B - In vitro assay testing the ability of purified TG2 to crosslink purified rhGFPT1 with BP . Samples were incubated for 3 h at 37 ° C before separation by SDS - PAGE . Proteins were transferred to a membrane and BP was revealed using HRP - conjugated streptavidin . rhGFPT1 is 77 . 5 kDa .

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

B - In vitro assay testing the ability of purified TG2 to crosslink purified rhGFPT1 with BP. Samples were incubated for 3 h at 37°C before separation by SDS-PAGE. Proteins were transferred to a membrane and BP was revealed using HRP-conjugated streptavidin. rhGFPT1 is 77.5 kDa.


D - In vitro assay was performed as described in B using wild type rhGFPT1 ( WT ) , rhGFPT1 Q58N , rhGFPT1 Q328N or rhGFPT1 Q555N as substrates . The reaction was performed at 37 ° C for 30 min . After probing with HRP - Streptavidin the membrane was washed and probed with anti - GFPT antibodies followed with HRP - conjugated secondary antibodies . The ratio of modified protein ( streptavidin signal ) to the total GFPT is shown , normalized to its value with WT rhGFPT1 . The mean ± SD of five independent experiments is shown , the p - value of the Student ' s ratio - paired t - test is indicated when < 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D - In vitro assay was performed as described in B using wild type rhGFPT1 (WT), rhGFPT1 Q58N, rhGFPT1 Q328N or rhGFPT1 Q555N as substrates. The reaction was performed at 37 °C for 30 min. After probing with HRP-Streptavidin the membrane was washed and probed with anti-GFPT antibodies followed with HRP-conjugated secondary antibodies. The ratio of modified protein (streptavidin signal) to the total GFPT is shown, normalized to its value with WT rhGFPT1. The mean ± SD of five independent experiments is shown, the p-value of the Student's ratio-paired t-test is indicated when <0.05.


E - Lysates of cells treated or not for 6 h with ionomycin were incubated at 37 ° C for 45 min with fructose - 6 - P and glutamine . The production of glucosamine - 6 - P was measured using HPAEC - PAD . Results of three independent experiments are shown , with mean ± SD , and p - value of the Student ' s paired t - test is indicated ( * P < 0 . 05 ) .

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 B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-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

E - Lysates of cells treated or not for 6 h with ionomycin were incubated at 37 °C for 45 min with fructose-6-P and glutamine. The production of glucosamine-6-P was measured using HPAEC-PAD. Results of three independent experiments are shown, with mean ± SD, and p-value of the Student's paired t-test is indicated (*P < 0.05).


B - Endocervical epithelial cells were pre - treated or not with 40 μM CP4d for 2 h before addition of the indicated concentration of ionomycin ( or an equivalent volume of DMSO ) and 0 . 5 µM BP . Six hours later whole cell lysates were analyzed by western blot . The membrane was first blotted with HRP - conjugated streptavidin to detect TG2 activity , then extensively washed and probed with anti - O - GlcNAcylation antibody followed with HRP - conjugated secondary antibodies . Last the membrane was probed with anti - actin as a loading control .

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 B-CONTROLLED_VAR 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

B - Endocervical epithelial cells were pre-treated or not with 40 μM CP4d for 2 h before addition of the indicated concentration of ionomycin (or an equivalent volume of DMSO) and 0.5 µM BP. Six hours later whole cell lysates were analyzed by western blot. The membrane was first blotted with HRP-conjugated streptavidin to detect TG2 activity, then extensively washed and probed with anti-O-GlcNAcylation antibody followed with HRP-conjugated secondary antibodies. Last the membrane was probed with anti-actin as a loading control.


C - The same experimental procedure as described in ( B ) was applied to HeLa cells treated for 48 h prior to ionomycin treatment ( 8 µM ) with siRNA control or directed against TG2 .

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

C - The same experimental procedure as described in (B) was applied to HeLa cells treated for 48 h prior to ionomycin treatment (8 µM) with siRNA control or directed against TG2.


A - HeLa cells were infected or not ( NI ) with C . trachomatis ( MOI = 1 ) , then lysed 24 or 48 hpi . After separation with SDS - PAGE , proteins were transferred to a membrane , probed with anti - O - GlcNAcylation antibody followed with HRP - conjugated secondary antibodies . After extensive washes the membrane was blotted again with anti - GFPT and anti - actin antibodies before revelation with HRP - conjugated secondary antibodies .

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

A - HeLa cells were infected or not (NI) with C. trachomatis (MOI = 1), then lysed 24 or 48 hpi. After separation with SDS-PAGE, proteins were transferred to a membrane, probed with anti-O-GlcNAcylation antibody followed with HRP-conjugated secondary antibodies. After extensive washes the membrane was blotted again with anti-GFPT and anti-actin antibodies before revelation with HRP-conjugated secondary antibodies.


B - HeLa cells were infected or not with C . trachomatis ( MOI = 1 ) . Twenty - four hours later , 8 µM ionomycin ( or DMSO alone ) and 0 . 5 mM BP were added . After 6 h of treatment , cells were lysed and proteins revealed as in ( A ) .

O O 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

B - HeLa cells were infected or not with C. trachomatis (MOI = 1). Twenty-four hours later, 8 µM ionomycin (or DMSO alone) and 0.5 mM BP were added. After 6 h of treatment, cells were lysed and proteins revealed as in (A).


C - HeLa cells treated for 72 h with siRNA targeting GFPT1 or GFPT2 were lysed . After separation with SDS - PAGE , proteins were transferred to a membrane , probed with anti - GFPT and anti - actin antibody before revelation with HRP - conjugated secondary antibodies .

O O O O O O O O O O O O O O O O O O O O O O O O O O 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 - HeLa cells treated for 72 h with siRNA targeting GFPT1 or GFPT2 were lysed. After separation with SDS-PAGE, proteins were transferred to a membrane, probed with anti-GFPT and anti-actin antibody before revelation with HRP-conjugated secondary antibodies.


D - HeLa cells were transfected with control siRNA or siRNAs against GFPT or TG2 . Two days later the cells were infected for the indicated times ( MOI = 0 . 3 ) before fixation , rupture of the cells and measurement of bacterial diameter . The mean diameter ± SD and p - values of Student ' s paired t - test on 4 independent experiments are shown .

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

D - HeLa cells were transfected with control siRNA or siRNAs against GFPT or TG2. Two days later the cells were infected for the indicated times (MOI=0.3) before fixation, rupture of the cells and measurement of bacterial diameter. The mean diameter ± SD and p-values of Student's paired t-test on 4 independent experiments are shown.


E - HeLa cells treated for 48 h with siRNA targeting GFPT1 or not ( siCTRL ) were infected with C . trachomatis ( MOI = 0 . 15 ) . Thirty hours later cells were fixed and analyzed by flow cytometry . The percentage of infected cells ( top ) and the mean fluorescence of the infected population ( middle ) ) ± SD are shown for at least four independent experiments . Duplicate wells were lysed and used to re - infect fresh HeLa cells to determine the bacterial titer ( bottom ) . P - values of Student ' s ratio - paired t - test are indicated .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 - HeLa cells treated for 48 h with siRNA targeting GFPT1 or not (siCTRL) were infected with C. trachomatis (MOI = 0.15). Thirty hours later cells were fixed and analyzed by flow cytometry. The percentage of infected cells (top) and the mean fluorescence of the infected population (middle) ) ± SD are shown for at least four independent experiments. Duplicate wells were lysed and used to re-infect fresh HeLa cells to determine the bacterial titer (bottom). P-values of Student's ratio-paired t-test are indicated.


IFN - β promoter activity ( A ) in Myc empty vector ( 200 ng ) or Myc - SIRT5 ( 200 ng ) - transfected HEK293T cells with or without SeV infection ( SeV or UI ) for 18 ~ 24 h . Data information : Graphs represent fold - induction relative to the luciferase activity in the control cells . UI , uninfected . All data are presented as the mean values based on three independent experiments , and error bars indicate S . E . M . ( unpaired two - tailed Student ' s t test ) .

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

IFN-β promoter activity (A) in Myc empty vector (200 ng) or Myc-SIRT5 (200 ng)-transfected HEK293T cells with or without SeV infection (SeV or UI) for 18~24 h. Data information: Graphs represent fold-induction relative to the luciferase activity in the control cells. UI, uninfected. All data are presented as the mean values based on three independent experiments, and error bars indicate S.E.M. (unpaired two-tailed Student's t test).


ISRE reporter activity ( B ) in Myc empty vector ( 200 ng ) or Myc - SIRT5 ( 200 ng ) - transfected HEK293T cells with or without SeV infection ( SeV or UI ) for 18 ~ 24 h . Data information : Graphs represent fold - induction relative to the luciferase activity in the control cells . UI , uninfected . All data are presented as the mean values based on three independent experiments , and error bars indicate S . E . M . ( unpaired two - tailed Student ' s t test ) .

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

ISRE reporter activity (B) in Myc empty vector (200 ng) or Myc-SIRT5 (200 ng)-transfected HEK293T cells with or without SeV infection (SeV or UI) for 18~24 h. Data information: Graphs represent fold-induction relative to the luciferase activity in the control cells. UI, uninfected. All data are presented as the mean values based on three independent experiments, and error bars indicate S.E.M. (unpaired two-tailed Student's t test).


IFN - β promoter activity ( C ) in Myc empty vector ( 200 ng ) or Myc - SIRT5 ( 200 ng ) - transfected H1299 cells with or without SeV infection ( SeV or UI ) for 18 ~ 24 h . Data information : Graphs represent fold - induction relative to the luciferase activity in the control cells . UI , uninfected . All data are presented as the mean values based on three independent experiments , and error bars indicate S . E . M . ( unpaired two - tailed Student ' s t test ) .

IFN-β promoter activity (C) in Myc empty vector (200 ng) or Myc-SIRT5 (200 ng)-transfected H1299 cells with or without SeV infection (SeV or UI) for 18~24 h. Data information: Graphs represent fold-induction relative to the luciferase activity in the control cells. UI, uninfected. All data are presented as the mean values based on three independent experiments, and error bars indicate S.E.M. (unpaired two-tailed Student's t test).


ISRE reporter activity ( D ) in Myc empty vector ( 200 ng ) or Myc - SIRT5 ( 200 ng ) - transfected H1299 cells with or without SeV infection ( SeV or UI ) for 18 ~ 24 h . Data information : Graphs represent fold - induction relative to the luciferase activity in the control cells . UI , uninfected . All data are presented as the mean values based on three independent experiments , and error bars indicate S . E . M . ( unpaired two - tailed Student ' s t test ) .

