F . Loss of pancreatic islet area in Rb ∆ K7 mice correlates with reduced proliferation and H3K36me3 histone marks , but not with increased cell death . TUNEL analysis showing apoptosis in 10 day - old Rb ∆ K7 islets is even lower than in control littermates . Arrow points to a rare apoptotic cell in control islet . Serine800 / 804 phosphorylation of pRb in β - cells from 10 day - old control but not Rb ∆ K7 islets with a Ser - to - Ala substitution at this site . BrdU incorporation and PH3 staining showing reduced S - phase and mitotic cells , respectively , in 2 - and 3 - week old Rb ∆ K7 vs control islets . Reduced H3K36me3 staining in pancreatic islets from 3 - week old Rb ∆ K7 mice compared to control littermates ( n > 3 for each experiment ) . Scale bar , 100 μm .

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F. Loss of pancreatic islet area in Rb∆K7 mice correlates with reduced proliferation and H3K36me3 histone marks, but not with increased cell death. TUNEL analysis showing apoptosis in 10 day-old Rb∆K7 islets is even lower than in control littermates. Arrow points to a rare apoptotic cell in control islet. Serine800/804 phosphorylation of pRb in β-cells from 10 day-old control but not Rb∆K7 islets with a Ser-to-Ala substitution at this site. BrdU incorporation and PH3 staining showing reduced S-phase and mitotic cells, respectively, in 2- and 3-week old Rb∆K7 vs control islets. Reduced H3K36me3 staining in pancreatic islets from 3-week old Rb∆K7 mice compared to control littermates (n>3 for each experiment). Scale bar, 100 μm.


H . Reduced ability of Rb ∆ K7 β - cell to re - enter the cell cycle in response to pregnancy ( top ) or mitogenic ( bottom ) signals . Top , cell proliferation in P14 . 5 pregnant Rb ∆ K7 islet β - cell vs . control was determined by double immunostaining with BrdU and insulin ( left ) ; quantification ( right ) . Results were normalized for the number of BrdU / Insulin positive cells in control islets . An independent experiment using ki67 to quantify cell cycle re - entry top . Bottom , cell proliferation in Rb ∆ K7 β - cell vs . control islets following Exendin4 treatment was determined by immunostaining for Ki67 ; results were normalized for the number of positive cells in control islets . Mean ± SD , P values by two - tailed unpaired student ' s t - test . Scale bar , 100 μm .

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 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 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 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

H. Reduced ability of Rb∆K7 β-cell to re-enter the cell cycle in response to pregnancy (top) or mitogenic (bottom) signals. Top, cell proliferation in P14.5 pregnant Rb∆K7 islet β-cell vs. control was determined by double immunostaining with BrdU and insulin (left); quantification (right). Results were normalized for the number of BrdU/Insulin positive cells in control islets. An independent experiment using ki67 to quantify cell cycle re-entry top. Bottom, cell proliferation in Rb∆K7 β-cell vs. control islets following Exendin4 treatment was determined by immunostaining for Ki67; results were normalized for the number of positive cells in control islets. Mean ±SD, P values by two-tailed unpaired student's t-test. Scale bar, 100 μm.


I . Representative immunostaining showing induction of PDX1 ( islet ) , SOX9 ( pancreatic epithelium ) and MCM4 ( islet ) expression in Rb ∆ K7 vs control mice . Scale bar , 100 μm .

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I. Representative immunostaining showing induction of PDX1 (islet), SOX9 (pancreatic epithelium) and MCM4 (islet) expression in Rb∆K7 vs control mice. Scale bar, 100 μm.


D . Representative image of a pancreatic islet from a 9 - month old Rb ∆ K7 mouse immunestained for the SASP marker IL - 6 ( red ) and insulin ( green ) . DAPI was used to stain nuclei ( blue ) . Arrows point to IL - 6 + , insulin + pancreatic β - cells . Scale bar , 20 μm .

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D. Representative image of a pancreatic islet from a 9-month old Rb∆K7 mouse immunestained for the SASP marker IL-6 (red) and insulin (green). DAPI was used to stain nuclei (blue). Arrows point to IL-6+, insulin+ pancreatic β-cells. Scale bar, 20 μm.


E . Representative immunostaining for the DDR marker γ - H2A in islets from 3 - week old Rb ∆ K7 and control mice ( left ) ; statistical analysis ( right ; n = 5 each ) . Arrows point to pancreatic γ - H2A - positive Rb ∆ K7 islet cells . Error bars represent SD ; P value calculated by one - way ANOVA , Tukey ' s multiple comparisons test . Scale bar , 100 μm .

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E. Representative immunostaining for the DDR marker γ-H2A in islets from 3-week old Rb∆K7 and control mice (left); statistical analysis (right; n=5 each). Arrows point to pancreatic γ-H2A-positive Rb∆K7 islet cells. Error bars represent SD; P value calculated by one-way ANOVA, Tukey's multiple comparisons test. Scale bar, 100 μm.


H . Vitamin C diet increases 5hmC expression and improves β - cell morphology and islet size in Rb ∆ K7 mice . Scale bar , 100 μm . I . Vitamin C diet reduces the difference in γ − H2A + β - cells in Rb ∆ K7 vs control pancreatic islets relative to regular diet by 3 . 74 folds . Bars indicate mean ± SD . Adjusted P values by one - way ANOVA , Tukey ' s multiple comparisons test . J . Vitamin C diet increases pancreatic islet cell cycle re - entry during pregnancy of Rb ∆ K7 mice relative to control pregnant mice ( no statistical difference , n . s . ) or relative to pregnant Rb ∆ K7 vs control mice fed on regular diet ( P < 0 . 0001 ) by 3 . 8 folds . Bars represent mean ± SD . Adjusted P values by one - way ANOVA , Tukey ' s multiple comparisons test .

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

H. Vitamin C diet increases 5hmC expression and improves β-cell morphology and islet size in Rb∆K7 mice. Scale bar, 100 μm. I. Vitamin C diet reduces the difference in γ−H2A+ β-cells in Rb∆K7 vs control pancreatic islets relative to regular diet by 3.74 folds. Bars indicate mean ±SD. Adjusted P values by one-way ANOVA, Tukey's multiple comparisons test. J. Vitamin C diet increases pancreatic islet cell cycle re-entry during pregnancy of Rb∆K7 mice relative to control pregnant mice (no statistical difference, n.s.) or relative to pregnant Rb∆K7 vs control mice fed on regular diet (P<0.0001) by 3.8 folds. Bars represent mean ±SD. Adjusted P values by one-way ANOVA, Tukey's multiple comparisons test.


B . Left , top , effect of retrovirus - mediated transduction of under - phosphorylated Rb ∆ K11 ( rRb ∆ K11 ) on Rb - deficient mammary tumor cell proliferation as determined by MTT assay ( n = 4 - 5 technical replicates ) . Left , bottom , quantification of SA - βGAL in rRb ∆ K11 - transduced cells . Bars represent mean ± SD ; P value calculated by two - tailed unpaired student ' s t - test . Right , representative images of SA - βGAL staining showing robust senescence ( arrows ) in rRb ∆ K11 - transduced cells . Original magnification : 200x .

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B. Left, top, effect of retrovirus-mediated transduction of under-phosphorylated Rb∆K11 (rRb∆K11) on Rb-deficient mammary tumor cell proliferation as determined by MTT assay (n=4-5 technical replicates). Left, bottom, quantification of SA-βGAL in rRb∆K11-transduced cells. Bars represent mean ±SD; P value calculated by two-tailed unpaired student's t-test. Right, representative images of SA-βGAL staining showing robust senescence (arrows) in rRb∆K11-transduced cells. Original magnification: 200x.


F . Vitamin C reduces cellular senescence induced by retroviral - mediated over - expression of Rb ∆ K11 . Top - images of SA - βGAL staining in human diploid cells , IMR90 , transduced with control or rRb ∆ K11 virus with or without vitamin C added at the time of infection . Arrows point to SA - βGAL - positive cells . Bottom left , quantification of SA - βGAL staining ( bars represent mean ± SEM ; n = 4 biological replicates ) . P values by one - way ANOVA , Tukey ' s multiple comparisons test . Bottom right , dot - blot analysis with antibody for 5hmC on DNA extracted from IMR90 cells treated or not with vitamin C . Original magnification : 200x .

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F. Vitamin C reduces cellular senescence induced by retroviral-mediated over-expression of Rb∆K11. Top - images of SA-βGAL staining in human diploid cells, IMR90, transduced with control or rRb∆K11 virus with or without vitamin C added at the time of infection. Arrows point to SA-βGAL-positive cells. Bottom left, quantification of SA-βGAL staining (bars represent mean ±SEM; n=4 biological replicates). P values by one-way ANOVA, Tukey's multiple comparisons test. Bottom right, dot-blot analysis with antibody for 5hmC on DNA extracted from IMR90 cells treated or not with vitamin C. Original magnification: 200x.


Associations between CSF T - tau , Ng and NFL ( panels A - C ) . Black squares : CN ( n = 109 ) , red circles : MCI ( n = 187 ) , blue triangles : AD ( n = 93 ) . Associations are shown for Spearman correlations . Aβ42 and T - tau were measured using the the INNOBIA AlzBio3 kit ( Fujirebio , Ghent , Belgium ) , Ng was measured using an in - house immunoassay for Ng ( Portelius et al , 2015 ) and NFL was measured using the NF - light ® ELISA kit ( Uman Diagnostics , Umeå , Sweden ) .

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Associations between CSF T-tau, Ng and NFL (panels A-C). Black squares: CN (n=109), red circles: MCI (n=187), blue triangles: AD (n=93). Associations are shown for Spearman correlations. Aβ42 and T-tau were measured using the the INNOBIA AlzBio3 kit (Fujirebio, Ghent, Belgium), Ng was measured using an in-house immunoassay for Ng (Portelius et al, 2015) and NFL was measured using the NF-light® ELISA kit (Uman Diagnostics, Umeå, Sweden).


