Effect of NMDA on total - surface TrkB - FL . Cultures were incubated with NMDA ( 100 µM ) or BDNF ( 100 ng / ml ) for 1 h . were labeled purified compared to corresponding total lysates . TrkB - FL are expressed relative to untreated .

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

Effect of NMDA on total and cell-surface TrkB-FL levels. Cultures were incubated with NMDA (100 µM) or BDNF (100 ng/ml) for 1 h. Membrane proteins were labeled and purified and compared to corresponding total lysates. TrkB-FL levels are expressed relative to untreated cells.


Effect of TFL457 on decrease of TrkB - FL surface induced by NMDA . was performed as before in incubated with CPPs ( 25 μM , 30 min ) NMDA - treated ( 1 h ) . Results obtained in excitotoxic cultures are expressed relative to treated with same peptide but no NMDA .

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

Effect of TFL457 on the decrease of TrkB-FL surface levels induced by NMDA. Analysis was performed as before in cells incubated with CPPs (25 μM, 30 min) and NMDA-treated (1 h). Results obtained in excitotoxic cultures are expressed relative to cells treated with the same peptide but no NMDA.


Effect of endocytosis inhibition on TrkB - FL downregulation . infarcted of hemisphere ( I ) was compared to corresponding region of ( C ) . Results from representative mice injected with dynasore or vehicle are shown . Different exposures are presented to facilitate visualization of dynasore effects on TrkB - FL f32 . Quantitation of TrkB - FL TrkB - T1 in infarcted . Normalized are expressed relative to those of corresponding region .

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

Effect of endocytosis inhibition on TrkB-FL downregulation. The infarcted area of the ipsilateral hemisphere (I) was compared to the corresponding region of the contralateral area (C). Results from representative mice injected with dynasore or vehicle are shown. Different exposures are presented to facilitate visualization of dynasore effects on TrkB-FL and f32. Quantitation of TrkB-FL and TrkB-T1 in the infarcted area. Normalized protein levels are expressed relative to those of the corresponding contralateral region.


Confirmation of Bio - TFL457 delivery to mice cortex . Biotinylated TFL457 ( Bio - TFL457 , 4 nMole / g , retro - orbitally injected ) was detected in coronal sections stained with NeuN DAPI . Representative confocal microscopy of cortical areas correspond to single sections . Scale bar , 10 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 B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O

Confirmation of Bio-TFL457 delivery to mice cortex. Biotinylated TFL457 (Bio- TFL457, 4 nMole/g, retro-orbitally injected) was detected in coronal sections stained with NeuN and DAPI. Representative confocal microscopy images of cortical areas correspond to single sections. Scale bar, 10 µm.


Representative of TrkB - FL at 3 h of damage . Comparison of infarcted ( I ) corresponding region ( C ) is shown . Quantitation of TrkB - FL . Means ± of normalized TrkB - FL in infarcted relative to corresponding

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

Representative analysis of TrkB-FL at 3 h of damage. Comparison of the infarcted area (I) and the corresponding contralateral region (C) is shown. Quantitation of TrkB-FL levels. Means ± SEM of normalized TrkB-FL levels in the infarcted area relative to the corresponding contralateral region


Representative 1 mm brain coronal slices stained with TTC after 24 h of insult corresponding to injected with MTMyc or MTFL457 .

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

Representative 1 mm brain coronal slices stained with TTC after 24 h of insult corresponding to animals injected with MTMyc or MTFL457.


Infarct volume of injected with MTMyc or MTFL457 expressed as a of hemisphere volume . Means ± are given . Differences were analyzed by Student ' s t - ( * * * p = 0 . 00008 ; n = 14 ) . Results for MTFL457 are also expressed as of infarct volume in injected with MTMyc .

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

Infarct volume of animals injected with MTMyc or MTFL457 expressed as a percentage of the hemisphere volume. Means ± SEM are given. Differences were analyzed by Student's t-test (***p = 0.00008; n = 14). Results for MTFL457 are also expressed as percentage of the infarct volume in animals injected with MTMyc.


Evaluation of balance motor coordination . Number of hind paw slips were measured after 24 h of damage . Data spread is presented by box whisker plots showing interquartile range , median , minimum maximum values .

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

Evaluation of balance and motor coordination. Number of contralateral hind paw slips were measured after 24 h of damage. Data spread is presented by box and whisker plots showing interquartile range, median, minimum and maximum values.


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

B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 with or without SeV infection ( SeV or UI ) for 18 ~ 24 h . Data information : Graphs represent fold - induction relative to luciferase activity in control . UI , uninfected . data are presented as mean values on three independent experiments , error bars indicate S . E . M . ( unpaired two - Student ' s t ) .

B-MEASURED_VAR 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 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 with or without SeV infection ( SeV or UI ) for 18 ~ 24 h . Data information : Graphs represent fold - induction relative to luciferase activity in control . UI , uninfected . data are presented as mean values on three independent experiments , error bars indicate S . E . M . ( unpaired two - Student ' s t ) .

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 with or without SeV infection ( SeV or UI ) for 18 ~ 24 h . Data information : Graphs represent fold - induction relative to luciferase activity in control . UI , uninfected . data are presented as mean values on three independent experiments , error bars indicate S . E . M . ( unpaired two - Student ' s t ) .

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 indicated siRNA - transfected H1299 ( si - NC , si - SIRT5 # 1 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 luciferase activity in control . UI , uninfected . data are presented as mean values on three independent experiments , error bars indicate S . E . M . ( unpaired two - Student ' s t ) .

B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O 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 (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 indicated siRNA - transfected H1299 ( si - NC , si - SIRT5 # 1 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 luciferase activity in control . UI , uninfected . data are presented as mean values on three independent experiments , error bars indicate S . E . M . ( unpaired two - Student ' s t ) .

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

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

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

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

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

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 with or without poly ( I : C ) transfection for 18 ~ 24 h . Data information : Graphs represent fold - induction relative to luciferase activity in control . UI , uninfected . data are presented as mean values on three independent experiments , error bars indicate S . E . M . ( unpaired two - Student ' s t ) .

