A Doughnut chart for RNA - seq analysis showing the differentially regulated alternative splicing events ( ASEs ) and their corresponding genes in ESRP1 knockdown cells compared to control cells ; SE : skipped exon , RI : retained intron , MXE : mutually exclusive exons , A5SS : alternative 5 ' splice site , and A3SS : alternative 3 ' splice site ) . The " percent spliced in " ( PSI or ψ ) value was estimated . Differences in inclusion levels ( ∆ ψ ) between samples and their significance were calculated ( P < 0 . 05 and IΔψI > 0 . 1 ) . Circle 1 : all ASEs ; Circle 2 : all genes corresponding to all ASEs ; Circle 3 : significant ASEs ; Circle 4 : significant genes corresponding to significantly altered ASEs ; Circle 5 : significant ASEs with ESRP1 motifs ; Circle 6 : significant genes corresponding to significantly altered ASEs with ESRP1 motifs . ESRP - binding motifs were obtained from Dittmar et al . 31 [ ] and analyzed based on the following assumption : the presence of the motif in the downstream region of an exon results in inclusion of the exon , while upstream or intraexonic location leads to exclusion of the exon .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Doughnut chart for RNA-seq analysis showing the differentially regulated alternative splicing events (ASEs) and their corresponding genes in ESRP1 knockdown cells compared to control cells; SE: skipped exon, RI: retained intron, MXE: mutually exclusive exons, A5SS: alternative 5' splice site, and A3SS: alternative 3' splice site). The "percent spliced in" (PSI or ψ) value was estimated. Differences in inclusion levels (∆ψ) between samples and their significance were calculated (P<0.05 and IΔψI>0.1). Circle 1: all ASEs; Circle 2: all genes corresponding to all ASEs; Circle 3: significant ASEs; Circle 4: significant genes corresponding to significantly altered ASEs; Circle 5: significant ASEs with ESRP1 motifs; Circle 6: significant genes corresponding to significantly altered ASEs with ESRP1 motifs. ESRP-binding motifs were obtained from Dittmar et al.31[] and analyzed based on the following assumption: the presence of the motif in the downstream region of an exon results in inclusion of the exon, while upstream or intraexonic location leads to exclusion of the exon.


B Radar plot showing that the EMT gene signature was not altered at the gene level in the ESRP1 knockdown model ( LCC2 set ) . C Radar plot showing that the EMT gene signature was altered at the ASEs level ( SE ) in the ESRP1 knockdown model ( LCC2 set ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Radar plot showing that the EMT gene signature was not altered at the gene level in the ESRP1 knockdown model (LCC2 set). C Radar plot showing that the EMT gene signature was altered at the ASEs level (SE) in the ESRP1 knockdown model (LCC2 set).


D Radar plot showing that the EMT gene signature was not altered at the gene level in the ESRP1 knockdown model ( LCC9 set ) . E Radar plots showing that the EMT gene signature was altered at the ASEs level ( SE ) in the ESRP1 knockdown model ( LCC9 set ) .

D Radar plot showing that the EMT gene signature was not altered at the gene level in the ESRP1 knockdown model (LCC9 set). E Radar plots showing that the EMT gene signature was altered at the ASEs level (SE) in the ESRP1 knockdown model (LCC9 set).


B TCGA Splicegraphs for the EMT signature genes in ESRP1high versus ESRP1low tumors . A splice graph of the gene ' s exons is shaded based on expression level and shows the selected splice event outlined in red .

O O O O O O O O O O O O O O O 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 TCGA Splicegraphs for the EMT signature genes in ESRP1high versus ESRP1low tumors. A splice graph of the gene's exons is shaded based on expression level and shows the selected splice event outlined in red.


A Volcano plots showing the distribution of significant gene expression changes using the HTA 2 . 0 platform .

A Volcano plots showing the distribution of significant gene expression changes using the HTA 2.0 platform.


C Validation of the protein levels of FASN , SCD1 and PHGDH protein levels in ESRP1 knockdown versus control resistant cell lines using Western blot analysis . GAPDH was used as the loading control . The data are representative of three individual biological replicates .

O O O O O O O O O O O O O O O O O O O O O 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 Validation of the protein levels of FASN, SCD1 and PHGDH protein levels in ESRP1 knockdown versus control resistant cell lines using Western blot analysis. GAPDH was used as the loading control. The data are representative of three individual biological replicates.


( A ) Mass spectrometry ( MS ) assay of indicated electrophoretic bands in Coomassie blue staining ( left panel ) , and proteins pulled down by Flag - tagged SIRT5 from 293T cells were indicated on the right .

O O O O O O O O O O O O O O O O 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) Mass spectrometry (MS) assay of indicated electrophoretic bands in Coomassie blue staining (left panel), and proteins pulled down by Flag-tagged SIRT5 from 293T cells were indicated on the right.


( B ) Lysates from 293T cells transfected with HA - TRIM21 and Flag - SIRT5 or vector control ( - ) as indicated were immunoprecipitated ( IP ) using anti - Flag antibody , and bound proteins were analyzed by western blotting ( WB ) . Data information : All IP and WB data in this work otherwise indicated are representative of at least three independent experiments .

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

(B) Lysates from 293T cells transfected with HA-TRIM21 and Flag-SIRT5 or vector control (-) as indicated were immunoprecipitated (IP) using anti-Flag antibody, and bound proteins were analyzed by western blotting (WB). Data information: All IP and WB data in this work otherwise indicated are representative of at least three independent experiments.


( C , Lysates from BMDMs were immunoprecipitated with anti - SIRT5 ( C ) or control IgG antibodies as indicated and then analyzed by western blotting . Data information : All IP and WB data in this work otherwise indicated are representative of at least three independent experiments .

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

(C, Lysates from BMDMs were immunoprecipitated with anti-SIRT5 (C) or control IgG antibodies as indicated and then analyzed by western blotting. Data information: All IP and WB data in this work otherwise indicated are representative of at least three independent experiments.


D ) Lysates from BMDMs were immunoprecipitated with anti - TRIM21 ( D ) or control IgG antibodies as indicated and then analyzed by western blotting . Data information : All IP and WB data in this work otherwise indicated are representative of at least three independent experiments .

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

D) Lysates from BMDMs were immunoprecipitated with anti-TRIM21 (D) or control IgG antibodies as indicated and then analyzed by western blotting. Data information: All IP and WB data in this work otherwise indicated are representative of at least three independent experiments.


( E ) Bacterially expressed and purified Flag - SIRT5 were incubated with purified HA - TRIM21 proteins prior to pull - down with anti - HA antibody followed by western blot analysis . Data information : All WB data in this work otherwise indicated are representative of at least three independent experiments .

(E) Bacterially expressed and purified Flag-SIRT5 were incubated with purified HA-TRIM21 proteins prior to pull-down with anti-HA antibody followed by western blot analysis. Data information: All WB data in this work otherwise indicated are representative of at least three independent experiments.


( F ) Immunoblot ( IB ) analysis of whole - cell lysates ( WCLs ) and immunoprecipitates ( IPs ) derived from 293T cells transfected with Flag - SIRT5 together with HA - TRIM21 or indicated HA - tagged mutant TRIM21 constructs . Cells were treated with 10 μM MG132 for 4 hours before harvesting . HA , hemagglutinin .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Immunoblot (IB) analysis of whole-cell lysates (WCLs) and immunoprecipitates (IPs) derived from 293T cells transfected with Flag-SIRT5 together with HA-TRIM21 or indicated HA-tagged mutant TRIM21 constructs. Cells were treated with 10 μM MG132 for 4 hours before harvesting. HA, hemagglutinin.


( G ) IB analysis of WCLs derived from 293T cells transfected with increasing amounts of Flag - TRIM21 construct . Where indicated , 10 μM MG132 was added for 4 hours before harvesting .

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

(G) IB analysis of WCLs derived from 293T cells transfected with increasing amounts of Flag-TRIM21 construct. Where indicated, 10 μM MG132 was added for 4 hours before harvesting.


( H ) 293T cells transfected with control or TRIM21 siRNA for 48 hours were then treated with 100 ng / mL CHX for the indicated time period before harvesting . Protein expression was analyzed by western blotting ( left panel ) . The SIRT5 protein abundance was quantified by ImageJ and plotted as indicated ( right panel ) . Data information : All WB data in this work otherwise indicated are representative of at least three independent experiments .

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

(H) 293T cells transfected with control or TRIM21 siRNA for 48 hours were then treated with 100 ng/mL CHX for the indicated time period before harvesting. Protein expression was analyzed by western blotting (left panel). The SIRT5 protein abundance was quantified by ImageJ and plotted as indicated (right panel). Data information: All WB data in this work otherwise indicated are representative of at least three independent experiments.


( I ) IB analysis of WCLs derived from TRIM21 + / + and TRIM21 - / - BMDMs treated with 100 ng / ml CHX for the indicated time period before harvesting ( left panel ) . The SIRT5 protein abundance was quantified ( right panel ) .

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

(I) IB analysis of WCLs derived from TRIM21+/+ and TRIM21-/- BMDMs treated with 100 ng/ml CHX for the indicated time period before harvesting (left panel). The SIRT5 protein abundance was quantified (right panel).


( J ) IB analysis of WCLs derived from TRIM21 + / + and TRIM21 - / - BMDMs treated with 100 ng / mL LPS for the indicated time period before harvesting ( top panel ) . The SIRT5 protein abundance was quantified ( bottom panel ) .

(J) IB analysis of WCLs derived from TRIM21+/+ and TRIM21-/- BMDMs treated with 100 ng/mL LPS for the indicated time period before harvesting (top panel). The SIRT5 protein abundance was quantified (bottom panel).


( A ) Immunoblot analysis of WCLs and anti - GST immunoprecipitates derived from 293T cells transfected with the indicated plasmids for 48 hours , cells were then treated with the proteasome inhibitor MG132 for another 4 hours prior to harvesting .

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

(A) Immunoblot analysis of WCLs and anti-GST immunoprecipitates derived from 293T cells transfected with the indicated plasmids for 48 hours, cells were then treated with the proteasome inhibitor MG132 for another 4 hours prior to harvesting.


( B ) WCLs from TRIM21 + / + and TRIM21 - / - BMDMs treated with 100 ng / mL LPS for 4 hours were immunoprecipitated with anti - SIRT5 antibody and analyzed by western blotting . 10 μM MG132 was added for 4 hours before harvesting . Data information : All IP and WB data in this work otherwise indicated are representative of at least three independent experiments .

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

(B) WCLs from TRIM21+/+ and TRIM21-/- BMDMs treated with 100 ng/mL LPS for 4 hours were immunoprecipitated with anti-SIRT5 antibody and analyzed by western blotting. 10 μM MG132 was added for 4 hours before harvesting. Data information: All IP and WB data in this work otherwise indicated are representative of at least three independent experiments.


