Human bronchial epithelial BEAS - 2B were infected with influenza A / Scotland / 20 / 74 ( H3N2 ) ( IAV ) at MOI = 1 for 4 h , then washed treated or not with succinate ( Suc ; 4 mg / mL ) for 20 h . ( a Expression of IAV ( i . e . NP , NS1 ) was analyzed by confocal microscopy using specific antibodies . Viral are stained in green , in blue actin in red . For a given antibody , pictures were taken on exact same day with same power settings , Pictures are representative of five independent experiments . Scale bar 10 µM . ( b ) Relative of NS1 or NP was determined by using Nuclei Cytoplasm Tool . Seven ( NS1 ) or nine ( NP ) random were collected per treatment with at least 5 per field . Data are represented as mean ± . Statistical was performed using Mann - Whitney , ( * P < 0 . 05 ) .

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

Human bronchial epithelial BEAS-2B cells were infected with influenza A/Scotland/20/74 (H3N2) virus (IAV) at MOI=1 for 4 h, then washed and treated or not with succinate (Suc; 4 mg/mL) for 20 h. (a Expression of IAV proteins (i.e. NP, NS1) was analyzed by confocal microscopy using specific antibodies. Viral proteins are stained in green, DNA in blue and actin in red. For a given antibody, pictures were taken on the exact same day with the same laser power settings, Pictures are representative of five independent experiments. Scale bar 10 µM. (b) Relative nuclear intensity of NS1 or NP was determined by using the Intensity Ratio Nuclei Cytoplasm Tool. Seven (NS1) or nine (NP) random images were collected per treatment with at least 5 cells per field. Data are represented as the mean ± SEM. Statistical analysis was performed using the Mann-Whitney test, (*P < 0.05).


Human bronchial epithelial BEAS - 2B were infected with influenza A / Scotland / 20 / 74 ( H3N2 ) ( IAV ) at MOI = 1 for 4 h , subsequently treated or not with 4 mg / mL of succinate ( Suc ) for 20 h . ( a , b ) were stained using a monoclonal antibody that specifically recognizes " NP - vRNA " complexes , but not - free NP , confocal microscopy ( a ) was performed ( green ) . value raw integrated density ( RawIntDen , which is sum of pixel values in ROI ( region of interest ) ) was further measured ( b ) . Data information : Data are represented as mean ± of 3 biological replicates or without ( a 2 technical replicates each . 8 ( b ) random were analysed per treatment with a minimum of 5 per field . Statistical was performed using Mann - Whitney ( b , ( * P < 0 . 05 ) .

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

Human bronchial epithelial BEAS-2B cells were infected with the influenza A/Scotland/20/74 (H3N2) virus (IAV) at MOI=1 for 4 h, and subsequently treated or not with 4 mg/mL of succinate (Suc) for 20 h. (a, b) Cells were stained using a monoclonal antibody that specifically recognizes "NP-vRNA" complexes, but not RNA-free NP, and confocal microscopy (a) was performed (green staining). The value raw integrated density (RawIntDen, which is the sum of all pixel values in the ROI (region of interest)) was further measured (b). Data information: Data are represented as the mean ± SEM of 3 biological replicates or without (a 2 technical replicates each. 8 (b) random images were analysed per treatment with a minimum of 5 cells per field. Statistical analysis was performed using the Mann-Whitney test (b, (*P < 0.05).