Associations between CSF T - tau , Ng and NFL and between these biomarkers and CSF A42 ( panels D - F ) . Black squares : CN ( n = 109 ) , red circles : MCI ( n = 187 ) , blue triangles : AD ( n = 93 ) . Associations are shown for Spearman correlations . Aβ42 and T - tau were measured using the the INNOBIA AlzBio3 kit ( Fujirebio , Ghent , Belgium ) , Ng was measured using an in - house immunoassay for Ng ( Portelius et al , 2015 ) and NFL was measured using the NF - light ® ELISA kit ( Uman Diagnostics , Umeå , Sweden ) .

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

Associations between CSF T-tau, Ng and NFL and between these biomarkers and CSF A42 (panels D-F). Black squares: CN (n=109), red circles: MCI (n=187), blue triangles: AD (n=93). Associations are shown for Spearman correlations. Aβ42 and T-tau were measured using the the INNOBIA AlzBio3 kit (Fujirebio, Ghent, Belgium), Ng was measured using an in-house immunoassay for Ng (Portelius et al, 2015) and NFL was measured using the NF-light® ELISA kit (Uman Diagnostics, Umeå, Sweden).


CSF T - tau ( panel A ) , Ng ( panel B ) and NFL ( panel C ) in different combinations of clinical diagnosis and A pathology ( CN Aβ - , n = 69 ; CN Aβ + , n = 40 ; MCI Aβ - , n = 50 ; MCI Aβ + , n = 137 ; AD Aβ - , n = 8 ; AD Aβ + , n = 85 ) . See Table 5 for comparisons between groups . Biomarker levels are standardized to z - scores and shown in boxplots ( indicating median and interquartile ranges ; whiskers are defined as quartiles 1 and 3 + / - 1 . 5 * interquartile range , respectively ) . Aβ42 and T - tau were measured using the the INNOBIA AlzBio3 kit ( Fujirebio , Ghent , Belgium ) , Ng was measured using an in - house immunoassay for Ng ( Portelius et al , 2015 ) and NFL was measured using the NF - light ® ELISA kit ( Uman Diagnostics , Umeå , Sweden ) .

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

CSF T-tau (panel A), Ng (panel B) and NFL (panel C) in different combinations of clinical diagnosis and A pathology (CN Aβ-, n=69; CN Aβ+, n=40; MCI Aβ-, n=50; MCI Aβ+, n=137; AD Aβ-, n=8; AD Aβ+, n=85). See Table 5 for comparisons between groups. Biomarker levels are standardized to z-scores and shown in boxplots (indicating median and interquartile ranges; whiskers are defined as quartiles 1 and 3 +/- 1.5 * interquartile range, respectively). Aβ42 and T-tau were measured using the the INNOBIA AlzBio3 kit (Fujirebio, Ghent, Belgium), Ng was measured using an in-house immunoassay for Ng (Portelius et al, 2015) and NFL was measured using the NF-light® ELISA kit (Uman Diagnostics, Umeå, Sweden).


Baseline associations between CSF T - tau , Ng and NFL and other AD traits . Data are estimates ( - coefficients ) from linear mixed effects models , with 95 % confidence intervals . The estimates are the main effects of the biomarkers , capturing the effects at study baseline . Effects were significant ( * ) for MMSE : T - tau ( p = . 034 ) and NFL ( p < . 0001 ) in A - negative . Note also that the range of the y - axes differs for the different outcomes , for visualization purposes . Models were adjusted for age and sex , and education ( for cognitive measures ) , and intracranial volume ( for MRI measures ) . Aβ42 and T - tau were measured using the the INNOBIA AlzBio3 kit ( Fujirebio , Ghent , Belgium ) , Ng was measured using an in - house immunoassay for Ng ( Portelius et al , 2015 ) and NFL was measured using the NF - light ® ELISA kit ( Uman Diagnostics , Umeå , Sweden ) .

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

Baseline associations between CSF T-tau, Ng and NFL and other AD traits. Data are estimates (-coefficients) from linear mixed effects models, with 95 % confidence intervals. The estimates are the main effects of the biomarkers, capturing the effects at study baseline. Effects were significant (*) for MMSE: T-tau (p=.034) and NFL (p<.0001) in A-negative. Note also that the range of the y-axes differs for the different outcomes, for visualization purposes. Models were adjusted for age and sex, and education (for cognitive measures), and intracranial volume (for MRI measures). Aβ42 and T-tau were measured using the the INNOBIA AlzBio3 kit (Fujirebio, Ghent, Belgium), Ng was measured using an in-house immunoassay for Ng (Portelius et al, 2015) and NFL was measured using the NF-light® ELISA kit (Uman Diagnostics, Umeå, Sweden).


Baseline associations between CSF T - tau , Ng and NFL and other AD traits . Data are estimates ( - coefficients ) from linear mixed effects models , with 95 % confidence intervals . The estimates are the main effects of the biomarkers , capturing the effects at study baseline . Effects were significant ( * ) for ADAS - cog : NFL in A - negative ( p = . 0013 ) and A - positive ( p = . 024 ) . Biomarkers and outcomes were standardized to z - scores . Note that the y - axes for ADAS - cog are flipped , so that the lower ranges constantly reflect " worse " outcomes . Note also that the range of the y - axes differs for the different outcomes , for visualization purposes . Models were adjusted for age and sex , and education ( for cognitive measures ) , and intracranial volume ( for MRI measures ) . Aβ42 and T - tau were measured using the the INNOBIA AlzBio3 kit ( Fujirebio , Ghent , Belgium ) , Ng was measured using an in - house immunoassay for Ng ( Portelius et al , 2015 ) and NFL was measured using the NF - light ® ELISA kit ( Uman Diagnostics , Umeå , Sweden ) .

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

Baseline associations between CSF T-tau, Ng and NFL and other AD traits. Data are estimates (-coefficients) from linear mixed effects models, with 95 % confidence intervals. The estimates are the main effects of the biomarkers, capturing the effects at study baseline. Effects were significant (*) for ADAS-cog: NFL in A-negative (p=.0013) and A-positive (p=.024). Biomarkers and outcomes were standardized to z-scores. Note that the y-axes for ADAS-cog are flipped, so that the lower ranges constantly reflect "worse" outcomes. Note also that the range of the y-axes differs for the different outcomes, for visualization purposes. Models were adjusted for age and sex, and education (for cognitive measures), and intracranial volume (for MRI measures). Aβ42 and T-tau were measured using the the INNOBIA AlzBio3 kit (Fujirebio, Ghent, Belgium), Ng was measured using an in-house immunoassay for Ng (Portelius et al, 2015) and NFL was measured using the NF-light® ELISA kit (Uman Diagnostics, Umeå, Sweden).


Baseline associations between CSF T - tau , Ng and NFL and other AD traits . Data are estimates ( - coefficients ) from linear mixed effects models , with 95 % confidence intervals . The estimates are the main effects of the biomarkers , capturing the effects at study baseline . Effects were significant ( * ) for hippocampal volume : NFL in A - negative ( p < . 0001 ) . Biomarkers and outcomes were standardized to z - scores . Note also that the range of the y - axes differs for the different outcomes , for visualization purposes . Models were adjusted for age and sex , and education ( for cognitive measures ) , and intracranial volume ( for MRI measures ) . Aβ42 and T - tau were measured using the the INNOBIA AlzBio3 kit ( Fujirebio , Ghent , Belgium ) , Ng was measured using an in - house immunoassay for Ng ( Portelius et al , 2015 ) and NFL was measured using the NF - light ® ELISA kit ( Uman Diagnostics , Umeå , Sweden ) .

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

Baseline associations between CSF T-tau, Ng and NFL and other AD traits. Data are estimates (-coefficients) from linear mixed effects models, with 95 % confidence intervals. The estimates are the main effects of the biomarkers, capturing the effects at study baseline. Effects were significant (*) for hippocampal volume: NFL in A-negative (p<.0001). Biomarkers and outcomes were standardized to z-scores. Note also that the range of the y-axes differs for the different outcomes, for visualization purposes. Models were adjusted for age and sex, and education (for cognitive measures), and intracranial volume (for MRI measures). Aβ42 and T-tau were measured using the the INNOBIA AlzBio3 kit (Fujirebio, Ghent, Belgium), Ng was measured using an in-house immunoassay for Ng (Portelius et al, 2015) and NFL was measured using the NF-light® ELISA kit (Uman Diagnostics, Umeå, Sweden).


Baseline associations between CSF T - tau , Ng and NFL and other AD traits . Data are estimates ( - coefficients ) from linear mixed effects models , with 95 % confidence intervals . The estimates are the main effects of the biomarkers , capturing the effects at study baseline . Effects were significant ( * ) for lateral ventricles : T - tau ( p = . 0019 ) , Ng ( p = . 00092 ) and NFL ( p = . 0024 ) in A - negative and T - tau ( p = . 00083 ) and Ng ( p < . 0001 ) in A - positive . Biomarkers and outcomes were standardized to z - scores . Note that the y - axes for ventricle size are flipped , so that the lower ranges constantly reflect " worse " outcomes . Note also that the range of the y - axes differs for the different outcomes , for visualization purposes . Models were adjusted for age and sex , and education ( for cognitive measures ) , and intracranial volume ( for MRI measures ) . Aβ42 and T - tau were measured using the the INNOBIA AlzBio3 kit ( Fujirebio , Ghent , Belgium ) , Ng was measured using an in - house immunoassay for Ng ( Portelius et al , 2015 ) and NFL was measured using the NF - light ® ELISA kit ( Uman Diagnostics , Umeå , Sweden ) .

