( A ) Wild - type , ULK1 / 2 double knockout ( dKO ) , or IKKα KO mouse embryonic fibroblasts ( MEFs ) were incubated with either complete medium , acid - deficient DMEM , or HBSS for 1 hour . Samples were immunoblotted using indicated antibodies . Data information : Unless otherwise indicated experiments were performed three times . Data are represented as mean ± standard deviation p values were determined by Student ' s T - .

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

(A) Wild-type, ULK1/2 double knockout (dKO), or IKKα KO mouse embryonic fibroblasts (MEFs) were incubated with either complete medium, amino acid-deficient DMEM, or HBSS for 1 hour. Samples were immunoblotted using the indicated antibodies. Data information: Unless otherwise indicated experiments were performed three times. Data are represented as mean ± standard deviation and p values were determined by Student's T-Test.


( B ) Wild - type , ULK1 / 2 dKO , or IKKα KO MEFs were infected with log phase Salmonella for 2 hours ; - containing media was then removed were incubated with gentamycin ( 50 µg / mL ) - containing DMEM for 2 hours . Samples were immunoblotted using indicated antibodies . Data information : Unless otherwise indicated experiments were performed three times . Data are represented as mean ± standard deviation p values were determined by Student ' s T - .

O O O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Wild-type, ULK1/2 dKO, or IKKα KO MEFs were infected with log phase Salmonella for 2 hours; bacteria-containing media was then removed and cells were incubated with gentamycin (50 µg/mL)-containing DMEM for 2 hours. Samples were immunoblotted using the indicated antibodies. Data information: Unless otherwise indicated experiments were performed three times. Data are represented as mean ± standard deviation and p values were determined by Student's T-Test.


( C ) Wild - type , ULK1 / 2 dKO , or IKKα KO MEFs were infected with Salmonella for 1 hours . Autophagic capture of Salmonella was analyzed by immunostaining for LPS LC3B . Representative are shown ( scale bars , 10 μm 3 μm ) . was generated from 8 fields of view from a representative experiment . experiments were repeated twice . Data information : Unless otherwise indicated experiments were performed three times . Data are represented as mean ± standard deviation p values were determined by Student ' s T - .

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

(C) Wild-type, ULK1/2 dKO, or IKKα KO MEFs cells were infected with Salmonella for 1 hours. Autophagic capture of Salmonella was analyzed by immunostaining for LPS and LC3B. Representative images are shown (scale bars, 10 μm and 3 μm). Quantification was generated from 8 fields of view from a representative experiment. The experiments were repeated twice. Data information: Unless otherwise indicated experiments were performed three times. Data are represented as mean ± standard deviation and p values were determined by Student's T-Test.


( D ) Wild - type , ULK1 / 2 dKO IKKα KO MEFs were infected with Salmonella for 1 hour . Xenophagy rates were examined through Colony Forming Unit ( CFU ) assays . of infection rates by immunofluorescence is demonstrated in right panel . Data information : Unless otherwise indicated experiments were performed three times . Data are represented as mean ± standard deviation p values were determined by Student ' s T - .

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

(D) Wild-type, ULK1/2 dKO and IKKα KO MEFs were infected with Salmonella for 1 hour. Xenophagy rates were examined through Colony Forming Unit (CFU) assays. Quantification of infection rates by immunofluorescence is demonstrated in the right panel. Data information: Unless otherwise indicated experiments were performed three times. Data are represented as mean ± standard deviation and p values were determined by Student's T-Test.


( A ) HEK293A were transfected with either flag - tagged WT ATG16L1 or T300A ATG16L1 . ULK1 was co - transfected in increasing amounts where indicated . Cleavage of ATG16L1 was analyzed by WB of whole lysates . of ATG16L1 cleavage were quantified from 3 biological repeats ( right panel ) . Data information : Unless otherwise indicated experiments were performed three times . Data are represented as mean ± standard deviation p values were determined by Student ' s T - .

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

(A) HEK293A cells were transfected with either flag-tagged WT ATG16L1 or T300A ATG16L1. ULK1 was co-transfected in increasing amounts where indicated. Cleavage of ATG16L1 was analyzed by WB of whole cell lysates. Levels of ATG16L1 cleavage were quantified from 3 biological repeats (right panel). Data information: Unless otherwise indicated experiments were performed three times. Data are represented as mean ± standard deviation and p values were determined by Student's T-Test.


( B ) HEK293A were transfected with either tagged wild - type , T300A , or S278 / T300A ATG16L1 in presence or absence of ULK1 . Cleavage of ATG16L1 was analyzed by WB . of ATG16L1 cleavage were measured from 3 biological repeats ( right panel ) . Data information : Unless otherwise indicated experiments were performed three times . Data are represented as mean ± standard deviation p values were determined by Student ' s T - .

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

(B) HEK293A cells were transfected with either tagged wild-type, T300A, or S278/T300A ATG16L1 in the presence or absence of ULK1. Cleavage of ATG16L1 was analyzed by WB. Levels of ATG16L1 cleavage were measured from 3 biological repeats (right panel). Data information: Unless otherwise indicated experiments were performed three times. Data are represented as mean ± standard deviation and p values were determined by Student's T-Test.


( C ) HEK293A were transfected with indicated plasmids in presence or absence of a pan - caspase inhibitor Z - VAD - FMK ( 15 µM ) for 4 hours . Cleavage of ATG16L1 was analyzed by WB of 3 biological repeats . Data information : Unless otherwise indicated experiments were performed three times . Data are represented as mean ± standard deviation p values were determined by Student ' s T - .

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

(C) HEK293A cells were transfected with the indicated plasmids in the presence or absence of a pan-caspase inhibitor Z-VAD-FMK (15 µM) for 4 hours. Cleavage of ATG16L1 was analyzed by WB of 3 biological repeats. Data information: Unless otherwise indicated experiments were performed three times. Data are represented as mean ± standard deviation and p values were determined by Student's T-Test.


( D ) Wild - type or T300A - expressing HEK293A were treated with Salmonella in presence or absence of ULK1 / 2 inhibitor ( 16 μM ) for indicated time points . Expression of ATG16L1 was analysed by WB . experiments were performed twice .

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

(D) Wild-type or T300A-expressing HEK293A were treated with Salmonella in the presence or absence of ULK1/2 inhibitor (16 μM) for the indicated time points. Expression of ATG16L1 was analysed by WB. The experiments were performed twice.


( E ) ATG16L1 knock - out HEK293A transfected with indicated HA GST ATG16L1 plasmids were infected with Salmonella for 1 hour . Xenophagy rates were examined through CFU assays . of infection rates by immunofluorescence is demonstrated in right panel . Data information : Unless otherwise indicated experiments were performed three times . Data are represented as mean ± standard deviation p values were determined by Student ' s T - .

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

(E) ATG16L1 knock-out HEK293A cells transfected with the indicated HA GST ATG16L1 plasmids were infected with Salmonella for 1 hour. Xenophagy rates were examined through CFU assays. Quantification of infection rates by immunofluorescence is demonstrated in the right panel. Data information: Unless otherwise indicated experiments were performed three times. Data are represented as mean ± standard deviation and p values were determined by Student's T-Test.


( A ) Wild - type MEF were infected with Salmonella for 25 minutes . Phospho - ATG16L1 , total ATG16L1 , LPS were stained analysed by immunofluorescence . Representative immunofluorescent are shown ( scale bars , 10 μm 1 μm ) . Data information : Unless otherwise indicated experiments were performed twice . Data are represented as mean p values were determined by Student ' s T - .

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

(A) Wild-type MEF cells were infected with Salmonella for 25 minutes. Phospho-ATG16L1, total ATG16L1, and LPS were stained and analysed by immunofluorescence. Representative immunofluorescent images are shown (scale bars, 10 μm and 1 μm). Data information: Unless otherwise indicated experiments were performed twice. Data are represented as mean and p values were determined by Student's T-Test.


( B ) Wild - type ULK1 / 2 dKO were infected with Salmonella for 25 minutes . Immunofluorescence was performed using antibodies against LPS ATG16L1 . Representative immunofluorescent are shown on left panel ( scale bars , 10 μm 2 μm ) . of ATG16L1 - positive from 7 fields of view from a representative experiment is shown in right panel . Data information : Unless otherwise indicated experiments were performed twice . Data are represented as mean p values were determined by Student ' s T - .

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

(B) Wild-type and ULK1/2 dKO were infected with Salmonella for 25 minutes. Immunofluorescence was performed using antibodies against LPS and ATG16L1. Representative immunofluorescent images are shown on the left panel (scale bars, 10 μm and 2 μm). Quantification of ATG16L1-positive bacteria from 7 fields of view from a representative experiment is shown in the right panel. Data information: Unless otherwise indicated experiments were performed twice. Data are represented as mean and p values were determined by Student's T-Test.


( C ) ATG16L1 knock - out HCT116 transfected with indicated GST HA ATG16L1 were infected with Salmonella for 1 hour . were stained using anti - LPS antibodies to analyze localization in addition to ATG16L1 . Representative immunofluorescent of ATG16L1 LPS are shown ( scale bars , 5μm 1 μm ) . of ATG16L1 localizing to from 7 fields of view from a representative experiment is shown in lower panel . Data information : Unless otherwise indicated experiments were performed twice . Data are represented as mean p values were determined by Student ' s T - .

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

(C) ATG16L1 knock-out HCT116 transfected with the indicated GST HA ATG16L1 were infected with Salmonella for 1 hour. Bacteria were stained using anti-LPS antibodies to analyze localization in addition to ATG16L1. Representative immunofluorescent images of ATG16L1 and LPS are shown (scale bars, 5μm and 1 μm). Quantification of ATG16L1 localizing to bacteria from 7 fields of view from a representative experiment is shown in the lower panel. Data information: Unless otherwise indicated experiments were performed twice. Data are represented as mean and p values were determined by Student's T-Test.


