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

O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O O O O B-CELL_LINE O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


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

O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O B-CELL_LINE O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O O B-GENEPROD O O B-SUBCELLULAR O B-SMALL_MOLECULE O O O O O O O

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


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

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O B-CELL_LINE O O O O O O O O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O O B-EXP_ASSAY O O B-EXP_ASSAY O O O B-EXP_ASSAY O O O O O O O O B-EXP_ASSAY O B-EXP_ASSAY O O O O O O O

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


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

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

O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O B-GENEPROD O O O O B-CELL_LINE O O O O B-GENEPROD O O O O B-EXP_ASSAY O O O B-EXP_ASSAY O O O B-EXP_ASSAY O O O B-GENEPROD O O O O O B-EXP_ASSAY O B-EXP_ASSAY O O O O O O O

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


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

O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O B-ORGANISM I-ORGANISM O O O O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-EXP_ASSAY O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE O

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


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

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O B-CELL_LINE O O O O O O O O O O O O B-GENEPROD O O O O O O O B-EXP_ASSAY O O B-EXP_ASSAY O O O B-EXP_ASSAY O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY O B-EXP_ASSAY O O O O O O O

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


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

O O O B-GENEPROD O O B-EXP_ASSAY O O O B-GENEPROD O B-GENEPROD O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O B-GENEPROD O O B-GENEPROD O O O O O O B-CELL_LINE O O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O B-EXP_ASSAY O O O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE O

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


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

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


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

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


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

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


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

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


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

O O O B-GENEPROD O B-CELL_LINE O O B-EXP_ASSAY O B-GENEPROD O B-SMALL_MOLECULE O O O O O O B-GENEPROD O O B-CELL_LINE O O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-GENEPROD O O B-CELL_LINE O O O B-GENEPROD O O B-CELL_LINE O O B-EXP_ASSAY O O O B-GENEPROD O O O O O O O O B-EXP_ASSAY O O O B-EXP_ASSAY O O O B-GENEPROD O O O O O B-EXP_ASSAY O B-EXP_ASSAY O O O O O O O

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


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

O O O O B-GENEPROD O B-GENEPROD O O B-CELL_LINE O O B-GENEPROD O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O B-CELL_LINE O O O O O O O O B-EXP_ASSAY O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O O O O O B-GENEPROD O O O B-CELL_LINE O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O B-EXP_ASSAY O O O B-GENEPROD O O O O O O O B-EXP_ASSAY O O O B-EXP_ASSAY O O O B-GENEPROD O O O O O B-EXP_ASSAY O B-EXP_ASSAY O O O O O O O

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


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

O O O O O B-GENEPROD O B-GENEPROD B-EXP_ASSAY O B-ORGANISM B-TISSUE O O O O B-TISSUE O O O O O O O B-TISSUE O O O O B-TISSUE O O O O B-GENEPROD O O O O B-ORGANISM O O O O O O O O O O O O O O B-GENEPROD B-EXP_ASSAY O O O O O B-EXP_ASSAY O O O B-GENEPROD O O O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O

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


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

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O O O B-CELL_LINE O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O B-EXP_ASSAY O O O B-ORGANISM O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


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

O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O B-CELL_LINE O O B-GENEPROD O O O O B-GENEPROD O O O O B-EXP_ASSAY O O O B-ORGANISM O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


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

O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O O O B-GENEPROD O O O O B-CELL_LINE O O B-GENEPROD O O O O B-GENEPROD O O O O B-EXP_ASSAY O O O B-ORGANISM O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


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

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


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

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


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

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


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

O O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O O B-CELL_LINE O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


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

B-GENEPROD O O O O B-CELL_LINE O O B-GENEPROD O O O O B-GENEPROD O O O O O B-EXP_ASSAY O B-ORGANISM O B-GENEPROD O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY O O O

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


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

B-GENEPROD O O O O B-CELL_LINE O O B-GENEPROD O O O O B-GENEPROD O O O O O B-EXP_ASSAY O B-ORGANISM O B-GENEPROD O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


