( 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 ) .

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(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 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 ) .

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

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(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 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 ) .

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(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 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 ) .

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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 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 .

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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 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 .

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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 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 .

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

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


( 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 .

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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 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 .

(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 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 .

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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 ) 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 .

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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 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 .

(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 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 .

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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 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 .

(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 TEM micrographs of nonspecific - or PIP5K1B - RNAi - transfected NRK cells 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 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 .

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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 . The outline indicates the 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 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 .

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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 cells were starved for 0 or 4 h and 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 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 .

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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 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 .

(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 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 .

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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 ) 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 .

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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 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 .

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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 ) 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 .

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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 TEM . 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 and 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 . The outline indicates the 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 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 .

(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 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 .

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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 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 .

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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 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 .

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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 TEM micrographs of nonspecific - or AP2 - RNAi - transfected NRK cells 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 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 .

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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 . The outline indicates the 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 and then analysed by western blot with antibodies against AP2α and 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 and then incubated with rat brain cytosol . Autolysosomes were washed and then analysed by western blotting with antibodies against AP2α and 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 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 .

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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 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 .

(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 TEM micrographs of nonspecific - or AP4 - RNAi - transfected NRK cells 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 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 .

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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 and LAMP1 . Scale bar , 2 μm . The bottom row shows enlarged regions of interest from the top panel .

(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 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 .

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(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.


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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 . The trypsin - treated ( stripped ) autolysosomes were incubated with rat brain cytosol and reaction mixture , and 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 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 .

(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 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 .

O O O O O O O O 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 I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 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 .

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(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 ) 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 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 .

(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 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 .

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(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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 ) 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 .

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(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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 ) 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 .

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(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 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 .

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

(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 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 .

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

(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 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 .

O O O O O O O O O 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 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

(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 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 .

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

(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 ) 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

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

(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 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 .

O O O O O O O O O O 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 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

(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 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 ) .

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 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

(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 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 .

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 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

(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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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), 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 ) 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O 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 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

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

(g) 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 ) 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 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 .

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

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

G) 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 patients with low ( red ) or high ( blue ) expression of H2AFZ ( H2A . Z . 1 ) obtained from R2 genomics .

O O O O O O O O O O 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) 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-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.


H In situ hybridization ( ISH ) showing the expression pattern of Sox2 in the initiation stage from E50 to E90 ; right figure panels are magnifications of boxed regions in left panels . Dashed lines mark the position of the successional dental lamina ( SDL ) ; green arrowhead indicates positive staining of Sox2 at the tip of the SDL . n = 3 . Scale bars = 100 µm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 In situ hybridization (ISH) showing the expression pattern of Sox2 in the initiation stage from E50 to E90; right figure panels are magnifications of boxed regions in left panels. Dashed lines mark the position of the successional dental lamina (SDL); green arrowhead indicates positive staining of Sox2 at the tip of the SDL. n = 3. Scale bars = 100 µm.


I Immunofluorescence ( IF ) of Ki67 at the SDL at E60 , E70 , E90 ; right figure panels are magnifications of boxed regions in left panels . n = 3 . Scale bars = 100 µm .

I Immunofluorescence (IF) of Ki67 at the SDL at E60, E70, E90; right figure panels are magnifications of boxed regions in left panels. n = 3. Scale bars = 100 µm.


J Semi - quantification and comparison of Ki67 expression levels during E60 , E70 , and E90 stages . n = 3 . Data information : Data represent the means ± SEM . * P < 0 . 05 ( one - way ANOVA and Newman - Keuls post - hoc tests ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Semi-quantification and comparison of Ki67 expression levels during E60, E70, and E90 stages. n = 3. Data information: Data represent the means ± SEM. *P < 0.05 (one-way ANOVA and Newman-Keuls post-hoc tests).


K TUNEL assay showing the apoptosis status of the SDL at E60 , E70 , E90 ; right figure panels are magnifications of boxed regions in left panels . n = 3 . Scale bars = 100 µm .

K TUNEL assay showing the apoptosis status of the SDL at E60, E70, E90; right figure panels are magnifications of boxed regions in left panels. n = 3. Scale bars = 100 µm.


