( C , D ) MDA - MB - 231 overexpressing PTPRN2 ( C ) , PLCβ1 ( D ) or control vector were treated with carrier alone or carrier incubated with PI ( 4 , 5 ) P2 for one hour then immediately subjected to migration . N = 5 inserts / group .

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(C,D) MDA-MB-231 cells overexpressing PTPRN2 (C), PLCβ1 (D) or control vector were treated with carrier alone or carrier incubated with PI(4,5)P2 for one hour and then immediately subjected to the migration assay. N = 5 inserts/group.


( E ) Kaplan - Meier curve representing distal metastasis - free survival of a cohort of breast cancer ( N = 1609 ) as a function of their ' s mean PIP5K1A , PIP5KB , PIP5KC expression ( Data from KMPlot ( Gyorffy et al . , 2010 ) ) . ' tumors ' combined PIP5K expression were classified as low ( blue ) or high ( red ) expression .

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(E) Kaplan-Meier curve representing distal metastasis-free survival of a cohort of breast cancer patients (N = 1609) as a function of their primary tumor's mean PIP5K1A, PIP5KB, and PIP5KC expression levels (Data from KMPlot (Gyorffy et al., 2010)). Patients' primary tumors' combined PIP5K expression levels were classified as low (blue) or high (red) expression.


( F , G ) Migration ( F ) Matrigel invasion ( G ) of LM2 transduced with a retroviral vector overexpressing PIP5K1A or control vector . Data normalized to control values . N = 5 inserts / group .

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(F,G) Migration (F) and Matrigel invasion (G) of LM2 cells transduced with a retroviral vector overexpressing PIP5K1A or control vector. Data normalized to control values. N = 5 inserts/group.


( A ) Kaplan - Meier curve representing distal metastasis - free survival of a cohort of breast cancer ( N = 1609 ) as a function of their ' s CFL1 expression ( Data from KMPlot ( Gyorffy et al . , 2010 ) ) . ' tumors ' CFL1 expression were classified as low ( blue ) or high ( red ) expression .

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(A) Kaplan-Meier curve representing distal metastasis-free survival of a cohort of breast cancer patients (N = 1609) as a function of their primary tumor's CFL1 expression levels (Data from KMPlot (Gyorffy et al., 2010)). Patients' primary tumors' CFL1 expression levels were classified as low (blue) or high (red) expression.


( B , C ) - associated were purified from transfected with siRNA targeting PTPRN2 ( B ) or PLCβ1 ( C ) or control siRNA . Fractions were subjected to Western blot for CFL1 EGFR . Right , densitometry of CFL1 normalized to EGFR .

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(B,C) Membrane and membrane-associated proteins were purified from cells transfected with siRNA targeting PTPRN2 (B) or PLCβ1 (C) or control siRNA. Fractions were subjected to Western blot analysis for CFL1 and EGFR levels. Right, densitometry analysis of CFL1 levels normalized to EGFR levels.


( D , E ) - associated were purified from overexpressing PTPRN2 ( D ) , PLCβ1 ( E ) or a control vector . Fractions were subjected to Western blot for CFL1 EGFR . Right , densitometry of CFL1 normalized to EGFR .

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(D,E) Membrane and membrane-associated proteins were purified from cells overexpressing PTPRN2 (D), PLCβ1 (E) or a control vector. Fractions were subjected to Western blot analysis for CFL1 and EGFR levels. Right, densitometry analysis of CFL1 levels normalized to EGFR levels.


( F ) LM2 transfected with siRNA targeting PTPRN2 , PLCβ1 , or control siRNA were partially permeabilized incubated with biotin - actin monomers . were stained for incorporated biotin - actin monomers using Streptavidin - 555 ( red ) DAPI ( blue ) . Right , of mean of incorporated biotin - actin monomers . N = 100 / group . Scale bar , 20 μm .

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(F) LM2 cells transfected with siRNA targeting PTPRN2, PLCβ1, or control siRNA were partially permeabilized and incubated with biotin-actin monomers. Cells were stained for incorporated biotin-actin monomers using Streptavidin-555 (red) and DAPI (blue). Right, quantification of mean fluorescence intensity of incorporated biotin-actin monomers. N = 100 cells/group. Scale bar, 20 μm.


( A ) LM2 transfected with siRNA targeting PTPRN2 , PLCβ1 , or control siRNA were stained with phalloidin ( red ) DAPI ( blue ) analyzed using microscopy . Right , mean of whole phalloidin signal . N = 40 / group . Scale bar , 10 μm .

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(A) LM2 cells transfected with siRNA targeting PTPRN2, PLCβ1, or control siRNA were stained with phalloidin (red) and DAPI (blue) and analyzed using fluorescence microscopy. Right, mean fluorescence intensity quantification of whole cell phalloidin signal. N = 40 cells/group. Scale bar, 10 μm.


( B ) Mean of whole phalloidin signal in MDA - MB - 231 overexpressing PTPRN2 , PLCβ1 , or control vector . N = 40 / group .