( C ) TRIM21 ubiquitinates SIRT5 in vitro . Immunopurified SIRT5 were incubated with purified recombinant TRIM21 or mutant TRIM21 proteins , E1 and E2 ( UBE2D2 ) , and ubiquitin as indicated in 30 ℃ for 90 min . The ubiquitination reaction products were resolved by SDS - PAGE and probed with the indicated antibodies .

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

(C) TRIM21 ubiquitinates SIRT5 in vitro. Immunopurified SIRT5 were incubated with purified recombinant TRIM21 or mutant TRIM21 proteins, E1 and E2 (UBE2D2), and ubiquitin as indicated in 30℃ for 90 min. The ubiquitination reaction products were resolved by SDS-PAGE and probed with the indicated antibodies.


( E ) Purified SIRT5 and mutant SIRT5 - 10KR proteins were incubated with purified E1 and E2 ( UBE2D2 ) , ubiquitin and / or purified recombinant TRIM21 or control mIgG or GST proteins as indicated at 30 ℃ for 90 min . SIRT5 ubiquitination and TRIM21 protein levels were analyzed by western blotting with the indicated antibodies . Data information : All WB data in this work otherwise indicated are representative of at least three independent experiments .

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

(E) Purified SIRT5 and mutant SIRT5-10KR proteins were incubated with purified E1 and E2 (UBE2D2), ubiquitin and/or purified recombinant TRIM21 or control mIgG or GST proteins as indicated at 30℃ for 90 min. SIRT5 ubiquitination and TRIM21 protein levels were analyzed by western blotting with the indicated antibodies. Data information: All WB data in this work otherwise indicated are representative of at least three independent experiments.


( A ) In vivo ubiquitination assay analysis of WCLs and GST - tagged immunoprecipitated products derived from 293T cells transfected with the indicated plasmids . 10 μM MG132 was added for 4 hours before harvesting .

O O O O O O O O O O O O O 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) In vivo ubiquitination assay analysis of WCLs and GST-tagged immunoprecipitated products derived from 293T cells transfected with the indicated plasmids. 10 μM MG132 was added for 4 hours before harvesting.


( B ) IB analysis of WCLs and anti - HA immunoprecipitates of 293T cells transfected with the indicated plasmids . 48 hours posttransfection , cells were treated with 10 μM MG132 for 4 hours before harvesting .

O O O O O O O O O O O O O O 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) IB analysis of WCLs and anti-HA immunoprecipitates of 293T cells transfected with the indicated plasmids. 48 hours posttransfection, cells were treated with 10 μM MG132 for 4 hours before harvesting.


( C ) Immunopurified SIRT5 proteins were incubated with purified recombinant E1 and ubiquitin ( Ub ) or mutant Ub - K48 or Ub - K63 in the presence or absence of purified E2 ( UBE2D2 ) and / or TRIM21 proteins as indicated in 30 ℃ for 90 min . The ubiquitination reaction products were resolved by SDS - PAGE and probed with the indicated antibodies .

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

(C) Immunopurified SIRT5 proteins were incubated with purified recombinant E1 and ubiquitin (Ub) or mutant Ub-K48 or Ub-K63 in the presence or absence of purified E2 (UBE2D2) and/or TRIM21 proteins as indicated in 30℃ for 90 min. The ubiquitination reaction products were resolved by SDS-PAGE and probed with the indicated antibodies.


( D ) Immunopurified SIRT5 proteins were incubated with purified recombinant TRIM21 , E1 and E2 ( UBE2D2 ) , and ubiquitin ( Ub ) or mutant Ub - K48R or Ub - K63R proteins as indicated for 45 min at 30 ℃ . The ubiquitination reaction products were resolved by SDS - PAGE and probed with the indicated antibodies .

(D) Immunopurified SIRT5 proteins were incubated with purified recombinant TRIM21, E1 and E2 (UBE2D2), and ubiquitin (Ub) or mutant Ub-K48R or Ub-K63R proteins as indicated for 45 min at 30℃. The ubiquitination reaction products were resolved by SDS-PAGE and probed with the indicated antibodies.


( E ) WCLs from TRIM21 + / + and TRIM21 - / - BMDMs treated with 100 ng / mL LPS for 4 hours were immunoprecipitated with anti - SIRT5 antibody and analyzed by western blotting using antibodies specifically against Ub - K48 and Ub - K63 . 10 μM MG132 was added for 4 hours before harvesting . Data information : All IP and WB data in this work otherwise indicated are representative of at least three independent experiments .

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

(E) WCLs from TRIM21+/+ and TRIM21-/- BMDMs treated with 100 ng/mL LPS for 4 hours were immunoprecipitated with anti-SIRT5 antibody and analyzed by western blotting using antibodies specifically against Ub-K48 and Ub-K63. 10 μM MG132 was added for 4 hours before harvesting. Data information: All IP and WB data in this work otherwise indicated are representative of at least three independent experiments.


( A ) Lysates from 293T cells transfected with HA - HAUSP and Flag - SIRT5 or vector control ( - ) as indicated were immunoprecipitated ( IP ) using anti - Flag antibody , and bound proteins were analyzed by western blotting ( WB ) . Data information : All IP and WB data in this work otherwise indicated are representative of at least three independent experiments .

(A) Lysates from 293T cells transfected with HA-HAUSP and Flag-SIRT5 or vector control (-) as indicated were immunoprecipitated (IP) using anti-Flag antibody, and bound proteins were analyzed by western blotting (WB). Data information: All IP and WB data in this work otherwise indicated are representative of at least three independent experiments.


( B , C ) Lysates from BMDMs were immunoprecipitated with anti - HAUSP ( B ) , anti - SIRT5 ( C ) or control IgG antibodies as indicated and then analyzed by western blotting . Data information : All IP and WB data in this work otherwise indicated are representative of at least three independent experiments .

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

(B, C) Lysates from BMDMs were immunoprecipitated with anti-HAUSP (B), anti-SIRT5 (C) or control IgG antibodies as indicated and then analyzed by western blotting. Data information: All IP and WB data in this work otherwise indicated are representative of at least three independent experiments.


( D ) IB analysis of WCLs and anti - SIRT5 immunoprecipitates of BMDMs treated with 100 ng / ml LPS for 4 hours . Cells were treated with 10 μM MG132 for 4 hours before harvesting .

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

(D) IB analysis of WCLs and anti-SIRT5 immunoprecipitates of BMDMs treated with 100 ng/ml LPS for 4 hours. Cells were treated with 10 μM MG132 for 4 hours before harvesting.


( E ) 293T cells were transfected with Flag - tagged HAUSP , mutant HAUSP - CS ( C223S ) or vector control as indicated for 48 hours . protein expression was determined by western blot analysis using indicated antibodies . Data information : All WB data in this work otherwise indicated are representative of at least three independent experiments .

(E) 293T cells were transfected with Flag-tagged HAUSP, mutant HAUSP-CS (C223S) or vector control as indicated for 48 hours. protein expression was determined by western blot analysis using indicated antibodies. Data information: All WB data in this work otherwise indicated are representative of at least three independent experiments.


( F ) IB analysis of WCLs derived from 293T cells transfected with HA - SIRT5 together with Flag - HAUSP or vector control constructs ( left panel ) . Cells were treated with 100 ng / ml CHX for the indicated time period prior to harvesting . The SIRT5 protein abundance was quantified ( right panel ) .

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

(F) IB analysis of WCLs derived from 293T cells transfected with HA-SIRT5 together with Flag-HAUSP or vector control constructs (left panel). Cells were treated with 100 ng/ml CHX for the indicated time period prior to harvesting. The SIRT5 protein abundance was quantified (right panel).


( G , H ) WCLs from BMDMs treated with increasing amounts of P5091 ( G ) or P22077 ( H ) in the presence or absence of 100 ng / mL LPS were subjected to IB analysis using indicated antibodies ( left panel ) . The SIRT5 protein abundance was quantified ( right panel ) .

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

(G, H) WCLs from BMDMs treated with increasing amounts of P5091(G) or P22077 (H) in the presence or absence of 100 ng/mL LPS were subjected to IB analysis using indicated antibodies (left panel). The SIRT5 protein abundance was quantified (right panel).


( B ) IB analysis of WCLs and anti - GST immunoprecipitates of 293T cells transfected with the indicated plasmids in the presence or absence of 100 µM P22077 for 24 hours . Cells were treated with 10 μM MG132 for 4 hours before harvesting .

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

(B) IB analysis of WCLs and anti-GST immunoprecipitates of 293T cells transfected with the indicated plasmids in the presence or absence of 100 µM P22077 for 24 hours. Cells were treated with 10 μM MG132 for 4 hours before harvesting.


( C , IB analysis of WCLs and GST - tagged immunoprecipitated products derived from 293T cells transfected with the indicated plasmids . 10 μM MG132 was added for 4 hours before harvesting .

O O O O O O O O O O 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, IB analysis of WCLs and GST-tagged immunoprecipitated products derived from 293T cells transfected with the indicated plasmids. 10 μM MG132 was added for 4 hours before harvesting.


D ) IB analysis of WCLs and GST - tagged immunoprecipitated products derived from 293T cells transfected with the indicated plasmids . 10 μM MG132 was added for 4 hours before harvesting .

O O O O O O O O O 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) IB analysis of WCLs and GST-tagged immunoprecipitated products derived from 293T cells transfected with the indicated plasmids. 10 μM MG132 was added for 4 hours before harvesting.


( E ) In vitro SIRT5 deubiquitination assay . Ubiquitinated SIRT5 was pulled down from 293T cells cotransfected HA - Ub , and then incubated with purified wild - type HAUSP or the catalytically inactive HAUSP mutant ( HAUSP - CS ) 30 ℃ for 90 min . The reaction products were resolved by SDS - PAGE and probed with the indicated antibodies .

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

(E) In vitro SIRT5 deubiquitination assay. Ubiquitinated SIRT5 was pulled down from 293T cells cotransfected HA-Ub, and then incubated with purified wild-type HAUSP or the catalytically inactive HAUSP mutant (HAUSP-CS) 30℃ for 90 min. The reaction products were resolved by SDS-PAGE and probed with the indicated antibodies.


( F ) IB analysis of WCLs and anti - SIRT5 immunoprecipitated products derived from BMDMs transfected with control or HAUSP siRNA for 48 hours . 10 μM MG132 was added for 6 hours before harvesting .

(F) IB analysis of WCLs and anti-SIRT5 immunoprecipitated products derived from BMDMs transfected with control or HAUSP siRNA for 48 hours. 10 μM MG132 was added for 6 hours before harvesting.


( G ) BMDMs transfected with control ( - ) or HAUSP siRNA for 48 hours were then treated with 100 ng / mL mM LPS for 4 hours , and 10 μM MG132 was added for 4 hours before harvesting . WCLs and anti - SIRT5 immunoprecipitated products were prepared and subjected to IB analysis using indicated antibodies .