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

B-GENEPROD O O O O B-CELL_LINE O O B-GENEPROD O O O O B-GENEPROD O O O O O B-EXP_ASSAY O B-ORGANISM O B-GENEPROD O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O

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


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

O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O B-CELL_TYPE O O B-GENEPROD O O O O B-GENEPROD O O O O B-EXP_ASSAY O O O B-ORGANISM O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


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

O O O B-GENEPROD O O B-CELL_TYPE O O B-GENEPROD O O O O B-GENEPROD O O O O O B-EXP_ASSAY O O O B-ORGANISM O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O

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


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

O O O B-GENEPROD O O B-CELL_TYPE O O B-GENEPROD O O O O B-GENEPROD O O O O O B-EXP_ASSAY O O O B-ORGANISM O O O O O B-EXP_ASSAY O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-EXP_ASSAY O O O B-GENEPROD O O O O B-EXP_ASSAY O O O B-GENEPROD I-GENEPROD I-GENEPROD O O

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


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

O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD B-SUBCELLULAR O O O B-GENEPROD O O B-CELL_TYPE O O B-GENEPROD O O O O B-GENEPROD O O O O B-EXP_ASSAY O B-ORGANISM O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O

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


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

O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O B-CELL_TYPE O O B-GENEPROD O O O O B-GENEPROD O O O O B-EXP_ASSAY O O O B-ORGANISM O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 cells ( Sirt5 + / + or Sirt5 - / - ) were infected with or without VSV - GFP viruses for 12 h , and viral infectivity was detected by fluorescence microscopy ( P )

O O B-GENEPROD O O B-CELL_TYPE O O B-GENEPROD O O O O B-GENEPROD O O O O O B-EXP_ASSAY O O O B-ORGANISM O B-GENEPROD O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY O O O

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


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

O O B-GENEPROD O O B-CELL_TYPE O O B-GENEPROD O O O O B-GENEPROD O O O O O B-EXP_ASSAY O O O B-ORGANISM O B-GENEPROD O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O

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


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

O O B-GENEPROD O O B-CELL_TYPE O O B-GENEPROD O O O O B-GENEPROD O O O O O B-EXP_ASSAY O O O B-ORGANISM O B-GENEPROD O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


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

B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD O O O O O O O B-GENEPROD O O B-CELL_TYPE O O B-GENEPROD O O O O B-GENEPROD O O O O B-EXP_ASSAY O O O B-ORGANISM O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


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

B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O O B-GENEPROD O O B-CELL_TYPE O O B-GENEPROD O O O O B-GENEPROD O O O O B-EXP_ASSAY O O O B-ORGANISM O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


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

B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O B-GENEPROD O O O O O O O B-GENEPROD O O B-CELL_TYPE O O B-GENEPROD O O O O B-GENEPROD O O O O B-EXP_ASSAY O O O B-ORGANISM O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


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

B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-GENEPROD O O B-CELL_TYPE O O B-GENEPROD O O O O B-GENEPROD O O O O B-EXP_ASSAY O O O B-ORGANISM O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


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

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


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

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

O O O B-ORGANISM B-EXP_ASSAY O B-GENEPROD B-EXP_ASSAY O B-ORGANISM B-TISSUE O O O O B-TISSUE O O O O O O O B-TISSUE O O O O B-TISSUE O O O O O B-ORGANISM O O O O O O B-ORGANISM O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O B-GENEPROD B-EXP_ASSAY O O O O O B-EXP_ASSAY O O O B-GENEPROD O O O B-EXP_ASSAY O O O O O O O O

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


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

O O O O O B-GENEPROD O B-GENEPROD B-EXP_ASSAY O B-ORGANISM B-TISSUE O O O O B-TISSUE O O O O B-ORGANISM B-EXP_ASSAY O O O O B-TISSUE O O O O B-TISSUE O O O O B-GENEPROD O O O O B-ORGANISM O B-GENEPROD O O O O B-GENEPROD O O O O O O O B-ORGANISM O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O B-GENEPROD B-EXP_ASSAY O O O O O B-EXP_ASSAY O O O B-GENEPROD O O O B-EXP_ASSAY O O O O O O O O