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(B) Mean fluorescence intensity quantification of whole cell phalloidin signal in MDA-MB-231 cells overexpressing PTPRN2, PLCβ1, or control vector. N = 40 cells/group.


( C , D ) MDA - MB - 231 were transfected with siRNAs targeting CFL1 or a control siRNA in setting of control or PTPRN2 overexpression subjected to migration ( C ) or invasion ( D ) assays . N = 5 inserts / group .

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(C,D) MDA-MB-231 cells were transfected with siRNAs targeting CFL1 or a control siRNA in the setting of control or PTPRN2 overexpression and subjected to the migration (C) or invasion (D) assays. N = 5 inserts/group.


( E ) MDA - MB - 231 were transfected with siRNAs targeting CFL1 or a control siRNA in setting of control or PLCβ1 overexpression were subjected to migration . N = 5 inserts / group .

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(E) MDA-MB-231 cells were transfected with siRNAs targeting CFL1 or a control siRNA in the setting of control or PLCβ1 overexpression were subjected to the migration assay. N = 5 inserts/group.


( F ) Bioluminescence imaging of 40 , 000 MDA - MB - 231 overexpressing PTPRN2 or control vector transfected with control siRNA or siRNA targeting CFL1 . Right , H & amp ; amp ; E of representative lung sections . N = 5 mice / group .

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(F) Bioluminescence imaging quantification of 40,000 MDA-MB-231 cells overexpressing PTPRN2 or control vector and transfected with control siRNA or siRNA targeting CFL1. Right, H&amp;E staining of representative lung sections. N = 5 mice/group.


( G , H ) MDA - MB - 231 overexpressing PTPRN2 ( G ) or PLCβ1 ( H ) were transfected with siRNA targeting 3 ' UTR of CFL1 to deplete endogenous CFL1 . were further transfected with plasmids encoding either GFP - CFL1 - WT or GFP - CFL1 - Lck subjected to migration . N = 5 inserts / group .

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(G,H) MDA-MB-231 cells overexpressing PTPRN2 (G) or PLCβ1 (H) were transfected with siRNA targeting the 3'UTR of CFL1 to deplete endogenous CFL1. Cells were further transfected with plasmids encoding either GFP-CFL1-WT or GFP-CFL1-Lck and subjected to the migration assay. N = 5 inserts/group.


A . In situ hybridization for GemC1 McIdas mRNA was performed on sagittal sections of E10 . 5 dpc mouse brain . GemC1 McIdas mRNA is specifically detected in developing choroid plexuses of ventricles . Lower panels show higher magnifications . Scale bars , 100 µm .

A. In situ hybridization for GemC1 and McIdas mRNA was performed on sagittal sections of E10.5 dpc mouse brain. GemC1 and McIdas mRNA is specifically detected in the developing choroid plexuses of the ventricles. Lower panels show higher magnifications. Scale bars, 100 µm.


B . In situ hybridization on sagittal ( upper ) coronal ( middle lower ) sections of airways of E15 . 5 dpc mouse , depicting specific GemC1 McIdas mRNA expression in respiratory epithelium of nasal oral cavity in upper bronchial tree in mouse lungs . Inserts show higher magnification . Scale bars , 200 µm .

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B. In situ hybridization on sagittal (upper) and coronal (middle and lower) sections of the airways of E15.5 dpc mouse embryos, depicting specific GemC1 and McIdas mRNA expression in the respiratory epithelium of the nasal and oral cavity and in the upper bronchial tree in the mouse lungs. Inserts show higher magnification images. Scale bars, 200 µm.


C . MTECs were cultured ex - vivo transitioned to ALI culture condition . Quantitative real time PCR was used to measure expression of GemC1 , McIdas , FoxJ1 Geminin mRNA at different time points during multiciliogenesis . Values indicate expression ( + / - standard error , s . e . ) relative to ALI DAY 1 ( marked by * ) , following normalization against house - keeping HPRT mRNA . qPCR data were analyzed by - MCS beta software from at least three independent experiments .

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C. MTECs were cultured ex-vivo and transitioned to ALI culture condition. Quantitative real time PCR was used to measure the expression levels of GemC1, McIdas, FoxJ1 and Geminin mRNA levels at different time points during multiciliogenesis. Values indicate expression levels (+/- standard error, s.e.) relative to ALI DAY 1 (marked by *), following normalization against the house-keeping HPRT mRNA. qPCR data were analyzed by the REST-MCS beta software from at least three independent experiments.


A . B . MTEC cultures were infected with a lentivirus expressing either GFP - GemC1 ( GemC1 ) or GFP as a control two days later transitioned to ALI culture conditions . harvested at different time points during differentiation were co - stained with antibodies against GFP ( green ) to mark infected endogenous McIdas ( A ) or Foxj1 ( B ) ( red ) . Arrows indicate infected that are McIdas or Foxj1 positive . Scale bars , 10 µm . C . D . of infected expressing McIdas ( C ) or Foxj1 ( D ) at each time point was quantified . Data are presented as mean values of at least 10 independent fields obtained from at least two independent experiments for each condition . Error bars indicate ± . * p < 0 . 05 , * * * p < 0 . 001 . P - values were calculated by non - parametrical two - Mann - Whitney .