O B-MEASURED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 for 24 h . Data information : Graphs represent fold - induction relative to luciferase activity in control . UI , uninfected . data are presented as mean values on three independent experiments , error bars indicate S . E . M . ( unpaired two - Student ' s t ) .

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

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 for 24 h . Data information : Graphs represent fold - induction relative to luciferase activity in control . UI , uninfected . data are presented as mean values on three independent experiments , error bars indicate S . E . M . ( unpaired two - Student ' s t ) .

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

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 for 24 h . Data information : Graphs represent fold - induction relative to luciferase activity in control . UI , uninfected . data are presented as mean values on three independent experiments , error bars indicate S . E . M . ( unpaired two - Student ' s t ) .

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 for 24 h . Data information : Graphs represent fold - induction relative to luciferase activity in control . UI , uninfected . data are presented as mean values on three independent experiments , error bars indicate S . E . M . ( unpaired two - Student ' s t ) .

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 for 24 h . Data information : Graphs represent fold - induction relative to luciferase activity in control . UI , uninfected . data are presented as mean values on three independent experiments , error bars indicate S . E . M . ( unpaired two - Student ' s t ) .

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 for 24 h . Data information : Graphs represent fold - induction relative to luciferase activity in control . UI , uninfected . data are presented as mean values on three independent experiments , error bars indicate S . E . M . ( unpaired two - Student ' s t ) .

O B-MEASURED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 detected by immunofluorescence using anti - SIRT5 anti - MAVS antibodies . Mito , Mitotracker ; Scale bar = 8 µm .

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

O O O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 SIRT5 . Anti - MAVS antibody was used for immunoprecipitation , normal mouse IgG was used as a control . Data information : IP , immunoprecipitation ; TCL , total lysates .

O O O O B-MEASURED_VAR I-MEASURED_VAR 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

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

O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O B-MEASURED_VAR 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 for GST - tagged MAVS His - tagged SIRT5 . GST - tagged MAVS His - tagged SIRT5 were expressed in Escherichia coli ( BL21 ) , respectively . association of GST - MAVS with His - SIRT5 was detected by immunoblotting with anti - SIRT5 antibody . GST GST - MAVS were stained with Coomassie blue .

O O O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O B-CONTROLLED_VAR O O O B-MEASURED_VAR O O O O O O O O O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR 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 of HA - SIRT5 with Flag - MAVS - truncated mutants . HEK293T were co - transfected with indicated plasmids . Anti - Flag antibody conjugated agarose beads were used for immunoprecipitation interaction was analyzed by immunoblotting with indicated antibodies . Flag - MAVS fragments ( WT : full ; 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 lysates .

O O O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 ) purified from HEK293T was incubated with indicated concentrations of succinyl - CoA . succinylation was detected with anti - pan - succinyl - lysine antibody ; GST - MAVS or Flag - MAVS was stained by Coomassie blue .

O O O B-MEASURED_VAR O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O 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 B-MEASURED_VAR O O O B-MEASURED_VAR 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 succinylated residue in MAVS was identified by mass spectrometry .

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


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

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 B-MEASURED_VAR 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 was incubated with indicated concentrations of succinyl - CoA . succinylation of MAVS at lysine 7 was detected by anti - succ - K7 - MAVS antibody .

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 B-MEASURED_VAR O O O O O O O O O O O O B-MEASURED_VAR 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 was incubated with indicated concentrations of succinyl - CoA . succinylation of MAVS at lysine 7 was detected by anti - succ - K7 - MAVS antibody .

O O B-MEASURED_VAR 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 B-MEASURED_VAR 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 enhanced succinylation of MAVS at Lys 7 compared to that in SIRT5 - intact H1299 ( SIRT5 + / + ) ( 3 . 2 vs . 1 . 0 ) . lysates from SIRT5 - deficient H1299 or SIRT5 - intact H1299 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 lysates .

O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR 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 B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O B-MEASURED_VAR 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 ( 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 has no effect on reduction in succinylation of MAVS at Lys 7 ( 1 . 0 vs . 1 . 2 ) . SIRT5 - / - H1299 were transfected with Flag - SIRT5 or Flag - SIRT5 - H158Y , followed by immunoprecipitating with anti - MAVS antibody or mouse IgG control , immunoblotting with anti - succ - K7 - MAVS antibody . Data information : IP , immunoprecipitation ; TCL , total lysates .

O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-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 B-MEASURED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O B-MEASURED_VAR 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 ) lungs ( K ) . extracted from livers ( J ) lungs ( K ) of Sirt5 KO wildtype ( n = 3 per group ) were detected by indicated antibodies . MAVS succinylation was determined by anti - succ - K7 - MAVS antibody , anti - pan - succinyl - lysine antibody was used as positive controls .

O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR 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 B-MEASURED_VAR O O O O O O O O O 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 ( qPCR ) of IFNβ ( A ) , CXCL10 ( B ) IFIT1 ( C ) mRNA in HEK293T transfected with control plasmid ( Myc empty ) or 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 . graphs represent fold - induction relative to untreated . data are presented as mean values on three independent experiments , error bars indicate S . E . M . ( unpaired two - Student ' s t ) .

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

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

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

O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O B-CONTROLLED_VAR 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 O O O O O 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 of IFNβ mRNA in HEK293T transfected with control plasmid ( Myc empty ) , or 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 : data are presented as mean values on three independent experiments , error bars indicate S . E . M . ( unpaired two - Student ' s t ) .

O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 of IFNβ mRNA in SIRT5 - deficient H1299 ( SIRT5 - / - ) transfected with control plasmid ( Myc empty ) or 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 : data are presented as mean values on three independent experiments , error bars indicate S . E . M . ( unpaired two - Student ' s t ) .

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

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 - of MAVS in HEK293T 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 B-MEASURED_VAR O O O O O O O O O O O B-CONTROLLED_VAR 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

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

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

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

B-CONTROLLED_VAR 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

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

B-CONTROLLED_VAR 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 O 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 ( SIRT5 - / - or SIRT5 + / + ) were infected with VSV - GFP ( MOI = 0 . 1 ) for 12 h , viral infectivity was detected by flow cytometry ( n = 3 ) ( Q ) .