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

(G) BMDMs transfected with control (-) or HAUSP siRNA for 48 hours were then treated with 100 ng/mL mM LPS for 4 hours, and 10 μM MG132 was added for 4 hours before harvesting. WCLs and anti-SIRT5 immunoprecipitated products were prepared and subjected to IB analysis using indicated antibodies.


( A ) IB analysis of WCLs and anti - Flag immunoprecipitated products derived from 293T cells transfected with the indicated plasmids using anti - Flag , anti - Acetyllysine , anti - Succinyllysine , anti - Glutaryllysine and anti - Malonyllysine

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

(A) IB analysis of WCLs and anti-Flag immunoprecipitated products derived from 293T cells transfected with the indicated plasmids using anti-Flag, anti-Acetyllysine, anti-Succinyllysine, anti-Glutaryllysine and anti-Malonyllysine antibodies


( B ) IB analysis of WCLs and anti - TRIM21 immunoprecipitated products derived from 293T cells transfected with control or SIRT5 siRNA using anti - Flag , anti - acetyl Lysine ( AcK ) , anti - succinyl Lysine ( SulK ) , anti - glutaryl Lysine ( GluK ) and anti - malonyl Lysine ( MalK ) antibodies .

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

(B) IB analysis of WCLs and anti-TRIM21 immunoprecipitated products derived from 293T cells transfected with control or SIRT5 siRNA using anti-Flag, anti-acetyl Lysine (AcK), anti-succinyl Lysine (SulK), anti-glutaryl Lysine (GluK) and anti-malonyl Lysine (MalK) antibodies.


( C ) IB analysis of WCLs and anti - Flag immunoprecipitated products derived from 293T cells transfected with the indicated plasmids . 10 μM MG132 was added for 4 hours before harvesting .

(C) IB analysis of WCLs and anti-Flag immunoprecipitated products derived from 293T cells transfected with the indicated plasmids. 10 μM MG132 was added for 4 hours before harvesting.


( D ) IB analysis of WCLs and anti - TRIM21 immunoprecipitated products derived from BMDMs treated 100 ng / mL LPS or left untreated for 4 hours . 10 μM MG132 was added for 4 hours before harvesting .

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

(D) IB analysis of WCLs and anti-TRIM21 immunoprecipitated products derived from BMDMs treated 100 ng/mL LPS or left untreated for 4 hours. 10 μM MG132 was added for 4 hours before harvesting.


( E ) Protein expression in SIRT5 + / + and SIRT5 - / - BMDMs was determined by western blot analysis . Data information : All WB data in this work otherwise indicated are representative of at least three independent experiments .

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

(E) Protein expression in SIRT5+/+ and SIRT5-/- BMDMs was determined by western blot analysis. Data information: All WB data in this work otherwise indicated are representative of at least three independent experiments.


( F , IB analysis of WCLs and anti - Flag immunoprecipitated products derived from 293T cells transfected with the indicated plasmids . 10 μM MG132 was added for 4 hours before harvesting .

(F, IB analysis of WCLs and anti-Flag immunoprecipitated products derived from 293T cells transfected with the indicated plasmids. 10 μM MG132 was added for 4 hours before harvesting.


G ) IB analysis of WCLs and anti - Flag immunoprecipitated products derived from 293T cells transfected with the indicated plasmids . 10 μM MG132 was added for 4 hours before harvesting .

G) IB analysis of WCLs and anti-Flag immunoprecipitated products derived from 293T cells transfected with the indicated plasmids. 10 μM MG132 was added for 4 hours before harvesting.


( H ) IB analysis of WCLs and anti - GST immunoprecipitated products derived from 293T cells transfected with the indicated plasmids in the presence or absence of 3 mM NAM for 24 hours . 10 μM MG132 was added for 4 hours before harvesting .

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

(H) IB analysis of WCLs and anti-GST immunoprecipitated products derived from 293T cells transfected with the indicated plasmids in the presence or absence of 3 mM NAM for 24 hours. 10 μM MG132 was added for 4 hours before harvesting.


( I ) Purified TRIM21 , TRIM21 - K351R and TRIM21 - K385R proteins were incubated with purified E1 and E2 ( UBE2D2 ) , ubiquitin and SIRT5 proteins as indicated at 30 ℃ for 90 min . The ubiquitination reaction products were resolved by SDS - PAGE and probed with the indicated antibodies .

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(I) Purified TRIM21, TRIM21-K351R and TRIM21-K385R proteins were incubated with purified E1 and E2 (UBE2D2), ubiquitin and SIRT5 proteins as indicated at 30℃ for 90 min. The ubiquitination reaction products were resolved by SDS-PAGE and probed with the indicated antibodies.


( J ) IB analysis of WCLs and anti - GST immunoprecipitated products derived from 293T cells transfected with the indicated plasmids . 10 μM MG132 was added for 4 hours before harvesting .

(J) IB analysis of WCLs and anti-GST immunoprecipitated products derived from 293T cells transfected with the indicated plasmids. 10 μM MG132 was added for 4 hours before harvesting.


( E ) Genotyping of TRIM21 - / - , SIRT5 - / - and TRIM21 - / - SIRT5 - / - mice . DNA derived from the tail of wild type and knockout animals was analyzed by PCR . Protein expression of TRIM21 and SIRT5 in BMDMs derived from mice were also analyzed by western blotting . Data information : Western blots in E represent three independent experiments .

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(E) Genotyping of TRIM21-/-, SIRT5-/- and TRIM21-/-SIRT5-/- mice. DNA derived from the tail of wild type and knockout animals was analyzed by PCR. Protein expression of TRIM21 and SIRT5 in BMDMs derived from mice were also analyzed by western blotting. Data information: Western blots in E represent three independent experiments.


wild - type ( WT , n = 5 ) , TRIM21 - / - ( n = 5 ) , SIRT5 - / - ( n = 5 ) , and TRIM21 - / - SIRT5 - / - ( n = 5 ) mice were orally administrated with 2 . 5 % ( w / v ) DSS in drinking water for 7 days and regular drinking water for another 2 days . Colon - length ( G ) changes were measured . Data information : Data are means ± SD . n = 5 biological replicates . P values were determined by unpaired two - tailed Student ' s t - tests . ns , not significant ; * * * p < 0 . 001 , * * * * p < 0 . 0001 .

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wild-type (WT, n=5), TRIM21-/- (n=5), SIRT5-/-(n=5), and TRIM21-/-SIRT5-/- (n=5) mice were orally administrated with 2.5% (w/v) DSS in drinking water for 7 days and regular drinking water for another 2 days. Colon-length (G) changes were measured. Data information: Data are means ± SD. n=5 biological replicates. P values were determined by unpaired two-tailed Student's t-tests. ns, not significant; ***p < 0.001, ****p < 0.0001.


A Genome - wide nucleosome maps were generated from the frequency distribution of the read midpoint positions of pair - end sequenced MNase - resistant fragments in the size range 138 - 161 bp ( spanning the nucleosome footprint ) . Figure shows a section of a nucleosome map taken from chromosome 12 derived from pluripotent iPSC ( pl - iPSC ) to show examples of nucleosomes selected as highly positioned , marked * .

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A Genome-wide nucleosome maps were generated from the frequency distribution of the read midpoint positions of pair-end sequenced MNase-resistant fragments in the size range 138-161 bp (spanning the nucleosome footprint). Figure shows a section of a nucleosome map taken from chromosome 12 derived from pluripotent iPSC (pl-iPSC) to show examples of nucleosomes selected as highly positioned, marked *.


B The distribution of positioned nucleosomes ( derived 138 - 161 bp size class ) and sub - nucleosome fragments ( derived 112 - 137 bp size class ) for pl - iPSC and iPSC - derived NPC ( NPC ) . Actual calculated numbers are presented above each column .

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B The distribution of positioned nucleosomes (derived 138-161 bp size class) and sub-nucleosome fragments (derived 112-137 bp size class) for pl-iPSC and iPSC-derived NPC (NPC). Actual calculated numbers are presented above each column.


D Average frequency distribution of nucleosome positions relative to each other . Data was aligned to each mapped nucleosome in the genome and plotted for a 600 bp window for pl - iPSC , NPC and the chronic myelogenous leukaemia ( Cml ) derived cell line , K562 . In all cases , a prominent single nucleosome is presented with only minor flanking peaks , indicating that the majority of nucleosomes do not occur as evenly spaced arrays in human cells .

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D Average frequency distribution of nucleosome positions relative to each other. Data was aligned to each mapped nucleosome in the genome and plotted for a 600 bp window for pl-iPSC, NPC and the chronic myelogenous leukaemia (Cml) derived cell line, K562. In all cases, a prominent single nucleosome is presented with only minor flanking peaks, indicating that the majority of nucleosomes do not occur as evenly spaced arrays in human cells.


E Distribution of nucleosome array sizes for pl - iPSC , NPC and K562 , calculated as the number of nucleosomes within a distance of 150 - 200 bp of each other . Bars show the number of nucleosomes arrayed as singletons ( 1 ) , pairs ( 2 ) , triplets ( 3 ) and greater than 3 ( > 3 ) , displayed as log10 values and the percentage distribution within a cell line is shown above the column .

E Distribution of nucleosome array sizes for pl-iPSC, NPC and K562, calculated as the number of nucleosomes within a distance of 150-200 bp of each other. Bars show the number of nucleosomes arrayed as singletons (1), pairs (2), triplets (3) and greater than 3 (>3), displayed as log10 values and the percentage distribution within a cell line is shown above the column.


A Bar chart showing the number of nucleosomes that map to selected chromatin states [ 20 ] . Active ( State 1 ) ; Insulator ( State 8 ) ; Repressed , Rep , ( State 12 ) and heterochromatin , Hetero ( State 13 ) , and to open chromatin ( based on ATAC - seq ) . Shown are the observed number of nucleosomes per chromatin state ( black ) and the expected number calculated assuming a random distribution across the genome ( grey ) .

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A Bar chart showing the number of nucleosomes that map to selected chromatin states [20]. Active (State 1); Insulator (State 8); Repressed, Rep, (State 12) and heterochromatin, Hetero (State 13), and to open chromatin (based on ATAC-seq). Shown are the observed number of nucleosomes per chromatin state (black) and the expected number calculated assuming a random distribution across the genome (grey).


A , B Nucleosome maps at and surrounding , the TSS region of the NANOG and GRIA1 genes in pl - iPSC ( upper , black ) and in NPC cells ( lower , grey ) . The TSS is indicated by a dashed line at chr 12 : 7 , 941 , 991 for NANOG and chr5 : 152 , 870 , 105 for GRIA1 . * indicates the position of a highly positioned nucleosome associated with the TSS of the active gene .

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A, B Nucleosome maps at and surrounding, the TSS region of the NANOG and GRIA1 genes in pl-iPSC (upper, black) and in NPC cells (lower, grey). The TSS is indicated by a dashed line at chr 12: 7,941,991 for NANOG and chr5: 152,870,105 for GRIA1. * indicates the position of a highly positioned nucleosome associated with the TSS of the active gene.