( D ) ATG16L1 knock - out HCT116 transfected with indicated GST HA ATG16L1 were infected with Salmonella for 1 hour . were stained using anti - LPS antibodies to analyze localization in addition to autophagy marker LC3B . Representative immunofluorescent of LC3B LPS are shown ( scale bars , 5μm 1 μm ) . of undergoing autophagic clearance from 7 fields of view from a representative experiment is shown in lower panel . Data information : Unless otherwise indicated experiments were performed twice . Data are represented as mean p values were determined by Student ' s T - .

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

(D) ATG16L1 knock-out HCT116 transfected with the indicated GST HA ATG16L1 were infected with Salmonella for 1 hour. Bacteria were stained using anti-LPS antibodies to analyze localization in addition to the autophagy marker LC3B. Representative immunofluorescent images of LC3B and LPS are shown (scale bars, 5μm and 1 μm). Quantification of bacteria undergoing autophagic clearance from 7 fields of view from a representative experiment is shown in the lower panel. Data information: Unless otherwise indicated experiments were performed twice. Data are represented as mean and p values were determined by Student's T-Test.


( B ) HEK293T transiently expressing FLAG FLAG - tagged TEX264 were subjected to immunoprecipitation ( IP ) . Inputs ( 20 % of lysates ) immunoprecipitates ( from 80 % of lysates ) were analyzed by immunoblotting using indicated antibodies .

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

(B) HEK293T cells transiently expressing FLAG and FLAG-tagged TEX264 were subjected to immunoprecipitation (IP). Inputs (20% of the lysates) and immunoprecipitates (from 80% of the lysates) were analyzed by immunoblotting using the indicated antibodies.


( C ) HEK293T transiently expressing WT or mutated TEX264 - FLAG were subjected to immunoprecipitation with an anti - FLAG antibody detected with indicated antibodies .

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

(C) HEK293T cells transiently expressing WT or mutated TEX264-FLAG were subjected to immunoprecipitation with an anti-FLAG antibody and detected with the indicated antibodies.


( D E ) MEFs stably expressing TEX264 - GFP or its mutant were cultured in starvation media immunostained with an anti - LC3 antibody . Bars : 10 µm 1 µm ( insets ) ( D ) . of number of TEX264 per . Solid bars indicate medians , boxes interquartile range ( 25th to 75th percentile ) , whiskers 0th to 100th percentile . Differences were statistically analyzed by one - way ANOVA Tukey ' s multiple comparison . Data were collected from 48 for each type ( E ) .

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

(D and E) MEFs stably expressing TEX264-GFP or its mutant were cultured in starvation media and immunostained with an anti-LC3 antibody. Bars: 10 µm and 1 µm (insets) (D). Quantification of the number of TEX264 puncta per cell. Solid bars indicate the medians, boxes the interquartile range (25th to 75th percentile), and whiskers the 0th to 100th percentile. Differences were statistically analyzed by one-way ANOVA and Tukey's multiple comparison test. Data were collected from 48 cells for each cell type (E).


( F G ) WT TEX264 - KO HeLa ( with or without indicated TEX264 mutants with a C - terminal FLAG tag ) stably expressing ER - phagy reporter ( ss - RFP - GFP - KDEL ) were cultured in presence of doxycycline for 24 h to induce reporter . After doxycycline was removed , were cultured in starvation medium lacking serum for 9 h ( F ) . band intensities of RFP RFP - GFP were quantified of RFP : RFP - GFP ( normalized to WT ) is shown . Data represent mean ± standard error of mean ( ) of three independent experiments . Differences were statistically analyzed by one - way ANOVA Tukey ' s multiple comparison ( G ) .

O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 and G) WT and TEX264-KO HeLa cells (with or without the indicated TEX264 mutants with a C-terminal FLAG tag) stably expressing the ER-phagy reporter (ss-RFP-GFP-KDEL) were cultured in the presence of doxycycline for 24 h to induce the reporter. After doxycycline was removed, cells were cultured in starvation medium lacking amino acids and serum for 9 h (F). The band intensities of RFP and RFP-GFP were quantified and the ratio of RFP:RFP-GFP (normalized to WT) is shown. Data represent the mean ± standard error of the mean (SEM) of three independent experiments. Differences were statistically analyzed by one-way ANOVA and Tukey's multiple comparison test (G).


( A B ) MEFs stably expressing TEX264 - GFP were cultured in starvation media with or without bafilomycin A1 ( Baf A1 ) immunostained with anti - pTEX264 anti - LC3 antibodies . Bars : 10 µm 1 µm ( insets ) ( A ) . of number of pTEX264 per . Solid bars indicate medians , boxes interquartile range ( 25th to 75th percentile ) , whiskers 0th to 100th percentile . Differences were statistically analyzed by one - way ANOVA Tukey ' s multiple comparison . Data were collected from 35 for each type ( B ) .

O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 and B) MEFs stably expressing TEX264-GFP were cultured in starvation media with or without bafilomycin A1 (Baf A1) and immunostained with anti-pTEX264 and anti-LC3 antibodies. Bars: 10 µm and 1 µm (insets) (A). Quantification of the number of pTEX264 puncta per cell. Solid bars indicate the medians, boxes the interquartile range (25th to 75th percentile), and whiskers the 0th to 100th percentile. Differences were statistically analyzed by one-way ANOVA and Tukey's multiple comparison test. Data were collected from 35 cells for each cell type (B).


( C ) HeLa were treated with 250 nM Torin 1 Baf A1 for 16 h , homogenates were subjected to an OptiPrep flotation .

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(C) HeLa cells were treated with 250 nM Torin 1 and Baf A1 for 16 h, and cell homogenates were subjected to an OptiPrep membrane flotation analysis.


( D , E F ) WT , ATG3 - KO , FIP200 - KO HeLa were cultured in starvation media lacking serum with or without Baf A1 for 1 or 5 h . lysates were analyzed by immunoblotting using indicated antibodies ( D ) . Relative changes of of band intensities of phosphorylated TEX264 to HSP90 total TEX264 during starvation are shown ( E F ) . Data represent mean ± of three independent experiments .

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

(D, E and F) WT, ATG3-KO, and FIP200-KO HeLa cells were cultured in starvation media lacking amino acids and serum with or without Baf A1 for 1 or 5 h. Cell lysates were analyzed by immunoblotting using the indicated antibodies (D). Relative changes of the ratio of band intensities of phosphorylated TEX264 to HSP90 and total TEX264 during starvation are shown (E and F). Data represent the mean ± SEM of three independent experiments.


( A ) HeLa were cultured with 20 nM CK1 inhibitor ( D4476 ) or CK2 inhibitor ( CX4945 ) for 3 h , followed by starvation ( with inhibitors ) for 3 h . lysates were analyzed by immunoblotting using indicated antibodies .

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(A) HeLa cells were cultured with 20 nM CK1 inhibitor (D4476) or CK2 inhibitor (CX4945) for 3 h, followed by starvation (with the inhibitors) for 3 h. Cell lysates were analyzed by immunoblotting using the indicated antibodies.


( B ) Recombinant TEX264 - FLAG was incubated with or without CK2 CK2 inhibitor ( CX4945 ) in presence of 200 µM ATP for 1 h .

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

(B) Recombinant TEX264-FLAG was incubated with or without CK2 and the CK2 inhibitor (CX4945) in the presence of 200 µM ATP for 1 h.


( C ) HEK293T transiently expressing TEX264 - FLAG one of three Halo - CK2A isoforms or Halo - CK2B were subjected to immunoprecipitation with an anti - FLAG antibody detected with anti - Halo anti - FLAG antibodies .

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

(C) HEK293T cells transiently expressing TEX264-FLAG and one of the three Halo-CK2A isoforms or Halo-CK2B were subjected to immunoprecipitation with an anti-FLAG antibody and detected with anti-Halo and anti-FLAG antibodies.


( D E ) HEK293T transiently expressing WT or TEX264 - FLAG mutants were subjected to immunoprecipitation ( IP ) . Inputs ( 20 % of lysates ) immunoprecipitates ( from 80 % of lysates ) were analyzed by immunoblotting using indicated antibodies ( D ) . band intensities in IP fractions were quantified , phospho - serine : FLAG was calculated . Data represent mean ± of five independent experiments . Differences were statistically analyzed by one - way ANOVA Tukey ' s multiple comparison ( E ) .

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

(D and E) HEK293T cells transiently expressing WT or TEX264-FLAG mutants were subjected to immunoprecipitation (IP). Inputs (20% of the lysates) and immunoprecipitates (from 80% of the lysates) were analyzed by immunoblotting using the indicated antibodies (D). The band intensities in the IP fractions were quantified, and the phospho-serine:FLAG ratio was calculated. Data represent the mean ± SEM of five independent experiments. Differences were statistically analyzed by one-way ANOVA and Tukey's multiple comparison test (E).


( F G ) MEFs stably expressing TEX264 - GFP were cultured with CK2 inhibitor for 3 h followed by starvation ( with CK2 inhibitor ) immunostained with an anti - LC3 antibody . Scale bars represent 10 μm 1 μm ( insets ) ( F ) . of number of TEX264 per . Solid bars indicate medians , boxes interquartile range ( 25th to 75th percentile ) , whiskers 0th to 100th percentile . Differences were statistically analyzed by an unpaired two - Student ' s t - . Data were collected from 85 for each type ( G ) .

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

(F and G) MEFs stably expressing TEX264-GFP were cultured with the CK2 inhibitor for 3 h followed by starvation (with the CK2 inhibitor) and immunostained with an anti-LC3 antibody. Scale bars represent 10 μm and 1 μm (insets) (F). Quantification of the number of TEX264 puncta per cell. Solid bars indicate the medians, boxes the interquartile range (25th to 75th percentile), and whiskers the 0th to 100th percentile. Differences were statistically analyzed by an unpaired two-tailed Student's t-test. Data were collected from 85 cells for each cell type (G).