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

Baseline associations between CSF T-tau, Ng and NFL and other AD traits. Data are estimates (-coefficients) from linear mixed effects models, with 95 % confidence intervals. The estimates are the main effects of the biomarkers, capturing the effects at study baseline. Effects were significant (*) for lateral ventricles: T-tau (p=.0019), Ng (p=.00092) and NFL (p=.0024) in A-negative and T-tau (p=.00083) and Ng (p<.0001) in A-positive. Biomarkers and outcomes were standardized to z-scores. Note that the y-axes for ventricle size are flipped, so that the lower ranges constantly reflect "worse" outcomes. Note also that the range of the y-axes differs for the different outcomes, for visualization purposes. Models were adjusted for age and sex, and education (for cognitive measures), and intracranial volume (for MRI measures). Aβ42 and T-tau were measured using the the INNOBIA AlzBio3 kit (Fujirebio, Ghent, Belgium), Ng was measured using an in-house immunoassay for Ng (Portelius et al, 2015) and NFL was measured using the NF-light® ELISA kit (Uman Diagnostics, Umeå, Sweden).


Baseline associations between CSF T - tau , Ng and NFL and other AD traits . Data are estimates ( - coefficients ) from linear mixed effects models , with 95 % confidence intervals . The estimates are the main effects of the biomarkers , capturing the effects at study baseline . Effects were significant ( * ) for FDG - PET : NFL in A - negative ( p = . 046 ) and A - positive ( p = . 0041 ) . Biomarkers and outcomes were standardized to z - scores . Note also that the range of the y - axes differs for the different outcomes , for visualization purposes . Models were adjusted for age and sex , and education ( for cognitive measures ) , and intracranial volume ( for MRI measures ) . Aβ42 and T - tau were measured using the the INNOBIA AlzBio3 kit ( Fujirebio , Ghent , Belgium ) , Ng was measured using an in - house immunoassay for Ng ( Portelius et al , 2015 ) and NFL was measured using the NF - light ® ELISA kit ( Uman Diagnostics , Umeå , Sweden ) .

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

Baseline associations between CSF T-tau, Ng and NFL and other AD traits. Data are estimates (-coefficients) from linear mixed effects models, with 95 % confidence intervals. The estimates are the main effects of the biomarkers, capturing the effects at study baseline. Effects were significant (*) for FDG-PET: NFL in A-negative (p=.046) and A-positive (p=.0041). Biomarkers and outcomes were standardized to z-scores. Note also that the range of the y-axes differs for the different outcomes, for visualization purposes. Models were adjusted for age and sex, and education (for cognitive measures), and intracranial volume (for MRI measures). Aβ42 and T-tau were measured using the the INNOBIA AlzBio3 kit (Fujirebio, Ghent, Belgium), Ng was measured using an in-house immunoassay for Ng (Portelius et al, 2015) and NFL was measured using the NF-light® ELISA kit (Uman Diagnostics, Umeå, Sweden).


Baseline associations between CSF T - tau , Ng and NFL and other AD traits . Data are estimates ( - coefficients ) from linear mixed effects models , with 95 % confidence intervals . The estimates are the main effects of the biomarkers , capturing the effects at study baseline . Biomarkers and outcomes were standardized to z - scores . Note that the y - axes for WMH are flipped , so that the lower ranges constantly reflect " worse " outcomes . Note also that the range of the y - axes differs for the different outcomes , for visualization purposes . Models were adjusted for age and sex , and education ( for cognitive measures ) , and intracranial volume ( for MRI measures ) . Aβ42 and T - tau were measured using the the INNOBIA AlzBio3 kit ( Fujirebio , Ghent , Belgium ) , Ng was measured using an in - house immunoassay for Ng ( Portelius et al , 2015 ) and NFL was measured using the NF - light ® ELISA kit ( Uman Diagnostics , Umeå , Sweden ) .

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

Baseline associations between CSF T-tau, Ng and NFL and other AD traits. Data are estimates (-coefficients) from linear mixed effects models, with 95 % confidence intervals. The estimates are the main effects of the biomarkers, capturing the effects at study baseline. Biomarkers and outcomes were standardized to z-scores. Note that the y-axes for WMH are flipped, so that the lower ranges constantly reflect "worse" outcomes. Note also that the range of the y-axes differs for the different outcomes, for visualization purposes. Models were adjusted for age and sex, and education (for cognitive measures), and intracranial volume (for MRI measures). Aβ42 and T-tau were measured using the the INNOBIA AlzBio3 kit (Fujirebio, Ghent, Belgium), Ng was measured using an in-house immunoassay for Ng (Portelius et al, 2015) and NFL was measured using the NF-light® ELISA kit (Uman Diagnostics, Umeå, Sweden).


Longitudinal associations between CSF T - tau , Ng and NFL and other AD traits . Data are estimates ( - coefficients ) from linear mixed effects models , with 95 % confidence intervals . The estimates are the effect of time plus the biomarker by time interactions , capturing the longitudinal effects of the biomarkers . For each model , the " average " effect of time is also shown for comparison . Effects were significant ( * ) , meaning that biomarker levels affected the slopes of the outcome , for MMSE : T - tau ( p = . 021 ) and NFL ( p < . 0001 ) in A - negative and for T - tau ( p < . 0001 ) , Ng ( p = . 0031 ) and NFL ( p < . 0001 ) in A - positive . Biomarkers and outcomes were standardized to z - scores . Note also that the range of the y - axes differs for the different outcomes , for visualization purposes . Models were adjusted for age and sex , and education ( for cognitive measures ) , and intracranial volume ( for MRI measures ) . Aβ42 and T - tau were measured using the the INNOBIA AlzBio3 kit ( Fujirebio , Ghent , Belgium ) , Ng was measured using an in - house immunoassay for Ng ( Portelius et al , 2015 ) and NFL was measured using the NF - light ® ELISA kit ( Uman Diagnostics , Umeå , Sweden ) .

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

Longitudinal associations between CSF T-tau, Ng and NFL and other AD traits. Data are estimates (-coefficients) from linear mixed effects models, with 95 % confidence intervals. The estimates are the effect of time plus the biomarker by time interactions, capturing the longitudinal effects of the biomarkers. For each model, the "average" effect of time is also shown for comparison. Effects were significant (*), meaning that biomarker levels affected the slopes of the outcome, for MMSE: T-tau (p=.021) and NFL (p<.0001) in A-negative and for T-tau (p<.0001), Ng (p=.0031) and NFL (p<.0001) in A-positive. Biomarkers and outcomes were standardized to z-scores. Note also that the range of the y-axes differs for the different outcomes, for visualization purposes. Models were adjusted for age and sex, and education (for cognitive measures), and intracranial volume (for MRI measures). Aβ42 and T-tau were measured using the the INNOBIA AlzBio3 kit (Fujirebio, Ghent, Belgium), Ng was measured using an in-house immunoassay for Ng (Portelius et al, 2015) and NFL was measured using the NF-light® ELISA kit (Uman Diagnostics, Umeå, Sweden).


Longitudinal associations between CSF T - tau , Ng and NFL and other AD traits . Data are estimates ( - coefficients ) from linear mixed effects models , with 95 % confidence intervals . The estimates are the effect of time plus the biomarker by time interactions , capturing the longitudinal effects of the biomarkers . For each model , the " average " effect of time is also shown for comparison . Effects were significant ( * ) , meaning that biomarker levels affected the slopes of the outcome , for ADAS - cog : NFL in A - negative ( p < . 0001 ) and for T - tau ( p < . 0001 ) , Ng ( p = . 0015 ) and NFL ( p = . 0027 ) in A - positive . Biomarkers and outcomes were standardized to z - scores . Note that the y - axes for ADAS - cog are flipped , so that the lower ranges constantly reflect " worse " outcomes . Note also that the range of the y - axes differs for the different outcomes , for visualization purposes . Models were adjusted for age and sex , and education ( for cognitive measures ) , and intracranial volume ( for MRI measures ) . Aβ42 and T - tau were measured using the the INNOBIA AlzBio3 kit ( Fujirebio , Ghent , Belgium ) , Ng was measured using an in - house immunoassay for Ng ( Portelius et al , 2015 ) and NFL was measured using the NF - light ® ELISA kit ( Uman Diagnostics , Umeå , Sweden ) .

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

Longitudinal associations between CSF T-tau, Ng and NFL and other AD traits. Data are estimates (-coefficients) from linear mixed effects models, with 95 % confidence intervals. The estimates are the effect of time plus the biomarker by time interactions, capturing the longitudinal effects of the biomarkers. For each model, the "average" effect of time is also shown for comparison. Effects were significant (*), meaning that biomarker levels affected the slopes of the outcome, for ADAS-cog: NFL in A-negative (p<.0001) and for T-tau (p<.0001), Ng (p=.0015) and NFL (p=.0027) in A-positive. Biomarkers and outcomes were standardized to z-scores. Note that the y-axes for ADAS-cog are flipped, so that the lower ranges constantly reflect "worse" outcomes. Note also that the range of the y-axes differs for the different outcomes, for visualization purposes. Models were adjusted for age and sex, and education (for cognitive measures), and intracranial volume (for MRI measures). Aβ42 and T-tau were measured using the the INNOBIA AlzBio3 kit (Fujirebio, Ghent, Belgium), Ng was measured using an in-house immunoassay for Ng (Portelius et al, 2015) and NFL was measured using the NF-light® ELISA kit (Uman Diagnostics, Umeå, Sweden).


Longitudinal associations between CSF T - tau , Ng and NFL and other AD traits . Data are estimates ( - coefficients ) from linear mixed effects models , with 95 % confidence intervals . The estimates are the effect of time plus the biomarker by time interactions , capturing the longitudinal effects of the biomarkers . For each model , the " average " effect of time is also shown for comparison . Effects were significant ( * ) , meaning that biomarker levels affected the slopes of the outcome , for hippocampal volume : NFL in A - negative ( p = . 0048 ) and for T - tau ( p = . 0015 ) and Ng ( p = . 0027 ) in A - positive . Biomarkers and outcomes were standardized to z - scores . Note also that the range of the y - axes differs for the different outcomes , for visualization purposes . Models were adjusted for age and sex , and education ( for cognitive measures ) , and intracranial volume ( for MRI measures ) . Aβ42 and T - tau were measured using the the INNOBIA AlzBio3 kit ( Fujirebio , Ghent , Belgium ) , Ng was measured using an in - house immunoassay for Ng ( Portelius et al , 2015 ) and NFL was measured using the NF - light ® ELISA kit ( Uman Diagnostics , Umeå , Sweden ) .