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


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

O B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-ORGANISM O O O O O O B-GENEPROD O O B-ORGANISM O O O O O O O O O O O O B-ORGANISM O O O O O O B-ORGANISM O O O O O O O

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


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

O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-TISSUE O O O B-ORGANISM O O O O O O B-GENEPROD O O B-ORGANISM O O O O O O O O B-ORGANISM O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


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

O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O B-EXP_ASSAY I-EXP_ASSAY O B-TISSUE O O B-ORGANISM O O O O O O O O O

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


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

B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O B-TISSUE O O O B-GENEPROD O O B-ORGANISM O B-GENEPROD O O O O B-GENEPROD O O O O O O O B-ORGANISM O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


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

B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD O O O O O O B-TISSUE O O O B-GENEPROD O O B-ORGANISM O B-GENEPROD O O O O B-GENEPROD O O O O O O O B-ORGANISM O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


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

O B-EXP_ASSAY I-EXP_ASSAY O B-ORGANISM O O O B-TISSUE O O O B-GENEPROD O O B-ORGANISM O B-GENEPROD O O O O B-GENEPROD O O O O O O O B-ORGANISM O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

B-ORGANISM B-TISSUE B-CELL_TYPE I-CELL_TYPE B-EXP_ASSAY O O O O O B-GENEPROD O O O B-ORGANISM O O O O O O O B-TISSUE O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O B-GENEPROD O O O O B-CELL_TYPE O B-GENEPROD O O O O B-GENEPROD O O O B-SMALL_MOLECULE B-SUBCELLULAR B-EXP_ASSAY O O O O B-EXP_ASSAY B-CELL_TYPE I-CELL_TYPE B-GENEPROD O O B-GENEPROD 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 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 .

B-ORGANISM B-TISSUE B-CELL_TYPE I-CELL_TYPE B-EXP_ASSAY O O O O O B-GENEPROD O O O O B-ORGANISM O O O O O O O B-TISSUE O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O B-GENEPROD O O O O O B-CELL_TYPE O B-GENEPROD O O O O B-GENEPROD O O O O B-SMALL_MOLECULE B-SUBCELLULAR B-EXP_ASSAY O O O O B-EXP_ASSAY B-CELL_TYPE I-CELL_TYPE O B-GENEPROD O O B-GENEPROD 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 ) 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 .