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A. and B. MTEC cultures were infected with a lentivirus expressing either GFP-GemC1 (GemC1) or GFP as a control and two days later transitioned to ALI culture conditions. Cells harvested at different time points during differentiation were co-stained with antibodies against GFP (green) to mark infected cells and endogenous McIdas(A) or Foxj1(B) (red). Arrows indicate infected cells that are McIdas or Foxj1 positive. Scale bars, 10 µm.C. and D. The percentage of infected cells expressing McIdas(C) or Foxj1(D) at each time point was quantified. Data are presented as the mean values of at least 10 independent fields obtained from at least two independent experiments for each condition. Error bars indicate ± SEM. *p<0.05, ***p<0.001. P-values were calculated by the non-parametrical two-tailed Mann-Whitney test.


MCIDAS ( A ) regulatory elements ( see materials methods ) cloned upstream of luciferase gene were co - transfected into HEK293T with vectors expressing GemC1 , McIdas Geminin , as indicated or an empty vector ( - ) as a control . luciferase experiments ( A , B , D ) were normalized for transfection efficiency with an expression vector for Renilla luciferase . Fold induction is between normalized luciferase activity induced by expression constructs that induced by empty expression vector . Data are mean values of at least three independent experiments error bars indicate ± . * * p < 0 . 01 , * * * p < 0 . 001 . P - values were calculated by non - parametrical two - Mann - Whitney . Abbreviations : RLF : Relative Luciferase Fold induction .

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MCIDAS (A) regulatory elements (see materials and methods) cloned upstream of the luciferase gene were co-transfected into HEK293T cells with vectors expressing GemC1, McIdas and Geminin, as indicated or an empty vector (-) as a control. All luciferase experiments (A, B, D) were normalized for transfection efficiency with an expression vector for Renilla luciferase. Fold induction is the ratio between the normalized luciferase activity induced by the expression constructs and that induced by the empty expression vector. Data are the mean values of at least three independent experiments and error bars indicate ± SEM. **p<0.01, ***p<0.001. P-values were calculated by the non-parametrical two-tailed Mann-Whitney test. Abbreviations: RLF: Relative Luciferase Fold induction.


FOXJ1 ( B , D ) regulatory elements ( see materials methods ) cloned upstream of luciferase gene were co - transfected into HEK293T with vectors expressing GemC1 , McIdas Geminin , as indicated or an empty vector ( - ) as a control . luciferase experiments ( A , B , D ) were normalized for transfection efficiency with an expression vector for Renilla luciferase . Fold induction is between normalized luciferase activity induced by expression constructs that induced by empty expression vector . Data are mean values of at least three independent experiments error bars indicate ± . * * p < 0 . 01 , * * * p < 0 . 001 . P - values were calculated by non - parametrical two - Mann - Whitney . Abbreviations : RLF : Relative Luciferase Fold induction .

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FOXJ1 (B, D) regulatory elements (see materials and methods) cloned upstream of the luciferase gene were co-transfected into HEK293T cells with vectors expressing GemC1, McIdas and Geminin, as indicated or an empty vector (-) as a control. All luciferase experiments (A, B, D) were normalized for transfection efficiency with an expression vector for Renilla luciferase. Fold induction is the ratio between the normalized luciferase activity induced by the expression constructs and that induced by the empty expression vector. Data are the mean values of at least three independent experiments and error bars indicate ± SEM. **p<0.01, ***p<0.001. P-values were calculated by the non-parametrical two-tailed Mann-Whitney test. Abbreviations: RLF: Relative Luciferase Fold induction.


McIdasmRNA after McIdas RNAi were assessed by qPCR ( C ) .

McIdasmRNA levels after McIdas RNAi were assessed by qPCR (C).


FOXJ1 ( B , D ) regulatory elements ( see materials methods ) cloned upstream of luciferase gene were co - transfected into HEK293T with vectors expressing GemC1 , McIdas Geminin , as indicated or an empty vector ( - ) as a control . In D , McIdas siRNAs or a control siRNA were also co - transfected . luciferase experiments ( A , B , D ) were normalized for transfection efficiency with an expression vector for Renilla luciferase . Fold induction is between normalized luciferase activity induced by expression constructs that induced by empty expression vector . Data are mean values of at least three independent experiments error bars indicate ± . * * p < 0 . 01 , * * * p < 0 . 001 . P - values were calculated by non - parametrical two - Mann - Whitney . Abbreviations : RLF : Relative Luciferase Fold induction .

