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

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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 projections ) of ATG7KO / TAX1BP1KO K562 expressing tf - NBR1 . Selected region ( box ) of micrograph is shown as single merged channels from microscopy . RFP , magenta ; GFP , green ; merged , ; 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 electron microscopy ( CLEM ) of K562 ATG7KO / TAX1BP1KO expressing tf - NBR1 under basal conditions . workflow is indicated by green arrows . arrow indicates a structure of interest . box demarcates zoomed in bottom . 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 indicated K562 knock - out expressing tf - NBR1 were transfected with TagBFP or TagBFP - RavZ , BFP - positive were analyzed for red : green by flow cytometry . median red : green of each sample was used to calculate flux relative to WT + TagBFP . Bar graphs represent mean + / - SD from three independent experiments . n > 7 , 500 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 ) clonal deletion isolates were resolved by SDS - PAGE followed by immunoblotting ( IB ) with indicated antibodies . samples were normalized by total using a BCA 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 ( 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 indicated K562 knock - out were treated corresponding extracts were resolved by SDS - PAGE analyzed by immunoblotting ( IB ) with indicated antibodies . Shown are representative from one experiment with mean 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 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 - . * * , 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 deletion K562 expressing tf - NBR1 were normalized for total by BCA . NBR1 was immunoprecipitated using GFP - Trap dynabeads . Input 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 ( as maximum projections ) of wild - type K562 ATG9AKO expressing tf - NBR1 . Selected regions ( box ) of are shown as single merged channels from immunofluorescence microscopy against indicated . In conditions where were not observed , representative regions were selected to showcase diffuseness of signal . TAX1BP1 , SQSTM1 , or NDP52 , magenta ; NBR1 , green ; Hoechst , blue .

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(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 ( as maximum projections ) of K562 expressing tf - NDP52 ( or tf - TAX1BP1 ) transduced with sgRNAs targeting ATG9A ( sgATG9A ) or a negative control ( sgControl ) . Selected regions ( box ) of are shown as single merged channels from 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 were transfected with either myc - TAX1BP1WT or myc - NBR1WT indicated TagBFP - V5 - TAX1BP1 variants . Extracts derived from transfected were immunoprecipitated ( IP ) with anti - TagBFP magnetic beads . Input 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 were transfected with myc - NBR1WT indicated TagBFP - V5 - TAX1BP1 variants . Extracts derived from transfected were immunoprecipitated ( IP ) with anti - TagBFP magnetic beads . Input 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 ( as maximum projections ) from wild - type deletion K562 expressing tf - NBR1 . Selected regions ( box ) of are shown as single merged channels from immunofluorescence microscopy against indicated . P - TBK1 , magenta ; NBR1 , green ; Hoechst , blue . ( B ) Representative confocal ( as maximum projections ) from wild - type deletion K562 expressing tf - NBR1 . Selected regions ( box ) of are shown as single merged channels from immunofluorescence microscopy against indicated . 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 ( as maximum projections ) from wild - type , TAX1BP1KO , ATG7KO / TAX1BP1KO K562 expressing tf - NBR1 transduced with indicated sgRNA . Selected regions ( box ) of are shown as single merged channels from immunofluorescence microscopy against indicated . FIP200 , magenta ; NBR1 , green ; Hoechst , blue . ( D ) Quantitation of colocalization between NBR1 with FIP200 in wild - type , TAX1BP1KO , ATG7KO / TAX1BP1KO imaged in ( C ) Bar graphs represent mean + / - SD for three independently generated deletion lines ( dots ) . sgATG9A samples were compared using a one - way ANOVA ( p < 0 . 0001 ) with Tukey HSD post - . * * * , p < 0 . 001 . n > 200 NBR1 for each biological replicate . ( E ) Quantitation of FIP200 at NBR1 in wild - type , TAX1BP1KO , ATG7KO / TAX1BP1KO imaged in ( C ) . Bar graphs represent mean + / - SD for three independently generated deletion lines ( dots ) . n > 80 FIP200 - positive for each biological replicate . Samples were compared using a one - way ANOVA ( p < 0 . 0001 ) with Tukey HSD post - . * * * , p < 0 . 001 . a . u . , arbitrary 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 were isolated by microfluidic chip , then seeded ( 500 ) in 90 Pa soft 3D fibrin gel for 3 days . of colonies formed was calculated colony was recorded . Scale bar , 100 μm . Data information : Two - Paired Student ' s t 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 . Eight later , lung metastasis was analyzed by H & E . metastasis index was defined as of total metastatic nodule to total lung on calculation from 10 slides . Scale bar , 0 . 5 mm . n = 3 ( for B16 ) or 5 ( for MP - 1 ) mice with metastatic . Data information : Two - Paired Student ' s t 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.


