( B - D ) 293T cells were transfected with the indicated constructs , lysed and subjected to GST immunoprecipitation using agarose beads coupled to glutathione ( IP , immunoprecipitation ; TL , total lysate ) . Shown are WBs against the indicated molecules . ( B ) AU - ATG16L1 co ‐ precipitates with wild ‐ type ( WT ) TMEM59 - GST but not with a mutated version where the four essential amino acids were mutated to alanine ( TMEM59 - 4M - GST ) . ( C ) Full ‐ length TMEM59 and TMEM59 - Δ282 co ‐ precipitate with GST - ATG16L1 , whereas the respective 4M versions do not . ( D ) AU - ATG16L1 co ‐ precipitates with a fusion protein between GST and the minimal active peptide of TMEM59 ( GST - 263 - 281 ) , but not with a 4M version ( GST - 263 - 281 - 4M ) or a GST fusion protein with the inactive portion of TMEM59 - ID ( GST - 282 - 323 ) .

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

(B-D) 293T cells were transfected with the indicated constructs, lysed and subjected to GST immunoprecipitation using agarose beads coupled to glutathione (IP, immunoprecipitation; TL, total lysate). Shown are WBs against the indicated molecules. (B) AU-ATG16L1 co‐precipitates with wild‐type (WT) TMEM59-GST but not with a mutated version where the four essential amino acids were mutated to alanine (TMEM59-4M-GST). (C) Full‐length TMEM59 and TMEM59-Δ282 co‐precipitate with GST-ATG16L1, whereas the respective 4M versions do not. (D) AU-ATG16L1 co‐precipitates with a fusion protein between GST and the minimal active peptide of TMEM59 (GST-263-281), but not with a 4M version (GST-263-281-4M) or a GST fusion protein with the inactive portion of TMEM59-ID (GST-282-323).


( E ) HA - ATG16L1 expressed in bacteria co − precipitates with a GST - 263 - 281 recombinant protein purified from bacterial cultures , but not with a 4M version of the same construct or GST - 282 - 323 . The indicated GST partners were expressed in bacteria and purified using agarose beads coupled to glutathione . The loaded beads were then used for HA - ATG16L1 pull − down from crude bacterial lysates . Shown are WBs against the indicated molecules ( PD , pull − down ) . A Coomasie staining of a protein gel with the purified GST fusion proteins is shown . The right panel compares the amount of HA - ATG16L1 pulled down by GST - 263 - 281 with the signal provided by direct anti − HA immunoprecipitation . This result shows that about 20 - 25 % of the available HA - ATG16L1 protein is precipitated by GST - 263 - 281 . Asterisks indicate irrelevant bands in B and D . Source data for this figure is available on the online supplementary information page .

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

(E) HA-ATG16L1 expressed in bacteria co−precipitates with a GST-263-281 recombinant protein purified from bacterial cultures, but not with a 4M version of the same construct or GST-282-323. The indicated GST partners were expressed in bacteria and purified using agarose beads coupled to glutathione. The loaded beads were then used for HA-ATG16L1 pull−down from crude bacterial lysates. Shown are WBs against the indicated molecules (PD, pull−down). A Coomasie staining of a protein gel with the purified GST fusion proteins is shown. The right panel compares the amount of HA-ATG16L1 pulled down by GST-263-281 with the signal provided by direct anti−HA immunoprecipitation. This result shows that about 20-25% of the available HA-ATG16L1 protein is precipitated by GST-263-281. Asterisks indicate irrelevant bands in B and D.Source data for this figure is available on the online supplementary information page.


( A - F ) 293T cells were transfected with the indicated constructs , lysed and subjected to GST immunoprecipitation with agarose beads coupled to glutathione . Shown are WBs against the indicated molecules . ( A ) A deleted version of ATG16L1 lacking the WD domain ( HA - ATG16L1 - ΔWD ) does not co ‐ precipitate with TMEM59 - GST . ( B ) TMEM59 does not co ‐ precipitate with ATG16L1 - ΔWD fused to GST . ( C ) The WD domain of ATG16L1 ( HA - ATG16L1 - WD ) suffices to co ‐ precipitate with TMEM59 - GST . ( D ) TMEM59 co ‐ precipitates with ATG16L1 - WD fused to GST . ( E ) HA - ATG16L1 - WD does not bind a 4M version of TMEM59 - GST . ( F ) TMEM59 - 4M does not co ‐ precipitate with the ATG16L1 - WD fused to GST . Source data for this figure is available on the online supplementary information page .

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

(A-F) 293T cells were transfected with the indicated constructs, lysed and subjected to GST immunoprecipitation with agarose beads coupled to glutathione. Shown are WBs against the indicated molecules. (A) A deleted version of ATG16L1 lacking the WD domain (HA-ATG16L1-ΔWD) does not co‐precipitate with TMEM59-GST. (B) TMEM59 does not co‐precipitate with ATG16L1-ΔWD fused to GST. (C) The WD domain of ATG16L1 (HA-ATG16L1-WD) suffices to co‐precipitate with TMEM59-GST. (D) TMEM59 co‐precipitates with ATG16L1-WD fused to GST. (E) HA-ATG16L1-WD does not bind a 4M version of TMEM59-GST. (F) TMEM59-4M does not co‐precipitate with the ATG16L1-WD fused to GST.Source data for this figure is available on the online supplementary information page.


( C , D , E , G , H ) 293T cells were transfected with the indicated constructs , lysed and subjected to GST immunoprecipitation with agarose beads coupled to glutathione . Shown are WBs against the indicated molecules . ( C ) The N ‐ terminal CARD of NOD2 ( NOD2 - CARD1 - HA ) , but not the CARD of NOD1 ( NOD1 - CARD - HA ) , co ‐ precipitates with GST - ATG16L1 . ( D ) The ID of TLR2 ( HA - TLR2 - ID ) co ‐ precipitates with GST - ATG16L1 . ( E ) Mutated versions ( MUT ) of NOD2 - CARD1 - HA and HA - TLR2 - ID ( as shown ) do not co ‐ precipitate with GST - ATG16L1 . ( F ) Amino ‐ acid sequences of T3JAM and DEDD2 including the motif . Residues identified by the Prosite algorithm are highlighted . ( G ) Wild ‐ type ( WT ) versions of HA - T3JAM and HA - DEDD2 ( as indicated ) co ‐ precipitate with GST - ATG16L1 . ( H ) Mutated versions ( MUT ) of HA - T3JAM and HA - DEDD2 do not co ‐ precipitate with GST - ATG16L1 . Asterisks indicate irrelevant bands in C , D and G . Source data for this figure is available on the online supplementary information page .

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

(C, D, E, G, H) 293T cells were transfected with the indicated constructs, lysed and subjected to GST immunoprecipitation with agarose beads coupled to glutathione. Shown are WBs against the indicated molecules. (C) The N‐terminal CARD of NOD2 (NOD2-CARD1-HA), but not the CARD of NOD1 (NOD1-CARD-HA), co‐precipitates with GST-ATG16L1. (D) The ID of TLR2 (HA-TLR2-ID) co‐precipitates with GST-ATG16L1. (E) Mutated versions (MUT) of NOD2-CARD1-HA and HA-TLR2-ID (as shown) do not co‐precipitate with GST-ATG16L1. (F) Amino‐acid sequences of T3JAM and DEDD2 including the motif. Residues identified by the Prosite algorithm are highlighted. (G) Wild‐type (WT) versions of HA-T3JAM and HA-DEDD2 (as indicated) co‐precipitate with GST-ATG16L1. (H) Mutated versions (MUT) of HA-T3JAM and HA-DEDD2 do not co‐precipitate with GST-ATG16L1. Asterisks indicate irrelevant bands in C, D and G.Source data for this figure is available on the online supplementary information page.


( A ) The right panel shows successful TMEM59 depletion ( rabbit anti ‐ TMEM59immunoprecipitation plus chicken anti ‐ TMEM59WB ) .

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

(A) The right panel shows successful TMEM59 depletion (rabbit anti‐TMEM59immunoprecipitation plus chicken anti‐TMEM59WB).


( A ) Depletion of TMEM59 inhibits LC3II generation by SA at early infection times . HeLa cells were transfected with the indicated siRNAs and , 48 h later , infected with the bacteria ( SA ) for the shown times before lysing them for western blotting against the indicated molecules . The right panel shows successful TMEM59 depletion ( rabbit anti ‐ TMEM59 immunoprecipitation plus chicken anti ‐ TMEM59 WB ) .

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(A) Depletion of TMEM59 inhibits LC3II generation by SA at early infection times. HeLa cells were transfected with the indicated siRNAs and, 48 h later, infected with the bacteria (SA) for the shown times before lysing them for western blotting against the indicated molecules. The right panel shows successful TMEM59 depletion (rabbit anti‐TMEM59 immunoprecipitation plus chicken anti‐TMEM59 WB).


( B ) Colocalization events between bacteria ( GFP labelled ) , TMEM59 - HA and Cherry - LC3A in infected cells . HeLa cells stably expressing TMEM59 - HA and Cherry - LC3A were infected for the indicated times ( moi = 10 ) , and processed for anti ‐ HA immunofluorescence . Representative confocal images are shown . The GFP , anti ‐ HA ( Cy5 ) and DAPI signals were pseudocolored in blue , green and white , respectively . Asterisks indicate overlay images : ( * ) , Cherry - LC3A / TMEM59 ; ( * * ) , Cherry - LC3A / TMEM59 / bacteria ; ( * * * ) , Cherry - LC3A / TMEM59 / bacteria / DAPI .

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(B) Colocalization events between bacteria (GFP labelled), TMEM59-HA and Cherry-LC3A in infected cells. HeLa cells stably expressing TMEM59-HA and Cherry-LC3A were infected for the indicated times (moi=10), and processed for anti‐HA immunofluorescence. Representative confocal images are shown. The GFP, anti‐HA (Cy5) and DAPI signals were pseudocolored in blue, green and white, respectively. Asterisks indicate overlay images: (*), Cherry-LC3A/TMEM59; (**), Cherry-LC3A/TMEM59/bacteria; (***), Cherry-LC3A/TMEM59/bacteria/DAPI.