ISRE reporter activity (D) in Myc empty vector (200 ng) or Myc-SIRT5 (200 ng)-transfected H1299 cells with or without SeV infection (SeV or UI) for 18~24 h. Data information: Graphs represent fold-induction relative to the luciferase activity in the control cells. UI, uninfected. All data are presented as the mean values based on three independent experiments, and error bars indicate S.E.M. (unpaired two-tailed Student's t test).


IFN - β promoter activity ( E ) in the indicated siRNA - transfected H1299 cells ( si - NC , si - SIRT5 # 1 and si - SIRT5 # 2 ) with or without SeV infection ( SeV or UI ) for 18 ~ 24 h . NC , negative control . Data information : Graphs represent fold - induction relative to the luciferase activity in the control cells . UI , uninfected . All data are presented as the mean values based on three independent experiments , and error bars indicate S . E . M . ( unpaired two - tailed Student ' s t test ) .

IFN-β promoter activity (E) in the indicated siRNA-transfected H1299 cells (si-NC, si-SIRT5#1 and si-SIRT5#2) with or without SeV infection (SeV or UI) for 18~24 h. NC, negative control. Data information: Graphs represent fold-induction relative to the luciferase activity in the control cells. UI, uninfected. All data are presented as the mean values based on three independent experiments, and error bars indicate S.E.M. (unpaired two-tailed Student's t test).


ISRE reporter activity ( F ) in the indicated siRNA - transfected H1299 cells ( si - NC , si - SIRT5 # 1 and si - SIRT5 # 2 ) with or without SeV infection ( SeV or UI ) for 18 ~ 24 h . NC , negative control . Data information : Graphs represent fold - induction relative to the luciferase activity in the control cells . UI , uninfected . All data are presented as the mean values based on three independent experiments , and error bars indicate S . E . M . ( unpaired two - tailed Student ' s t test ) .

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

ISRE reporter activity (F) in the indicated siRNA-transfected H1299 cells (si-NC, si-SIRT5#1 and si-SIRT5#2) with or without SeV infection (SeV or UI) for 18~24 h. NC, negative control. Data information: Graphs represent fold-induction relative to the luciferase activity in the control cells. UI, uninfected. All data are presented as the mean values based on three independent experiments, and error bars indicate S.E.M. (unpaired two-tailed Student's t test).


IFN - β promoter activity ( G ) in SIRT5 - deficient H1299 cells ( SIRT5 - / - ) or the wildtype ( WT ) H1299 cells ( SIRT5 + / + ) with or without SeV infection ( SeV or UI ) for 18 ~ 24 h . Data information : Graphs represent fold - induction relative to the luciferase activity in the control cells . UI , uninfected . All data are presented as the mean values based on three independent experiments , and error bars indicate S . E . M . ( unpaired two - tailed Student ' s t test ) .

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

IFN-β promoter activity (G) in SIRT5-deficient H1299 cells (SIRT5 -/-) or the wildtype (WT) H1299 cells (SIRT5 +/+) with or without SeV infection (SeV or UI) for 18~24 h. Data information: Graphs represent fold-induction relative to the luciferase activity in the control cells. UI, uninfected. All data are presented as the mean values based on three independent experiments, and error bars indicate S.E.M. (unpaired two-tailed Student's t test).


ISRE reporter activity ( H ) in SIRT5 - deficient H1299 cells ( SIRT5 - / - ) or the wildtype ( WT ) H1299 cells ( SIRT5 + / + ) with or without SeV infection ( SeV or UI ) for 18 ~ 24 h . Data information : Graphs represent fold - induction relative to the luciferase activity in the control cells . UI , uninfected . All data are presented as the mean values based on three independent experiments , and error bars indicate S . E . M . ( unpaired two - tailed Student ' s t test ) .

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

ISRE reporter activity (H) in SIRT5-deficient H1299 cells (SIRT5 -/-) or the wildtype (WT) H1299 cells (SIRT5 +/+) with or without SeV infection (SeV or UI) for 18~24 h. Data information: Graphs represent fold-induction relative to the luciferase activity in the control cells. UI, uninfected. All data are presented as the mean values based on three independent experiments, and error bars indicate S.E.M. (unpaired two-tailed Student's t test).


I ISRE reporter activity in Myc - SIRT5 ( 0 , 100 ng or 200 ng ) - transfected HEK293T cells with or without poly ( I : C ) transfection for 18 ~ 24 h . Data information : Graphs represent fold - induction relative to the luciferase activity in the control cells . UI , uninfected . All data are presented as the mean values based on three independent experiments , and error bars indicate S . E . M . ( unpaired two - tailed Student ' s t test ) .

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

I ISRE reporter activity in Myc-SIRT5 (0, 100 ng or 200 ng)-transfected HEK293T cells with or without poly(I:C) transfection for 18~24 h. Data information: Graphs represent fold-induction relative to the luciferase activity in the control cells. UI, uninfected. All data are presented as the mean values based on three independent experiments, and error bars indicate S.E.M. (unpaired two-tailed Student's t test).


J ISRE reporter activity in co - transfection of Myc - RIG - I ( 200 ng ) together with Myc - SIRT5 ( 0 , 100 ng or 200ng ) - transfected HEK293T cells for 24 h . Data information : Graphs represent fold - induction relative to the luciferase activity in the control cells . UI , uninfected . All data are presented as the mean values based on three independent experiments , and error bars indicate S . E . M . ( unpaired two - tailed Student ' s t test ) .

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

J ISRE reporter activity in co-transfection of Myc-RIG-I (200 ng) together with Myc-SIRT5 (0, 100 ng or 200ng)-transfected HEK293T cells for 24 h. Data information: Graphs represent fold-induction relative to the luciferase activity in the control cells. UI, uninfected. All data are presented as the mean values based on three independent experiments, and error bars indicate S.E.M. (unpaired two-tailed Student's t test).


K ISRE reporter activity in co - transfection of Myc - MDA5 ( 200 ng ) together with Myc - SIRT5 ( 0 , 100 ng or 200 ng ) in HEK293T cells for 24 h . Data information : Graphs represent fold - induction relative to the luciferase activity in the control cells . UI , uninfected . All data are presented as the mean values based on three independent experiments , and error bars indicate S . E . M . ( unpaired two - tailed Student ' s t test ) .

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K ISRE reporter activity in co-transfection of Myc-MDA5 (200 ng) together with Myc-SIRT5 (0, 100 ng or 200 ng) in HEK293T cells for 24 h. Data information: Graphs represent fold-induction relative to the luciferase activity in the control cells. UI, uninfected. All data are presented as the mean values based on three independent experiments, and error bars indicate S.E.M. (unpaired two-tailed Student's t test).


L ISRE reporter activity in co - transfection of HA - MAVS ( 200 ng ) together with Myc - SIRT5 ( 0 , 100 ng or 200 ng ) in HEK293T cells for 24 h . Data information : Graphs represent fold - induction relative to the luciferase activity in the control cells . UI , uninfected . All data are presented as the mean values based on three independent experiments , and error bars indicate S . E . M . ( unpaired two - tailed Student ' s t test ) .

L ISRE reporter activity in co-transfection of HA-MAVS (200 ng) together with Myc-SIRT5 (0, 100 ng or 200 ng) in HEK293T cells for 24 h. Data information: Graphs represent fold-induction relative to the luciferase activity in the control cells. UI, uninfected. All data are presented as the mean values based on three independent experiments, and error bars indicate S.E.M. (unpaired two-tailed Student's t test).


M ISRE reporter activity in co - transfection of Myc - TBK1 ( 200 ng ) together with Myc - SIRT5 ( 0 , 100 ng or 200 ng ) in HEK293T cells for 24 h . Data information : Graphs represent fold - induction relative to the luciferase activity in the control cells . UI , uninfected . All data are presented as the mean values based on three independent experiments , and error bars indicate S . E . M . ( unpaired two - tailed Student ' s t test ) .

M ISRE reporter activity in co-transfection of Myc-TBK1 (200 ng) together with Myc-SIRT5 (0, 100 ng or 200 ng) in HEK293T cells for 24 h. Data information: Graphs represent fold-induction relative to the luciferase activity in the control cells. UI, uninfected. All data are presented as the mean values based on three independent experiments, and error bars indicate S.E.M. (unpaired two-tailed Student's t test).


N ISRE reporter activity in co - transfection of HA - IRF3 ( 200 ng ) together with Myc - SIRT5 ( 0 , 100 ng or 200 ng ) in HEK293T cells for 24 h . Data information : Graphs represent fold - induction relative to the luciferase activity in the control cells . UI , uninfected . All data are presented as the mean values based on three independent experiments , and error bars indicate S . E . M . ( unpaired two - tailed Student ' s t test ) .

N ISRE reporter activity in co-transfection of HA-IRF3 (200 ng) together with Myc-SIRT5 (0, 100 ng or 200 ng) in HEK293T cells for 24 h. Data information: Graphs represent fold-induction relative to the luciferase activity in the control cells. UI, uninfected. All data are presented as the mean values based on three independent experiments, and error bars indicate S.E.M. (unpaired two-tailed Student's t test).


O ISRE reporter activity in co - transfection of HA - MAVS ( 200 ng ) together with Myc - SIRT5 ( 200ng ) or Myc - SIRT5 - H158Y ( 200ng ) respectively in HEK293T cells for 24 h . Data information : Graphs represent fold - induction relative to the luciferase activity in the control cells . UI , uninfected . All data are presented as the mean values based on three independent experiments , and error bars indicate S . E . M . ( unpaired two - tailed Student ' s t test ) .

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

O ISRE reporter activity in co-transfection of HA-MAVS (200 ng) together with Myc-SIRT5 (200ng) or Myc-SIRT5-H158Y(200ng) respectively in HEK293T cells for 24 h. Data information: Graphs represent fold-induction relative to the luciferase activity in the control cells. UI, uninfected. All data are presented as the mean values based on three independent experiments, and error bars indicate S.E.M. (unpaired two-tailed Student's t test).


A Confocal microscopy image of endogenous SIRT5 co - localized with endogenous MAVS in H1299 cells detected by immunofluorescence staining using anti - SIRT5 and anti - MAVS antibodies . Mito , Mitotracker ; Scale bar = 8 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

A Confocal microscopy image of endogenous SIRT5 co-localized with endogenous MAVS in H1299 cells detected by immunofluorescence staining using anti-SIRT5 and anti-MAVS antibodies. Mito, Mitotracker; Scale bar = 8 µm.


