( C ) Lysosomes with high ( CMA + ) and low ( CMA − ) ability for CMA isolated from livers of fed or 48 h starved rats were processed as in ( A ) . Top left : Levels of two CMA components ( LAMP ‐ 2A and hsc70 ) and of an unrelated lysosomalmembrane protein ( LAMP ‐ 1 ) in each of the four groups of lysosomes . Bottom left : Percentage of total lysosomal LAMP ‐ 2A present in each fraction , determined by densitometric quantification of the immunoblots .

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(C) Lysosomes with high (CMA+) and low (CMA−) ability for CMA isolated from livers of fed or 48 h starved rats were processed as in (A). Top left: Levels of two CMA components (LAMP‐2A and hsc70) and of an unrelated lysosomalmembrane protein (LAMP‐1) in each of the four groups of lysosomes. Bottom left: Percentage of total lysosomal LAMP‐2A present in each fraction, determined by densitometric quantification of the immunoblots.


( A ) Degradation of long ‐ lived proteins in mouse fibroblasts ( NIH3T3 cells ) treated or not with 25 mM MBCD . Where indicated , 15 mM NH4Cl / 100 μM leupeptin ( NH4Cl + Leup ) were added into the medium . Values are the mean + s . e . of three experiments with triplicate samples .

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(A) Degradation of long‐lived proteins in mouse fibroblasts (NIH3T3 cells) treated or not with 25 mM MBCD. Where indicated, 15 mM NH4Cl/100 μM leupeptin (NH4Cl+Leup) were added into the medium. Values are the mean+s.e. of three experiments with triplicate samples.


( B ) Effect of MBCD on total rates of protein degradation , measured as in ( A ) , in wild ‐ type mouse fibroblasts and in fibroblasts stably RNA interfered for LAMP ‐ 2A ( LAMP ‐ 2A ( − ) ) . The ratio of degradation in MBCD ‐ treated and untreated cells is shown for cells maintained with or without 10 mM 3 ‐ methyladenine ( 3MA ) .

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(B) Effect of MBCD on total rates of protein degradation, measured as in (A), in wild‐type mouse fibroblasts and in fibroblasts stably RNA interfered for LAMP‐2A (LAMP‐2A(−)). The ratio of degradation in MBCD‐treated and untreated cells is shown for cells maintained with or without 10 mM 3‐methyladenine (3MA).


( C ) Proteolysis of [ 14C ] GAPDH or a pool of [ 3H ] labeled cytosolic proteins ( pool proteins ) by isolated rat liver lysosomes , treated or not with MBCD . Broken : lysosomes disrupted by a hypotonic shock before the incubation with the radiolabeled proteins . Values are expressed as percentage of the degradation by untreated lysosomes and are the mean + s . e . of three experiments with triplicate samples .

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(C) Proteolysis of [14C]GAPDH or a pool of [3H]labeled cytosolic proteins (pool proteins) by isolated rat liver lysosomes, treated or not with MBCD. Broken: lysosomes disrupted by a hypotonic shock before the incubation with the radiolabeled proteins. Values are expressed as percentage of the degradation by untreated lysosomes and are the mean+s.e. of three experiments with triplicate samples.


( D ) Lysosomal - mitochondrial fractions were isolated from mouse fibroblasts untreated or loaded with cholesterol ( 50 μM ) . Top : Distribution of LAMP ‐ 2A after Triton X ‐ 114 extraction and sucrose density gradient centrifugation . Bottom : Ability of intact or disrupted lysosomal - mitochondrial fractions to proteolize [ 14C ] GAPDH . Values are expressed as percentage of the degradation in fractions from untreated cells and are the mean ± s . e . of triplicate samples .

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(D) Lysosomal-mitochondrial fractions were isolated from mouse fibroblasts untreated or loaded with cholesterol (50 μM). Top: Distribution of LAMP‐2A after Triton X‐114 extraction and sucrose density gradient centrifugation. Bottom: Ability of intact or disrupted lysosomal-mitochondrial fractions to proteolize [14C]GAPDH. Values are expressed as percentage of the degradation in fractions from untreated cells and are the mean±s.e. of triplicate samples.


( A ) Lysosomes from mouse fibroblasts transfected with wild ‐ type hLAMP ‐ 2A and maintained in the presence ( Serum + ) or absence ( serum − ) of serum were collected by centrifugation and subjected to Triton X ‐ 114 extraction , sucrose density gradient centrifugation and immunobloting for human ( hL2A ) or mouse ( mL2A ) LAMP ‐ 2A . Lane 1 : homogenate .

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(A) Lysosomes from mouse fibroblasts transfected with wild‐type hLAMP‐2A and maintained in the presence (Serum+) or absence (serum−) of serum were collected by centrifugation and subjected to Triton X‐114 extraction, sucrose density gradient centrifugation and immunobloting for human (hL2A) or mouse (mL2A) LAMP‐2A. Lane 1: homogenate.


( B ) Amino - acid sequence of the transmembrane and cytosolic region of hLAMP - 2A and the mutations performed . Replaced amino acids are shadowed .

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(B) Amino-acid sequence of the transmembrane and cytosolic region of hLAMP-2A and the mutations performed. Replaced amino acids are shadowed.


( C ) Lysosomes from mouse fibroblasts transfected with the indicated forms of hLAMP ‐ 2A and maintained in the presence of serum were processed as in ( A ) .

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(C) Lysosomes from mouse fibroblasts transfected with the indicated forms of hLAMP‐2A and maintained in the presence of serum were processed as in (A).


( D ) The percentage of total lysosomal hLAMP ‐ 2A present in each fraction was determined by densitometric quantification of immunoblots as the ones shown here . Values , corrected for the amount of mLAMP ‐ 2A in each fraction , are mean + s . e . of 3 - 4 experiments .

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(D) The percentage of total lysosomal hLAMP‐2A present in each fraction was determined by densitometric quantification of immunoblots as the ones shown here. Values, corrected for the amount of mLAMP‐2A in each fraction, are mean+s.e. of 3-4 experiments.


( E ) Changes in hLAMP ‐ 2A distribution induced by serum removal . Values are expressed as times the values in cells maintained in the presence of serum and are the mean of two experiments . Changes in the three nonDR regions are shown together ( no DR ) .

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(E) Changes in hLAMP‐2A distribution induced by serum removal. Values are expressed as times the values in cells maintained in the presence of serum and are the mean of two experiments. Changes in the three nonDR regions are shown together (no DR).


( F ) Proteolysis of a pool of 3 [ H ] labeled cytosolic proteins by intact ( left ) or disrupted ( right ) lysosomes isolated from untransfected mouse fibroblasts ( none ) or fibroblasts transfected with wild ‐ type ( WT ) or the indicated mutant forms of hLAMP ‐ 2A . Values are the mean + s . e . of two experiments with triplicate samples .

(F) Proteolysis of a pool of 3[H]labeled cytosolic proteins by intact (left) or disrupted (right) lysosomes isolated from untransfected mouse fibroblasts (none) or fibroblasts transfected with wild‐type (WT) or the indicated mutant forms of hLAMP‐2A. Values are the mean+s.e. of two experiments with triplicate samples.


( G ) Part of the lysosomes from ( F ) was treated with MBCD before the incubation with the radiolabeled proteins . Values are expressed as percentage of degradation by untreated lysosomes and are the mean + s . e . of two experiments with triplicate samples .

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(G) Part of the lysosomes from (F) was treated with MBCD before the incubation with the radiolabeled proteins. Values are expressed as percentage of degradation by untreated lysosomes and are the mean+s.e. of two experiments with triplicate samples.


( A ) The Triton X ‐ 114 ‐ resistant and soluble regions of the lysosomalmembrane were subjected to centrifugation through a continuous ( 10 - 80 % ) sucrose density gradient , SDS - PAGE and immunoblot for LAMP ‐ 2A . The molecular weight range of each fraction is as follows : fraction 1 ( 100 kDa ) ; fraction 2 ( 100 - 200 kDa ) ; fraction 3 ( 200 - 400 kDa ) ; fraction 4 ( 400 - 600 kDa ) ; fraction 5 ( > 600 kDa ) .

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(A) The Triton X‐114‐resistant and soluble regions of the lysosomalmembrane were subjected to centrifugation through a continuous (10-80%) sucrose density gradient, SDS-PAGE and immunoblot for LAMP‐2A. The molecular weight range of each fraction is as follows: fraction 1 (100 kDa); fraction 2 (100-200 kDa); fraction 3 (200-400 kDa); fraction 4 (400-600 kDa); fraction 5 (>600 kDa).


( B ) Fed rat liver lysosomes were incubated with 1 mM CaCl2 ( + Ca2 + ) in an isotonic buffer in the absence or presence of a cocktail of protease inhibitors ( PI ) . Samples were subjected to Triton X ‐ 114 extraction , sucrose density gradients centrifugation and LAMP ‐ 2A immunoblot .

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(B) Fed rat liver lysosomes were incubated with 1 mM CaCl2 (+Ca2+) in an isotonic buffer in the absence or presence of a cocktail of protease inhibitors (PI). Samples were subjected to Triton X‐114 extraction, sucrose density gradients centrifugation and LAMP‐2A immunoblot.


( C ) Fed rat liver lysosomes treated or not with 25 mM MBCD were incubated in an isotonic buffer for 30 min and then subjected to SDS - PAGE and immunobloted with antibodies against the cytosolic ( Top ; L ‐ 2A ) and luminal ( Bottom ; L ‐ 2 ) regions of LAMP ‐ 2A . Arrow : LAMP ‐ 2A proteolytic fragment ( Cuervo and Dice , 2000a ) .

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(C) Fed rat liver lysosomes treated or not with 25 mM MBCD were incubated in an isotonic buffer for 30 min and then subjected to SDS-PAGE and immunobloted with antibodies against the cytosolic (Top; L‐2A) and luminal (Bottom; L‐2) regions of LAMP‐2A. Arrow: LAMP‐2A proteolytic fragment (Cuervo and Dice, 2000a).


( D , E ) Rat liver lysosomes incubated or not with 1 mM CaCl2 ( + Ca2 + ) were subjected to Triton X ‐ 114 extraction and sucrose density gradients centrifugation . Levels of cathepsin A ( D ) and carboxypeptidase A activity ( E ) were measured in each fraction by immunoblot or using a standardized colorimetric assay , respectively . Values in ( E ) are expressed as times the activity in untreated membranes and are mean + s . e . of two experiments .

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(D, E) Rat liver lysosomes incubated or not with 1 mM CaCl2 (+Ca2+) were subjected to Triton X‐114 extraction and sucrose density gradients centrifugation. Levels of cathepsin A (D) and carboxypeptidase A activity (E) were measured in each fraction by immunoblot or using a standardized colorimetric assay, respectively. Values in (E) are expressed as times the activity in untreated membranes and are mean+s.e. of two experiments.


