vehicle ( control ) , 30 µM of Bay U6571 ( GPi ) or 25 µg / ml of oligomycin for 2 h . Cells were then cultured in serum - and glucose free RPMI 1640 in the presence or absence of Bay U6571 or oligomycin for the indicated times . ( a ) p < 0 . 01 oligomycin time 0 versus control time 0 and ( b ) p < 0 . 01 versus time 0 at each treatment group .

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Effect of GPi (Bay U6571) and oligomycin on ATP production. A2780pcDNA transfected and Rab25 expressing cells were pretreated with vehicle (control), 30 µM of Bay U6571 (GPi) or 25 µg/ml of oligomycin for 2 h. Cells were then cultured in serum- and glucose free RPMI 1640 in the presence or absence of Bay U6571 or oligomycin for the indicated times. (a) p < 0.01 oligomycin time 0 versus control time 0 and (b) p < 0.01 versus time 0 at each treatment group.


Effect of Bay U6571 on cellular glycogen ( left panel ) and ATP ( right panel ) levels in HEY cells . ( a ) p < 0 . 01 versus pcDNA control time 0 at 5 % FBS and ( b ) p < 0 . 01 GPi versus control at each corresponding treatment .

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Effect of Bay U6571 on cellular glycogen (left panel) and ATP (right panel) levels in HEY cells. (a) p < 0.01 versus pcDNA control time 0 at 5% FBS and (b) p < 0.01 GPi versus control at each corresponding treatment.


Down regulation of Rab25 expression and inhibition of glycogen breakdown by GPi administration increases 2DG induced cell death * p < 0 . 05 versus shRNA control .

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Down regulation of Rab25 expression and inhibition of glycogen breakdown by GPi administration increases 2DG induced cell death *p < 0.05 versus shRNA control.


A . Rab25 expression increases phospho but not total AKT and GSK3 levels in A2780 cells leading to decreased phosphorylation of GS ( pGS ) .

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A. Rab25 expression increases phospho but not total AKT and GSK3 levels in A2780 cells leading to decreased phosphorylation of GS (pGS).


B . WB analysis of AKT and GSK3 phosphorylation confirming the inhibitory effect of PI3K pathways inhibitors PI103 , GPi and synthase kinase 3 inhibitor ( GSK3i ) in HEY cells .

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B. WB analysis of AKT and GSK3 phosphorylation confirming the inhibitory effect of PI3K pathways inhibitors PI103, GPi and synthase kinase 3 inhibitor (GSK3i) in HEY cells.


C . Inhibition of GSK3 activity increases total cellular glycogen levels . pcDNA transfected and Rab25 expressing ovarian cancer cells were cultured in complete media in the presence of 10 µM GSK3i for the indicated times . Total cellular glycogen content was measured and normalized with total protein content . ( a ) p < 0 . 001 versus no GSK3i treated pcDNA cells .

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C. Inhibition of GSK3 activity increases total cellular glycogen levels. pcDNA transfected and Rab25 expressing ovarian cancer cells were cultured in complete media in the presence of 10 µM GSK3i for the indicated times. Total cellular glycogen content was measured and normalized with total protein content. (a) p < 0.001 versus no GSK3i treated pcDNA cells.


D - E . Effect of PI3K inhibitor PI103 on cellular glycogen content and ATP production . Ovarian cancer cells were pretreated with PI103 for at least 2 h in complete media . Cells were then subjected to 2 hglucose and FBS withdrawal to deplete endogenous glycogen followed by culturing cells in RPMI - glucose media in the presence of PI103 for 2 h ( 2 hSF ) . After 2 h of nutrient stress , complete media containing PI103 was added to the cells for 30 min ( 5 % FBS ) for recovery . A second glucose and FBS withdrawal ( 15 min SF ) followed immediately to examine the effect on glycogen and ATP levels . a , p < 0 . 001 5 % FBS versus 2 hSF , b , p < 0 . 01 15 min SF versus 5 % FBS . D . There is an increase in glycogen content during recovery phase ( 5 % FBS ) and a decrease in glycogen when cells are subject to a second nutrient stress ( 15 min SF ) .