A Three - dimensional reconstruction of serial H & E frontal sections of miniature pig mandibles at embryonic day 60 ( E60 ) and E90 ; deciduous canine ( DC ) in purple , permanent canine ( PC ) in yellow , and alveolar socket in green . The red , blue , and green arrows indicate the width of the labial alveolar socket , lingual alveolar socket , and DC , respectively . n = 3 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Three-dimensional reconstruction of serial H&E frontal sections of miniature pig mandibles at embryonic day 60 (E60) and E90; deciduous canine (DC) in purple, permanent canine (PC) in yellow, and alveolar socket in green. The red, blue, and green arrows indicate the width of the labial alveolar socket, lingual alveolar socket, and DC, respectively. n = 3.


B DC and labial and lingual alveolar socket widths in the horizontal plane at the bottom of the PC during E60 and E90 . n = 3 . Data information : Data represent the means ± SEM . Unpaired t - tests , * P < . 05 , * * P < . 01 , * * * P < . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 DC and labial and lingual alveolar socket widths in the horizontal plane at the bottom of the PC during E60 and E90. n = 3. Data information: Data represent the means ± SEM. Unpaired t-tests, *P < .05, **P < .01, ***P < .001.


C The proportion of DC width relative to the total alveolar socket width during E60 and E90 . n = 3 . Data information : Data represent the means ± SEM . Unpaired t - tests , * P < . 05 , * * P < . 01 , * * * P < . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 proportion of DC width relative to the total alveolar socket width during E60 and E90. n = 3. Data information: Data represent the means ± SEM. Unpaired t-tests, *P < .05, **P < .01, ***P < .001.


D Micro - CT imaging of the whole mandible at E60 ; boxed region is magnified in D ' ( top panel ) . ( D ' ) Mandible slice isolated with Geomagic software . White dashed lines mark DC . E 3 - D color map ( left ) after alignment of mandible slices before and after ( sham ) surgery showing a comparison of the surface points . Coronal sections through cusp tips ( transparent squares , left ) were selected for 2 - D comparisons ( middle ) ; right panels are magnifications of yellow - boxed regions . The solid purple contour and dotted black contour indicate the pre - and post - surgery shapes , respectively . The distance between the two contours are the colored line segments showing the distance and direction of the movement . The colored ball in the 2 - D comparison marks the position of the maximum displacement . The PC position is indicated in pink . n = 3 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Micro-CT imaging of the whole mandible at E60; boxed region is magnified in D' (top panel). (D') Mandible slice isolated with Geomagic software. White dashed lines mark DC. E 3-D color map (left) after alignment of mandible slices before and after (sham) surgery showing a comparison of the surface points. Coronal sections through cusp tips (transparent squares, left) were selected for 2-D comparisons (middle); right panels are magnifications of yellow-boxed regions. The solid purple contour and dotted black contour indicate the pre- and post-surgery shapes, respectively. The distance between the two contours are the colored line segments showing the distance and direction of the movement. The colored ball in the 2-D comparison marks the position of the maximum displacement. The PC position is indicated in pink. n = 3.


F Dissected mandible slice with a " U " shape .

F Dissected mandible slice with a "U" shape.


A Diagram of the dissected miniature pig mandible slice illustrating the deciduous ( DC ) and permanent ( PC ) canine and application of compression in vitro using Flexcell FX - 5000 Compression System .

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A Diagram of the dissected miniature pig mandible slice illustrating the deciduous (DC) and permanent (PC) canine and application of compression in vitro using Flexcell FX-5000 Compression System.


B - D H & E staining of canine frontal sections from embryonic day 60 ( E60 ) ( B ) , after culturing for two days without stress ( 0 kPa ) ( C ) , or with stress ( 3 kPa ) ( D ) . ( B ' - D ' ) are magnifications of boxed regions in their corresponding figure panels . Scale bars = 50 µm . n = 6 / 6 in E60 group ; n = 12 / 12 in 0 - kPa group ; n = 4 / 8 in 3 - kPa group .