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

O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O B-CONTROLLED_VAR 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 O O O O O 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 ( Sirt5 + / + or Sirt5 - / - ) were infected with or without SeV for indicated times , lysates were analyzed by immunoblotting for monomeric ( Monomer ) dimeric ( Dimer ) Irf3 ( top ; native - PAGE ) , phosphorylated Irf3 ( p - Irf3 ) , total Irf3 , Sirt5 , GAPDH ( bottom ) .

O O O B-CONTROLLED_VAR 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 B-MEASURED_VAR O O O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O B-MEASURED_VAR O B-MEASURED_VAR 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 ( Sirt5 + / + or Sirt5 - / - ) were infected with or without SeV for 8 h , interaction between endogenous Mavs Rig - I was revealed by immunoprecipitation using anti - Mavs antibody , followed by immunoblotting with anti - Rig - I antibody .

O O O B-CONTROLLED_VAR 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 B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O B-MEASURED_VAR O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR 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 - of Mavs in WT or Sirt5 - deficient MEF ( Sirt5 + / + or Sirt5 - / - ) infected with SeV for 12 h . SDS - PAGE immunoblotting was used as a loading control .

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

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

O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O B-CONTROLLED_VAR 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 O O O O O O 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-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 ( Sirt5 + / + or Sirt5 - / - ) were infected with or without VSV - GFP for 12 h , viral infectivity was detected by microscopy ( P )

O O B-CONTROLLED_VAR 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

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 ( Sirt5 + / + or Sirt5 - / - ) were infected with or without VSV - GFP for 12 h , viral infectivity was detected by western blot ( Q )

O O B-CONTROLLED_VAR 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

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

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

O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O B-CONTROLLED_VAR 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 O O O O O O 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 of Cxcl11 ( G ) mRNA in WT or Sirt5 - deficient BMDC ( Sirt5 + / + or Sirt5 - / - ) infected with or without SeV ( Sev or UI ) for 8 h . Data information : UI , un - infected . graphs represent fold - induction relative to untreated . data are presented as mean values on three independent experiments , error bars indicate S . E . M . ( unpaired two - Student ' s t ) .

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

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

O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O B-CONTROLLED_VAR 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 O O O O O O 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 of Cxcl10 ( M ) Cxcl11 ( N ) mRNA in WT or Sirt5 - deficient BMDC ( Sirt5 + / + or Sirt5 - / - ) infected with or without VSV ( VSV or UI ) for 8 h . Data information : UI , un - infected . graphs represent fold - induction relative to untreated . data are presented as mean values on three independent experiments , error bars indicate S . E . M . ( unpaired two - Student ' s t ) .

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

O O O O O O O O O B-CONTROLLED_VAR 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 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 ) lungs ( B ) . in livers ( A ) lungs ( B ) of WT injected intraperitoneally with ( + ) VSV ( 1 × 107 plaque - forming units ( PFU ) per mouse ) or PBS control ( - ) ( n = 3 per group ) were detected with 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 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 B-MEASURED_VAR 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

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 ) lungs ( D ) upon VSV infection . in livers ( C ) lungs ( D ) of Sirt5 - deficient or WT ( 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 indicated antibodies . Mavs succinylation was determined by anti - succ - K7 - MAVS antibody . Relative succinylation level was quantified ( right panel ) .

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

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

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

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 of Ifnβ in serum from WT mice ( n = 4 ) 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 : graphs represent fold - induction relative to untreated WT mice . PBS , phosphate buffer saline . data are presented as mean values on three independent experiments , error bars indicate S . E . M . ( unpaired two - Student ' s t or two - way ANOVA ) .

O O O B-MEASURED_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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 - - eosin - stained 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

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


qPCR of Ifnβ ( H ) , Ifnα1 ( I ) , Ifit1 ( J ) , Isg15 ( K ) , Cxcl10 ( L ) , Cxcl11 ( M ) mRNA in 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 : graphs represent fold - induction relative to untreated WT mice . PBS , phosphate buffer saline . data are presented as mean values on three independent experiments , error bars indicate S . E . M . ( unpaired two - Student ' s t or two - way ANOVA ) .

O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O O O B-CONTROLLED_VAR 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 O O O O O O O O O O O O O O O O O O O O O O O O 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 of Rig - I ( N ) , Irf7 ( O ) mRNA in 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 : graphs represent fold - induction relative to untreated WT mice . PBS , phosphate buffer saline . data are presented as mean values on three independent experiments , error bars indicate S . E . M . ( unpaired two - Student ' s t or two - way ANOVA ) .

O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O O O B-CONTROLLED_VAR 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 O O O O O O O O O O O O O O O O O O O O O O O O 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 of VSV mRNA in 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 : graphs represent fold - induction relative to untreated WT mice . PBS , phosphate buffer saline . data are presented as mean values on three independent experiments , error bars indicate S . E . M . ( unpaired two - Student ' s t or two - way ANOVA ) .

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

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).


Human placental syncytiotrophoblast express markers of senescence , p16 Sections of normal human post - partum , third trimester , placenta were evaluated by immunofluorescence co - for senescence regulators ( A ) p16 Green label : syncytiotrophoblast marker βHCG , red label : p16 Blue label : DAPI stain . Arrowheads indicate positively stained syncytiotrophoblast βHCG + / p16 + ( A ) Scale bar , 50 µm .

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

Human placental syncytiotrophoblast cells express markers of cellular senescence, p16 Sections of normal human post-partum, third trimester, placenta were evaluated by immunofluorescence co-staining for senescence regulators (A) p16 Green label: the syncytiotrophoblast marker βHCG, red label: p16 Blue label: DAPI nuclear stain. Arrowheads indicate positively stained syncytiotrophoblast cells βHCG+/p16+ (A) Scale bar, 50 µm.