Human bronchial epithelial BEAS - 2B were infected with influenza A / Scotland / 20 / 74 ( H3N2 ) ( IAV ) at MOI = 1 for 4 h , subsequently treated or not with 4 mg / mL of succinate ( Suc ) for 20 h . ( e ) Human bronchial epithelial BEAS - 2B were infected with a A / Scotland / 20 / 74 ( H3N2 ) ( IAV ) carrying a wild - type NP ( NP WT ) or with corresponding mutated bearing a NP with a K87R substitution ( NP K87R ) . were infected by either at MOI = 1 for 4 h , then washed treated or not with 4 mg / mL of succinate for 20 h . Localization of NP was analyzed by confocal immunofluorescence microscopy . Scale bar : 10 µM . Pictures are representative of three independent experiments . ( f ) Relative of NP was determined by using Nuclei Cytoplasm Tool . Data information : Data are represented as mean ± of 3 biological replicates without 2 technical replicates each . at least 10 ( f ) random were analysed per treatment with a minimum of 5 per field . Statistical was performed using Mann - Whitney ( * P < 0 . 05 ) .

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

Human bronchial epithelial BEAS-2B cells were infected with the influenza A/Scotland/20/74 (H3N2) virus (IAV) at MOI=1 for 4 h, and subsequently treated or not with 4 mg/mL of succinate (Suc) for 20 h. (e) Human bronchial epithelial BEAS-2B cells were infected with a A/Scotland/20/74 (H3N2) virus (IAV) carrying a wild-type NP (NP WT) or with the corresponding mutated virus bearing a NP with a K87R substitution (NP K87R). Cells were infected by either virus at MOI=1 for 4 h, then washed and treated or not with 4 mg/mL of succinate for 20 h. Localization of NP proteins was analyzed by confocal immunofluorescence microscopy. Scale bar: 10 µM. Pictures are representative of three independent experiments. (f) Relative nuclear intensity of NP was determined by using the Intensity Ratio Nuclei Cytoplasm Tool. Data information: Data are represented as the mean ± SEM of 3 biological replicates without 2 technical replicates each. at least 10 (f) random images were analysed per treatment with a minimum of 5 cells per field. Statistical analysis was performed using the Mann-Whitney test (*P < 0.05).


( B ) number of metabolites that change significantly when wild - type yeast mutants in predicted target are treated with indicated drugs at a of 10 µM are shown . Drugs targeting GPR1 are written in black text , with drugs targeting SNF3 RGT2 written in purple green respectively . Orange filled bars are predicted antagonists for GPR1 .

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(B) The number of metabolites that change significantly when wild-type yeast and mutants in the predicted target are treated with the indicated drugs at a concentration of 10 µM are shown. Drugs targeting GPR1 are written in black text, with drugs targeting SNF3 and RGT2 written in purple and green respectively. Orange filled bars are predicted antagonists for GPR1.


( C ) of trehalose compared to a DMSO treated control are shown for both wild - type yeast gpr1 strains treated with predicted antagonists for GPR1 . bar height indicates mean values , error bars indicate standard deviation of mean . Asterisks indicate a p - value of less than 0 . 05 from a two - sided student ' s T - ( n = 4 biological replicates ) when comparing WT gpr1 reponses to drug . Results are representative of 3 independent experiments .

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(C) The levels of trehalose compared to a DMSO treated control are shown for both wild-type yeast and gpr1 strains treated with predicted antagonists for GPR1. The bar height indicates mean values, and error bars indicate the standard deviation of the mean. Asterisks indicate a p-value of less than 0.05 from a two-sided student's T-test (n = 4 biological replicates) when comparing The WT and gpr1 reponses to the drug. Results are representative of 3 independent experiments.


( C ) average quantified filamentous growth is shown for BY4741 Σ1278b yeast spotted onto media supplemented with either a vehicle control ( DMSO 0 . 1 % ( v / v ) or indicated compounds at a of 20 µM across four biological replicates . Error bars represent standard deviation of mean between averages of replicates , asterisks indicate p - value < 0 . 05 for a two - sided student ' s T - comparing normalized values to DMSO control ( n = 4 biological replicates ) .

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(C) The average quantified filamentous growth is shown for BY4741 and Σ1278b yeast spotted onto media supplemented with either a vehicle control (DMSO 0.1% (v/v) or the indicated compounds at a concentration of 20 µM across four biological replicates. Error bars represent the standard deviation of the mean between the averages of the replicates, and asterisks indicate p-value < 0.05 for a two-sided student's T-test comparing normalized values to the DMSO control (n = 4 biological replicates).