( C ) Colocalization between bacteria , TMEM59 - HA and Cherry - ATG16L1 . HeLa cells stably expressing TMEM59 - HA and Cherry - ATG16L1 were infected with SA ( 2 h , moi = 10 ) , and processed for anti ‐ HA immunofluorescence . Shown are confocal pictures displaying two representative examples . Asterisks indicate overlays as in B .

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(C) Colocalization between bacteria, TMEM59-HA and Cherry-ATG16L1. HeLa cells stably expressing TMEM59-HA and Cherry-ATG16L1 were infected with SA (2 h, moi=10), and processed for anti‐HA immunofluorescence. Shown are confocal pictures displaying two representative examples. Asterisks indicate overlays as in B.


( D ) SA promotes co ‐ precipitation of endogenous ATG16L1 with endogenous TMEM59 . HeLa cells were infected with SA ( 2 h , moi = 10 ) , lysed and subjected to immunoprecipitation with anti ‐ TMEM59 antibodies or irrelevant protein G beads ( as indicated ) . Immunoprecipitates were processed for western blotting . The right panel shows control WBs of the lysates used for immunoprecipitation .

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(D) SA promotes co‐precipitation of endogenous ATG16L1 with endogenous TMEM59. HeLa cells were infected with SA (2 h, moi=10), lysed and subjected to immunoprecipitation with anti‐TMEM59 antibodies or irrelevant protein G beads (as indicated). Immunoprecipitates were processed for western blotting. The right panel shows control WBs of the lysates used for immunoprecipitation.


( E ) Depletion of TMEM59 reduces recovery of SA from infected cells . HeLa cells were transfected with the indicated siRNAs , infected with SA 96 h later ( 5 h , moi = 0 . 1 ) and lysed for CFU evaluation . The experiment was carried out three independent times . The right panel shows successful TMEM59 depletion ( anti ‐ TMEM59 immunoprecipitation plus anti ‐ TMEM59 WB ) . Source data for this figure is available on the online supplementary information page .

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(E) Depletion of TMEM59 reduces recovery of SA from infected cells. HeLa cells were transfected with the indicated siRNAs, infected with SA 96 h later (5 h, moi=0.1) and lysed for CFU evaluation. The experiment was carried out three independent times. The right panel shows successful TMEM59 depletion (anti‐TMEM59 immunoprecipitation plus anti‐TMEM59 WB).Source data for this figure is available on the online supplementary information page.


( E ) Depletion of TMEM59 reduces recovery of SA from infected cells . HeLa cells were transfected with the indicated siRNAs , infected with SA 96 h later ( 5 h , moi = 0 . 1 ) and lysed for CFU evaluation . The experiment was carried out three independent times . The graph displays average CFU counts obtained for the 10 − 1 dilution of the relevant extracts , ± s . d . of the three data sets . Asterisks indicate significant differences ( P < 0 . 01 , paired Student ' s t ‐ test ) .

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(E) Depletion of TMEM59 reduces recovery of SA from infected cells. HeLa cells were transfected with the indicated siRNAs, infected with SA 96 h later (5 h, moi=0.1) and lysed for CFU evaluation. The experiment was carried out three independent times. The graph displays average CFU counts obtained for the 10−1 dilution of the relevant extracts, ±s.d. of the three data sets. Asterisks indicate significant differences (P<0.01, paired Student's t‐test).


Evaluation of expression by RNA - Seq and number of peptides required to detect an annotated protein by MS in M . pneumoniae . The plot represents the relationship between expression levels ( average expression from RNA - Seq data ) and number of possible unique tryptic peptides ( UTPs ) for two sets of studied proteins : detected ( blue dots ) and not detected ( orange dots ) by MS . B ) Evaluation of thresholds and artefactual signals in MS data . The histogram represents the total number of SEP proteins detected in 116 shotgun MS experiments with 1 UTP , 1 UTP and 1NUTP , or ≥ 2 UTPs for three categories . Color code : annotated ( blue bars ) , putative new ( orange bars ) , and decoy set ( gray bars ) . C ) Number of SEPs detected by increasing the number of experiments . Color code is the same as in panels B . Each line represents the accumulated number of different SEPs detected ( Y - axis ) when combining 1 to 116 MS datasets ( X - axis ) from M . pneumoniae . Each line has an associated error that is shaded and represents the standard deviation within combinations of datasets ( e . g . , x = 80 will present the average number of proteins detected taking every combination of datasets in groups of 80 samples ) .

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

Evaluation of expression by RNA-Seq and number of peptides required to detect an annotated protein by MS in M. pneumoniae. The plot represents the relationship between expression levels (average expression from RNA-Seq data) and number of possible unique tryptic peptides (UTPs) for two sets of studied proteins: detected (blue dots) and not detected (orange dots) by MS. B) Evaluation of thresholds and artefactual signals in MS data. The histogram represents the total number of SEP proteins detected in 116 shotgun MS experiments with 1 UTP, 1 UTP and 1NUTP, or ≥2 UTPs for three categories. Color code: annotated (blue bars), putative new (orange bars), and decoy set (gray bars). C) Number of SEPs detected by increasing the number of experiments. Color code is the same as in panels B. Each line represents the accumulated number of different SEPs detected (Y-axis) when combining 1 to 116 MS datasets (X-axis) from M. pneumoniae. Each line has an associated error that is shaded and represents the standard deviation within combinations of datasets (e.g., x=80 will present the average number of proteins detected taking every combination of datasets in groups of 80 samples).


( A ) PolyP accumulation in vivo . The polyP content of wild - type cells was set to 100 % . Knockout of Vtc1 , Vtc2 , Vtc3 or Vtc4 impacts cellular polyP levels . Data show the mean ± s . d ( biological replicates n = 3 ) .

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(A) PolyP accumulation in vivo. The polyP content of wild-type cells was set to 100%. Knockout of Vtc1, Vtc2, Vtc3 or Vtc4 impacts cellular polyP levels. Data show the mean±s.d (biological replicates n=3).


PolyP synthesis curves of purified endogenous Vtc4 / Vtc3 / Vtc1 and Vtc4 / Vtc2 / Vtc1 complexes at different ATP concentrations in the absence or presence of 10 mM InsP6 in vitro .

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PolyP synthesis curves of purified endogenous Vtc4/Vtc3/Vtc1 and Vtc4/Vtc2/Vtc1 complexes at different ATP concentrations in the absence or presence of 10 mM InsP6 in vitro.


PolyP synthesis by isolated vacuoles carrying VTC complexes in the absence or presence of 1 µM 5 - InsP7 in vitro .

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PolyP synthesis by isolated vacuoles carrying VTC complexes in the absence or presence of 1 µM 5-InsP7 in vitro.


Profile of the pore radius of the Vtc4 / Vtc3 / Vtc1 complex .

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Profile of the pore radius of the Vtc4/Vtc3/Vtc1 complex.


Cellular polyP content of Vtc4p point mutants expressed under the control of their native promoters in the vtc4Δ background . Data show the mean ± s . d ( biological replicates n = 3 ) .

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Cellular polyP content of Vtc4p point mutants expressed under the control of their native promoters in the vtc4Δ background. Data show the mean±s.d (biological replicates n=3).


Cellular polyP content of VTC4 and VTC3 point mutants expressed under the control of their native promoters in the vtc4Δ and vtc3Δ ( vtc2Δ ) backgrounds , respectively . Data show the mean ± s . d ( biological replicates n = 3 ) .

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Cellular polyP content of VTC4 and VTC3 point mutants expressed under the control of their native promoters in the vtc4Δ and vtc3Δ(vtc2Δ) backgrounds, respectively. Data show the mean±s.d (biological replicates n=3).


Cellular polyP content of VTC4 point mutants expressed under the control of their native promoters in the vtc4Δ backgrounds . Data show the mean ± s . d ( biological replicates n = 3 ) .

Cellular polyP content of VTC4 point mutants expressed under the control of their native promoters in the vtc4Δ backgrounds. Data show the mean±s.d (biological replicates n=3).


PolyP synthesis by isolated vacuoles carrying Vtc4 ( ΔSPX ) / Vtc3 / Vtc1 complex , Vtc4 / Vtc3 ( ΔSPX ) / Vtc1 complex or Vtc4 / Vtc3 / Vtc1 complex in the absence or presence of 1 μM 5 - InsP7 or 1 , 5 - InsP8 in vitro . The reaction system is detailed in Methods . Data show the mean ± s . d ( biological replicates n = 3 ) .

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

PolyP synthesis by isolated vacuoles carrying Vtc4(ΔSPX)/Vtc3/Vtc1 complex, Vtc4/Vtc3(ΔSPX)/Vtc1 complex or Vtc4/Vtc3/Vtc1 complex in the absence or presence of 1 μM 5-InsP7 or 1,5-InsP8 in vitro. The reaction system is detailed in Methods. Data show the mean±s.d (biological replicates n=3).


PolyP synthesis by isolated vacuoles carrying Vtc4 / Vtc3 ( R223E ) / Vtc1 complex , Vtc4 / Vtc3 ( R226E ) / Vtc1 complex or Vtc4 / Vtc3 / Vtc1 complex in the absence or presence of 1 μM 5 - InsP7 or 1 , 5 - InsP8 in vitro . The reaction system is detailed in Methods . Data show the mean ± s . d ( biological replicates n = 3 ) .

B-MEASURED_VAR 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 B-CONTROLLED_VAR 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 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

PolyP synthesis by isolated vacuoles carrying Vtc4/Vtc3(R223E)/Vtc1 complex, Vtc4/Vtc3(R226E)/Vtc1 complex or Vtc4/Vtc3/Vtc1 complex in the absence or presence of 1 μM 5-InsP7 or 1,5-InsP8 in vitro. The reaction system is detailed in Methods. Data show the mean±s.d (biological replicates n=3).