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B-D H&E staining of canine frontal sections from embryonic day 60 (E60) (B), after culturing for two days without stress (0 kPa) (C), or with stress (3 kPa) (D). (B'-D') are magnifications of boxed regions in their corresponding figure panels. Scale bars = 50 µm. n = 6/6 in E60 group; n = 12/12 in 0-kPa group; n = 4/8 in 3-kPa group.


F RT - qPCR of several mechanosensitive proteins ( TGFB1 , NFKB1 , EGR1 , and RUNX2 ) and of four mechanoreceptors ( ITGB1 , ITGB3 , ITGAV and ITGA2 ) in PC and surrounding soft tissue at E60 and after culturing for two days with ( 3 kPa ) or without extra mechanical stress ( 0 kPa ) . n = 3 for each group . Data represent the means ± SEM . One - way ANOVA ( Newman - Keuls test for post - hoc comparisons between two groups ) , * P < . 05 , * * P < . 01 , * * * P < . 001 ; NS , not significant . =

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F RT-qPCR of several mechanosensitive proteins (TGFB1, NFKB1, EGR1, and RUNX2) and of four mechanoreceptors (ITGB1, ITGB3, ITGAV and ITGA2) in PC and surrounding soft tissue at E60 and after culturing for two days with (3 kPa) or without extra mechanical stress (0 kPa). n = 3 for each group. Data represent the means ± SEM. One-way ANOVA (Newman-Keuls test for post-hoc comparisons between two groups), *P < .05, **P < .01, ***P < .001; NS, not significant. =


A - L IF of integrin β1 , ERK1 , and RUNX2 in miniature pig canine frontal sections at embryonic day 60 ( E60 ) , E90 , and E60 cultured under 3 kPa stress for two days and E60 cultured under 0 kPa stress for two days ; ( A ' - L ' ) are magnifications of boxed regions in the corresponding figure panels . M Relative IF expression levels of integrin β1 , ERK1 , and RUNX2 during E60 , E90 , and E60 cultured under 3 kPa stress for two days and E60 cultured under 0 kPa stress for two days . Data information : Data represent the means ± SEM . n = 3 . Scale bars = 25 µm ( A ' , G ' ) and 50 µm ( other panels ) . Unpaired t - tests for M , * P < . 05 , * * P < . 01 , * * * P < . 001 ; NS , not significant .

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A-L IF of integrin β1, ERK1, and RUNX2 in miniature pig canine frontal sections at embryonic day 60 (E60), E90, and E60 cultured under 3 kPa stress for two days and E60 cultured under 0 kPa stress for two days; (A'-L') are magnifications of boxed regions in the corresponding figure panels. M Relative IF expression levels of integrin β1, ERK1, and RUNX2 during E60, E90, and E60 cultured under 3 kPa stress for two days and E60 cultured under 0 kPa stress for two days. Data information: Data represent the means ± SEM. n = 3. Scale bars = 25 µm (A', G') and 50 µm (other panels). Unpaired t-tests for M , *P < .05, **P < .01, ***P < .001; NS, not significant.


N Morphology of the human PC at weeks 18 - 19 ( H & E staining ) ; ( N ' ) is magnification of the boxed region in ( N ) . DC , deciduous canine ; PC , permanent canine ; IEE , inner enamel epithelium ; OEE , outer enamel epithelium . Mesenchyme between DC and PC is indicated with a green arrow .

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N Morphology of the human PC at weeks 18-19 (H&E staining); (N') is magnification of the boxed region in (N). DC, deciduous canine; PC, permanent canine; IEE, inner enamel epithelium; OEE, outer enamel epithelium. Mesenchyme between DC and PC is indicated with a green arrow.


O - Q ISH expression patterns of ITGB1 , ERK1 , and RUNX2 in human PCs . Green arrows indicate the mesenchyme between DC and PC .

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O-Q ISH expression patterns of ITGB1, ERK1, and RUNX2 in human PCs. Green arrows indicate the mesenchyme between DC and PC.


R IF of RUNX2 in human PC . Mesenchyme between DC and PC is indicated with a red arrow . Red arrow indicates the mesenchyme between DC and PC .