C Global frequency distribution of nucleosome distributions at TSS of protein - coding genes . Alignment of nucleosome maps centred on TSS using the non - redundant list of 66 , 047 mapped human protein - coding gene TSS . The features corresponding to the nucleosome free region ( NFR ) and - 1 to + 2 nucleosomes are marked .

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C Global frequency distribution of nucleosome distributions at TSS of protein-coding genes. Alignment of nucleosome maps centred on TSS using the non-redundant list of 66,047 mapped human protein-coding gene TSS. The features corresponding to the nucleosome free region (NFR) and -1 to +2 nucleosomes are marked.


D Genome - wide positioning of nucleosomes within ± 300 bp of a TSS of an active gene . The number of nucleosomes observed are plotted for both pl - iPSC and NPC cell types for gene sets selected either as uniquely expressed in pl - iPSC ( n = 3 , 833 ) or NPC ( n = 2 , 082 ) . If positioned nucleosomes are a strong predictor of gene activity , high numbers of nucleosomes would be expected for pluripotent - specific genes in pl - iPSC , but not NPC stages , and for NPC specific genes in NPC , but not pl - iPSC stages . No correlation between gene activity and positioned nucleosomes was observed .

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D Genome-wide positioning of nucleosomes within ± 300 bp of a TSS of an active gene. The number of nucleosomes observed are plotted for both pl-iPSC and NPC cell types for gene sets selected either as uniquely expressed in pl-iPSC (n=3,833) or NPC (n=2,082). If positioned nucleosomes are a strong predictor of gene activity, high numbers of nucleosomes would be expected for pluripotent-specific genes in pl-iPSC, but not NPC stages, and for NPC specific genes in NPC, but not pl-iPSC stages. No correlation between gene activity and positioned nucleosomes was observed.


A The average distribution of nucleosomes centred on the RE1 site ( n = 871 ) , spanning 2 Kb of flanking genomic sequence .

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A The average distribution of nucleosomes centred on the RE1 site (n=871), spanning 2 Kb of flanking genomic sequence.


B , C Aligned pair - end sequence read data were stratified into two size classes , ( B ) 122 - 137 bp and ( C ) 162 - 188 bp for pl - iPSC and NPC . Plots show the average distribution of nucleosomes centred at and surrounding the RE1 site ( n = 871 ) . Peaks correspond to the NRSF / REST complex , and nucleosomes in B and C respectively .

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B, C Aligned pair-end sequence read data were stratified into two size classes, (B) 122-137 bp and (C) 162-188 bp for pl-iPSC and NPC. Plots show the average distribution of nucleosomes centred at and surrounding the RE1 site (n=871). Peaks correspond to the NRSF/REST complex, and nucleosomes in B and C respectively.


D Cluster analysis of nucleosome positioning centred on the RE1 site ± 300 bp . RE1 sites were clustered based on the 122 - 137 bp mid - point sequence read values ( NRSF / REST protein complex ) from pl - iPSC , shown as a frequency distribution in upper left panel . The frequency distribution data for the same sites was plotted using 122 - 137 bp data from NPC ( upper right panel ) and 162 - 188 bp data ( nucleosomes ; bottom panels ) for the strongest ( solid line ) and weakest ( dotted line ) clusters .

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D Cluster analysis of nucleosome positioning centred on the RE1 site ± 300 bp. RE1 sites were clustered based on the 122-137 bp mid-point sequence read values (NRSF/REST protein complex) from pl-iPSC, shown as a frequency distribution in upper left panel. The frequency distribution data for the same sites was plotted using 122-137 bp data from NPC (upper right panel) and 162-188 bp data (nucleosomes; bottom panels) for the strongest (solid line) and weakest (dotted line) clusters.


A Average frequency distribution for sequence read mid - point data in the size range of 122 - 137 bp centred on the CTCF binding site ( n = 9516 ) . This shows a distinctive peak corresponding to the CTCF protein complex , which is present in pl - iPSC , but reduced in NPC . B Average frequency distribution for sequence read mid - point data centred on the CTCF binding site in the size range of 162 - 188 bp , corresponding to larger nucleosome footprints , for pl - iPSC and NPC cells . This shows that the pattern of nucleosome positioning is independent of the amount CTCF protein complex . Although positioned nucleosomes are retained flanking CTCF sites , their positions are shifted closer to the CTCF site and their spacing is altered . C Average frequency distribution for sequence read mid - point data centred on the CTCF site for pl - iPSC ( 162 - 188 bp ) and K562 ( total MNase - seq data ) cells . Positioned nucleosomes are retained flanking CTCF sites in K562 cells , but with shifted positions and altered spacing .

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A Average frequency distribution for sequence read mid-point data in the size range of 122-137 bp centred on the CTCF binding site (n=9516). This shows a distinctive peak corresponding to the CTCF protein complex, which is present in pl-iPSC, but reduced in NPC. B Average frequency distribution for sequence read mid-point data centred on the CTCF binding site in the size range of 162-188 bp, corresponding to larger nucleosome footprints, for pl-iPSC and NPC cells. This shows that the pattern of nucleosome positioning is independent of the amount CTCF protein complex. Although positioned nucleosomes are retained flanking CTCF sites, their positions are shifted closer to the CTCF site and their spacing is altered. C Average frequency distribution for sequence read mid-point data centred on the CTCF site for pl-iPSC (162-188 bp) and K562 (total MNase-seq data) cells. Positioned nucleosomes are retained flanking CTCF sites in K562 cells, but with shifted positions and altered spacing.


D Average frequency distribution for sequence read mid - point data in the size range of 122 - 137 bp for pl - iPSC and NPC cells centred on the 2 , 522 CTCF binding sites identified from NPC specific ChIP - seq data . A distinctive peak corresponding to the CTCF protein complex is present in the NPC state , but reduced to background levels in pl - iPSC . E Average frequency distribution for sequence read mid - point data centred on the CTCF binding sites in the size range of 162 - 188 bp , corresponding to larger nucleosome footprints , for pl - iPSC and NPC centred on the 2 , 522 CTCF binding sites identified from NPC specific ChIP - seq data . Nucleosome arrays are present in both cell states , independent of the CTCF protein complex . Nucleosomes in the CTCF sites in pl - iPSC tend to position closer to the CTCF site than in NPC . The data derived from the unique NPC CTCF sites has greater noise due to the smaller number of sites analysed .

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D Average frequency distribution for sequence read mid-point data in the size range of 122-137 bp for pl-iPSC and NPC cells centred on the 2,522 CTCF binding sites identified from NPC specific ChIP-seq data. A distinctive peak corresponding to the CTCF protein complex is present in the NPC state, but reduced to background levels in pl-iPSC. E Average frequency distribution for sequence read mid-point data centred on the CTCF binding sites in the size range of 162-188 bp, corresponding to larger nucleosome footprints, for pl-iPSC and NPC centred on the 2,522 CTCF binding sites identified from NPC specific ChIP-seq data. Nucleosome arrays are present in both cell states, independent of the CTCF protein complex. Nucleosomes in the CTCF sites in pl-iPSC tend to position closer to the CTCF site than in NPC. The data derived from the unique NPC CTCF sites has greater noise due to the smaller number of sites analysed.


C Example images of TIRFM endpoint colocalization assays . Top panels show CEN DNA ( Top - left panel , blue circles ) or CDEIIIMUT CEN DNA ( top - right panel , blue circles ) visualized in lysates containing Ndc10 - mCherry . Middle panels show the visualized Ndc10 - mCherry on CEN DNA ( middle - left panel ) or CDEIIIMUT DNA ( middle - right panel ) with colocalization shown in relation to blue DNA circles . Bottom panels show overlay of DNA channel ( magenta ) with Ndc10 - mCherry ( yellow ) on CEN DNA ( bottom - left panel ) or CDEIIIMUT DNA ( bottom - right panel ) . Scale bars 3μm . Graph shows the quantification of Ndc10 endpoint colocalization on CEN DNA and on CDEIIIMUT CEN DNA ( 70 ± 7 . 6 % , 1 . 6 ± 0 . 3 % respectively , avg ± s . d . n = 4 experiments , each examining ~ 1 , 000 DNA molecules from different extracts ) .

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C Example images of TIRFM endpoint colocalization assays. Top panels show CEN DNA (Top-left panel, blue circles) or CDEIIIMUT CEN DNA (top-right panel, blue circles) visualized in lysates containing Ndc10-mCherry. Middle panels show the visualized Ndc10-mCherry on CEN DNA (middle-left panel) or CDEIIIMUT DNA (middle-right panel) with colocalization shown in relation to blue DNA circles. Bottom panels show overlay of DNA channel (magenta) with Ndc10-mCherry (yellow) on CEN DNA (bottom-left panel) or CDEIIIMUT DNA (bottom-right panel). Scale bars 3μm. Graph shows the quantification of Ndc10 endpoint colocalization on CEN DNA and on CDEIIIMUT CEN DNA (70 ± 7.6%, 1.6 ± 0.3% respectively, avg ± s.d. n=4 experiments, each examining ~1,000 DNA molecules from different extracts).


E Example images of TIRFM endpoint colocalization imaging . Top panels show CEN DNA ( top - left panel , blue circles ) or CDEIIIMUT CEN DNA ( top - middle panel , blue circles ) visualized in lysates that included Cse4CENP ‑ A - GFP or CEN DNA in lysates that lacked Scm3HJURP ( scm3 - AID ) ( top - right panel , blue circles ) . Middle panels show Cse4CENP ‑ A - GFP visualized on CEN DNA ( middle - left panel ) or CDEIIIMUT CEN DNA ( center panel ) or on CEN DNA in lysates lacking Scm3HJURP ( scm3 - AID ) ( middle - right panel ) with colocalization shown in relation to blue DNA circles . Bottom panels show overlay of CEN DNA channel ( magenta ) with Cse4CENP ‑ A - GFP ( green ) on CEN DNA ( bottom - left panel ) or CDEIIIMUT DNA ( bottom - middle panel ) or on CEN DNA in lysates lacking Scm3HJURP ( scm3 - AID ) ( bottom - right panel ) . Scale bars 3μm . Graph shows quantification of observed colocalization of Cse4 on CEN DNA and on CDEIIIMUT CEN DNA or on CEN DNA in lysates that lacked Scm3HJURP ( 47 ± 2 . 9 % , 3 . 5 ± 3 . 0 % and 0 . 6 ± 0 . 4 % respectively , avg ± s . d . n = 4 experiments , each examining ~ 1 , 000 DNA molecules from different extracts ) .