PolyP synthesis curves of purified endogenous Vtc4 / Vtc3 / Vtc1 and truncated Vtc4 / Vtc3 ( Δlp ) / Vtc1 complexes at different ATP concentrations in the absence or presence of 10 mM InsP6 in vitro . The reaction system is detailed in Methods . Data show the mean ± s . d ( biological replicates n = 3 ) . ( D ) PolyP synthesis by isolated vacuoles carrying Vtc4 / Vtc3 ( CL2 - Ala6 ) / Vtc1 complex , Vtc4 / Vtc3 ( CL2 - Asp6 ) / Vtc1 complex or Vtc4 / Vtc3 / Vtc1 complex in the absence or presence of 1 μM 5 - InsP7 or 1 , 5 - InsP8 in vitro . The reaction system is detailed in Methods . Data show the mean ± s . d ( biological replicates n = 3 ) .

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

PolyP synthesis curves of purified endogenous Vtc4/Vtc3/Vtc1 and truncated Vtc4/Vtc3(Δlp)/Vtc1 complexes at different ATP concentrations in the absence or presence of 10 mM InsP6 in vitro. The reaction system is detailed in Methods. Data show the mean±s.d (biological replicates n=3). (D) PolyP synthesis by isolated vacuoles carrying Vtc4/Vtc3(CL2-Ala6)/Vtc1 complex, Vtc4/Vtc3(CL2-Asp6)/Vtc1 complex or Vtc4/Vtc3/Vtc1 complex in the absence or presence of 1 μM 5-InsP7 or 1,5-InsP8 in vitro. The reaction system is detailed in Methods. Data show the mean±s.d (biological replicates n=3).


Cellular polyP content of VTC4 , VTC3 and VTC1 point mutants expressed under the control of their native promoters in the vtc4Δ , vtc3Δ ( vtc2Δ ) and vtc1Δ backgrounds , respectively . Δ indicates that the entire subunit was knocked out . FL indicates full length , indicating that the subunit has not been modified in any way . Data show the mean ± s . d ( biological replicates n = 3 ) .

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

Cellular polyP content of VTC4, VTC3 and VTC1 point mutants expressed under the control of their native promoters in the vtc4Δ, vtc3Δ(vtc2Δ) and vtc1Δ backgrounds, respectively. Δ indicates that the entire subunit was knocked out. FL indicates full length, indicating that the subunit has not been modified in any way. Data show the mean±s.d (biological replicates n=3).


( C ) Representative images of HeLa cells transiently transfected with GST - GFP - CENP - E2605 - 2701 and immunostained with PRC1 and CENP - C , scale bar 10μm ( n = 4 ) .

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(C) Representative images of HeLa cells transiently transfected with GST-GFP-CENP-E2605-2701 and immunostained with PRC1 and CENP-C, scale bar 10μm (n=4).


( E ) Top , Representative images of live HeLa cells in cytokinesis transiently transfected with GFP - CENP - E2605 - 2701 and mutants ( green ) , incubated with SiR - tubulin ( red ) . Scale bar , 10μm . Bottom , linescans showing the fluorescence intensity average and standard error of the mean ( SEM ) for the GFP - CENP - E2605 - 2701 and mutants and tubulin across the cell midbody . For GFP - CENP - E2605 - 2701 , n is the number of cells . n = 11 and for the GFP - CENP - E2605 - 2701 mutants FDN2661LTT , YF2660AA , FF2644AA , n = 15 , 16 and 25 respectively . Biological independent replicates were respectively 2 , 3 , 2 and 1 .

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(E) Top, Representative images of live HeLa cells in cytokinesis transiently transfected with GFP-CENP-E2605-2701 and mutants (green), incubated with SiR-tubulin (red). Scale bar, 10μm. Bottom, linescans showing the fluorescence intensity average and standard error of the mean (SEM) for the GFP-CENP-E2605-2701 and mutants and tubulin across the cell midbody. For GFP-CENP-E2605-2701, n is the number of cells. n=11 and for the GFP-CENP-E2605-2701 mutants FDN2661LTT, YF2660AA, FF2644AA, n= 15, 16 and 25 respectively. Biological independent replicates were respectively 2, 3, 2 and 1.


( A ) Top . Size - exclusion chromatography elution profile of MBP - CENP - E2605 - 2701 ( green ) , PRC11 - 168 ( pink ) and MBP - CENP - E2605 - 2701 / PRC11 - 168 ( orange ) . Bottom , Coomassie - stained gel showing the size - exclusion chromatography profile of PRC11 - 168 ( pink ) , MBP - CENP - E2605 - 2701 ( green ) and MBP - CENP - E2605 - 2701 / PRC11 - 168 ( orange ) . A shift in the elution volume was only seen in the presence of both CENP - E and PRC1 . ( B ) Top . Size - exclusion chromatography elution profile of MBP - CENP - E2605 - 2701FDN ( green ) and MBP - CENP - E2605 - 2701 FDN / PRC11 - 168 ( orange ) . Bottom , Coomassie - stained gel showing the size - exclusion chromatography profile of MBP - CENP - E2605 - 2701FDN ( green ) and MBP - CENP - E2605 - 2701 FDN / PRC11 - 168 ( orange ) . No shift in the elution profile was observed .

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

(A) Top. Size-exclusion chromatography elution profile of MBP-CENP-E2605-2701 (green), PRC11-168 (pink) and MBP-CENP-E2605-2701/PRC11-168 (orange). Bottom, Coomassie-stained gel showing the size-exclusion chromatography profile of PRC11-168 (pink), MBP-CENP-E2605-2701 (green) and MBP-CENP-E2605-2701/PRC11-168 (orange). A shift in the elution volume was only seen in the presence of both CENP-E and PRC1. (B) Top. Size-exclusion chromatography elution profile of MBP-CENP-E2605-2701FDN (green) and MBP-CENP-E2605-2701 FDN/PRC11-168 (orange). Bottom, Coomassie-stained gel showing the size-exclusion chromatography profile of MBP-CENP-E2605-2701FDN (green) and MBP-CENP-E2605-2701 FDN/PRC11-168 (orange). No shift in the elution profile was observed.


( B ) Representative images of live HeLa cells in mitosis transiently transfected with either GFP - Kif4A1133 - 1165 or GFP - Kif4A1133 - 1232 incubated with SiR - Tubulin . Scale bar , 10μm . Quantification of cells with GFP - Kif4A1133 - 1165 ( n = 12 ) or GFP - Kif4A1133 - 1232 ( n = 13 ) localization to the central spindle . Data represented from 2 independent experiments .

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(B) Representative images of live HeLa cells in mitosis transiently transfected with either GFP-Kif4A1133-1165 or GFP-Kif4A1133-1232 incubated with SiR-Tubulin. Scale bar, 10μm. Quantification of cells with GFP-Kif4A1133-1165 (n=12) or GFP-Kif4A1133-1232 (n=13) localization to the central spindle. Data represented from 2 independent experiments.


( C ) A shift in the elution volume was only seen in the presence of both Kif4A and PRC1 . Size - exclusion chromatography elution profile of GST - Kif4A1133 - 1232 ( green ) and PRC11 - 168 ( pink ) , or together ( orange ) . Bottom , Coomassie - stained gel showing the size - exclusion chromatography profile of GST - Kif4A1133 - 1232 ( green ) , PRC11 - 168 ( pink ) and GST - Kif4A1133 - 1232 / PRC11 - 168 ( orange ) .

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(C) A shift in the elution volume was only seen in the presence of both Kif4A and PRC1. Size-exclusion chromatography elution profile of GST-Kif4A1133-1232 (green) and PRC11-168 (pink), or together (orange). Bottom, Coomassie-stained gel showing the size-exclusion chromatography profile of GST-Kif4A1133-1232 (green), PRC11-168(pink) and GST-Kif4A1133-1232/PRC11-168 (orange).


( A ) Representative images of GFP - PRC1 ( magenta ) mixed with rhodamine - microtubules . Fire blue - green intensity LUT used to show tubulin intensity and microtubule overlaps . ( B ) Representative images of 647GST - CENP - E2639 - 2671 ( yellow ) mixed with rhodamine - microtubules .

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(A) Representative images of GFP-PRC1 (magenta) mixed with rhodamine-microtubules. Fire blue-green intensity LUT used to show tubulin intensity and microtubule overlaps. (B) Representative images of 647GST-CENP-E2639-2671 (yellow) mixed with rhodamine-microtubules.


( D ) Representative immunofluorescence images of HeLa cells stained for DNA , microtubules , PRC1 and with Alexa647 - labelled GST - CENP - E2639 - 2671 showing it also recognizes PRC1 in cells . Experiments were repeated > 3 times .

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(D) Representative immunofluorescence images of HeLa cells stained for DNA, microtubules, PRC1 and with Alexa647-labelled GST-CENP-E2639-2671 showing it also recognizes PRC1 in cells. Experiments were repeated >3 times.


( F ) Representative kymograph showing microtubule - microtubule sliding in the presence of 2 . 5 nM PRC1 and 50 nM full - length CENP - E . ( G ) Example kymograph showing a free microtubule sliding until reaching the end of the immobilized microtubule where it slows down to a stall .

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(F) Representative kymograph showing microtubule-microtubule sliding in the presence of 2.5 nM PRC1 and 50 nM full-length CENP-E. (G) Example kymograph showing a free microtubule sliding until reaching the end of the immobilized microtubule where it slows down to a stall.


( D ) Live - cell imaging of metaphase spindles in HeLa cells transiently transfected wild - type , phosphomimetic and non - phosphorylatable mutants of GFP - CENP - E2605 - 2701 ( monomeric ) and GFP - GST - CENP - E2605 - 2701 ( dimeric ) and stained for tubulin using SiR - Tubulin . The fraction of cells localizing to the overlapping microtubules is represented as a percentage . Scalebar , 10 μm .