( B ) Dot plot indicating fraction of tf - Reporters delivered to the lysosome by ATG7 - independent mechanisms . The median red : green ratio of each population was used to calculate fractional delivery according to the following formula : ( ratiosgATG7 - ratiosgATG9A ) / ( ratioNegativeCtl1 - ratiosgATG9A ) , with the assumption that sgATG9A yields a true autophagy - null phenotype . Mean values for each reporter are indicated by a black bar . n = 3 for each reporter .

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(B) Dot plot indicating fraction of tf-Reporters delivered to the lysosome by ATG7-independent mechanisms. The median red:green ratio of each population was used to calculate fractional delivery according to the following formula: (ratiosgATG7-ratiosgATG9A)/(ratioNegativeCtl1-ratiosgATG9A), with the assumption that sgATG9A yields a true autophagy-null phenotype. Mean values for each reporter are indicated by a black bar. n=3 for each reporter.


( C ) Wild - type ( WT ) K562 cells and indicated deletion isolates were treated with 100 nM Bafilomycin A1 ( BafA1 ) or DMSO for 18h and analyzed by flow cytometry for red : green ratio of tf - NBR1 . Median values for each sample are identified by a black line within each violin . The red dotted line across all samples corresponds to the red : green ratio in wild - type cells . The red solid line across all samples corresponds to the ratio observed under maximally inhibited conditions ( ATG9AKO cells ) . n = 10 , 000 cells .

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(C) Wild-type (WT) K562 cells and indicated deletion isolates were treated with 100 nM Bafilomycin A1 (BafA1) or DMSO for 18h and analyzed by flow cytometry for red:green ratio of tf-NBR1. Median values for each sample are identified by a black line within each violin. The red dotted line across all samples corresponds to the red:green ratio in wild-type cells. The red solid line across all samples corresponds to the ratio observed under maximally inhibited conditions (ATG9AKO cells). n = 10,000 cells.


( D ) Representative confocal micrographs ( as maximum intensity projections ) of wild - type K562 cells and indicated deletion isolates expressing tf - NBR1 . Selected regions ( white box ) of micrographs are shown as single and merged channels from fluorescence microscopy . Red fluorescent protein ( RFP ) , magenta ; Green fluorescent protein ( GFP ) , green ; Hoechst , blue . Scale bars : large panels , 5 µm ; small panels , 1 µm . All images scaled equally .

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(D) Representative confocal micrographs (as maximum intensity projections) of wild-type K562 cells and indicated deletion isolates expressing tf-NBR1. Selected regions (white box) of micrographs are shown as single and merged channels from fluorescence microscopy. Red fluorescent protein (RFP), magenta; Green fluorescent protein (GFP), green; Hoechst, blue. Scale bars: large panels, 5 µm; small panels, 1 µm. All images scaled equally.


( E ) Correlative light and electron microscopy ( CLEM ) of K562 ATG7KO cells expressing tf - NBR1 under basal conditions . Analysis workflow is indicated by green arrows . White arrows indicate representative structures of interest . White boxes demarcate zoomed areas in subsequent images . NBR1 , green ; Hoechst , blue . Scale bar ( small images ) , 2 . 5 µm . Scale bar ( large images ) , 250 nm .

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(E) Correlative light and electron microscopy (CLEM) of K562 ATG7KO cells expressing tf-NBR1 under basal conditions. Analysis workflow is indicated by green arrows. White arrows indicate representative structures of interest. White boxes demarcate zoomed areas in subsequent images. NBR1, green; Hoechst, blue. Scale bar (small images), 2.5 µm. Scale bar (large images), 250 nm.


( A ) Box plot of average beta scores ( similar to log - fold change ) for tf - NBR1 flux in indicated deletion cell lines . Median values ( central band ) , inner quartiles ( boxed regions ) , 1 . 5 interquartile ranges ( whiskers ) , and outliers ( dots ) are indicated . Representative hits across all three genetic backgrounds are indicated in blue .

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(A) Box plot of average beta scores (similar to log-fold change) for tf-NBR1 flux in indicated deletion cell lines. Median values (central band), inner quartiles (boxed regions), 1.5 interquartile ranges (whiskers), and outliers (dots) are indicated. Representative hits across all three genetic backgrounds are indicated in blue.


( B ) Gene correlation plot of average beta scores for lipidation dependent autophagy ( tf - NDP52 , from ( Shoemaker et al , 2019 ) ) and lipidation independent autophagy ( average of tf - NBR1 from ATG7KO , ATG10 KO and ATG3 KO cells Highlighted in red are genes with a beta score > 0 . 5 across all three lipidation - deficient cell lines . In black are lipidation components . Dashed lines , top 1 % of beta scores .

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(B) Gene correlation plot of average beta scores for lipidation dependent autophagy (tf-NDP52, from (Shoemaker et al, 2019)) and lipidation independent autophagy (average of tf-NBR1 from ATG7KO, ATG10 KO and ATG3 KO cells Highlighted in red are genes with a beta score > 0.5 across all three lipidation-deficient cell lines. In black are lipidation components. Dashed lines, top 1% of beta scores.


( C ) Wild - type K562 cells and indicated deletion isolates expressing Cas9 and tf - NDP52 or tf - NBR1 were transduced with individual sgRNAs for indicated genes . The median red : green ratio of each population was used to calculate the fold - repression according to the following formula : ( ratiosgGene - ratiosgATG9A ) / ( ratioNegativeCtl - ratiosgATG9A ) , with the assumption that sgATG9A yields a true autophagy - null phenotype . Deeper shades of red indicate stronger suppressor phenotypes . Genes were clustered on the basis of their patterns of genetic interactions .

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(C) Wild-type K562 cells and indicated deletion isolates expressing Cas9 and tf-NDP52 or tf-NBR1 were transduced with individual sgRNAs for indicated genes. The median red:green ratio of each population was used to calculate the fold-repression according to the following formula: (ratiosgGene-ratiosgATG9A)/(ratioNegativeCtl-ratiosgATG9A), with the assumption that sgATG9A yields a true autophagy-null phenotype. Deeper shades of red indicate stronger suppressor phenotypes. Genes were clustered on the basis of their patterns of genetic interactions.


( A ) Wild - type ( WT ) K562 cells and indicated deletion isolates were treated with 100nM Bafilomycin A1 ( BafA1 ) or DMSO for 18h and analyzed by flow cytometry for red : green ratio of tf - NBR1 . Median values for each sample are identified by a black line within each violin . The red dotted line across all samples corresponds to the red : green ratio in wild - type cells . The red solid line across all samples corresponds to the ratio observed under maximally inhibited conditions ( ATG9AKO cells ) . ( n > 10 , 000 cells ) .

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(A) Wild-type (WT) K562 cells and indicated deletion isolates were treated with 100nM Bafilomycin A1 (BafA1) or DMSO for 18h and analyzed by flow cytometry for red:green ratio of tf-NBR1. Median values for each sample are identified by a black line within each violin. The red dotted line across all samples corresponds to the red:green ratio in wild-type cells. The red solid line across all samples corresponds to the ratio observed under maximally inhibited conditions (ATG9AKO cells). (n > 10,000 cells).


( B ) Representative confocal micrograph ( as maximum intensity projections ) of ATG7KO / TAX1BP1KO K562 cells expressing tf - NBR1 . Selected region ( white box ) of micrograph is shown as single and merged channels from fluorescence microscopy . RFP , magenta ; GFP , green ; merged , white ; Hoechst , blue . Scale bars : large panel , 5 µm ; small panels , 1 µm .

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(B) Representative confocal micrograph (as maximum intensity projections) of ATG7KO/TAX1BP1KO K562 cells expressing tf-NBR1. Selected region (white box) of micrograph is shown as single and merged channels from fluorescence microscopy. RFP, magenta; GFP, green; merged, white; Hoechst, blue. Scale bars: large panel, 5 µm; small panels, 1 µm.


( C ) Correlative light and electron microscopy ( CLEM ) of K562 ATG7KO / TAX1BP1KO cells expressing tf - NBR1 under basal conditions . Analysis workflow is indicated by green arrows . White arrow indicates a structure of interest . White box demarcates zoomed area in bottom images . NBR1 , green ; Hoechst , blue .

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(C) Correlative light and electron microscopy (CLEM) of K562 ATG7KO/TAX1BP1KO cells expressing tf-NBR1 under basal conditions. Analysis workflow is indicated by green arrows. White arrow indicates a structure of interest. White box demarcates zoomed area in bottom images. NBR1, green; Hoechst, blue.


( A ) Wild - type and indicated K562 knock - out cells expressing tf - NBR1 were transfected with TagBFP or TagBFP - RavZ , and BFP - positive cells were analyzed for red : green ratio by flow cytometry . The median red : green ratio of each sample was used to calculate flux relative to WT + TagBFP . Bar graphs represent mean + / - SD from three independent experiments . n > 7 , 500 cells per sample .

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(A) Wild-type and indicated K562 knock-out cells expressing tf-NBR1 were transfected with TagBFP or TagBFP-RavZ, and BFP-positive cells were analyzed for red:green ratio by flow cytometry. The median red:green ratio of each sample was used to calculate flux relative to WT+TagBFP. Bar graphs represent mean +/- SD from three independent experiments. n > 7,500 cells per sample.


( B ) K562 - derived extracts prepared from wild - type ( WT ) and clonal deletion isolates were resolved by SDS - PAGE followed by immunoblotting ( IB ) with indicated antibodies . All samples were normalized by total protein using a BCA assay prior to loading . Bar graphs represent mean + / - SD from independent experiments ( n = 4 { NBR1 , NDP52 } ; n = 3 { SQSTM1 } ) . p values for WT vs ATG9AKO was determined using a one sample test ( theoretical mean = 1 ) with Bonferroni correction .

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(B) K562-derived extracts prepared from wild-type (WT) and clonal deletion isolates were resolved by SDS-PAGE followed by immunoblotting (IB) with indicated antibodies. All samples were normalized by total protein using a BCA assay prior to loading. Bar graphs represent mean +/- SD from independent experiments (n=4 {NBR1, NDP52}; n=3 {SQSTM1}). p values for WT vs ATG9AKO was determined using a one sample test (theoretical mean = 1) with Bonferroni correction.