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D-E. Effect of PI3K inhibitor PI103 on cellular glycogen content and ATP production. Ovarian cancer cells were pretreated with PI103 for at least 2 h in complete media. Cells were then subjected to 2 hglucose and FBS withdrawal to deplete endogenous glycogen followed by culturing cells in RPMI-glucose media in the presence of PI103 for 2 h (2 hSF). After 2 h of nutrient stress, complete media containing PI103 was added to the cells for 30 min (5% FBS) for recovery. A second glucose and FBS withdrawal (15 min SF) followed immediately to examine the effect on glycogen and ATP levels. a, p < 0.001 5% FBS versus 2 hSF, b, p < 0.01 15 min SF versus 5% FBS.D. There is an increase in glycogen content during recovery phase (5% FBS) and a decrease in glycogen when cells are subject to a second nutrient stress (15 min SF).


D - E . Effect of PI3K inhibitor PI103 on cellular glycogen content and ATP production . Ovarian cancer cells were pretreated with PI103 for at least 2 h in complete media . Cells were then subjected to 2 hglucose and FBS withdrawal to deplete endogenous glycogen followed by culturing cells in RPMI - glucose media in the presence of PI103 for 2 h ( 2 hSF ) . After 2 h of nutrient stress , complete media containing PI103 was added to the cells for 30 min ( 5 % FBS ) for recovery . A second glucose and FBS withdrawal ( 15 min SF ) followed immediately to examine the effect on glycogen and ATP levels . a , p < 0 . 001 5 % FBS versus 2 hSF , b , p < 0 . 01 15 min SF versus 5 % FBS . E . Utilization of glycogen to produce ATP .

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D-E. Effect of PI3K inhibitor PI103 on cellular glycogen content and ATP production. Ovarian cancer cells were pretreated with PI103 for at least 2 h in complete media. Cells were then subjected to 2 hglucose and FBS withdrawal to deplete endogenous glycogen followed by culturing cells in RPMI-glucose media in the presence of PI103 for 2 h (2 hSF). After 2 h of nutrient stress, complete media containing PI103 was added to the cells for 30 min (5% FBS) for recovery. A second glucose and FBS withdrawal (15 min SF) followed immediately to examine the effect on glycogen and ATP levels. a, p < 0.001 5% FBS versus 2 hSF, b, p < 0.01 15 min SF versus 5% FBS.E. Utilization of glycogen to produce ATP.


F . Addition of AKT inhibitor MK2206 ( AKTi ) abolishes Rab25 - dependent glycogen storage . ( a ) p < 0 . 05 versus shRNA control and ( b ) p < 0 . 01 SF + AKTi versus SF .

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F. Addition of AKT inhibitor MK2206 (AKTi) abolishes Rab25-dependent glycogen storage. (a) p < 0.05 versus shRNA control and (b) p < 0.01 SF + AKTi versus SF.


G . Inhibiting AKT pathway and glucose metabolism decreases cell viability . Cells were pretreated with 10 µM of PI103 or AKTi , 100 µM LND or 3BrPy for 2 h before switching to SF media in the presence of inhibitors for an addition16 h before cell titre blue viability assay . * p < 0 . 001 shRNA Rab25 versus shRNA control .

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G. Inhibiting AKT pathway and glucose metabolism decreases cell viability. Cells were pretreated with 10 µM of PI103 or AKTi, 100 µM LND or 3BrPy for 2 h before switching to SF media in the presence of inhibitors for an addition16 h before cell titre blue viability assay. *p < 0.001 shRNA Rab25 versus shRNA control.


A . Rab25 over - expression increases H3 - labelled 2DG uptake . ( a ) p < 0 . 001 Rab25 versus pcDNA . Results are mean ± s . d . of a triplicate in one of representative experiment .

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A. Rab25 over-expression increases H3-labelled 2DG uptake. (a) p < 0.001 Rab25 versus pcDNA. Results are mean ± s.d. of a triplicate in one of representative experiment.