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(D) Live-cell imaging of metaphase spindles in HeLa cells transiently transfected wild-type, phosphomimetic and non-phosphorylatable mutants of GFP-CENP-E2605-2701 (monomeric) and GFP-GST-CENP-E2605-2701 (dimeric) and stained for tubulin using SiR-Tubulin. The fraction of cells localizing to the overlapping microtubules is represented as a percentage. Scalebar, 10 μm.


( G ) Bar graph showing mean and standard error for GFP fluorescence intensity at peak fluorescence for GFP - GST - CENP - E2605 - 2701 and mutants at 9 . 7μm , quantified in F . Asterisks indicate ordinary One - way Anova test significance value . * * * * P < 0 . 0001 .

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(G) Bar graph showing mean and standard error for GFP fluorescence intensity at peak fluorescence for GFP-GST-CENP-E2605-2701 and mutants at 9.7μm, quantified in F. Asterisks indicate ordinary One-way Anova test significance value. ****P<0.0001.


( B , C ) Elution profile from a size - exclusion chromatography ( SEC , black line , left y - axis ) run with subsequent multi - angle light scattering ( MALS , right y - axis ) analysis for PRC11 - 168 and PRC11 - 168 MEE . Outcome of the MALS analysis for the peak is presented in pink ( molecular weight , right y - axis ) .

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(B, C) Elution profile from a size-exclusion chromatography (SEC, black line, left y-axis) run with subsequent multi-angle light scattering (MALS, right y-axis) analysis for PRC11-168 and PRC11-168 MEE. Outcome of the MALS analysis for the peak is presented in pink (molecular weight, right y-axis).


( D ) Fire purple - yellow intensity LUT used to show tubulin intensity and microtubule overlaps . Microtubules are shown alone or incubated with 2 . 5 nM full - length PRC1 WT or PRC1 MEE . Scalebar , 10 μm . Experiment was replicated 2 times .

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(D) Fire purple-yellow intensity LUT used to show tubulin intensity and microtubule overlaps. Microtubules are shown alone or incubated with 2.5 nM full-length PRC1 WT or PRC1 MEE. Scalebar, 10 μm. Experiment was replicated 2 times.


( A ) Representative immunofluorescence images of HeLa cells in late anaphase and telophase , expressing GFP - PRC1 - WT or - MEE and depleted for endogenous PRC1 using a PRC1 siRNA and a PRC1 sgRNA after doxycycline - induced Cas 9 expression . Microtubules , GFP and CENP - E are in white , green and red respectively . DNA is in blue . ( B ) Graph showing the percentage of cells with a PRC1 - localized at the site of abscission for cells treated in ( A ) . Mean and standard deviation are presented , n = 156 and 141 for cells expressing PRC1 - WT and - MEE respectively . Data from 4 biological replicates and 1 technical replicate . Asterisks indicate a T - test significance value . * * * * P < 0 . 0001 . ( C ) Graph showing the percentage of cells with CENP - E localized at the abscission site for cells treated in ( A ) , mean and standard deviation are presented , n = 58 and 66 for cells expressing PRC1 - WT and - MEE respectively . Data from 2 biological replicates . p - value ( p = 0 . 11 ) calculated for an unpaired T - test .

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(A) Representative immunofluorescence images of HeLa cells in late anaphase and telophase, expressing GFP-PRC1-WT or -MEE and depleted for endogenous PRC1 using a PRC1 siRNA and a PRC1 sgRNA after doxycycline-induced Cas 9 expression. Microtubules, GFP and CENP-E are in white, green and red respectively. DNA is in blue. (B) Graph showing the percentage of cells with a PRC1-localized at the site of abscission for cells treated in (A). Mean and standard deviation are presented, n= 156 and 141 for cells expressing PRC1-WT and -MEE respectively. Data from 4 biological replicates and 1 technical replicate. Asterisks indicate a T-test significance value. ****P<0.0001. (C) Graph showing the percentage of cells with CENP-E localized at the abscission site for cells treated in (A), mean and standard deviation are presented, n= 58 and 66 for cells expressing PRC1-WT and -MEE respectively. Data from 2 biological replicates. p-value (p=0.11) calculated for an unpaired T-test.


( G ) Representative live - cell images of HeLa cells expressing GFP - PRC1 - WT or - MEE and depleted for endogenous PRC1 using a PRC1 siRNA and a PRC1 sgRNA after doxycycline - induced Cas 9 expression . Microtubules ( SiR - Tubulin ) and DNA ( SPY650 ) are shown in red . The cell membrane ( CellMask ) is highlighted in white and GFP - PRC1 is in green . Scalebar , 10 μm .

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(G) Representative live-cell images of HeLa cells expressing GFP-PRC1-WT or -MEE and depleted for endogenous PRC1 using a PRC1 siRNA and a PRC1 sgRNA after doxycycline-induced Cas 9 expression. Microtubules (SiR-Tubulin) and DNA (SPY650) are shown in red. The cell membrane (CellMask) is highlighted in white and GFP-PRC1 is in green. Scalebar, 10 μm.


( J ) Representative immunofluorescence images of cells expressing GFP - PRC1 - WT or - MEE after 72 hours siRNA depletion and doxycycline - induced knockout of endogenous PRC1 . DNA , microtubules and GFP - PRC1 / GFP - PRC1 - MEE are in blue , red and green respectively . Scalebar , 10μm .

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(J) Representative immunofluorescence images of cells expressing GFP-PRC1-WT or -MEE after 72 hours siRNA depletion and doxycycline-induced knockout of endogenous PRC1. DNA, microtubules and GFP-PRC1/GFP-PRC1-MEE are in blue, red and green respectively. Scalebar, 10μm.


G . Endogenous MITOL enhances the K48 - polyubiquitin chains of Parkin . WT or MITOL KO HeLa cells stably expressing HA - Parkin were treated with CCCP ( 10 µM ) for indicated time . MG132 ( 30 μM ) was added 3 h after CCCP treatment . Lysates of cells were fractionated into cytosolic , mitochondrial and ER fractions , and then subjected to an IP - IB assay with the indicated antibodies . K48 - polyubiquitin chains were immunoblotted with K48 - specific ubiquitin antibody .

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G. Endogenous MITOL enhances the K48-polyubiquitin chains of Parkin. WT or MITOL KO HeLa cells stably expressing HA-Parkin were treated with CCCP (10 µM) for indicated time. MG132 (30 μM) was added 3 h after CCCP treatment. Lysates of cells were fractionated into cytosolic, mitochondrial and ER fractions, and then subjected to an IP-IB assay with the indicated antibodies. K48-polyubiquitin chains were immunoblotted with K48-specific ubiquitin antibody.


E . MITOL regulates mitophagy . MITOL WT HeLa cells , MITOL KO HeLa cells and KO HeLa cells transfected with MITOL WT plasmids 0 . 5µg ( + ) and 5µg ( + + ) stably expressing HA ‐ Parkin and mt ‐ Keima were treated with CCCP ( 10µM ) for 8h . Then , mKeima was measured at 488 ( pH 7 ) and 561 ( pH 4 ) nm lasers using Flow Cytometer . Percentages of mitophagy were calculated from 30 , 000 cells in each independent experiment . Data represent the mean ± SD of three independent experiments . For statistical analysis , a one - way ANOVA with Tukey post - test was performed , * * P < 0 . 01 .

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

E. MITOL regulates mitophagy. MITOL WT HeLa cells, MITOL KO HeLa cells and KO HeLa cells transfected with MITOL WT plasmids 0.5µg (+) and 5µg (++) stably expressing HA‐Parkin and mt‐Keima were treated with CCCP (10µM) for 8h. Then, mKeima was measured at 488 (pH 7) and 561 (pH 4) nm lasers using Flow Cytometer. Percentages of mitophagy were calculated from 30,000 cells in each independent experiment. Data represent the mean ± SD of three independent experiments. For statistical analysis, a one-way ANOVA with Tukey post-test was performed, **P < 0.01.


C . Endogenous MITOL translocates to the ER in later phase of mitophagy . EGFP - MITOL knock in HeLa cells were transfected with HA - Parkin and treated with DMSO or CCCP ( 10 µM ) as indicated times . Cells were fixed , permeabilized and subjected to immunofluorescence analysis with the indicated antibodies . Colocalization was quantified by Manders ' s coefficient . Means ± SEM of more than 10 cells obtained from three independent experiments . For statistical analysis , a one - way ANOVA with Tukey ' s multiple comparisons test were performed , * * * * P < 0 . 0001 . Scale bar represents 1 μm .

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C. Endogenous MITOL translocates to the ER in later phase of mitophagy. EGFP-MITOL knock in HeLa cells were transfected with HA-Parkin and treated with DMSO or CCCP (10 µM) as indicated times. Cells were fixed, permeabilized and subjected to immunofluorescence analysis with the indicated antibodies. Colocalization was quantified by Manders's coefficient. Means ± SEM of more than 10 cells obtained from three independent experiments. For statistical analysis, a one-way ANOVA with Tukey's multiple comparisons test were performed, ****P < 0.0001. Scale bar represents 1 μm.


E . ER - localized MITOL is transported from the mitochondria , and is not newly synthesized protein . HeLa cells stably expressing HA - Parkin were co - transfected with vectors for MITOL - KikGR and FLAG - FKBP38 . After ultraviolet light ( 365 nm ) exposure to the whole plate , cells were incubated with or without CCCP ( 10 µM ) for indicated time , and monitored for red and green KikGR fluorescence . Scale bar , 10 μm . Higher magnification images of the boxed regions are shown in the small panel . Right panel is schematic experimental model for KikGR in this study .

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E. ER-localized MITOL is transported from the mitochondria, and is not newly synthesized protein. HeLa cells stably expressing HA-Parkin were co-transfected with vectors for MITOL-KikGR and FLAG-FKBP38. After ultraviolet light (365 nm) exposure to the whole plate, cells were incubated with or without CCCP (10 µM) for indicated time, and monitored for red and green KikGR fluorescence. Scale bar, 10 μm. Higher magnification images of the boxed regions are shown in the small panel. Right panel is schematic experimental model for KikGR in this study.


