( E ) Quantification of the phenotype in D . The number of GFP - LC3 ‐ positive vesicles per transfected cell was scored for at least fifty cells . Data are expressed as means ± s . d . of one representative experiment of three repetitions . Source data for this figure is available on the online supplementary information page .

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(E) Quantification of the phenotype in D. The number of GFP-LC3‐positive vesicles per transfected cell was scored for at least fifty cells. Data are expressed as means ±s.d. of one representative experiment of three repetitions.Source data for this figure is available on the online supplementary information page.


( A ) TMEM59 ID is required for HA - LC3 lipidation . 293 cells were transfected with full ‐ length TMEM59 ( FL ) or a deleted version lacking the ID ( ΔID , Δ263 - 323 ) , HA - LC3A ( left ) or HA - LC3B ( right ) and GST . Cells were lysed for western blotting against the shown molecules .

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(A) TMEM59 ID is required for HA-LC3 lipidation. 293 cells were transfected with full‐length TMEM59 (FL) or a deleted version lacking the ID (ΔID, Δ263-323), HA-LC3A (left) or HA-LC3B (right) and GST. Cells were lysed for western blotting against the shown molecules.


( B ) The ID is necessary for GFP - LC3 activation . 293 cells were transfected with the indicated TMEM59 constructs and GFP - LC3A or GFP - LC3B ( as indicated ) . Representative confocal pictures are shown .

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(B) The ID is necessary for GFP-LC3 activation. 293 cells were transfected with the indicated TMEM59 constructs and GFP-LC3A or GFP-LC3B (as indicated). Representative confocal pictures are shown.


( C ) Quantification of the phenotype in B . Scoring and data expression were as in Figure 1E .

(C) Quantification of the phenotype in B. Scoring and data expression were as in Figure 1E.


( D ) TMEM59 ID ( amino acids 263 - 323 ) suffices for HA - LC3A lipidation . 293 cells were transfected with the indicated CD16 : 7 chimera ( Control : empty chimera ) and vectors encoding HA - LC3A and GST , subjected to aggregation with the shown amounts of anti ‐ CD16 antibody and lysed for western blotting . The right panel shows control WBs demonstrating equal loading ( ACTIN ) , transfection ( GST ) and chimera expression ( CD16 ) in unaggregated samples , as all experimental points per chimera derive from a single transfection .

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(D) TMEM59 ID (amino acids 263-323) suffices for HA-LC3A lipidation. 293 cells were transfected with the indicated CD16:7 chimera (Control: empty chimera) and vectors encoding HA-LC3A and GST, subjected to aggregation with the shown amounts of anti‐CD16 antibody and lysed for western blotting. The right panel shows control WBs demonstrating equal loading (ACTIN), transfection (GST) and chimera expression (CD16) in unaggregated samples, as all experimental points per chimera derive from a single transfection.


( E ) Comparable surface levels of CD16 : 7 constructs . Transfected 293 cells were processed for anti ‐ CD16 flow cytometry . The graph displays percentages of positive cells ( left axis ) and means of fluorescence of positive cells ( MF , right axis ) obtained from triplicates . Data are expressed as means ± s . d . of the triplicates .

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(E) Comparable surface levels of CD16:7 constructs. Transfected 293 cells were processed for anti‐CD16 flow cytometry. The graph displays percentages of positive cells (left axis) and means of fluorescence of positive cells (MF, right axis) obtained from triplicates. Data are expressed as means ±s.d. of the triplicates.


( F ) The ID suffices for HA - LC3B lipidation .

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(F) The ID suffices for HA-LC3B lipidation.


( G ) The ID suffices for GFP - LC3 activation . 293 cells were transfected with the indicated chimeras and GFP - LC3A or GFP - LC3B , aggregated and mounted . Representative confocal microscopy pictures are shown . Activated GFP - LC3 appears as a collapsed mass of indiscernible vacuoles .

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(G) The ID suffices for GFP-LC3 activation. 293 cells were transfected with the indicated chimeras and GFP-LC3A or GFP-LC3B, aggregated and mounted. Representative confocal microscopy pictures are shown. Activated GFP-LC3 appears as a collapsed mass of indiscernible vacuoles.