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FOXJ1 (B, D) regulatory elements (see materials and methods) cloned upstream of the luciferase gene were co-transfected into HEK293T cells with vectors expressing GemC1, McIdas and Geminin, as indicated or an empty vector (-) as a control. In D, McIdas siRNAs or a control siRNA were also co-transfected. All luciferase experiments (A, B, D) were normalized for transfection efficiency with an expression vector for Renilla luciferase. Fold induction is the ratio between the normalized luciferase activity induced by the expression constructs and that induced by the empty expression vector. Data are the mean values of at least three independent experiments and error bars indicate ± SEM. **p<0.01, ***p<0.001. P-values were calculated by the non-parametrical two-tailed Mann-Whitney test. Abbreviations: RLF: Relative Luciferase Fold induction.


A . B . MCIDAS ( A ) FOXJ1 ( B ) regulatory elements cloned upstream of luciferase gene were co - transfected into HEK293T with vectors expressing GemC1 , McIdas , Geminin E2F5 , as indicated , or an empty vector ( - ) as a control . Luciferase values were normalized against co - transfected Renilla luciferase depicted as fold induction over control empty vector . Data shown are mean values of at least three independent experiments error bars indicate ± . * * p < 0 . 01 , * * * p < 0 . 001 . P - values were calculated by non - parametrical two - Mann - Whitney . Abbreviations : RLF : Relative Luciferase Fold induction .

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A. and B. MCIDAS (A) and FOXJ1 (B) regulatory elements cloned upstream of the luciferase gene were co-transfected into HEK293T cells with vectors expressing GemC1, McIdas, Geminin and E2F5, as indicated, or an empty vector (-) as a control. Luciferase values were normalized against co-transfected Renilla luciferase and depicted as fold induction over the control empty vector. Data shown are mean values of at least three independent experiments and error bars indicate ± SEM. **p<0.01, ***p<0.001. P-values were calculated by the non-parametrical two-tailed Mann-Whitney test. Abbreviations: RLF: Relative Luciferase Fold induction.


C . D . immunoprecipitation of MCIDAS FOXJ1 promoter fragments by GemC1 . HEK293T were co - transfected with vectors expressing GemC1 - GFP ( GemC1 ) or GFP as a control , E2F5 DP1 , together with regulatory elements of either MCIDAS ( C ) or FOXJ1 ( D ) . Following immunoprecipitation , fragments of MCIDAS FOXJ1 promoters were detected in immunoprecipitates by qPCR . Data from a representative experiment are shown as fold enrichment of MCIDAS FOXJ1 promoter fragments in anti - GFP immunoprecipitates over control IgG immunoprecipitates . At least two independent experiments were performed .

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C. and D. Chromatin immunoprecipitation of MCIDAS and FOXJ1 promoter fragments by GemC1. HEK293T cells were co-transfected with vectors expressing GemC1-GFP (GemC1) or GFP as a control, E2F5 and DP1, together with the regulatory elements of either MCIDAS (C) or FOXJ1 (D). Following chromatin immunoprecipitation, DNA fragments of the MCIDAS and FOXJ1 promoters were detected in immunoprecipitates by qPCR. Data from a representative experiment are shown as fold enrichment of MCIDAS and FOXJ1 promoter fragments in anti-GFP immunoprecipitates over control IgG immunoprecipitates. At least two independent experiments were performed.


B . GemC1KO / KO mice compared to their wild type ( GemC1KO / WT ) at P5 .

B. GemC1KO/KO mice compared to their wild type littermates (GemC1KO/WT) at P5.


C . Survival plot of mice homozygous for GemC1 deletion ( GemC1KO / KO , n = 8 ) control wild type or heterozygous GemC1 ( GemC1WT , n = 14 ) . GemC1 knock - out mice exhibit markedly reduced growth after birth die within first postnatal week .

C. Survival plot of mice homozygous for the GemC1 deletion (GemC1KO/KO, n=8) and control wild type or heterozygous littermates GemC1 (GemC1WT, n=14). GemC1 knock-out mice exhibit markedly reduced growth after birth and die within the first postnatal week.


A . , B . Transverse sections from trachea isolated from GemC1WT / WT ( upper ) or GemC1KO / KO ( lower ) P0 mice , immunostained with antibodies against acetylated y - tubulin ( ACT , cilia marker , red ) , pericentrin ( PCNT , marker for nascent centrioles , green ) , γ tubulin ( centriole marker , green ) E - cadherin ( marker for boundaries , blue ) . Serial have been merged to show complete trachea section ( left ) . Inserts show higher magnification for acetylated y - tubulin pericentrin in GemC1WT / WT GemC1KO / KO trachea , respectively . Scale bars , 10 µm .

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A., B. Transverse sections from trachea isolated from GemC1WT/WT (upper) or GemC1KO/KO (lower) P0 mice, immunostained with antibodies against acetylated y-tubulin (ACT, cilia marker, red), pericentrin (PCNT, marker for nascent centrioles, green), γ tubulin (centriole marker, green) and E-cadherin (marker for cell boundaries, blue). Serial images have been merged to show the complete trachea section (left). Inserts show higher magnification images for acetylated y-tubulin and pericentrin staining in GemC1WT/WT and GemC1KO/KO trachea, respectively. Scale bars, 10 µm.