B - C Co - immunoprecipitation of Myc - SIRT5 with HA - MAVS and vice versa . HEK293T cells were co - transfected with indicated plasmids for 24 h . Anti - Myc ( B ) or anti - HA antibody conjugated agarose beads ( C ) were used for immunoprecipitation and the interaction was detected by immunoblotting with the indicated antibodies . Data information : IP , immunoprecipitation ; TCL , total cell lysates .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-C Co-immunoprecipitation of Myc-SIRT5 with HA-MAVS and vice versa. HEK293T cells were co-transfected with indicated plasmids for 24 h. Anti-Myc (B) or anti-HA antibody conjugated agarose beads (C) were used for immunoprecipitation and the interaction was detected by immunoblotting with the indicated antibodies. Data information: IP, immunoprecipitation; TCL, total cell lysates.


D Endogenous interaction between MAVS and SIRT5 . Anti - MAVS antibody was used for immunoprecipitation , and normal mouse IgG was used as a control . Data information : IP , immunoprecipitation ; TCL , total cell lysates .

D Endogenous interaction between MAVS and SIRT5. Anti-MAVS antibody was used for immunoprecipitation, and normal mouse IgG was used as a control. Data information: IP, immunoprecipitation; TCL, total cell lysates.


E Endogenous interaction between MAVS and SIRT5 in the wildtype ( WT ) ( MAVS + / + ) or SIRT5 - deficient ( MAVS - / - ) H1299 cells . Anti - MAVS antibody was used for immunoprecipitation , and the interaction was detected by immunoblotting with anti - SIRT5 antibody . Data information : IP , immunoprecipitation ; TCL , total cell lysates .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Endogenous interaction between MAVS and SIRT5 in the wildtype (WT) (MAVS +/+) or SIRT5-deficient (MAVS -/-) H1299 cells. Anti-MAVS antibody was used for immunoprecipitation, and the interaction was detected by immunoblotting with anti-SIRT5 antibody. Data information: IP, immunoprecipitation; TCL, total cell lysates.


F GST pull - down assay for GST - tagged MAVS and His - tagged SIRT5 . GST - tagged MAVS and His - tagged SIRT5 were expressed in Escherichia coli ( BL21 ) , respectively . The association of GST - MAVS with His - SIRT5 was detected by immunoblotting with anti - SIRT5 antibody . GST and GST - MAVS proteins were stained with Coomassie blue .

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

F GST pull-down assay for GST-tagged MAVS and His-tagged SIRT5. GST-tagged MAVS and His-tagged SIRT5 were expressed in Escherichia coli (BL21), respectively. The association of GST-MAVS with His-SIRT5 was detected by immunoblotting with anti-SIRT5 antibody. GST and GST-MAVS proteins were stained with Coomassie blue.


H - I Co - immunoprecipitation analysis of HA - SIRT5 with Flag - MAVS - truncated mutants . HEK293T cells were co - transfected with the indicated plasmids . Anti - Flag antibody conjugated agarose beads were used for immunoprecipitation and the interaction was analyzed by immunoblotting with the indicated antibodies . Flag - MAVS fragments ( WT : full length ; ΔC1 , 1 - 173 aa ; ΔC2 , 1 - 513 aa ; ΔN1 , 91 - 540 aa ; ΔN2 , 173 - 540 aa ; ΔCARD , Δ10 - 77 aa ; ΔPR , Δ103 - 173 aa ; ΔTM , Δ514 - 535 aa ) . Data information : IP , immunoprecipitation ; TCL , total cell lysates .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-I Co-immunoprecipitation analysis of HA-SIRT5 with Flag-MAVS-truncated mutants. HEK293T cells were co-transfected with the indicated plasmids. Anti-Flag antibody conjugated agarose beads were used for immunoprecipitation and the interaction was analyzed by immunoblotting with the indicated antibodies. Flag-MAVS fragments (WT: full length; ΔC1, 1-173 aa; ΔC2, 1-513 aa; ΔN1, 91-540 aa; ΔN2, 173-540 aa; ΔCARD, Δ10-77 aa; ΔPR, Δ103-173 aa; ΔTM, Δ514-535 aa). Data information: IP, immunoprecipitation; TCL, total cell lysates.


A - B MAVS could be succinylated in vitro . GST - MAVS ( A ) extracted from E . coli or Flag - tagged MAVS ( B ) protein purified from HEK293T cells was incubated with the indicated concentrations of succinyl - CoA . Protein succinylation was detected with anti - pan - succinyl - lysine antibody ; GST - MAVS or Flag - MAVS was stained by Coomassie blue .

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

A-B MAVS could be succinylated in vitro. GST-MAVS (A) extracted from E.coli or Flag-tagged MAVS (B) protein purified from HEK293T cells was incubated with the indicated concentrations of succinyl-CoA. Protein succinylation was detected with anti-pan-succinyl-lysine antibody; GST-MAVS or Flag-MAVS was stained by Coomassie blue.


C The succinylated residue in MAVS was identified by mass spectrometry analysis .

C The succinylated residue in MAVS was identified by mass spectrometry analysis.


E Dot - blot assay for the specificity of anti - succ - K7 - MAVS antibody . Equal amounts of succinyl - peptides or the control peptides were immunoblotted with the indicated dilutions of anti - succ - K7 - MAVS antibody .

O O O O O O O O O O O O O O O O O O O O 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 Dot-blot assay for the specificity of anti-succ-K7-MAVS antibody. Equal amounts of succinyl-peptides or the control peptides were immunoblotted with the indicated dilutions of anti-succ-K7-MAVS antibody.


GST - MAVS ( F ) extracted from E . coli cells was incubated with the indicated concentrations of succinyl - CoA . The succinylation of MAVS at lysine 7 was detected by anti - succ - K7 - MAVS antibody .

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

GST-MAVS (F) extracted from E.coli cells was incubated with the indicated concentrations of succinyl-CoA. The succinylation of MAVS at lysine 7 was detected by anti-succ-K7-MAVS antibody.


Flag - MAVS ( G ) purified from HEK293T cells was incubated with the indicated concentrations of succinyl - CoA . The succinylation of MAVS at lysine 7 was detected by anti - succ - K7 - MAVS antibody .

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

Flag-MAVS (G) purified from HEK293T cells was incubated with the indicated concentrations of succinyl-CoA. The succinylation of MAVS at lysine 7 was detected by anti-succ-K7-MAVS antibody.


H Disruption of SIRT5 in H1299 cells enhanced succinylation of MAVS at Lys 7 compared to that in the SIRT5 - intact H1299 cells ( SIRT5 + / + ) ( 3 . 2 vs . 1 . 0 ) . The cell lysates from SIRT5 - deficient H1299 cells or the SIRT5 - intact H1299 cells were immunoprecipitated with anti - MAVS antibody or mouse IgG control , followed by immunoblotting with anti - succ - K7 - MAVS antibody . Data information : IP , immunoprecipitation ; TCL , total cell lysates .

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

H Disruption of SIRT5 in H1299 cells enhanced succinylation of MAVS at Lys 7 compared to that in the SIRT5-intact H1299 cells (SIRT5 +/+) (3.2 vs. 1.0). The cell lysates from SIRT5-deficient H1299 cells or the SIRT5-intact H1299 cells were immunoprecipitated with anti-MAVS antibody or mouse IgG control, followed by immunoblotting with anti-succ-K7-MAVS antibody. Data information: IP, immunoprecipitation; TCL, total cell lysates.


I Reconstitution of WT SIRT5 in SIRT5 - deficient H1299 cells ( SIRT5 - / - ) caused a significant reduction in succinylation of MAVS at Lys 7 ( 1 . 0 vs . 0 . 2 ) ; but overexpression of SIRT5 - H158Y in SIRT5 - / - H1299 cells has no effect on the reduction in succinylation of MAVS at Lys 7 ( 1 . 0 vs . 1 . 2 ) . The SIRT5 - / - H1299 cells were transfected with Flag - SIRT5 or Flag - SIRT5 - H158Y , followed by immunoprecipitating with anti - MAVS antibody or mouse IgG control , and immunoblotting with anti - succ - K7 - MAVS antibody . Data information : IP , immunoprecipitation ; TCL , total cell lysates .

O O 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 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

I Reconstitution of WT SIRT5 in SIRT5-deficient H1299 cells (SIRT5 -/-) caused a significant reduction in succinylation of MAVS at Lys 7 (1.0 vs. 0.2); but overexpression of SIRT5-H158Y in SIRT5 -/- H1299 cells has no effect on the reduction in succinylation of MAVS at Lys 7 (1.0 vs. 1.2). The SIRT5 -/- H1299 cells were transfected with Flag-SIRT5 or Flag-SIRT5-H158Y, followed by immunoprecipitating with anti-MAVS antibody or mouse IgG control, and immunoblotting with anti-succ-K7-MAVS antibody. Data information: IP, immunoprecipitation; TCL, total cell lysates.


J - K Knockout of Sirt5 increased Mavs succinylation in mouse livers ( J ) and lungs ( K ) . Proteins extracted from livers ( J ) and lungs ( K ) of Sirt5 KO and the wildtype littermates ( n = 3 per group ) were detected by the indicated antibodies . MAVS succinylation was determined by anti - succ - K7 - MAVS antibody , and anti - pan - succinyl - lysine antibody was used as positive controls .

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

J-K Knockout of Sirt5 increased Mavs succinylation in mouse livers (J) and lungs (K). Proteins extracted from livers (J) and lungs (K) of Sirt5 KO and the wildtype littermates (n = 3 per group) were detected by the indicated antibodies. MAVS succinylation was determined by anti-succ-K7-MAVS antibody, and anti-pan-succinyl-lysine antibody was used as positive controls.


A - C Quantitative real time PCR analysis ( qPCR ) of IFNβ ( A ) , CXCL10 ( B ) and IFIT1 ( C ) mRNA in HEK293T cells transfected with the control plasmid ( Myc empty ) or the plasmid expressing Myc - SIRT5 ( Myc - SIRT5 ) for 24 h , followed by infection with or without SeV ( SeV or UI ) for 8h . Data information : UI , un - infected . The graphs represent fold - induction relative to the untreated cells . All data are presented as the mean values based on three independent experiments , and error bars indicate S . E . M . ( unpaired two - tailed Student ' s t test ) .