Human placental syncytiotrophoblast express markers of senescence , p21 . Sections of normal human post - partum , third trimester , placenta were evaluated by immunofluorescence co - for senescence regulators ( B ) p21 . Green label : syncytiotrophoblast marker βHCG , red label : p21 . Blue label : DAPI stain . Arrowheads indicate positively stained syncytiotrophoblast , βHCG + / p21 + ( B ) . Scale bar , 50 µm .

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

Human placental syncytiotrophoblast cells express markers of cellular senescence, p21. Sections of normal human post-partum, third trimester, placenta were evaluated by immunofluorescence co-staining for senescence regulators (B) p21. Green label: the syncytiotrophoblast marker βHCG, red label: p21. Blue label: DAPI nuclear stain. Arrowheads indicate positively stained syncytiotrophoblast cells , βHCG+/p21+ (B). Scale bar, 50 µm.


( C - F ) Sections of human normal third - trimester placentas post - partum ( n = 4 normal placentas ) IUGR - complicated placentas of same gestational ( n = 4 IUGR placentas ) were stained for senescence - related markers of p15 ( C ) , p16 ( D ) , p21 ( E ) , p53 ( F ) in syncytiotrophoblast ( n = 3 sections , derived from each normal IUGR placenta ) . Percentages of positively stained syncytia were quantified ( n = at least 12 fields of view from sections ) . Scale bar , 100 µm . Arrowheads indicate syncytiotrophoblast . Data information : Values are means + of at least three experimental repeats . Statistical significance was determined by unpaired two - Student ' s t - ( * ) p < 0 . 05 ; ( * * ) p < 0 . 01 ; ( * * * ) p < 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O 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 B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-F) Sections of human normal third-trimester placentas post-partum (n=4 normal placentas) and IUGR-complicated placentas of the same gestational age (n=4 IUGR placentas) were stained for senescence-related markers of p15 (C), p16 (D), p21 (E), and p53 (F) in the syncytiotrophoblast (n=3 sections, derived from each normal and IUGR placenta). Percentages of positively stained syncytia were quantified (n= at least 12 fields of view from all sections). Scale bar, 100 µm. Arrowheads indicate syncytiotrophoblast cells. Data information: Values are means + SEM of at least three experimental repeats. Statistical significance was determined by unpaired two-tailed Student's t-test (*) p < 0.05; (**) p < 0.01; (***) p < 0.001.


( G ) Immunoblot of content of p16 , p21 , p53 DCR in normal ( n = 4 placentas ) in IUGR - complicated placentas ( n = 4 placentas ) . Each lane represents one independent placenta .

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

(G) Immunoblot analysis of the protein content of p16, p21, p53 and DCR in normal (n=4 placentas) and in IUGR-complicated placentas (n=4 placentas). Each lane represents one independent placenta.


( H ) Quantitative RT - PCR of p16 , p21 DCR2 expression in IUGR - complicated in normal placentas . Results were obtained from four human normal four IUGR placentas . Data information : Values are means + of at least three experimental repeats . Statistical significance was determined by unpaired two - Student ' s t - ( * ) p < 0 . 05 ; ( * * ) p < 0 . 01 ; ( * * * ) p < 0 . 001 .

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

(H) Quantitative RT-PCR analysis of p16, p21 and DCR2 expression in IUGR-complicated and in normal placentas. Results were obtained from four human normal and four IUGR placentas. Data information: Values are means + SEM of at least three experimental repeats. Statistical significance was determined by unpaired two-tailed Student's t-test (*) p < 0.05; (**) p < 0.01; (***) p < 0.001.


DCE - MRI was performed on pregnant mice of WT , Cdkn1a , p53 , Cdkn2a Cdkn2a ; p53 genotypes on day E14 . 5 . Mice were injected i . v . with an albumin - labeled contrast agent ( biotin - BSA - GdDTPA ; 10 mg / mouse ) placental enhancement was monitored at 9 . 4T MRI for 60 min . ( A ) Representative T1 - weighted image of a pregnant WT mouse a selection of four different placentas ( outlined by colored lines ) .

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

DCE-MRI was performed on pregnant mice of WT, Cdkn1a, p53, Cdkn2a and Cdkn2a;p53 genotypes on day E14.5. Mice were injected i.v. with an albumin-labeled contrast agent (biotin-BSA-GdDTPA; 10 mg/mouse) and placental enhancement was monitored at 9.4T MRI for 60 min. (A) Representative T1-weighted image of a pregnant WT mouse and a selection of four different placentas (outlined by colored lines).


DCE - MRI was performed on pregnant mice of WT , Cdkn1a , p53 , Cdkn2a Cdkn2a ; p53 genotypes on day E14 . 5 . Mice were injected i . v . with an albumin - labeled contrast agent ( biotin - BSA - GdDTPA ; 10 mg / mouse ) placental enhancement was monitored at 9 . 4T MRI for 60 min . ( B ) Representative SI dynamics of a single WT placenta ( marked in A ) , following biotin - BSA - GdDTPA administered for 60 min . SI parameters of Initial Enhancement Recovery are illustrated .

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

DCE-MRI was performed on pregnant mice of WT, Cdkn1a, p53, Cdkn2a and Cdkn2a;p53 genotypes on day E14.5. Mice were injected i.v. with an albumin-labeled contrast agent (biotin-BSA-GdDTPA; 10 mg/mouse) and placental enhancement was monitored at 9.4T MRI for 60 min. (B) Representative SI dynamics of a single WT placenta (marked in A), following biotin-BSA-GdDTPA administered for 60 min. SI parameters of Initial Enhancement and Recovery are illustrated.


DCE - MRI was performed on pregnant mice of WT , Cdkn1a , p53 , Cdkn2a Cdkn2a ; p53 genotypes on day E14 . 5 . Mice were injected i . v . with an albumin - labeled contrast agent ( biotin - BSA - GdDTPA ; 10 mg / mouse ) placental enhancement was monitored at 9 . 4T MRI for 60 min . ( C ) Representative SI dynamics of a single WT placenta ( marked in A ) over time . Scale bar , 2 . 5 mm .