( D ) Wild - type and indicated K562 knock - out cells were treated The corresponding cell extracts were resolved by SDS - PAGE and analyzed by immunoblotting ( IB ) with indicated antibodies . Shown are representative images from one experiment with mean intensity levels from ( E ) indicated below each image ( NBR1 : n = 4 ; TAX1BP1 : n = 3 , NDP52 : n = 4 , tubulin : n = 4 ) . ( E ) Quantitation of protease - protection data from experiments in ( D ) . Bar graphs show the mean + / - SD of each sample from independent experiments . Trypsin - treated samples were compared using a one - way ANOVA ( NBR1 : n = 4 , p = 0 . 0001 ; TAX1BP1 : n = 3 , p = 0 . 0098 ; NDP52 : n = 4 , p < 0 . 0001 ) with Tukey HSD post - test . * * , p < 0 . 01 ; * , p < 0 . 05 ; ns , not

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(D) Wild-type and indicated K562 knock-out cells were treated The corresponding cell extracts were resolved by SDS-PAGE and analyzed by immunoblotting (IB) with indicated antibodies. Shown are representative images from one experiment with mean intensity levels from (E) indicated below each image (NBR1: n = 4; TAX1BP1: n = 3, NDP52: n = 4, tubulin: n = 4). (E) Quantitation of protease-protection data from experiments in (D). Bar graphs show the mean +/- SD of each sample from independent experiments. Trypsin-treated samples were compared using a one-way ANOVA (NBR1: n = 4, p = 0.0001; TAX1BP1: n = 3, p = 0.0098; NDP52: n = 4, p < 0.0001) with Tukey HSD post-test. **, p < 0.01; *, p < 0.05; ns, not significant


( A ) Extracts derived from wild - type and deletion K562 cells expressing tf - NBR1 were normalized for total protein by BCA . NBR1 was immunoprecipitated using GFP - Trap dynabeads . Input and eluate were resolved by SDS - PAGE followed by immunoblotting ( IB ) with indicated antibodies .

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(A) Extracts derived from wild-type and deletion K562 cells expressing tf-NBR1 were normalized for total protein by BCA. NBR1 was immunoprecipitated using GFP-Trap dynabeads. Input and eluate were resolved by SDS-PAGE followed by immunoblotting (IB) with indicated antibodies.


( B ) Representative confocal micrographs ( as maximum intensity projections ) of wild - type K562 and ATG9AKO cells expressing tf - NBR1 . Selected regions ( white box ) of micrographs are shown as single and merged channels from fluorescence and immunofluorescence microscopy against indicated proteins . In conditions where puncta were not observed , representative cytoplasmic regions were selected to showcase diffuseness of signal . TAX1BP1 , SQSTM1 , or NDP52 , magenta ; NBR1 , green ; Hoechst , blue .

(B) Representative confocal micrographs (as maximum intensity projections) of wild-type K562 and ATG9AKO cells expressing tf-NBR1. Selected regions (white box) of micrographs are shown as single and merged channels from fluorescence and immunofluorescence microscopy against indicated proteins. In conditions where puncta were not observed, representative cytoplasmic regions were selected to showcase diffuseness of signal. TAX1BP1, SQSTM1, or NDP52, magenta; NBR1, green; Hoechst, blue.


( C ) Shown are representative confocal micrographs ( as maximum intensity projections ) of K562 cells expressing tf - NDP52 ( or tf - TAX1BP1 ) and transduced with sgRNAs targeting ATG9A ( sgATG9A ) or a negative control ( sgControl ) . Selected regions ( white box ) of micrographs are shown as single and merged channels from fluorescence microscopy . RFP , magenta ; GFP , green ; Hoechst , blue .

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(C) Shown are representative confocal micrographs (as maximum intensity projections) of K562 cells expressing tf-NDP52 (or tf-TAX1BP1) and transduced with sgRNAs targeting ATG9A (sgATG9A) or a negative control (sgControl). Selected regions (white box) of micrographs are shown as single and merged channels from fluorescence microscopy. RFP, magenta; GFP, green; Hoechst, blue.


( C ) HEK293T cells were transfected with either myc - TAX1BP1WT or myc - NBR1WT and indicated TagBFP - V5 - TAX1BP1 variants . Extracts derived from transfected cells were immunoprecipitated ( IP ) with anti - TagBFP magnetic beads . Input and eluates were resolved by SDS - PAGE followed by immunoblotting ( IB ) with indicated antibodies .

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(C) HEK293T cells were transfected with either myc-TAX1BP1WT or myc-NBR1WT and indicated TagBFP-V5-TAX1BP1 variants. Extracts derived from transfected cells were immunoprecipitated (IP) with anti-TagBFP magnetic beads. Input and eluates were resolved by SDS-PAGE followed by immunoblotting (IB) with indicated antibodies.


( D ) HEK293T cells were transfected with myc - NBR1WT and indicated TagBFP - V5 - TAX1BP1 variants . Extracts derived from transfected cells were immunoprecipitated ( IP ) with anti - TagBFP magnetic beads . Input and eluates were resolved by SDS - PAGE followed by immunoblotting ( IB ) with indicated antibodies .

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(D) HEK293T cells were transfected with myc-NBR1WT and indicated TagBFP-V5-TAX1BP1 variants. Extracts derived from transfected cells were immunoprecipitated (IP) with anti-TagBFP magnetic beads. Input and eluates were resolved by SDS-PAGE followed by immunoblotting (IB) with indicated antibodies.


( A ) Representative confocal micrographs ( as maximum intensity projections ) from wild - type and deletion K562 cells expressing tf - NBR1 . Selected regions ( white box ) of micrographs are shown as single and merged channels from fluorescence and immunofluorescence microscopy against indicated proteins . P - TBK1 , magenta ; NBR1 , green ; Hoechst , blue . ( B ) Representative confocal micrographs ( as maximum intensity projections ) from wild - type and deletion K562 cells expressing tf - NBR1 . Selected regions ( white box ) of micrographs are shown as single and merged channels from fluorescence and immunofluorescence microscopy against indicated proteins . FIP200 , magenta ; NBR1 , green ; Hoechst , blue .

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(A) Representative confocal micrographs (as maximum intensity projections) from wild-type and deletion K562 cells expressing tf-NBR1. Selected regions (white box) of micrographs are shown as single and merged channels from fluorescence and immunofluorescence microscopy against indicated proteins. P-TBK1, magenta; NBR1, green; Hoechst, blue. (B) Representative confocal micrographs (as maximum intensity projections) from wild-type and deletion K562 cells expressing tf-NBR1. Selected regions (white box) of micrographs are shown as single and merged channels from fluorescence and immunofluorescence microscopy against indicated proteins. FIP200, magenta; NBR1, green; Hoechst, blue.


( C ) Representative confocal micrographs ( as maximum intensity projections ) from wild - type , TAX1BP1KO , and ATG7KO / TAX1BP1KO K562 cells expressing tf - NBR1 and transduced with indicated sgRNA . Selected regions ( white box ) of micrographs are shown as single and merged channels from fluorescence and immunofluorescence microscopy against indicated proteins . FIP200 , magenta ; NBR1 , green ; Hoechst , blue . ( D ) Quantitation of colocalization between NBR1 with FIP200 in wild - type , TAX1BP1KO , and ATG7KO / TAX1BP1KO cells imaged in ( C ) Bar graphs represent mean + / - SD for three independently generated deletion cell lines ( dots ) . sgATG9A samples were compared using a one - way ANOVA ( p < 0 . 0001 ) with Tukey HSD post - test . * * * , p < 0 . 001 . n > 200 NBR1 puncta for each biological replicate . ( E ) Quantitation of FIP200 intensity at NBR1 puncta in wild - type , TAX1BP1KO , and ATG7KO / TAX1BP1KO cells imaged in ( C ) . Bar graphs represent mean + / - SD for three independently generated deletion cell lines ( dots ) . n > 80 FIP200 - positive puncta for each biological replicate . Samples were compared using a one - way ANOVA ( p < 0 . 0001 ) with Tukey HSD post - test . * * * , p < 0 . 001 . a . u . , arbitrary intensity units .

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(C) Representative confocal micrographs (as maximum intensity projections) from wild-type, TAX1BP1KO, and ATG7KO/TAX1BP1KO K562 cells expressing tf-NBR1 and transduced with indicated sgRNA. Selected regions (white box) of micrographs are shown as single and merged channels from fluorescence and immunofluorescence microscopy against indicated proteins. FIP200, magenta; NBR1, green; Hoechst, blue. (D) Quantitation of colocalization between NBR1 with FIP200 in wild-type, TAX1BP1KO, and ATG7KO/TAX1BP1KO cells imaged in (C) Bar graphs represent mean +/- SD for three independently generated deletion cell lines (dots). sgATG9A samples were compared using a one-way ANOVA (p < 0.0001) with Tukey HSD post-test. ***, p < 0.001. n > 200 NBR1 puncta for each biological replicate. (E) Quantitation of FIP200 intensity at NBR1 puncta in wild-type, TAX1BP1KO, and ATG7KO/TAX1BP1KO cells imaged in (C). Bar graphs represent mean +/- SD for three independently generated deletion cell lines (dots). n > 80 FIP200-positive puncta for each biological replicate. Samples were compared using a one-way ANOVA (p < 0.0001) with Tukey HSD post-test. ***, p < 0.001. a.u., arbitrary intensity units.


( A ) Soft or stiff 4T1 or MCF - 7 cells were isolated by the microfluidic chip , and then seeded ( 500 cells ) in 90 Pa soft 3D fibrin gel for 3 days . The percentage of colonies formed was calculated and the colony size was recorded . Scale bar , 100 μm . Data information : Two - tailed Paired Student ' s t test The data represent mean ± SD . n = 3 independent experiments in ( A ) .

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(A) Soft or stiff 4T1 or MCF-7 cells were isolated by the microfluidic chip, and then seeded (500 cells) in 90 Pa soft 3D fibrin gel for 3 days. The percentage of colonies formed was calculated and the colony size was recorded. Scale bar, 100 μm. Data information: Two-tailed Paired Student's t test The data represent mean ± SD. n = 3 independent experiments in (A).


( F ) NSG mice were intravenously injected with 100 soft or stiff B16 or MP - 1 cells . Eight weeks later , the lung metastasis was analyzed by H & E staining . The metastasis index was defined as the percentage of total metastatic nodule area to the total lung area based on the calculation from 10 slides . Scale bar , 0 . 5 mm . n = 3 ( for B16 ) or 5 ( for MP - 1 ) mice with metastatic tumor . Data information : Two - tailed Paired Student ' s t test The data represent mean ± SD .

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(F) NSG mice were intravenously injected with 100 soft or stiff B16 or MP-1 cells. Eight weeks later, the lung metastasis was analyzed by H&E staining. The metastasis index was defined as the percentage of total metastatic nodule area to the total lung area based on the calculation from 10 slides. Scale bar, 0.5 mm. n = 3 (for B16) or 5 (for MP-1) mice with metastatic tumor. Data information: Two-tailed Paired Student's t test The data represent mean ± SD.