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

Longitudinal associations between CSF T-tau, Ng and NFL and other AD traits. Data are estimates (-coefficients) from linear mixed effects models, with 95 % confidence intervals. The estimates are the effect of time plus the biomarker by time interactions, capturing the longitudinal effects of the biomarkers. For each model, the "average" effect of time is also shown for comparison. Effects were significant (*), meaning that biomarker levels affected the slopes of the outcome, for hippocampal volume: NFL in A-negative (p=.0048) and for T-tau (p=.0015) and Ng (p=.0027) in A-positive. Biomarkers and outcomes were standardized to z-scores. Note also that the range of the y-axes differs for the different outcomes, for visualization purposes. Models were adjusted for age and sex, and education (for cognitive measures), and intracranial volume (for MRI measures). Aβ42 and T-tau were measured using the the INNOBIA AlzBio3 kit (Fujirebio, Ghent, Belgium), Ng was measured using an in-house immunoassay for Ng (Portelius et al, 2015) and NFL was measured using the NF-light® ELISA kit (Uman Diagnostics, Umeå, Sweden).


Longitudinal associations between CSF T - tau , Ng and NFL and other AD traits . Data are estimates ( - coefficients ) from linear mixed effects models , with 95 % confidence intervals . The estimates are the effect of time plus the biomarker by time interactions , capturing the longitudinal effects of the biomarkers . For each model , the " average " effect of time is also shown for comparison . Effects were significant ( * ) , meaning that biomarker levels affected the slopes of the outcome , for lateral ventricles : Ng ( p = . 0051 ) and NFL ( p < . 0001 ) in A - negative and NFL ( p < . 0001 ) in A - positive . Biomarkers and outcomes were standardized to z - scores . Note that the y - axes for ventricle size are flipped , so that the lower ranges constantly reflect " worse " outcomes . Note also that the range of the y - axes differs for the different outcomes , for visualization purposes . Models were adjusted for age and sex , and education ( for cognitive measures ) , and intracranial volume ( for MRI measures ) . Aβ42 and T - tau were measured using the the INNOBIA AlzBio3 kit ( Fujirebio , Ghent , Belgium ) , Ng was measured using an in - house immunoassay for Ng ( Portelius et al , 2015 ) and NFL was measured using the NF - light ® ELISA kit ( Uman Diagnostics , Umeå , Sweden ) .

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

Longitudinal associations between CSF T-tau, Ng and NFL and other AD traits. Data are estimates (-coefficients) from linear mixed effects models, with 95 % confidence intervals. The estimates are the effect of time plus the biomarker by time interactions, capturing the longitudinal effects of the biomarkers. For each model, the "average" effect of time is also shown for comparison. Effects were significant (*), meaning that biomarker levels affected the slopes of the outcome, for lateral ventricles: Ng (p=.0051) and NFL (p<.0001) in A-negative and NFL (p<.0001) in A-positive. Biomarkers and outcomes were standardized to z-scores. Note that the y-axes for ventricle size are flipped, so that the lower ranges constantly reflect "worse" outcomes. Note also that the range of the y-axes differs for the different outcomes, for visualization purposes. Models were adjusted for age and sex, and education (for cognitive measures), and intracranial volume (for MRI measures). Aβ42 and T-tau were measured using the the INNOBIA AlzBio3 kit (Fujirebio, Ghent, Belgium), Ng was measured using an in-house immunoassay for Ng (Portelius et al, 2015) and NFL was measured using the NF-light® ELISA kit (Uman Diagnostics, Umeå, Sweden).


Longitudinal associations between CSF T - tau , Ng and NFL and other AD traits . Data are estimates ( - coefficients ) from linear mixed effects models , with 95 % confidence intervals . The estimates are the effect of time plus the biomarker by time interactions , capturing the longitudinal effects of the biomarkers . For each model , the " average " effect of time is also shown for comparison . Effects were significant ( * ) , meaning that biomarker levels affected the slopes of the outcome , for FDG - PET : T - tau ( p = . 00038 ) and Ng ( p = . 0019 ) in A - positive . Biomarkers and outcomes were standardized to z - scores . Note also that the range of the y - axes differs for the different outcomes , for visualization purposes . Models were adjusted for age and sex , and education ( for cognitive measures ) , and intracranial volume ( for MRI measures ) . Aβ42 and T - tau were measured using the the INNOBIA AlzBio3 kit ( Fujirebio , Ghent , Belgium ) , Ng was measured using an in - house immunoassay for Ng ( Portelius et al , 2015 ) and NFL was measured using the NF - light ® ELISA kit ( Uman Diagnostics , Umeå , Sweden ) .

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

Longitudinal associations between CSF T-tau, Ng and NFL and other AD traits. Data are estimates (-coefficients) from linear mixed effects models, with 95 % confidence intervals. The estimates are the effect of time plus the biomarker by time interactions, capturing the longitudinal effects of the biomarkers. For each model, the "average" effect of time is also shown for comparison. Effects were significant (*), meaning that biomarker levels affected the slopes of the outcome, for FDG-PET: T-tau (p=.00038) and Ng (p=.0019) in A-positive. Biomarkers and outcomes were standardized to z-scores. Note also that the range of the y-axes differs for the different outcomes, for visualization purposes. Models were adjusted for age and sex, and education (for cognitive measures), and intracranial volume (for MRI measures). Aβ42 and T-tau were measured using the the INNOBIA AlzBio3 kit (Fujirebio, Ghent, Belgium), Ng was measured using an in-house immunoassay for Ng (Portelius et al, 2015) and NFL was measured using the NF-light® ELISA kit (Uman Diagnostics, Umeå, Sweden).


Longitudinal associations between CSF T - tau , Ng and NFL and other AD traits . Data are estimates ( - coefficients ) from linear mixed effects models , with 95 % confidence intervals . The estimates are the effect of time plus the biomarker by time interactions , capturing the longitudinal effects of the biomarkers . For each model , the " average " effect of time is also shown for comparison . Biomarkers and outcomes were standardized to z - scores . Note that the y - axes for WMH are flipped , so that the lower ranges constantly reflect " worse " outcomes . Note also that the range of the y - axes differs for the different outcomes , for visualization purposes . Models were adjusted for age and sex , and education ( for cognitive measures ) , and intracranial volume ( for MRI measures ) . Aβ42 and T - tau were measured using the the INNOBIA AlzBio3 kit ( Fujirebio , Ghent , Belgium ) , Ng was measured using an in - house immunoassay for Ng ( Portelius et al , 2015 ) and NFL was measured using the NF - light ® ELISA kit ( Uman Diagnostics , Umeå , Sweden ) .

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

Longitudinal associations between CSF T-tau, Ng and NFL and other AD traits. Data are estimates (-coefficients) from linear mixed effects models, with 95 % confidence intervals. The estimates are the effect of time plus the biomarker by time interactions, capturing the longitudinal effects of the biomarkers. For each model, the "average" effect of time is also shown for comparison. Biomarkers and outcomes were standardized to z-scores. Note that the y-axes for WMH are flipped, so that the lower ranges constantly reflect "worse" outcomes. Note also that the range of the y-axes differs for the different outcomes, for visualization purposes. Models were adjusted for age and sex, and education (for cognitive measures), and intracranial volume (for MRI measures). Aβ42 and T-tau were measured using the the INNOBIA AlzBio3 kit (Fujirebio, Ghent, Belgium), Ng was measured using an in-house immunoassay for Ng (Portelius et al, 2015) and NFL was measured using the NF-light® ELISA kit (Uman Diagnostics, Umeå, Sweden).


( B ) GST , GST - MCM2N ( human ) and GST - Spt5N ( yeast ) were mixed as indicated with yeast histones H3 - H4 , in the presence of glutathione agarose beads ( see STAR method for further details ) . The input and pulldown samples were analysed by SDS - PAGE before staining with colloidal Coomassie blue . The asterisks denote truncations of GST - Spt5N . ( C ) Similar experiment to ( B ) but with histones H2A - H2B . ( D ) Similar experiment to ( B ) to compare histone binding by GST - MCM2N and GST - MCM2N - 2A ( with Y81A Y90A mutations of conserved residues ) .

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

(B) GST, GST-MCM2N (human) and GST-Spt5N (yeast) were mixed as indicated with yeast histones H3-H4, in the presence of glutathione agarose beads (see STAR method for further details). The input and pulldown samples were analysed by SDS-PAGE before staining with colloidal Coomassie blue. The asterisks denote truncations of GST-Spt5N. (C) Similar experiment to (B) but with histones H2A-H2B. (D) Similar experiment to (B) to compare histone binding by GST-MCM2N and GST-MCM2N-2A (with Y81A Y90A mutations of conserved residues).


( H ) . Budding yeast cells expressing fusions of Spt5N and Spt5N - 3A to Protein A were grown in parallel with cells expressing the equivalent wild type and mutant versions of fission yeast Spt5N ( Sp Spt5N and Sp Spt5N - 3A - the latter contained the F146A , E150A and V153A mutations ) .

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

(H). Budding yeast cells expressing fusions of Spt5N and Spt5N-3A to Protein A were grown in parallel with cells expressing the equivalent wild type and mutant versions of fission yeast Spt5N (Sp Spt5N and Sp Spt5N-3A - the latter contained the F146A, E150A and V153A mutations).


( C ) Yeast cells were generated that expressed Protein A - tagged versions of wild type Spt5 ( full length or the indicated truncations ) , or full length Spt5 - 3A , under the control of the GAL1 , 10 promoter . Cells were grown in medium containing galactose and the ProteinA - tagged Spt5 proteins were isolated from cell extracts on magnetic beads coated with IgG . Association of the tagged Spt5 proteins with the elongating form of Pol II was monitored by immunoblotting , using antibodies specific to the indicated phosphorylation sites in the C - Terminal Domain repeats of the Rpb1 subunit of Pol II .