O O O O O O O B-ORGANISM O O O O B-TISSUE O O O O O O O O B-TISSUE O O B-DISEASE O O B-TISSUE O O O O O O O O O B-DISEASE B-TISSUE O O B-EXP_ASSAY O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O B-CELL_TYPE O O O O O O O O O O O B-DISEASE B-TISSUE O O O O O B-EXP_ASSAY B-CELL_TYPE 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-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O B-TISSUE O O O B-DISEASE O O B-TISSUE O O O O B-TISSUE O O O O O O O B-TISSUE 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 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 .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD B-EXP_ASSAY O B-DISEASE O O O O O B-TISSUE O O O O O O B-ORGANISM O O O B-DISEASE B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 ) .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-ORGANISM O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O B-ORGANISM O O O O O O O O B-GENEPROD O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-ORGANISM O O B-TISSUE O O O O O O O B-EXP_ASSAY O O O O O O O O O O O B-EXP_ASSAY O O O O B-ORGANISM O O O O O O B-TISSUE 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 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 .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-ORGANISM O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O B-ORGANISM O O O O O O O O B-GENEPROD O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-ORGANISM O O B-TISSUE O O O O O O O B-EXP_ASSAY O O O O O O O O B-EXP_ASSAY O O O O O B-TISSUE O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-EXP_ASSAY 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 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 .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-ORGANISM O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O B-ORGANISM O O O O O O O O B-GENEPROD O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-ORGANISM O O B-TISSUE O O O O O O O B-EXP_ASSAY O O O O O O O O B-EXP_ASSAY O O O O O B-TISSUE 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 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 .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-ORGANISM O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O B-ORGANISM O O O O O O O O B-GENEPROD O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-ORGANISM O O B-TISSUE O O O O O O O B-EXP_ASSAY O O O O O O O O O O B-EXP_ASSAY O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O B-TISSUE O O O O O O O B-ORGANISM O O O O O O O O O B-EXP_ASSAY O O O O O O B-EXP_ASSAY O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O O O B-GENEPROD O O O O B-GENEPROD O O O B-TISSUE O B-EXP_ASSAY O O O O O O O O O B-ORGANISM O O O O O O B-TISSUE B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-TISSUE I-TISSUE O B-ORGANISM B-GENEPROD O O O O O O O B-TISSUE O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O O O O O O B-TISSUE O O O O O O O O O B-TISSUE O O O O O O O O O O O O B-TISSUE I-TISSUE O O O O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O B-TISSUE O O O O O O B-EXP_ASSAY B-SUBCELLULAR O O O B-TISSUE O B-EXP_ASSAY O O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O B-TISSUE O O O O O B-TISSUE O O O B-TISSUE O O B-TISSUE O O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O B-TISSUE O B-TISSUE O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O B-TISSUE O O O O O O O O B-TISSUE O O O B-TISSUE O B-EXP_ASSAY O O B-TISSUE 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 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 .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-TISSUE I-TISSUE O B-ORGANISM B-GENEPROD O O O O O O O B-TISSUE O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-TISSUE O O O O O O O O O B-TISSUE O O O O O O O O O O O B-GENEPROD B-EXP_ASSAY O O B-TISSUE I-TISSUE O B-ORGANISM O O B-GENEPROD O O O O B-GENEPROD O O O O O B-GENEPROD O O O O B-GENEPROD O O O B-TISSUE O O O O O O O O O O B-TISSUE O O O O O O O O O B-EXP_ASSAY O B-GENEPROD B-EXP_ASSAY O O B-TISSUE I-TISSUE O O O O B-GENEPROD B-CELL_TYPE I-CELL_TYPE O O O O O O O B-TISSUE O O O O B-EXP_ASSAY O O O O O O O O O O B-TISSUE O O O O O O B-GENEPROD B-EXP_ASSAY O O B-GENEPROD B-EXP_ASSAY O O B-TISSUE I-TISSUE O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O 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 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 .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-TISSUE I-TISSUE O B-ORGANISM B-GENEPROD O O O O O O O B-TISSUE O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O B-TISSUE O O O O O O O O O B-TISSUE O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O O B-TISSUE I-TISSUE O O O B-GENEPROD O O O O O B-GENEPROD O O O O B-GENEPROD O O O B-TISSUE O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O B-TISSUE O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

B-ORGANISM O B-TISSUE O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY O B-SMALL_MOLECULE O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O O B-SMALL_MOLECULE 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 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 .

B-ORGANISM O B-TISSUE O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O B-CELL_TYPE B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM B-TISSUE O O O O

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 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 .

B-ORGANISM O B-TISSUE O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O B-ORGANISM B-GENEPROD O O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O B-CELL_TYPE 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 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 )

B-ORGANISM O B-TISSUE O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O O O B-ORGANISM B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

B-ORGANISM O B-TISSUE O O O B-CELL_TYPE I-CELL_TYPE O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY O O O O O O O O O B-CELL_TYPE O O O B-ORGANISM B-TISSUE O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O 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 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 .

B-ORGANISM O B-TISSUE O O O B-CELL_TYPE I-CELL_TYPE O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O B-CELL_TYPE O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY O O O O O O O O O B-CELL_TYPE O O O B-ORGANISM B-TISSUE O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O 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 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 .

B-ORGANISM O B-TISSUE O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O B-EXP_ASSAY O O O O O O B-ORGANISM B-CELL_TYPE I-CELL_TYPE O O O O O B-TISSUE 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 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 .

B-ORGANISM O B-TISSUE O O O B-CELL_TYPE I-CELL_TYPE O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-ORGANISM B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY O O O O O O O O O B-CELL_TYPE O O O B-ORGANISM B-TISSUE O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O 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 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 .