B . Down - regulation of Rab25 or AKT expression , or inhibition AKT activity by MK2206 decreases glucose uptake in HEY cells ( a ) p < 0 . 001 versus NT siRNA control , ( b ) p < 0 . 05 versus siRab25 and ( c ) p < 0 . 05 versus siAKT .

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B. Down-regulation of Rab25 or AKT expression, or inhibition AKT activity by MK2206 decreases glucose uptake in HEY cells (a) p < 0.001 versus NT siRNA control, (b) p < 0.05 versus siRab25 and (c) p < 0.05 versus siAKT.


C . Effect of down regulation of Rab25 and AKT inhibitor MK2206 ( AKTi ) on glucose uptake . Cells were either pretreated for 2 h with AKTi before assessing glucose uptake . ( a ) p < 0 . 05 versus shRNA control and ( b ) p < 0 . 05 versus shRab25 .

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C. Effect of down regulation of Rab25 and AKT inhibitor MK2206 (AKTi) on glucose uptake. Cells were either pretreated for 2 h with AKTi before assessing glucose uptake. (a) p < 0.05 versus shRNA control and (b) p < 0.05 versus shRab25.


D . Rab25 co - localization with GLUT1 in A2780 cells . Immunofluorescence microscopy staining of GLUT1 ( red ) and Rab25 ( green ) . Co - localization of GLUT1 and Rab25 ( i . e . yellow ) is indicated by arrows . Single scale bar , 10 µM .

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D. Rab25 co-localization with GLUT1 in A2780 cells. Immunofluorescence microscopy staining of GLUT1 (red) and Rab25 (green). Co-localization of GLUT1 and Rab25 (i.e. yellow) is indicated by arrows. Single scale bar, 10 µM.


E - G . Resistance to 2DG induced cell death in Rab25 cells is mediated through the PI3K / AKT pathway . Inhibition of the PI3K / AKT pathway was achieved by addition of 10 µM of PI103 or MK2206 . E . AKT inhibition increases sensitivity to 2DG induced cell death . * p < 0 . 001 versus HEYpcDNA cells .

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E-G. Resistance to 2DG induced cell death in Rab25 cells is mediated through the PI3K/AKT pathway. Inhibition of the PI3K/AKT pathway was achieved by addition of 10 µM of PI103 or MK2206.E. AKT inhibition increases sensitivity to 2DG induced cell death. *p < 0.001 versus HEYpcDNA cells.


E - G . Resistance to 2DG induced cell death in Rab25 cells is mediated through the PI3K / AKT pathway . Inhibition of the PI3K / AKT pathway was achieved by addition of 10 µM of PI103 or MK2206 . F . Down - regulation of AKT by siRNA specific to AKT .

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E-G. Resistance to 2DG induced cell death in Rab25 cells is mediated through the PI3K/AKT pathway. Inhibition of the PI3K/AKT pathway was achieved by addition of 10 µM of PI103 or MK2206.F. Down-regulation of AKT by siRNA specific to AKT.


E - G . Resistance to 2DG induced cell death in Rab25 cells is mediated through the PI3K / AKT pathway . Inhibition of the PI3K / AKT pathway was achieved by addition of 10 µM of PI103 or MK2206 . G . Or down - regulation of Rab25 by shRNA specific to Rab25 enhances 2DG induced cell death . * p < 0 . 001 versus RNAi control cells .

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E-G. Resistance to 2DG induced cell death in Rab25 cells is mediated through the PI3K/AKT pathway. Inhibition of the PI3K/AKT pathway was achieved by addition of 10 µM of PI103 or MK2206.G. Or down-regulation of Rab25 by shRNA specific to Rab25 enhances 2DG induced cell death. *p < 0.001 versus RNAi control cells.