C . McIdas Foxj1 specific antibodies were used for immunofluorescence on trachea from GemC1WT / WT ( upper ) or GemC1KO / KO ( lower ) P0 mice . Arrows show representative positive for McIdas ( green ) FoxJ1 ( red ) in GemC1WT / WT mice . was stained with Draq - 5 ( blue ) . Scale bars , 10 µm .

C. McIdas and Foxj1 specific antibodies were used for immunofluorescence on trachea from GemC1WT/WT (upper) or GemC1KO/KO (lower) P0 mice. Arrows show representative positive cells for McIdas (green) and FoxJ1 (red) in the GemC1WT/WT mice. DNA was stained with Draq-5 (blue). Scale bars, 10 µm.


D . Transverse sections from P0 GemC1WT / WT ( upper ) or GemC1KO / KO ( lower ) trachea , immunostained with antibodies against Keratin 5 ( Krt5 , basal marker , green ) , acetylated y - tubulin ( ACT , cilia marker , red ) . was stained with Draq - 5 ( blue ) . Serial have been merged to show complete trachea section on left . Higher magnification are shown on right . Scale bars , 10 µm .

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D. Transverse sections from P0 GemC1WT/WT (upper) or GemC1KO/KO (lower) trachea, immunostained with antibodies against Keratin 5 (Krt5, basal cell marker, green), and acetylated y-tubulin (ACT, cilia marker, red). DNA was stained with Draq-5 (blue). Serial images have been merged to show the complete trachea section on the left. Higher magnification images are shown on the right. Scale bars, 10 µm.


E . Transverse sections from P0 GemC1WT / WT ( left two ) or GemC1KO / KO ( right two ) trachea , immunostained with antibodies against Muc5ac ( marker for mucus - expressing , red ) E - cadherin ( marker for boundaries , green ) . was stained with Draq - 5 ( blue ) . Serial have been merged to show complete trachea section ( left ) . Higher magnifications from same section are shown on right . Scale bars , 10 µm .

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E. Transverse sections from P0 GemC1WT/WT (left two images) or GemC1KO/KO (right two images) trachea, immunostained with antibodies against Muc5ac (marker for mucus-expressing cells, red) and E-cadherin (marker for cell boundaries, green). DNA was stained with Draq-5 (blue). Serial images have been merged to show the complete trachea section (left). Higher magnifications from the same section are shown on the right. Scale bars, 10 µm.


C . Representative flow cytometric profiles of thymocytes from 6 - week old Rb ∆ K4 control mice for CD3 - , CD4 - CD8 - positive populations . Bottom , average of CD3 - , CD4 - CD8 single - double - positive in Rb ∆ K4 vs . control thymocytes showing no significant ( n . s . ) difference by student ' s t - . Bars represent mean ± SD ( standard deviation ) ; n = 6 biological replicates ) .

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C. Representative flow cytometric profiles of thymocytes from 6-week old Rb∆K4 and control mice for CD3-, CD4- and CD8-positive cell populations. Bottom, average of CD3-, CD4- and CD8 single- and double-positive cells in Rb∆K4 vs. control thymocytes showing no significant (n.s.) difference by student's t-test. Bars represent mean ±SD (standard deviation); n=6 biological replicates).


D . Short telomeres in Rb ∆ K4 mice . of splenocyte metaphase spreads hybridized with telomere repeat probe ( pink ) . Note shorter telomeres in Rb ∆ K4 splenocytes ( arrows ) . Bottom , quantitative of 10 splenocyte metaphases from one pair of Rb ∆ K4 control littermate ; ( P < 0 . 001 by two - Mann - Whitney - Wilcoxon ) .

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D. Short telomeres in Rb∆K4 mice. Images of splenocyte metaphase spreads hybridized with the telomere repeat probe (pink). Note shorter telomeres in Rb∆K4 splenocytes (arrows). Bottom, quantitative analysis of 10 splenocyte metaphases from one pair of Rb∆K4 and control littermate; (P<0.001 by two-tailed Mann-Whitney-Wilcoxon test).


C . Left , representative flow cytometric for CD3 , CD4 CD8 , showing near normal T development in 10 - day - old Rb ∆ K7 mice . Right , average of CD3 , CD4 CD8 single - double - positive populations in Rb ∆ K7 vs control thymocytes with no significant difference ( n . s . ) by student ' s t - . Results are presented as mean ± SD , n = 4 biological replicates ) .

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C. Left, representative flow cytometric analysis for CD3, CD4 and CD8, showing near normal T cell development in 10-day-old Rb∆K7 mice. Right, average of CD3, CD4 and CD8 single- and double-positive cell populations in Rb∆K7 vs control thymocytes with no significant difference (n.s.) by student's t-test. Results are presented as mean ±SD, n=4 biological replicates).


D . Left , showing apparent normal division of retinal progenitors in Rb ∆ K7 mice . Mice were labeled with BrdU for 2 hrs ; retinal sections from indicated genotypes were stained with DAPI ( blue ) , Ki67 ( green ) , PH3 ( red ) or BrdU ( red ) to mark nuclei , dividing , mitotic or S - phase , respectively . Right , of showing no significant difference among genotypes . Bars denote mean ± SD ; n = 3 biological replicates ) . Scale bar : 20 μm .