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R IF of RUNX2 in human PC. Mesenchyme between DC and PC is indicated with a red arrow. Red arrow indicates the mesenchyme between DC and PC.


S PC epithelium areas in E60 , E90 , and E60 cultured under 3 kPa stress for two days and E60 cultured under 0 kPa stress for two days . Data information : Data represent the means ± SEM . n = 3 . , * P < . 05 , * * P < . 01 , * * * P < . 001 ; NS , not significant .

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S PC epithelium areas in E60, E90, and E60 cultured under 3 kPa stress for two days and E60 cultured under 0 kPa stress for two days. Data information: Data represent the means ± SEM. n = 3. , *P < .05, **P < .01, ***P < .001; NS, not significant.


IF of RUNX2 and phospho - ERK1 / 2 ( p - ERK1 / 2 ) upon force loading with 0 or 1 . 0 g / cm2 for 2 h or after force was removed and the cells cultured for an additional 1 , 2 , and 4 hours .

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IF of RUNX2 and phospho-ERK1/2 (p-ERK1/2) upon force loading with 0 or 1.0 g/cm2 for 2 h or after force was removed and the cells cultured for an additional 1, 2, and 4 hours.


V Relative IF expression levels of RUNX2 and p - ERK1 / 2 between groups Data information : Data represent the means ± SEM . n = 3 . One - way ANOVA ( Newman - Keuls test for post - hoc comparisons between two groups ) for V , * P < . 05 , * * P < . 01 , * * * P < . 001 ; NS , not significant .

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V Relative IF expression levels of RUNX2 and p-ERK1/2 between groups Data information: Data represent the means ± SEM. n = 3. One-way ANOVA (Newman-Keuls test for post-hoc comparisons between two groups) for V , *P < .05, **P < .01, ***P < .001; NS, not significant.


W Western blots of p - ERK1 / 2 and RUNX2 levels after 1 . 0 g / cm2 was applied for 2 h , with or without anti - integrin β1 antibody and ERK1 inhibitor ( U0126 ) . X Relative expression levels of p - ERK1 / 2 , RUNX2 , and Actin in ( W ) . Data information : Data represent the means ± SEM . n = 3 . Unpaired t - tests for M ; One - way ANOVA ( Newman - Keuls test for post - hoc comparisons between two groups ) for X , * P < . 05 , * * P < . 01 , * * * P < . 001 ; NS , not significant .

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W Western blots of p-ERK1/2 and RUNX2 levels after 1.0 g/cm2 was applied for 2 h, with or without anti-integrin β1 antibody and ERK1 inhibitor (U0126). X Relative expression levels of p-ERK1/2, RUNX2, and Actin in (W). Data information: Data represent the means ± SEM. n = 3. Unpaired t-tests for M; One-way ANOVA (Newman-Keuls test for post-hoc comparisons between two groups) for X, *P < .05, **P < .01, ***P < .001; NS, not significant.


A - C H & E staining of miniature pig canine frontal sections from embryonic day 60 ( E60 group ) ( A ) and after the mandible slices were infected with RUNX2 overexpression lentiviral vector with a Myc tag ( overexpression group ) ( B ) or overexpression control lentiviral vector with a Myc tag ( O - control group ) ( C ) ; ( A ' - C ' ) are magnifications of boxed regions in the corresponding figure panels . Data information : Data represent the means ± SEM . n = 3 for all experiments . Scale bars = 50

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A-C H&E staining of miniature pig canine frontal sections from embryonic day 60 (E60 group) (A) and after the mandible slices were infected with RUNX2 overexpression lentiviral vector with a Myc tag (overexpression group) (B) or overexpression control lentiviral vector with a Myc tag (O-control group) (C); (A'-C') are magnifications of boxed regions in the corresponding figure panels. Data information: Data represent the means ± SEM. n = 3 for all experiments. Scale bars = 50 µm


D - F Immunohistochemical ( IHC ) staining of Myc expression in mandible slices from the E60 , overexpression , and O - control groups . Data information : Data represent the means ± SEM . n = 3 for all experiments . Scale bars = 100 µm ( D - F )