100 stiff ALDH + or soft ALDH - 4T1 were injected into mammary fat pads of NSG mice . Eight later , lung sections were H & E stained . metastasis index was calculated ( D ) . Scale bar , 0 . 5 mm . n = 5 mice with metastatic . Data information : two - Paired Student ' s t 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 ) of stemness association genes expression determined by mRNA sequencing comparing stiff soft MCF - 7 . n = 3 . unit for 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 ) expression of BCL9L or β - catenin in stiff or soft 4T1 , MCF - 7 , B16 or MP - 1 was determined by western blot . ( B ) expression of β - catenin BCL9L from SGGFP , BCL9L - SGs - 4T1 , MCF - 7 , B16 or MP - 1 were determined by western blot . Data information n = 3 independent experiments in ( A 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 ) 100 soft SGGFP or BCL9L - SGs - 4T1 , B16 or MP - 1 were injected into mammary fat pads ( 4T1 ) of BALB / c mice or tail vein ( B16 MP - 1 ) of NSG mice . formation was recorded ( E ) . Six to eight later , lung sections were H & E stained . metastatic micronodules in lung were counted metastasis index was calculated ( F ) . Scale bar , 0 . 5 mm . n = 3 ( for B16 - F1 ) or 5 ( for MP - 1 ) mice with metastatic . Data information : Bonferroni 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 ) stiffness of breast ( HBC , n = 3 ) colon ( HCC , n = 3 ) cancer screened from outlet or backflow inlet was measured by AFM . n = 100 from each . Data information : Mann - Whitney ( A ) 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 ) stiff soft were isolated from 3 with breast cancer stained with anti - BCL9L antibody . expression of BCL9L was observed under confocal microscope . Scale bar , 10 μ Data information : Paired Student ' s t ( C ) 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 ) correlation between expression of BCL9L from 18 melanoma grade of melanoma . Data information : Pearson ' s correlation E ) 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 BCL9L level in breast cancer ( BRCA , n = 536 ) or melanoma ( n = 376 ) . Data information : Log - rank survival ( F ) . 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 LINC00665 locus . MCF10A were either mock - treated or treated with TGF - β 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 of CIP2A - BP were determined by polysome profiling qRT - PCR in MCF - 10A TNBC either mock treated or treated with TGF - β . Relative of LINC00665 were determined by qRT - PCR in MCF - 10A TNBC either mock treated or treated with TGF - β .

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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 : ORFs were constructed into pcDNA3 . 1 vector transfected to MCF - 10A for 24h . ORFs - His fusion 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.