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

DCE-MRI was performed on pregnant mice of WT, Cdkn1a, p53, Cdkn2a and Cdkn2a;p53 genotypes on day E14.5. Mice were injected i.v. with an albumin-labeled contrast agent (biotin-BSA-GdDTPA; 10 mg/mouse) and placental enhancement was monitored at 9.4T MRI for 60 min. (C) Representative SI dynamics of a single WT placenta (marked in A) over time. Scale bar, 2.5 mm.


DCE - MRI was performed on pregnant mice of WT , Cdkn1a , p53 , Cdkn2a Cdkn2a ; p53 genotypes on day E14 . 5 . Mice were injected i . v . with an albumin - labeled contrast agent ( biotin - BSA - GdDTPA ; 10 mg / mouse ) placental enhancement was monitored at 9 . 4T MRI for 60 min . ( D − G ) Representative SI plots of Cdkn1a - / - ( D ) , p53 - / - ( E ) , Cdkn2a - / - ( F ) , Cdkn2a - / - ; p53 - / - ( G ) placentas , compared to their WT or HET . ( H − I ) of SI Initial Enhancement ( H ) SI Recovery ( I ) in WT , Cdkn1a - / - , p53 - / - , Cdkn2a - / - , Cdkn2a - / - ; p53 - / - placentas . MRI experiments were repeated at least three times for each murine genotype ( n > = 3 placental SI measurements from each genotype ) . Values are means + ; Statistical significance was determined by unpaired two - Student ' s t - ( * ) p < 0 . 05 ; ( * * ) p < 0 . 01 ; ( * * * ) p < 0 . 001 .

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

DCE-MRI was performed on pregnant mice of WT, Cdkn1a, p53, Cdkn2a and Cdkn2a;p53 genotypes on day E14.5. Mice were injected i.v. with an albumin-labeled contrast agent (biotin-BSA-GdDTPA; 10 mg/mouse) and placental enhancement was monitored at 9.4T MRI for 60 min. (D−G) Representative SI plots of Cdkn1a-/- (D), p53-/- (E), Cdkn2a-/- (F), and Cdkn2a-/-;p53-/- (G) placentas, compared to their WT or HET littermates. (H−I) Quantification of the SI Initial Enhancement (H) and SI Recovery (I) in WT, Cdkn1a-/-, p53-/-, Cdkn2a-/-, and Cdkn2a-/-;p53-/- placentas. MRI experiments were repeated at least three times for each murine genotype (n>=3 placental SI measurements from each genotype). Values are means +SEM; Statistical significance was determined by unpaired two-tailed Student's t-test (*) p < 0.05; (**) p < 0.01; (***) p < 0.001.


Histological evaluation of H & E - stained sections in labyrinth zone of murine Cdkn2a - / - ( n = 5 placentas ) , Cdkn2a - / - ; p53 - / - ( n = 5 ) , p53 - / - ( n = 4 ) , Cdkn1a - / - ( n = 3 ) placentas on day E14 . 5 , compared to WT placentas ( n = 6 ) . ( A − E ) labyrinth zone of WT ( A ) differs from those of Cdkn2a - / - ( B ) Cdkn2a - / - ; p53 - / - ( C ) placentas . Black arrows indicate regular - sized nuclei in WT placenta polyploidy in Cdkn2a - / - Cdkn2a - / - ; p53 - / - placentas . Blue arrows indicate normal vasculature in WT placenta collapsed vasculature in Cdkn2a - / - Cdkn2a - / - ; p53 - / - placentas . Labyrinths of p53 - / - ( D ) Cdkn1a - / - ( E ) placentas did not differ from that of WT placenta . L , labyrinth . for each placenta was performed in triplicates .

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

Histological evaluation of H&E-stained sections in the labyrinth zone of murine Cdkn2a-/- (n=5 placentas), Cdkn2a-/-;p53-/- (n=5), p53-/- (n=4), and Cdkn1a-/- (n=3) placentas on day E14.5, compared to WT placentas (n=6). (A−E) The labyrinth zone of the WT (A) differs from those of Cdkn2a-/- (B) and Cdkn2a-/-;p53-/- (C) placentas. Black arrows indicate regular-sized nuclei in the WT placenta and polyploidy in the Cdkn2a-/- and Cdkn2a-/-;p53-/- placentas. Blue arrows indicate normal vasculature in the WT placenta and collapsed vasculature in the Cdkn2a-/- and Cdkn2a-/-;p53-/- placentas. Labyrinths of p53-/- (D) and Cdkn1a-/- (E) placentas did not differ from that of the WT placenta. L, labyrinth. Staining for each placenta was performed in triplicates.


Histological evaluation of H & E - stained sections in labyrinth zone of murine Cdkn2a - / - ( n = 5 placentas ) , Cdkn2a - / - ; p53 - / - ( n = 5 ) , p53 - / - ( n = 4 ) , Cdkn1a - / - ( n = 3 ) placentas on day E14 . 5 , compared to WT placentas ( n = 6 ) . ( F − G ) Ki67 immunostaining in labyrinth zones of murine WT , p53 - / - , Cdkn2a - / - , Cdkn2a - / - ; p53 - / - placentas , on day E14 . 5 ( n = 3 placentas from each genotype ) . ( F ) Representative of Ki67 in labyrinth zone are shown . Positive Ki67 trophoblast are indicated with arrows . L , labyrinth . ( G ) Proliferation scores ( n = 3 scores , derived from three placentas of each genotype ) on Ki67 show enhanced Ki67 expression in placental labyrinths of Cdkn2a - / - Cdkn2a - / - ; p53 - / - placentas relative to WT . Values are means + of three scores for each genotype . * P < 0 . 05 by a one - unpaired Student ' s t - . Data information : Scale bars , 50 µm .