Ovarian cancer HEY cells ( upper panel ) were co - transfected with AKT - IFPN and Rab25 - IFPC or with AKT - IFPC and Rab25 - IFPN . Cells become fluorescent due to an interaction between AKT and Rab25 bringing the two halves of the fluorescent protein into proximity creating a stable complex and restoring fluorescence . Hela cells stably expressing AKT - IFPN ( lower panel ) were transfected with Rab25 - IFPC or PDK - IFPC as control . A brightfield image corresponding to the fluorescent image is shown and transfected cells indicated by an arrow .

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Ovarian cancer HEY cells (upper panel) were co-transfected with AKT-IFPN and Rab25-IFPC or with AKT-IFPC and Rab25-IFPN. Cells become fluorescent due to an interaction between AKT and Rab25 bringing the two halves of the fluorescent protein into proximity creating a stable complex and restoring fluorescence. Hela cells stably expressing AKT-IFPN (lower panel) were transfected with Rab25-IFPC or PDK-IFPC as control. A brightfield image corresponding to the fluorescent image is shown and transfected cells indicated by an arrow.


Detection of Rab25 and AKT interaction by immunoprecipitation . Ovarian cells expressing HA - tagged Rab25 were IP with anti - HA antibody . The resultant complex was separated by gel electrophoresis and detected by WB using anti - AKT antibody .

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Detection of Rab25 and AKT interaction by immunoprecipitation. Ovarian cells expressing HA-tagged Rab25 were IP with anti-HA antibody. The resultant complex was separated by gel electrophoresis and detected by WB using anti-AKT antibody.


Generation of Rab25 deletion mutants . Expression of the deletion mutants was detected by immunofluorescence staining ( upper panel ) and WB ( middle panel ) using anti - GFP antibody which interacts with YFP . Diagrammatic representation of the structure of the mutants is shown in the lower panel .

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Generation of Rab25 deletion mutants. Expression of the deletion mutants was detected by immunofluorescence staining (upper panel) and WB (middle panel) using anti-GFP antibody which interacts with YFP. Diagrammatic representation of the structure of the mutants is shown in the lower panel.


Rab25 deletion mutants do not alter cellular glycogen and ATP levels . Ovarian cancer cell lysates were collected 24 h post transfection . ( a ) p < 0 . 05 versus empty vector transfected cells .

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Rab25 deletion mutants do not alter cellular glycogen and ATP levels. Ovarian cancer cell lysates were collected 24 h post transfection. (a) p < 0.05 versus empty vector transfected cells.


A . Rab25 expression correlates with glycogen levels in patienttumours . Total RNA and total cellular extracts , isolated from 31 ovarian cancer patient specimens , were subjected to Rab25 gene expression and glycogen content analysis using qPCR and glycogen assay , respectively .

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A. Rab25 expression correlates with glycogen levels in patienttumours. Total RNA and total cellular extracts, isolated from 31 ovarian cancer patient specimens, were subjected to Rab25 gene expression and glycogen content analysis using qPCR and glycogen assay, respectively.


B - C . The Rab25 expression signature identifies patients with a poor prognosis . Ovarian cancer patients were classified as either mirroring the Rab25 - associated gene expression signature ( Rab25 signature ) or not ( non - Rab25 signature ) using linear discriminant analysis in BRB tools , in two independent published ovarian datasets . Progression free survival curvesfor patients with advanced disease ( Stage II to IV ) are shown . Univariate analyses were plotted using Kaplan - Meier method and Coxplots used for multivariate analyses ( co - variates included stage , grade , histology and residual disease ) . B . Tothill et al , 2008 . C . Dressman et al , 2007 .

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B-C. The Rab25 expression signature identifies patients with a poor prognosis. Ovarian cancer patients were classified as either mirroring the Rab25-associated gene expression signature (Rab25 signature) or not (non-Rab25 signature) using linear discriminant analysis in BRB tools, in two independent published ovarian datasets. Progression free survival curvesfor patients with advanced disease (Stage II to IV) are shown. Univariate analyses were plotted using Kaplan-Meier method and Coxplots used for multivariate analyses (co-variates included stage, grade, histology and residual disease).B. Tothill et al, 2008.C. Dressman et al, 2007.