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D. Left, images showing apparent normal division of retinal progenitors in Rb∆K7 mice. Mice were labeled with BrdU for 2 hrs; retinal sections from indicated genotypes were stained with DAPI (blue), Ki67 (green), PH3 (red) or BrdU (red) to mark nuclei, all dividing cells, mitotic or S-phase cells, respectively. Right, quantification of staining showing no significant difference among the genotypes. Bars denote mean ±SD; n=3 biological replicates). Scale bar: 20 μm.


E . Left , top , western blot of Rb ∆ K7 thymocytes with an antibody that recognizes both hyper - phosphorylated ( ppRb ) hypo - phosphorylated pRb species . Right , phospho - specific immunoblots showing absence of phosphorylation at upstream Thr350 Ser601 or substituted Ser773Ala site in Rb ∆ K7 thymocytes . Right , immuno - precipitation ( IP ) - Western blot demonstrating phosphorylation of pRb ∆ K7 on Ser243 / Thr246 ( top ) Ser605 ( bottom ) . n . s . , non - specific band .

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E. Left, top, western blot analysis of Rb∆K7 thymocytes with an antibody that recognizes both hyper-phosphorylated (ppRb) and hypo-phosphorylated pRb species. Right, phospho-specific immunoblots showing absence of phosphorylation at upstream Thr350 and Ser601 or the substituted Ser773Ala site in Rb∆K7 thymocytes. Right, immuno-precipitation (IP)-Western blot analysis demonstrating phosphorylation of pRb∆K7 on Ser243/Thr246 (top) and Ser605 (bottom). n.s., non-specific band.


B . Increased resistance of Rb ∆ K7 vs . control MEFs to CDK2 inhibition ( P = 0 . 008 by two - unpaired student ' s t - ) . Effect of a pan CDK inhibitor was near significance ( P = 0 . 07 ) . Bars represent mean ± SD , n = 3 independent MEFs .

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B. Increased resistance of Rb∆K7 vs. control MEFs to CDK2 inhibition (P=0.008 by two-tailed unpaired student's t-test). Effect of a pan CDK inhibitor was near significance (P=0.07). Bars represent mean ±SD, n=3 independent MEFs.


C . Left , representative flow cytometric with 7 - AAD AnnexinV , showing a significant decrease in apoptosis in Rb ∆ K7 MEFs relative to control . Right , significant differences in average of live ( 7 - AAD - : AnnexinV - ) vs . apoptotic ( 7 - AAD + : AnnexinV + ) . Bars represent mean ± SD ; P value calculated by two - unpaired student ' s t - ; n = 3 independent MEFs .

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C. Left, representative flow cytometric analysis with 7-AAD and AnnexinV, showing a significant decrease in apoptosis in Rb∆K7 MEFs relative to control. Right, significant differences in average percentage of live (7-AAD-:AnnexinV-) vs. apoptotic (7-AAD+:AnnexinV+) cells. Bars represent mean ±SD; P value calculated by two-tailed unpaired student's t-test; n=3 independent MEFs.


F - I . Delayed hair re - growth in eight - month old Rb ∆ K7 mice two post hair plucking ( F ) . ( G ) Abnormal skin histology in Rb ∆ K7 mice relative to control . FCL , subcutaneous fat layer , T , telogen ; A , anagen . ( H - I ) Representative immunostaining for keratin 14 PCNA showing reduced expression in Rb ∆ K7 hair follicles vs controls . Scale bar , 50 μm .

F-I. Delayed hair re-growth in eight-month old Rb∆K7 mice two weeks post hair plucking (F). (G) Abnormal skin histology in Rb∆K7 mice relative to control. FCL, subcutaneous fat cell layer, T, telogen; A, anagen. (H-I) Representative immunostaining for keratin 14 and PCNA showing reduced expression in Rb∆K7 hair follicles vs controls. Scale bar, 50 μm.


J . Hyperglycemia in Rb ∆ K7 but not Rb ∆ K4 mice . Blood glucose in 10 - 12 - month old fasting Rb ∆ K7 ( left , n = 11 ) or 18 - month old fasting Rb ∆ K4 ( right , n = 15 ) mice versus control ( n = 6 n = 8 , respectively ) . Mean ± SD ; P values by two - unpaired student ' s t - .

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J. Hyperglycemia in Rb∆K7 but not Rb∆K4 mice. Blood glucose in 10-12-month old fasting Rb∆K7 (left, n=11) or 18-month old fasting Rb∆K4 (right, n=15) mice versus control littermates (n=6 and n=8, respectively). Mean ±SD; P values by two-tailed unpaired student's t-test.