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E Example images of TIRFM endpoint colocalization imaging. Top panels show CEN DNA (top-left panel, blue circles) or CDEIIIMUT CEN DNA (top-middle panel, blue circles) visualized in lysates that included Cse4CENP‑A-GFP or CEN DNA in lysates that lacked Scm3HJURP (scm3-AID) (top-right panel, blue circles). Middle panels show Cse4CENP‑A-GFP visualized on CEN DNA (middle-left panel) or CDEIIIMUT CEN DNA (center panel) or on CEN DNA in lysates lacking Scm3HJURP (scm3-AID) (middle-right panel) with colocalization shown in relation to blue DNA circles. Bottom panels show overlay of CEN DNA channel (magenta) with Cse4CENP‑A-GFP (green) on CEN DNA (bottom-left panel) or CDEIIIMUT DNA (bottom-middle panel) or on CEN DNA in lysates lacking Scm3HJURP (scm3-AID) (bottom-right panel). Scale bars 3μm. Graph shows quantification of observed colocalization of Cse4 on CEN DNA and on CDEIIIMUT CEN DNA or on CEN DNA in lysates that lacked Scm3HJURP (47 ± 2.9%, 3.5 ± 3.0% and 0.6 ± 0.4% respectively, avg ± s.d. n=4 experiments, each examining ~1,000 DNA molecules from different extracts).


B Estimated survival function plots of Kaplan - Meier analysis of CEN DNA residence lifetimes of Ndc10 ( magenta - median lifetime of 102 s , n = 3231 over 3 experiments of ~ 1000 DNA molecules using different extracts ) and Cse4CENP ‑ A ( red - median lifetime of 79 s , n = 1049 over 3 experiments of ~ 1000 DNA molecules using different extracts ) . Significant difference between ternary Ndc10 residence lifetime survival plots ( * * * ) compared to Cse4CENP ‑ A ( two - tailed p - value of 0 as determined by log - rank test ) . 95 % confidence intervals indicated ( dashed lines ) , right - censored lifetimes ( plus icons ) were included and unweighted in survival function estimates .

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B Estimated survival function plots of Kaplan-Meier analysis of CEN DNA residence lifetimes of Ndc10 (magenta - median lifetime of 102 s, n=3231 over 3 experiments of ~1000 DNA molecules using different extracts) and Cse4CENP‑A (red - median lifetime of 79 s, n=1049 over 3 experiments of ~1000 DNA molecules using different extracts). Significant difference between ternary Ndc10 residence lifetime survival plots (***) compared to Cse4CENP‑A (two-tailed p-value of 0 as determined by log-rank test). 95% confidence intervals indicated (dashed lines), right-censored lifetimes (plus icons) were included and unweighted in survival function estimates.


E Kaplan - Meier analysis of TernaryNdc10 residence lifetimes of Cse4CENP ‑ A on CEN DNA ( purple - median lifetime of 88 s , n = 539 over 3 experiments of ~ 1000 DNA molecules using different extracts ) and of Non - TernaryNdc10 Cse4CENP ‑ A residence lifetimes ( teal - median lifetime of 69 s , n = 612 over 3 experiments of ~ 1000 DNA molecules using different extracts ) . There is a significant difference ( * * ) between TernaryNdc10 and Non - TernaryNdc10 lifetime survival plots ( two - tailed p - value of 7 . 4e - 4 as determined by log - rank test ) . 95 % confidence intervals indicated ( dashed lines ) , right - censored lifetimes ( plus icons ) were included and unweighted in survival function estimates .

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E Kaplan-Meier analysis of TernaryNdc10 residence lifetimes of Cse4CENP‑A on CEN DNA (purple - median lifetime of 88 s, n=539 over 3 experiments of ~1000 DNA molecules using different extracts) and of Non-TernaryNdc10 Cse4CENP‑A residence lifetimes (teal - median lifetime of 69 s, n=612 over 3 experiments of ~1000 DNA molecules using different extracts). There is a significant difference (**) between TernaryNdc10 and Non-TernaryNdc10 lifetime survival plots (two-tailed p-value of 7.4e-4 as determined by log-rank test). 95% confidence intervals indicated (dashed lines), right-censored lifetimes (plus icons) were included and unweighted in survival function estimates.


G Timing of all non - simultaneous Ndc10 and Cse4CENP ‑ A ternary residence events . The proportion when Ndc10 precedes Cse4CENP ‑ A is 0 . 83 ± . 06 and when Cse4CENP ‑ A precedes Ndc10 is 0 . 17 ± . 06 ( avg ± s . d . , n = 504 over 3 experiments of ~ 1000 DNA molecules using different extracts ) . Simultaneous arrival defined as residence initiation within 5 s of each other and comprise . 07 ± . 04 of all ternary residence events ( avg ± s . d . , n = 35 over 3 experiments of ~ 1000 DNA molecules using different extracts ) .

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G Timing of all non-simultaneous Ndc10 and Cse4CENP‑A ternary residence events. The proportion when Ndc10 precedes Cse4CENP‑A is 0.83 ± .06 and when Cse4CENP‑A precedes Ndc10 is 0.17 ± .06 (avg ± s.d., n=504 over 3 experiments of ~1000 DNA molecules using different extracts). Simultaneous arrival defined as residence initiation within 5 s of each other and comprise .07 ± .04 of all ternary residence events (avg ± s.d., n=35 over 3 experiments of ~1000 DNA molecules using different extracts).


F Longer residence lifetimes are measured for Cse4CENP ‑ A after Scm3HJURP colocalizes . Estimated survival function plots of Kaplan - Meier analysis of the lifetimes of TernaryScm3 Cse4CENP ‑ A residences on CEN DNA ( purple - median lifetime of 108 s , n = 305 over 3 experiments of ~ 1000 DNA molecules using different extracts ) and Non - TernaryScm3 Cse4CENP ‑ A residences on CEN DNA ( green - of 79 s , n = 1230 over 3 experiments of ~ 1000 DNA molecules using different extracts ) . Significant difference ( * * ) between TernaryScm3 and Non - TernaryScm3 lifetime survival plots ( two - tailed p - value of 2 . 1e - 3 as determined by log - rank test ) . 95 % confidence intervals indicated ( dashed lines ) , right - censored lifetimes ( plus icons ) were included and unweighted in survival function estimates .

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

F Longer residence lifetimes are measured for Cse4CENP‑A after Scm3HJURP colocalizes. Estimated survival function plots of Kaplan-Meier analysis of the lifetimes of TernaryScm3 Cse4CENP‑A residences on CEN DNA (purple - median lifetime of 108 s, n=305 over 3 experiments of ~1000 DNA molecules using different extracts) and Non-TernaryScm3 Cse4CENP‑A residences on CEN DNA (green - of 79 s, n=1230 over 3 experiments of ~1000 DNA molecules using different extracts). Significant difference (**) between TernaryScm3 and Non-TernaryScm3 lifetime survival plots (two-tailed p-value of 2.1e-3 as determined by log-rank test). 95% confidence intervals indicated (dashed lines), right-censored lifetimes (plus icons) were included and unweighted in survival function estimates.


B Kinetochores were assembled on beads containing either a 250 bp single - tether , a 250 bp double - tether or a 250 bp CDEIIImut CEN DNA . They were then treated with MNase , and the remaining DNA was visualized on an agarose gel . Black arrow indicates Cse4CENP ‑ A nucleosome protected DNA ( ~ 150 bp ) ; white arrow indicates theoretical location of undigested template DNA ( 250 bp ) .

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

B Kinetochores were assembled on beads containing either a 250 bp single-tether, a 250 bp double-tether or a 250 bp CDEIIImut CEN DNA. They were then treated with MNase, and the remaining DNA was visualized on an agarose gel. Black arrow indicates Cse4CENP‑A nucleosome protected DNA (~150 bp); white arrow indicates theoretical location of undigested template DNA (250 bp).


C Example images of TIRFM endpoint colocalization assays . Top panels show Cse4CENP ‑ A / Ndc10 ternary colocalizations visualized on single - tethered CEN DNA ( top - left panel ) or on double - tethered CEN DNA ( top - right panel ) with colocalization shown in relation to Ndc10 in yellow circles . Bottom panels show overlay of Ndc10 channel ( magenta ) with Cse4CENP ‑ A ( green ) . Scale bars 3 μm . D Quantification of observed ternary colocalization of Cse4CENP ‑ A with Ndc10 ( right ) on single - tethered CEN DNA containing Ndc10 ( 19 . 9 ± 2 . 4 % , avg ± s . d . n = 4 experiments , each examining ~ 1 , 000 DNA molecules from different extracts ) and on double - tethered CEN DNA ( 7 . 3 ± 1 . 1 % , avg ± s . d . n = 4 experiments , each examining ~ 1 , 000 DNA molecules from different extracts ) .

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

C Example images of TIRFM endpoint colocalization assays. Top panels show Cse4CENP‑A/Ndc10 ternary colocalizations visualized on single-tethered CEN DNA (top-left panel) or on double-tethered CEN DNA (top-right panel) with colocalization shown in relation to Ndc10 in yellow circles. Bottom panels show overlay of Ndc10 channel (magenta) with Cse4CENP‑A (green). Scale bars 3 μm. D Quantification of observed ternary colocalization of Cse4CENP‑A with Ndc10 (right) on single-tethered CEN DNA containing Ndc10 (19.9± 2.4%, avg ± s.d. n=4 experiments, each examining ~1,000 DNA molecules from different extracts) and on double-tethered CEN DNA (7.3 ± 1.1%, avg ± s.d. n=4 experiments, each examining ~1,000 DNA molecules from different extracts).


F Residence times for Cse4CENP ‑ A on double - tethered CEN DNAs are shorter than on single - tethered CEN DNAs and equivalent to those on non - functional mutant CEN DNAs . Estimated survival function plots of Kaplan - Meier analysis of ternary residences of Cse4CENP ‑ A with Ndc10 on single - tethered CEN DNA ( blue - median lifetime of 79 s , n = 675 over 3 experiments of ~ 1000 DNA molecules using different extracts ) , on double - tethered CEN DNA ( red - median lifetime of 67 sec , n = 3175 over 3 experiments of ~ 1000 DNA molecules using different extracts ) or Cse4CENP ‑ A lifetimes on CDEIII - 80 bp CEN DNA ( purple - median lifetime of 71 sec , n = 901 over 3 experiments of ~ 1000 DNA molecules using different extracts ) . No significant difference ( n . s . ) between double - tethered 250 bp CEN DNA and 80 bp CDEIII CEN DNA lifetime survival plots ( two - tailed p - value of 0 . 06 as determined by log - rank test ) . Significant difference ( * * * ) between double - tethered 250 bp CEN DNA and 250 bp single - tether DNA lifetime survival plots ( two - tailed p - value of 1 . 4e - 10 as determined by log - rank test ) . 95 % confidence intervals indicated ( dashed lines ) , right - censored lifetimes ( plus icons ) were included and unweighted in survival function estimates .