( H I ) WT HeLa stably expressing ER - phagy reporter were cultured in presence of doxycycline for 24 h to induce reporter . After doxycycline was removed , were cultured with 20 nM CK2 inhibitor for 3 h followed by starvation ( with CK2 inhibitor ) for 6 h ( H ) . band intensities of RFP RFP - GFP were quantified of RFP : RFP - GFP ( normalized to WT ) is shown . Data represent mean ± of six independent experiments . Differences were statistically analyzed by an unpaired two - Student ' s t - ( I ) .

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

(H and I) WT HeLa cells stably expressing the ER-phagy reporter were cultured in the presence of doxycycline for 24 h to induce the reporter. After doxycycline was removed, cells were cultured with 20 nM CK2 inhibitor for 3 h followed by starvation (with the CK2 inhibitor) for 6 h (H). The band intensities of RFP and RFP-GFP were quantified and the ratio of RFP:RFP-GFP (normalized to WT) is shown. Data represent the mean ± SEM of six independent experiments. Differences were statistically analyzed by an unpaired two-tailed Student's t-test (I).


( B ) HEK293T transiently expressing WT or mutated TEX264 - FLAG were subjected to immunoprecipitation with an anti - FLAG antibody detected with indicated antibodies .

(B) HEK293T cells transiently expressing WT or mutated TEX264-FLAG were subjected to immunoprecipitation with an anti-FLAG antibody and detected with the indicated antibodies.


( C D ) MEFs stably expressing TEX264 - GFP or its mutants were cultured in starvation media immunostained with an anti - LC3 antibody . Bars : 10 µm 5 µm ( insets ) ( C ) . of GFP signal under starvation conditions was quantified at TEX264 - GFP ER . : ER signal was calculated . Solid bars indicate medians , boxes interquartile range ( 25th to 75th percentile ) , whiskers 0th to 100th percentile . Differences were statistically analyzed by one - way ANOVA Tukey ' s multiple comparison . Data were collected from 70 for each type ( D ) .

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

(C and D) MEFs stably expressing TEX264-GFP or its mutants were cultured in starvation media and immunostained with an anti-LC3 antibody. Bars: 10 µm and 5 µm (insets) (C). The intensity of the GFP signal under starvation conditions was quantified at TEX264-GFP puncta and the ER. The puncta:ER signal ratio was calculated. Solid bars indicate the medians, boxes the interquartile range (25th to 75th percentile), and whiskers the 0th to 100th percentile. Differences were statistically analyzed by one-way ANOVA and Tukey's multiple comparison test. Data were collected from 70 puncta for each cell type (D).


( E F ) WT TEX264 - KO ( expressing WT or TEX264 - FLAG mutants ) HeLa stably expressing ER - phagy reporter were cultured in presence of doxycycline for 24 h to induce reporter . After doxycycline was removed , were cultured in starvation medium lacking serum for 9 h ( E ) . band intensities of RFP RFP - GFP were quantified of RFP : RFP - GFP ( normalized to WT ) is shown . Data represent mean ± of four independent experiments . Differences were statistically analyzed by one - way ANOVA Tukey ' s multiple comparison ( F ) .

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(E and F) WT and TEX264-KO (expressing WT or TEX264-FLAG mutants) HeLa cells stably expressing the ER-phagy reporter were cultured in the presence of doxycycline for 24 h to induce the reporter. After doxycycline was removed, the cells were cultured in starvation medium lacking amino acids and serum for 9 h (E). The band intensities of RFP and RFP-GFP were quantified and the ratio of RFP:RFP-GFP (normalized to WT) is shown. Data represent the mean ± SEM of four independent experiments. Differences were statistically analyzed by one-way ANOVA and Tukey's multiple comparison test (F).


( b ) Density gradient fractions of NRK starved for 8 h were analysed by transmission electron microscopy ( upper panel ) immunoblotted for LC3 or LAMP1 ( lower panel ) . in fraction 1 were identified by mass spectrometry . Three replicates were carried out from three independent purifications . Scale bar , 2 μm .

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(b) Density gradient fractions of NRK cells starved for 8 h were analysed by transmission electron microscopy (upper panel) and immunoblotted for LC3 or LAMP1 (lower panel). Proteins in fraction 1 were identified by mass spectrometry analysis. Three replicates were carried out from three independent purifications. Scale bar, 2 μm.


( e ) NRK grown on SAMCell were transfected with FAM - RNAi by reverse transfection transfection efficiency was monitored by microscopy . Left panel , FAM - RNAi - positive ; right panel , differential interference contrast ( DIC ) image of in left panel .

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(e) NRK cells grown on the SAMCell were transfected with FAM-RNAi by reverse transfection and the transfection efficiency was monitored by microscopy. Left panel, FAM-RNAi-positive cells; right panel, differential interference contrast (DIC) image of the cells in the left panel.


( a ) NRK stably expressing CFP - LC3 ; LAMP1 - YFP were transfected with nonspecific ( NS ) - or Cltc ( clathrin heavy chain ) - RNAi starved for 12 h . Scale bar , 5 μm . ( b ) from a were assessed for enlarged autolysosomes after starvation quantified . n = 100 from three independent experiments . Error bars indicate s . d .

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(a) NRK cells stably expressing CFP-LC3; LAMP1-YFP were transfected with nonspecific (NS)- or Cltc (clathrin heavy chain)-RNAi and starved for 12 h. Scale bar, 5 μm. (b) Cells from a were assessed for enlarged autolysosomes after starvation and quantified. n = 100 cells from three independent experiments. Error bars indicate the s.d.


( c ) Representative of nonspecific - or Cltc - RNAi - transfected NRK after 12 h of serum starvation . Scale bar , 5 μm .

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(c) Representative TEM micrographs of nonspecific- or Cltc-RNAi-transfected NRK cells after 12 h of serum starvation. Scale bar, 5 μm.


( d ) NRK lines stably expressing LAMP1 - Cherry ; clathrin - GFP were starved for 0 or 4 h observed by confocal microscopy . As a control , clathrin - GFP - expressing were starved for 0 or 4 h then stained with MitoTracker red . Scale bar , 5 μm . insets in a , c d represent enlarged regions of interest . ( e ) from d were analysed for Pearson ' s co - localization coefficent ( R ( r ) ) . n = 30 from three independent experiments . Error bars indicate s . d .

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(d) NRK cell lines stably expressing LAMP1-Cherry; clathrin-GFP were starved for 0 or 4 h and observed by confocal microscopy. As a control, clathrin-GFP-expressing cells were starved for 0 or 4 h and then stained with MitoTracker red. Scale bar, 5 μm. The insets in a, c and d represent enlarged regions of interest. (e) Cells from d were analysed for Pearson's co-localization coefficent (R(r)). n = 30 cells from three independent experiments. Error bars indicate the s.d.


( f ) NRK stably expressing LAMP1 - Cherry ; clathrin - GFP were starved for 4 h time - lapse were acquired by spinning - disc microscopy . Scale bar , 500 nm . arrows indicate a clathrin punctum moving along a LAMP1 - positive structure .

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(f) NRK cells stably expressing LAMP1-Cherry; clathrin-GFP were starved for 4 h and time-lapse images were acquired by spinning-disc microscopy. Scale bar, 500 nm. The white arrows indicate a clathrin punctum moving along a LAMP1-positive structure.


( g ) NRK were starved for 0 , 4 or 6 h then subjected to fractionation . Fractions were analysed by western blotting with antibodies against LAMP2 clathrin heavy chain . 1 is top fraction . outline indicates Lamp2 - positive fraction .

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(g) NRK cells were starved for 0, 4 or 6 h and then subjected to cell fractionation. Fractions were analysed by western blotting with antibodies against LAMP2 and clathrin heavy chain. 1 is the top fraction. The outline indicates the Lamp2-positive fraction.


( h ) lines stably expressing clathrin - GFP were starved for 4 h , stained with antibodies against GFP LAMP1 , analysed by 3D - SIM . Scale bar , 2 . 5 μm . right panels show enlarged regions of interest from left panel : T , top view ; L , lateral view ; B , bottom view .

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(h) Cell lines stably expressing clathrin-GFP were starved for 4 h, stained with antibodies against GFP and LAMP1, analysed by 3D-SIM. Scale bar, 2.5 μm. The right panels show enlarged regions of interest from the left panel: T, top view; L, lateral view; B, bottom view.


( i ) Left panel , representative of purified autolysosomes from starved clathrin - GFP - expressing . Scale bar , 250 nm . Right panel , SIM image of purified autolysosomes stained with GFP LAMP1 from same experiment . Scale bar , 5 μm .

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(i) Left panel, representative TEM micrographs of purified autolysosomes from starved clathrin-GFP-expressing cells. Scale bar, 250 nm. Right panel, SIM image of purified autolysosomes stained with GFP and LAMP1 from the same experiment. Scale bar, 5 μm.


( j ) NRK or stable clathrin - GFP - expressing NRK lines were analysed by pre - embedding - immuno - electron microscopy using antibodies against GFP . Scale bar , 100 nm . ( k ) of average number of gold particles per image from control or clathrin - GFP . n = 25 from three independent experiments . error bars indicate s . d . Uncropped of blots are shown in Supplementary Fig . S6 .

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(j) NRK or stable clathrin-GFP-expressing NRK cell lines were analysed by pre-embedding-immuno-electron microscopy using antibodies against GFP. Scale bar, 100 nm. (k) Quantification of the average number of gold particles per image from control or clathrin-GFP cells. n = 25 images from three independent experiments. The error bars indicate the s.d. Uncropped images of blots are shown in Supplementary Fig. S6.