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

A-C Quantitative real time PCR analysis (qPCR) of IFNβ (A), CXCL10 (B) and IFIT1 (C) mRNA in HEK293T cells transfected with the control plasmid (Myc empty) or the plasmid expressing Myc-SIRT5 (Myc-SIRT5) for 24 h, followed by infection with or without SeV (SeV or UI) for 8h. Data information: UI, un-infected. The graphs represent fold-induction relative to the untreated cells. All data are presented as the mean values based on three independent experiments, and error bars indicate S.E.M. (unpaired two-tailed Student's t test).


D - F qPCR analysis of IFNβ ( D ) , CXCL10 ( E ) , and IFIT1 ( F ) mRNA in SIRT5 - deficient or WT H1299 cells ( SIRT5 - / - or SIRT5 + / + ) infected with or without SeV ( SeV or UI ) for 8 h . Data information : UI , un - infected . The graphs represent fold - induction relative to the untreated cells . All data are presented as the mean values based on three independent experiments , and error bars indicate S . E . M . ( unpaired two - tailed Student ' s t test ) .

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

D-F qPCR analysis of IFNβ (D), CXCL10 (E), and IFIT1 (F) mRNA in SIRT5-deficient or WT H1299 cells (SIRT5 -/- or SIRT5 +/+) infected with or without SeV (SeV or UI) for 8 h. Data information: UI, un-infected. The graphs represent fold-induction relative to the untreated cells. All data are presented as the mean values based on three independent experiments, and error bars indicate S.E.M. (unpaired two-tailed Student's t test).


G - I qPCR analysis of IFNβ ( G ) , CXCL10 ( H ) and IFIT1 ( I ) mRNA in SIRT5 - deficient or WT H1299 cells ( SIRT5 - / - or SIRT5 + / + ) infected with or without VSV ( VSV or UI ) for 8h . Data information : UI , un - infected . The graphs represent fold - induction relative to the untreated cells . All data are presented as the mean values based on three independent experiments , and error bars indicate S . E . M . ( unpaired two - tailed Student ' s t test ) .

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

G-I qPCR analysis of IFNβ (G), CXCL10 (H) and IFIT1 (I) mRNA in SIRT5-deficient or WT H1299 cells (SIRT5 -/- or SIRT5 +/+) infected with or without VSV (VSV or UI) for 8h. Data information: UI, un-infected. The graphs represent fold-induction relative to the untreated cells. All data are presented as the mean values based on three independent experiments, and error bars indicate S.E.M. (unpaired two-tailed Student's t test).


J qPCR analysis of IFNβ mRNA in HEK293T cells transfected with the control plasmid ( Myc empty ) , or the plasmid expressing Myc - SIRT5 ( Myc - SIRT5 ) , or its enzyme - deficient mutant H158Y ( Myc - SIRT5 - H158Y ) for 24 h , followed by stimulation with or without poly ( I : C ) for 8 h . Data information : All data are presented as the mean values based on three independent experiments , and error bars indicate S . E . M . ( unpaired two - tailed Student ' s t test ) .

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

J qPCR analysis of IFNβ mRNA in HEK293T cells transfected with the control plasmid (Myc empty), or the plasmid expressing Myc-SIRT5 (Myc-SIRT5), or its enzyme-deficient mutant H158Y (Myc-SIRT5-H158Y) for 24 h, followed by stimulation with or without poly(I:C) for 8 h. Data information: All data are presented as the mean values based on three independent experiments, and error bars indicate S.E.M. (unpaired two-tailed Student's t test).


K qPCR analysis of IFNβ mRNA in SIRT5 - deficient H1299 cells ( SIRT5 - / - ) transfected with the control plasmid ( Myc empty ) or the plasmid expressing Myc - SIRT5 ( Myc - SIRT5 ) or its enzyme - deficient mutant H158Y ( Myc - SIRT5 - H158Y ) for 24 h , followed by infection with ( + ) or without ( - ) SeV for 8 h . Data information : All data are presented as the mean values based on three independent experiments , and error bars indicate S . E . M . ( unpaired two - tailed Student ' s t test ) .

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

K qPCR analysis of IFNβ mRNA in SIRT5-deficient H1299 cells (SIRT5-/-) transfected with the control plasmid (Myc empty) or the plasmid expressing Myc-SIRT5 (Myc-SIRT5) or its enzyme-deficient mutant H158Y (Myc-SIRT5-H158Y) for 24 h, followed by infection with (+) or without (-) SeV for 8 h. Data information: All data are presented as the mean values based on three independent experiments, and error bars indicate S.E.M. (unpaired two-tailed Student's t test).


L SDD - AGE analysis of MAVS aggregates in HEK293T cells transfected HA - MAVS together with WT Myc - SIRT5 or its enzyme - deficient mutant ( Myc - SIRT5 - H158Y ) after infected with SeV for 12h . SDS - PAGE immunoblotting was used as a loading control .

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

L SDD-AGE analysis of MAVS aggregates in HEK293T cells transfected HA-MAVS together with WT Myc-SIRT5 or its enzyme-deficient mutant (Myc-SIRT5-H158Y) after infected with SeV for 12h. SDS-PAGE immunoblotting was used as a loading control.


M - N IFN - β promoter activity ( M ) and ISRE reporter activity ( N ) in HEK293T cells co - transfected Myc - SIRT5 together with HA - MAVS or HA - MAVS - K7R . Data information All data are presented as the mean values based on three independent experiments , and error bars indicate S . E . M . ( unpaired two - tailed Student ' s t test ) .

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

M-N IFN-β promoter activity (M) and ISRE reporter activity (N) in HEK293T cells co-transfected Myc-SIRT5 together with HA-MAVS or HA-MAVS-K7R. Data information All data are presented as the mean values based on three independent experiments, and error bars indicate S.E.M. (unpaired two-tailed Student's t test).


SIRT5 - deficient or WT H1299 cells ( SIRT5 - / - or SIRT5 + / + ) were infected with VSV - GFP viruses ( MOI = 0 . 1 ) for 12 h , and viral infectivity was detected by fluorescence microscopy ( O )

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

SIRT5-deficient or WT H1299 cells (SIRT5 -/- or SIRT5 +/+) were infected with VSV-GFP viruses (MOI = 0.1) for 12 h, and viral infectivity was detected by fluorescence microscopy (O)


SIRT5 - deficient or WT H1299 cells ( SIRT5 - / - or SIRT5 + / + ) were infected with VSV - GFP viruses ( MOI = 0 . 1 ) for 12 h and viral infectivity western blot analysis ( P ) Data information : ll data are presented as the mean values based on three independent experiments , and error bars indicate S . E . M . ( unpaired two - tailed Student ' s t test ) .

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

SIRT5-deficient or WT H1299 cells (SIRT5 -/- or SIRT5 +/+) were infected with VSV-GFP viruses (MOI = 0.1) for 12 h and viral infectivity western blot analysis (P) Data information: ll data are presented as the mean values based on three independent experiments, and error bars indicate S.E.M. (unpaired two-tailed Student's t test).


SIRT5 - deficient or WT H1299 cells ( SIRT5 - / - or SIRT5 + / + ) were infected with VSV - GFP viruses ( MOI = 0 . 1 ) for 12 h , and viral infectivity was detected by flow cytometry analysis ( n = 3 ) ( Q ) .

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

SIRT5-deficient or WT H1299 cells (SIRT5 -/- or SIRT5 +/+) were infected with VSV-GFP viruses (MOI = 0.1) for 12 h, and viral infectivity was detected by flow cytometry analysis (n = 3) (Q).


A - F qPCR analysis of Ifnβ ( A ) , Ifnα1 ( B ) , Ifnα4 ( C ) , Ifit1 ( D ) , Cxcl10 ( E ) , and Cxcl11 ( F ) mRNA in WT or Sirt5 - deficient MEF cells ( Sirt5 + / + or Sirt5 - / - ) infected with or without SeV ( SeV or UI ) for 8h . Data information : UI , un - infected . The graphs represent fold - induction relative to the untreated cells . All data are presented as the mean values based on three independent experiments , and error bars indicate S . E . M . ( unpaired two - tailed Student ' s t test ) .

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

A-F qPCR analysis of Ifnβ (A), Ifnα1 (B), Ifnα4 (C), Ifit1 (D), Cxcl10 (E), and Cxcl11 (F) mRNA in WT or Sirt5-deficient MEF cells (Sirt5 +/+ or Sirt5 -/-) infected with or without SeV (SeV or UI) for 8h. Data information: UI, un-infected. The graphs represent fold-induction relative to the untreated cells. All data are presented as the mean values based on three independent experiments, and error bars indicate S.E.M. (unpaired two-tailed Student's t test).


G WT or Sirt5 - deficient MEF cells ( Sirt5 + / + or Sirt5 - / - ) were infected with or without SeV for the indicated times , and the cell lysates were analyzed by immunoblotting for monomeric ( Monomer ) and dimeric ( Dimer ) Irf3 ( top ; native - PAGE ) , phosphorylated Irf3 ( p - Irf3 ) , total Irf3 , Sirt5 , and GAPDH ( bottom ) .

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

G WT or Sirt5-deficient MEF cells (Sirt5 +/+ or Sirt5 -/-) were infected with or without SeV for the indicated times, and the cell lysates were analyzed by immunoblotting for monomeric (Monomer) and dimeric (Dimer) Irf3 (top; native-PAGE), phosphorylated Irf3 (p-Irf3), total Irf3, Sirt5, and GAPDH (bottom).


H WT or Sirt5 - deficient MEF cells ( Sirt5 + / + or Sirt5 - / - ) were infected with or without SeV for 8 h , the interaction between endogenous Mavs and Rig - I was revealed by immunoprecipitation using anti - Mavs antibody , followed by immunoblotting with anti - Rig - I antibody .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 WT or Sirt5-deficient MEF cells (Sirt5 +/+ or Sirt5 -/-) were infected with or without SeV for 8 h, the interaction between endogenous Mavs and Rig-I was revealed by immunoprecipitation using anti-Mavs antibody, followed by immunoblotting with anti-Rig-I antibody.


I SDD - AGE analysis of Mavs aggregates in WT or Sirt5 - deficient MEF cells ( Sirt5 + / + or Sirt5 - / - ) infected with SeV for 12 h . SDS - PAGE immunoblotting was used as a loading control .

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

I SDD-AGE analysis of Mavs aggregates in WT or Sirt5-deficient MEF cells (Sirt5 +/+ or Sirt5 -/-) infected with SeV for 12 h. SDS-PAGE immunoblotting was used as a loading control.