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

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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 His fusion constructs . start codon ATG of LINC00665 ORF is mutated to ATT . indicated constructs were stably expressed in MDA - MB - 231 , micropeptide CIP2A - BP was immuno - stained using anti - His ( I ) anti - CIP2A - BP antibodies ( K ) respectively ; CIP2A - BP - His fusion were determined by western blotting with anti - His 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 incubated in absence or presence of puromycin ( 200μM ) were fractionated on sucrose gradients , 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 MDA - MB - 231 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 were determined by western blot with anti - CIP2A - BP antibody in MCF - 10A TNBC 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 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 were detected by western blot with anti - CIP2A - BP antibody in indicated non - metastatic TNBC tissues ( non - metastasis ) metastatic TNBC tissues ( metastasis ) . Micropeptide CIP2A - BP expression in MCF - 10A was used as 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 with high or low micropeptide CIP2A - BP expression in Suzhou cohort ( discovery set , n = 112 ) 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 invasion of TNBC transfected with LINC00665 ORF control lentiviruses for 48h . Right panel is of TNBC that migrated through without Matrigel ( upper ) invaded through Matrigel - coated ( 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 invasion of wild - type CIP2A - BP knockout TNBC CIP2A - BP re - expressed Hs578TKO MDA - MB - 231KO . Right panel is of that migrated through without Matrigel ( upper ) invaded through Matrigel - coated ( lower ) .

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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 on Hs578T MDA - MB - 231 transfected with LINC00665 ORF expressing control lentiviruses . wound edges were photographed at indicated time points after wounding . Right panel is of relative wound healing .

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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 CIP2A - BP knockout TNBC CIP2A - BP re - expressed Hs578TKO MDA - MB - 231KO were seeded on culture inserts for wound - healing . wound edges were photographed at indicated time points after wounding . Right panel is of relative wound healing .

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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 histological of lungs of nude mice injected with indicated Hs578T ( G ) MDA - MB - 231 ( H ) ( 1 . 0 × 105 - 106 / mouse ) via tail vein ; lung metastatic nodules were visualized 8 post - transplantation ( 5 mice per group ) . Right panel is 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 of expression of indicated in TNBC cultured with or without TGF - β .

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


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

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

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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 binding to eIF4E by TGF - β . TNBC were treated with TGF - β equal amounts of were subjected to immunoprecipitation / or examined by immunoblotting .

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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 of expression of micropeptide CIP2A - BP β - actin in TNBC 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 corresponding control transfected TNBC were cultured with or without TGF - β . indicated were determined by Immunoblotting .

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

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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 corresponding control transfected TNBC were cultured with or without TGF - β . indicated were determined by immunoblotting .

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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 . Whole - lysates were subjected to co - immunoprecipitation using anti - His antibody immunoblot with CIP2A antibody to verify that CIP2A bound to CIP2A - BP .

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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 . Whole - lysates were subjected to co - immunoprecipitation by CIP2A antibody immunoblot with CIP2A - BP antibodies to verify that CIP2A - BP bound to CIP2A .

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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 CIP2A were co - immunoprecipitated in MCF - 10A .

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


LINC00665 ORF - His plasmid was transfected into MDA - MB - 231 . Whole - lysates were treated with 30 mg / mL RNase A for 30 min , indicated complexes were co - immunoprecipitated by anti - His or anti - CIP2A antibody , 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 domain structure of full CIP2A . Red : RIP = Required for Interaction with PPP2R5C ; Orange : RH = Required for Homodimerization ; Blue , CC = Coiled coil . Lower : Co - immunoprecipitation coupled western blot assays revealed interaction of CIP2A - BP with CIP2A via N - terminus domain of CIP2A . Upper : A diagram of domain structure of CIP2A fragment ( 1 - 245 ) . Red : RIP = Required for Interaction with PPP2R5C . Lower : Co - immunoprecipitation coupled western blot assays revealed interaction of CIP2A - BP with CIP2A via N - terminus domain of CIP2A ( 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 interaction between CIP2A B56γ in TNBC 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.


expression of indicated were detected by immunoblotting in CIP2A - BP KO , LINC00665 ORF overexpressed ( OE ) respective controls of TNBC .

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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 phosphatase kit . PP2A activity was performed to evaluate PP2A activity in CIP2A - BP KO , LINC00665 ORF overexpressed ( OE ) respective control TNBC .

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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 ( ) of p - AKT from TNBC ( n = 20 ) with high or low micropeptide CIP2A - BP expression . Lower panels show higher - magnification of insets in upper panels . Right panel is of 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 were transfected with anti - CIP2A siRNAs . Whole - lysates of these were subjected to immunoblot with CIP2A , CIP2A - BP , PP2Ac , p - AKT , AKT β - actin antibodies .