( D ) average quantified filamentous growth is shown for BY4741 Σ1278b yeast spotted onto media supplemented with either a vehicle control ( DMSO 0 . 1 % ( v / v ) or indicated concentrations of ibuprofen . Error bars represent standard deviation of mean between averages of replicates , asterisks indicate p - value < 0 . 05 for a two - sided student ' s T - comparing normalized values to DMSO control ( n = 4 biological replicates ) .

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(D) The average quantified filamentous growth is shown for BY4741 and Σ1278b yeast spotted onto media supplemented with either a vehicle control (DMSO 0.1% (v/v) or the indicated concentrations of ibuprofen. Error bars represent the standard deviation of the mean between the averages of the replicates, and asterisks indicate p-value < 0.05 for a two-sided student's T-test comparing normalized values to the DMSO control (n = 4 biological replicates).


( E ) average relative amount of trehalose in ibuprofen - treated compared to a DMSO treatment is shown . bar height indicates mean values , error bars indicate standard deviation of mean , asterisks indicate p - value < 0 . 05 for a two - sided student ' s T - comparing to WT control ( n = 8 biological replicates ) . Results are representative of 3 independent experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) The average relative amount of trehalose in ibuprofen-treated cells compared to a DMSO treatment is shown. The bar height indicates mean values, error bars indicate the standard deviation of the mean, and asterisks indicate p-value < 0.05 for a two-sided student's T-test comparing to the WT control (n = 8 biological replicates). Results are representative of 3 independent experiments.


( A , B ) PTPRN2 ( A ) PLCB1 ( B ) expression were determined by qRT - PCR .

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(A,B) PTPRN2 (A) and PLCB1 (B) expression levels were determined by qRT-PCR.


( C ) Bioluminescence imaging of lung colonization by 40 , 000 LM2 breast cancer transduced with shRNAs targeting PTPRN2 or a control hairpin . For shCntrl , sh1PTPRN2 , N = 5 mice / group . For sh2PTPRN2 N = 6 mice . Right , H & amp ; amp ; E of representative lung sections .

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(C) Bioluminescence imaging quantification of lung colonization by 40,000 LM2 breast cancer cells transduced with shRNAs targeting PTPRN2 or a control hairpin. For shCntrl, sh1PTPRN2, N = 5 mice/group. For sh2PTPRN2 N = 6 mice. Right, H&amp;E staining of representative lung sections.


( D ) Bioluminescence imaging of lung colonization by 40 , 000 LM2 transfected with siRNA targeting PLCβ1 or a control siRNA . For siCntrl , N = 5 mice . For si1PLCβ1 , si2PLCβ1 , N = 6 mice / group . Right , H & amp ; amp ; E of representative lung sections .

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(D) Bioluminescence imaging quantification of lung colonization by 40,000 LM2 cells transfected with siRNA targeting PLCβ1 or a control siRNA. For siCntrl, N = 5 mice. For si1PLCβ1, si2PLCβ1, N = 6 mice/group. Right, H&amp;E staining of representative lung sections.


( E , F ) PTPRN2 ( E ) PLCB1 ( F ) were analyzed in human breast cancers ( stages I - IV ) normal breast tissue from TissueScan qPCR Array Breast Cancer Panels II III ( Origene , N = 97 ) . Expression were normalized to in normal tissue for each gene .

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(E,F) PTPRN2 (E) and PLCB1 (F) levels were analyzed in human breast cancers (stages I-IV) and normal breast tissue from TissueScan qPCR Array Breast Cancer Panels II and III (Origene, N = 97). Expression levels were normalized to levels in normal tissue for each gene.