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

F Residence times for Cse4CENP‑A on double-tethered CEN DNAs are shorter than on single-tethered CEN DNAs and equivalent to those on non-functional mutant CEN DNAs. Estimated survival function plots of Kaplan-Meier analysis of ternary residences of Cse4CENP‑A with Ndc10 on single-tethered CEN DNA (blue - median lifetime of 79 s, n=675 over 3 experiments of ~1000 DNA molecules using different extracts), on double-tethered CEN DNA (red - median lifetime of 67 sec, n=3175 over 3 experiments of ~1000 DNA molecules using different extracts) or Cse4CENP‑A lifetimes on CDEIII-80 bp CEN DNA (purple - median lifetime of 71 sec, n=901 over 3 experiments of ~1000 DNA molecules using different extracts). No significant difference (n.s.) between double-tethered 250 bp CEN DNA and 80 bp CDEIII CEN DNA lifetime survival plots (two-tailed p-value of 0.06 as determined by log-rank test). Significant difference (***) between double-tethered 250 bp CEN DNA and 250 bp single-tether DNA lifetime survival plots (two-tailed p-value of 1.4e-10 as determined by log-rank test). 95% confidence intervals indicated (dashed lines), right-censored lifetimes (plus icons) were included and unweighted in survival function estimates.


B Immunoblot analysis of whole cell extracts from WT , chl4 - K13S and okp1 - AID cells using indicated antibodies .

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

B Immunoblot analysis of whole cell extracts from WT, chl4-K13S and okp1-AID cells using indicated antibodies.


C Example images of TIRFM endpoint colocalization assays . Top panels show visualized Cse4CENP ‑ A - GFP on CEN DNA in extracts from chl4 - K13S ( top - left panel ) or auxin - treated okp1 - AID strains ( okp1 - AID , top - right panel ) with colocalization shown in relation to identified CEN DNA in blue circles . Bottom panels show overlay of CEN DNA channel ( magenta ) with Cse4CENP ‑ A - GFP ( green ) , Scale bars 3 μm . D Quantification of Cse4CENP ‑ A endpoint colocalization with CEN DNA in extracts from WT , chl4 - K13S , or okp1 - AID genetic backgrounds ( 19 ± 1 . 1 % , 8 ± 0 . 7 % , 5 ± 0 . 9 % , avg ± s . d . n = 4 experiments , each examining ~ 1 , 000 DNA molecules from different extracts ) .

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

C Example images of TIRFM endpoint colocalization assays. Top panels show visualized Cse4CENP‑A-GFP on CEN DNA in extracts from chl4-K13S (top-left panel) or auxin-treated okp1-AID strains (okp1-AID, top-right panel) with colocalization shown in relation to identified CEN DNA in blue circles. Bottom panels show overlay of CEN DNA channel (magenta) with Cse4CENP‑A-GFP (green), Scale bars 3 μm. D Quantification of Cse4CENP‑A endpoint colocalization with CEN DNA in extracts from WT, chl4-K13S, or okp1-AID genetic backgrounds (19 ± 1.1%, 8 ± 0.7%, 5 ± 0.9%, avg ± s.d. n=4 experiments, each examining ~1,000 DNA molecules from different extracts).


F Kaplan - Meier analysis of Cse4CENP ‑ A residence lifetimes on CEN DNA in extracts from WT ( blue - median lifetime of 82 s , n = 1419 over 3 experiments of ~ 1000 DNA molecules using different extracts ) , chl4 - K13S ( red - median lifetime of 70 s , n = 546 over 3 experiments of ~ 1000 DNA molecules using different extracts ) and okp1 - AID ( purple - median lifetime of 61 s , n = 348 over 3 experiments of ~ 1000 DNA molecules using different extracts ) genetic backgrounds . Significant difference ( * * * ) between WT extract and chl4 - K13S extract residence lifetime plots ( two - tailed p - value of 3 . 4e - 5 as determined by log - rank test ) . No significant difference ( n . s . ) between chl4 - K13S and okp1 - AID residence lifetimes in ( two - tailed p - value of . 40 as determined by log - rank test ) . 95 % confidence intervals indicated ( dashed lines ) , right - censored lifetimes ( plus icons ) were included and unweighted in survival function estimates .

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

F Kaplan-Meier analysis of Cse4CENP‑A residence lifetimes on CEN DNA in extracts from WT (blue - median lifetime of 82 s, n=1419 over 3 experiments of ~1000 DNA molecules using different extracts), chl4-K13S (red - median lifetime of 70 s, n=546 over 3 experiments of ~1000 DNA molecules using different extracts) and okp1-AID (purple - median lifetime of 61 s, n=348 over 3 experiments of ~1000 DNA molecules using different extracts) genetic backgrounds. Significant difference (***) between WT extract and chl4-K13S extract residence lifetime plots (two-tailed p-value of 3.4e-5 as determined by log-rank test). No significant difference (n.s.) between chl4-K13S and okp1-AID residence lifetimes in (two-tailed p-value of .40 as determined by log-rank test). 95% confidence intervals indicated (dashed lines), right-censored lifetimes (plus icons) were included and unweighted in survival function estimates.


B Example images of TIRFM endpoint colocalization assays . Visualized Cse4CENP ‑ A - GFP on unstable1 CDEII - mutant DNA ( top panel ) or on v - unstable1 CDEII - mutant DNA ( bottom panel ) with colocalization shown in relation to identified CEN DNA in blue circles . Scale bars 3 μm . C Quantification of endpoint colocalization of Cse4CENP ‑ A on CEN , unstable1 , unstable2 , v - unstable1 , and v - unstable2 CEN DNA ( 19 . 3 ± 1 . 1 % , 13 . 8 ± 0 . 6 % , 7 . 8 ± 0 . 5 % , 5 . 5 ± 1 . 0 % , 5 . 5 ± 1 . 1 % , avg ± s . d . n = 4 experiments , each examining ~ 1 , 000 DNA molecules from different extracts ) .

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

B Example images of TIRFM endpoint colocalization assays. Visualized Cse4CENP‑A-GFP on unstable1 CDEII-mutant DNA (top panel) or on v-unstable1 CDEII-mutant DNA (bottom panel) with colocalization shown in relation to identified CEN DNA in blue circles. Scale bars 3 μm. C Quantification of endpoint colocalization of Cse4CENP‑A on CEN, unstable1, unstable2, v-unstable1, and v-unstable2 CEN DNA (19.3 ± 1.1%, 13.8 ± 0.6%, 7.8 ± 0.5%, 5.5 ± 1.0%, 5.5 ± 1.1%, avg ± s.d. n=4 experiments, each examining ~1,000 DNA molecules from different extracts).


E Very unstable CDEII mutants have reduced average Cse4CENP ‑ A binding when compared to unstable counterparts . Average residences of Cse4CENP ‑ A per CEN DNA ( left ) on CEN , unstable1 , unstable2 , v - unstable1 , and v - unstable2 CEN DNA ( 1 . 03 ± 0 . 12 , 0 . 88 ± 0 . 22 , 0 . 58 ± 0 . 41 , 0 . 61 ± 0 . 18 , 0 . 41 ± 0 . 09 , avg ± s . e . m . n = 3 experiments of ~ 1000 DNA molecules using different extracts ) and average residences of Scm3HJURP per CEN DNA ( right ) on CEN , unstable1 , unstable2 , v - unstable1 , and v - unstable2 CEN DNA ( 0 . 57 ± 0 . 07 , 0 . 35 ± 0 . 10 , 0 . 52 ± 0 . 16 , 0 . 53 ± 0 . 12 , 0 . 46 ± 0 . 02 , avg ± s . e . m . n = 3 experiments of ~ 1000 DNA molecules using different extracts ) .

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

E Very unstable CDEII mutants have reduced average Cse4CENP‑A binding when compared to unstable counterparts. Average residences of Cse4CENP‑A per CEN DNA (left) on CEN, unstable1, unstable2, v-unstable1, and v-unstable2 CEN DNA (1.03 ± 0.12, 0.88 ± 0.22, 0.58 ± 0.41, 0.61 ± 0.18, 0.41 ± 0.09, avg ± s.e.m. n=3 experiments of ~1000 DNA molecules using different extracts) and average residences of Scm3HJURP per CEN DNA (right) on CEN, unstable1, unstable2, v-unstable1, and v-unstable2 CEN DNA (0.57 ± 0.07, 0.35 ± 0.10, 0.52 ± 0.16, 0.53 ± 0.12, 0.46 ± 0.02, avg ± s.e.m. n=3 experiments of ~1000 DNA molecules using different extracts).


TP53INP1 - KO ( − / − ) and WT ( + / + ) male mice were subjected to a high - fat diet ( HFD , 60 % fat ) or a control diet ( CTRL ) for 16 weeks . Mice drank tap water or tap water supplemented with NAC ( 10 mg / ml or 1 % ) . Curves show mice body weight recorded every week . CTRL : P ( − / − versus + / + ; t = 8w ) = 0 . 047 ; P ( − / − versus + / + ; t = 9w ) = 0 . 023 . HFD : P ( − / − versus + / + ; t = 7w ) = 0 . 039 ; P ( − / − versus + / + ; t = 8w ) = 0 . 029 ; P ( − / − versus + / + ; t = 9w ) = 0 . 021 ; P ( − / − versus + / + ; t = 10w ) = 0 . 014 ; P ( − / − versus + / + ; t = 11w ) = 0 . 0046 ; P ( − / − versus + / + ; t = 12w ) = 0 . 0028 ; P ( − / − versus + / + ; t = 13w ) = 0 . 0025 ; P ( − / − versus + / + ; t = 14w ) = 0 . 00051 ; P ( − / − versus + / + ; t = 15w ) = 0 . 00027 ; P ( − / − versus + / + ; t = 16w ) = 0 . 00013 .

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

TP53INP1-KO (−/−) and WT (+/+) male mice were subjected to a high-fat diet (HFD, 60% fat) or a control diet (CTRL) for 16 weeks. Mice drank tap water or tap water supplemented with NAC (10 mg/ml or 1%).Curves show mice body weight recorded every week. CTRL: P (−/− versus +/+; t = 8w) = 0.047; P (−/− versus +/+; t = 9w) = 0.023. HFD: P (−/− versus +/+; t = 7w) = 0.039; P (−/− versus +/+; t = 8w) = 0.029; P (−/− versus +/+; t = 9w) = 0.021; P (−/− versus +/+; t = 10w) = 0.014; P (−/− versus +/+; t = 11w) = 0.0046; P (−/− versus +/+; t = 12w) = 0.0028; P (−/− versus +/+; t = 13w) = 0.0025; P (−/− versus +/+; t = 14w) = 0.00051; P (−/− versus +/+; t = 15w) = 0.00027; P (−/− versus +/+; t = 16w) = 0.00013.