The 100 stiff ALDH + or soft ALDH - 4T1 cells were injected into the mammary fat pads of NSG mice . Eight weeks later , the lung sections were H & E stained . metastasis index was calculated ( D ) . Scale bar , 0 . 5 mm . n = 5 mice with metastatic tumor . Data information : two - tailed Paired Student ' s t test data represent mean ± SD .

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The 100 stiff ALDH+ or soft ALDH- 4T1 cells were injected into the mammary fat pads of NSG mice. Eight weeks later, the lung sections were H&E stained. metastasis index was calculated (D). Scale bar, 0.5 mm. n = 5 mice with metastatic tumor. Data information: two-tailed Paired Student's t test data represent mean ± SD.


( E ) Heatmap of stemness association genes expression determined by mRNA sequencing comparing stiff and soft MCF - 7 cells . n = 3 . The unit for the color scale was z - score of log2 expression data shown .

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(E) Heatmap of stemness association genes expression determined by mRNA sequencing comparing stiff and soft MCF-7 cells. n = 3. The unit for the color scale was z-score of log2 expression data shown.


( A ) The expression of BCL9L or β - catenin in stiff or soft 4T1 , MCF - 7 , B16 or MP - 1 cells was determined by western blot . ( B ) The expression of β - catenin and BCL9L from SGGFP , BCL9L - SGs - 4T1 , MCF - 7 , B16 or MP - 1 cells were determined by western blot . Data information n = 3 independent experiments in ( A and B ) .

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(A) The expression of BCL9L or β-catenin in stiff or soft 4T1, MCF-7, B16 or MP-1 cells was determined by western blot. (B) The expression of β-catenin and BCL9L from SGGFP, BCL9L-SGs-4T1, MCF-7, B16 or MP-1 cells were determined by western blot. Data information n = 3 independent experiments in (A and B).


( E , F ) The 100 soft SGGFP or BCL9L - SGs - 4T1 , B16 or MP - 1 cells were injected into the mammary fat pads ( 4T1 ) of BALB / c mice or tail vein ( B16 and MP - 1 ) of NSG mice . The tumor formation was recorded ( E ) . Six to eight weeks later , the lung sections were H & E stained . The metastatic micronodules in the lung were counted and the metastasis index was calculated ( F ) . Scale bar , 0 . 5 mm . n = 3 ( for B16 - F1 ) or 5 ( for MP - 1 ) mice with metastatic tumor . Data information : Bonferroni test The data represent mean ± SD .

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(E, F) The 100 soft SGGFP or BCL9L-SGs- 4T1, B16 or MP-1 cells were injected into the mammary fat pads (4T1) of BALB/c mice or tail vein (B16 and MP-1) of NSG mice. The tumor formation was recorded (E). Six to eight weeks later, the lung sections were H&E stained. The metastatic micronodules in the lung were counted and the metastasis index was calculated (F). Scale bar, 0.5 mm. n = 3 (for B16-F1) or 5 (for MP-1) mice with metastatic tumor. Data information: Bonferroni test The data represent mean ± SD.


( A ) The stiffness of primary breast ( HBC , n = 3 ) and colon ( HCC , n = 3 ) cancer cells screened from the outlet or backflow inlet was measured by AFM . n = 100 from each patient . Data information : Mann - Whitney test ( A ) The data represent mean ± SD .

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(A) The stiffness of primary breast (HBC, n = 3) and colon (HCC, n = 3) cancer cells screened from the outlet or backflow inlet was measured by AFM. n = 100 from each patient. Data information: Mann-Whitney test (A) The data represent mean ± SD.


( C ) The stiff and soft tumor cells were isolated from 3 patients with breast cancer and stained with anti - BCL9L antibody . The expression of BCL9L was observed under confocal microscope . Scale bar , 10 μ Data information : Paired Student ' s t test ( C ) The data represent mean ± SD .

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(C) The stiff and soft tumor cells were isolated from 3 patients with breast cancer and stained with anti-BCL9L antibody. The expression of BCL9L was observed under confocal microscope. Scale bar, 10 μ Data information: Paired Student's t test (C) The data represent mean ± SD.


( E ) The correlation between the expression of BCL9L from 18 melanoma patients and the grade of melanoma . Data information : Pearson ' s correlation test E ) The data represent mean ± SD .

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(E) The correlation between the expression of BCL9L from 18 melanoma patients and the grade of melanoma. Data information: Pearson's correlation test E) The data represent mean ± SD.


( F ) Overall survival compared to the BCL9L level in breast cancer ( BRCA , n = 536 ) or melanoma ( n = 376 ) patients . Data information : Log - rank survival analysis ( F ) . The data represent mean ± SD .

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(F) Overall survival compared to the BCL9L level in breast cancer (BRCA, n = 536) or melanoma (n = 376) patients. Data information: Log-rank survival analysis (F). The data represent mean ± SD.


Ribosome occupancy map at the LINC00665 locus . MCF10A were either mock - treated or treated with TGF - β and Ribosome profiling on three biological replicates were shown .

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Ribosome occupancy map at the LINC00665 locus. MCF10A were either mock-treated or treated with TGF-β and Ribosome profiling on three biological replicates were shown.


Relative levels of CIP2A - BP were determined by polysome profiling and qRT - PCR in MCF - 10A and TNBC cells either mock treated or treated with TGF - β . Relative levels of LINC00665 were determined by qRT - PCR in MCF - 10A and TNBC cells either mock treated or treated with TGF - β .

Relative levels of CIP2A-BP were determined by polysome profiling and qRT-PCR in MCF-10A and TNBC cells either mock treated or treated with TGF-β. Relative levels of LINC00665 were determined by qRT-PCR in MCF-10A and TNBC cells either mock treated or treated with TGF-β.


Upper : putative ORFs in LINC00665 . Lower : The ORFs were constructed into pcDNA3 . 1 vector and transfected to MCF - 10A cells for 24h . The ORFs - His fusion proteins were determined by western blot with anti - His antibody .

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Upper: putative ORFs in LINC00665. Lower: The ORFs were constructed into pcDNA3.1 vector and transfected to MCF-10A cells for 24h. The ORFs-His fusion proteins were determined by western blot with anti-His antibody.


The indicated constructs were transfected into MCF - 10A cells for 24h , then the GFP fluorescence was detected using fluorescence microscope ( F ) and fusion protein levels were determined by western blot with anti - GFP and anti - CIP2A - BP antibodies

The indicated constructs were transfected into MCF-10A cells for 24h, then the GFP fluorescence was detected using fluorescence microscope (F) and fusion protein levels were determined by western blot with anti-GFP and anti-CIP2A-BP antibodies respectively


Diagram of the His fusion constructs . The start codon ATG of the LINC00665 ORF is mutated to ATT . The indicated constructs were stably expressed in MDA - MB - 231 cells , micropeptide CIP2A - BP was immuno - stained using anti - His ( I ) and anti - CIP2A - BP antibodies ( K ) respectively ; CIP2A - BP - His fusion protein levels were determined by western blotting with anti - His and CIP2A - BP

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Diagram of the His fusion constructs. The start codon ATG of the LINC00665 ORF is mutated to ATT. The indicated constructs were stably expressed in MDA-MB-231 cells, micropeptide CIP2A-BP was immuno-stained using anti-His (I) and anti-CIP2A-BP antibodies (K) respectively; CIP2A-BP-His fusion protein levels were determined by western blotting with anti-His and CIP2A-BP antibodies


MCF - 10A cells incubated in the absence or presence of puromycin ( 200μM ) were fractionated on sucrose gradients , and distribution of CIP2A - BP transcripts was quantified by qPCR .

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MCF-10A cells incubated in the absence or presence of puromycin (200μM) were fractionated on sucrose gradients, and distribution of CIP2A-BP transcripts was quantified by qPCR.


Micropeptide CIP2A - BP was immune - stained with anti - CIP2A - BP antibody in MCF - 10A and MDA - MB - 231 cells either mock treated or treated with TGF - β .

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Micropeptide CIP2A-BP was immune-stained with anti-CIP2A-BP antibody in MCF-10A and MDA-MB-231 cells either mock treated or treated with TGF-β.


Micropeptide CIP2A - BP levels were determined by western blot with anti - CIP2A - BP antibody in MCF - 10A and TNBC cells either mock treated or treated with TGF - β .

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Micropeptide CIP2A-BP levels were determined by western blot with anti-CIP2A-BP antibody in MCF-10A and TNBC cells either mock treated or treated with TGF-β.


MCF - 10A cells were first transfected with anti - LINC00665 translation - blocking antisense oligo , then micropeptide CIP2A - BP was detected .

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MCF-10A cells were first transfected with anti-LINC00665 translation-blocking antisense oligo, then micropeptide CIP2A-BP was detected.


Micropeptide CIP2A - BP levels were detected by western blot with anti - CIP2A - BP antibody in the indicated non - metastatic TNBC tissues ( non - metastasis ) and metastatic TNBC tissues ( metastasis ) . Micropeptide CIP2A - BP expression in MCF - 10A cells was used as the positive control .

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Micropeptide CIP2A-BP levels were detected by western blot with anti-CIP2A-BP antibody in the indicated non-metastatic TNBC tissues (non-metastasis) and metastatic TNBC tissues (metastasis). Micropeptide CIP2A-BP expression in MCF-10A cells was used as the positive control.


Kaplan - Meier overall survival curves for TNBC patients with high or low micropeptide CIP2A - BP expression in Suzhou cohort ( discovery set , n = 112 ) and Guangzhou cohort ( validation set , n = 105 ) .

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Kaplan-Meier overall survival curves for TNBC patients with high or low micropeptide CIP2A-BP expression in Suzhou cohort (discovery set, n = 112) and Guangzhou cohort (validation set, n = 105).


Migration and invasion of TNBC cells transfected with LINC00665 ORF and control lentiviruses for 48h . Right panel is the quantification of TNBC cells that migrated through the membrane without the Matrigel ( upper ) and invaded through Matrigel - coated membrane ( lower ) .

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Migration and invasion of TNBC cells transfected with LINC00665 ORF and control lentiviruses for 48h. Right panel is the quantification of TNBC cells that migrated through the membrane without the Matrigel (upper) and invaded through Matrigel-coated membrane (lower).


Migration and invasion of wild - type and CIP2A - BP knockout TNBC cells and CIP2A - BP re - expressed Hs578TKO and MDA - MB - 231KO cells . Right panel is the quantification of cells that migrated through the membrane without the Matrigel ( upper ) and invaded through Matrigel - coated membrane ( lower ) .

Migration and invasion of wild-type and CIP2A-BP knockout TNBC cells and CIP2A-BP re-expressed Hs578TKO and MDA-MB-231KO cells. Right panel is the quantification of cells that migrated through the membrane without the Matrigel (upper) and invaded through Matrigel-coated membrane (lower).