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(C) Yeast cells were generated that expressed Protein A-tagged versions of wild type Spt5 (full length or the indicated truncations), or full length Spt5-3A, under the control of the GAL1,10 promoter. Cells were grown in medium containing galactose and the ProteinA-tagged Spt5 proteins were isolated from cell extracts on magnetic beads coated with IgG. Association of the tagged Spt5 proteins with the elongating form of Pol II was monitored by immunoblotting, using antibodies specific to the indicated phosphorylation sites in the C-Terminal Domain repeats of the Rpb1 subunit of Pol II.


( E ) Serial dilutions of control cells or spt5 - AID cells , expressing the indicated versions of Spt5 from the GAL1 , 10 promoter , were grown on the indicated medium .

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(E) Serial dilutions of control cells or spt5-AID cells, expressing the indicated versions of Spt5 from the GAL1,10 promoter, were grown on the indicated medium.


( B ) Immunoblot analysis at the indicated times for the samples described in ( A ) .

(B) Immunoblot analysis at the indicated times for the samples described in (A).


( C ) Cells from the end of the experiment described in ( A ) were fixed and processed for ChIP - Seq , using antibodies specific for phosphorylated Serine 5 in the C - Terminal Domain of the Pol II catalytic subunit Rpb1 ( CTD - Ser5P ) . For each of the three indicated strains , the histograms represent the normalized read density ( DNA sequence read count per million reads , or RPM ) , across a sample gene body .

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(C) Cells from the end of the experiment described in (A) were fixed and processed for ChIP-Seq, using antibodies specific for phosphorylated Serine 5 in the C-Terminal Domain of the Pol II catalytic subunit Rpb1 (CTD-Ser5P). For each of the three indicated strains, the histograms represent the normalized read density (DNA sequence read count per million reads, or RPM), across a sample gene body.


( A ) Samples from the experiment described in Figure 3A were also processed for MNase - Seq . For each of the three indicated strains , the histograms represent the normalized DNA sequence read count per million reads ( RPM ) , across a sample gene body .

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(A) Samples from the experiment described in Figure 3A were also processed for MNase-Seq. For each of the three indicated strains, the histograms represent the normalized DNA sequence read count per million reads (RPM), across a sample gene body.


( C ) Samples from the same experiment were further processed for ChIP - Seq using antibodies to histone H3K4me3 . For each of the three indicated strains , the normalised read count per million reads is presented across a sample gene body .

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(C) Samples from the same experiment were further processed for ChIP-Seq using antibodies to histone H3K4me3. For each of the three indicated strains, the normalised read count per million reads is presented across a sample gene body.


Decreasing ∆ ψm from ∆ III to ρ0 cells . WT and mutant yeast cells were cultured to log phase and stained with TMRM for FACS analysis .

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Decreasing ∆ψm from ∆III to ρ0 cells. WT and mutant yeast cells were cultured to log phase and stained with TMRM for FACS analysis.


Decreasing proliferation rates from ∆ III to ρ0 cells . Cells were cultured from 0 . 05 OD600 in SCD . OD600 was measured every 2 hours .

Decreasing proliferation rates from ∆III to ρ0 cells. Cells were cultured from 0.05 OD600 in SCD. OD600 was measured every 2 hours.


Transcriptome analysis of OXPHOS mutants . WT and mutant yeast cells were cultured to log phase . RNAseq was performed with three biological replicates per genotype ( Dataset EV1 ) . All the transcripts and four categories of transcripts ( gene lists in Table EV1B ) are shown . Significantly changed genes ( P value < 0 . 05 ; fold change > 1 . 5 or < 0 . 67 ) are highlighted with dashed lines ; numbers of significantly changed genes are indicated .

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Transcriptome analysis of OXPHOS mutants. WT and mutant yeast cells were cultured to log phase. RNAseq was performed with three biological replicates per genotype (Dataset EV1). All the transcripts and four categories of transcripts (gene lists in Table EV1B) are shown. Significantly changed genes (P value < 0.05; fold change > 1.5 or < 0.67) are highlighted with dashed lines; numbers of significantly changed genes are indicated.


qRT - PCR analysis of selected transcripts in ρ0 cells .

qRT-PCR analysis of selected transcripts in ρ0 cells.


SILAC analysis of mitochondrial proteome of ∆ IV and ρ0 cells . Equal amount of WT cells labeled with heavy amino acids and unlabeled mutant cells were mixed to extract mitochondrial fraction for mass - spectrometry analysis . Three biological replicates were prepared for each pair of strains ( Dataset EV2 ) . 568 high fidelity mitochondrial proteins ( gene / protein list in Table EV1A ) in ∆ IV cells and 572 such proteins in ρ0 cells were quantitatively measured . Significantly changed proteins ( P value < 0 . 05 ; fold change > 1 . 5 or < 0 . 67 ) are highlighted with dashed lines ; numbers of significantly changed proteins are indicated .

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SILAC analysis of mitochondrial proteome of ∆IV and ρ0 cells. Equal amount of WT cells labeled with heavy amino acids and unlabeled mutant cells were mixed to extract mitochondrial fraction for mass-spectrometry analysis. Three biological replicates were prepared for each pair of strains (Dataset EV2). 568 high fidelity mitochondrial proteins (gene/protein list in Table EV1A) in ∆IV cells and 572 such proteins in ρ0 cells were quantitatively measured. Significantly changed proteins (P value < 0.05; fold change > 1.5 or < 0.67) are highlighted with dashed lines; numbers of significantly changed proteins are indicated.


Western blot analysis of representative mitochondrial proteins endogenously tagged with HA . The selection markers were removed by the Cre - loxP system . Whole cell lysates were extracted and equal amount of proteins was loaded for western blot .

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Western blot analysis of representative mitochondrial proteins endogenously tagged with HA. The selection markers were removed by the Cre-loxP system. Whole cell lysates were extracted and equal amount of proteins was loaded for western blot.


Polysome fractionation analysis of OXPHOS mutants . Polysomes were fractionated by sucrose gradients ( 10 % - 50 % ) , and the gradients were separated and measured at A254 .

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Polysome fractionation analysis of OXPHOS mutants. Polysomes were fractionated by sucrose gradients (10%-50%), and the gradients were separated and measured at A254.


qRT - PCR analysis of INH1 mRNA levels . Data are mean ± SD from three biological replicates .

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qRT-PCR analysis of INH1 mRNA levels. Data are mean ± SD from three biological replicates.


Western blot analysis of Inh1 protein levels . Whole cell lysates were extracted and equal amount of proteins was loaded for western blot .

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Western blot analysis of Inh1 protein levels. Whole cell lysates were extracted and equal amount of proteins was loaded for western blot.


Deletion of INH1 benefits the proliferation of ρ0 cells at 37 ℃ . Serial dilutions ( tenfold dilution ) of the indicated strains were analyzed on SCD plates at 30 ℃ and 37 ℃ for 2 days .

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Deletion of INH1 benefits the proliferation of ρ0 cells at 37 ℃. Serial dilutions (tenfold dilution) of the indicated strains were analyzed on SCD plates at 30℃ and 37 ℃ for 2 days.


Western blot analysis of copper - inducible overexpression of Inh1 . A cassette expressing Inh1 under the control of CUP1 promoter was inserted into the HO locus . Inh1 overexpression was induced by 100 μM CuSO4 for 12 hours .

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Western blot analysis of copper-inducible overexpression of Inh1. A cassette expressing Inh1 under the control of CUP1 promoter was inserted into the HO locus. Inh1 overexpression was induced by 100 μM CuSO4 for 12 hours.


Overexpression of Inh1 decreases ∆ ψm in ρ0 cells . Cells were cultured in SCD or SCD plus 100 μM CuSO4 for 12 hours to mid - log phase . Cells were then stained with 125 nM TMRM for FACS analysis .

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Overexpression of Inh1 decreases ∆ψm in ρ0 cells. Cells were cultured in SCD or SCD plus 100 μM CuSO4 for 12 hours to mid-log phase. Cells were then stained with 125 nM TMRM for FACS analysis.


Overexpression of Inh1 represses the proliferation of ρ0 cells . Serial dilutions ( tenfold dilution ) of the indicated strains were analyzed on SCD or SCD plus 100 μM CuSO4 plates at 30 ℃ for 2 days .

Overexpression of Inh1 represses the proliferation of ρ0 cells. Serial dilutions (tenfold dilution) of the indicated strains were analyzed on SCD or SCD plus 100 μM CuSO4 plates at 30℃ for 2 days.


Overexpression of Inh1 reduces the protein levels of mitochondrial ISC biosynthesis proteins ( Nfs1 and Yah1 ) and nuclear ISC - containing protein Pol3 . Cells were cultured in SCD or SCD plus 100 μM CuSO4 for 12 hours to mid - log phase . Whole cell lysates were extracted and equal amount of proteins was loaded for western blot . Nfs1 , Yah1 were endogenously tagged with FLAG , and Pol3 was endogenously tagged with HA .

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Overexpression of Inh1 reduces the protein levels of mitochondrial ISC biosynthesis proteins (Nfs1 and Yah1) and nuclear ISC-containing protein Pol3. Cells were cultured in SCD or SCD plus 100 μM CuSO4 for 12 hours to mid-log phase. Whole cell lysates were extracted and equal amount of proteins was loaded for western blot. Nfs1, Yah1 were endogenously tagged with FLAG, and Pol3 was endogenously tagged with HA.


The changes of Puf3 - associated transcripts encoding mitochondrial proteins in OXPHOS mutants . The RNAseq results in Figure 1C ( Dataset EV1 ) are re - analyzed . Transcripts encoding high fidelity mitochondrial proteins ( gene list in Table EV1A ) and the Puf3 - associated transcripts encoding mitochondrial proteins ( gene list in Table EV1D ) are shown . The significantly changed transcripts ( P value < 0 . 05 ; fold change > 1 . 5 or < 0 . 67 ) are highlighted with dashed lines ; numbers of significantly changed transcripts are indicated .