J - O qPCR analysis of Ifnβ ( J ) , Ifnα1 ( K ) , Ifnα4 ( L ) , Ifit1 ( M ) , Cxcl10 ( N ) , and Cxcl11 ( O ) mRNA in WT or Sirt5 - deficient MEF cells ( Sirt5 + / + or Sirt5 - / - ) infected with or without VSV ( VSV or UI ) for 8 h . Data information : UI , un - infected . The graphs represent fold - induction relative to the untreated cells . All data are presented as the mean values based on three independent experiments , and error bars indicate S . E . M . ( unpaired two - tailed Student ' s t test ) .

J-O qPCR analysis of Ifnβ (J), Ifnα1 (K), Ifnα4 (L), Ifit1 (M), Cxcl10 (N), and Cxcl11 (O) mRNA in WT or Sirt5-deficient MEF cells (Sirt5 +/+ or Sirt5 -/-) infected with or without VSV (VSV or UI) for 8 h. Data information: UI, un-infected. The graphs represent fold-induction relative to the untreated cells. All data are presented as the mean values based on three independent experiments, and error bars indicate S.E.M. (unpaired two-tailed Student's t test).


WT or Sirt5 - deficient MEF cells ( Sirt5 + / + or Sirt5 - / - ) were infected with or without VSV - GFP viruses for 12 h , and viral infectivity was detected by fluorescence microscopy ( P )

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

WT or Sirt5-deficient MEF cells (Sirt5 +/+ or Sirt5 -/-) were infected with or without VSV-GFP viruses for 12 h, and viral infectivity was detected by fluorescence microscopy (P)


WT or Sirt5 - deficient MEF cells ( Sirt5 + / + or Sirt5 - / - ) were infected with or without VSV - GFP viruses for 12 h , and viral infectivity was detected by western blot analysis ( Q )

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

WT or Sirt5-deficient MEF cells (Sirt5 +/+ or Sirt5 -/-) were infected with or without VSV-GFP viruses for 12 h, and viral infectivity was detected by western blot analysis (Q)


WT or Sirt5 - deficient MEF cells ( Sirt5 + / + or Sirt5 - / - ) were infected with or without VSV - GFP viruses for 12 h , and viral infectivity was detected by flow cytometry analysis ( n = 3 ) ( R ) . Data information : All data are presented as the mean values based on three independent experiments , and error bars indicate S . E . M . ( unpaired two - tailed Student ' s t test ) .

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

WT or Sirt5-deficient MEF cells (Sirt5 +/+ or Sirt5 -/-) were infected with or without VSV-GFP viruses for 12 h, and viral infectivity was detected by flow cytometry analysis (n = 3) (R). Data information: All data are presented as the mean values based on three independent experiments, and error bars indicate S.E.M. (unpaired two-tailed Student's t test).


qPCR analysis of Ifnβ ( A ) , Ifnα1 ( B ) , Ifnα4 ( C ) , Ifit1 ( D ) , Rig - I ( E ) , Cxcl10 ( F ) mRNA in WT or Sirt5 - deficient BMDC cells ( Sirt5 + / + or Sirt5 - / - ) infected with or without SeV ( Sev or UI ) for 8 h . Data information : UI , un - infected . The graphs represent fold - induction relative to the untreated cells . All data are presented as the mean values based on three independent experiments , and error bars indicate S . E . M . ( unpaired two - tailed Student ' s t test ) .

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

qPCR analysis of Ifnβ (A), Ifnα1 (B), Ifnα4 (C), Ifit1 (D), Rig-I (E), Cxcl10 (F) mRNA in WT or Sirt5-deficient BMDC cells (Sirt5 +/+ or Sirt5 -/-) infected with or without SeV (Sev or UI) for 8 h. Data information: UI, un-infected. The graphs represent fold-induction relative to the untreated cells. All data are presented as the mean values based on three independent experiments, and error bars indicate S.E.M. (unpaired two-tailed Student's t test).


qPCR analysis of Cxcl11 ( G ) mRNA in WT or Sirt5 - deficient BMDC cells ( Sirt5 + / + or Sirt5 - / - ) infected with or without SeV ( Sev or UI ) for 8 h . Data information : UI , un - infected . The graphs represent fold - induction relative to the untreated cells . All data are presented as the mean values based on three independent experiments , and error bars indicate S . E . M . ( unpaired two - tailed Student ' s t test ) .

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

qPCR analysis of Cxcl11 (G) mRNA in WT or Sirt5-deficient BMDC cells (Sirt5 +/+ or Sirt5 -/-) infected with or without SeV (Sev or UI) for 8 h. Data information: UI, un-infected. The graphs represent fold-induction relative to the untreated cells. All data are presented as the mean values based on three independent experiments, and error bars indicate S.E.M. (unpaired two-tailed Student's t test).


qPCR analysis of Ifnβ ( H ) , Ifnα1 ( I ) mRNA in WT or Sirt5 - deficient BMDC cells ( Sirt5 + / + or Sirt5 - / - ) infected with or without VSV viruses ( VSV or UI ) for 8 h . Data information : UI , un - infected . The graphs represent fold - induction relative to the untreated cells . All data are presented as the mean values based on three independent experiments , and error bars indicate S . E . M . ( unpaired two - tailed Student ' s t test ) .

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

qPCR analysis of Ifnβ (H), Ifnα1 (I) mRNA in WT or Sirt5-deficient BMDC cells (Sirt5 +/+ or Sirt5 -/-) infected with or without VSV viruses (VSV or UI) for 8 h. Data information: UI, un-infected. The graphs represent fold-induction relative to the untreated cells. All data are presented as the mean values based on three independent experiments, and error bars indicate S.E.M. (unpaired two-tailed Student's t test).


qPCR analysis of Ifnα4 ( J ) , Ifit1 ( K ) , Rig - I ( L ) mRNA in WT or Sirt5 - deficient BMDC cells ( Sirt5 + / + or Sirt5 - / - ) infected with or without VSV viruses ( VSV or UI ) for 8 h . Data information : UI , un - infected . The graphs represent fold - induction relative to the untreated cells . All data are presented as the mean values based on three independent experiments , and error bars indicate S . E . M . ( unpaired two - tailed Student ' s t test ) .

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

qPCR analysis of Ifnα4 (J), Ifit1 (K), Rig-I (L) mRNA in WT or Sirt5-deficient BMDC cells (Sirt5 +/+ or Sirt5 -/-) infected with or without VSV viruses (VSV or UI) for 8 h. Data information: UI, un-infected. The graphs represent fold-induction relative to the untreated cells. All data are presented as the mean values based on three independent experiments, and error bars indicate S.E.M. (unpaired two-tailed Student's t test).


qPCR analysis of Cxcl10 ( M ) and Cxcl11 ( N ) mRNA in WT or Sirt5 - deficient BMDC cells ( Sirt5 + / + or Sirt5 - / - ) infected with or without VSV viruses ( VSV or UI ) for 8 h . Data information : UI , un - infected . The graphs represent fold - induction relative to the untreated cells . All data are presented as the mean values based on three independent experiments , and error bars indicate S . E . M . ( unpaired two - tailed Student ' s t test ) .

qPCR analysis of Cxcl10 (M) and Cxcl11 (N) mRNA in WT or Sirt5-deficient BMDC cells (Sirt5 +/+ or Sirt5 -/-) infected with or without VSV viruses (VSV or UI) for 8 h. Data information: UI, un-infected. The graphs represent fold-induction relative to the untreated cells. All data are presented as the mean values based on three independent experiments, and error bars indicate S.E.M. (unpaired two-tailed Student's t test).


O Confocal fluorescence microscopy image of replication in WT or Sirt5 - deficient BMDC cells ( Sirt5 + / + or Sirt5 - / - ) after infected with VSV - GFP viruses for 12 h ( MOI = 1 ) ( fluorescence , upper ; bright - field , bottom ) . Scale bar = 75 µm .

O Confocal fluorescence microscopy image of replication in WT or Sirt5-deficient BMDC cells (Sirt5 +/+ or Sirt5 -/-) after infected with VSV-GFP viruses for 12 h (MOI = 1) (fluorescence, upper; bright-field, bottom). Scale bar = 75 µm.


A - B VSV infection increased Mavs succinylation in mouse livers ( A ) and lungs ( B ) . Proteins in the livers ( A ) and lungs ( B ) of WT littermates injected intraperitoneally with ( + ) VSV ( 1 × 107 plaque - forming units ( PFU ) per mouse ) or PBS control ( - ) ( n = 3 per group ) were detected with the indicated antibodies . Mavs succinylation was determined by anti - succ - K7 - MAVS antibody . Relative succinylation level was quantified ( right panel ) .

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

A-B VSV infection increased Mavs succinylation in mouse livers (A) and lungs (B). Proteins in the livers (A) and lungs (B) of WT littermates injected intraperitoneally with (+) VSV (1 × 107 plaque-forming units (PFU) per mouse) or PBS control (-) (n = 3 per group) were detected with the indicated antibodies. Mavs succinylation was determined by anti-succ-K7-MAVS antibody. Relative succinylation level was quantified (right panel).


C - D Knockout of Sirt5 increased Mavs succinylation in mouse livers ( C ) and lungs ( D ) upon VSV infection . Proteins in the livers ( C ) and lungs ( D ) of Sirt5 - deficient or WT littermates ( Sirt5 - / - or Sirt5 + / + ) injected intraperitoneally with VSV ( 1 × 107 plaque - forming units ( PFU ) per mouse ) ) ( n = 3 per group ) for 24 h were detected with the indicated antibodies . Mavs succinylation was determined by anti - succ - K7 - MAVS antibody . Relative succinylation level was quantified ( right panel ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-D Knockout of Sirt5 increased Mavs succinylation in mouse livers (C) and lungs (D) upon VSV infection. Proteins in the livers (C) and lungs (D) of Sirt5-deficient or WT littermates (Sirt5 -/- or Sirt5 +/+) injected intraperitoneally with VSV (1 × 107 plaque-forming units (PFU) per mouse)) (n = 3 per group) for 24 h were detected with the indicated antibodies. Mavs succinylation was determined by anti-succ-K7-MAVS antibody. Relative succinylation level was quantified (right panel).


E Survival ( Kaplan - Meier curve ) of WT mice ( n = 7 ) and Sirt5 - deficient mice ( n = 7 ) injected intraperitoneally with a high dose of VSV ( 5 × 107 PFU per mouse ) and monitored for 10 d .