( a ) NRK lines stably expressing CFP - LC3 ; LAMP1 - YFP were transfected with nonspecific ( NS ) - or PIP5K1B - RNAi starved for 12 h . Scale bars , 5 μm . ( b ) from a were assessed for enlarged autolysosomes in a blind fashion after starvation quantified . n = 100 from three independent experiments . Error bars indicate s . d .

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(a) NRK cell lines stably expressing CFP-LC3; LAMP1-YFP were transfected with nonspecific (NS)- or PIP5K1B-RNAi and starved for 12 h. Scale bars, 5 μm. (b) Cells from a were assessed for enlarged autolysosomes in a blind fashion after starvation and quantified. n = 100 cells from three independent experiments. Error bars indicate the s.d.


( c ) Representative of nonspecific - or PIP5K1B - RNAi - transfected NRK after 12 h of serum starvation . Scale bar , 5 μm .

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(c) Representative TEM micrographs of nonspecific- or PIP5K1B-RNAi-transfected NRK cells after 12 h of serum starvation. Scale bar, 5 μm.


( d ) NRK were starved for 4 h , stained with antibodies against PIP5K1B LAMP1 , then analysed by 3D - SIM . Scale bar , 2 . 5 μm . right panels show enlarged regions of interest from left panel : T , top view ; L , lateral view ; B , bottom view .

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(d) NRK cells were starved for 4 h, stained with antibodies against PIP5K1B and LAMP1, and then analysed by 3D-SIM. Scale bar, 2.5 μm. The right panels show enlarged regions of interest from the left panel: T, top view; L, lateral view; B, bottom view.


( e ) Fractions from Fig . 2f were analysed by western blotting with an antibody against PIP5K1B . outline indicates LAMP2 - positive fraction .

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(e) Fractions from Fig. 2f were analysed by western blotting with an antibody against PIP5K1B. The outline indicates the LAMP2-positive fraction.


( f ) NRK were starved for 4 h , autolysosomes were purified by density fractionation quality of purified autolysosomes was monitored by ( left panel ) . Right panel , purified autolysosomes stained with antibodies against PI ( 4 ) P PtdIns ( 4 , 5 ) P2 . Scale bar , 500 nm .

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(f) NRK cells were starved for 4 h, autolysosomes were purified by density fractionation and the quality of purified autolysosomes was monitored by TEM (left panel). Right panel, purified autolysosomes stained with antibodies against PI(4)P and PtdIns(4,5)P2. Scale bar, 500 nm.


( g ) NRK were starved for 0 or 4 h stained with antibody against PI ( 4 ) P . Scale bar , 5 μm .

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(g) NRK cells were starved for 0 or 4 h and stained with antibody against PI(4)P. Scale bar, 5 μm.


( h ) NRK lines stably expressing OSBP - PH - GFP ; LAMP1 - Cherry were starved for 0 , 4 , or 8 h observed by confocal microscopy . Scale bars , 5 μm .

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(h) NRK cells lines stably expressing OSBP-PH-GFP;LAMP1-Cherry were starved for 0, 4, or 8 h and observed by confocal microscopy. Scale bars, 5 μm.


( i ) NRK lines stably expressing OSBP - PH - GFP ; LAMP1 - Cherry were transfected with PIP5K1B - RNAi . At 60 h after transfection , were starved for 12 h analysed by confocal microscopy . Scale bar , 5 μm .

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(i) NRK cell lines stably expressing OSBP-PH-GFP;LAMP1-Cherry were transfected with PIP5K1B-RNAi. At 60 h after transfection, cells were starved for 12 h and analysed by confocal microscopy. Scale bar, 5 μm.


( j ) NRK lines stably expressing LAMP1 - Cherry ; clathrin - GFP were transfected with nonspecific - or PIP5K1B - RNAi starved for 12 h , then observed by confocal microscopy . Scale bars , 5 μm . insets in a , h - j represent enlarged regions of interest .

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(j) NRK cell lines stably expressing LAMP1-Cherry;clathrin-GFP were transfected with nonspecific- or PIP5K1B-RNAi and starved for 12 h, and then observed by confocal microscopy. Scale bars, 5 μm. The insets in a, h-j represent enlarged regions of interest.


( k ) from g were analysed for Pearson ' s co - localization coefficent ( R ( r ) ) by Olympus Fluoview . n = 30 from three independent experiments . Error bars indicate s . d . Uncropped of blots are shown in Supplementary Fig . S6 .

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(k) Cells from g were analysed for Pearson's co-localization coefficent (R(r)) by Olympus Fluoview. n = 30 cells from three independent experiments. Error bars indicate the s.d. Uncropped images of blots are shown in Supplementary Fig. S6.


( a ) NRK stably expressing CFP - LC3 ; LAMP1 - YFP were transfected with nonspecific ( NS ) - or PIP5K1A - RNAi starved for 10 h . insets represent enlarged regions of interest . Scale bar , 5 μm . ( b ) from a were assessed for presence of elongated reformation tubules in a blind fashion after starvation quantified . n = 100 from three independent experiments . Error bars indicate s . d . ( c ) from a were assessed for of reformation tubules in a blind fashion after starvation quantified . 50 were counted . average tubule is indicated above each column . ( d ) Diagram of reformation tubules . R , radius of main ; L , . ( e ) Representative image of reformation tubules in nonspecific - or PIP5K1A - RNAi - transfected . top bottom panels show two randomly selected of respective samples .

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(a) NRK cells stably expressing CFP-LC3;LAMP1-YFP were transfected with nonspecific (NS)- or PIP5K1A-RNAi and starved for 10 h. The insets represent enlarged regions of interest. Scale bar, 5 μm. (b) Cells from a were assessed for the presence of elongated reformation tubules in a blind fashion after starvation and quantified. n = 100 cells from three independent experiments. Error bars indicate the s.d. (c) Cells from a were assessed for the length of reformation tubules in a blind fashion after starvation and quantified. 50 cells were counted. The average tubule length is indicated above each column. (d) Diagram of reformation tubules. R, radius of main body; L, length. (e) Representative image of reformation tubules in nonspecific- or PIP5K1A-RNAi-transfected cells. The top and bottom panels show two randomly selected images of the respective samples.


( f ) from a were assessed for L / R of reformation tubules in a blind fashion after starvation quantified . 25 were counted . average L / R is indicated above each column .

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(f) Cells from a were assessed for the L/R ratio of reformation tubules in a blind fashion after starvation and quantified. 25 cells were counted. The average L/R is indicated above each column.


( g ) Purified reformation tubules were monitored by . Scale bar , 250 nm .

(g) Purified reformation tubules were monitored by TEM. Scale bar, 250 nm.


( h ) Purified reformation tubules stained with antibodies against PI ( 4 ) P , PtdIns ( 4 , 5 ) P2 PIP5K1A . Scale bar , 250 nm .

(h) Purified reformation tubules stained with antibodies against PI(4)P, PtdIns(4,5)P2 and PIP5K1A. Scale bar, 250 nm.


( i ) Fractions from Fig . 2f were analysed by western blotting with an antibody against PIP5K1A . outline indicates LAMP2 - positive fraction .

(i) Fractions from Fig. 2f were analysed by western blotting with an antibody against PIP5K1A. The outline indicates the LAMP2-positive fraction.


( j ) NRK stably expressing CFP - LC3 ; LAMP1 - YFP were starved for 8 h , budding of proto - lysosomes was monitored by spinning - disc microscopy . Scale bar , 100 nm .

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(j) NRK cells stably expressing CFP-LC3;LAMP1-YFP were starved for 8 h, and the budding of proto-lysosomes was monitored by spinning-disc microscopy. Scale bar, 100 nm.


( k ) NRK lines stably expressing LAMP1 - Cherry ; clathrin - GFP were starved for 8 h observed by confocal microscopy . right panels show enlarged regions of interest . Scale bar , 5 μm .

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(k) NRK cell lines stably expressing LAMP1-Cherry;clathrin-GFP were starved for 8 h and observed by confocal microscopy. The right panels show enlarged regions of interest. Scale bar, 5 μm.


( l ) NRK lines stably expressing LAMP1 - Cherry ; clathrin - GFP were transfected with nonspecific - or PIP5K1A - RNAi starved for 8 h . Scale bar , 5 μm . middle panels show enlarged regions of interest from upper panels . between clathrin dots on each reformation tubule in upper panels is shown in diagrammatic form in lower panels . Black : reformation tubules ; red dot : clathrin . ( m ) between two clathrin dots on reformation tubules ( RTs ) in from l was accessed in a blind fashion . Twenty - five were counted . average between two clathrin dots is indicated above each column . Uncropped of blots are shown in Supplementary Fig . S6 .

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(l) NRK cell lines stably expressing LAMP1-Cherry;clathrin-GFP were transfected with nonspecific- or PIP5K1A-RNAi and starved for 8 h. Scale bar, 5 μm. The middle panels show enlarged regions of interest from the upper panels. The distance between clathrin dots on each reformation tubule in the upper panels is shown in diagrammatic form in the lower panels. Black line: reformation tubules; red dot: clathrin. (m) The distance between two clathrin dots on reformation tubules (RTs) in cells from l was accessed in a blind fashion. Twenty-five cells were counted. The average distance between two clathrin dots is indicated above each column. Uncropped images of blots are shown in Supplementary Fig. S6.


( a ) NRK stably expressing CFP - LC3 ; LAMP1 - YFP were transfected with nonspecific ( NS ) - or AP2 - RNAi starved for 12 h . Scale bars , 5 μm . ( b ) from a were assessed for enlarged autolysosomes in a blind fashion after starvation quantified . n = 100 from three independent experiments . Error bars indicate s . d .