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

Histological evaluation of H&E-stained sections in the labyrinth zone of murine Cdkn2a-/- (n=5 placentas), Cdkn2a-/-;p53-/- (n=5), p53-/- (n=4), and Cdkn1a-/- (n=3) placentas on day E14.5, compared to WT placentas (n=6). (F−G) Ki67 immunostaining in the labyrinth zones of murine WT , p53-/- ,Cdkn2a-/-, and Cdkn2a-/-;p53-/- placentas, on day E14.5 (n=3 placentas from each genotype). (F) Representative images of Ki67 staining in the labyrinth zone are shown. Positive Ki67 trophoblast cells are indicated with arrows. L, labyrinth. (G) Proliferation scores (n=3 scores, derived from three placentas of each genotype) based on Ki67 staining show enhanced Ki67 expression in the placental labyrinths of Cdkn2a-/- and Cdkn2a-/-;p53-/- placentas relative to WT. Values are means +SEM of three scores for each genotype. *P < 0.05 by a one-tailed unpaired Student's t-test. Data information: Scale bars, 50 µm.


Histological evaluation of H & E - stained sections in labyrinth zone of murine Cdkn2a - / - ( n = 5 placentas ) , Cdkn2a - / - ; p53 - / - ( n = 5 ) placentas on day E14 . 5 , compared to WT placentas ( n = 6 ) . ( H ) Representative of SA - β - gal in labyrinth zones of WT , Cdkn2a - / - , Cdkn2a - / - ; p53 - / - placentas on day E14 . 5 . ( I ) SA - β - gal activity , quantified by ImageJ sofware in Cdkn2a - / - Cdkn2a - / - ; p53 - / - labyrinths , is significantly reduced relative to those in WT labyrinth ( n > = 7 fields of view from each genotype ) . Values are means + . ( * * * ) p < 0 . 001 by a two - unpaired Student ' s t - . Data information : Scale bars , 50 µm .

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

Histological evaluation of H&E-stained sections in the labyrinth zone of murine Cdkn2a-/- (n=5 placentas), Cdkn2a-/-;p53-/- (n=5) placentas on day E14.5, compared to WT placentas (n=6). (H) Representative images of SA-β-gal staining in the labyrinth zones of WT, Cdkn2a-/-, and Cdkn2a-/-;p53-/- placentas on day E14.5. (I) SA-β-gal activity, quantified by ImageJ sofware in Cdkn2a-/- and Cdkn2a-/-;p53-/- labyrinths, is significantly reduced relative to those in WT labyrinth (n>=7 fields of view from each genotype). Values are means + SEM. (***) p < 0.001 by a two-tailed unpaired Student's t-test. Data information: Scale bars, 50 µm.


Human term placentas were dissected cytotrophoblast were extracted seeded . On days 1 5 after seeding , fusion was monitored by for phalloidin ( F - actin ) ( green ) DAPI ( blue ) . 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 O O O O O O O O O O O O O O O O O O O

Human term placentas were dissected and cytotrophoblast cells were extracted and seeded. On days 1 and 5 after seeding, cell fusion was monitored by staining for phalloidin (F-actin) (green) and DAPI (blue). Scale bar, 50 μm.


Human term placentas were dissected cytotrophoblast were extracted seeded . ( B ) Trophoblast fusion was quantified on days 1 , 3 , 5 post - seeding . Values are means + , derived independently from human placentas of three pregnancies .

Human term placentas were dissected and cytotrophoblast cells were extracted and seeded. (B) Trophoblast cell fusion was quantified on days 1, 3, and 5 post-seeding. Values are means + SEM, derived independently from human placentas of three pregnancies.


Human term placentas were dissected cytotrophoblast were extracted seeded . ( C ) of human βhCG hormone , normally secreted by functional syncytiotrophoblast , in medium of a representative trophoblast culture on post - delivery days 2 , 3 5 .

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

Human term placentas were dissected and cytotrophoblast cells were extracted and seeded. (C) Quantification of the human βhCG hormone, normally secreted by functional syncytiotrophoblast cells, in the medium of a representative trophoblast culture on post-delivery days 2, 3 and 5.


Human term placentas were dissected cytotrophoblast were extracted seeded . ( D ) Gene set enrichment ( GSEA ) of published datasets from human trophoblast cultures on days 5 2 ( n = 4 trophoblast culture of day 5 day 2 ) . Gene sets related to pregnancy , aging , immune response , cytokine activity , MAPK , JAK - STAT , NF - κB metalloproteinase activity were found to be enriched in day 5 cultures , whereas on same day gene sets related to cycle , E2F targets , mitosis replication were downregulated . Statistical significance was determined by permutation testing with normalized enrichment score ( NES )

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

Human term placentas were dissected and cytotrophoblast cells were extracted and seeded. (D) Gene set enrichment analysis (GSEA) of published datasets from human primary trophoblast cultures on days 5 and 2 (n=4 trophoblast culture of day 5 and day 2). Gene sets related to pregnancy, aging, immune response, cytokine activity, MAPK, JAK-STAT, NF-κB and metalloproteinase activity were found to be enriched in day 5 cultures, whereas on the same day gene sets related to cell cycle, E2F targets, mitosis and DNA replication were downregulated. Statistical significance was determined by permutation testing with normalized enrichment score (NES)


Human term placentas were dissected cytotrophoblast were extracted seeded . Quantitative RT - PCR of expression of cycle - related genes p16 , p21 CCNE1 Data information : Quantitative RT - PCR values are expressed as means + . Results were obtained from trophoblast cultures derived from human placentas of four independent pregnancies . RT - PCR experiments were performed in triplicates repeated at least three times . Statistical significance was determined by unpaired two - Student ' s t - ( * ) p < 0 . 05 ; ( * * ) p < 0 . 01 .

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

Human term placentas were dissected and cytotrophoblast cells were extracted and seeded. Quantitative RT-PCR analysis of expression of the cell cycle-related genes p16, p21 and CCNE1 Data information: Quantitative RT-PCR values are expressed as means +SEM. Results were obtained from trophoblast cultures derived from human placentas of four independent pregnancies. All RT-PCR experiments were performed in triplicates and repeated at least three times. Statistical significance was determined by unpaired two-tailed Student's t-test (*) p < 0.05; (**) p < 0.01.