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The changes of Puf3-associated transcripts encoding mitochondrial proteins in OXPHOS mutants. The RNAseq results in Figure 1C (Dataset EV1) are re-analyzed. Transcripts encoding high fidelity mitochondrial proteins (gene list in Table EV1A) and the Puf3-associated transcripts encoding mitochondrial proteins (gene list in Table EV1D) are shown. The significantly changed transcripts (P value < 0.05; fold change > 1.5 or < 0.67) are highlighted with dashed lines; numbers of significantly changed transcripts are indicated.


B and C , Deletion of PUF3 upregulates mitochondrial transcripts in WT cells ( B ) , whereas downregulates mitochondrial transcripts in ρ0 cells ( C ) . WT and mutant yeast cells were cultured to log phase . RNAseq was performed with three biological replicates per genotype ( Dataset EV4 ) . All the transcripts and the Puf3 - associated transcripts encoding mitochondrial proteins are shown . The significantly changed transcripts ( P value < 0 . 05 ; fold change > 1 . 5 or < 0 . 67 ) are highlighted with dashed lines ; numbers of significantly changed transcripts are indicated .

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B and C, Deletion of PUF3 upregulates mitochondrial transcripts in WT cells (B), whereas downregulates mitochondrial transcripts in ρ0 cells (C). WT and mutant yeast cells were cultured to log phase. RNAseq was performed with three biological replicates per genotype (Dataset EV4). All the transcripts and the Puf3-associated transcripts encoding mitochondrial proteins are shown. The significantly changed transcripts (P value < 0.05; fold change > 1.5 or < 0.67) are highlighted with dashed lines; numbers of significantly changed transcripts are indicated.


D . Deletion of PUF3 in ρ0 cells downregulates mitochondrial proteins . SILAC analysis of mitochondrial fractions from ρ0 and ρ0 puf3 ∆ cells are shown . Equal amount of ρ0 cells labeled with heavy amino acids and unlabeled ρ0 puf3 ∆ cells were mixed to extract mitochondrial fraction for mass - spectrometry analysis . Three biological replicates were prepared for each pair of strains ( Dataset EV5 ) . Mitochondrial proteins encoded by Puf3 - associated mRNAs are highlighted in red . Mia40 substrates are highlighted in green ( gene list in Table EV1E ) . Mia40 substrates encoded by Puf3 associated transcripts are highlighted in green with red circle . The significantly changed proteins ( P value < 0 . 05 ; fold change > 1 . 5 or < 0 . 67 ) are highlighted with dashed lines ; numbers of significantly changed proteins are indicated .

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D. Deletion of PUF3 in ρ0 cells downregulates mitochondrial proteins. SILAC analysis of mitochondrial fractions from ρ0 and ρ0 puf3∆ cells are shown. Equal amount of ρ0 cells labeled with heavy amino acids and unlabeled ρ0 puf3∆ cells were mixed to extract mitochondrial fraction for mass-spectrometry analysis. Three biological replicates were prepared for each pair of strains (Dataset EV5). Mitochondrial proteins encoded by Puf3-associated mRNAs are highlighted in red. Mia40 substrates are highlighted in green (gene list in Table EV1E). Mia40 substrates encoded by Puf3 associated transcripts are highlighted in green with red circle. The significantly changed proteins (P value < 0.05; fold change > 1.5 or < 0.67) are highlighted with dashed lines; numbers of significantly changed proteins are indicated.


E . Correlation plots of fold changes ( ρ0 puf3 ∆ versus ρ0 ) between mitochondrial proteins and their transcripts . Puf3 - associated transcripts encoding mitochondrial proteins are highlighted in red . Mia40 substrates are highlighted in green . Mia40 substrates encoded by Puf3 associated transcripts are highlighted in green with red circle .

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E. Correlation plots of fold changes (ρ0 puf3∆ versus ρ0) between mitochondrial proteins and their transcripts. Puf3-associated transcripts encoding mitochondrial proteins are highlighted in red. Mia40 substrates are highlighted in green. Mia40 substrates encoded by Puf3 associated transcripts are highlighted in green with red circle.


F . Deletion of PUF3 decreases ∆ ψm in ρ0 cells . Cells were cultured to mid - log phase , and stained with 125 nM TMRM for FACS analysis

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F. Deletion of PUF3 decreases ∆ψm in ρ0 cells. Cells were cultured to mid-log phase, and stained with 125 nM TMRM for FACS analysis


F . Deletion of PUF3 decreases ∆ ψm in ρ0 cells . Cells were cultured to mid - log phase , and stained with 125 nM TMRM for FACS

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G . Deletion of PUF3 decreases the proliferation of ρ0 cells . Serial dilutions ( tenfold dilution ) of the indicated strains were analyzed on SCD plates at 30 ℃ for 2 days .

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G. Deletion of PUF3 decreases the proliferation of ρ0 cells. Serial dilutions (tenfold dilution) of the indicated strains were analyzed on SCD plates at 30℃ for 2 days.


H . Schematic illustration of the consensus Puf3 - binding motif Lapointe et al . , 2015 ( ) and the motif at Mia40 3 ' - UTR . Mutating the Puf3 - binding motif of MIA40 mRNA abolishes Mia40 upregulation in ρ0 cells . Mia40 was endogenously tagged with FLAG and its 3 ' - UTR was edited as indicated . The selection markers were removed by the Cre - loxP system . Whole cell lysates were extracted and equal amount of proteins was loaded for western blot . RC : reverse complement . DE : deletion .

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H. Schematic illustration of the consensus Puf3-binding motif Lapointe et al., 2015() and the motif at Mia40 3'-UTR. Mutating the Puf3-binding motif of MIA40 mRNA abolishes Mia40 upregulation in ρ0 cells. Mia40 was endogenously tagged with FLAG and its 3'-UTR was edited as indicated. The selection markers were removed by the Cre-loxP system. Whole cell lysates were extracted and equal amount of proteins was loaded for western blot. RC: reverse complement. DE: deletion.


I . Mutating the Puf3 - binding motif of MIA40 mRNA decreases ∆ ψm of ρ0 cells . J . Mutating the Puf3 - binding motif of MIA40 mRNA impairs the proliferation of ρ0 cells . Cells were cultured from 0 . 05 OD in SCD , and OD600 was measured every 2 hours . Cells were cultured to mid - log phase , and stained with 125 nM TMRM for FACS

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I. Mutating the Puf3-binding motif of MIA40 mRNA decreases ∆ψm of ρ0 cells. J. Mutating the Puf3-binding motif of MIA40 mRNA impairs the proliferation of ρ0 cells. Cells were cultured from 0.05 OD in SCD, and OD600 was measured every 2 hours. Cells were cultured to mid-log phase, and stained with 125 nM TMRM for FACS analysis


I . Mutating the Puf3 - binding motif of MIA40 mRNA decreases ∆ ψm of ρ0 cells .

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I. Mutating the Puf3-binding motif of MIA40 mRNA decreases ∆ψm of ρ0 cells.


K . Overexpression of MIA40 rescues ∆ ψm of ρ0 puf3 ∆ cells . Single copy plasmid expressing Mia40 driven by the MIA40 promoter was transformed into the indicated strains . L . Overexpression of MIA40 rescues the proliferation of ρ0 puf3 ∆ cells . Strains were prepared as in ( K ) . Cells were cultured from 0 . 05 OD in SCD , and OD600 was measured every 2 hours . Cells were cultured to mid - log phase , and stained with 125 nM TMRM for FACS

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K. Overexpression of MIA40 rescues ∆ψm of ρ0 puf3∆ cells. Single copy plasmid expressing Mia40 driven by the MIA40 promoter was transformed into the indicated strains. L. Overexpression of MIA40 rescues the proliferation of ρ0 puf3∆ cells. Strains were prepared as in (K). Cells were cultured from 0.05 OD in SCD, and OD600 was measured every 2 hours. Cells were cultured to mid-log phase, and stained with 125 nM TMRM for FACS analysis


K . Overexpression of MIA40 rescues ∆ ψm of ρ0 puf3 ∆ cells . Single copy plasmid expressing Mia40 driven by the MIA40 promoter was transformed into the indicated strains .

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K. Overexpression of MIA40 rescues ∆ψm of ρ0 puf3∆ cells. Single copy plasmid expressing Mia40 driven by the MIA40 promoter was transformed into the indicated strains.


Puf3 is phosphorylated in WT cells and is hyper - phosphorylated in ρ0 cells . Puf3 - FLAG was immunoprecipitated from the indicated strains and treated with / without λ - phosphatase , and analyzed by regular and Phos - tag SDS - PAGE .

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Puf3 is phosphorylated in WT cells and is hyper-phosphorylated in ρ0 cells. Puf3-FLAG was immunoprecipitated from the indicated strains and treated with/without λ-phosphatase, and analyzed by regular and Phos-tag SDS-PAGE.


Puf3 phosphorylation in response to OXPHOS inhibitors and in OXPHOS mutants . WT cells were treated with antimycin ( 10 μM ) , KCN ( 10 μM ) or oligomycin ( 10 μM ) for 3 hours , or shifted from SCD to SCEG medium for 3 hours . Whole cell lysates were extracted and equal amount of proteins was loaded for western blot . Treatments and strains with Puf3 hyper - phosphorylation was highlighted in red . Glucose depletion was used as a positive control . Asterisk indicates a non - specific band .

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Puf3 phosphorylation in response to OXPHOS inhibitors and in OXPHOS mutants. WT cells were treated with antimycin (10 μM), KCN (10 μM) or oligomycin (10 μM) for 3 hours, or shifted from SCD to SCEG medium for 3 hours. Whole cell lysates were extracted and equal amount of proteins was loaded for western blot. Treatments and strains with Puf3 hyper-phosphorylation was highlighted in red. Glucose depletion was used as a positive control. Asterisk indicates a non-specific band.


Puf3 - 15A mutation inhibits Puf3 hyper - phosphorylation in ρ0 cells . A cassette expressing WT or mutant Puf3 - FLAG ( 11A or 15A ) under the control of PUF3 promoter was inserted into the HO site in puf3 ∆ cells . The 15A mutant that blocks Puf3 hyper - phosphorylation is highlighted in red . Puf3 - FLAG was immunoprecipitated from the indicated strains and analyzed by regular and Phos - tag SDS - PAGE .