O O O O O O O O O O O O O O O O O O O O O O O O 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 Survival (Kaplan-Meier curve) of WT mice (n = 7) and Sirt5-deficient mice (n = 7) injected intraperitoneally with a high dose of VSV (5 × 107 PFU per mouse) and monitored for 10 d.


F ELISA assay of Ifnβ in serum from the WT mice ( n = 4 ) and Sirt5 - deficient mice ( n = 4 ) injected intraperitoneally with VSV ( 1 × 107 plaque - forming units ( PFU ) per mouse ) ) or PBS control for 24 h . Data information : The graphs represent fold - induction relative to the untreated WT mice . PBS , phosphate buffer saline . All data are presented as the mean values based on three independent experiments , and error bars indicate S . E . M . ( unpaired two - tailed Student ' s t test or two - way ANOVA analysis ) .

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

F ELISA assay of Ifnβ in serum from the WT mice (n = 4) and Sirt5-deficient mice (n = 4) injected intraperitoneally with VSV (1 × 107 plaque-forming units (PFU) per mouse)) or PBS control for 24 h. Data information: The graphs represent fold-induction relative to the untreated WT mice. PBS, phosphate buffer saline. All data are presented as the mean values based on three independent experiments, and error bars indicate S.E.M. (unpaired two-tailed Student's t test or two-way ANOVA analysis).


G Hematoxylin - and - eosin - stained images of lung sections from mice in F . Scale bar = 50 µm .

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

G Hematoxylin-and-eosin-stained images of lung sections from mice in F. Scale bar = 50 µm.


qPCR analysis of Ifnβ ( H ) , Ifnα1 ( I ) , Ifit1 ( J ) , Isg15 ( K ) , Cxcl10 ( L ) , Cxcl11 ( M ) mRNA in the lungs of WT or Sirt5 - deficient mice ( Sirt5 + / + or Sirt5 - / - ) injected intraperitoneally with VSV ( 1 × 107 plaque - forming units ( PFU ) per mouse ) or PBS control for 24 h . Data information : The graphs represent fold - induction relative to the untreated WT mice . PBS , phosphate buffer saline . All data are presented as the mean values based on three independent experiments , and error bars indicate S . E . M . ( unpaired two - tailed Student ' s t test or two - way ANOVA analysis ) .

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

qPCR analysis of Ifnβ (H), Ifnα1 (I), Ifit1 (J), Isg15 (K), Cxcl10 (L), Cxcl11 (M) mRNA in the lungs of WT or Sirt5-deficient mice (Sirt5 +/+ or Sirt5 -/-) injected intraperitoneally with VSV (1 × 107 plaque-forming units (PFU) per mouse) or PBS control for 24 h. Data information: The graphs represent fold-induction relative to the untreated WT mice. PBS, phosphate buffer saline. All data are presented as the mean values based on three independent experiments, and error bars indicate S.E.M. (unpaired two-tailed Student's t test or two-way ANOVA analysis).


qPCR analysis of Rig - I ( N ) , and Irf7 ( O ) mRNA in the lungs of WT or Sirt5 - deficient mice ( Sirt5 + / + or Sirt5 - / - ) injected intraperitoneally with VSV ( 1 × 107 plaque - forming units ( PFU ) per mouse ) or PBS control for 24 h . Data information : The graphs represent fold - induction relative to the untreated WT mice . PBS , phosphate buffer saline . All data are presented as the mean values based on three independent experiments , and error bars indicate S . E . M . ( unpaired two - tailed Student ' s t test or two - way ANOVA analysis ) .

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

qPCR analysis of Rig-I (N), and Irf7 (O) mRNA in the lungs of WT or Sirt5-deficient mice (Sirt5 +/+ or Sirt5 -/-) injected intraperitoneally with VSV (1 × 107 plaque-forming units (PFU) per mouse) or PBS control for 24 h. Data information: The graphs represent fold-induction relative to the untreated WT mice. PBS, phosphate buffer saline. All data are presented as the mean values based on three independent experiments, and error bars indicate S.E.M. (unpaired two-tailed Student's t test or two-way ANOVA analysis).


P qPCR analysis of VSV mRNA in the lungs of WT or Sirt5 - deficient mice ( Sirt5 + / + or Sirt5 - / - ) injected intraperitoneally with VSV ( 1 × 107 plaque - forming units ( PFU ) per mouse ) or PBS control for 24 h . Data information : The graphs represent fold - induction relative to the untreated WT mice . PBS , phosphate buffer saline . All data are presented as the mean values based on three independent experiments , and error bars indicate S . E . M . ( unpaired two - tailed Student ' s t test or two - way ANOVA analysis ) .

P qPCR analysis of VSV mRNA in the lungs of WT or Sirt5-deficient mice (Sirt5 +/+ or Sirt5 -/-) injected intraperitoneally with VSV (1 × 107 plaque-forming units (PFU) per mouse) or PBS control for 24 h. Data information: The graphs represent fold-induction relative to the untreated WT mice. PBS, phosphate buffer saline. All data are presented as the mean values based on three independent experiments, and error bars indicate S.E.M. (unpaired two-tailed Student's t test or two-way ANOVA analysis).


IIA1 . 6 B lymphoma cells were stimulated with BCR - ligand - ( IgM ) or BCR - ligand + ( IgG ) beads for 60 min , fixed and stained for F - actin ( top ) and α - tubulin ( bottom ) . Scale bar : 3 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

IIA1.6 B lymphoma cells were stimulated with BCR-ligand- (IgM) or BCR-ligand+ (IgG) beads for 60 min, fixed and stained for F-actin (top) and α-tubulin (bottom). Scale bar: 3 µm.


Histograms show the quantifications of the polymerized tubulin and filamentous F - actin at the centrosome ( dashed outline on the image , values correspond to the fraction of fluorescence in a 2 - micron - wide area around the centrosome relative to the total fluorescence in the cell ) and the total amount of polymerized fluorescent tubulin ( bottom right , values were normalized with respect to the mean of control condition ) . Measurements were pooled from 3 independent experiments ; IgM ( BCR - ligand - ) : n = 88 ; IgG ( BCR - ligand + ) : n = 93 . Error bars correspond to standard deviations . P values were calculated with Mann - Whitney test . Scale bar : 2 µm .

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

Histograms show the quantifications of the polymerized tubulin and filamentous F-actin at the centrosome (dashed outline on the image, values correspond to the fraction of fluorescence in a 2-micron-wide area around the centrosome relative to the total fluorescence in the cell) and the total amount of polymerized fluorescent tubulin (bottom right, values were normalized with respect to the mean of control condition). Measurements were pooled from 3 independent experiments; IgM (BCR-ligand-): n= 88; IgG (BCR-ligand+): n= 93. Error bars correspond to standard deviations. P values were calculated with Mann-Whitney test. Scale bar: 2 µm.


Percentage differences of centrosomal F - actin and centrosomal microtubule fluorescence intensities in cells stimulated with BCR - ligand + beads with respect to cells stimulated with BCR - ligand - beads . The data set is identical to panel ( B ) . Measurements were pooled from 3 independent experiments ; IgM ( BCR - ligand - ) : n = 88 ; IgG ( BCR - ligand + ) : n = 93 . Error bars correspond to standard deviations . P values were calculated with one - sample t - test ( i . e . comparison to a theoretical mean of " 0 " ) .

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

Percentage differences of centrosomal F-actin and centrosomal microtubule fluorescence intensities in cells stimulated with BCR-ligand+ beads with respect to cells stimulated with BCR-ligand- beads. The data set is identical to panel (B). Measurements were pooled from 3 independent experiments; IgM (BCR-ligand-): n= 88; IgG (BCR-ligand+): n= 93. Error bars correspond to standard deviations. P values were calculated with one-sample t-test (i.e. comparison to a theoretical mean of "0").


The graph shows the variations of the total amount of tubulin per cell with respect to the content of actin at the centrosome in an XY representation of individual measurements . The two lines correspond to linear regressions of the two sets of data relative to cells stimulated with BCR - ligand + ( activated cells ) or BCR - ligand - ( resting cells ) beads .

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

The graph shows the variations of the total amount of tubulin per cell with respect to the content of actin at the centrosome in an XY representation of individual measurements. The two lines correspond to linear regressions of the two sets of data relative to cells stimulated with BCR-ligand+ (activated cells) or BCR-ligand- (resting cells) beads.


IIA1 . 6 B lymphoma cells were fixed and immuno - stained for F - actin ( red ) and microtubules ( green ) . Images show the projection of maximal intensity of three confocal slices spaced by 0 . 5 µm apart from the centrosome . Scale bars : 2 µm ( 0 . 5 µm in the zoomed insets ) .

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

IIA1.6 B lymphoma cells were fixed and immuno-stained for F-actin (red) and microtubules (green). Images show the projection of maximal intensity of three confocal slices spaced by 0.5 µm apart from the centrosome. Scale bars: 2 µm (0.5 µm in the zoomed insets).


IIA1 . 6 B lymphoma cells were treated 45 min with indicated inhibitors ( CK666 at 25 µM , SMIFH2 at 25µM ) or DMSO as control prior to being fixed and stained for α - tubulin ( left column ) and F - actin ( right column ) . Scale bar : 3 µm .

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

IIA1.6 B lymphoma cells were treated 45 min with indicated inhibitors (CK666 at 25 µM, SMIFH2 at 25µM) or DMSO as control prior to being fixed and stained for α-tubulin (left column) and F-actin (right column). Scale bar: 3 µm.


Histograms show the quantifications of the amount of polymerized fluorescent tubulin ( right , values were normalized with respect to the mean of control condition ) and filamentous actin at the centrosome ( left , values correspond to the fraction of fluorescence in a 2 - micron - wide area around the centrosome relative to the total fluorescence in the cell ) . Measurements were pooled from 3 independent experiments ; DMSO : n = 91 , CK666 : n = 82 , SMIFH2 : n = 74 , latrunculinA : n = 96 . Error bars correspond to standard deviations . P values were calculated with Mann - Whitney test .

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

Histograms show the quantifications of the amount of polymerized fluorescent tubulin (right, values were normalized with respect to the mean of control condition) and filamentous actin at the centrosome (left, values correspond to the fraction of fluorescence in a 2-micron-wide area around the centrosome relative to the total fluorescence in the cell). Measurements were pooled from 3 independent experiments; DMSO: n= 91, CK666: n= 82, SMIFH2: n= 74, latrunculinA: n= 96. Error bars correspond to standard deviations. P values were calculated with Mann-Whitney test.