C . Phosphorylated Parkin accumulates in the ER fraction in the absence of MITOL . WT or MITOL KO HeLa cells stably expressing HA - Parkin were treated with DMSO or CCCP ( 10 µM ) for 48 h . Lysates of cells were fractionated into cytosolic , mitochondrial and ER fractions , and then subjected to an IB assay with the indicated antibodies . Phospho - Ser65 - Parkin is detected by using specific antibody . Right panels are the quantification of IB about Phospho - Ser65 - Parkin protein levels of the ER fraction . The data represent the mean ± SD for three independent experiments . For statistical analysis , a one - way ANOVA with Tukey post - test was performed , * * P < 0 . 01 .

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

C. Phosphorylated Parkin accumulates in the ER fraction in the absence of MITOL. WT or MITOL KO HeLa cells stably expressing HA-Parkin were treated with DMSO or CCCP (10 µM) for 48 h. Lysates of cells were fractionated into cytosolic, mitochondrial and ER fractions, and then subjected to an IB assay with the indicated antibodies. Phospho-Ser65-Parkin is detected by using specific antibody. Right panels are the quantification of IB about Phospho-Ser65-Parkin protein levels of the ER fraction. The data represent the mean ± SD for three independent experiments. For statistical analysis, a one-way ANOVA with Tukey post-test was performed, **P < 0.01.


D . Parkin is detected at the ER in the absence of MITOL . HeLa cells were transfected with vectors for KikGR - Parkin . After ultraviolet light ( 365 nm ) exposure to the whole plate , cells were incubated with or without CCCP ( 10 µM ) for indicated time , and monitored for red and green KikGR fluorescence . Scale bar , 10 μm . Higher magnification images of the boxed regions are shown in the small panel .

O O B-MEASURED_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 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

D. Parkin is detected at the ER in the absence of MITOL. HeLa cells were transfected with vectors for KikGR-Parkin. After ultraviolet light (365 nm) exposure to the whole plate, cells were incubated with or without CCCP (10 µM) for indicated time, and monitored for red and green KikGR fluorescence. Scale bar, 10 μm. Higher magnification images of the boxed regions are shown in the small panel.


D . MITOL KO increases degradation of ER targeting FKBP38 . HeLa cells stably expressing HA - Parkin were transfected with mitochondrial targeting FLAG - FKBP38 ( FKBP38ActA ) and ER targeting FLAG - FKBP38 ( FKBP38IYFFT ) , and treated with DMSO or CCCP ( 10 µM ) for 24 h . Lysates of cells were subjected to an IB assay with FLAG antibodies for organelle targeting FKBP38 or indicated antibodies . Another cell lysates were subsequently separated on a Phos - tag gel , followed by IB with anti - Parkin antibodies ( Phos - tag ) .

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D. MITOL KO increases degradation of ER targeting FKBP38. HeLa cells stably expressing HA-Parkin were transfected with mitochondrial targeting FLAG-FKBP38 (FKBP38ActA) and ER targeting FLAG-FKBP38 (FKBP38IYFFT), and treated with DMSO or CCCP (10 µM) for 24 h. Lysates of cells were subjected to an IB assay with FLAG antibodies for organelle targeting FKBP38 or indicated antibodies. Another cell lysates were subsequently separated on a Phos-tag gel, followed by IB with anti-Parkin antibodies (Phos-tag).


E - G . MITOL partly prevents CCCP - induced cell death by reducing Parkin - mediated degradation of FKBP38 . WT or MITOL KO HeLa cells stably expressing HA - Parkin and HeLa cells were transfected with the indicated vectors , and treated with DMSO or CCCP ( 10 µM ) for 24 h . Cells were stained with propidium iodide ( PI ) for counting the percentage of dead cells using fluorescence microscopy . Data represent the mean ± SD of five independent experiments ( > 100 individual cells were counted ) . For statistical analysis , a one - way ANOVA with Tukey post - test was performed , * * P < 0 . 01 . KR , FKBP38 K271 / 273R .

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E-G. MITOL partly prevents CCCP-induced cell death by reducing Parkin-mediated degradation of FKBP38. WT or MITOL KO HeLa cells stably expressing HA-Parkin and HeLa cells were transfected with the indicated vectors, and treated with DMSO or CCCP (10 µM) for 24 h. Cells were stained with propidium iodide (PI) for counting the percentage of dead cells using fluorescence microscopy. Data represent the mean ± SD of five independent experiments (>100 individual cells were counted). For statistical analysis, a one-way ANOVA with Tukey post-test was performed, **P < 0.01. KR, FKBP38 K271/273R.


A . Mislocalization of Bcl - 2 is observed in MITOL KO cells in mitophagy . WT or MITOL KO HeLa cells stably expressing HA - Parkin were transfected with FLAG - Bcl2 and treated with DMSO or CCCP ( 10 µM ) for the indicated times . Cells were immunostained with indicated antibody . Scale bar , 1 μm .

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A. Mislocalization of Bcl-2 is observed in MITOL KO cells in mitophagy. WT or MITOL KO HeLa cells stably expressing HA-Parkin were transfected with FLAG-Bcl2 and treated with DMSO or CCCP (10 µM) for the indicated times. Cells were immunostained with indicated antibody. Scale bar, 1 μm.


B . MITOL prevents caspase - dependent cell death in mitophagy . WT or MITOL KO HeLa cells stably expressing HA - Parkin and HeLa cells were treated with DMSO or CCCP ( 10 µM ) with or without Z - VAD - FMK ( 10 μM ) for 24 h . Cells were stained with propidium iodide ( PI ) for counting fluorescence microscopy of the percentage of dead cells . Data represent the mean ± SD of three independent experiments ( > 100 individual cells were counted ) . For statistical analysis , a one - way ANOVA with Tukey post - test was performed , * * P < 0 . 01 . C . Cell death in mitophagy caused by MITOL deficiency depends on calcium leak . WT and MITOL KO HeLa cells stably expressing HA - Parkin or HeLa cells were treated with DMSO or CCCP ( 10 µM ) with or without BAPTA - AM ( 10 μM ) for 24 h . Cells were stained with propidium iodide ( PI ) for counting fluorescence microscopy of the percentage of dead cells . Data represent the mean ± SD of three independent experiments ( > 100 individual cells were counted ) . For statistical analysis , a one - way ANOVA with Tukey post - test was performed , * * P < 0 . 01 .

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

B. MITOL prevents caspase-dependent cell death in mitophagy. WT or MITOL KO HeLa cells stably expressing HA-Parkin and HeLa cells were treated with DMSO or CCCP (10 µM) with or without Z-VAD-FMK (10 μM) for 24 h. Cells were stained with propidium iodide (PI) for counting fluorescence microscopy of the percentage of dead cells. Data represent the mean ± SD of three independent experiments (>100 individual cells were counted). For statistical analysis, a one-way ANOVA with Tukey post-test was performed, **P < 0.01. C. Cell death in mitophagy caused by MITOL deficiency depends on calcium leak. WT and MITOL KO HeLa cells stably expressing HA-Parkin or HeLa cells were treated with DMSO or CCCP (10 µM) with or without BAPTA-AM (10 μM) for 24 h. Cells were stained with propidium iodide (PI) for counting fluorescence microscopy of the percentage of dead cells. Data represent the mean ± SD of three independent experiments (>100 individual cells were counted). For statistical analysis, a one-way ANOVA with Tukey post-test was performed, **P < 0.01.


( E ) Northern blot analysis of circBtnl1 in different tissues of two - month - old mice , 18S RNA served as a loading control .

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(E) Northern blot analysis of circBtnl1 in different tissues of two-month-old mice, 18S RNA served as a loading control.


( F ， G ) RNA fluorescence in situ hybridization of circBtnl1 in small intestine tissues ( F ) and organoid ( G ) . Green region shows the distribution of circBtnl1 using antisense probe , gray region shows the nuclei staining by DAPI . For ( F ) ， scale bars ， 5um ; for ( G ) ， scale bars ， 20 um .

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(F， G) RNA fluorescence in situ hybridization of circBtnl1 in small intestine tissues (F) and organoid (G). Green region shows the distribution of circBtnl1 using antisense probe, gray region shows the nuclei staining by DAPI. For (F)，scale bars，5um; for (G)，scale bars，20 um.


( C ) Representative images of Ki67 staining of in small intestinal tissues from circBtnl1 + / + and circBtnl1 - / - mice . For all representative images , at least three independent experiments were performed with similar results . For every microscope field , average Ki67 + ISC numbers per crypt are shown as means ± SD . * * P < 0 . 01 by two - tailed Student ' s t - test in right panel . Scale bar , 50 μm .

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(C) Representative images of Ki67 staining of in small intestinal tissues from circBtnl1+/+ and circBtnl1-/- mice. For all representative images, at least three independent experiments were performed with similar results. For every microscope field, average Ki67+ ISC numbers per crypt are shown as means ± SD. **P < 0.01 by two-tailed Student's t-test in right panel. Scale bar, 50 μm.


( E ) H & E staining of small intestine tissues from indicated mice at different time points ( D0 - D7 ) after 8 Gy radiation damage . Number of crypts were calculated as means ± SD . * * P < 0 . 01 by two - tailed Student ' s t - test in right panel . n = 5 fields were calculated for each group . Scale bar , 50 μm .

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(E) H&E staining of small intestine tissues from indicated mice at different time points (D0 - D7) after 8 Gy radiation damage. Number of crypts were calculated as means ± SD. **P < 0.01 by two-tailed Student's t-test in right panel. n = 5 fields were calculated for each group. Scale bar, 50 μm.


( G ) Confocal microscopy of ISCs makers Olmf4 in organoids from circBtnl1 + / + and circBtnl1 - / - mice . Scale bars , 30 μm . Three independent experiments were performed with similar results , and representative experiments are shown . For every microscope field , average Olmf4 + ISC numbers per crypt are shown as means ± SD . * * P < 0 . 01 by two - tailed Student ' s t - test in right panel in right panel .