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Puf3-15A mutation inhibits Puf3 hyper-phosphorylation in ρ0 cells. A cassette expressing WT or mutant Puf3-FLAG (11A or 15A) under the control of PUF3 promoter was inserted into the HO site in puf3∆ cells. The 15A mutant that blocks Puf3 hyper-phosphorylation is highlighted in red. Puf3-FLAG was immunoprecipitated from the indicated strains and analyzed by regular and Phos-tag SDS-PAGE.


Puf3 - 15A mutation inhibits Puf3 hyper - phosphorylation upon oligomycin treatment and in ∆ V and ρ0 cells . WT cells were treated with antimycin ( 10 μM ) , KCN ( 10 μM ) or oligomycin ( 10 μM ) for 3 hours , or shifted from SCD to SCEG medium for 3 hours . Whole cell lysates were extracted and equal amount of proteins was loaded for western blot . Asterisk indicates a non - specific band .

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Puf3-15A mutation inhibits Puf3 hyper-phosphorylation upon oligomycin treatment and in ∆V and ρ0 cells. WT cells were treated with antimycin (10 μM), KCN (10 μM) or oligomycin (10 μM) for 3 hours, or shifted from SCD to SCEG medium for 3 hours. Whole cell lysates were extracted and equal amount of proteins was loaded for western blot. Asterisk indicates a non-specific band.


Puf3 - 15A mutation downregulates Puf3 - associated mitochondrial transcripts in ρ0 cells . ρ0 and ρ0 puf3 - 15A cells were cultured to log phase . RNAseq was performed with three biological replicates per genotype ( Dataset EV7 ) . All the transcripts and the Puf3 - associated transcripts encoding mitochondrial proteins ( gene lists in Table EV1D ) are shown . The significantly changed transcripts ( P value < 0 . 05 ; fold change > 1 . 5 or < 0 . 67 ) are highlighted with dashed lines ; numbers of significantly changed transcripts are indicated .

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Puf3-15A mutation downregulates Puf3-associated mitochondrial transcripts in ρ0 cells. ρ0 and ρ0 puf3-15A cells were cultured to log phase. RNAseq was performed with three biological replicates per genotype (Dataset EV7). All the transcripts and the Puf3-associated transcripts encoding mitochondrial proteins (gene lists in Table EV1D) are shown. The significantly changed transcripts (P value < 0.05; fold change > 1.5 or < 0.67) are highlighted with dashed lines; numbers of significantly changed transcripts are indicated.


Puf3 - 15A mutation inhibits the upregulation of Mia40 in ∆ V and ρ0 cells . Mia40 was endogenously tagged with FLAG . The selection markers were removed by the Cre - loxP system . Whole cell lysates were extracted and equal amount of proteins was loaded for western blot .

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Puf3-15A mutation inhibits the upregulation of Mia40 in ∆V and ρ0 cells. Mia40 was endogenously tagged with FLAG. The selection markers were removed by the Cre-loxP system. Whole cell lysates were extracted and equal amount of proteins was loaded for western blot.


Puf3 - 15A mutation decreases ∆ ψm in ρ0 cells . Cells were cultured to mid - log phase and stained with 125 nM TMRM for FACS analysis . The red ] highlight two cell lines for comparison .

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Puf3-15A mutation decreases ∆ψm in ρ0 cells. Cells were cultured to mid-log phase and stained with 125 nM TMRM for FACS analysis. The red ] highlight two cell lines for comparison.


Puf3 - 15A mutation has no effect on the proliferation of WT cells . Puf3 - 15A mutation inhibits the proliferation of ρ0 cells . Mia40 overexpression rescues the proliferation defect of ρ0 puf3 - 15A cells . Single copy plasmid expressing Mia40 driven by MIA40 promoter was transformed into the indicated strains .

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Puf3-15A mutation has no effect on the proliferation of WT cells.   Puf3-15A mutation inhibits the proliferation of ρ0 cells.   Mia40 overexpression rescues the proliferation defect of ρ0 puf3-15A cells. Single copy plasmid expressing Mia40 driven by MIA40 promoter was transformed into the indicated strains.


Puf3 - 15A mutation has no effect on the proliferation of WT cells .

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

Puf3-15A mutation has no effect on the proliferation of WT cells.


Snf1 maintains glycolysis and glucose transporter gene expression in both WT and ρ0 cells , and represses ribosome and Ribi gene expression in ρ0 cells . ρ0 snf1 ∆ cells were prepared by acute EtBr ( 25 µg / ml ) treatment of snf1 ∆ cells in SCD for 2 days . RNAseq was performed with three biological replicates per genotype ( Dataset EV8 ) . All the transcripts and two categories of transcripts ( gene lists in Table EV1B ) are shown . Significantly changed genes ( P value < 0 . 05 ; fold change > 1 . 5 or < 0 . 67 ) are highlighted with dashed lines ; numbers of significantly changed genes are indicated .

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

Snf1 maintains glycolysis and glucose transporter gene expression in both WT and ρ0 cells, and represses ribosome and Ribi gene expression in ρ0 cells. ρ0 snf1∆ cells were prepared by acute EtBr (25 µg/ml) treatment of snf1∆ cells in SCD for 2 days. RNAseq was performed with three biological replicates per genotype (Dataset EV8). All the transcripts and two categories of transcripts (gene lists in Table EV1B) are shown. Significantly changed genes (P value < 0.05; fold change > 1.5 or < 0.67) are highlighted with dashed lines; numbers of significantly changed genes are indicated.


Snf1 represses ribosome biogenesis in WT and ρ0 cells . Polysomes of the indicated strains were fractionated by sucrose gradients ( 10 % - 50 % ) , and the gradients were separated and measured at A254 .

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Snf1 represses ribosome biogenesis in WT and ρ0 cells. Polysomes of the indicated strains were fractionated by sucrose gradients (10%-50%), and the gradients were separated and measured at A254.


Deletion of SNF1 decreases ∆ ψm in both ∆ V and ρ0 cells . Cells were cultured to mid - log phase and stained with 125 nM TMRM for FACS analysis

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Deletion of SNF1 decreases ∆ψm in both ∆V and ρ0 cells. Cells were cultured to mid-log phase and stained with 125 nM TMRM for FACS analysis


Deletion of SNF1 decreases ∆ ψm in both ∆ V and ρ0 cells . Cells were cultured to mid - log phase and stained with 125 nM TMRM for FACS

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Deletion of SNF1 decreases the proliferation of both ∆ V and ρ0 cells . Serial dilutions of the indicated strains were analyzed on SCD plates at 30 ℃ for 2 days .

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Deletion of SNF1 decreases the proliferation of both ∆V and ρ0 cells. Serial dilutions of the indicated strains were analyzed on SCD plates at 30℃ for 2 days.


Deletion of MIG1 in ρ0 snf1 ∆ cells rescues ∆ ψm . Cells were cultured to mid - log phase and stained with 125 nM TMRM for FACS analysis

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Deletion of MIG1 in ρ0 snf1∆ cells rescues ∆ψm. Cells were cultured to mid-log phase and stained with 125 nM TMRM for FACS analysis


Deletion of MIG1 in ρ0 snf1 ∆ cells rescues ∆ ψm . Cells were cultured to mid - log phase and stained with 125 nM TMRM for FACS

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Deletion of MIG1 in ρ0 snf1 ∆ cells partially rescues cell proliferation . A plasmid overexpressing MIG1 under the control of MIG1 promoter was transformed into the indicated strains . Serial dilutions ( tenfold dilution ) were analyzed on SCD plates at 30 ℃ for 2 days . V : empty vector .

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Deletion of MIG1 in ρ0 snf1∆ cells partially rescues cell proliferation. A plasmid overexpressing MIG1 under the control of MIG1 promoter was transformed into the indicated strains. Serial dilutions (tenfold dilution) were analyzed on SCD plates at 30℃ for 2 days. V: empty vector.


Combining MIG1 deletion and NOG2 overexpression in ρ0 snf1 ∆ cells fully rescues cell proliferation . A plasmid overexpressing NOG2 under the control of TEF1 promoter was transformed into the indicated strains . Serial dilutions ( tenfold dilution ) were analyzed on SCD plates at 30 ℃ for 2 days . V : empty vector .

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Combining MIG1 deletion and NOG2 overexpression in ρ0 snf1∆ cells fully rescues cell proliferation. A plasmid overexpressing NOG2 under the control of TEF1 promoter was transformed into the indicated strains. Serial dilutions (tenfold dilution) were analyzed on SCD plates at 30℃ for 2 days. V: empty vector.


Heat map plots of the mRNA level changes of chaperones , ubiquitin - proteasome pathway , and autophagy genes ( gene list in Table EV1F ) in the indicated strains ( data from Dataset EV1 and Dataset EV10 ) . Selected genes for growth analysis in B are highlighted in red .

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Heat map plots of the mRNA level changes of chaperones, ubiquitin-proteasome pathway, and autophagy genes (gene list in Table EV1F) in the indicated strains (data from Dataset EV1 and Dataset EV10). Selected genes for growth analysis in B are highlighted in red.


Deletion of HSC82 selectively impairs the proliferation of ρ0 puf3 ∆ cells . Cells were cultured from 0 . 05 OD in SCD , and OD600 was measured every 2 hours . Data are mean ± SD from three biological replicates .

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Deletion of HSC82 selectively impairs the proliferation of ρ0 puf3∆ cells. Cells were cultured from 0.05 OD in SCD, and OD600 was measured every 2 hours. Data are mean ± SD from three biological replicates.


Deletion of HSC82 selectively decreases ∆ ψm in ρ0 puf3 ∆ cells . Cells were cultured to mid - log phase and stained with 125 nM TMRM for FACS analysis . The red ] highlight two cell lines for comparison .

O O B-CONTROLLED_VAR 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

Deletion of HSC82 selectively decreases ∆ψm in ρ0 puf3∆ cells. Cells were cultured to mid-log phase and stained with 125 nM TMRM for FACS analysis. The red ] highlight two cell lines for comparison.