( G ) Kaplan - Meier curve representing overall survival of a cohort of breast cancer ( N = 528 ) as a function of their ' s PTPRN2 PLCB1 expression ( data from TCGA Research Network ( Cancer Genome Atlas , 2012 ) ) . whose tumors ' PTPRN2 PLCB1 expression were higher or lower than median of population were classified as low ( blue ) or high ( red ) expression .

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(G) Kaplan-Meier curve representing overall survival of a cohort of breast cancer patients (N = 528) as a function of their primary tumor's PTPRN2 and PLCB1 expression levels (data from the TCGA Research Network (Cancer Genome Atlas, 2012)). Patients whose primary tumors' PTPRN2 and PLCB1 expression levels were higher or lower than the median of the population were classified as low (blue) or high (red) expression.


( H ) Kaplan - Meier curve representing distal metastasis - free survival of a cohort of breast cancer ( N = 1609 ) as a function of their ' s PTPRN2 PLCB1 expression ( Data from KMPlot ( Gyorffy et al . , 2010 ) ) . ' tumors ' PTPRN2 PLCB1 expression were classified as low ( blue ) or high ( red ) expression .

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(H) 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 PTPRN2 and PLCB1 expression levels (Data from KMPlot (Gyorffy et al., 2010)). Patients' primary tumors' PTPRN2 and PLCB1 expression levels were classified as low (blue) or high (red) expression.


( A ) Matrigel invasion by 50 , 000 LM2 transfected with siRNA targeting PTPRN2 , PLCβ1 , or control siRNA . Data normalized to control values . N = 6 inserts / group .

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(A) Matrigel invasion by 50,000 LM2 cells transfected with siRNA targeting PTPRN2, PLCβ1, or control siRNA. Data normalized to control values. N = 6 inserts/group.


( B ) Migration by 100 , 000 LM2 transfected with siRNA targeting PTPRN2 , PLCβ1 , or control siRNA . Data normalized to control values . N = 6 inserts / group . Right , representative of migration . Scale bar , 100 μm .

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(B) Migration assay by 100,000 LM2 cells transfected with siRNA targeting PTPRN2, PLCβ1, or control siRNA. Data normalized to control values. N = 6 inserts/group. Right, representative images of the migration assay. Scale bar, 100 μm.


( C ) of covered by 24 h after a scratch was made through confluent transfected with siRNA targeting PTPRN2 , PLCβ1 , or control siRNA . N = 5 wells / group . Right , representative of scratch .

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(C) Quantification of area covered by cells 24 h after a scratch was made through confluent cells transfected with siRNA targeting PTPRN2, PLCβ1, or control siRNA. N = 5 wells/group. Right, representative images of the scratch assay.


( D , E ) MDA - MB - 231 transduced with PTPRN2 , PTPRN2C945A , or control vector were subjected to Matrigel invasion ( D ) migration assays ( E ) . N = 5 inserts / group .

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(D,E) MDA-MB-231 cells transduced with PTPRN2, PTPRN2C945A, or control vector were subjected to the Matrigel invasion (D) and migration assays (E). N = 5 inserts/group.


( F ) Bioluminescence imaging of lung colonization by 40 , 000 MDA - MB - 231 overexpressing PTPRN2 , PTPRN2C945A , or control vector . N = 5 mice / group . Right , H & amp ; amp ; E of representative lung sections .

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(F) Bioluminescence imaging quantification of lung colonization by 40,000 MDA-MB-231 cells overexpressing PTPRN2, PTPRN2C945A, or control vector. N = 5 mice/group. Right, H&amp;E staining of representative lung sections.


( G , H ) MDA - MB - 231 transduced with PLCβ1 , PLCβ1H331Q , or control vector were subjected to Matrigel invasion ( G ) migration assays ( H ) . N = 5 inserts / group .

(G,H) MDA-MB-231 cells transduced with PLCβ1, PLCβ1H331Q, or control vector were subjected to the Matrigel invasion (G) and migration assays (H). N = 5 inserts/group.