( H ) Quantification of the phenotype in G . As individual vesicles could not be counted , quantification was done as the percentage of transfected cells showing redistributed GFP - LC3 . At least 10 different fields were counted ( about 400 cells ) . The experiment was repeated three times . Data are expressed as means ± s . d . of the triplicates . Source data for this figure is available on the online supplementary information page .

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(H) Quantification of the phenotype in G. As individual vesicles could not be counted, quantification was done as the percentage of transfected cells showing redistributed GFP-LC3. At least 10 different fields were counted (about 400 cells). The experiment was repeated three times. Data are expressed as means ±s.d. of the triplicates.Source data for this figure is available on the online supplementary information page.


( A , B ) Ability of serial C ‐ terminal deletions of TMEM59 to activate LC3 . 293 cells were transfected with the indicated TMEM59 C ‐ terminal deletions , HA - LC3A ( A ) or HA - LC3B ( B ) and GST , and lysed for western blotting against the indicated molecules .

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(A, B) Ability of serial C‐terminal deletions of TMEM59 to activate LC3. 293 cells were transfected with the indicated TMEM59 C‐terminal deletions, HA-LC3A (A) or HA-LC3B (B) and GST, and lysed for western blotting against the indicated molecules.


( C ) Amino acids 263 - 281 retain the full potential of TMEM59 ID to promote HA - LC3 conversion . 293 cells were transfected with the shown CD16 : 7 chimeras , HA - LC3A or HA - LC3B ( as indicated ) and GST , and subjected to anti ‐ CD16 aggregation before lysing them for western blotting . The lower panels show control WBs ( unaggregated samples ) .

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(C) Amino acids 263-281 retain the full potential of TMEM59 ID to promote HA-LC3 conversion. 293 cells were transfected with the shown CD16:7 chimeras, HA-LC3A or HA-LC3B (as indicated) and GST, and subjected to anti‐CD16 aggregation before lysing them for western blotting. The lower panels show control WBs (unaggregated samples).


D Amino acids 263 - 281 suffice for GFP - LC3 activation and retain the full potential of TMEM59 ID to promote GFP - LC3 activation . 293 cells were transfected with the indicated CD16 : 7 chimeras and GFP - LC3A or GFP - LC3B ( as indicated ) , and subjected to anti ‐ CD16 aggregation before fixing them for microscopy . The graph shows percentages of transfected cells exhibiting redistributed GFP - LC3 . Quantification and data expression were done as in Figure 2H .

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D Amino acids 263-281 suffice for GFP-LC3 activation and retain the full potential of TMEM59 ID to promote GFP-LC3 activation. 293 cells were transfected with the indicated CD16:7 chimeras and GFP-LC3A or GFP-LC3B (as indicated), and subjected to anti‐CD16 aggregation before fixing them for microscopy. The graph shows percentages of transfected cells exhibiting redistributed GFP-LC3. Quantification and data expression were done as in Figure 2H.


( E ) Amino acids 282 - 323 of TMEM59 lack LC3 activation potential . 293 cells were transfected with the shown chimeras , HA - LC3A or HA - LC3B and GST , and processed for western blotting against the indicated molecules . Control WBs of unaggregated samples are shown in the lower panels .

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(E) Amino acids 282-323 of TMEM59 lack LC3 activation potential. 293 cells were transfected with the shown chimeras, HA-LC3A or HA-LC3B and GST, and processed for western blotting against the indicated molecules. Control WBs of unaggregated samples are shown in the lower panels.


( F ) Surface expression levels of CD16 : 7 chimeras . Procedures and data expression were as in Figure 2E . The figure shows that the functional differences observed between CD16 : 7 chimeras ( C - E ) are not caused by differential surface expression .

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(F) Surface expression levels of CD16:7 chimeras. Procedures and data expression were as in Figure 2E. The figure shows that the functional differences observed between CD16:7 chimeras (C-E) are not caused by differential surface expression.


( A ) Endocytic vesicles containing aggregated CD16 : 7 - 263 - 281 become labelled with GFP - LC3 . JAR cells were transfected with vectors expressing the indicated CD16 : 7 chimeras and GFP - LC3A , and subjected to anti ‐ CD16 aggregation for 8 h before staining for the endocytosed chimera ( red ) . Representative confocal pictures are shown . The inset highlights a vesicle where the peripheral GFP - LC3 staining is particularly distinguishable . The right panel displays control WBs showing that chimera aggregation for 8 h activates HA - LC3A conversion in JAR cells .