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(a) NRK cells stably expressing CFP-LC3; LAMP1-YFP were transfected with nonspecific (NS)- or AP2-RNAi and starved for 12 h. Scale bars, 5 μm. (b) Cells from a were assessed for enlarged autolysosomes in a blind fashion after starvation and quantified. n = 100 cells from three independent experiments. Error bars indicate the s.d.


( c ) Representative of nonspecific - or AP2 - RNAi - transfected NRK after 12 h of serum starvation . Scale bars , 5 μm .

(c) Representative TEM micrographs of nonspecific- or AP2-RNAi-transfected NRK cells after 12 h of serum starvation. Scale bars, 5 μm.


( d ) NRK were starved for 4 h , stained with antibodies against AP2 LAMP1 , then analysed by 3D - SIM . Scale bar , 5 μm . right panels show enlarged regions of interest from left panel : T , top view ; L , lateral view ; B , bottom view .

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(d) NRK cells were starved for 4 h, stained with antibodies against AP2 and LAMP1, and then analysed by 3D-SIM. Scale bar, 5 μm. The right panels show enlarged regions of interest from the left panel: T, top view; L, lateral view; B, bottom view.


( e ) Fractions from Fig . 2f were analysed by western blotting with an antibody against AP2α . outline indicates LAMP2 - positive fraction .

(e) Fractions from Fig. 2f were analysed by western blotting with an antibody against AP2α. The outline indicates the LAMP2-positive fraction.


( g ) Purified autolysosomes were treated with trypsin , incubated with rat brain cytosol , washed then analysed by western blot with antibodies against AP2α LAMP2 .

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(g) Purified autolysosomes were treated with trypsin, incubated with rat brain cytosol, washed and then analysed by western blot with antibodies against AP2α and LAMP2.


( h ) Stripped autolysosomes were pretreated with GFP or PtdIns ( 4 , 5 ) P2 antibody then incubated with rat brain cytosol . Autolysosomes were washed then analysed by western blotting with antibodies against AP2α LAMP2 .

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(h) Stripped autolysosomes were pretreated with GFP or PtdIns(4,5)P2 antibody and then incubated with rat brain cytosol. Autolysosomes were washed and then analysed by western blotting with antibodies against AP2α and LAMP2.


( i ) NRK lines stably expressing LAMP1 - Cherry ; clathrin - GFP were transfected with nonspecific - or AP2 - RNAi starved for 12 h . Scale bar , 5 μm . insets in a , c i represent enlarged regions of interest . ( j ) from i were analysed for Pearson ' s co - localization coefficent ( R ( r ) ) by Olympus Fluoview . n = 30 from three independent experiments ) . Error bars indicate s . d . Uncropped of blots are shown in Supplementary Fig . S6 .

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(i) NRK cell lines stably expressing LAMP1-Cherry;clathrin-GFP were transfected with nonspecific- or AP2-RNAi and starved for 12 h. Scale bar, 5 μm. The insets in a, c and i represent enlarged regions of interest. (j) Cells from i were analysed for the Pearson's co-localization coefficent (R(r)) by Olympus Fluoview. n = 30 cells from three independent experiments). Error bars indicate the s.d. Uncropped images of blots are shown in Supplementary Fig. S6.


( a ) NRK stably expressing CFP - LC3 ; LAMP1 - YFP were transfected with nonspecific ( NS ) - or AP4 - RNAi starved for 12 h . Scale bars , 5 μm . ( b ) from a were assessed for enlarged autolysosomes in a blind fashion after starvation quantified . n = 100 from three independent experiments . Error bars indicate s . d .

(a) NRK cells stably expressing CFP-LC3; LAMP1-YFP were transfected with nonspecific (NS)- or AP4-RNAi and starved for 12 h. Scale bars, 5 μm. (b) Cells from a were assessed for enlarged autolysosomes in a blind fashion after starvation and quantified. n = 100 cells from three independent experiments. Error bars indicate the s.d.


( c ) Representative of nonspecific - or AP4 - RNAi - transfected NRK after 12 h of serum starvation . Scale bars , 5 μm .

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(c) Representative TEM micrographs of nonspecific- or AP4-RNAi-transfected NRK cells after 12 h of serum starvation. Scale bars, 5 μm.


( d ) NRK were starved for 0 , 4 , 8 or 12 h , isolated lysosomes / autolysosome were analysed by western blotting using antibodies against AP4 or LAMP2 .

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(d) NRK cells were starved for 0, 4, 8 or 12 h, and isolated lysosomes/autolysosome were analysed by western blotting using antibodies against AP4 or LAMP2.


( e ) Purified reformation tubules were stained with antibodies against AP4 LAMP1 . Scale bar , 2 μm . bottom row shows enlarged regions of interest from top panel .

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

(e) Purified reformation tubules were stained with antibodies against AP4 and LAMP1. Scale bar, 2 μm. The bottom row shows enlarged regions of interest from the top panel.


( f ) NRK were transfected with GFP LAMP1 - GFP . At 20 h after transfection , were starved for 0 or 8 h , then collected analysed by immunoprecipitation ( IP ) western blotting ( WB ) of as indicated , using stably expressed LAMP1 - GFP as bait . TCL , total lysate .

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

(f) NRK cells were transfected with GFP and LAMP1-GFP. At 20 h after transfection, cells were starved for 0 or 8 h, and then collected and analysed by immunoprecipitation (IP) and western blotting (WB) of proteins as indicated, using stably expressed LAMP1-GFP as bait. TCL, total cell lysate.


insets in a , c g represent enlarged regions of interest . ( g ) NRK lines stably expressing LAMP1 - Cherry ; clathrin - GFP were transfected with nonspecific - or AP4 - RNAi starved for 12 h . Scale bars , 5 μm . ( h ) from g were analysed for Pearson ' s co - localization coefficent ( R ( r ) ) by Olympus Fluoview . n = 30 from three independent experiments . Error bars indicate s . d . Uncropped of blots are shown in Supplementary Fig . S6 .

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

The insets in a, c and g represent enlarged regions of interest. (g) NRK cell lines stably expressing LAMP1-Cherry; clathrin-GFP were transfected with nonspecific- or AP4-RNAi and starved for 12 h. Scale bars, 5 μm. (h) Cells from g were analysed for the Pearson's co-localization coefficent (R(r)) by Olympus Fluoview. n = 30 cells from three independent experiments. Error bars indicate the s.d. Uncropped images of blots are shown in Supplementary Fig. S6.


( a ) Purified autolysosomes were treated with trypsin . trypsin - treated ( stripped ) autolysosomes were incubated with rat brain cytosol reaction mixture , then analysed by western blotting . HC , heavy chain .

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(a) Purified autolysosomes were treated with trypsin. The trypsin-treated (stripped) autolysosomes were incubated with rat brain cytosol and reaction mixture, and then analysed by western blotting. HC, heavy chain.


( b ) Scanning electron of purified autolysosomes , rat brain cytosol , trypsin - treated purified autolysosomes trypsin - treated purified autolysosomes after budding . Scale bar , 250 nm . arrows indicate buds .

(b) Scanning electron micrographs of purified autolysosomes, rat brain cytosol, trypsin-treated purified autolysosomes and trypsin-treated purified autolysosomes after the membrane budding assay. Scale bar, 250 nm. The arrows indicate buds.


( c ) Trypsin - treated autolysosomes were incubated with rat brain cytosol reaction mixture in presence of antibodies against GFP , clathrin , AP2 , AP3 , AP4 PtdIns ( 4 , 5 ) P2 , then analysed by western blotting .

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

(c) Trypsin-treated autolysosomes were incubated with rat brain cytosol and reaction mixture in the presence of antibodies against GFP, clathrin, AP2, AP3, AP4 and PtdIns(4,5)P2, and then analysed by western blotting.


( d ) Reactions from c were visualized by scanning electron microscopy . Scale bar , 250 nm . Arrow indicates bud . ( e ) Samples from d were quantified for number of buds on each autolysosome . Error bars represent s . d . n = 50 autolysosomes from three independent experiments . Uncropped of blots are shown in Supplementary Fig . S6 .

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(d) Reactions from c were visualized by scanning electron microscopy. Scale bar, 250 nm. Arrow indicates bud. (e) Samples from d were quantified for the number of buds on each autolysosome. Error bars represent s.d. n = 50 autolysosomes from three independent experiments. Uncropped images of blots are shown in Supplementary Fig. S6.


( a ) NRK were transfected with nonspecific ( NS ) - or Cltc ( clathrin heavy chain ) - RNAi starved for 0 , 4 , 8 or 12 h . Scale bar , 5 mm . were stained with antibodies against LC3 . ( b ) from a were assessed for LC3 in a blind fashion after starvation quantified . n = 100 from three independent experiments . Error bars indicate s . d .

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

(a) NRK cells were transfected with nonspecific (NS)- or Cltc (clathrin heavy chain)-RNAi and starved for 0, 4, 8 or 12 h. Scale bar, 5 mm. Cells were stained with antibodies against LC3. (b) Cells from a were assessed for LC3 puncta in a blind fashion after starvation and quantified. n = 100 cells from three independent experiments. Error bars indicate the s.d.


( a ) Diagrams of deletion - mutation constructs of Keap1 ( left ) corresponding immunoprecipitation assays ( right ) . Each Flag - tagged mouse Keap1 mutant was expressed in HEK293T . At 22 h after transfection , lysates were prepared immunoprecipitated with anti - Flag antibody . resulting immunoprecipitates were subjected to SDS - PAGE analysed by immunoblotting with anti - Flag , anti - p62 , anti - Nrf2 anti - actin antibodies . Data are representative of three experiments .

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

(a) Diagrams of the deletion-mutation constructs of Keap1 (left) and the corresponding immunoprecipitation assays (right). Each Flag-tagged mouse Keap1 and mutant was expressed in HEK293T cells. At 22 h after transfection, lysates were prepared and immunoprecipitated with anti-Flag antibody. The resulting immunoprecipitates were subjected to SDS-PAGE and analysed by immunoblotting with anti-Flag, anti-p62, anti-Nrf2 and anti-actin antibodies. Data are representative of three individual experiments.