Wound - healing assay on Hs578T and MDA - MB - 231 cells transfected with LINC00665 ORF expressing and control lentiviruses . The wound edges were photographed at the indicated time points after wounding . Right panel is the quantification of the relative wound healing area .

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Wound-healing assay on Hs578T and MDA-MB-231 cells transfected with LINC00665 ORF expressing and control lentiviruses. The wound edges were photographed at the indicated time points after wounding. Right panel is the quantification of the relative wound healing area.


Wild - type and CIP2A - BP knockout TNBC cells and CIP2A - BP re - expressed Hs578TKO and MDA - MB - 231KO were seeded on cell culture inserts for wound - healing assay . The wound edges were photographed at the indicated time points after wounding . Right panel is the quantification of the relative wound healing area .

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Wild-type and CIP2A-BP knockout TNBC cells and CIP2A-BP re-expressed Hs578TKO and MDA-MB-231KO were seeded on cell culture inserts for wound-healing assay. The wound edges were photographed at the indicated time points after wounding. Right panel is the quantification of the relative wound healing area.


Macroscopic and histological analysis of the lungs of nude mice injected with the indicated Hs578T ( G ) and MDA - MB - 231 cells ( H ) ( 1 . 0 × 105 - 106 cells / mouse ) via tail vein ; lung metastatic nodules were visualized 8 weeks post - transplantation ( 5 mice per group ) . Right panel is the quantification of pulmonary metastases .

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Macroscopic and histological analysis of the lungs of nude mice injected with the indicated Hs578T (G) and MDA-MB-231 cells (H) (1.0×105-106 cells/mouse) via tail vein; lung metastatic nodules were visualized 8 weeks post-transplantation (5 mice per group). Right panel is the quantification of pulmonary metastases.


Immunoblotting analysis of expression of the indicated proteins in TNBC cells cultured with or without TGF - β .

Immunoblotting analysis of expression of the indicated proteins in TNBC cells cultured with or without TGF-β.


Chromatin immunoprecipitation showing Smad4 occupancy at the 4E - BP1 locus in TNBC cells . Co - precipitated DNA was analyzed for amplicons A - E by qPCR . Values represent the enrichment of bound protein fractions relative to input .

Chromatin immunoprecipitation showing Smad4 occupancy at the 4E-BP1 locus in TNBC cells. Co-precipitated DNA was analyzed for amplicons A-E by qPCR. Values represent the enrichment of bound protein fractions relative to input.


Luciferase reporter assay was performed in Hs578T ( F ) and MDA - MB - 231 ( G ) cells following co - transfection with wildtype or mutant 4E - BP1 promoter fragment and Smad4 over - expressed , or knocked down and respective controls for 36h and treatment with TGF - β for 24h . The reporter constructs were expressing the luciferase gene under 4E - BP1 promoter segment or 4E - BP1 promoter deleted 0 to + 200 region .

Luciferase reporter assay was performed in Hs578T (F) and MDA-MB-231 (G) cells following co-transfection with wildtype or mutant 4E-BP1 promoter fragment and Smad4 over-expressed, or knocked down and respective controls for 36h and treatment with TGF-β for 24h. The reporter constructs were expressing the luciferase gene under 4E-BP1 promoter segment or 4E-BP1 promoter deleted 0 to +200 region.


Regulation of 4E - BP1 expression and binding to eIF4E by TGF - β . TNBC cells were treated with TGF - β and equal amounts of proteins were subjected to immunoprecipitation and / or examined by immunoblotting analysis .

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Regulation of 4E-BP1 expression and binding to eIF4E by TGF-β. TNBC cells were treated with TGF-β and equal amounts of proteins were subjected to immunoprecipitation and/or examined by immunoblotting analysis.


Immunoblotting analysis of expression of micropeptide CIP2A - BP and β - actin in TNBC cells cultured with or without TGF - β .

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Immunoblotting analysis of expression of micropeptide CIP2A-BP and β-actin in TNBC cells cultured with or without TGF-β.


Smad4 siRNA and corresponding control transfected TNBC cells were cultured with or without TGF - β . The indicated proteins were determined by Immunoblotting analysis .

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Smad4 siRNA and corresponding control transfected TNBC cells were cultured with or without TGF-β. The indicated proteins were determined by Immunoblotting analysis.


TNBC cells were pretreated with specific antagonist against Smad3 ( SIS3 , 3μM ) for 1h and then cultured with or without TGF - β . The indicated proteins were determined by immunoblotting analysis .

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TNBC cells were pretreated with specific antagonist against Smad3 (SIS3, 3μM) for 1h and then cultured with or without TGF-β. The indicated proteins were determined by immunoblotting analysis.


4E - BP1 siRNA and corresponding control transfected TNBC cells were cultured with or without TGF - β . The indicated proteins were determined by immunoblotting analysis .

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4E-BP1 siRNA and corresponding control transfected TNBC cells were cultured with or without TGF-β. The indicated proteins were determined by immunoblotting analysis.


LINC00665 ORF - His plasmid was transfected into MDA - MB - 231 cells . Whole - cell lysates were subjected to co - immunoprecipitation using anti - His antibody and immunoblot analysis with CIP2A antibody to verify that CIP2A bound to CIP2A - BP .

LINC00665 ORF-His plasmid was transfected into MDA-MB-231 cells. Whole-cell lysates were subjected to co-immunoprecipitation using anti-His antibody and immunoblot analysis with CIP2A antibody to verify that CIP2A bound to CIP2A-BP.


LINC00665 ORF - His plasmid was transfected into MDA - MB - 231 cells . Whole - cell lysates were subjected to co - immunoprecipitation by CIP2A antibody and immunoblot analysis with CIP2A - BP antibodies to verify that CIP2A - BP bound to CIP2A .

LINC00665 ORF-His plasmid was transfected into MDA-MB-231 cells. Whole-cell lysates were subjected to co-immunoprecipitation by CIP2A antibody and immunoblot analysis with CIP2A-BP antibodies to verify that CIP2A-BP bound to CIP2A.


Endogenous CIP2A - BP and CIP2A were co - immunoprecipitated in MCF - 10A cell line .

Endogenous CIP2A-BP and CIP2A were co-immunoprecipitated in MCF-10A cell line.


LINC00665 ORF - His plasmid was transfected into MDA - MB - 231 cells . Whole - cell lysates were treated with 30 mg / mL RNase A for 30 min , the indicated complexes were co - immunoprecipitated by anti - His or anti - CIP2A antibody , and CIP2A or micropeptide CIP2A - BP were detected .

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LINC00665 ORF-His plasmid was transfected into MDA-MB-231 cells. Whole-cell lysates were treated with 30 mg/mL RNase A for 30 min, the indicated complexes were co-immunoprecipitated by anti-His or anti-CIP2A antibody, and CIP2A or micropeptide CIP2A-BP were detected.


Upper : A diagram of the domain structure of full length CIP2A protein . Red : RIP = Required for Interaction with PPP2R5C ; Orange : RH = Required for Homodimerization ; Blue , CC = Coiled coil . Lower : Co - immunoprecipitation coupled western blot analysis assays revealed the interaction of CIP2A - BP with CIP2A via the N - terminus domain of CIP2A . Upper : A diagram of the domain structure of CIP2A protein fragment ( 1 - 245 ) . Red : RIP = Required for Interaction with PPP2R5C . Lower : Co - immunoprecipitation coupled western blot analysis assays revealed the interaction of CIP2A - BP with CIP2A via the N - terminus domain of CIP2A ( amino acid 159 - 245 ) .

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Upper: A diagram of the domain structure of full length CIP2A protein. Red: RIP=Required for Interaction with PPP2R5C; Orange: RH=Required for Homodimerization; Blue, CC=Coiled coil. Lower: Co-immunoprecipitation coupled western blot analysis assays revealed the interaction of CIP2A-BP with CIP2A via the N-terminus domain of CIP2A. Upper: A diagram of the domain structure of CIP2A protein fragment (1-245). Red: RIP=Required for Interaction with PPP2R5C. Lower: Co-immunoprecipitation coupled western blot analysis assays revealed the interaction of CIP2A-BP with CIP2A via the N-terminus domain of CIP2A (amino acid 159-245).


Co - immunoprecipitation assays showed that the interaction between CIP2A and B56γ in TNBC cells was affected by CIP2A - BP expression .

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Co-immunoprecipitation assays showed that the interaction between CIP2A and B56γ in TNBC cells was affected by CIP2A-BP expression.


The expression levels of the indicated proteins were detected by immunoblotting analysis in CIP2A - BP KO , LINC00665 ORF overexpressed ( OE ) and respective controls of TNBC cells .

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The expression levels of the indicated proteins were detected by immunoblotting analysis in CIP2A-BP KO, LINC00665 ORF overexpressed (OE) and respective controls of TNBC cells.


PP2A activity was measured using a protein phosphatase assay kit . PP2A activity assay was performed to evaluate PP2A activity in CIP2A - BP KO , LINC00665 ORF overexpressed ( OE ) and respective control TNBC cells .

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PP2A activity was measured using a protein phosphatase assay kit. PP2A activity assay was performed to evaluate PP2A activity in CIP2A-BP KO, LINC00665 ORF overexpressed (OE) and respective control TNBC cells.


Immunohistochemistry ( IHC ) staining of p - AKT from TNBC primary tumor ( n = 20 ) with high or low micropeptide CIP2A - BP expression . Lower panels show higher - magnification images of insets in upper panels . Right panel is the quantification of IHC staining p - AKT .

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Immunohistochemistry (IHC) staining of p-AKT from TNBC primary tumor (n = 20) with high or low micropeptide CIP2A-BP expression. Lower panels show higher-magnification images of insets in upper panels. Right panel is the quantification of IHC staining p-AKT.


MDA - MB - 231 cells were transfected with anti - CIP2A siRNAs . Whole - cell lysates of these cells were subjected to immunoblot analysis with CIP2A , CIP2A - BP , PP2Ac , p - AKT , AKT and β - actin antibodies .

MDA-MB-231 cells were transfected with anti-CIP2A siRNAs. Whole-cell lysates of these cells were subjected to immunoblot analysis with CIP2A, CIP2A-BP, PP2Ac, p-AKT, AKT and β-actin antibodies.


CIP2A - BP KO , LINC00665 ORF overexpressed ( OE ) and respective control MDA - MB - 231 cells were transfected with anti - CIP2A siRNAs . PP2A activity assay was performed to evaluate PP2A activity in the indicated cells .

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CIP2A-BP KO, LINC00665 ORF overexpressed (OE) and respective control MDA-MB-231 cells were transfected with anti-CIP2A siRNAs. PP2A activity assay was performed to evaluate PP2A activity in the indicated cells.


The indicated cells were pretreated with specific antagonist against AKT ( MK - 2206 ， 3μM ) for 1h . Then whole lysates of these cells were subjected to immunoblot analysis .