At the end of protocol , mice were sacrificed ; liver and epididymal and renal fat masses were taken and weighed . Histograms show organ weight . Liver : P ( − / − versus + / + ; HFD ) = 0 . 014 ; P ( HFD versus CTRL ; + / + ) = 0 . 00063 ; P ( HFD versus CTRL ; − / − ) = 0 . 0010 ; P ( HFD versus CTRL ; + / + NAC ) = 0 . 034 ; P ( HFD versus CTRL ; − / − NAC ) = 0 . 027 ; P ( NAC versus no NAC ; − / − HFD ) = 0 . 014 . Epididymal fat mass : P ( − / − versus + / + ; HFD ) = 0 . 011 ; P ( HFD versus CTRL ; + / + ) = 0 . 028 ; P ( HFD versus CTRL ; − / − ) = 0 . 000017 ; P ( HFD versus CTRL ; + / + NAC ) = 0 . 019 ; P ( HFD versus CTRL ; − / − NAC ) = 0 . 0054 ; P ( NAC versus no NAC ; + / + CTRL ) = 0 . 037 ; P ( NAC versus no NAC ; − / − HFD ) = 0 . 0025 . Renal fat mass : P ( − / − versus + / + ; HFD ) = 0 . 0041 ; P ( HFD versus CTRL ; + / + ) = 0 . 028 ; P ( HFD versus CTRL ; − / − ) = 0 . 000013 ; P ( HFD versus CTRL ; + / + NAC ) = 0 . 019 ; P ( HFD versus CTRL ; − / − NAC ) = 0 . 0078 ; P ( NAC versus no NAC ; − / − HFD ) = 0 . 0047 .

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

At the end of protocol, mice were sacrificed; liver and epididymal and renal fat masses were taken and weighed. Histograms show organ weight. Liver: P (−/− versus +/+; HFD) = 0.014; P (HFD versus CTRL; +/+) = 0.00063; P (HFD versus CTRL; −/−) = 0.0010; P (HFD versus CTRL; +/+ NAC) = 0.034; P (HFD versus CTRL; −/− NAC) = 0.027; P (NAC versus no NAC; −/− HFD) = 0.014. Epididymal fat mass: P (−/− versus +/+; HFD) = 0.011; P (HFD versus CTRL; +/+) = 0.028; P (HFD versus CTRL; −/−) = 0.000017; P (HFD versus CTRL; +/+ NAC) = 0.019; P (HFD versus CTRL; −/− NAC) = 0.0054; P (NAC versus no NAC; +/+ CTRL) = 0.037; P (NAC versus no NAC; −/− HFD) = 0.0025. Renal fat mass: P (−/− versus +/+; HFD) = 0.0041; P (HFD versus CTRL; +/+) = 0.028; P (HFD versus CTRL; −/−) = 0.000013; P (HFD versus CTRL; +/+ NAC) = 0.019; P (HFD versus CTRL; −/− NAC) = 0.0078; P (NAC versus no NAC; −/− HFD) = 0.0047.


A , B Histograms show bloodglucose ( A ) or plasmainsulin ( B ) levels of 6 - h - fasted mice at the beginning ( Week 0 ) and / or at the end of the protocol ( Week 16 ) . Fasting bloodglucose week 16 : P ( − / − versus + / + ; HFD ) = 0 . 0052 ; P ( CTRL versus HFD ; − / − ) = 0 . 000081 ; P ( NAC versus no NAC ; − / − HFD ) = 0 . 0019 ; P ( w16 versus w0 ; + / + CTRL ) = 0 . 012 ; P ( w16 versus w0 ; − / − CTRL ) = 0 . 050 ; P ( w16 versus w0 ; − / − HFD ) = 0 . 0023 . Fasting plasmainsulin : P ( − / − versus + / + ; HFD ) = 0 . 043 ; P ( CTRL versus HFD ; + / + ) = 0 . 028 ; P ( CTRL versus HFD ; − / − ) = 0 . 013 ; P ( CTRL versus HFD ; + / + NAC ) = 0 . 011 ; P ( CTRL versus HFD ; − / − NAC ) = 0 . 0015 ; P ( NAC versus no NAC ; − / − HFD ) = 0 . 038 .

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

A, B Histograms show bloodglucose (A) or plasmainsulin (B) levels of 6-h-fasted mice at the beginning (Week 0) and/or at the end of the protocol (Week 16). Fasting bloodglucose week 16: P (−/− versus +/+; HFD) = 0.0052; P (CTRL versus HFD; −/−) = 0.000081; P (NAC versus no NAC; −/− HFD) = 0.0019; P (w16 versus w0; +/+ CTRL) = 0.012; P (w16 versus w0; −/− CTRL) = 0.050; P (w16 versus w0; −/− HFD) = 0.0023. Fasting plasmainsulin: P (−/− versus +/+; HFD) = 0.043; P (CTRL versus HFD; +/+) = 0.028; P (CTRL versus HFD; −/−) = 0.013; P (CTRL versus HFD; +/+ NAC) = 0.011; P (CTRL versus HFD; −/− NAC) = 0.0015; P (NAC versus no NAC; −/− HFD) = 0.038.


C Glucose tolerance test ( GTT ) was performed on 6 - h - fasted mice during 120 min after injection of 1 g glucose / kg of body weight . Curves on the left show blood glucose level monitored after injection of glucose . Histograms on the right show area under curve ( AUC ) . Fasting blood glucose : P ( − / − versus + / + ; CTRL ; t = 0 min ) = 0 . 045 ; P ( − / − versus + / + ; CTRL ; t = 15 min ) = 0 . 013 ; P ( − / − versus + / + ; CTRL ; t = 30 min ) = 0 . 016 ; P ( − / − versus + / + ; CTRL ; t = 60 min ) = 0 . 030 ; P ( − / − versus + / + ; CTRL ; t = 90 min ) = 0 . 017 ; P ( − / − versus + / + ; HFD ; t = 60 min ) = 0 . 041 ; P ( − / − versus + / + ; HFD ; t = 90 min ) = 0 . 043 ; P ( − / − versus + / + ; HFD ; t = 120 min ) = 0 . 034 ; P ( HFD versus CTRL ; + / + ; t = 15 min ) = 0 . 0076 ; P ( HFD versus CTRL ; + / + ; t = 30 min ) = 0 . 0067 ; P ( HFD versus CTRL ; + / + ; t = 60 min ) = 0 . 00058 ; P ( HFD versus CTRL ; + / + ; t = 90 min ) = 0 . 0010 ; P ( HFD versus CTRL ; + / + ; t = 120 min ) = 0 . 023 ; P ( HFD versus CTRL ; − / − ; t = 60 min ) = 0 . 032 . AUC : P ( − / − versus + / + ; CTRL ) = 0 . 023 ; P ( − / − versus + / + ; HFD ) = 0 . 035 ; P ( HFD versus CTRL ; + / + ) = 0 . 042 . D Insulin tolerance test ( ITT ) was performed on 6 - h - fasted mice during 150 min after injection of 0 . 70 U insulin / kg of body weight . Curves on the left show blood glucose level monitored after injection of insulin . Histograms on the right show area above curve ( AAC ) . Fasting blood glucose : P ( − / − versus + / + ; CTRL ; t = 15 min = 0 . 012 ; P ( − / − versus + / + ; CTRL ; t = 30 min ) = 0 . 022 ; P ( − / − versus + / + ; HFD ; t = 0 min ) = 0 . 027 ; P ( − / − versus + / + ; HFD ; t = 15 min ) = 0 . 011 ; P ( − / − versus + / + ; HFD ; t = 30 min ) = 0 . 0037 ; P ( − / − versus + / + ; HFD ; t = 60 min ) = 0 . 0028 ; P ( − / − versus + / + ; HFD ; t = 90 min ) = 0 . 041 ; P ( − / − versus + / + ; HFD ; t = 120 min ) = 0 . 0032 ; P ( − / − versus + / + ; HFD ; t = 150 min ) = 0 . 0025 ; P ( HFD versus CTRL ; + / + ; t = 15 min ) = 0 . 0082 ; P ( HFD versus CTRL ; + / + ; t = 30 min ) = 0 . 033 ; P ( HFD versus CTRL ; + / + ; t = 90 min ) = 0 . 047 ; P ( HFD versus CTRL ; + / + ; t = 150 min ) = 0 . 028 ; P ( HFD versus CTRL ; − / − ; t = 15 min ) = 0 . 026 ; P ( HFD versus CTRL ; − / − ; t = 30 min ) = 0 . 0095 ; P ( HFD versus CTRL ; − / − ; t = 60 min ) = 0 . 0031 ; P ( HFD versus CTRL ; − / − ; t = 90 min ) = 0 . 033 ; P ( HFD versus CTRL ; − / − ; t = 120 min ) = 0 . 0068 ; P ( HFD versus CTRL ; − / − ; t = 150 min ) = 0 . 0082 . AAC : P ( − / − versus + / + ; HFD ) = 0 . 030 ; P ( HFD versus CTRL ; − / − ) = 0 . 037 .

O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Glucose tolerance test (GTT) was performed on 6-h-fasted mice during 120 min after injection of 1 g glucose/kg of body weight. Curves on the left show blood glucose level monitored after injection of glucose. Histograms on the right show area under curve (AUC). Fasting blood glucose: P (−/− versus +/+; CTRL; t = 0 min) = 0.045; P (−/− versus +/+; CTRL; t = 15 min) = 0.013; P (−/− versus +/+; CTRL; t = 30 min) = 0.016; P (−/− versus +/+; CTRL; t = 60 min) = 0.030; P (−/− versus +/+; CTRL; t = 90 min) = 0.017; P (−/− versus +/+; HFD; t = 60 min) = 0.041; P (−/− versus +/+; HFD; t = 90 min) = 0.043; P (−/− versus +/+; HFD; t = 120 min) = 0.034; P (HFD versus CTRL; +/+; t = 15 min) = 0.0076; P (HFD versus CTRL; +/+; t = 30 min) = 0.0067; P (HFD versus CTRL; +/+; t = 60 min) = 0.00058; P (HFD versus CTRL; +/+; t = 90 min) = 0.0010; P (HFD versus CTRL; +/+; t = 120 min) = 0.023; P (HFD versus CTRL; −/−; t = 60 min) = 0.032. AUC: P (−/− versus +/+; CTRL) = 0.023; P (−/− versus +/+; HFD) = 0.035; P (HFD versus CTRL; +/+) = 0.042.D Insulin tolerance test (ITT) was performed on 6-h-fasted mice during 150 min after injection of 0.70 U insulin/kg of body weight. Curves on the left show blood glucose level monitored after injection of insulin. Histograms on the right show area above curve (AAC). Fasting blood glucose: P (−/− versus +/+; CTRL; t = 15 min = 0.012; P (−/− versus +/+; CTRL; t = 30 min) = 0.022; P (−/− versus +/+; HFD; t = 0 min) = 0.027; P (−/− versus +/+; HFD; t = 15 min) = 0.011; P (−/− versus +/+; HFD; t = 30 min) = 0.0037; P (−/− versus +/+; HFD; t = 60 min) = 0.0028; P (−/− versus +/+; HFD; t = 90 min) = 0.041; P (−/− versus +/+; HFD; t = 120 min) = 0.0032; P (−/− versus +/+; HFD; t = 150 min) = 0.0025; P (HFD versus CTRL; +/+; t = 15 min) = 0.0082; P (HFD versus CTRL; +/+; t = 30 min) = 0.033; P (HFD versus CTRL; +/+; t = 90 min) = 0.047; P (HFD versus CTRL; +/+; t = 150 min) = 0.028; P (HFD versus CTRL; −/−; t = 15 min) = 0.026; P (HFD versus CTRL; −/−; t = 30 min) = 0.0095; P (HFD versus CTRL; −/−; t = 60 min) = 0.0031; P (HFD versus CTRL; −/−; t = 90 min) = 0.033; P (HFD versus CTRL; −/−; t = 120 min) = 0.0068; P (HFD versus CTRL; −/−; t = 150 min) = 0.0082. AAC: P (−/− versus +/+; HFD) = 0.030; P (HFD versus CTRL; −/−) = 0.037.