B-ORGANISM O B-TISSUE O O O B-CELL_TYPE I-CELL_TYPE O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O B-ORGANISM B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY O O O O O O O O O B-CELL_TYPE O O O B-ORGANISM B-TISSUE O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O 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 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 ) .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O B-TISSUE O O O O O O O O O B-TISSUE 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 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 .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY O B-GENEPROD O B-ORGANISM B-GENEPROD O O O O B-GENEPROD O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

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 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 .

O O O O O O B-EXP_ASSAY O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-TISSUE I-TISSUE O B-ORGANISM O O B-GENEPROD O O O O B-GENEPROD O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

O O O O O O B-EXP_ASSAY O O O O O O O O O O O O B-EXP_ASSAY O B-ORGANISM O O O O O O B-TISSUE O B-TISSUE O B-DISEASE O O O O O O O O O O O O O O O O O O O B-DISEASE B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O B-TISSUE O O O O O O B-TISSUE O B-DISEASE O O O O O O O O O O O O O O B-TISSUE 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 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 .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O O O B-TISSUE O B-TISSUE O B-DISEASE O O O O O O O O B-ORGANISM O B-TISSUE O O B-TISSUE O B-DISEASE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

O 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-EXP_ASSAY I-EXP_ASSAY O O O 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-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O B-GENEPROD 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 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 .

O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD 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.


( D ) Same as in ( B ) except MEFs were co - transduced with lentiviruses expressing all possible pairwise combinations of shRNAs for the nine TRs . Left : Heat map depicting the effects of paired KD combinations in the Reprogramming Efficiency ( RE % ) . Shown are the average REs values from at least two independent experiments . The REs of the double KDs were evaluated against the RE of the corresponding single KDs . Right : Alkaline Phosphatase ( AP ) stained cultures of terminal reprogrammed MEFs upon representative single and double KDs of our shRNA - based screens .

O O O O O O O O O O B-CELL_TYPE O O O O O B-ORGANISM O 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-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O 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-GENEPROD I-GENEPROD O B-GENEPROD O B-EXP_ASSAY O O O O B-CELL_TYPE O O O O O O O O O O O O O

(D) Same as in (B) except MEFs were co-transduced with lentiviruses expressing all possible pairwise combinations of shRNAs for the nine TRs. Left: Heat map depicting the effects of paired KD combinations in the Reprogramming Efficiency (RE%). Shown are the average REs values from at least two independent experiments. The REs of the double KDs were evaluated against the RE of the corresponding single KDs. Right: Alkaline Phosphatase (AP) stained cultures of terminal reprogrammed MEFs upon representative single and double KDs of our shRNA-based screens.


( A ) RNA in situ hybridization depicting the expression patterns of the nine TRs in days 3 and 6 of MEFs reprogramming . The boundaries of the early - iPSCs formations are indicated by a blue dashed line . Scale bars : 100 μm .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O B-CELL_TYPE O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O

(A) RNA in situ hybridization depicting the expression patterns of the nine TRs in days 3 and 6 of MEFs reprogramming. The boundaries of the early-iPSCs formations are indicated by a blue dashed line. Scale bars: 100 μm.


( C ) Bar graph showing the percentage of cells isolated from early - iPSCs colonies or outside of the colonies ( rest of population ) expressing each of the 9 TRs . Data are shown as mean ± SEM of at least two independent experiments . 0 . 05 < ( * ) p < 0 . 1 , * p < 0 . 05 , * * p < 0 . 01 by unpaired Student ' s t - test or Welch ' s t - test , when appropriate .

O O O O O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Bar graph showing the percentage of cells isolated from early-iPSCs colonies or outside of the colonies (rest of population) expressing each of the 9 TRs. Data are shown as mean ± SEM of at least two independent experiments. 0.05 < (*)p < 0.1, *p < 0.05, **p < 0.01 by unpaired Student's t-test or Welch's t-test, when appropriate.