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The indicated cells were pretreated with specific antagonist against AKT (MK-2206，3μM) for 1h. Then whole lysates of these cells were subjected to immunoblot analysis.


Macroscopic and histological analysis of the lungs of 140 days old MMTV - PyMT mice ; lung metastatic nodules were visualized 8 weeks post - transplantation ( 5 mice per group ) . Right panel is the quantification of pulmonary metastases .

Macroscopic and histological analysis of the lungs of 140 days old MMTV-PyMT mice; lung metastatic nodules were visualized 8 weeks post-transplantation (5 mice per group). Right panel is the quantification of pulmonary metastases.


Immunohistochemistry ( IHC ) staining of p - AKT of mammary tumors from MMTV - PyMT ; CIP2A - BP + / + or MMTV - PyMT ; CIP2A - BP - / - mice . Lower panels show higher - magnification images of insets in upper panels . Right panel is the quantification of IHC staining p - AKT .

Immunohistochemistry (IHC) staining of p-AKT of mammary tumors from MMTV-PyMT;CIP2A-BP+/+ or MMTV-PyMT;CIP2A-BP-/- mice. Lower panels show higher-magnification images of insets in upper panels. Right panel is the quantification of IHC staining p-AKT .


MMTV - PyMT mice were injected with CIP2A - BP or svCIP2A - BP via mammary fat pad . Macroscopic and histological analysis of the lungs of 140 days old MMTV - PyMT mice ; lung metastatic nodules were visualized 8 weeks post - transplantation ( 5 mice per group ) . Right panel is the quantification of pulmonary metastases .

MMTV-PyMT mice were injected with CIP2A-BP or svCIP2A-BP via mammary fat pad. Macroscopic and histological analysis of the lungs of 140 days old MMTV-PyMT mice; lung metastatic nodules were visualized 8 weeks post-transplantation (5 mice per group). Right panel is the quantification of pulmonary metastases.


Representative IHC images of p - AKT of mammary tumors from MMTV - PyMT mice that were injected with CIP2A - BP or svCIP2A - BP via mammary fat pad ; Lower panels show higher - magnification images of insets in upper panels . Right panel is the quantification of IHC staining p - AKT .

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Representative IHC images of p-AKT of mammary tumors from MMTV-PyMT mice that were injected with CIP2A-BP or svCIP2A-BP via mammary fat pad; Lower panels show higher-magnification images of insets in upper panels. Right panel is the quantification of IHC staining p-AKT.


Kaplan - Meier survival curves of nude mice transplanted with MDA - MB - 231 cells and injected with CIP2A - BP or svCIP2A - BP ( 10 mice per group ) . The horizontal line indicates the time after the mice were injected with the cells via the tail vein .

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Kaplan-Meier survival curves of nude mice transplanted with MDA-MB-231 cells and injected with CIP2A-BP or svCIP2A-BP (10 mice per group). The horizontal line indicates the time after the mice were injected with the cells via the tail vein.


Macroscopic and histological analysis of the lungs of nude mice injected with MDA - MB - 231 cells and CIP2A - BP or svCIP2A - BP via tail vein ; lung metastatic nodules were visualized 8 weeks post - transplantation ( 5 mice per group ) . Right panel is the quantification of pulmonary metastases .

Macroscopic and histological analysis of the lungs of nude mice injected with MDA-MB-231 cells and CIP2A-BP or svCIP2A-BP via tail vein; lung metastatic nodules were visualized 8 weeks post-transplantation (5 mice per group). Right panel is the quantification of pulmonary metastases.


( A ) Live - cell nanoscopy of HeLa cells . COX8A - SNAP was labeled with SNAP - cell SiR and imaged with STED nanoscopy .

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(A) Live-cell nanoscopy of HeLa cells. COX8A-SNAP was labeled with SNAP-cell SiR and imaged with STED nanoscopy.


( B ) 3D architecture of cristae in HeLa cells visualized by FIB - SEM . The OM and the IBM are together shown as a transparent surface . Cristae of two mitochondria are depicted in light and dark blue . A twisted crista is shown in grey .

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(B) 3D architecture of cristae in HeLa cells visualized by FIB-SEM. The OM and the IBM are together shown as a transparent surface. Cristae of two mitochondria are depicted in light and dark blue. A twisted crista is shown in grey.


( C ) Live - cell recording as in ( A ) . In addition , mitochondrial DNA was stained with PicoGreen and imaged by confocal laser scanning microscopy . Arrows mark sites where nucleoids but no lamellar cristae are present .

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(C) Live-cell recording as in (A). In addition, mitochondrial DNA was stained with PicoGreen and imaged by confocal laser scanning microscopy. Arrows mark sites where nucleoids but no lamellar cristae are present.


( D ) STED nanoscopy of mitochondria immunolabeled for Mic60 and dsDNA . Arrows mark sites where nucleoids are present .

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(D) STED nanoscopy of mitochondria immunolabeled for Mic60 and dsDNA. Arrows mark sites where nucleoids are present.


( E ) Dual - color STED nanoscopy of cells labeled for COX8A - SNAP and immunolabeled for Mic60 . Arrows mark Mic60 clusters in the absence of fully developed lamellar cristae .

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(E) Dual-color STED nanoscopy of cells labeled for COX8A-SNAP and immunolabeled for Mic60. Arrows mark Mic60 clusters in the absence of fully developed lamellar cristae.


( A ) Composition of MICOS in WT cells and MICOS mutants . Mic60 specific antibodies were used to pull - down Mic60 and interacting proteins from insolated mitochondria . * * Unspecific band , due to the cross reaction of the anti - Mic25 antibody with Mic19 .

(A) Composition of MICOS in WT cells and MICOS mutants. Mic60 specific antibodies were used to pull-down Mic60 and interacting proteins from insolated mitochondria. ** Unspecific band, due to the cross reaction of the anti-Mic25 antibody with Mic19.


( B ) Oxygen consumption rate ( OCR ) of WT cells and MICOS mutants as analyzed by Seahorse Analyzer . Basal and maximal OCR are shown relative to the WT . Error bars : SEM , n = 6 .

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(B) Oxygen consumption rate (OCR) of WT cells and MICOS mutants as analyzed by Seahorse Analyzer. Basal and maximal OCR are shown relative to the WT. Error bars: SEM, n=6.


( C ) Mitochondrial networks of WT and MICOS mutant cells . Cells were immunolabeled for TOM20 and visualized by confocal microscopy . The inset shows a magnification of the respective overview image . Scale bar : 10 µm . ( D ) Quantification of the mitochondrial networks as shown in ( C ) . The evaluation was performed manually in a blinded approach based on pre - defined morphology criteria . Average and SD of 3 independent biological replicates are shown ( > 170 cells per sample and replicate ) .

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(C) Mitochondrial networks of WT and MICOS mutant cells. Cells were immunolabeled for TOM20 and visualized by confocal microscopy. The inset shows a magnification of the respective overview image. Scale bar: 10 µm. (D) Quantification of the mitochondrial networks as shown in (C). The evaluation was performed manually in a blinded approach based on pre-defined morphology criteria. Average and SD of 3 independent biological replicates are shown (>170 cells per sample and replicate).


( A ) Representative TEM recordings of WT cells and MICOS - KO mutants , as indicated .

(A) Representative TEM recordings of WT cells and MICOS-KO mutants, as indicated.


( B ) Quantification of the overall cristae morphology on TEM recordings . Data information : n : number of mitochondrial

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(B) Quantification of the overall cristae morphology on TEM recordings. Data information: n: number of mitochondrial sections


( C ) Quantification of detailed morphology of CMs in MICOS mutants ( left ) . TEM recordings were assigned to 8 classes ( right ) . Data information : n : number of mitochondrial

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(C) Quantification of detailed morphology of CMs in MICOS mutants (left). TEM recordings were assigned to 8 classes (right). Data information: n: number of mitochondrial sections


( D ) CJ frequency on TEM recordings . The number of CJs was manually determined and normalized to the length of the OM . Samples were compared to WT by a One - way ANOVA test . Error bars : SEM . Data information : n : number of

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(D) CJ frequency on TEM recordings. The number of CJs was manually determined and normalized to the length of the OM. Samples were compared to WT by a One-way ANOVA test. Error bars: SEM. Data information: n: number of CJs


( E ) Diameter of CJs . CJs were manually measured on TEM recordings . Red line : Average . Error bars : SD . Samples were compared to WT by a One - way ANOVA test . Data information : n : number of mitochondrial

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(E) Diameter of CJs. CJs were manually measured on TEM recordings. Red line: Average. Error bars: SD. Samples were compared to WT by a One-way ANOVA test. Data information: n: number of mitochondrial sections


( F ) Representative ET reconstructions of mitochondria from a WT cell ( left ) , a Mic10 - KO cell ( center ) and a Mic60 - KO cell ( right ) . The OM is displayed in clear grey , the side of the IM that faces the matrix is shown in dark blue . The IM side that faces the IMS is shown in light blue .

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(F) Representative ET reconstructions of mitochondria from a WT cell (left), a Mic10-KO cell (center) and a Mic60-KO cell (right). The OM is displayed in clear grey, the side of the IM that faces the matrix is shown in dark blue. The IM side that faces the IMS is shown in light blue.


( A Nanoscopy of mitochondria of WT and Mic10 - KO cells , as indicated . ( A ) Mitochondria were immunolabeled for Mic60 and ATPB and visualized by 2D STED nanoscopy .

(A Nanoscopy of mitochondria of WT and Mic10-KO cells, as indicated. (A) Mitochondria were immunolabeled for Mic60 and ATPB and visualized by 2D STED nanoscopy.


( B ) Mitochondria were immunolabeled for TOM20 and ATPB and recorded with 3D STED nanoscopy . Image data were deconvolved . Depicted are cross - sections of a mitochondrion ( side view ) .

(B) Mitochondria were immunolabeled for TOM20 and ATPB and recorded with 3D STED nanoscopy. Image data were deconvolved. Depicted are cross-sections of a mitochondrion (side view).


( C ) 3D MINFLUX nanoscopy . Mic10 - KO cells were immunolabeled for Mic60 using a directly labeled antibody . Colors encode depth information .

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(C) 3D MINFLUX nanoscopy. Mic10-KO cells were immunolabeled for Mic60 using a directly labeled antibody. Colors encode depth information.


( D ) 3D SIM of living WT and Mic10 - KO cells . Cells were labeled with Mitotracker Green . Images show maximum intensity projections .

(D) 3D SIM of living WT and Mic10-KO cells. Cells were labeled with Mitotracker Green. Images show maximum intensity projections.


( E ) FIB - SEM of Mic10 - KO cells . Shown is a representative reconstructed mitochondrion . Dashed box : Orthoslice view on the smaller mitochondrion . The OM and IBM are displayed together in clear grey , the CM in is shown in blue .