B . Glucose tolerance ( GTT ) demonstrating progressive defect in clearance of blood glucose in Rb ∆ K7 mice . 6 - 7 week ( left ) or 6 month ( right ) old fasting Rb ∆ K7 mice control ( n = 5 - 6 each per group ) were injected with glucose , blood glucose was determined at indicated intervals . Error bars represent SD .

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B. Glucose tolerance Test (GTT) demonstrating progressive defect in clearance of blood glucose in Rb∆K7 mice. 6-7 week (left) or 6 month (right) old fasting Rb∆K7 mice and control littermates (n=5-6 each per group) were injected with glucose, and blood glucose was determined at indicated intervals. Error bars represent SD.


F . Loss of pancreatic islet in Rb ∆ K7 mice correlates with reduced proliferation H3K36me3 histone marks , but not with increased . TUNEL showing apoptosis in 10 day - old Rb ∆ K7 islets is even lower than in control . Arrow points to a rare apoptotic in control islet . Serine800 / 804 phosphorylation of pRb in β - from 10 day - old control but not Rb ∆ K7 islets with a Ser - to - Ala substitution at this site . BrdU incorporation PH3 showing reduced S - phase mitotic , respectively , in 2 - 3 - week old Rb ∆ K7 vs control islets . Reduced H3K36me3 in pancreatic islets from 3 - week old Rb ∆ K7 mice compared to control ( 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 - to re - enter cycle in response to pregnancy ( top ) or mitogenic ( bottom ) signals . Top , proliferation in P14 . 5 pregnant Rb ∆ K7 islet β - vs . control was determined by double immunostaining with BrdU insulin ( left ) ; ( right ) . Results were normalized for number of BrdU / Insulin positive in control islets . An independent experiment using ki67 to quantify cycle re - entry top . Bottom , proliferation in Rb ∆ K7 β - vs . control islets following Exendin4 treatment was determined by immunostaining for Ki67 ; results were normalized for number of positive in control islets . Mean ± SD , P values by two - unpaired student ' s t - . Scale bar , 100 μm .

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

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 ) 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 SASP marker IL - 6 ( red ) insulin ( green ) . DAPI was used to stain nuclei ( blue ) . Arrows point to IL - 6 + , insulin + pancreatic β - . 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 DDR marker γ - H2A in islets from 3 - week old Rb ∆ K7 control mice ( left ) ; statistical ( right ; n = 5 each ) . Arrows point to pancreatic γ - H2A - positive Rb ∆ K7 islet . Error bars represent SD ; P value calculated by one - way ANOVA , Tukey ' s multiple comparisons . 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 improves β - islet in Rb ∆ K7 mice . Scale bar , 100 μm . I . Vitamin C diet reduces difference in γ − H2A + β - 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 . J . Vitamin C diet increases pancreatic islet 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 .

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

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 proliferation as determined by MTT ( n = 4 - 5 technical replicates ) . Left , bottom , of SA - βGAL in rRb ∆ K11 - transduced . Bars represent mean ± SD ; P value calculated by two - unpaired student ' s t - . Right , representative of SA - βGAL showing robust senescence ( arrows ) in rRb ∆ K11 - transduced . 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 senescence induced by retroviral - mediated over - expression of Rb ∆ K11 . Top - of SA - βGAL in human diploid , IMR90 , transduced with control or rRb ∆ K11 with or without vitamin C added at time of infection . Arrows point to SA - βGAL - positive . Bottom left , of SA - βGAL ( bars represent mean ± ; n = 4 biological replicates ) . P values by one - way ANOVA , Tukey ' s multiple comparisons . Bottom right , dot - blot with antibody for 5hmC on extracted from IMR90 treated or not with vitamin C . Original magnification : 200x .

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

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 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 T - tau were measured using INNOBIA AlzBio3 kit ( Fujirebio , Ghent , Belgium ) , Ng was measured using an in - house immunoassay for Ng ( Portelius et al , 2015 ) NFL was measured using NF - ® 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 NFL between these biomarkers 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 T - tau were measured using INNOBIA AlzBio3 kit ( Fujirebio , Ghent , Belgium ) , Ng was measured using an in - house immunoassay for Ng ( Portelius et al , 2015 ) NFL was measured using NF - ® ELISA kit ( Uman Diagnostics , Umeå , Sweden ) .

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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 ) NFL ( panel C ) in different combinations of clinical diagnosis 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 are standardized to z - scores shown in boxplots ( indicating median interquartile ranges ; whiskers are defined as quartiles 1 3 + / - 1 . 5 * interquartile range , respectively ) . Aβ42 T - tau were measured using INNOBIA AlzBio3 kit ( Fujirebio , Ghent , Belgium ) , Ng was measured using an in - house immunoassay for Ng ( Portelius et al , 2015 ) NFL was measured using NF - ® ELISA kit ( Uman Diagnostics , Umeå , Sweden ) .

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

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

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

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

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

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


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

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

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

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

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

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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 ) GST - Spt5N ( yeast ) were mixed as indicated with yeast histones H3 - H4 , in presence of glutathione agarose beads ( see STAR method for further details ) . input pulldown samples were analysed by SDS - PAGE before with colloidal Coomassie blue . 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 GST - MCM2N - 2A ( with Y81A Y90A mutations of conserved residues ) .