D . Prediction of breast cancer overall survival in two independent published breast datasets ( upper panel ) Pawitan et al , 2005 and ( lower panel ) Chin et al , 2006 , based on Rab25 - associated gene signature . Breast patient classification was determined by BRB tool class prediction function to identify patient with Rab25 - associated gene expression signature ( Rab25 - signature ) or without ( non - Rab25 signature ) . All patients from the datasets were included in class prediction .

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D. Prediction of breast cancer overall survival in two independent published breast datasets (upper panel) Pawitan et al, 2005 and (lower panel) Chin et al, 2006, based on Rab25-associated gene signature. Breast patient classification was determined by BRB tool class prediction function to identify patient with Rab25-associated gene expression signature (Rab25-signature) or without (non-Rab25 signature). All patients from the datasets were included in class prediction.


Figure 1 . Extensive identification of Mmi1 RNA targets by combining RNA - IP sequencing and computational approaches . ( A ) Box - plot of the enrichment of the RNAs identified by Mmi1 RNA - IPs coupled to high throughput sequencing . The log of the average enrichments obtained from two independent RNA - IPs is plotted . The enrichment is relative to the no antibody RNA - IPs conducted in parallel of Mmi1 RNA - IPs . The mRNAs and lncRNAs enriched at least two folds in both Mmi1 RNA - IPs are shown in blue and green , respectively . Crosses represent newly identified Mmi1 targets and dots known targets .

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Figure 1. Extensive identification of Mmi1 RNA targets by combining RNA-IP sequencing and computational approaches. (A) Box-plot of the enrichment of the RNAs identified by Mmi1 RNA-IPs coupled to high throughput sequencing. The log of the average enrichments obtained from two independent RNA-IPs is plotted. The enrichment is relative to the no antibody RNA-IPs conducted in parallel of Mmi1 RNA-IPs. The mRNAs and lncRNAs enriched at least two folds in both Mmi1 RNA-IPs are shown in blue and green, respectively. Crosses represent newly identified Mmi1 targets and dots known targets.


Figure 1 . Extensive identification of Mmi1 RNA targets by combining RNA - IP sequencing and computational approaches . ( D ) RNA - IPs showing the impact of Mmi1 YTH domain point mutations on Mmi1 ' s binding to ssm4 mRNA . The lower part shows a Western blot monitoring the protein level of WT and mutant Mmi1 proteins in the cells used for the RNA - IPs . Loading was monitored using an anti - Tub1 ( tubulin ) antibody .

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Figure 1. Extensive identification of Mmi1 RNA targets by combining RNA-IP sequencing and computational approaches. (D) RNA-IPs showing the impact of Mmi1 YTH domain point mutations on Mmi1's binding to ssm4 mRNA. The lower part shows a Western blot monitoring the protein level of WT and mutant Mmi1 proteins in the cells used for the RNA-IPs. Loading was monitored using an anti-Tub1 (tubulin) antibody.


Figure 1 . Extensive identification of Mmi1 RNA targets by combining RNA - IP sequencing and computational approaches . ( E , F ) RNA - IPs showing the YTH - dependent association of Mmi1 to the lncRNAs identified in ( A ) and ( B ) . Average fold enrichment is shown with error bars that indicate mean average deviations for three independent experiments for ( D , E and F ) .

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Figure 1. Extensive identification of Mmi1 RNA targets by combining RNA-IP sequencing and computational approaches. (E, F) RNA-IPs showing the YTH-dependent association of Mmi1 to the lncRNAs identified in (A) and (B). Average fold enrichment is shown with error bars that indicate mean average deviations for three independent experiments for (D, E and F).


Figure 2 . Mmi1 binding to nam1 lncRNA controls MAPK - mediated entry into sexual differentiation . ( A ) Upper part , scheme of S . pombe sexual differentiation . Lower part , microscopy images showing WT and mmi1 ∆ cells , and mmi1 ∆ cells expressing Mmi1 - R351E or Mmi1 - R381E mutant proteins . Images were taken after 24 hours of induction of sexual differentiation by growth on SPAS medium . The percentage of cells that underwent differentiation and iodine vapor assays , conducted on the corresponding patches , are shown at the bottom of each image .