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(A) Endocytic vesicles containing aggregated CD16:7-263-281 become labelled with GFP-LC3. JAR cells were transfected with vectors expressing the indicated CD16:7 chimeras and GFP-LC3A, and subjected to anti‐CD16 aggregation for 8 h before staining for the endocytosed chimera (red). Representative confocal pictures are shown. The inset highlights a vesicle where the peripheral GFP-LC3 staining is particularly distinguishable. The right panel displays control WBs showing that chimera aggregation for 8 h activates HA-LC3A conversion in JAR cells.


( B ) Colocalization of endocytosed CD16 : 7 - 263 - 281 with EEA1 and CD63 . JAR cells were transfected with CD16 : 7 - 263 - 281 and subjected to aggregation before staining them for the endocytosed chimera ( red ) and EEA1 or CD63 ( green ; FITC ‐ coupled primary antibodies ) , as indicated . Representative confocal microscopy pictures are shown .

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(B) Colocalization of endocytosed CD16:7-263-281 with EEA1 and CD63. JAR cells were transfected with CD16:7-263-281 and subjected to aggregation before staining them for the endocytosed chimera (red) and EEA1 or CD63 (green; FITC‐coupled primary antibodies), as indicated. Representative confocal microscopy pictures are shown.


( C ) Immunoelectron microscopy assay showing that endocytosed CD16 : 7 - 263 - 281 localizes to LC3 − labelled , single − membrane vesicles . JAR cells were transfected with the CD16 : 7 - 263 - 281 chimera and a construct expressing human IgG1 fused to LC3A . Cells were subjected to anti − CD16 aggregation for 8 h and processed for immunoelectron microscopy . Thick gold signal ( 18 nm ) : aggregated , endocytosed chimera ; thin gold signal ( 12 nm ) : IgG1 - LC3 . Arrows indicate single membrane ( black ) or IgG1 - LC3A ( white ) . Scale bar : 400 nm .

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(C) Immunoelectron microscopy assay showing that endocytosed CD16:7-263-281 localizes to LC3−labelled, single−membrane vesicles. JAR cells were transfected with the CD16:7-263-281 chimera and a construct expressing human IgG1 fused to LC3A. Cells were subjected to anti−CD16 aggregation for 8 h and processed for immunoelectron microscopy. Thick gold signal (18 nm): aggregated, endocytosed chimera; thin gold signal (12 nm): IgG1-LC3. Arrows indicate single membrane (black) or IgG1-LC3A (white). Scale bar: 400 nm.


( D ) Lysosomal inhibition increases HA - LC3II levels promoted by CD16 : 7 - 263 - 281 . 293 cells were transfected with the indicated chimera and HA - LC3A , and subjected to anti − CD16 aggregation in the absence or presence of bafilomycin ( 200 nM , added 4 h post aggregation ) before lysing them for western blotting against the indicated molecules .

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(D) Lysosomal inhibition increases HA-LC3II levels promoted by CD16:7-263-281. 293 cells were transfected with the indicated chimera and HA-LC3A, and subjected to anti−CD16 aggregation in the absence or presence of bafilomycin (200 nM, added 4 h post aggregation) before lysing them for western blotting against the indicated molecules.


( E ) Aggregation of CD16 : 7 - 263 - 281 promotes its own degradation . 293 cells were transfected with the indicated constructs , aggregated and lysed for western blotting . Shown are overexposed anti ‐ CD16 WBs .

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(E) Aggregation of CD16:7-263-281 promotes its own degradation. 293 cells were transfected with the indicated constructs, aggregated and lysed for western blotting. Shown are overexposed anti‐CD16 WBs.


( F ) Degradation of CD16 : 7 - 263 - 281 is inhibited by ATG5 depletion . 293 cells were transfected with the indicated siRNAs and subsequently with the shown CD16 : 7 constructs , aggregated and lysed for anti ‐ CD16 western blotting ( left panel ) . The right panel displays control WBs showing ATG5 depletion . Source data for this figure is available on the online supplementary information page .