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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 ) respective control MDA - MB - 231 were transfected with anti - CIP2A siRNAs . PP2A activity was performed to evaluate PP2A activity in indicated .

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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.


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

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

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

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

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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 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 of insets in upper panels . Right panel is of 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 injected with CIP2A - BP or svCIP2A - BP ( 10 mice per group ) . horizontal indicates time after mice were injected with via 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 histological of lungs of nude mice injected with MDA - MB - 231 CIP2A - BP or svCIP2A - BP via tail vein ; lung metastatic nodules were visualized 8 post - transplantation ( 5 mice per group ) . Right panel is of pulmonary metastases .

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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 - nanoscopy of HeLa . COX8A - SNAP was labeled with SNAP - SiR 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 visualized by FIB - . OM IBM are together shown as a transparent surface . Cristae of two mitochondria are depicted in 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 - recording as in ( A ) . In addition , mitochondrial was stained with PicoGreen imaged by confocal 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 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 labeled for COX8A - SNAP immunolabeled for Mic60 . Arrows mark Mic60 clusters in 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 MICOS mutants . Mic60 specific antibodies were used to pull - down Mic60 interacting from insolated mitochondria . * * Unspecific band , due to cross reaction of anti - Mic25 antibody with Mic19 .

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(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 MICOS mutants as analyzed by Seahorse Analyzer . Basal maximal OCR are shown relative to WT . Error bars : , n = 6 .

(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 MICOS mutant . were immunolabeled for TOM20 visualized by confocal microscopy . inset shows a magnification of respective overview image . Scale bar : 10 µm . ( D ) of mitochondrial networks as shown in ( C ) . evaluation was performed manually in a blinded approach on pre - defined criteria . Average SD of 3 independent biological replicates are shown ( > 170 per sample 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 recordings of WT MICOS - KO mutants , as indicated .

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


( B ) of overall cristae on recordings . Data information : n : number of mitochondrial

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


( C ) of detailed of CMs in MICOS mutants ( left ) . 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 recordings . number of CJs was manually determined normalized to of OM . Samples were compared to WT by a One - way ANOVA . Error bars : . 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 recordings . Red : Average . Error bars : SD . Samples were compared to WT by a One - way ANOVA . Data information : n : number of mitochondrial

(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 ( left ) , a Mic10 - KO ( center ) a Mic60 - KO ( right ) . OM is displayed in clear grey , side of IM that faces matrix is shown in blue . IM side that faces IMS is shown in 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 Mic10 - KO , as indicated . ( A ) Mitochondria were immunolabeled for Mic60 ATPB visualized by 2D STED nanoscopy .

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

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(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 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 Mic10 - KO . were labeled with Mitotracker Green . show maximum projections .

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


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

(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 were transfected with a plasmid for Mic10 - FLAG expression under control of a tetracycline - inducible promoter . Left panel : Mitochondria from noninduced . Right : Mitochondria from induced expressing Mic10 - FLAG .

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(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 ) were immunolabeled for Mic60 visualized by confocal microscopy before after induction of Mic60 expression for 48 h .