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D-F Immunohistochemical (IHC) staining of Myc expression in mandible slices from the E60, overexpression, and O-control groups. Data information: Data represent the means ± SEM. n = 3 for all experiments. Scale bars = 100 µm (D-F)


G - I IF staining of RUNX2 expression ( yellow arrows ) in mandible slices of the E60 , overexpression , and O - control groups . Data information : Data represent the means ± SEM . n = 3 for all experiments . Scale bars = 50

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G-I IF staining of RUNX2 expression (yellow arrows) in mandible slices of the E60, overexpression, and O-control groups. Data information: Data represent the means ± SEM. n = 3 for all experiments. Scale bars = 50 µm


J , K Relative expression levels of Myc and RUNX2 in the E60 , O - control , and overexpression groups . Data information : Data represent the means ± SEM . n = 3 for all experiments . One - way ANOVA ( Newman - Keuls test for post - hoc comparisons between two groups ) for J , K , * P < . 05 , * * P < . 01 , * * * P < . 001 .

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J, K Relative expression levels of Myc and RUNX2 in the E60, O-control, and overexpression groups. Data information: Data represent the means ± SEM. n = 3 for all experiments. One-way ANOVA (Newman-Keuls test for post-hoc comparisons between two groups) for J, K , *P < .05, **P < .01, ***P < .001.


L , M IF staining of RUNX2 expression after infecting mandible slices with scrambled shRNA lentiviral vector ( knockdown control , K - control ) ( L ) or RUNX2 shRNA lentiviral vector ( knockdown ) ( M ) ; ( L ' - M ' ) are magnifications of boxed regions in the corresponding figure panels . Data information : Data represent the means ± SEM . n = 3 for all experiments . Scale bars = 50 µm ( other panels ) .

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L, M IF staining of RUNX2 expression after infecting mandible slices with scrambled shRNA lentiviral vector (knockdown control, K-control) (L) or RUNX2 shRNA lentiviral vector (knockdown) (M); (L'-M') are magnifications of boxed regions in the corresponding figure panels. Data information: Data represent the means ± SEM. n = 3 for all experiments. Scale bars = 50 µm (other panels).


N Relative RUNX2 expression levels between K - control and knockdown groups . Data information : Data represent the means ± SEM . n = 3 for all experiments . Unpaired t - tests for N , * P < . 05 , * * P < . 01 , * * * P < . 001 .

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N Relative RUNX2 expression levels between K-control and knockdown groups. Data information: Data represent the means ± SEM. n = 3 for all experiments. Unpaired t-tests for N , *P < .05, **P < .01, ***P < .001.


O - Q H & E staining of mandible slice sections from the K - control , knockdown + force ( 3 kPa ) , and K - control + force ( 3 kPa ) groups . ( O ' - Q ' ) are magnifications of boxed regions in the corresponding figure panels . Data information : Data represent the means ± SEM . n = 3 for all experiments . Scale bars = 50

O-Q H&E staining of mandible slice sections from the K-control, knockdown + force (3 kPa), and K-control + force (3 kPa) groups. (O'-Q') are magnifications of boxed regions in the corresponding figure panels. Data information: Data represent the means ± SEM. n = 3 for all experiments. Scale bars = 50 µm


R - T IF staining of RUNX2 expression ( yellow arrows ) of mandible slice sections from the K - control , knockdown + force ( 3 kPa ) , and K - control + force ( 3 kPa ) groups . ( R ' - T ' ) are magnifications of boxed regions in the corresponding figure panels . Data information : Data represent the means ± SEM . n = 3 for all experiments . Scale bars = 25 µm ( R ' - T ' )

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R-T IF staining of RUNX2 expression (yellow arrows) of mandible slice sections from the K-control, knockdown + force (3 kPa), and K-control + force (3 kPa) groups. (R'-T') are magnifications of boxed regions in the corresponding figure panels. Data information: Data represent the means ± SEM. n = 3 for all experiments. Scale bars = 25 µm (R'-T')


U Relative RUNX2 expression levels among the three groups Data information : Data represent the means ± SEM . n = 3 for all experiments . One - way ANOVA ( Newman - Keuls test for post - hoc comparisons between two groups ) for U , * P < . 05 , * * P < . 01 , * * * P < . 001 .