A , B cRNA or water as a control was injected into oocytes and uptake was assessed two days after injection for 14h in N free MBS . Data information : biological replicates : n = 5 with 2 oocytes in each sample . Asterisks denote significant difference : * * P < 0 . 01 , * P < 0 . 05 ( Student ' s t - test ) .

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A, B cRNA or water as a control was injected into oocytes and uptake was assessed two days after injection for 14h in N free MBS. Data information: biological replicates: n = 5 with 2 oocytes in each sample. Asterisks denote significant difference: **P<0.01, *P<0.05 (Student's t-test).


A , B The oocytes were exposed to different concentrations of NO3 - in N free MBS for 1 . 5 h at pH5 . 5 . Net NO3 - accumulation was fitted with both Michaelis - Menten and Linear Regression in A for high affinity range and Linear Regression in B for low affinity . Values are the mean ± SE ( biological replicates : n = 3 ) with 3 - 4 oocytes in each sample .

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A, B The oocytes were exposed to different concentrations of NO3- in N free MBS for 1.5 h at pH5.5. Net NO3- accumulation was fitted with both Michaelis-Menten and Linear Regression in A for high affinity range and Linear Regression in B for low affinity. Values are the mean ± SE (biological replicates: n=3) with 3-4 oocytes in each sample.


A Histochemical stained roots from composite plants transformed with proMtNPF6 . 5 : GUS ( left column ) , proMtNPF6 . 7 : GUS ( middle column ) , proMtNPF6 . 6 : GUS ( right column ) .

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A Histochemical stained roots from composite plants transformed with proMtNPF6.5:GUS (left column), proMtNPF6.7:GUS (middle column), proMtNPF6.6:GUS (right column).


B Expression of MtNPFs in roots after transfer to media containing different levels of NO3 - for 30 minutes . C = KCl control , N = KNO3 ( Biological replicates : n = 3 ( 3 pooled root organs in each replicate ) , asterisk denotes significance p < 0 . 05 , Student ' s t - test ) . The means of all samples were compared to that of their concentration - matched KCl controls . Expression is shown relative to Ubiquitin .

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B Expression of MtNPFs in roots after transfer to media containing different levels of NO3- for 30 minutes. C=KCl control, N=KNO3 (Biological replicates: n = 3 (3 pooled root organs in each replicate), asterisk denotes significance p<0.05, Student's t-test). The means of all samples were compared to that of their concentration-matched KCl controls. Expression is shown relative to Ubiquitin.


A , B I - V responses in oocytes ( subtracted of the water injected controls ) exposed to 40 mM Cl at pH5 . 8 ( A ) and pH7 . 4 ( B ) . The currents were measured while the oocyte plasma membrane was clamped at - 60 mv to between 20 and - 140 mv for 120 ms with - 20 mv increments for each Cl - concentration . The Cl - - elicited current values in D were obtained by subtracting the currents in water injected oocytes from the MtNPF6 . 5 injected oocytes at each Cl - concentration Data information : Value = mean ± SEM , biological replicates : n =

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

A, B I-V responses in oocytes (subtracted of the water injected controls) exposed to 40 mM Cl at pH5.8 (A) and pH7.4 (B). The currents were measured while the oocyte plasma membrane was clamped at -60 mv to between 20 and -140 mv for 120 ms with -20 mv increments for each Cl- concentration. The Cl- -elicited current values in D were obtained by subtracting the currents in water injected oocytes from the MtNPF6.5 injected oocytes at each Cl- concentration Data information: Value = mean ± SEM, biological replicates: n = 4


C , D I - V responses in the ( C ) water and ( D ) MtNPF6 . 5 ( subtracted of the water injected controls ) injected oocytes exposed to different concentrations of Cl - at pH5 . 8 . The currents were measured while the oocyte plasma membrane was clamped at - 60 mv to between 20 and - 140 mv for 120 ms with - 20 mv increments for each Cl - concentration . The Cl - - elicited current values in D were obtained by subtracting the currents in water injected oocytes from the MtNPF6 . 5 injected oocytes at each Cl - concentration . Data information : Value = mean ± SEM , biological replicates : n =

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

C, D I-V responses in the (C) water and (D) MtNPF6.5 (subtracted of the water injected controls) injected oocytes exposed to different concentrations of Cl- at pH5.8. The currents were measured while the oocyte plasma membrane was clamped at -60 mv to between 20 and -140 mv for 120 ms with -20 mv increments for each Cl- concentration. The Cl- -elicited current values in D were obtained by subtracting the currents in water injected oocytes from the MtNPF6.5 injected oocytes at each Cl- concentration. Data information: Value = mean ± SEM, biological replicates: n = 6


C Oocytes were exposed to 1 . 17 mM 36Cl - for 2h with 1 and 10 mM NO3 - . Biological replicates : n = 6 ( 4 oocytes in each sample ) . D The same data as in C is shown without AtNPF6 . 3 to allow for better comparison between MtNPFs . Biological replicates : n = 6 ( 4 oocytes in each sample ) . Data information : Asterisks denote significant difference : * * P < 0 . 01 , * P < 0 . 05 ( Student ' s t - test ) . In the boxplots , horizontal black bars represent the median , and black cross represent the mean . Boxes represent the middle 50 % of the distribution , and whiskers represent the entire spread of the data . Means were compared using Student ' s t - test , α = 0 . 05 .

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C Oocytes were exposed to 1.17 mM 36Cl- for 2h with 1 and 10 mM NO3-. Biological replicates: n=6 (4 oocytes in each sample). D The same data as in C is shown without AtNPF6.3 to allow for better comparison between MtNPFs. Biological replicates: n=6 (4 oocytes in each sample). Data information: Asterisks denote significant difference: **P<0.01, *P<0.05 (Student's t-test). In the boxplots, horizontal black bars represent the median, and black cross represent the mean. Boxes represent the middle 50% of the distribution, and whiskers represent the entire spread of the data. Means were compared using Student's t-test, α= 0.05.


A , B Relative expression of NPFs in M . truncatula roots grown in FP and exposed to different NaCl treatments . Data information : * p < 0 . 05 , Student ' s t - test ; biological replicates : n = 3 ( 3 pooled root organs in each replicate ) ±

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A, B Relative expression of NPFs in M. truncatula roots grown in FP and exposed to different NaCl treatments. Data information: *p<0.05, Student's t-test; biological replicates: n=3 (3 pooled root organs in each replicate) ±SEM


D Relative expression of NPFs in M . truncatula roots grown in FP and exposed to 100mM KCl treatments for different times . Data information : * p < 0 . 05 , Student ' s t - test ; biological replicates : n = 3 ( 3 pooled root organs in each replicate ) ± SEM

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D Relative expression of NPFs in M. truncatula roots grown in FP and exposed to 100mM KCl treatments for different times. Data information: *p<0.05, Student's t-test; biological replicates: n=3 (3 pooled root organs in each replicate) ±SEM


A Seedling Cl - content determined by ion chromatography 2 h after treatment with 50mM NaCl . * p < 0 . 05 , Student ' s t - test ; biological replicates : n = 4 . B Cl - content determined by ion chromatography of seedlings supplemented with 5mM KNO3 or NH4NO3 . * p < 0 . 05 , Student ' s t - test ; biological replicates : n = 4 .

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A Seedling Cl- content determined by ion chromatography 2 h after treatment with 50mM NaCl. *p<0.05, Student's t-test; biological replicates: n=4. B Cl- content determined by ion chromatography of seedlings supplemented with 5mM KNO3 or NH4NO3. *p<0.05, Student's t-test; biological replicates: n=4.


C On the left , relative Cl - content determined by XRF of NO3 - starved seedlings after KNO3 supplementation . * p < 0 . 05 , Student ' s t - test ; biological replicates : n = 3 - 5 . On the right , a representative XRF image of Cl - signal across WT and mutant roots at the 0h timepoint . Five roots per genotype are shown . The outlines indicate the measurement areas . The color legend indicates relative Cl - concentration .

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C On the left, relative Cl- content determined by XRF of NO3- starved seedlings after KNO3 supplementation. *p<0.05, Student's t-test; biological replicates: n=3-5. On the right, a representative XRF image of Cl- signal across WT and mutant roots at the 0h timepoint. Five roots per genotype are shown. The outlines indicate the measurement areas. The color legend indicates relative Cl- concentration.


A Biomass ( fresh weight ) of seedlings grown on FP or FP supplemented with 50 or 100 mM NaCl . * p < 0 . 05 , Student ' s t - test ; biological replicates : n = 3 ( 2 - 5 pooled organs in each replicate ) .

A Biomass (fresh weight) of seedlings grown on FP or FP supplemented with 50 or 100 mM NaCl. *p<0.05, Student's t-test; biological replicates: n=3 (2-5 pooled organs in each replicate).


B Image of seedlings grown on FP or 50 mM NaCl . Data information : roots were re - arranged to better display their length , they did not exhibit curling / agravitropic growth .

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B Image of seedlings grown on FP or 50 mM NaCl. Data information: roots were re-arranged to better display their length, they did not exhibit curling/agravitropic growth.


B Changes in NO3 - and Cl - content ( mol / g dry weight ) of NO3 - starved plants after NO3 - provision Data information : , * p < 0 . 05 , Student ' s t - test ; biological replicates : n = 4 ± SEM .

B Changes in NO3- and Cl- content (mol/g dry weight) of NO3- starved plants after NO3- provision Data information: , *p< 0.05, Student's t-test; biological replicates: n=4 ±SEM.


E Relative MtNPF expression following treatment with 5mM KNO3 + 100mM NaCl Data information : expression shown is relative to Ubiquitin ; * p < 0 . 05 , Student ' s t - test ; biological replicates : n = 3 ( 3 pooled root organs in each replicate ) ± SEM .

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E Relative MtNPF expression following treatment with 5mM KNO3 +100mM NaCl Data information: expression shown is relative to Ubiquitin; *p< 0.05, Student's t-test; biological replicates: n= 3 (3 pooled root organs in each replicate) ±SEM.