Human term placentas were dissected cytotrophoblast were extracted seeded . Quantitative RT - PCR of expression of syncytiotrophoblast markers GCM1 , CGA CGB ( F ) . Data information : Quantitative RT - PCR values are expressed as means + . Results were obtained from trophoblast cultures derived from human placentas of four independent pregnancies . RT - PCR experiments were performed in triplicates repeated at least three times . Statistical significance was determined by unpaired two - Student ' s t - ( * ) p < 0 . 05 ; ( * * ) p < 0 . 01 .

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

Human term placentas were dissected and cytotrophoblast cells were extracted and seeded. Quantitative RT-PCR analysis of expression of the syncytiotrophoblast markers GCM1, CGA and CGB (F). Data information: Quantitative RT-PCR values are expressed as means +SEM. Results were obtained from trophoblast cultures derived from human placentas of four independent pregnancies. All RT-PCR experiments were performed in triplicates and repeated at least three times. Statistical significance was determined by unpaired two-tailed Student's t-test (*) p < 0.05; (**) p < 0.01.


Human term placentas were dissected cytotrophoblast were extracted seeded . ( G ) Immunoblot of indicated in human trophoblast , derived independently from two placentas , on days 2 5 .

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

Human term placentas were dissected and cytotrophoblast cells were extracted and seeded. (G) Immunoblot analysis of the indicated proteins in human trophoblast cells, derived independently from two placentas, on days 2 and 5.


Human term placentas were dissected cytotrophoblast were extracted seeded . Quantitative RT - PCR of expression of SASP components CCL2 , CCL5 , CCL8 , IL6 , IL1β , TGF - β PDG - FC ( H ) in human trophoblast cultures on days 5 2 . Data information : Quantitative RT - PCR values are expressed as means + . Results were obtained from trophoblast cultures derived from human placentas of four independent pregnancies . RT - PCR experiments were performed in triplicates repeated at least three times . Statistical significance was determined by unpaired two - Student ' s t - ( * ) p < 0 . 05 ; ( * * ) p < 0 . 01 .

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

Human term placentas were dissected and cytotrophoblast cells were extracted and seeded. Quantitative RT-PCR analysis of expression of the SASP components CCL2, CCL5, CCL8, IL6, IL1β, TGF-β and PDG-FC (H) in human primary trophoblast cultures on days 5 and 2. Data information: Quantitative RT-PCR values are expressed as means +SEM. Results were obtained from trophoblast cultures derived from human placentas of four independent pregnancies. All RT-PCR experiments were performed in triplicates and repeated at least three times. Statistical significance was determined by unpaired two-tailed Student's t-test (*) p < 0.05; (**) p < 0.01.


Human term placentas were dissected cytotrophoblast were extracted seeded . Quantitative RT - PCR of expression of metalloproteases MMP2 , MMP3 MMP9 ( I ) , in human trophoblast cultures on days 5 2 . Data information : Quantitative RT - PCR values are expressed as means + . Results were obtained from trophoblast cultures derived from human placentas of four independent pregnancies . RT - PCR experiments were performed in triplicates repeated at least three times . Statistical significance was determined by unpaired two - Student ' s t - ( * ) p < 0 . 05 ; ( * * ) p < 0 . 01 .

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

Human term placentas were dissected and cytotrophoblast cells were extracted and seeded. Quantitative RT-PCR analysis of expression of the metalloproteases MMP2, MMP3 and MMP9 (I), in human primary trophoblast cultures on days 5 and 2. Data information: Quantitative RT-PCR values are expressed as means +SEM. Results were obtained from trophoblast cultures derived from human placentas of four independent pregnancies. All RT-PCR experiments were performed in triplicates and repeated at least three times. Statistical significance was determined by unpaired two-tailed Student's t-test (*) p < 0.05; (**) p < 0.01.


( A ) Immunoblot of indicated in WT Cdkn2a - / - ; p53 - / - placentas , on day E14 . 5 ( two independent placentas out of four from each genotype are shown ) .

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

(A) Immunoblot analysis of the indicated proteins in WT and Cdkn2a-/-;p53-/- placentas, on day E14.5 (two independent placentas out of four from each genotype are shown).


RT - PCR of expression of Mmp2 in murine Cdkn2a - / - ; p53 - / - in WT placentas on day E14 . 5 ( n > = 4 fields of view for each genotype ) . Values are means + of at least three experimental repeats . Data information : Values are means + of at least three experimental repeats . Statistical significance was determined by unpaired two - Student ' s t - ( * ) p < 0 . 05 ; ( * * ) p < 0 . 01 ; ( * * * ) P < 0 . 001 .

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

RT-PCR analysis of expression of Mmp2 in murine Cdkn2a-/-;p53-/- and in WT placentas on day E14.5 (n>=4 fields of view for each genotype). Values are means + SEM of at least three experimental repeats. Data information: Values are means + SEM of at least three experimental repeats. Statistical significance was determined by unpaired two-tailed Student's t-test (*) p < 0.05; (**) p < 0.01; (***) P<0.001.


RT - PCR of expression of Mmp9 in murine Cdkn2a - / - ; p53 - / - in WT placentas on day E14 . 5 ( n > = 4 fields of view for each genotype ) . Values are means + of at least three experimental repeats . Data information : Values are means + of at least three experimental repeats . Statistical significance was determined by unpaired two - Student ' s t - ( * ) p < 0 . 05 ; ( * * ) p < 0 . 01 ; ( * * * ) P < 0 . 001 .

RT-PCR analysis of expression of Mmp9 in murine Cdkn2a-/-;p53-/- and in WT placentas on day E14.5 (n>=4 fields of view for each genotype). Values are means + SEM of at least three experimental repeats. Data information: Values are means + SEM of at least three experimental repeats. Statistical significance was determined by unpaired two-tailed Student's t-test (*) p < 0.05; (**) p < 0.01; (***) P<0.001.