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(E) FIB-SEM of Mic10-KO cells. Shown is a representative reconstructed mitochondrion. Dashed box: Orthoslice view on the smaller mitochondrion. The OM and IBM are displayed together in clear grey, the CM in is shown in blue.


( F ) 2D STED nanoscopy of mitochondria . Mic19 - KO cells were transfected with a plasmid for Mic10 - FLAG expression under the control of a tetracycline - inducible promoter . Left panel : Mitochondria from noninduced cell . Right : Mitochondria from induced cell expressing Mic10 - FLAG .

(F) 2D STED nanoscopy of mitochondria. Mic19-KO cells were transfected with a plasmid for Mic10-FLAG expression under the control of a tetracycline-inducible promoter. Left panel: Mitochondria from noninduced cell. Right: Mitochondria from induced cell expressing Mic10-FLAG.


( A ) Cells were immunolabeled for Mic60 and DNA and visualized by confocal fluorescence microscopy before and after induction of Mic60 expression for 48 h .

(A) Cells were immunolabeled for Mic60 and DNA and visualized by confocal fluorescence microscopy before and after induction of Mic60 expression for 48 h.


( B ) Protein levels of MICOS proteins after Mic60 induction . Cell lysates were analyzed by western blotting at the indicated time points .

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(B) Protein levels of MICOS proteins after Mic60 induction. Cell lysates were analyzed by western blotting at the indicated time points.


( C ) Recovery of mitochondrial networks upon Mic60 re - expression . Cells were induced with doxycycline hyclate for 72 h , immunolabeled for TOM20 and visualized by confocal microscopy .

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(C) Recovery of mitochondrial networks upon Mic60 re-expression. Cells were induced with doxycycline hyclate for 72 h, immunolabeled for TOM20 and visualized by confocal microscopy.


( D - G ) TEM of Mic60 - TO cells before and after induction . ( D ) TEM recordings of Mic60 - TO cells at the indicated time points . ( E ) Quantification of the cristae morphology . ( F ) CJ frequency . The number of CJs on TEM recordings was normalized to the length of the OM . At least 90 mitochondrial sections were analyzed for each sample . ( G ) Quantification of the cristae morphology of Mic60 - TO cells . Only mitochondria exhibiting at least one CJ were analyzed . Data information : n : Number of mitochondrial sections . Scale bars : 20 µm ( A ) , 10 µm ( C ) , 500 nm ( D ) .

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(D-G) TEM of Mic60-TO cells before and after induction. (D) TEM recordings of Mic60-TO cells at the indicated time points. (E) Quantification of the cristae morphology. (F) CJ frequency. The number of CJs on TEM recordings was normalized to the length of the OM. At least 90 mitochondrial sections were analyzed for each sample. (G) Quantification of the cristae morphology of Mic60-TO cells. Only mitochondria exhibiting at least one CJ were analyzed. Data information: n: Number of mitochondrial sections. Scale bars: 20 µm (A), 10 µm (C), 500 nm (D).


( A , B ) Live - cell nanoscopy of Mic10 - TO cells . Cells expressing COX8A - SNAP were labeled with SNAP - cell SiR and recorded with STED nanoscopy . ( A ) Cristae architecture in noninduced cells . ( B ) Cristae architecture after Mic10 re - expression ( 24 h induction with doxycycline ) . STED image data were deconvolved .

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(A,B) Live-cell nanoscopy of Mic10-TO cells. Cells expressing COX8A-SNAP were labeled with SNAP-cell SiR and recorded with STED nanoscopy. (A) Cristae architecture in noninduced cells. (B) Cristae architecture after Mic10 re-expression (24 h induction with doxycycline). STED image data were deconvolved.


( C ) STED nanoscopy of fixed Mic10 - TO cells . Cells were induced for 24 h and immunolabeled for Mic60 and Mic10 - FLAG . Upper row : Cell with weak Mic10 expression level . Lower row : Cell with strong Mic10 expression level .

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(C) STED nanoscopy of fixed Mic10-TO cells. Cells were induced for 24 h and immunolabeled for Mic60 and Mic10-FLAG. Upper row: Cell with weak Mic10 expression level. Lower row: Cell with strong Mic10 expression level.


( D - G ) TEM of Mic10 - TO cells . ( D ) Cells induced for the indicated period of time were recorded with TEM . Arrow indicates an intermediately shaped crista . ( E ) Quantification of the cristae morphology . n : Number of mitochondrial sections . ( F ) CJ frequency in Mic10 - TO cells . Number of CJs was normalized to the length of the OM . The same sections were analyzed as in ( E ) . ( G ) Diameter of CJs estimated on TEM recordings . Data information : n : Number of analyzed mitochondrial sections ( E ) or CJs ( G ) . .

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(D-G) TEM of Mic10-TO cells. (D) Cells induced for the indicated period of time were recorded with TEM. Arrow indicates an intermediately shaped crista. (E) Quantification of the cristae morphology. n: Number of mitochondrial sections. (F) CJ frequency in Mic10-TO cells. Number of CJs was normalized to the length of the OM. The same sections were analyzed as in (E). (G) Diameter of CJs estimated on TEM recordings. Data information: n: Number of analyzed mitochondrial sections (E) or CJs (G)..


( A ) FIB - SEM of Mic10 - TO cells . Cells were induced for 0 h , 16 h and 24 h . The representative reconstructions show mitochondria from noninduced , rescued and intermediate cells , as indicated . The OM is shown together with the IBM in clear grey , the CM in blue .

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(A) FIB-SEM of Mic10-TO cells. Cells were induced for 0 h, 16 h and 24 h. The representative reconstructions show mitochondria from noninduced, rescued and intermediate cells, as indicated. The OM is shown together with the IBM in clear grey, the CM in blue.


( B ) ET of Mic10 - TO cells . Mic10 expression was induced for 0 h and 16 h . Shown are reconstructions of representative noninduced and almost completely rescued mitochondria . The OM is displayed in clear grey , the side of the IM that faces the matrix is shown in dark blue . The IM side that faces the IMS is shown in light blue .

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(B) ET of Mic10-TO cells. Mic10 expression was induced for 0 h and 16 h. Shown are reconstructions of representative noninduced and almost completely rescued mitochondria. The OM is displayed in clear grey, the side of the IM that faces the matrix is shown in dark blue. The IM side that faces the IMS is shown in light blue.


Knockdown ( KD ) of OPA1 in HeLa cells . WT and Mic60 - KO cells were transfected with a scrambled control ( Ctrl . ) or siRNA pools against OPA1 ( OPA1 - KD ) for 5 d . ( A ) Cell lysates were analyzed by western blotting .

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Knockdown (KD) of OPA1 in HeLa cells. WT and Mic60-KO cells were transfected with a scrambled control (Ctrl.) or siRNA pools against OPA1 (OPA1-KD) for 5 d. (A) Cell lysates were analyzed by western blotting.


( B ) TEM recordings from OPA1 depleted WT and Mic60 - KO cells . Arrows point to a septum .

(B) TEM recordings from OPA1 depleted WT and Mic60-KO cells. Arrows point to a septum.


( C , D ) Quantification of the cristae morphology of OPA1 deficient WT ( C ) and Mic60 - KO ( D ) cells . The number of CJs was normalized to the length of the OM ( C ) . The number of septated mitochondria in Mic60 - KO cells was estimated ( D ) .

(C,D) Quantification of the cristae morphology of OPA1 deficient WT (C) and Mic60-KO (D) cells. The number of CJs was normalized to the length of the OM (C). The number of septated mitochondria in Mic60-KO cells was estimated (D).


( E , F ) ET of mitochondria from OPA1 - KD cells . WT cells were transfected with a scrambled control ( E ) or siRNA pools against OPA1 ( F ) for 48 h . Reconstructions of representative mitochondria are displayed . The OM is displayed in clear grey , the side of the IM that faces the matrix is shown in dark blue . The IM side that faces the IMS is shown in light blue .

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(E,F) ET of mitochondria from OPA1-KD cells. WT cells were transfected with a scrambled control (E) or siRNA pools against OPA1 (F) for 48 h. Reconstructions of representative mitochondria are displayed. The OM is displayed in clear grey, the side of the IM that faces the matrix is shown in dark blue. The IM side that faces the IMS is shown in light blue.


WT and Mic10 - KO cells were transfected with a scrambled control ( Ctrl . ) or siRNA pools against OPA1 ( OPA1 - KD ) for 72 h . ( A , B ) STED nanoscopy of OPA1 depleted WT ( A ) and Mic10 - KO ( B ) cells . Cells were immunolabeled for Mic60 and ATPB .

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

WT and Mic10-KO cells were transfected with a scrambled control (Ctrl.) or siRNA pools against OPA1 (OPA1-KD) for 72 h. (A,B) STED nanoscopy of OPA1 depleted WT (A) and Mic10-KO (B) cells. Cells were immunolabeled for Mic60 and ATPB.


( C ) TEM of OPA1 depleted Mic10 - KO cells . Left : Representative TEM recordings showing CJs marked by arrows in a control cell and a similar view for an OPA1 - KD cell . The CMs are colored in green and the OM and IBM together in magenta . Right : Quantification of mitochondria containing CJs or septa junctions . Sections that contained CJ - like structures were analyzed . Of these , the number of sections exhibiting septa or cristae structures ( other ) was quantified .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) TEM of OPA1 depleted Mic10-KO cells. Left: Representative TEM recordings showing CJs marked by arrows in a control cell and a similar view for an OPA1-KD cell. The CMs are colored in green and the OM and IBM together in magenta. Right: Quantification of mitochondria containing CJs or septa junctions. Sections that contained CJ-like structures were analyzed. Of these, the number of sections exhibiting septa or cristae structures (other) was quantified.


( D Depletion of ATP5ME in HeLa cells . Cells were transfected with a scrambled control or siRNA pools against ATP5ME for 4 d . TEM recording ( left ) and quantification of the number of cristae per mitochondrial section ( right ) .

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

(D Depletion of ATP5ME in HeLa cells. Cells were transfected with a scrambled control or siRNA pools against ATP5ME for 4 d. TEM recording (left) and quantification of the number of cristae per mitochondrial section (right).


( E ) Cells depleted of ATP5ME were immunolabeled for Mic60 and recorded with STED . The arrows point to crossing points of the opposite Mic60 distribution bands .

O O O O O 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) Cells depleted of ATP5ME were immunolabeled for Mic60 and recorded with STED. The arrows point to crossing points of the opposite Mic60 distribution bands.