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Figure 2. Mmi1 binding to nam1 lncRNA controls MAPK-mediated entry into sexual differentiation. (A) Upper part, scheme of S. pombe sexual differentiation. Lower part, microscopy images showing WT and mmi1∆ cells, and mmi1∆ cells expressing Mmi1-R351E or Mmi1-R381E mutant proteins. Images were taken after 24 hours of induction of sexual differentiation by growth on SPAS medium. The percentage of cells that underwent differentiation and iodine vapor assays, conducted on the corresponding patches, are shown at the bottom of each image.


Figure 2 . Mmi1 binding to nam1 lncRNA controls MAPK - mediated entry into sexual differentiation . ( B ) Mating assay showing the percentage of zygotes forming over time in the same cells as in ( A ) .

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Figure 2. Mmi1 binding to nam1 lncRNA controls MAPK-mediated entry into sexual differentiation. (B) Mating assay showing the percentage of zygotes forming over time in the same cells as in (A).


Figure 2 . Mmi1 binding to nam1 lncRNA controls MAPK - mediated entry into sexual differentiation . ( E ) Mmi1 RNA - IPs showing the specific loss of binding of Mmi1 to nam1 - 1 lncRNA but not to mei4 mRNA , another target of Mmi1 .

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Figure 2. Mmi1 binding to nam1 lncRNA controls MAPK-mediated entry into sexual differentiation. (E) Mmi1 RNA-IPs showing the specific loss of binding of Mmi1 to nam1-1 lncRNA but not to mei4 mRNA, another target of Mmi1.


Figure 2 . Mmi1 binding to nam1 lncRNA controls MAPK - mediated entry into sexual differentiation . ( F ) Mating assay showing the percentage of zygotes formed over time in WT and nam1 - 1 cells . Average fold enrichment is shown with error bars that indicate mean average deviations ( n = 3 ) ( B , E and F ) .

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Figure 2. Mmi1 binding to nam1 lncRNA controls MAPK-mediated entry into sexual differentiation. (F) Mating assay showing the percentage of zygotes formed over time in WT and nam1-1 cells. Average fold enrichment is shown with error bars that indicate mean average deviations (n=3) (B, E and F).


Figure 2 . Mmi1 binding to nam1 lncRNA controls MAPK - mediated entry into sexual differentiation . ( G ) Western blots showing the level of Flag - Byr2 protein over the first 4 hours of sexual differentiation in WT and nam1 - 1 cells . Tubulin ( Tub1 ) level was used as a loading control .

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Figure 2. Mmi1 binding to nam1 lncRNA controls MAPK-mediated entry into sexual differentiation. (G) Western blots showing the level of Flag-Byr2 protein over the first 4 hours of sexual differentiation in WT and nam1-1 cells. Tubulin (Tub1) level was used as a loading control.


Figure 2 . Mmi1 binding to nam1 lncRNA controls MAPK - mediated entry into sexual differentiation . ( H ) Microscopy images of WT ( h90 ) cells transformed with an empty plasmid ( Control ) and nam1 - 1 cells transformed with either an empty plasmid ( Control ) or a plasmid expressing Byr2 protein ( Byr2 ) , after 24 hours of induction of sexual differentiation . Scale bar , 10 µm .

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Figure 2. Mmi1 binding to nam1 lncRNA controls MAPK-mediated entry into sexual differentiation. (H) Microscopy images of WT (h90) cells transformed with an empty plasmid (Control) and nam1-1 cells transformed with either an empty plasmid (Control) or a plasmid expressing Byr2 protein (Byr2), after 24 hours of induction of sexual differentiation. Scale bar, 10 µm.