Percentage differences of centrosomal F - actin and microtubule fluorescence intensities in cells treated with cytoskeleton inhibitors in comparison with the respective densities in cells treated with DMSO . Errors bars represent standard deviations . P values were calculated with one - sample t - test ( i . e . comparison to a theoretical mean of " 0 " ) .

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

Percentage differences of centrosomal F-actin and microtubule fluorescence intensities in cells treated with cytoskeleton inhibitors in comparison with the respective densities in cells treated with DMSO. Errors bars represent standard deviations. P values were calculated with one-sample t-test (i.e. comparison to a theoretical mean of "0").


The graph shows the same measurements as in panel ( B ) in an XY representation of individual measurements . The three lines correspond to linear regressions of the three sets of data relative to cells stimulated with each actin drug .

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

The graph shows the same measurements as in panel (B) in an XY representation of individual measurements. The three lines correspond to linear regressions of the three sets of data relative to cells stimulated with each actin drug.


IIA1 . 6 B lymphoma cells were transfected to transiently express centrin1 - GFP ( red ) and EB3 - mCherry ( green ) and video - recorded at the contact site with the glass coverslip ( left ) and at the centrosome ( right ) . Scale bar : 3 µm .

IIA1.6 B lymphoma cells were transfected to transiently express centrin1-GFP (red) and EB3-mCherry (green) and video-recorded at the contact site with the glass coverslip (left) and at the centrosome (right). Scale bar: 3 µm.


The duration of EB3 - positive comets presence in the bottom plane was measured in DMSO and CK666 treated cells ( left ) . Error bars correspond to standard deviations . The number of EB3 - positive comets exiting the a 2 - µm wide centrosomal area was also compared between the two conditions ( right ) . In both cases P values were calculated with Mann - Whitney test .

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

The duration of EB3-positive comets presence in the bottom plane was measured in DMSO and CK666 treated cells (left). Error bars correspond to standard deviations. The number of EB3-positive comets exiting the a 2-µm wide centrosomal area was also compared between the two conditions (right). In both cases P values were calculated with Mann-Whitney test.


IIA1 . 6 B lymphoma cells were transfected to transiently express centrin1 - VCA - GFP ( bottom ) or centrin1 - GFP ( top ) as control prior to be fixed and stained for α - tubulin ( left column ) and F - actin ( middle column ) . The GFP signal of centrin1 or centrin1 - VCA is shown in the right column to illustrate the proper centrosome targeting . Scale bar : 3 µm .

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

IIA1.6 B lymphoma cells were transfected to transiently express centrin1-VCA-GFP (bottom) or centrin1-GFP (top) as control prior to be fixed and stained for α-tubulin (left column) and F-actin (middle column). The GFP signal of centrin1 or centrin1-VCA is shown in the right column to illustrate the proper centrosome targeting. Scale bar: 3 µm.


Histograms show the quantifications of the amount of polymerized fluorescent tubulin ( right ) and filamentous actin at the centrosome ( left ) . Values correspond to the fraction of fluorescence in a 2 - micron - wide area around the centrosome relative to the total fluorescence in the cell . Measurements were pooled from 3 independent experiments ; centrin1 - GFP : n = 88 , centrin1 - VCA - GFP : n = 87 . Errors bars represent standard deviations . P values were calculated with Mann - Whitney test .

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

Histograms show the quantifications of the amount of polymerized fluorescent tubulin (right) and filamentous actin at the centrosome (left). Values correspond to the fraction of fluorescence in a 2-micron-wide area around the centrosome relative to the total fluorescence in the cell. Measurements were pooled from 3 independent experiments; centrin1-GFP: n= 88, centrin1-VCA-GFP: n= 87. Errors bars represent standard deviations. P values were calculated with Mann-Whitney test.


Percentage differences of F - actin and microtubule fluorescence intensities at the centrosome were compared in cells transfected either with centrin1 - VCA - GFP or with centrin1 - GFP . Errors bars represent standard deviations . P values were calculated with one - sample t - test ( i . e . comparison to a theoretical mean of " 0 " ) .

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

Percentage differences of F-actin and microtubule fluorescence intensities at the centrosome were compared in cells transfected either with centrin1-VCA-GFP or with centrin1-GFP. Errors bars represent standard deviations. P values were calculated with one-sample t-test (i.e. comparison to a theoretical mean of "0").


The graph shows the variations of the total amount of tubulin per cell with respect to the content of actin at the centrosome . The two lines correspond to linear regressions of the two sets of data relative to cells transfected with centrin1 - VCA - GFP or centrin1 - GFP .

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

The graph shows the variations of the total amount of tubulin per cell with respect to the content of actin at the centrosome. The two lines correspond to linear regressions of the two sets of data relative to cells transfected with centrin1-VCA-GFP or centrin1-GFP.


Two sets of representative images showing fluorescent microtubules and actin filaments assembled from isolated centrosomes . Centrosomes were isolated from Jurkat cells expressing centrin1 - GFP . Upper and lower lines show actin filaments and microtubules radiating from two distinct centrosomes with low ( top ) and high ( bottom ) densities of actin filaments . Scale bars : 10 μm .

Two sets of representative images showing fluorescent microtubules and actin filaments assembled from isolated centrosomes. Centrosomes were isolated from Jurkat cells expressing centrin1-GFP. Upper and lower lines show actin filaments and microtubules radiating from two distinct centrosomes with low (top) and high (bottom) densities of actin filaments. Scale bars: 10 μm.


The graph shows the number of microtubules per centrosome relative to the density of actin filaments . Inset shows actin filaments at the centrosome with a FIRE look - up table and a 20 µm - wide circle in which actin fluorescence intensity is measured . Measurements were pooled from 5 independent experiments ; n = 50 .

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

The graph shows the number of microtubules per centrosome relative to the density of actin filaments. Inset shows actin filaments at the centrosome with a FIRE look-up table and a 20 µm-wide circle in which actin fluorescence intensity is measured. Measurements were pooled from 5 independent experiments; n= 50.


Microtubules ( top line ) and actin filaments ( bottom line ) assembly from isolated centrosomes in the presence of increasing concentration of monomeric actin ( from left to right ) . Scale bar : 20 μm .

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

Microtubules (top line) and actin filaments (bottom line) assembly from isolated centrosomes in the presence of increasing concentration of monomeric actin (from left to right). Scale bar: 20 μm.


The graph shows the number of microtubules per centrosome in response to increasing concentrations of monomeric actin . Data were pooled from 2 independent experiments ; 0 µM : n = 21 ; 0 . 3 µM : n = 17 ; 0 . 5 µM : n = 17 ; 1 . 0 µM : n = 17 . * * * * : P < 0 . 001 Mann - Whitney test .

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The graph shows the number of microtubules per centrosome in response to increasing concentrations of monomeric actin. Data were pooled from 2 independent experiments; 0 µM: n=21; 0.3 µM: n=17; 0.5 µM: n=17; 1.0 µM: n=17. ****:P<0.001 Mann-Whitney test.


The image shows the density of actin filaments ( in the presence of 1µM of actin monomers ) at the centrosome color - coded with the FIRE look - up table and the definition of central , peripheral and distal regions corresponding to decreasing concentrations of actin filaments . Scale bar : 20 μm . The graph shows the measurements of microtubule growth rate in each region . Data were pooled from 3 independent experiments : central zone : n = 58 , peripheral : n = 104 , outside : n = 61 ; n . s . means no statistical difference between the data set according to Mann - Whitney test .

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The image shows the density of actin filaments (in the presence of 1µM of actin monomers) at the centrosome color-coded with the FIRE look-up table and the definition of central, peripheral and distal regions corresponding to decreasing concentrations of actin filaments. Scale bar: 20 μm. The graph shows the measurements of microtubule growth rate in each region. Data were pooled from 3 independent experiments: central zone: n=58, peripheral: n=104, outside: n=61; n. s. means no statistical difference between the data set according to Mann-Whitney test.


The graphs show the various intensities of centrosome immuno - staining with antibodies against gamma - tubulin on the same coverslip depending on the presence / absence of actin filaments ( left ) or on the amount of actin filaments ( right ) . Data were pooled from 3 independent experiments . Left graph : without actin n = 69 , with actin n = 26 , right graph n = 26 . N . s . means no statistical difference between the data set according to Mann - Whitney test .

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The graphs show the various intensities of centrosome immuno-staining with antibodies against gamma-tubulin on the same coverslip depending on the presence/absence of actin filaments (left) or on the amount of actin filaments (right). Data were pooled from 3 independent experiments. Left graph: without actin n=69, with actin n=26, right graph n=26. N.s. means no statistical difference between the data set according to Mann-Whitney test.


Schematic illustration of the first dynamic assay : sequential addition of tubulin followed by tubulin and actin on isolated centrosomes . Representative images showing microtubules ( top line ) and the merged images of actin and microtubules ( bottom line ) for the two steps of the assay ; in the presence of tubulin only ( left column ) and in the presence of tubulin and actin ( right column ) . Scale bar : 10 µm . Quantification of the differences in the number of microtubules per centrosome between the two stages of the experiment described above on centrosomes capable ( first condition ) , or not ( second condition ) , to grow actin filaments . Data were collected from a single experiment ; asters without actin filaments : n = 29 ; asters with actin filaments : n = 13 . Data were analyzed using Mann - Whitney test .

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Schematic illustration of the first dynamic assay: sequential addition of tubulin followed by tubulin and actin on isolated centrosomes. Representative images showing microtubules (top line) and the merged images of actin and microtubules (bottom line) for the two steps of the assay; in the presence of tubulin only (left column) and in the presence of tubulin and actin (right column). Scale bar: 10 µm. Quantification of the differences in the number of microtubules per centrosome between the two stages of the experiment described above on centrosomes capable (first condition), or not (second condition), to grow actin filaments. Data were collected from a single experiment; asters without actin filaments: n= 29; asters with actin filaments: n= 13. Data were analyzed using Mann-Whitney test.


Schematic illustration of the second dynamic assay : tubulin is added to measure centrosome nucleation capacity and washed out . Then actin is added followed by actin and tubulin . Representative images showing microtubules ( top line ) and the merged images of actin and microtubule ( bottom line ) during the three steps of the assay ; in the presence of tubulin only ( left column ) , in the absence of tubulin and presence of actin ( middle column ) and in the presence of tubulin and actin ( right column ) . Scale bar : 10 µm . Arrowheads indicate microtubules unable to re - grow after assembly of actin filaments . Quantification of the differences in the number of microtubules per centrosome between the first and last steps of the experiment described above ( panels D and E ) on centrosomes capable ( first condition ) , or not ( second condition ) , to grow actin filaments . Data were pooled from 2 independent experiments ; asters without actin filaments : n = 24 ; asters with actin filaments : n = 13 . * * * * : P < 0 . 001 Student t - test .