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(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 ) of MICOS after Mic60 induction . lysates were analyzed by western blotting at 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 . were induced with doxycycline hyclate for 72 h , immunolabeled for TOM20 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 ) of Mic60 - TO before after induction . ( D ) recordings of Mic60 - TO at indicated time points . ( E ) of cristae . ( F ) CJ frequency . number of CJs on recordings was normalized to of OM . At least 90 mitochondrial sections were analyzed for each sample . ( G ) of cristae of Mic60 - TO . 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 - nanoscopy of Mic10 - TO . expressing COX8A - SNAP were labeled with SNAP - SiR recorded with STED nanoscopy . ( A ) Cristae architecture in noninduced . ( 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 . were induced for 24 h immunolabeled for Mic60 Mic10 - FLAG . Upper row : with weak Mic10 expression level . Lower row : 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 ) of Mic10 - TO . ( D ) induced for indicated period of time were recorded with . Arrow indicates an intermediately shaped crista . ( E ) of cristae . n : Number of mitochondrial sections . ( F ) CJ frequency in Mic10 - TO . Number of CJs was normalized to of OM . same sections were analyzed as in ( E ) . ( G ) Diameter of CJs estimated on 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 - of Mic10 - TO . were induced for 0 h , 16 h 24 h . representative reconstructions show mitochondria from noninduced , rescued intermediate , as indicated . OM is shown together with IBM in clear grey , 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 . Mic10 expression was induced for 0 h 16 h . Shown are reconstructions of representative noninduced almost completely rescued mitochondria . OM is displayed in clear grey , side of IM that faces matrix is shown in blue . IM side that faces IMS is shown in 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 . WT Mic60 - KO were transfected with a scrambled control ( Ctrl . ) or siRNA pools against OPA1 ( OPA1 - KD ) for 5 d . ( A ) 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 ) recordings from OPA1 depleted WT Mic60 - KO . Arrows point to a septum .

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


( C , D ) of cristae of OPA1 deficient WT ( C ) Mic60 - KO ( D ) . number of CJs was normalized to of OM ( C ) . number of septated mitochondria in Mic60 - KO was estimated ( D ) .

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(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 . WT were transfected with a scrambled control ( E ) or siRNA pools against OPA1 ( F ) for 48 h . Reconstructions of representative mitochondria are displayed . OM is displayed in clear grey , side of IM that faces matrix is shown in blue . IM side that faces IMS is shown in 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 Mic10 - KO 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 ) Mic10 - KO ( B ) . were immunolabeled for Mic60 ATPB .

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

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(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 . were transfected with a scrambled control or siRNA pools against ATP5ME for 4 d . recording ( left ) of number of cristae per mitochondrial section ( right ) .

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

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

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(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 indicated from two independent experiments . Bottom : 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 .

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(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 ) lysosomal fractions ( Lys ) isolated from SH - SY5Y ( c ) or indicated mouse brain regions ( d ) , immunoblotted for indicated .

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

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

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(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 uptake calculated from of LRRK2 in immunoblots of lysosomes from starved rat livers , untreated or pretreated with protease inhibitors ( PI ) 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 without PI .

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(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 degradation of a radiolabeled pool of by intact lysosomes . Values are of degradation without LRRK2 ( n = 5 or 6 independent experiments ) .

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

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(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 RNase A ( e ) or LRRK2 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 : association expressed as a of 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 ) .

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(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 ) .

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

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

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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), 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 expressing GFP - tagged WT or G / S LRRK2 pulsed 24 h with doxycycline , 48 h later ( no longer undergoing de novo synthesis ) , ( b ) Same as in a , untreated ( Ctr ) or treated with interference for LAMP - 2A ( L2A ( − ) ) , pulsed as in a , sequentially collected immunoblotted . Top left panels : representative immunoblots for GFP . Actin is shown as a loading control . Bottom left panels , , corrected by of knockdown loading control ( n = 3 or 4 independent experiments ) . Top right : immunoblot for LAMP - 2A . Bottom right : of LRRK2 degraded per hour in 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 uptake calculated from of LRRK2 in immunoblots of lysosomes from starved rat livers , untreated or pretreated with protease inhibitors ( PI ) incubated with WT or G / S LRRK2 . Inset : representative immunoblot ( n = 9 independent experiments ) .

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(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 presence of increasing concentrations of WT or G / S LRRK2 . Inset : representative immunoblot n = 4 - 6 .

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(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 G / S LRRK2 to lysosomes in presence of GAPDH ( e ) or RNase A ( f ) . Top : representative immunoblots . Bottom : lysosome - bound LRRK2 expressed as a multiple of 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.


( g ) Immunoblot of rat brain lysosomes ( Lysosom ) incubated with WT or G / S LRRK2 as in c . Inputs , one - tenth of added .