A , B ( A , B ) Immunocytofluorescent staining of TP53INP1 ( red ) and insulin ( green ) in mouse pancreatic sections ( A ) and single human islet beta cell ( B ) . Scale bars represent 50 μm ( A ) and 10 μm ( B ) .

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

A, B (A, B) Immunocytofluorescent staining of TP53INP1 (red) and insulin (green) in mouse pancreatic sections (A) and single human islet beta cell (B). Scale bars represent 50 μm (A) and 10 μm (B).


C - E Quantitative PCR for Tp53inp1 mRNA levels in tissues and cells from rat ( C ) and C57BL / 6Jmice fed with a normal diet ( 5 % fat ; ND ) or an high - fat diet ( 45 % fat ; HFD ) ( D , E ) . Results are expressed as the mean ± SEM and are representative of two independent experiments . n = 2 for ratliver , islets and mousespleen ; n = 4 for INS - 1E cells ; n = 5 for mouseexocrine pancreas ; n = 6 for Min6 cells and ND and HFD islets ; n = 11 for mouseislets . * P = 0 . 035 for HFD versus ND .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-E Quantitative PCR for Tp53inp1 mRNA levels in tissues and cells from rat (C) and C57BL/6Jmice fed with a normal diet (5% fat; ND) or an high-fat diet (45% fat; HFD) (D, E). Results are expressed as the mean ± SEM and are representative of two independent experiments. n = 2 for ratliver, islets and mousespleen; n = 4 for INS-1E cells; n = 5 for mouseexocrine pancreas; n = 6 for Min6 cells and ND and HFD islets; n = 11 for mouseislets. *P = 0.035 for HFD versus ND.


Immortalized MEFs ( MEFi ) deficient ( − / − ) or not ( + / + ) for TP53INP1 were left untreated ( NT ) or treated with 3 - methyladenine ( 3 - MA , 5 mM ) during 4 h . Histograms show mt ROS level measured by flow cytometry upon MitoSox staining . P ( − / − versus + / + ; NT ) = 0 . 019 ; P ( − / − versus + / + ; 3 - MA ) = 0 . 025 ; P ( 3 - MA versus NT ; + / + ) = 0 . 031 . Histograms show mt mass evaluated by flow cytometry in KO or WT MEFi using MitoTracker staining . P ( − / − versus + / + ; NT ) = 0 . 012 ; P ( 3 - MA versus NT ; + / + ) = 0 . 036 . Histogram shows MitoSox fluorescence normalized with MitoTracker fluorescence . P ( − / − versus + / + ; NT ) = 0 . 041 ; P ( − / − versus + / + ; 3 - MA ) = 0 . 030 ; P ( 3 - MA versus NT ; + / + ) = 0 . 045 .

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

Immortalized MEFs (MEFi) deficient (−/−) or not (+/+) for TP53INP1 were left untreated (NT) or treated with 3-methyladenine (3-MA, 5 mM) during 4 h.Histograms show mt ROS level measured by flow cytometry upon MitoSox staining. P (−/− versus +/+; NT) = 0.019; P (−/− versus +/+; 3-MA) = 0.025; P (3-MA versus NT; +/+) = 0.031.Histograms show mt mass evaluated by flow cytometry in KO or WT MEFi using MitoTracker staining. P (−/− versus +/+; NT) = 0.012; P (3-MA versus NT; +/+) = 0.036.Histogram shows MitoSox fluorescence normalized with MitoTracker fluorescence. P (−/− versus +/+; NT) = 0.041; P (−/− versus +/+; 3-MA) = 0.030; P (3-MA versus NT; +/+) = 0.045.


After 4 h recovering in normal media , H2O2 ( 1 h , 100 μM ) or non - treated ( NT ) MEFi deficient ( − / − ) or not ( + / + ) for TP53INP1 were observed by transmission electron microscopy ( TEM ) . N = nucleus ; white arrow = mitophagic vacuoles . Scale bar represents 0 . 5 μm . Mean size of mitochondrion ( area ) , number of mitochondria and mitophagic vacuoles normalized by cytoplasmic surface area were quantified . Size : P ( − / − versus + / + ; H2O2 ) = 0 . 000027 ; P ( H2O2 versus NT ; + / + ) = 0 . 0070 ; P ( H2O2 versus NT ; − / − ) = 0 . 013 . Nb mito . : P ( − / − versus + / + ; NT ) = 0 . 035 ; P ( − / − versus + / + ; H2O2 ) = 0 . 016 . Nb vacuoles . : P ( − / − versus + / + ; NT ) = 0 . 047 .

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

After 4 h recovering in normal media, H2O2 (1 h, 100 μM) or non-treated (NT) MEFi deficient (−/−) or not (+/+) for TP53INP1 were observed by transmission electron microscopy (TEM). N = nucleus; white arrow = mitophagic vacuoles. Scale bar represents 0.5 μm.Mean size of mitochondrion (area), number of mitochondria and mitophagic vacuoles normalized by cytoplasmic surface area were quantified. Size: P (−/− versus +/+; H2O2) = 0.000027; P (H2O2 versus NT; +/+) = 0.0070; P (H2O2 versus NT; −/−) = 0.013. Nb mito.: P (−/− versus +/+; NT) = 0.035; P (−/− versus +/+; H2O2) = 0.016. Nb vacuoles.: P (−/− versus +/+; NT) = 0.047.


After 4 h recovering in normal media , TCLs from H2O2 - ( 1 h , 100 μM ) , NAC - ( 24 h , 10 mM ) or non - treated ( NT ) MEFi deficient ( − / − ) or not ( + / + ) for TP53INP1 were analyzed by immunoblotting for PGC - 1α , PINK1 , PARKIN , BNIP3 , BNIP3L / NIX , VDAC1 and β - tubulin .

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

After 4 h recovering in normal media, TCLs from H2O2- (1 h, 100 μM), NAC- (24 h, 10 mM) or non-treated (NT) MEFi deficient (−/−) or not (+/+) for TP53INP1 were analyzed by immunoblotting for PGC-1α, PINK1, PARKIN, BNIP3, BNIP3L/NIX, VDAC1 and β-tubulin.


HEK293T cells were cotransfected with plasmids encoding TP53INP1α - NTAP or TP53INP1β - NTAP . TP53INP1α - or β - NTAP was precipitated with a streptavidin - containing resin ( P ) , resolved by PAGE and Western blots developed with anti - TP53INP1 ( TP53INP1 precipitation control ) , anti - PINK1 , anti - PARKIN , anti - BNIP3 or anti - BNIP3L / NIX antibody . Western blot on TCL ( on the right ) served as a transfection control .

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

HEK293T cells were cotransfected with plasmids encoding TP53INP1α-NTAP or TP53INP1β-NTAP. TP53INP1α- or β-NTAP was precipitated with a streptavidin-containing resin (P), resolved by PAGE and Western blots developed with anti-TP53INP1 (TP53INP1 precipitation control), anti-PINK1, anti-PARKIN, anti-BNIP3 or anti-BNIP3L/NIX antibody. Western blot on TCL (on the right) served as a transfection control.


Three - month - old TP53INP1 - deficient and WT male mice were sacrificed and their livers harvested . Mitochondrial lysates ( Mito ) were purified from total liver lysates ( TCL ) , and both were analyzed by immunoblotting for TP53INP1 , PINK1 , PARKIN , VDAC1 and β - tubulin .

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

Three-month-old TP53INP1-deficient and WT male mice were sacrificed and their livers harvested. Mitochondrial lysates (Mito) were purified from total liver lysates (TCL), and both were analyzed by immunoblotting for TP53INP1, PINK1, PARKIN, VDAC1 and β-tubulin.


A - D High - resolution respirometry was performed on permeabilized MEFi TP53INP1 deficient ( − / − , black bars ) or not ( + / + , white bars ) using glucose ( A ) , lipid - related substrates ( C ) or complex IV substrate ( D ) . High - resolution respirometry was also performed on intact MEFi ( B ) . For different respiratory state details ( routine , 3 , leak , and ETS capacity ) , please refer to the Materials and Methods section . Results are expressed as the mean ± SEM and are representative of three independent experiments . In ( A ) : P ( − / − versus + / + ; State 3 ) = 0 . 0079 ; P ( − / − versus + / + ; ETS ) = 0 . 0079 ; n = 5 in each group . In ( B ) : P ( − / − versus + / + ; routine ) = 0 . 0159 ; P ( − / − versus + / + ; leak ) = 0 . 0159 ; P ( − / − versus + / + ; ETS ) = 0 . 0159 ; n = 5 in each group . In ( C ) : P ( − / − versus + / + ; State 3 ) = 0 . 029 ; P ( − / − versus + / + ; ETS ) = 0 . 0079 ; n = 5 in each group . In ( D ) : P ( − / − versus + / + ; ETS ) = 0 . 0259 ; n = 5 in each group . * TP53INP1 − / − versus TP53INP1 + / + ; 1 character : P < 0 . 05 ; 2 characters : P < 0 . 01 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-D High-resolution respirometry was performed on permeabilized MEFi TP53INP1 deficient (−/−, black bars) or not (+/+, white bars) using glucose (A), lipid-related substrates (C) or complex IV substrate (D). High-resolution respirometry was also performed on intact MEFi (B). For different respiratory state details (routine, 3, leak, and ETS capacity), please refer to the Materials and Methods section. Results are expressed as the mean ± SEM and are representative of three independent experiments. In (A): P (−/− versus +/+; State 3) = 0.0079; P (−/− versus +/+; ETS) = 0.0079; n = 5 in each group. In (B): P (−/− versus +/+; routine) = 0.0159; P (−/− versus +/+; leak) = 0.0159; P (−/− versus +/+; ETS) = 0.0159; n = 5 in each group. In (C): P (−/− versus +/+; State 3) = 0.029; P (−/− versus +/+; ETS) = 0.0079; n = 5 in each group. In (D): P (−/− versus +/+; ETS) = 0.0259; n = 5 in each group. *TP53INP1−/− versus TP53INP1 +/+; 1 character: P < 0.05; 2 characters: P < 0.01.


E TCLs from MEFi were analyzed by immunoblotting for CPT1A , CPT1B and β - tubulin . Results are representative of three independent experiments .

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

E TCLs from MEFi were analyzed by immunoblotting for CPT1A, CPT1B and β-tubulin. Results are representative of three independent experiments.