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Schematic illustration of the second dynamic assay: tubulin is added to measure centrosome nucleation capacity and washed out. Then actin is added followed by actin and tubulin. Representative images showing microtubules (top line) and the merged images of actin and microtubule (bottom line) during the three steps of the assay; in the presence of tubulin only (left column), in the absence of tubulin and presence of actin (middle column) and in the presence of tubulin and actin (right column). Scale bar:10 µm. Arrowheads indicate microtubules unable to re-grow after assembly of actin filaments. Quantification of the differences in the number of microtubules per centrosome between the first and last steps of the experiment described above (panels D and E) on centrosomes capable (first condition), or not (second condition), to grow actin filaments. Data were pooled from 2 independent experiments; asters without actin filaments: n= 24; asters with actin filaments: n= 13. ****:P<0.001 Student t-test.


Representative images of microtubules ( top ) and actin filaments ( bottom ) growth from micropatterns . Scale bars : 20 μm .

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Representative images of microtubules (top) and actin filaments (bottom) growth from micropatterns. Scale bars: 20 μm.


Schematic illustration of the assay on micropatterned substrate . Tubulin was first added alone to measure the nucleation capacity of each micropattern , and then washed out . Later on , actin was added followed by actin and tubulin . Finally , actin was rinsed out and gelsolin was added to fully disassemble actin filaments . Representative images showing microtubules ( top line ) and the merged images of actin and microtubules ( bottom line ) during the four steps of the assay ; in the presence of tubulin only , in the absence of tubulin and presence of actin , in the presence of tubulin and actin , and finally in the presence of tubulin and gelsolin but in the absence of actin filaments ( from left to right ) . Scale bar : 10 μm .

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Schematic illustration of the assay on micropatterned substrate. Tubulin was first added alone to measure the nucleation capacity of each micropattern, and then washed out. Later on, actin was added followed by actin and tubulin. Finally, actin was rinsed out and gelsolin was added to fully disassemble actin filaments. Representative images showing microtubules (top line) and the merged images of actin and microtubules (bottom line) during the four steps of the assay; in the presence of tubulin only, in the absence of tubulin and presence of actin, in the presence of tubulin and actin, and finally in the presence of tubulin and gelsolin but in the absence of actin filaments (from left to right). Scale bar: 10 μm.


Quantification of the number of microtubules per micropattern in the presence of tubulin only ( left ) , actin and tubulin ( middle ) and tubulin only after actin filaments disassembly ( right ) . Data were pooled from 2 independent experiments ; n = 133 . * * * * : P < 0 . 001 Student t - test . The graph shows the same measurements as in panel ( E ) in an XY representation of individual measurements . It illustrates the differences in the number of microtubules per micropattern between the first to the second step ( tubulin only versus actin and tubulin together ) with respect to the density of actin filaments per micropattern .

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Quantification of the number of microtubules per micropattern in the presence of tubulin only (left), actin and tubulin (middle) and tubulin only after actin filaments disassembly (right). Data were pooled from 2 independent experiments; n= 133. ****:P<0.001 Student t-test. The graph shows the same measurements as in panel (E) in an XY representation of individual measurements. It illustrates the differences in the number of microtubules per micropattern between the first to the second step (tubulin only versus actin and tubulin together) with respect to the density of actin filaments per micropattern.


RPE1 cells stably expressing centrin1 - GFP were plated for 3 h on coverslips coated with different ratios ( 100 : 0 ; 50 : 50 or 1 : 99 ) of fibronectin and PLL - PEG prior to fixation and staining for F - actin ( top line and magnified views around centrosome below . Scale bars : 10 µm and 2 µm , respectively ) and α - tubulin ( bottom line . Scale bar : 10 µm ) .

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RPE1 cells stably expressing centrin1-GFP were plated for 3 h on coverslips coated with different ratios (100:0; 50:50 or 1:99) of fibronectin and PLL-PEG prior to fixation and staining for F-actin (top line and magnified views around centrosome below. Scale bars: 10 µm and 2 µm, respectively) and α-tubulin (bottom line. Scale bar: 10 µm).


IIA1 . 6 B lymphoma cells were plated for 60 min on poly - L - lysine , fibronectin or ICAM - 1 coated cover slides prior to be fixed and stained for F - actin ( top line ) and α - tubulin ( bottom line ) . Scale bar : 3 µm .

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IIA1.6 B lymphoma cells were plated for 60 min on poly-L-lysine, fibronectin or ICAM-1 coated cover slides prior to be fixed and stained for F-actin (top line) and α-tubulin (bottom line). Scale bar: 3 µm.


Quantification of the area occupied by RPE1 cells on the substrate ( top left ) , F - actin content at the centrosome ( top right ) polymerized tubulin at the centrosome ( bottom left ) and in the entire cell ( bottom right ) for the three conditions of cell adhesion described in ( B ) . Measurements came from 3 independent experiments with more than 60 analyzed cells in each . Errors bars represent standard deviations . F - actin and microtubule contents were compared using Mann - Whitney test , and variations of the cell area were compared using unpaired t - test .

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

Quantification of the area occupied by RPE1 cells on the substrate (top left), F-actin content at the centrosome (top right) polymerized tubulin at the centrosome (bottom left) and in the entire cell (bottom right) for the three conditions of cell adhesion described in (B). Measurements came from 3 independent experiments with more than 60 analyzed cells in each. Errors bars represent standard deviations. F-actin and microtubule contents were compared using Mann-Whitney test, and variations of the cell area were compared using unpaired t-test.


Quantification of the area occupied by B lymphoma cells on the substrate ( top left ) , F - actin content at the centrosome ( top right ) polymerized tubulin at the centrosome ( bottom left ) and in the entire cell ( bottom right ) for the three conditions of cell adhesion described in ( D ) . Measurements came from 3 independent experiments with more than 80 analyzed cells in each . Errors bars represent standard deviations . F - actin and microtubule contents were compared using Mann - Whitney test , and variations of the cell area were compared using unpaired t - test .

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

Quantification of the area occupied by B lymphoma cells on the substrate (top left), F-actin content at the centrosome (top right) polymerized tubulin at the centrosome (bottom left) and in the entire cell (bottom right) for the three conditions of cell adhesion described in (D). Measurements came from 3 independent experiments with more than 80 analyzed cells in each. Errors bars represent standard deviations. F-actin and microtubule contents were compared using Mann-Whitney test, and variations of the cell area were compared using unpaired t-test.


B HeLa cells expressing GFP - parkin ( green ) , OCT - DsRed2 ( red ) and CFP - Drp1K38E were left untreated ( untreated ) or treated for 2 h with 10 µM CCCP ( CCCP ) , then fixed and stained for TOM20 ( blue ) . Scale bars , 30 µm .

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B HeLa cells expressing GFP-parkin (green), OCT-DsRed2 (red) and CFP-Drp1K38E were left untreated (untreated) or treated for 2 h with 10 µM CCCP (CCCP), then fixed and stained for TOM20 (blue). Scale bars, 30 µm.


C Cells prepared as in ( B ) were treated with 50 µM antimycin A for 2 h . Arrows indicate OCT - DsRed2 - positive / TOM20 - negative MDVs that colocalize with GFP - parkin , while circles indicate MDVs that are parkin - negative . Arrowheads indicate nascent vesicles , adjacent to mitochondria , exhibiting cargo selectivity and parkin recruitment . Open arrowheads indicate parkin - negative MDVs containing the reciprocal cargo ( TOM20 - positive / OCT - DsRed2 - negative ) . Cell boundaries are delineated in the GFP - parkin single - channel image . Scale bars , 30 µm .

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C Cells prepared as in (B) were treated with 50 µM antimycin A for 2 h. Arrows indicate OCT-DsRed2-positive/TOM20-negative MDVs that colocalize with GFP-parkin, while circles indicate MDVs that are parkin-negative. Arrowheads indicate nascent vesicles, adjacent to mitochondria, exhibiting cargo selectivity and parkin recruitment. Open arrowheads indicate parkin-negative MDVs containing the reciprocal cargo (TOM20-positive/OCT-DsRed2-negative). Cell boundaries are delineated in the GFP-parkin single-channel image. Scale bars, 30 µm.


A Quantification of the number of OCTμDsRed2μpositive / TOM20μnegative vesicles in cells expressing GFP , GFPμparkinWT , GFPμparkinR42P , GFPμparkinK211N , or GFPμparkinC431F , treated with or without antimycin A ( anti A ) as in ( C ) ; both the total number ( white bars ) and the number colocalizing with GFPμparkin ( gray bars ) are indicated . Bars represent the mean ± s . e . m . Pμvalues are given first for GFPμ / GFPμparkinμpositive vesicles , then for total vesicle number ( n = 49 - 68 cells in 2 - 3 experiments ) ; ns , not significant ; * * P < 0 . 01 ; * * * P < 0 . 001 ) .

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A Quantification of the number of OCTμDsRed2μpositive/TOM20μnegative vesicles in cells expressing GFP, GFPμparkinWT, GFPμparkinR42P, GFPμparkinK211N, or GFPμparkinC431F, treated with or without antimycin A (anti A) as in (C); both the total number (white bars) and the number colocalizing with GFPμparkin (gray bars) are indicated. Bars represent the mean ± s.e.m. Pμvalues are given first for GFPμ/GFPμparkinμpositive vesicles, then for total vesicle number (n = 49-68 cells in 2-3 experiments); ns, not significant; **P < 0.01; ***P < 0.001).


C HeLa cells expressing various GFP - parkin mutant contructs ( green ) , pOCT - DsRed2 ( red ) and CFP - Drp1K38E were treated with 50 µM antimycin A for 2 h , then fixed and immunostained against TOM20 . Arrows indicate matrix - positive / TOM20 - negative structures colocalizing with GFP - parkin . Scale bars , 30 µm .

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C HeLa cells expressing various GFP-parkin mutant contructs (green), pOCT-DsRed2 (red) and CFP-Drp1K38E were treated with 50 µM antimycin A for 2 h, then fixed and immunostained against TOM20. Arrows indicate matrix-positive/TOM20-negative structures colocalizing with GFP-parkin. Scale bars, 30 µm.