( b ) Diagrams of deletion - mutation constructs of p62 ( left ) corresponding input ( upper right ) pull - down ( lower right ) . MBP - tagged mouse p62 deletion mutants conjugated to amylose ( AM ) resins were incubated with purified GST - tagged mouse Keap1 - DC . pulled - down complexes with MBP - p62 mutants were subjected to SDS - PAGE revealed by with Coomassie brilliant blue . bands corresponding to MBP - p62 its mutants are indicated by black dots . Red arrowheads indicate band corresponding to GST - Keap1 - DC . For details of construct 14 see Supplementary Information , Fig . S3 .

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

(b) Diagrams of the deletion-mutation constructs of p62 (left) and the corresponding input (upper right) and pull-down assay (lower right). The MBP-tagged mouse p62 deletion mutants conjugated to amylose (AM) resins were incubated with purified GST-tagged mouse Keap1-DC. The pulled-down complexes with the MBP-p62 mutants were subjected to SDS-PAGE and revealed by staining with Coomassie brilliant blue. The bands corresponding to MBP-p62 and its mutants are indicated by black dots. Red arrowheads indicate the band corresponding to GST-Keap1-DC. For details of construct 14 see Supplementary Information, Fig. S3.


( d ) Immunoprecipitation assays . Flag - tagged p62 , KIR - deleted p62 ( p62 ΔKIR ) a p62 mutant defective in oligomerization ( p62 K7AD69A ) were expressed in mouse hepatocytes by adenovirus system ( left ) or in HEK293T by transfection ( right ) . lysates were immunoprecipitated with anti - Flag antibody . resulting immunoprecipitates were subjected to SDS - PAGE analysed by immunoblotting with anti - p62 anti - Keap1 antibodies . bands corresponding to Flag - p62 , endogenous p62 , Keap1 actin are indicated . data shown are representative of three separate experiments .

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

(d) Immunoprecipitation assays. Flag-tagged p62, KIR-deleted p62 (p62 ΔKIR) and a p62 mutant defective in oligomerization (p62 K7AD69A) were expressed in primary mouse hepatocytes by the adenovirus system (left) or in HEK293T cells by transfection (right). Cell lysates were immunoprecipitated with anti-Flag antibody. The resulting immunoprecipitates were subjected to SDS-PAGE and analysed by immunoblotting with anti-p62 and anti-Keap1 antibodies. The bands corresponding to Flag-p62, endogenous p62, Keap1 and actin are indicated. The data shown are representative of three separate experiments.


( e ) Interaction of endogenous p62 with Keap1 . Lysates prepared from human hepatocellular carcinoma Huh - 1 were immunoprecipitated with anti - p62 antibody or anti - Flag antibody ( negative control ) followed by immunoblotting with antibodies against p62 Keap1 . bands corresponding to endogenous p62 , Keap1 IgG heavy chain ( IgG H . C . ) are indicated . data shown are representative of three separate experiments . Uncropped of blots are shown in Supplementary Information , Fig . S11 .

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

(e) Interaction of endogenous p62 with Keap1. Lysates prepared from the human hepatocellular carcinoma cell line Huh-1 were immunoprecipitated with anti-p62 antibody or anti-Flag antibody (negative control) followed by immunoblotting with antibodies against p62 and Keap1. The bands corresponding to endogenous p62, Keap1 and IgG heavy chain (IgG H.C.) are indicated. The data shown are representative of three separate experiments. Uncropped images of blots are shown in Supplementary Information, Fig. S11.


( a ) A representative ITC profile of titration of Keap1 - DC with p62M7 ( residues 168 - 391 ) . upper panel shows raw ITC thermograms lower panel shows fitted binding isotherms .

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

(a) A representative ITC profile of the titration of Keap1-DC with p62M7 (residues 168-391). The upper panel shows the raw ITC thermograms and the lower panel shows the fitted binding isotherms.


( b ) Immunoprecipitation assays . Flag - tagged Keap1 was co - expressed with increasing concentrations of green ( GFP ) - p62 ( lanes 3 - 6 ) in HEK293T . lysates were immunoprecipitated with anti - Flag antibody . resulting immunoprecipitates were subjected to SDS - PAGE analysed by immunoblotting with anti - Flag , anti - p62 anti - Nrf2 antibodies . bands corresponding to Flag - Keap1 , endogenous p62 , Nrf2 actin are indicated . Data are representative of three independent experiments .

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

(b) Immunoprecipitation assays. Flag-tagged Keap1 was co-expressed with increasing concentrations of green fluorescent protein (GFP)-p62 (lanes 3-6) in HEK293T cells. Cell lysates were immunoprecipitated with anti-Flag antibody. The resulting immunoprecipitates were subjected to SDS-PAGE and analysed by immunoblotting with anti-Flag, anti-p62 and anti-Nrf2 antibodies. The bands corresponding to Flag-Keap1, endogenous p62, Nrf2 and actin are indicated. Data are representative of three independent experiments.


( c ) competitive p62 activity against Keap1 was measured by luciferase . expression plasmids for Nrf2 , Keap1 p62 wild - type ( w . t . ) or its mutants were transfected into Hepa1 along with pNqo1 - ARE reporter plasmid pRL - TK as an internal control . At 36 h after transfection , luciferase activity was measured in accordance with instructions provided by manufacturer . Assays were performed twice in triplicate . Data are means s . d . for six determinations .

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

(c) The competitive p62 activity against Keap1 was measured by luciferase assay. The expression plasmids for Nrf2, Keap1 and p62 wild-type (w.t.) or its mutants were transfected into Hepa1 cells along with pNqo1-ARE reporter plasmid and pRL-TK as an internal control. At 36 h after transfection, the luciferase activity was measured in accordance with the instructions provided by the manufacturer. Assays were performed twice in triplicate. Data are means and s.d. for six determinations.


( d ) Immunoblot . Flag - tagged p62 its mutants defective in interacting with Keap1 were overproduced in wild - type p62 − / − mouse hepatocytes by adenovirus system . At 48 h after infection , total lysates fractions were prepared subjected to immunoblot with antibodies specified . bands corresponding to Flag - p62 , endogenous p62 , Keap1 , Nrf2 , Nqo1 , actin Lamin B are shown . Data are representative of three independent experiments . Uncropped of blots are shown in Supplementary Information , Fig . S11 .

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

(d) Immunoblot analysis. Flag-tagged p62 and its mutants defective in interacting with Keap1 were overproduced in wild-type and p62−/− primary mouse hepatocytes by the adenovirus system. At 48 h after infection, total cell lysates and nuclear fractions were prepared and subjected to immunoblot analysis with the antibodies specified. The bands corresponding to Flag-p62, endogenous p62, Keap1, Nrf2, Nqo1, actin and Lamin B are shown. Data are representative of three independent experiments. Uncropped images of blots are shown in Supplementary Information, Fig. S11.


( e ) of mRNA of detoxification enzymes Nqo1 , Gstm1 Cyp2a5 in hepatocytes overexpressing Flag - p62 its mutants . Total RNAs were prepared from non - infected or infected hepatocytes reverse transcribed into their respective cDNAs , which were used as templates in real - time PCR . Values were normalized to amount of each mRNA in non - infected hepatocytes . experiments were performed three times . Data are means ± s . d . for three experiments .

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

(e) Quantification of mRNA levels of the detoxification enzymes Nqo1, Gstm1 and Cyp2a5 in hepatocytes overexpressing Flag-p62 and its mutants. Total RNAs were prepared from non-infected or infected hepatocytes and reverse transcribed into their respective cDNAs, which were used as templates in real-time PCR analysis. Values were normalized to the amount of each mRNA in the non-infected hepatocytes. The experiments were performed three times. Data are means ± s.d. for three experiments.


( a ) Insolubilization of Keap1 in Atg7 - deficient hepatocytes . Liver homogenates from Atg7F / F : Mx1 mice on various days after injection of poly ( I ) • poly ( C ) were separated into detergent - soluble detergent - insoluble fractions with 0 . 5 % Triton X - 100 . Each fraction was subjected to SDS - PAGE analysed by immunoblotting with indicated antibodies . data displayed are representative of three separate experiments .

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

(a) Insolubilization of Keap1 in Atg7-deficient hepatocytes. Liver homogenates from Atg7F/F:Mx1 mice on various days after injection of poly(I)•poly(C) were separated into detergent-soluble and detergent-insoluble fractions with 0.5% Triton X-100. Each fraction was subjected to SDS-PAGE and analysed by immunoblotting with the indicated antibodies. The data displayed are representative of three separate experiments.


( b ) Immunoblot of Atg7 - deficient ( Atg7F / F : Mx1 ; Atg7F / F shown here as F / F ) Atg7 p62 - deficient ( Atg7F / F : Mx1 : p62 − / − ) livers . Liver homogenates from mice of stated genotypes at 12 days after injection of poly ( I ) • poly ( C ) were separated into detergent - soluble detergent - insoluble fractions . Each fraction was subjected to SDS - PAGE analysed by immunoblotting with indicated antibodies . Atg7F / F mice in which Atg7 is efficiently expressed at a level similar to that in wild - type mice were used as control . Data shown are representative of three separate experiments . Uncropped of blots are shown in Supplementary Information , Fig . S11 .

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

(b) Immunoblot analysis of Atg7-deficient (Atg7F/F:Mx1; Atg7F/F shown here as F/F) and Atg7 p62-deficient (Atg7F/F:Mx1:p62−/−) livers. Liver homogenates from mice of the stated genotypes at 12 days after injection of poly(I)•poly(C) were separated into detergent-soluble and detergent-insoluble fractions. Each fraction was subjected to SDS-PAGE and analysed by immunoblotting with the indicated antibodies. Atg7F/F mice in which Atg7 is efficiently expressed at a level similar to that in the wild-type mice were used as control. Data shown are representative of three separate experiments. Uncropped images of blots are shown in Supplementary Information, Fig. S11.