( D ) Scatter plot depicting a visual unbiased representation of the percentile of cells expressing each of the nine TRs . Each dot depicts the percentage of cells expressing the indicated TRs in individual experiments . Data are shown as mean ± SEM of at least two independent experiments . Anova test p < 0 . 0001 .

O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O B-EXP_ASSAY O O O O O 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) Scatter plot depicting a visual unbiased representation of the percentile of cells expressing each of the nine TRs. Each dot depicts the percentage of cells expressing the indicated TRs in individual experiments. Data are shown as mean ± SEM of at least two independent experiments. Anova test p<0.0001.


( E ) Schematic representation of the statistical analysis estimating the probability of co - expression of the nine TRs per cell within the early - iPSCs colonies . Each row consists of 9 tiles representing a single cell and each column shows the expression status of each of the nine TRs ( shown with different color ) in 282 cells ( right side legend ) . The co - expression of gradually increasing numbers of TRs is grouped from the bottom to the top of the figure . The left part has been generated by estimating the percentages of expression of any single TR per cell as derived from single gene expression in single cells , while the right part depicts the co - expression of combinations of the nine TRs based on double and triple single - cell experiments .

O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O B-EXP_ASSAY O 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-EXP_ASSAY O O O O O 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-EXP_ASSAY O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O

(E) Schematic representation of the statistical analysis estimating the probability of co-expression of the nine TRs per cell within the early-iPSCs colonies. Each row consists of 9 tiles representing a single cell and each column shows the expression status of each of the nine TRs (shown with different color) in 282 cells (right side legend). The co-expression of gradually increasing numbers of TRs is grouped from the bottom to the top of the figure. The left part has been generated by estimating the percentages of expression of any single TR per cell as derived from single gene expression in single cells, while the right part depicts the co-expression of combinations of the nine TRs based on double and triple single-cell experiments.


( A - I ) Shown are ChIP - seq big wig files in the IGV browser depicting the binding of Oct4 ( Red ) , Sox2 ( Blue ) , Klf4 ( Green ) and c - Myc ( Black ) to putative regulatory regions in the nine TRs genes in MEFs undergoing reprogramming ( 18 hrs , D3 and D5 ) and in control MEFs , mESCs and miPSCs . All peaks have been normalized against input DNA . The relative sizes of the represented genomic loci and the corresponding TSSs are also indicated . The side color bar depicts the expression levels of each TR at the indicated time points .

O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O 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-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O

(A-I) Shown are ChIP-seq big wig files in the IGV browser depicting the binding of Oct4 (Red), Sox2 (Blue), Klf4 (Green) and c-Myc (Black) to putative regulatory regions in the nine TRs genes in MEFs undergoing reprogramming (18 hrs, D3 and D5) and in control MEFs, mESCs and miPSCs. All peaks have been normalized against input DNA. The relative sizes of the represented genomic loci and the corresponding TSSs are also indicated. The side color bar depicts the expression levels of each TR at the indicated time points.


( C ) Heatmap summarizing the effects of each of the nine TRs single knockdowns on the expression of the other TRs on day 6 ; The white color denotes no change in expression levels .

O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O B-EXP_ASSAY O O

(C) Heatmap summarizing the effects of each of the nine TRs single knockdowns on the expression of the other TRs on day 6; The white color denotes no change in expression levels.


( D ) Scatter plots depicting the extent of co - expression of either the broadly - or early - iPSCs - specifically expressed TRs in individual cells obtained from the early - iPSCs colonies or from the rest of the population ( left and right panel respectively ) . Each dot depicts the percentage of cells co - expressing a unique combination of two or three TRs . Data are shown as mean ± SEM of at least two independent experiments .

O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O B-CELL_TYPE O O B-EXP_ASSAY O O O O O O O O O B-CELL_TYPE O 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-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O

(D) Scatter plots depicting the extent of co-expression of either the broadly- or early-iPSCs-specifically expressed TRs in individual cells obtained from the early-iPSCs colonies or from the rest of the population (left and right panel respectively). Each dot depicts the percentage of cells co-expressing a unique combination of two or three TRs. Data are shown as mean ± SEM of at least two independent experiments.