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(G) Confocal microscopy of ISCs makers Olmf4 in organoids from circBtnl1+/+ and circBtnl1-/- mice. Scale bars, 30 μm. Three independent experiments were performed with similar results, and representative experiments are shown. For every microscope field, average Olmf4+ ISC numbers per crypt are shown as means ± SD. **P < 0.01 by two-tailed Student's t-test in right panel in right panel.


( B , C ) CircBtnl1 was co - localized with Ddx3y protein in small intestine tissue ( B ) and organoid ( C ) by RNA FISH , followed by immunofluorescence staining . Scale bars , 5 μm .

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(B, C) CircBtnl1 was co-localized with Ddx3y protein in small intestine tissue (B) and organoid (C) by RNA FISH, followed by immunofluorescence staining. Scale bars, 5 μm.


( G ) Electrophoretic mobility shift assay ( EMSA ) using biotin - labeled circBtnl1 and recombinant Ddx3y .

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(G) Electrophoretic mobility shift assay (EMSA) using biotin-labeled circBtnl1 and recombinant Ddx3y.


( H ) Truncated fragments of biotinylated circBtnl1 were incubated with small intestine crypts lysates , followed by RNA pulldown assay and Western blot .

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(H) Truncated fragments of biotinylated circBtnl1 were incubated with small intestine crypts lysates, followed by RNA pulldown assay and Western blot.


( B ) Small intestine crypt cells were isolated from circBtnl1 + / + and circBtnl1 - / - two - month - old mice , followed by RNA extraction and q - PCR analysis for top ten upregulated transcription factors . n = 3 independent samples . Results of relative fold changes are shown as means ± SD . * * P < 0 . 01 by two - tailed Student ' s t - test .

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(B) Small intestine crypt cells were isolated from circBtnl1+/+ and circBtnl1-/- two-month-old mice, followed by RNA extraction and q-PCR analysis for top ten upregulated transcription factors. n = 3 independent samples. Results of relative fold changes are shown as means ± SD. **P < 0.01 by two-tailed Student's t-test.


( D ) Immunoblotting analysis of Ddx3y in RNA pull - down samples by Atf4 sense , antisense and control probes in mice ISCs . ( E ) Immunoblotting analysis of Ddx3y in RNA pull - down samples by Atf4 and control probes in circBtnl1 + / + and circBtnl1 - / - mice ISCs .

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(D) Immunoblotting analysis of Ddx3y in RNA pull-down samples by Atf4 sense, antisense and control probes in mice ISCs. (E) Immunoblotting analysis of Ddx3y in RNA pull-down samples by Atf4 and control probes in circBtnl1+/+ and circBtnl1-/- mice ISCs.


( J ) Confocal microscopy of ATF4 in Ddx3y knockdown and overexpression organoids from circBtnl1 + / + and circBtnl1 - / - mice . Scale bars , 20 μm .

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(J) Confocal microscopy of ATF4 in Ddx3y knockdown and overexpression organoids from circBtnl1+/+ and circBtnl1-/- mice. Scale bars, 20 μm.


( K , L ) Expression levels of Ddx3y and Atf4 in circBtnl1 + / + and circBtnl1 - / - ISCs were examined by real - time PCR ( K ) and Western blot ( L ) , three independent experiments were performed . Results of relative fold changes are shown as means ± SD . * * P < 0 . 01 , NS , not significant , by two - tailed Student ' s t - test .

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(K, L) Expression levels of Ddx3y and Atf4 in circBtnl1+/+ and circBtnl1-/- ISCs were examined by real-time PCR (K) and Western blot (L), three independent experiments were performed. Results of relative fold changes are shown as means ± SD. ** P < 0.01, NS, not significant, by two-tailed Student's t-test.


( D , E ) ChIP assay was performed using anti - H3K4me3 ( D ) or anti - H3K27me3 ( E ) with circBtnl1 + / + and circBtnl1 - / - ISCs lysates . * * P < 0 . 01 by two - tailed Student ' s t - test . Data are representative of at least three independent experiments .

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(D, E) ChIP assay was performed using anti-H3K4me3 (D) or anti-H3K27me3 (E) with circBtnl1+/+ and circBtnl1-/- ISCs lysates. **P < 0.01 by two-tailed Student's t-test. Data are representative of at least three independent experiments.


( H ) Confocal microscopy of Sox9 in Atf4 knockdown ( Atf4 KD ) organoids from circBtnl1 + / + and circBtnl1 - / - mice . Scale bars , 30 μm .

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(H) Confocal microscopy of Sox9 in Atf4 knockdown (Atf4 KD) organoids from circBtnl1+/+ and circBtnl1-/- mice. Scale bars, 30 μm.


( D ) H & E staining of small intestinal tissues from indicated mice . Two - month - old mice were used and n = 3 fields were taken for each group . Scale bar , 100 μm . Number of crypts were calculated as means ± SD . * P < 0 . 05 , * * P < 0 . 01 by two - tailed Student ' s t - test in right panel .

(D) H&E staining of small intestinal tissues from indicated mice. Two-month-old mice were used and n = 3 fields were taken for each group. Scale bar, 100 μm. Number of crypts were calculated as means ± SD. *P < 0.05, **P < 0.01 by two-tailed Student's t-test in right panel.


( E ) H & E staining of intestinal tissues from indicated mice at different time points after 8 Gy of radiation damage . Two - month - old mice were used and n = 3 fields were taken for each group . Number of crypts were calculated as means ± SD . * P < 0 . 05 , * * P < 0 . 01 by two - tailed Student ' s t - test in right panel . Scale bar , 50 μm .

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(E) H&E staining of intestinal tissues from indicated mice at different time points after 8 Gy of radiation damage. Two-month-old mice were used and n = 3 fields were taken for each group. Number of crypts were calculated as means ± SD. *P < 0.05, **P < 0.01 by two-tailed Student's t-test in right panel. Scale bar, 50 μm.


( F ) Representative organoid images of circBtnl1 + / + ; sgSox9 and circBtnl1 - / - ; sgSox9 mice , sgCtrl served as control . Organoid formation ratios ( n = 3 ) are shown . Organoid formation ratios are shown as mean ± s . d . * P < 0 . 05 by two - tailed Student ' s t - test . Scale bars , 200 μm .

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(F) Representative organoid images of circBtnl1+/+; sgSox9 and circBtnl1-/-; sgSox9 mice, sgCtrl served as control. Organoid formation ratios (n = 3) are shown. Organoid formation ratios are shown as mean ± s. d. *P < 0.05 by two-tailed Student's t-test. Scale bars, 200 μm.


( a ) Kaplan - Meier survival curves of combined male and female WT and Atg5Tg mice no . 25 ( P0 . 001 ; n = 65 for WT mice and n = 70 for Atg5Tg mice ) . ( b , c ) Lifespan extension in male Atg5Tg mice no . 25 ( P0 . 001 ; n = 32 for WT mice and n = 36 for Atg5Tg mice no . 25 ) ( b ) and female Atg5Tg mice no . 25 ( P0 . 001 ; n = 33 for WT mice and n = 34 for Atg5Tg mice no . 25 ) ( c ) . ( d - f ) Kaplan - Meier survival curves of combined male and female WT and Atg5Tg mice no . 43 ( d ) , no . 47 ( e ) and no . 471 ( f ) ( P0 . 001 ; n = 25 for WT mice and n = 30 for Atg5Tg mice ) . P - values were calculated using the log - rank test .

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(a) Kaplan-Meier survival curves of combined male and female WT and Atg5Tg mice no. 25 (P0.001; n=65 for WT mice and n=70 for Atg5Tg mice). (b,c) Lifespan extension in male Atg5Tg mice no. 25 (P0.001; n=32 for WT mice and n=36 for Atg5Tg mice no. 25) (b) and female Atg5Tg mice no. 25 (P0.001; n=33 for WT mice and n=34 for Atg5Tg mice no. 25) (c). (d-f) Kaplan-Meier survival curves of combined male and female WT and Atg5Tg mice no. 43 (d), no. 47 (e) and no. 471 (f) (P0.001; n=25 for WT mice and n=30 for Atg5Tg mice). P-values were calculated using the log-rank test.


( g ) Expression levels of Atg5 and Atg12 - Atg5 conjugate , the conversion of LC3 I into LC3 II , and p62 level in the hearts of Atg5 Tg mice . Whole - tissue extracts were prepared from 3 - month - old Atg5 Tg mice and WT littermates , and analysed by western blotting using the indicated antibodies . β - Actin served as a control . ( h ) Densitometric analysis of the signals on the blots shown in ( g ) . The bars represent the mean ± s . d . ( n = 3 ) .

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(g) Expression levels of Atg5 and Atg12-Atg5 conjugate, the conversion of LC3 I into LC3 II, and p62 level in the hearts of Atg5 Tg mice. Whole-tissue extracts were prepared from 3-month-old Atg5 Tg mice and WT littermates, and analysed by western blotting using the indicated antibodies. β-Actin served as a control. (h) Densitometric analysis of the signals on the blots shown in (g). The bars represent the mean±s.d. (n=3).


( a ) Western blot analysis showing enhanced levels of Atg5 , Atg12 - Atg5 conjugate and LC3 conversion , and reduced p62 levels in the indicated tissues of 18 - month - old Atg5 Tg mice no . 25 ( T ) and WT mice ( W ) . ( b ) Densitometric analysis of the signals on the blots shown in ( a ) .

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(a) Western blot analysis showing enhanced levels of Atg5, Atg12-Atg5 conjugate and LC3 conversion, and reduced p62 levels in the indicated tissues of 18-month-old Atg5 Tg mice no. 25 (T) and WT mice (W). (b) Densitometric analysis of the signals on the blots shown in (a).


( c ) Electron microscopy images of autophagosomes ( arrows ) and late - autophagic compartments ( arrowheads ) in the liver tissues of 18 - month - old Atg5 Tg mice no . 25 and WT mice . Scale bars are indicated . ( d ) The numbers of autophagosomes and autolysosomes per cell ( n = 50 ) in the electron microscopic images shown in ( c ) were counted . The bars represent the mean ± s . d . * * * P0 . 0001 versus control ; Student ' s t - test .