( c ) Quantitative real - time PCR analyses of Nqo1 Gstm1 in mouse livers . Total RNAs were prepared from livers of indicated genotypes at 12 days after injection of poly ( I ) • poly ( C ) . Values were normalized to amount of mRNA in Atg7F / F liver . Data are means ± s . d . for three experiments .

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

(c) Quantitative real-time PCR analyses of Nqo1 and Gstm1 in mouse livers. Total RNAs were prepared from livers of the indicated genotypes at 12 days after injection of poly(I)•poly(C). Values were normalized to the amount of mRNA in Atg7F/F liver. Data are means ± s.d. for three experiments.


( d ) Immunofluorescence of localization of p62 Keap1 . Liver sections from mice of indicated genotypes at 28 days after injection of poly ( I ) • poly ( C ) were immunostained with anti - Keap1 ( top ) anti - p62 ( middle ) antibodies . Bottom : merged of Keap1 ( green ) p62 ( red ) . Each inset in Atg7 - deficient liver panels is a magnified image of boxed region . Scale bars , 20 μm .

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

(d) Immunofluorescence analysis of the cellular localization of p62 and Keap1. Liver sections from mice of the indicated genotypes at 28 days after injection of poly(I)•poly(C) were immunostained with anti-Keap1 (top) and anti-p62 (middle) antibodies. Bottom: merged images of Keap1 (green) and p62 (red). Each inset in the Atg7-deficient liver panels is a magnified image of the boxed region. Scale bars, 20 μm.


( a ) Immunoblotting of Atg7 - deficient ( Atg7F / F : Mx1 ; Atg7F / F shown here as F / F ) Atg7 Nrf2 - deficient ( Atg7F / F : Mx1 : Nrf2 − / − ) livers . Liver homogenates from mice of assigned genotypes at 28 days after injection of poly ( I ) • poly ( C ) were separated into detergent - soluble detergent - insoluble fractions . Total , soluble insoluble fractions were subjected to SDS - PAGE analysed by immunoblotting with indicated antibodies ( top section ) . Total lysates were subjected to SDS - PAGE analysed by immunoblotting with antibodies against Nqo1 , Gstm1 actin ( bottom left section ) . fractions were prepared from livers of indicated genotypes at 28 days after injection of poly ( I ) • poly ( C ) , subjected to SDS - PAGE analysed by immunoblotting with antibodies against Nrf2 Lamin B ( as control ) ( bottom right section ) . Data were obtained from three independent experiments . Uncropped of blots are shown in Supplementary Information , Fig . S11 .

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

(a) Immunoblotting of Atg7-deficient (Atg7F/F:Mx1; Atg7F/F shown here as F/F) and Atg7 Nrf2-deficient (Atg7F/F:Mx1:Nrf2−/−) livers. Liver homogenates from mice of the assigned genotypes at 28 days after injection of poly(I)•poly(C) were separated into detergent-soluble and detergent-insoluble fractions. Total, soluble and insoluble fractions were subjected to SDS-PAGE and analysed by immunoblotting with the indicated antibodies (top section). Total lysates were subjected to SDS-PAGE and analysed by immunoblotting with antibodies against Nqo1, Gstm1 and actin (bottom left section). Nuclear fractions were prepared from the livers of the indicated genotypes at 28 days after injection of poly(I)•poly(C), subjected to SDS-PAGE and analysed by immunoblotting with antibodies against Nrf2 and Lamin B (as control) (bottom right section). Data were obtained from three independent experiments. Uncropped images of blots are shown in Supplementary Information, Fig. S11.


( b ) Immunofluorescence of localization of p62 Keap1 . Liver sections from mice of indicated genotypes at 28 days after injection of poly ( I ) • poly ( C ) were immunostained with anti - Keap1 ( left ) anti - p62 ( middle ) antibodies . Right : merged of Keap1 ( green ) p62 ( red ) . Each inset in Atg7 - deficient Atg7 Nrf2 - deficient liver panels is a magnified image of boxed region . Scale bars , 20 μm .

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

(b) Immunofluorescence analysis of the cellular localization of p62 and Keap1. Liver sections from mice of the indicated genotypes at 28 days after injection of poly(I)•poly(C) were immunostained with anti-Keap1 (left) and anti-p62 (middle) antibodies. Right: merged images of Keap1 (green) and p62 (red). Each inset in the Atg7-deficient and Atg7 Nrf2-deficient liver panels is a magnified image of the boxed region. Scale bars, 20 μm.


( c ) Quantitative real - time PCR analyses of Nqo1 , Gstm1 Cyp2a5 in mouselivers . Total RNAs were prepared from livers of indicated genotypes at 28 days after injection of poly ( I ) • poly ( C ) . Values were normalized to amount of mRNA in Atg7F / F liver . Data are means ± s . d . for three experiments .

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

(c) Quantitative real-time PCR analyses of Nqo1, Gstm1 and Cyp2a5 in mouselivers. Total RNAs were prepared from livers of the indicated genotypes at 28 days after injection of poly(I)•poly(C). Values were normalized to the amount of mRNA in the Atg7F/F liver. Data are means ± s.d. for three experiments.


( d ) Liver . weights of mouse livers of different genotypes shown at 28 days after injection of poly ( I ) • poly ( C ) were measured . Data are means ± s . d . for five mice from each group . Three asterisks , P 0 . 001 ( Student ' s t - ) .

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

(d) Liver weight. The weights of the mouse livers of the different genotypes shown at 28 days after injection of poly(I)•poly(C) were measured. Data are means ± s.d. for five mice from each group. Three asterisks, P 0.001 (Student's t-test).


( e ) Histological of mouse liver of indicated genotypes . At 28 days after injection of poly ( I ) • poly ( C ) , livers were processed for haematoxylin / eosin . Higher - magnification views are shown in bottom panels . CV , central vein ; P , portal vein . Scale bars , 100 μm .

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

(e) Histological analysis of the mouse liver of the indicated genotypes. At 28 days after injection of poly(I)•poly(C), the livers were processed for haematoxylin/eosin staining. Higher-magnification views are shown in the bottom panels. CV, central vein; P, portal vein. Scale bars, 100 μm.


( f ) Liver function of mice used in d . serum of aspartate aminotransferase ( AST ) , alanine aminotransferase ( ALT ) alkaline phosphatase ( ALP ) were measured . ( IU / L , international unit per liter ) . Data are means ± s . d . for seven mice from each group . Asterisk , P 0 . 05 ; three asterisks , P 0 . 001 .

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(f) Liver function tests of the mice used in d. The serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT) and alkaline phosphatase (ALP) were measured. (IU/L, international unit per liter). Data are means ± s.d. for seven mice from each group. Asterisk, P 0.05; three asterisks, P 0.001.


( a ) Immunoblotting of Keap1 - deficient ( Keap1F / F : Alb ) Keap1 Atg7 - deficient ( Keap1F / F : Atg7F / F : Alb ) livers . Liver homogenates from 8 - week - old mice of these genotypes were separated into detergent - soluble detergent - insoluble fractions . Total , soluble insoluble fractions were subjected to SDS - PAGE analysed by immunoblotting with indicated antibodies ( top section ) . Total lysates were subjected to SDS - PAGE analysed by immunoblotting with antibodies against Nqo1 actin ( bottom section ) . Data were obtained from three independent experiments . Uncropped of blots are shown in Supplementary Information , Fig . S11 .

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

(a) Immunoblotting of Keap1-deficient (Keap1F/F:Alb) and Keap1 Atg7-deficient (Keap1F/F:Atg7F/F:Alb) livers. Liver homogenates from 8-week-old mice of these genotypes were separated into detergent-soluble and detergent-insoluble fractions. Total, soluble and insoluble fractions were subjected to SDS-PAGE and analysed by immunoblotting with the indicated antibodies (top section). Total lysates were subjected to SDS-PAGE and analysed by immunoblotting with antibodies against Nqo1 and actin (bottom section). Data were obtained from three independent experiments. Uncropped images of blots are shown in Supplementary Information, Fig. S11.


( b ) Quantitative real - time PCR analyses of Nqo1 , Mrp4 , p62 Ho - 1 in mouse livers . Total RNAs were prepared from livers of 9 - week - old indicated genotypes . Values were normalized to amount of mRNA in Atg7F / F liver ( control ) . Data are means ± s . d . for three experiments .

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

(b) Quantitative real-time PCR analyses of Nqo1, Mrp4, p62 and Ho-1 in mouse livers. Total RNAs were prepared from livers of 9-week-old indicated genotypes. Values were normalized to the amount of mRNA in the Atg7F/F liver (control). Data are means ± s.d. for three experiments.


( c ) Kaplan - Meier curves of survival of Atg7F / F : Alb Keap1F / F : Atg7F / F : Alb mice . survival of control ( n = 33 ) , Keap1F / F : Alb ( n = 15 ) , Atg7F / F : Alb ( n = 12 ) Keap1F / F : Atg7F / F : Alb mice ( n = 14 ) was on a 16 - week follow - up period .

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(c) Kaplan-Meier curves of survival of Atg7F/F:Alb and Keap1F/F:Atg7F/F:Alb mice. The survival analysis of control (n = 33), Keap1F/F:Alb (n = 15), Atg7F/F:Alb (n = 12) and Keap1F/F:Atg7F/F:Alb mice (n = 14) was based on a 16-week follow-up period.