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U Relative RUNX2 expression levels among the three groups Data information: Data represent the means ± SEM. n = 3 for all experiments. One-way ANOVA (Newman-Keuls test for post-hoc comparisons between two groups) for U, *P < .05, **P < .01, ***P < .001.


V PC epithelium areas in the overexpression , O - control , knockdown , knockdown + force , K - control , and K - control + force groups . Data information : Data represent the means ± SEM . n = 3 for all experiments . Unpaired t - tests for V ; , * P < . 05 , * * P < . 01 , * * * P < . 001 .

V PC epithelium areas in the overexpression, O-control, knockdown, knockdown + force, K-control, and K-control + force groups. Data information: Data represent the means ± SEM. n = 3 for all experiments. Unpaired t-tests for V; , *P < .05, **P < .01, ***P < .001.


A , B ISH of miniature pig canine frontal sections showing expression patterns of RUNX2 at embryonic day 60 ( E60 ) and E90 . ( A ' , B ' ) are magnifications of boxed regions in the corresponding figure panels . Data information : Scale bars = 50 µm .

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A, B ISH of miniature pig canine frontal sections showing expression patterns of RUNX2 at embryonic day 60 (E60) and E90. (A', B') are magnifications of boxed regions in the corresponding figure panels. Data information: Scale bars = 50 µm.


C Relative ISH expression levels of RUNX2 in epithelium ( Epi . ) and mesenchyme ( Mes . ) at E60 and E90 . Data information Data represent the means ± SEM . n = 3 for all experiments . Unpaired t - tests for C , * P < . 05 , * * P < . 01 , * * * P < . 001 .

C Relative ISH expression levels of RUNX2 in epithelium (Epi.) and mesenchyme (Mes.) at E60 and E90. Data information Data represent the means ± SEM. n = 3 for all experiments. Unpaired t-tests for C , *P < .05, **P < .01, ***P < .001.


D , E ISH of miniature pig canine frontal sections showing expression patterns of β - catenin at E60 and E90 . ( D ' , E ' ) are magnifications of boxed regions in the corresponding figure panels . Data information : Scale bars = 50 µm .

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D, E ISH of miniature pig canine frontal sections showing expression patterns of β-catenin at E60 and E90. (D', E') are magnifications of boxed regions in the corresponding figure panels. Data information: Scale bars = 50 µm.


F Relative ISH expression levels of β - catenin in epithelium ( Epi . ) and mesenchyme ( Mes . ) at E60 and E90 . Data information : Data represent the means ± SEM . n = 3 for all experiments . Unpaired t - tests , * P < . 05 , * * P < . 01 , * * * P < . 001 .

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F Relative ISH expression levels of β-catenin in epithelium (Epi.) and mesenchyme (Mes.) at E60 and E90. Data information: Data represent the means ± SEM. n = 3 for all experiments. Unpaired t-tests , *P < .05, **P < .01, ***P < .001.


G , H IF of Lef1 at E60 and E90 . ( G , H ' ) are magnifications of boxed regions in the corresponding figure panels . Data information : Scale bars = 50 µm .

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G, H IF of Lef1 at E60 and E90. (G, H') are magnifications of boxed regions in the corresponding figure panels. Data information: Scale bars = 50 µm.


I Relative IF expression levels of Lef1 in epithelium ( Epi . ) and mesenchyme ( Mes . ) at E60 and E90 . Data represent the means ± SEM . n = 3 for all experiments . Unpaired t - tests , * P < . 05 , * * P < . 01 , * * * P < . 001 .

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I Relative IF expression levels of Lef1 in epithelium (Epi.) and mesenchyme (Mes.) at E60 and E90. Data represent the means ± SEM. n = 3 for all experiments. Unpaired t-tests , *P < .05, **P < .01, ***P < .001.