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(c) Electron microscopy images of autophagosomes (arrows) and late-autophagic compartments (arrowheads) in the liver tissues of 18-month-old Atg5 Tg mice no. 25 and WT mice. Scale bars are indicated. (d) The numbers of autophagosomes and autolysosomes per cell (n=50) in the electron microscopic images shown in (c) were counted. The bars represent the mean±s.d. ***P0.0001 versus control; Student's t-test.


( e ) Fluorogenic comparison of basal autophagy activity between GFP - LC3 Tg mice and Atg5 / GFP - LC3 double Tg mice . Whole bodies of 8 - week - old male ( left ) and female ( right ) GFP - LC3 Tg ( LC3 ) , and LC3 / LC3 - LC3Tg mice ( Atg5 / LC3 ) were visualized using a Kodak Image Station 4000MM equipped with a filter set for FITC ( Kodak Molecular Imaging Software , Version 4 . 0 ) .

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(e) Fluorogenic comparison of basal autophagy activity between GFP-LC3 Tg mice and Atg5/GFP-LC3 double Tg mice. Whole bodies of 8-week-old male (left) and female (right) GFP-LC3 Tg (LC3), and LC3/LC3-LC3Tg mice (Atg5/LC3) were visualized using a Kodak Image Station 4000MM equipped with a filter set for FITC (Kodak Molecular Imaging Software, Version 4.0).


( f ) Western blot analysis showing autophagy markers in the liver of GFP - LC3 mice . GFP - LC3 mice were injected for 5 days with siRNA - Atg5 or Atg5 - HA cDNA through the tail vein . Whole - liver tissue lysates were prepared and subjected to immunoblotting using the indicated antibodies . β - Actin served as a control .

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(f) Western blot analysis showing autophagy markers in the liver of GFP-LC3 mice. GFP-LC3 mice were injected for 5 days with siRNA-Atg5 or Atg5-HA cDNA through the tail vein. Whole-liver tissue lysates were prepared and subjected to immunoblotting using the indicated antibodies. β-Actin served as a control.


( a ) Representative physical pictures of 18 - month - old WT and Atg5 Tg mice fed with a regular diet . ( b ) Body weight chart of ageing - induced obesity in WT and Atg5 Tg mice . The mice were fed with a regular diet for 24 months ( n = 180 - - 250 ) and their body weights were monitored monthly . The bars represent the mean ± s . d . * * * P0 . 0001 versus control ; Student ' s t - test .

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(a) Representative physical pictures of 18-month-old WT and Atg5 Tg mice fed with a regular diet. (b) Body weight chart of ageing-induced obesity in WT and Atg5 Tg mice. The mice were fed with a regular diet for 24 months (n=180--250) and their body weights were monitored monthly. The bars represent the mean±s.d. ***P0.0001 versus control; Student's t-test.


. ( c ) Average daily oral food intake normalized to body weight , measured on regular diet . The food intake of mice ( n = 18 - 24 ) was measured for 2 weeks at 3 - , 6 - , 12 - and 18 - month - old age .

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. (c) Average daily oral food intake normalized to body weight, measured on regular diet. The food intake of mice (n=18-24) was measured for 2 weeks at 3-, 6-, 12- and 18-month-old age.


d ) Quantification of gonadal fat pads from 18 - month - old mice ( n = 11 ) . * * * P0 . 0001 versus control ; Student ' s t - test . ( e , f ) Quantification of visceral ( e ) and subcutaneous ( f ) fat pads of 18 - month - old mice ( n = 6 ) . * * * P0 . 0001 versus control ; Student ' s t - test .

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d) Quantification of gonadal fat pads from 18-month-old mice (n=11). ***P0.0001 versus control; Student's t-test. (e,f) Quantification of visceral (e) and subcutaneous (f) fat pads of 18-month-old mice (n=6). ***P0.0001 versus control; Student's t-test.


( g ) Enhanced glucose tolerance of Atg5 Tg mice . WT and Atg5 Tg mice were starved overnight and then given with an i . p injection of glucose ( 1 mg g − 1 body weight ) . The results are the mean ± s . d . of 12 - week - old WT and Atg5 Tg mice ( n = 12 ) . * P0 . 01 , * * P0 . 001 , * * * P0 . 0001 versus control ; Student ' s t - test .

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(g) Enhanced glucose tolerance of Atg5 Tg mice. WT and Atg5 Tg mice were starved overnight and then given with an i.p injection of glucose (1 mg g−1 body weight). The results are the mean±s.d. of 12-week-old WT and Atg5 Tg mice (n=12). *P0.01, **P0.001, ***P0.0001 versus control; Student's t-test.


( h ) Enhanced insulin sensitivity of Atg5 Tg mice . WT and Atg5 Tg mice were starved for 6 h and then i . p injected with 0 . 75 IU soluble insulin . * P0 . 01 , * * P0 . 001 versus control ; Student ' s t - test .

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(h) Enhanced insulin sensitivity of Atg5 Tg mice. WT and Atg5 Tg mice were starved for 6 h and then i.p injected with 0.75 IU soluble insulin. *P0.01, **P0.001 versus control; Student's t-test.


( i ) Plasma levels of triglycerol ( TG ) in WT and Atg5 Tg mice . The mice were fasted for 3 h before measurement . Young mice ( 4 - week old , n = 8 ) ; old mice ( 18 - month old , n = 8 ) . * * * P0 . 0001 versus control ; Student ' s t -

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(i) Plasma levels of triglycerol (TG) in WT and Atg5 Tg mice. The mice were fasted for 3 h before measurement. Young mice (4-week old, n=8); old mice (18-month old, n=8). ***P0.0001 versus control; Student's t-test


( j ) Plasma leptin levels in WT and Atg5 Tg mice ( n = 8 - 12 ) . * * * P0 . 0001 versus control ; Student ' s t - test . (

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(j) Plasma leptin levels in WT and Atg5 Tg mice (n=8-12). ***P0.0001 versus control; Student's t-test. (


( k ) GSH / GSSG ratio in the liver ( n = 4 males , three times trial ) . * * P0 . 001 , * * * P0 . 0001 versus control ; Student ' s t - test .

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(k) GSH/GSSG ratio in the liver (n=4 males, three times trial). **P0.001, ***P0.0001 versus control; Student's t-test.


( l ) Respiration rates of mitochondria in WT and Atg5 Tg MEFs ( n = 2 ) .

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(l) Respiration rates of mitochondria in WT and Atg5 Tg MEFs (n=2).


( m ) Wire - hang endurance test of Atg5 Tg and WT mice ( n = 10 ) . All the bars represent the mean ± s . d . * * * P0 . 0001 versus control ; Student ' s t - test .

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(m) Wire-hang endurance test of Atg5 Tg and WT mice (n=10). All the bars represent the mean±s.d. ***P0.0001 versus control; Student's t-test.


( a ) Comparison of Atg12 - Atg5 conjugate levels in WT and Atg5 Tg MEFs by western blot analysis . MEFs from WT and Atg5 Tg mice were cultured at embryonic day 13 , after which cell extracts were examined by western blot analysis using anti - Atg5 and anti - β - actin antibodies .

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(a) Comparison of Atg12-Atg5 conjugate levels in WT and Atg5 Tg MEFs by western blot analysis. MEFs from WT and Atg5 Tg mice were cultured at embryonic day 13, after which cell extracts were examined by western blot analysis using anti-Atg5 and anti-β-actin antibodies.


( b ) Increased conversion of LC3 and reduced expression of p62 in Atg5 Tg MEFs no . 6 by rapamycin and / or Baf . A1 . WT MEFs no . 1 and Atg5 Tg MEFs no . 6 in passage number 3 were incubated for 48 h with 10 μM rapamycin in the absence or presence of Baf . A1 , and cell extracts were then analysed by western blotting . The signals on the blot were quantified by densitometric analysis and represented as the ratio of p62 to β - actin .

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(b) Increased conversion of LC3 and reduced expression of p62 in Atg5 Tg MEFs no. 6 by rapamycin and/or Baf.A1. WT MEFs no. 1 and Atg5 Tg MEFs no. 6 in passage number 3 were incubated for 48 h with 10 μM rapamycin in the absence or presence of Baf.A1, and cell extracts were then analysed by western blotting. The signals on the blot were quantified by densitometric analysis and represented as the ratio of p62 to β-actin.


( c , d ) Increased resistance of Atg5 Tg MEFs to oxidative stress . WT MEFs no . 1 and Atg5 Tg MEFs no . 6 were treated for 24 h with 50 , 100 , 200 and 300 μM H2O2 ( c ) or 300 μM H2O2 in the absence or presence of 5 mM 3 - MA or 20 nM Baf . A1 ( Baf . A1 ) ( d ) . Cell viability was assessed by propidium iodide staining . The values are the mean ± s . e . m . ( n = 3 ) . * * P0 . 001 , * * * P0 . 0001 versus control ; Student ' s t - test .

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(c,d) Increased resistance of Atg5 Tg MEFs to oxidative stress. WT MEFs no. 1 and Atg5 Tg MEFs no. 6 were treated for 24 h with 50, 100, 200 and 300 μM H2O2 (c) or 300 μM H2O2 in the absence or presence of 5 mM 3-MA or 20 nM Baf.A1 (Baf.A1) (d). Cell viability was assessed by propidium iodide staining. The values are the mean±s.e.m. (n=3). **P0.001, ***P0.0001 versus control; Student's t-test.


( e ) Representative photographs showing the resistance of Atg5 Tg MEFs to oxidative stress . Primary cultured WT and Atg5 Tg MEFs were treated with 300 μM H2O2 for 24 h and then observed under a light microscope . Scale bars , 50 μm .

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(e) Representative photographs showing the resistance of Atg5 Tg MEFs to oxidative stress. Primary cultured WT and Atg5 Tg MEFs were treated with 300 μM H2O2 for 24 h and then observed under a light microscope. Scale bars, 50 μm.