( d ) Liver relative to was measured for different genotypes . Data are means ± s . d . for Atg7F / F ( control ) ( n = 24 ) , Keap1F / F : Alb ( n = 13 ) , Atg7F / F : Alb ( n = 9 ) Keap1F / F : Atg7F / F : Alb mice ( n = 7 ) . Two asterisks , P 0 . 01 ( Student ' s t - ) .

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(d) Liver weight relative to body weight was measured for the different genotypes. Data are means ± s.d. for Atg7F/F (control) (n = 24), Keap1F/F:Alb (n = 13), Atg7F/F:Alb (n = 9) and Keap1F/F:Atg7F/F:Alb mice (n = 7). Two asterisks, P 0.01 (Student's t-test).


( e ) Histological of mouse liver of indicated genotypes . livers from 9 - week - old mice were processed for haematoxylin / eosin . Scale bar , 50 μm ( left bottom panel ) ; 20 μm ( other panels ) .

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(e) Histological analysis of mouse liver of the indicated genotypes. The livers from 9-week-old mice were processed for haematoxylin/eosin staining. Scale bar, 50 μm (left bottom panel); 20 μm (other panels).


( f ) Liver function of mice used in d . serum of aspartate aminotransferase ( AST ) alanine aminotransferase ( ALT ) were measured . Data are means ± s . d . for Atg7F / F ( control ) ( n = 24 ) , Keap1F / F : Alb ( n = 13 ) , Atg7F / F : Alb ( n = 9 ) Keap1F / F : Atg7F / F : Alb mice ( n = 7 ) . Two asterisks , P 0 . 01 .

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

(f) Liver function tests of the mice used in d. The serum levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were measured. Data are means ± s.d. for Atg7F/F (control) (n = 24), Keap1F/F:Alb (n = 13), Atg7F/F:Alb (n = 9) and Keap1F/F:Atg7F/F:Alb mice (n = 7). Two asterisks, P 0.01.


( g ) Immunohistochemical of localization of p62 Keap1 . Paraffin sections of liver from 9 - week - old mice of indicated genotypes were immunolabelled with anti - p62 ( left ) anti - Keap1 ( right ) antibodies . Scale bars , 20 μm .

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

(g) Immunohistochemical analysis of cellular localization of p62 and Keap1. Paraffin sections of the liver from 9-week-old mice of the indicated genotypes were immunolabelled with anti-p62 (left) and anti-Keap1 (right) antibodies. Scale bars, 20 μm.


E ) Representative of prometaphase chromosomes from HeLa co - transfected with H2A . Z . 2 siRNA each of constructs in D ( green ) . Scale bar : 5μm . F ) of of with micronuclei from experiment ( E ) . error bars represent SD of three biological replicates ( Control si N = 887 ; H2A . Z . 2 si N = 1005 ; H2A . Z . 2 si + wt N = 760 ; H2A . Z . 2 si + KR N = 479 ) . Data sets were statistically analysed using Chi - square .

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

E) Representative images of prometaphase chromosomes from HeLa cells co-transfected with H2A.Z.2 siRNA and each of the constructs in D (green). Scale bar: 5μm. F) Quantification of the percentage of cells with micronuclei from experiment (E). The error bars represent the SD of three biological replicates (Control si N=887; H2A.Z.2 si N=1005; H2A.Z.2 si + wt N=760; H2A.Z.2 si + KR N=479). Data sets were statistically analysed using Chi-square test.


B ) Representative of metaphases from YFP : CENP - A ( green ) HeLa treated with Control ( top ) or H2A . Z . 2 ( bottom ) siRNA . arrowheads indicate mis - aligned chromosomes . Scale bar : 10 μm . C ) Representative of metaphase spreads from Control ( top ) or H2A . Z . 2 ( bottom ) siRNA - transfected HeLa after FISH with a Chr17 centromeric probe ( green ) . arrowheads indicate separated sister chromatids . Scale bar : 10 μm .

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

B) Representative images of metaphases from YFP:CENP-A (green) HeLa cells treated with Control (top) or H2A.Z.2 (bottom) siRNA. The white arrowheads indicate the mis-aligned chromosomes. Scale bar: 10 μm. C) Representative images of metaphase spreads from Control (top) or H2A.Z.2 (bottom) siRNA-transfected HeLa cells after FISH with a Chr17 centromeric probe (green). The arrowheads indicate the separated sister chromatids. Scale bar: 10 μm.


D ) Representative of HeLa transfected with Control ( top ) or H2A . Z . 2 ( bottom ) siRNA , fixed , stained for Sgo1 ( green ) . Scale bar : 10 μm . E ) of Sgo1 localisation in pro - metaphase from experiment in ( D ) . Error bar represents SD of two biological replicates . 35 pro - metaphase were analysed .

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

D) Representative images of HeLa cells transfected with Control (top) or H2A.Z.2 (bottom) siRNA, fixed, and stained for Sgo1 (green). Scale bar: 10 μm. E) Quantification of Sgo1 localisation in pro-metaphase cells from the experiment in (D). Error bar represents SD of two biological replicates. 35 pro-metaphase cells were analysed.


J ) Representative of HeLa YFP : CENP - A ( green ) mitotic after Control ( top ) or H2A . Z . 2 ( bottom ) siRNA treatment . Scale bar : 5 μm . K ) Violin plot of centromeric CENP - A of pro - metaphase / metaphase from experiments in J . ( Control si N = 197 H2A . Z . 2 si N = 260 ) . bar represents median . Data sets were statistically analysed using Wilcoxon rank in R . * * = p < 0 . 01 . L ) Representative of HeLa mitotic stained for CENP - C after Control ( top ) or H2A . Z . 2 ( bottom ) siRNA treatment . Scale bar : 5 μm . M ) Violin plot of centromeric CENP - C of pro - metaphase / metaphase from experiments in L . ( Control si N = 1083 H2A . Z . 2 si N = 686 , from 3 biological replicas ) . bars represent median . Data sets were statistically analysed using Wilcoxon rank in R . * * * = p < 0 . 0001 .

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

J) Representative images of HeLa YFP:CENP-A (green) mitotic cells after Control (top) or H2A.Z.2 (bottom) siRNA treatment. Scale bar: 5 μm. K) Violin plot of centromeric CENP-A intensity of pro-metaphase/metaphase cells from the experiments in J. (Control si N=197 H2A.Z.2 si N=260). The bar represents the median. Data sets were statistically analysed using the Wilcoxon rank test in R. **=p<0.01. L) Representative images of HeLa mitotic cells stained for CENP-C after Control (top) or H2A.Z.2 (bottom) siRNA treatment. Scale bar: 5 μm. M) Violin plot of centromeric CENP-C intensity of pro-metaphase/metaphase cells from the experiments in L. (Control si N=1083 H2A.Z.2 si N=686, from 3 biological replicas). The bars represent the median. Data sets were statistically analysed using the Wilcoxon rank test in R. ***=p<0.0001.


D ) IGV analyses of H2A . Z localisation ( from ENCODE ) on Aurora B ( green ) c - MYC ( blue ) genes showing H2A . Z enrichment at TSS at upstream region respectively ( bottom panels ) . IGV analyses of ATAC seq peaks for Control si H2A . Z . 1 si for Aurora B MYC genes . number in red represents log2Ratio H2AZ . 1 si / Control si .

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

D) IGV analyses of H2A.Z localisation (from ENCODE) on Aurora B (green) and c-MYC (blue) genes showing H2A.Z enrichment at the TSS and at the upstream region respectively (bottom panels). IGV analyses of ATAC seq peaks for Control si and H2A.Z.1 si for Aurora B and MYC genes. The number in red represents the log2Ratio H2AZ.1 si/Control si.


E ) Representative of Hela stained for Ki67 . arrows indicate different patterns quantified : strong signal ( grey arrow ) , weak signal ( brown arrow ) no signal ( brown arrow ( scale bar 10μm ) . F ) of experiment in ( E ) . Error bars indicate standard deviation from 3 biological replicates Data sets were statistically analysed using a Chi square . * * * = p < 0 . 001 .

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

E) Representative images of Hela cell stained for Ki67. The arrows indicate the different staining patterns quantified: strong signal (grey arrow), weak signal (light brown arrow) and no signal (dark brown arrow (scale bar 10μm). F) Quantification of the experiment in (E). Error bars indicate the standard deviation from 3 biological replicates Data sets were statistically analysed using a Chi square test. ***=p<0.001.


G ) Representative of HeLa transfected with control ( top ) or H2A . Z . 1 ( bottom ) siRNA stained for Lamin A / C ( green ) . Scale bar = 10μm .

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

G) Representative images of HeLa cells transfected with control (top) or H2A.Z.1 (bottom) siRNA and stained for Lamin A/C (green). Scale bar = 10μm.


C ) Kaplan Meier survival curve for neuroblastoma with low ( red ) or high ( blue ) expression of H2AFZ ( H2A . Z . 1 ) obtained from R2 genomics .

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C) Kaplan Meier survival curve for neuroblastoma patients with low (red) or high (blue) expression of H2AFZ (H2A.Z.1) obtained from R2 genomics.


A - G Hematoxylin eosin ( H & E ) of frontal sections of miniature pig mandible slices showing morphological changes during permanent canine ( PC ) initiation stage from embryonic day 50 ( E50 ) to postnatal day 10 ( PN10 ) ; ( A ' - F ' ) are magnifications of boxed regions in their corresponding figure panel . DC , deciduous canine ; PC , permanent canine ; SDL , successional dental lamina ; n = 3 . Scale bars = 100 µm .

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A-G Hematoxylin and eosin (H&E) staining of the frontal sections of miniature pig mandible slices showing morphological changes during the permanent canine (PC) initiation stage from embryonic day 50 (E50) to postnatal day 10 (PN10); (A'-F') are magnifications of boxed regions in their corresponding figure panel. DC, deciduous canine; PC, permanent canine; SDL, successional dental lamina; n = 3. Scale bars = 100 µm.