( a ) Top : immunoblots for the indicated proteins from two independent experiments . Bottom : quantification of changes in total LRRK2 ( n = 6 independent experiments ) . SH - SY5Y cells were untreated or incubated with 5 μM lactacystin ( Lact ) , 10 mM 3MA or 20 mM NH4Cl and 100 μM leupeptin ( NL ) for 9 . 5 h .

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

(a) Top: immunoblots for the indicated proteins from two independent experiments. Bottom: quantification of changes in total LRRK2 (n = 6 independent experiments). SH-SY5Y cells were untreated or incubated with 5 μM lactacystin (Lact), 10 mM 3MA or 20 mM NH4Cl and 100 μM leupeptin (NL) for 9.5 h.


( b ) Top : representative immunoblots for the indicated proteins from two independent experiments . Bottom : quantification of LRRK2 relative to that in untreated samples ( n = 3 or 4 independent experiments ) . Brain slices from mouse cortex , midbrain or cerebellum were incubated without additions or with NL or 3MA for 2 h at 37 ° C .

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

(b) Top: representative immunoblots for the indicated proteins from two independent experiments. Bottom: quantification of LRRK2 relative to that in untreated samples (n = 3 or 4 independent experiments). Brain slices from mouse cortex, midbrain or cerebellum were incubated without additions or with NL or 3MA for 2 h at 37 °C.


( c , d ) Homogenate ( Hom ) and lysosomal fractions ( Lys ) isolated from SH - SY5Y cells ( c ) or the indicated mouse brain regions ( d ) , immunoblotted for the indicated proteins .

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

(c,d) Homogenate (Hom) and lysosomal fractions (Lys) isolated from SH-SY5Y cells (c) or the indicated mouse brain regions (d), immunoblotted for the indicated proteins.


( e ) HEK293 cells expressing GFP - tagged LRRK2 and transduced with lentivirus control ( Ctr ) or carrying shRNA against LAMP - 2A ( L2A ( − ) ) , incubated or not with NL for 12 h and immunoblotted for the indicated proteins .

(e) HEK293 cells expressing GFP-tagged LRRK2 and transduced with lentivirus control (Ctr) or carrying shRNA against LAMP-2A (L2A(−)), incubated or not with NL for 12 h and immunoblotted for the indicated proteins.


( f ) Immunoblots for LRRK2 and LAMP - 2A of homogenates and lysosome - enriched fractions isolated from control fibroblasts ( Ctr ) or fibroblasts stably interfered for LAMP - 2A ( L2A ( − ) ) , maintained in the presence or absence of serum . Actin and LAMP - 1 ( L - 1 ) are shown as loading controls of homogenate and lysosomes , respectively . All values are mean + s . e . m . ; * P 0 . 05 , unpaired t - test . Full - length blots and gels in Supplementary Figure 12 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Immunoblots for LRRK2 and LAMP-2A of homogenates and lysosome-enriched fractions isolated from control fibroblasts (Ctr) or fibroblasts stably interfered for LAMP-2A (L2A(−)), maintained in the presence or absence of serum. Actin and LAMP-1 (L-1) are shown as loading controls of homogenate and lysosomes, respectively. All values are mean + s.e.m.; *P 0.05, unpaired t-test. Full-length blots and gels in Supplementary Figure 12.


( b ) Binding , association and uptake calculated from quantification of LRRK2 in immunoblots of lysosomes from starved rat livers , untreated or pretreated with protease inhibitors ( PI ) and incubated with LRRK2 . Inset : representative immunoblot . LAMP - 2A is shown as lysosomal loading control . Input ( Inp ) , one - fifth of LRRK2 added . n = 7 independent experiments . Uptake : difference between LRRK2 associated with lysosomes with and without PI .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Binding, association and uptake calculated from quantification of LRRK2 in immunoblots of lysosomes from starved rat livers, untreated or pretreated with protease inhibitors (PI) and incubated with LRRK2. Inset: representative immunoblot. LAMP-2A is shown as lysosomal loading control. Input (Inp), one-fifth of LRRK2 added. n = 7 independent experiments. Uptake: difference between LRRK2 associated with lysosomes with and without PI.


( c ) Effect of increasing concentrations of LRRK2 on the degradation of a radiolabeled pool of cytosolic proteins by intact lysosomes . Values are percentage of the degradation without LRRK2 ( n = 5 or 6 independent experiments ) .

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

(c) Effect of increasing concentrations of LRRK2 on the degradation of a radiolabeled pool of cytosolic proteins by intact lysosomes. Values are percentage of the degradation without LRRK2 (n = 5 or 6 independent experiments).


( d ) Association of GAPDH to rat liver lysosomes in the presence of increasing concentrations of LRRK2 . Values are percentage of the GAPDH associated with lysosomes in the absence of LRRK2 ( n = 4 - 6 independent experiments in duplicate ) . Inset : representative immunoblot . Inp : 1 μg of

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Association of GAPDH to rat liver lysosomes in the presence of increasing concentrations of LRRK2. Values are percentage of the GAPDH associated with lysosomes in the absence of LRRK2 (n = 4-6 independent experiments in duplicate). Inset: representative immunoblot. Inp: 1 μg of GAPDH


( e , f ) Association of LRRK2 and RNase A ( e ) or LRRK2 and GAPDH ( f ) with PI - treated rat liver lysosomes incubated with increasing concentrations of GAPDH ( e ) or RNase A ( f ) or ovalbumin ( Ov ) . Left : representative immunoblots . Right : protein association expressed as a percentage of the value when incubated with lysosomes alone ( n = 5 or 6 independent experiments ) . Inp : RNase A ( 0 . 4 μg ) , LRRK2 ( 0 . 04 μg ) or GAPDH ( 1 μg ) .

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

(e,f) Association of LRRK2 and RNase A (e) or LRRK2 and GAPDH (f) with PI-treated rat liver lysosomes incubated with increasing concentrations of GAPDH (e) or RNase A (f) or ovalbumin (Ov). Left: representative immunoblots. Right: protein association expressed as a percentage of the value when incubated with lysosomes alone (n = 5 or 6 independent experiments). Inp: RNase A (0.4 μg), LRRK2 (0.04 μg) or GAPDH (1 μg).


( g , h ) Association of LRRK2 to lysosomes incubated with GAPDH ( g ) or RNase A ( h ) at 4 ° C ( n = 3 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

(g,h) Association of LRRK2 to lysosomes incubated with GAPDH (g) or RNase A (h) at 4 °C (n = 3 independent experiments).


( i , j ) Immunoblots of brain lysosomes incubated with LRRK2 and increasing concentrations of GAPDH ( i ) or RNase A ( j ) . All values are mean + s . e . m . ; differences with lysosomes incubated with the protein alone were significant at * P 0 . 05 , unpaired t - test . Full - length blots and gels in Supplementary Figure 12 .

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

(i,j) Immunoblots of brain lysosomes incubated with LRRK2 and increasing concentrations of GAPDH (i) or RNase A (j). All values are mean + s.e.m.; differences with lysosomes incubated with the protein alone were significant at *P 0.05, unpaired t-test. Full-length blots and gels in Supplementary Figure 12.


( a ) Top : immunoblot for GFP of tet - on HEK293 cells expressing GFP - tagged WT or G / S LRRK2 pulsed 24 h with doxycycline and , 48 h later ( no longer undergoing de novo synthesis ) , incubated without additions ( − ) or with 20 mM NH4Cl and 100 μM leupeptin ( NL ) , 5 μM MG132 ( MG ) or 10 mM 3MA . Bottom : percentage inhibition ( n = 3 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 B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O

(a) Top: immunoblot for GFP of tet-on HEK293 cells expressing GFP-tagged WT or G/S LRRK2 pulsed 24 h with doxycycline and, 48 h later (no longer undergoing de novo synthesis), incubated without additions (−) or with 20 mM NH4Cl and 100 μM leupeptin (NL), 5 μM MG132 (MG) or 10 mM 3MA. Bottom: percentage inhibition (n = 3 independent experiments).


( a ) Top : immunoblot for GFP of tet - on HEK293 cells expressing GFP - tagged WT or G / S LRRK2 pulsed 24 h with doxycycline and , 48 h later ( no longer undergoing de novo synthesis ) , ( b ) Same cells as in a , untreated ( Ctr ) or treated with RNA interference for LAMP - 2A ( L2A ( − ) ) , pulsed as in a , sequentially collected and immunoblotted . Top left panels : representative immunoblots for GFP . Actin is shown as a loading control . Bottom left panels , quantification , corrected by levels of knockdown and loading control ( n = 3 or 4 independent experiments ) . Top right : immunoblot for LAMP - 2A . Bottom right : percentage of LRRK2 degraded per hour in the same samples .

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(a) Top: immunoblot for GFP of tet-on HEK293 cells expressing GFP-tagged WT or G/S LRRK2 pulsed 24 h with doxycycline and, 48 h later (no longer undergoing de novo synthesis), (b) Same cells as in a, untreated (Ctr) or treated with RNA interference for LAMP-2A (L2A(−)), pulsed as in a, sequentially collected and immunoblotted. Top left panels: representative immunoblots for GFP. Actin is shown as a loading control. Bottom left panels, quantification, corrected by levels of knockdown and loading control (n = 3 or 4 independent experiments). Top right: immunoblot for LAMP-2A. Bottom right: percentage of LRRK2 degraded per hour in the same samples.


( c ) Binding , association and uptake calculated from quantification of LRRK2 in immunoblots of lysosomes from starved rat livers , untreated or pretreated with protease inhibitors ( PI ) and incubated with WT or G / S LRRK2 . Inset : representative immunoblot ( n = 9 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

(c) Binding, association and uptake calculated from quantification of LRRK2 in immunoblots of lysosomes from starved rat livers, untreated or pretreated with protease inhibitors (PI) and incubated with WT or G/S LRRK2. Inset: representative immunoblot (n = 9 independent experiments).


( d ) Association of GAPDH ( 5 μg ) to starved rat liver lysosomes in the presence of increasing concentrations of WT or G / S LRRK2 . Inset : representative immunoblot n = 4 - 6 .

O O O O O O O O O O O O O O O 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) Association of GAPDH (5 μg) to starved rat liver lysosomes in the presence of increasing concentrations of WT or G/S LRRK2. Inset: representative immunoblot n = 4-6.


( e , f ) Binding of WT and G / S LRRK2 to lysosomes in the presence of GAPDH ( e ) or RNase A ( f ) . Top : representative immunoblots . Bottom : lysosome - bound LRRK2 expressed as a multiple of the amount bound when added alone . Trends of mean values ( n = 2 independent experiments ) ; error bars indicate range .

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(e,f) Binding of WT and G/S LRRK2 to lysosomes in the presence of GAPDH (e) or RNase A (f). Top: representative immunoblots. Bottom: lysosome-bound LRRK2 expressed as a multiple of the amount bound when added alone. Trends of mean values (n = 2 independent experiments); error bars indicate range.