( f ) Effect of oxidative stress on the autophagy activity in Atg5 Tg MEFs . WT MEFs no . 1 and Atg5 Tg MEFs no . 6 were treated with 100 μM H2O2 for 24 h in the presence or absence of 20 nM Baf . A1 , and cell extracts were analysed by western blotting . About 10 and 30 μg of proteins were used for LC3 ( upper panel ) and p62 ( bottom panel ) detection , respectively . β - Actin served as a control .

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(f) Effect of oxidative stress on the autophagy activity in Atg5 Tg MEFs. WT MEFs no. 1 and Atg5 Tg MEFs no. 6 were treated with 100 μM H2O2 for 24 h in the presence or absence of 20 nM Baf.A1, and cell extracts were analysed by western blotting. About 10 and 30 μg of proteins were used for LC3 (upper panel) and p62 (bottom panel) detection, respectively. β-Actin served as a control.


Representative images for the TIF analysis shown in ( C ) . Shown are POT1Y223C / - hESCs and an unedited wild - type hESC clone ( WT / WT ) isolated in parallel , as well as a POT1 WT / Q623H hESC and a contemporaneously isolated hECS clones ( WT / WT ) . Scale bars = 10µm . Box and whisker plot ( showing min , max and average ) quantifying telomere - dysfunction induced foci ( TIF ) in two POT1 Y223C / - hESC lines , one POT1 Q623H / Q623H , and one POT1WT / Q623H hESC lines compared to wild - type unedited control hESCs . Cells were analyzed 3 months post targeting . Cells were stained for TRF1 and γ - H2Ax . POT1Y223C / - cells have increased TIFs compared to an unedited wild - type cell clone , isolated and cultured in parallel ( P < 0 . 01 , two - tailed Mann - Whitney test , one biological replicate ) .

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Representative images for the TIF analysis shown in (C). Shown are POT1Y223C/- hESCs and an unedited wild-type hESC clone (WT/WT) isolated in parallel, as well as a POT1 WT/Q623H hESC and a contemporaneously isolated hECS clones (WT/WT). Scale bars = 10µm.   Box and whisker plot (showing min, max and average) quantifying telomere-dysfunction induced foci (TIF) in two POT1 Y223C/- hESC lines, one POT1 Q623H/Q623H, and one POT1WT/Q623H hESC lines compared to wild-type unedited control hESCs. Cells were analyzed 3 months post targeting. Cells were stained for TRF1 and γ-H2Ax. POT1Y223C/- cells have increased TIFs compared to an unedited wild-type cell clone, isolated and cultured in parallel (P<0.01, two-tailed Mann-Whitney test, one biological replicate).


Images of telomere and centromere FISH on metaphase spreads of cell lines as in ( B ) . Cy3 Tel - C PNA probes were used . Arrows indicate chromosomal abnormalities . E ) Quantification of chromosomal abnormalities as determined by FISH analysis in ( D ) .

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Images of telomere and centromere FISH on metaphase spreads of cell lines as in (B). Cy3 Tel-C PNA probes were used. Arrows indicate chromosomal abnormalities. E) Quantification of chromosomal abnormalities as determined by FISH analysis in (D).


B ) Quantification of telomere length data from two STELA experiments using the same genomic DNA , shown in ( A ) and ( Appendix Figure 2C ) . Indicated in red is the mean telomere length and the error bars indicate the standard deviation .

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B) Quantification of telomere length data from two STELA experiments using the same genomic DNA, shown in (A) and (Appendix Figure 2C). Indicated in red is the mean telomere length and the error bars indicate the standard deviation.


B ) TIF analysis of wild - type , POT1WT / Y223C hESCs , and the indicated controls using antibodies against TRF1 and γ - H2AX . Scale bars = 10µm . Lentiviral expression of TPP1 ∆ PBD served as a positive control to demonstrate that cells have an intact telomere DNA damage response . TPP1 ∆ PBD infected cells were analyzed 72h post infection and without selection .

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B) TIF analysis of wild-type, POT1WT/Y223C hESCs, and the indicated controls using antibodies against TRF1 and γ-H2AX. Scale bars = 10µm. Lentiviral expression of TPP1∆PBD served as a positive control to demonstrate that cells have an intact telomere DNA damage response. TPP1∆PBD infected cells were analyzed 72h post infection and without selection.


E ) Quantification of two STELA experiments , including that shown in ( D ) . Indicated in red is the mean telomere length and the error bars indicate the standard deviation .

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E) Quantification of two STELA experiments, including that shown in (D). Indicated in red is the mean telomere length and the error bars indicate the standard deviation.


D ) Quantification of edited or unedited colonies isolated in the in vitro differentiation described in ( C ) WT : wild - type , MT : mutant .

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D) Quantification of edited or unedited colonies isolated in the in vitro differentiation described in (C) WT: wild-type, MT: mutant.


C ) Representative genotyping outcomes of hCD34 + cells nucleofected sgRNA - 1 , xenografted and isolated from the bone marrow 16 weeks after transplantation into mice determined by NGS . Data information : The vertical dashed line shows the site of CAS9 cut and the base pairs noted in red show insertion . A dash inside a gray box indicates a deleted base pair .

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C) Representative genotyping outcomes of hCD34+ cells nucleofected sgRNA-1, xenografted and isolated from the bone marrow 16 weeks after transplantation into mice determined by NGS. Data information: The vertical dashed line shows the site of CAS9 cut and the base pairs noted in red show insertion. A dash inside a gray box indicates a deleted base pair.


B ) Telomere restriction fragment assay for the five subjects shown in ( A ) . Mean telomere length is indicated .

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B) Telomere restriction fragment assay for the five subjects shown in (A). Mean telomere length is indicated.


( A ) Representative micrographs of human skin sections stained with hematoxylin - eosin ( H & E ) ( left ) and anti - IL - 38 antibody ( right ) from normal patients ( n = 11 ) and tumors of cSCC patients ( n = 13 ) . Scale bars represent 100 μm . The graph shows the quantification of mean IL - 38 expression per high - powered field in tissues . ( B ) Relative expression of IL - 38 in human normal tissues ( n = 9 ) and cSCC ( n = 18 ) were analyzed using Geo Datasets ( GSE98767 ) . Error bars represent the mean ± SD . All data are biological replicates . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; p values were calculated using Student ' s t test .

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(A) Representative micrographs of human skin sections stained with hematoxylin-eosin (H&E) (left) and anti-IL-38 antibody (right) from normal patients (n = 11) and tumors of cSCC patients (n = 13). Scale bars represent 100 μm. The graph shows the quantification of mean IL-38 expression per high-powered field in tissues. (B) Relative expression of IL-38 in human normal tissues (n = 9) and cSCC (n = 18) were analyzed using Geo Datasets (GSE98767). Error bars represent the mean ± SD. All data are biological replicates. *p < 0.05; **p < 0.01; ***p < 0.001; p values were calculated using Student's t test.


( C and D ) The dorsal hair of normal C57 / BL6 mice were shaved and treated with DMBA / TPA twice a week for 32 weeks to induce skin tumors . ( C ) Representative micrographs of mouse normal skin ( n = 6 ) and tumor ( n = 6 ) sections stained with anti - IL - 38 antibody . The graph shows the quantification of mean IL - 38 expression per high - powered field in tissues . Scale bars represent 100 μm . ( D ) Relative expression levels of Il - 38 in normal skin ( n = 5 ) and tumors ( n = 5 ) of mice were quantified using qPCR . Error bars represent the mean ± SD . All data are biological replicates . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; p values were calculated using Student ' s t test .

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(C and D) The dorsal hair of normal C57/BL6 mice were shaved and treated with DMBA/TPA twice a week for 32 weeks to induce skin tumors. (C) Representative micrographs of mouse normal skin (n = 6) and tumor (n = 6) sections stained with anti-IL-38 antibody. The graph shows the quantification of mean IL-38 expression per high-powered field in tissues. Scale bars represent 100 μm. (D) Relative expression levels of Il-38 in normal skin (n = 5) and tumors (n = 5) of mice were quantified using qPCR. Error bars represent the mean ± SD. All data are biological replicates. *p < 0.05; **p < 0.01; ***p < 0.001; p values were calculated using Student's t test.


( F ) Representative western blot bands indicating IL - 38 in mouse normal skin ( n = 3 ) and DMBA / TPA - induced tumors ( n = 3 ) . The graph shows the quantification of mean IL - 38 expression in tissues . Error bars represent the mean ± SD . All data are biological replicates . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; p values were calculated using Student ' s t test .

O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Representative western blot bands indicating IL-38 in mouse normal skin (n = 3) and DMBA/TPA-induced tumors (n = 3). The graph shows the quantification of mean IL-38 expression in tissues. Error bars represent the mean ± SD. All data are biological replicates. *p < 0.05; **p < 0.01; ***p < 0.001; p values were calculated using Student's t test.


( B ) Representative photographs of Il - 38f / f ( n = 12 ) and K14Cre / + - Il - 38f / f mice ( n = 8 ) treated with DMBA / TPA for 32 weeks . White triangles indicate the tumors on the back of mice . ( C Average tumor number ( C ) per mouse from Il - 38f / f ( n = 12 ) and K14Cre / + - Il - 38f / f mice ( n = 8 ) treated with DMBA / TPA . . Error bars represent the mean ± SD ( C ) All data are biological replicates . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; p values were calculated using Student ' s t test (

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

(B) Representative photographs of Il-38f/f (n = 12) and K14Cre/+-Il-38f/f mice (n = 8) treated with DMBA/TPA for 32 weeks. White triangles indicate the tumors on the back of mice. (C Average tumor number (C) per mouse from Il-38f/f (n = 12) and K14Cre/+-Il-38f/f mice (n = 8) treated with DMBA/TPA.. Error bars represent the mean ± SD (C) All data are biological replicates. *p < 0.05; **p < 0.01; ***p < 0.001; p values were calculated using Student's t test (C