( D , E ) Representative ( D ) ( E ) of in - situ gelatin zymography of labyrinth zone of murine WT Cdkn2a - / - ; p53 - / - placentas on day E14 . 5 . Scale bar , 50 μm . Data information : Values are means + of at least three experimental repeats . Statistical significance was determined by unpaired two - Student ' s t - ( * ) p < 0 . 05 ; ( * * ) p < 0 . 01 ; ( * * * ) P < 0 . 001 .

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

(D, E) Representative images (D) and quantification (E) of in-situ gelatin zymography of the labyrinth zone of murine WT and Cdkn2a-/-;p53-/- placentas on day E14.5. Scale bar, 50 μm. Data information: Values are means + SEM of at least three experimental repeats. Statistical significance was determined by unpaired two-tailed Student's t-test (*) p < 0.05; (**) p < 0.01; (***) P<0.001.


( F , G ) Representative ( F ) ( G ) of in - situ zymography of human normal third trimester post - partum placentas placentas from IUGR - complicated pregnancies of same gestational ( n > = 9 fields of view of normal IUGR placentas ) . Scale bar , 100 µm . Data information : Values are means + of at least three experimental repeats . Statistical significance was determined by unpaired two - Student ' s t - ( * ) p < 0 . 05 ; ( * * ) p < 0 . 01 ; ( * * * ) P < 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, G) Representative images (F) and quantification (G) of in-situ zymography of human normal third trimester post-partum placentas and placentas from IUGR-complicated pregnancies of the same gestational age (n>=9 fields of view of normal and IUGR placentas). Scale bar, 100 µm. Data information: Values are means + SEM of at least three experimental repeats. Statistical significance was determined by unpaired two-tailed Student's t-test (*) p < 0.05; (**) p < 0.01; (***) P<0.001.


( H ) Immunoblot of content of p - p65 , p65 , p - STAT3 STAT3 in normal placentas ( n = 4 ) placentas from IUGR - complicated pregnancies ( n = 4 ) . Each lane represents one independent placenta .

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

(H) Immunoblot analysis of the protein content of p-p65, p65, p-STAT3 and STAT3 in normal placentas (n=4) and placentas from IUGR-complicated pregnancies (n=4). Each lane represents one independent placenta.


( I ) Quantitative RT - PCR of expression of MMP2 , MMP9 , IL6 , JAK - STAT targets IFNAR1 IFNAR2 , in normal placentas placentas from IUGR - complicated pregnancies . Results are from four human normal placentas four placentas from IUGR - complicated pregnancies . Data information : Values are means + of at least three experimental repeats . Statistical significance was determined by unpaired two - Student ' s t - ( * ) p < 0 . 05 ; ( * * ) p < 0 . 01 ; ( * * * ) P < 0 . 001 .

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

(I) Quantitative RT-PCR analysis of expression of MMP2, MMP9, IL6, and the JAK-STAT targets IFNAR1 and IFNAR2, in normal placentas and placentas from IUGR-complicated pregnancies. Results are from four human normal placentas and four placentas from IUGR-complicated pregnancies. Data information: Values are means + SEM of at least three experimental repeats. Statistical significance was determined by unpaired two-tailed Student's t-test (*) p < 0.05; (**) p < 0.01; (***) P<0.001.


( B ) Bar graph summarizing efficiency of single RNAi knockdowns for 28 out of 30 TRs , their effects in reprogramming efficiency in comparison to control expressing scramble shRNA . efficiency of each TR ' s knockdown ( KD ) does not correlate with its effect in reprogramming efficiency . knockdown of 9 ( Class III ) out of 28 TRs decreased efficiency of reprogramming in a statistically significant manner ( unpaired two - Student ' s t - , p - value < 0 . 05 ) . Data are shown as mean ± of at least two independent experiments . Class I corresponds to one TR inhibiting reprogramming , whereas Class II corresponds to 18 TRs having a weak or no effect in repogramming . Shown are effects of 28 out of 30 TRs , because we did not succeed in knocking down Cphx Phox2a .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Bar graph summarizing the efficiency of single RNAi knockdowns for 28 out of the 30 TRs, and their effects in the reprogramming efficiency in comparison to control cells expressing scramble shRNA. The efficiency of each TR's knockdown (KD) does not correlate with its effect in reprogramming efficiency. The knockdown of 9 (Class III) out of the 28 TRs decreased the efficiency of the reprogramming in a statistically significant manner (unpaired two-tailed Student's t-test, p-value <0.05). Data are shown as mean ± SEM of at least two independent experiments. Class I corresponds to one TR inhibiting reprogramming, whereas Class II corresponds to 18 TRs having a weak or no effect in repogramming. Shown are the effects of 28 out of 30 TRs, because we did not succeed in knocking down Cphx and Phox2a.


( C ) Left : Shown is a graph depicting normalized mRNA expression of 9 TRs at indicated time points during reprogramming of MEFs . data were plotted after normalization with endogenous Gapdh using " ΔCt method " . grey vertical dashed depicts day 6 in which expression of nearly TRs peaks . Notice that y - axis is broken to accommodate large spread of expression . Right : Shown are western blots using antibodies specific for Irf6 , Tead4 , Taf1c , Rcan1 Tfap4 . Whole extracts were prepared from MEFs at day 6 of reprogramming run side - by - side with extracts from control samples ( day 0 ) . Gapdh , mTOR b - Tubulin were used as loading 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 O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) Left: Shown is a line graph depicting normalized mRNA expression levels of the 9 TRs at the indicated time points during the reprogramming of MEFs. The data were plotted after normalization with the endogenous Gapdh using the "ΔCt method". The grey vertical dashed line depicts day 6 in which the expression levels of nearly all TRs peaks. Notice that the y-axis is broken to accommodate the large spread of expression levels. Right: Shown are western blots using antibodies specific for Irf6, Tead4, Taf1c, Rcan1 and Tfap4. Whole cell extracts were prepared from MEFs at day 6 of reprogramming and run side-by-side with extracts from control samples (day 0). Gapdh, mTOR and b-Tubulin were used as loading controls.