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(F) Degradation of CD16:7-263-281 is inhibited by ATG5 depletion. 293 cells were transfected with the indicated siRNAs and subsequently with the shown CD16:7 constructs, aggregated and lysed for anti‐CD16 western blotting (left panel). The right panel displays control WBs showing ATG5 depletion.Source data for this figure is available on the online supplementary information page.


( A ) Identification of critical amino acids for HA - LC3 conversion . 293 cells were transfected with the indicated CD16 : 7 - 263 - 281 mutants and HA - LC3A , subjected to anti ‐ CD16 aggregation and lysed for western blotting against the indicated molecules . Asterisks mark mutations with reduced activity . Shown is one representative experiment of six repetitions .

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(A) Identification of critical amino acids for HA-LC3 conversion. 293 cells were transfected with the indicated CD16:7-263-281 mutants and HA-LC3A, subjected to anti‐CD16 aggregation and lysed for western blotting against the indicated molecules. Asterisks mark mutations with reduced activity. Shown is one representative experiment of six repetitions.


( B ) Identification of critical amino acids for GFP - LC3 activation and colocalization with the endocytosed chimera . JAR cells were transfected with the indicated CD16 : 7 - 263 - 281 mutants and GFP - LC3A , aggregated and stained for the endocytosed chimera ( red ) . Preparations were scored by blindly counting the number of red vesicles ( chimera ) and green vesicles ( GFP - LC3A ) per cell , as well as the number of red vesicles colocalizing with green ones . The percentage of green vesicles colocalizing with red ones was close to 100 % for all mutants , that is , virtually no GFP - LC3A vesicles were unrelated to endocytosed chimera ( not shown ) . At least 50 cells were scored per experimental point . The experiment was repeated three times . The graph shows the number of GFP - LC3A vesicles per cell expressed as the percentage of the value obtained for the wild − type chimera ( left axis ) , and the percentage of chimera vesicles labelled with GFP - LC3A ( right axis ) . Data are expressed as means ± s . d . of the triplicates . Asterisks indicate significant differences with respect to wild − type values ( paired Student ' s t − test ; P < 0 . 01 ) .

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(B) Identification of critical amino acids for GFP-LC3 activation and colocalization with the endocytosed chimera. JAR cells were transfected with the indicated CD16:7-263-281 mutants and GFP-LC3A, aggregated and stained for the endocytosed chimera (red). Preparations were scored by blindly counting the number of red vesicles (chimera) and green vesicles (GFP-LC3A) per cell, as well as the number of red vesicles colocalizing with green ones. The percentage of green vesicles colocalizing with red ones was close to 100% for all mutants, that is, virtually no GFP-LC3A vesicles were unrelated to endocytosed chimera (not shown). At least 50 cells were scored per experimental point. The experiment was repeated three times. The graph shows the number of GFP-LC3A vesicles per cell expressed as the percentage of the value obtained for the wild−type chimera (left axis), and the percentage of chimera vesicles labelled with GFP-LC3A (right axis). Data are expressed as means ±s.d. of the triplicates. Asterisks indicate significant differences with respect to wild−type values (paired Student's t−test; P<0.01).


( C ) Representative confocal microscopy pictures of the phenotype produced by the indicated mutations . Procedures were as in B .

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(C) Representative confocal microscopy pictures of the phenotype produced by the indicated mutations. Procedures were as in B.


( D ) Simultaneous mutation of the four essential amino acids to alanine blocks HA - LC3 conversion induced by CD16 : 7 - 263 - 281 . 293 cells were transfected with the indicated chimeras ( 4M , quadruple mutant ) and HA - LC3A , aggregated and lysed for western blotting .

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(D) Simultaneous mutation of the four essential amino acids to alanine blocks HA-LC3 conversion induced by CD16:7-263-281. 293 cells were transfected with the indicated chimeras (4M, quadruple mutant) and HA-LC3A, aggregated and lysed for western blotting.


( E ) Simultaneous mutation of the four essential amino acids blocks GFP - LC3 activation and colocalization with the endocytosed chimera . JAR cells were transfected with the indicated CD16 : 7 - 263 - 281 constructs and GFP - LC3A , aggregated and stained for the endocytosed chimera ( red ) . Representative confocal microscopy images are shown .