( I ) Bioluminescence imaging of lung colonization by 40 , 000 MDA - MB - 231 overexpressing PLCβ1 , PLCβ1H331Q , or control vector . For Cntrl PLCβ1H331Q OE , N = 6 mice / group . For PLCβ1 OE , N = 5 mice . Right , H & amp ; amp ; E of representative lung sections .

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(I) Bioluminescence imaging quantification of lung colonization by 40,000 MDA-MB-231 cells overexpressing PLCβ1, PLCβ1H331Q, or control vector. For Cntrl and PLCβ1H331Q OE, N = 6 mice/group. For PLCβ1 OE, N = 5 mice. Right, H&amp;E staining of representative lung sections.


( A ) Mean of PI ( 4 , 5 ) P2 was analyzed in MDA - MB - 231 LM2 immunostained with PI ( 4 , 5 ) P2 antibody using microscopy . N = 50 / group . Scale bar , 10 μm .

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(A) Mean fluorescence intensity of membrane PI(4,5)P2 was analyzed in MDA-MB-231 and LM2 cells immunostained with PI(4,5)P2 antibody using fluorescence microscopy. N = 50 cells/group. Scale bar, 10 μm.


( B ) Bioluminescence imaging of lung colonization by 40 , 000 LM2 treated with carrier incubated with PI ( 4 , 5 ) P2 or carrier alone for one hour then immediately injected . Right , H & amp ; amp ; E of representative lung sections . N = 6 mice / group .

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(B) Bioluminescence imaging quantification of lung colonization by 40,000 LM2 cells treated with carrier incubated with PI(4,5)P2 or carrier alone for one hour and then immediately injected. Right, H&amp;E staining of representative lung sections. N=6 mice/group.


( C , D ) MDA - MB - 231 overexpressing PTPRN2 , PLCβ1 , or a control vector ( C ) or LM2 transfected with siRNA targeting PTPRN2 , PLCβ1 , or a control siRNA ( D ) were immunostained for PI ( 4 , 5 ) P2 analyzed by microscopy . Mean of plasma of lipid were quantified . N = 50 / group . Left , representative immunofluorescence of stained with anti - PI ( 4 , 5 ) P2 antibody ( red ) 4 ' , 6 - diamidino - 2 - phenylindole ( DAPI , blue ) . Scale bar , 10 μm .

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(C,D) MDA-MB-231 cells overexpressing PTPRN2, PLCβ1, or a control vector (C) or LM2 cells transfected with siRNA targeting PTPRN2, PLCβ1, or a control siRNA (D) were immunostained for PI(4,5)P2 levels and analyzed by fluorescence microscopy. Mean fluorescence intensity of plasma membrane levels of the lipid were quantified. N = 50 cells/group. Left, representative immunofluorescence images of cells stained with anti-PI(4,5)P2 antibody (red) and 4',6-diamidino-2-phenylindole (DAPI, blue). Scale bar, 10 μm.


( A , B ) LM2 transfected with siRNA targeting PTPRN2 ( A ) , PLCβ1 ( B ) or a control siRNA were transfected with Lyn11 - FRB INPP5E - FKBP , treated with either DMSO or 100 nm rapamycin subjected to migration . N = 5 inserts / group .

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(A,B) LM2 cells transfected with siRNA targeting PTPRN2 (A), PLCβ1 (B) or a control siRNA were transfected with Lyn11-FRB and INPP5E-FKBP, treated with either DMSO or 100 nm rapamycin and subjected to the migration assay. N = 5 inserts/group.


( 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.


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( 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.


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( 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.


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( 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 .

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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 ) .

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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 .

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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 .

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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 .

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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 .

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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 .

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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 .

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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 .

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


Associations between CSF T - tau , Ng 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 ) .

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

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

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

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

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

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 ) .

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

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

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

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

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).