Figure 3 . Mmi1 promotes transcription termination of nam1 non - coding gene and prevents nam1 read - through transcription from repressing the downstream MAPKKK gene byr2 . ( A ) Northern blots showing nam1 and byr2 RNA levels during the first 4 hours of sexual differentiation . Ribosomal RNAs ( rRNAs ) stained with ethidium bromide were used as loading controls . Black lines indicate probes used to detect nam1 and byr2 RNAs .

Figure 3. Mmi1 promotes transcription termination of nam1 non-coding gene and prevents nam1 read-through transcription from repressing the downstream MAPKKK gene byr2. (A) Northern blots showing nam1 and byr2 RNA levels during the first 4 hours of sexual differentiation. Ribosomal RNAs (rRNAs) stained with ethidium bromide were used as loading controls. Black lines indicate probes used to detect nam1 and byr2 RNAs.


Figure 3 . Mmi1 promotes transcription termination of nam1 non - coding gene and prevents nam1 read - through transcription from repressing the downstream MAPKKK gene byr2 . ( B ) ChIPs showing the occupancy of the elongating RNAPII ( RNAPII - S2P ) over nam1 - byr2 locus , in WT and nam1 - 1 cells . RNAPII - S2P was immunoprecipitated with an antibody recognizing the heptameric repeats ( present in the C - terminal domain of the polymerase ) when it is phosphorylated on its serine 2 . Black lines , genomic regions investigated . Average fold enrichment is shown with error bars that indicate mean average deviations ( n = 3 ) for ( B , C , E and F ) .

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Figure 3. Mmi1 promotes transcription termination of nam1 non-coding gene and prevents nam1 read-through transcription from repressing the downstream MAPKKK gene byr2. (B) ChIPs showing the occupancy of the elongating RNAPII (RNAPII-S2P) over nam1-byr2 locus, in WT and nam1-1 cells. RNAPII-S2P was immunoprecipitated with an antibody recognizing the heptameric repeats (present in the C-terminal domain of the polymerase) when it is phosphorylated on its serine 2. Black lines, genomic regions investigated. Average fold enrichment is shown with error bars that indicate mean average deviations (n=3) for (B, C, E and F).


Figure 3 . Mmi1 promotes transcription termination of nam1 non - coding gene and prevents nam1 read - through transcription from repressing the downstream MAPKKK gene byr2 . ( C ) RT - qPCRs showing the accumulation of nam1 read - through transcripts ( RT1 - qPCR ) and nam1 lncRNAs ( RT2 - qPCR ) in cells with or without the transcription terminator Ttef inserted at the 3 ' end of nam1 ( scheme ) . Black arrow , primer used for the strand - specific reverse transcription ( RT ) ; Black line , location of the region amplified by PCR . Average fold enrichment is shown with error bars that indicate mean average deviations ( n = 3 ) for ( B , C , E and F ) .

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Figure 3. Mmi1 promotes transcription termination of nam1 non-coding gene and prevents nam1 read-through transcription from repressing the downstream MAPKKK gene byr2. (C) RT-qPCRs showing the accumulation of nam1 read-through transcripts (RT1-qPCR) and nam1 lncRNAs (RT2-qPCR) in cells with or without the transcription terminator Ttef inserted at the 3' end of nam1 (scheme). Black arrow, primer used for the strand-specific reverse transcription (RT); Black line, location of the region amplified by PCR. Average fold enrichment is shown with error bars that indicate mean average deviations (n=3) for (B, C, E and F).


Figure 3 . Mmi1 promotes transcription termination of nam1 non - coding gene and prevents nam1 read - through transcription from repressing the downstream MAPKKK gene byr2 . ( D ) Microscopy images of , respectively , WT ( h90 ) , nam1 - 1 and nam1 - 1 - Ttef cells , after induction of sexual differentiation for 24 hours . The percentage of sporulation and iodine vapor assays are shown at the bottom of the images . Scale bar , 10 µm .

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Figure 3. Mmi1 promotes transcription termination of nam1 non-coding gene and prevents nam1 read-through transcription from repressing the downstream MAPKKK gene byr2. (D) Microscopy images of, respectively, WT (h90), nam1-1 and nam1-1-Ttef cells, after induction of sexual differentiation for 24 hours. The percentage of sporulation and iodine vapor assays are shown at the bottom of the images. Scale bar, 10 µm.