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(E) Simultaneous mutation of the four essential amino acids blocks GFP-LC3 activation and colocalization with the endocytosed chimera. JAR cells were transfected with the indicated CD16:7-263-281 constructs and GFP-LC3A, aggregated and stained for the endocytosed chimera (red). Representative confocal microscopy images are shown.


( F ) Quantification of the phenotype in E . Data gathering and expression were as in B . Source data for this figure is available on the online supplementary information page .

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(F) Quantification of the phenotype in E. Data gathering and expression were as in B.Source data for this figure is available on the online supplementary information page.


( A ) Apposition events between GFP - ATG16L1 and endocytosed CD16 : 7 - 263 - 281 . JAR cells were transfected with CD16 : 7 - 263 - 281 and GFP - ATG16L1 or GFP - BECLIN ( as indicated ) , aggregated for 4 h and stained for the endocytosed chimera ( red ) . Representative confocal microscopy pictures are shown . Two different examples are provided for GFP - ATG16L1 .

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(A) Apposition events between GFP-ATG16L1 and endocytosed CD16:7-263-281. JAR cells were transfected with CD16:7-263-281 and GFP-ATG16L1 or GFP-BECLIN (as indicated), aggregated for 4 h and stained for the endocytosed chimera (red). Representative confocal microscopy pictures are shown. Two different examples are provided for GFP-ATG16L1.


( 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 ) .

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(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 B-GENEPROD O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-GENEPROD O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD B-EXP_ASSAY O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O 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 B-CELL_LINE O O O O O O O O O O O O B-GENEPROD B-EXP_ASSAY O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O B-GENEPROD 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 .

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(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-GENEPROD O O O O O B-GENEPROD O O O O B-GENEPROD 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 ) .

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

(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 .

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

(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 .

O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-CELL_LINE O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-ORGANISM O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O

(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 .

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

(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 B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O B-EXP_ASSAY O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O B-EXP_ASSAY O O O B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 ) .

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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-SMALL_MOLECULE O O O B-SUBCELLULAR O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O 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-SMALL_MOLECULE O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O O B-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE O O O O O O 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-SMALL_MOLECULE O O O B-SUBCELLULAR O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O 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-SMALL_MOLECULE O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 ) .

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

(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 .

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

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

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

(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 ) .

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

(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 .

O O O O O O B-GENEPROD O B-GENEPROD O O O O O B-SMALL_MOLECULE O B-SUBCELLULAR O O O O O O O O O O B-GENEPROD O O B-SUBCELLULAR O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-SMALL_MOLECULE O B-SUBCELLULAR O

(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 .

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

(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 .

O O O O B-EXP_ASSAY O B-SUBCELLULAR O B-SUBCELLULAR O O O O O O O O O B-GENEPROD O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-EXP_ASSAY O O O B-SUBCELLULAR O O O O O O O O B-SUBCELLULAR O O O O O O O O

(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 .

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

(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 .

O O O O O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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 ) .

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

(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 .

O O O O O O O O O O O O B-GENEPROD O O B-SUBCELLULAR O O B-SUBCELLULAR O O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O

(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 .

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

(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 .

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

(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 .

O O O O B-EXP_ASSAY O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O B-SMALL_MOLECULE O O O O O B-GENEPROD O O O B-SUBCELLULAR O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O

(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 .

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

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

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 .

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

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 .

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

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

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-GENEPROD O O O O B-SUBCELLULAR O O O O B-GENEPROD O B-CELL_LINE O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-GENEPROD B-EXP_ASSAY 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 ) .

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

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 .

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

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 .

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

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

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 .

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

(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 .

O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-TISSUE I-TISSUE O 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-SUBCELLULAR B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O

(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 .

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

(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 .

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

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

(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 .

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

(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 .

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

(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 .

O O O O O O O O O O O B-TISSUE I-TISSUE I-TISSUE O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O

(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 .

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

(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 .

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

(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 .

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

(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 .

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

(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 .

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

(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 .

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

(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 .

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

(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 .

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

(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 .

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

(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 .

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

(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.