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

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

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

(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 or spt5 - AID , expressing indicated versions of Spt5 from GAL1 , 10 promoter , were grown on indicated medium .

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

(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 at indicated times for samples described in ( A ) .

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


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

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

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

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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 . WT mutant yeast were cultured to log phase stained with TMRM for FACS .

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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 . were cultured from 0 . 05 OD600 in SCD . OD600 was measured every 2 hours .

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


Transcriptome of OXPHOS mutants . WT mutant yeast were cultured to log phase . RNAseq was performed with three biological replicates per genotype ( Dataset EV1 ) . transcripts 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 of selected transcripts in ρ0 .

qRT-PCR analysis of selected transcripts in ρ0 cells.


SILAC of mitochondrial proteome of ∆ IV ρ0 . Equal amount of WT labeled with heavy unlabeled mutant were mixed to extract mitochondrial fraction for mass - spectrometry . Three biological replicates were prepared for each pair of strains ( Dataset EV2 ) . 568 high fidelity mitochondrial ( gene / list in Table EV1A ) in ∆ IV 572 such in ρ0 were quantitatively measured . Significantly changed ( P value < 0 . 05 ; fold change > 1 . 5 or < 0 . 67 ) are highlighted with dashed lines ; numbers of significantly changed 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 of representative mitochondrial endogenously tagged with HA . selection markers were removed by Cre - loxP system . Whole lysates were extracted equal amount of 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 of OXPHOS mutants . Polysomes were fractionated by sucrose gradients ( 10 % - 50 % ) , gradients were separated 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 of INH1 mRNA . Data are mean ± SD from three biological replicates .

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


Western blot of Inh1 . Whole lysates were extracted equal amount of was loaded for western blot .

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 proliferation of ρ0 at 37 ℃ . Serial dilutions ( tenfold dilution ) of indicated strains were analyzed on SCD plates at 30 ℃ 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 of copper - inducible overexpression of Inh1 . A cassette expressing Inh1 under control of CUP1 promoter was inserted into 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 . were cultured in SCD or SCD plus 100 μM CuSO4 for 12 hours to mid - log phase . were then stained with 125 nM TMRM for FACS .

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

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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 of mitochondrial ISC biosynthesis ( Nfs1 Yah1 ) ISC - containing Pol3 . were cultured in SCD or SCD plus 100 μM CuSO4 for 12 hours to mid - log phase . Whole lysates were extracted equal amount of was loaded for western blot . Nfs1 , Yah1 were endogenously tagged with FLAG , 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.


changes of Puf3 - associated transcripts encoding mitochondrial in OXPHOS mutants . RNAseq results in Figure 1C ( Dataset EV1 ) are re - analyzed . Transcripts encoding high fidelity mitochondrial ( gene list in Table EV1A ) Puf3 - associated transcripts encoding mitochondrial ( gene list in Table EV1D ) are shown . 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 C , Deletion of PUF3 upregulates mitochondrial transcripts in WT ( B ) , whereas downregulates mitochondrial transcripts in ρ0 ( C ) . WT mutant yeast were cultured to log phase . RNAseq was performed with three biological replicates per genotype ( Dataset EV4 ) . transcripts Puf3 - associated transcripts encoding mitochondrial are shown . 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 downregulates mitochondrial . SILAC of mitochondrial fractions from ρ0 ρ0 puf3 ∆ are shown . Equal amount of ρ0 labeled with heavy unlabeled ρ0 puf3 ∆ were mixed to extract mitochondrial fraction for mass - spectrometry . Three biological replicates were prepared for each pair of strains ( Dataset EV5 ) . Mitochondrial 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 . significantly changed ( P value < 0 . 05 ; fold change > 1 . 5 or < 0 . 67 ) are highlighted with dashed lines ; numbers of significantly changed 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 their transcripts . Puf3 - associated transcripts encoding mitochondrial 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 . were cultured to mid - log phase , stained with 125 nM TMRM for FACS

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


G . Deletion of PUF3 decreases proliferation of ρ0 . Serial dilutions ( tenfold dilution ) of 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 consensus Puf3 - binding motif Lapointe et al . , 2015 ( ) motif at Mia40 3 ' - UTR . Mutating Puf3 - binding motif of MIA40 mRNA abolishes Mia40 upregulation in ρ0 . Mia40 was endogenously tagged with FLAG its 3 ' - UTR was edited as indicated . selection markers were removed by Cre - loxP system . Whole lysates were extracted equal amount of 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 Puf3 - binding motif of MIA40 mRNA decreases ∆ ψm of ρ0 . J . Mutating Puf3 - binding motif of MIA40 mRNA impairs proliferation of ρ0 . were cultured from 0 . 05 OD in SCD , OD600 was measured every 2 hours . were cultured to mid - log phase , 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 Puf3 - binding motif of MIA40 mRNA decreases ∆ ψm of ρ0 .

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


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