Figure 3 . Mmi1 promotes transcription termination of nam1 non - coding gene and prevents nam1 read - through transcription from repressing the downstream MAPKKK gene byr2 . ( E ) RT - qPCRs showing the accumulation of nam1 read - through transcripts in mmi1 ∆ and rrp6 ∆ cells . Black arrow and line as in ( C ) . . Average fold enrichment is shown with error bars that indicate mean average deviations ( n = 3 ) for ( B , C , E and F ) .

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Figure 3. Mmi1 promotes transcription termination of nam1 non-coding gene and prevents nam1 read-through transcription from repressing the downstream MAPKKK gene byr2. (E) RT-qPCRs showing the accumulation of nam1 read-through transcripts in mmi1∆ and rrp6∆ cells. Black arrow and line as in (C). . Average fold enrichment is shown with error bars that indicate mean average deviations (n=3) for (B, C, E and F).


Figure 3 . Mmi1 promotes transcription termination of nam1 non - coding gene and prevents nam1 read - through transcription from repressing the downstream MAPKKK gene byr2 . ( F ) ChIPs monitoring the enrichment of H3K9me2 over nam1 - byr2 locus and mei4 gene in WT and rrp6 ∆ cells . Average fold enrichment is shown with error bars that indicate mean average deviations ( n = 3 ) for ( B , C , E and F ) .

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Figure 3. Mmi1 promotes transcription termination of nam1 non-coding gene and prevents nam1 read-through transcription from repressing the downstream MAPKKK gene byr2. (F) ChIPs monitoring the enrichment of H3K9me2 over nam1-byr2 locus and mei4 gene in WT and rrp6∆ cells. Average fold enrichment is shown with error bars that indicate mean average deviations (n=3) for (B, C, E and F).


Figure 4 . Mmi1 drives Rrp6 - dependent heterochromatin gene silencing at pericentromeric regions . ( B ) Northern blot showing the level of nam5 / 6 / 7 lncRNA population in mmi1 - ts3 , dcr1 ∆ , clr4 ∆ single mutant cells and in mmi1 - ts3 dcr1 ∆ and mmi1 - ts3 clr4 ∆ double mutant cells , at the permissive ( 25ºC ) and restrictive ( 36ºC ) temperatures .

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Figure 4. Mmi1 drives Rrp6-dependent heterochromatin gene silencing at pericentromeric regions. (B) Northern blot showing the level of nam5/6/7 lncRNA population in mmi1-ts3, dcr1∆, clr4∆ single mutant cells and in mmi1-ts3 dcr1∆ and mmi1-ts3 clr4∆ double mutant cells, at the permissive (25ºC) and restrictive (36ºC) temperatures.


Figure 4 . Mmi1 drives Rrp6 - dependent heterochromatin gene silencing at pericentromeric regions . ( C ) RT - PCRs monitoring the accumulation of nam7 lncRNAs and nam7 - L read - through transcripts , in the same cells and conditions as in ( B ) . Red arrow heads point to the expected PCR products while the other bands correspond to non - specific PCR products . From the scheme and the agarose gels : black arrows , primers used for the three different reverse transcriptions ; black lines and red numbers , expected PCR products of the three different RT - PCRs ; M , DNA ladder Markers ; tub1 , tubulin control .

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Figure 4. Mmi1 drives Rrp6-dependent heterochromatin gene silencing at pericentromeric regions. (C) RT-PCRs monitoring the accumulation of nam7 lncRNAs and nam7-L read-through transcripts, in the same cells and conditions as in (B). Red arrow heads point to the expected PCR products while the other bands correspond to non-specific PCR products. From the scheme and the agarose gels: black arrows, primers used for the three different reverse transcriptions; black lines and red numbers, expected PCR products of the three different RT-PCRs; M, DNA ladder Markers; tub1, tubulin control.