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(g) Immunoblot of rat brain lysosomes (Lysosom) incubated with WT or G/S LRRK2 as in c. Inputs, one-tenth of protein added.


( h , i ) Rat brain lysosomes incubated as in d e , respectively . Left : representative immunoblots . Right : quantifications ( n = 3 or 4 independent experiments ) . values expressed as mean + s . e . m . ; differences with untreated samples ( * ) between WT LRRK2 G / S LRRK2 ( § ) were significant at P 0 . 05 ; n . s . , not significant ; ANOVA Bonferroni . Full - blots gels in Supplementary Figure 12 .

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(h,i) Rat brain lysosomes incubated as in d and e, respectively. Left: representative immunoblots. Right: quantifications (n = 3 or 4 independent experiments). All values expressed as mean + s.e.m.; differences with untreated samples (*) and between WT LRRK2 and G/S LRRK2 (§) were significant at P 0.05; n.s., not significant; ANOVA and Bonferroni test. Full-length blots and gels in Supplementary Figure 12.


( a ) Proteolysis in HEK293 stably transfected with an empty plasmid ( Ctr ) or plasmids expressing WT or G / S LRRK2 under control of a tet promoter , treated with doxycycline to activate expression then labeled with [ 3H ] leucine for 48 h . After extensive washing , degradation at indicated times was measured , in maintained either in presence ( + ) or absence ( - ) of serum , as amount of acid - precipitable radioactivity ( small peptides ) released into media . Values are expressed as of radioactivity in ( acid precipitable ) at time 0 ( n = 4 independent experiments , each in triplicate ) .

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(a) Proteolysis in HEK293 cells stably transfected with an empty plasmid (Ctr) or plasmids expressing WT or G/S LRRK2 under the control of a tet promoter, treated with doxycycline to activate protein expression and then labeled with [3H]leucine for 48 h. After extensive washing, protein degradation at the indicated times was measured, in cells maintained either in the presence (+) or absence (-) of serum, as the amount of acid-precipitable radioactivity (amino acids and small peptides) released into the media. Values are expressed as percentage of radioactivity in proteins (acid precipitable) at time 0 (n = 4 independent experiments, each in triplicate).


( b ) same , transiently transfected with a KFERQ - PA - mCherry1 reporter for CMA activity . After photoactivation , were maintained for 16 h in presence or absence of serum . Left : representative ; right column shows higher magnification of outlined regions . Arrows denote . Right : average number of per in each condition , measured in > 150 .

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(b) The same cells, transiently transfected with a KFERQ-PA-mCherry1 fluorescent reporter for CMA activity. After photoactivation, cells were maintained for 16 h in the presence or absence of serum. Left: representative micrographs; right column shows higher magnification images of outlined regions. Arrows denote puncta. Right: average number of puncta per cell in each condition, measured in >150 cells.


( c ) Cultured ventral midbrain dopaminergic neurons from Ctr from WT - LRRK2 G / S - LRRK2 transgenic mice , transduced with same reporter as in b co - stained with antibody to tyrosine hydroxylase ( TH ) . Nuclei are highlighted with DAPI ( 4 ′ , 6 - diamidino - 2 - phenylindole ) in blue . Left : representative . Right : average number of per in > 50 TH - positive . Scale bars , 10 μm . values are mean + s . e . m . ; differences with Ctr ( * ) between WT LRRK2 G / S LRRK2 ( § ) were significant at P 0 . 05 ; unpaired t - in a ANOVA Bonferroni in b , c .

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(c) Cultured ventral midbrain dopaminergic neurons from Ctr and from WT-LRRK2 and G/S-LRRK2 transgenic mice, transduced with the same reporter as in b and co-stained with antibody to tyrosine hydroxylase (TH). Nuclei are highlighted with DAPI (4′,6-diamidino-2-phenylindole) in blue. Left: representative images. Right: average number of puncta per cell in >50 TH-positive cells. Scale bars, 10 μm. All values are mean + s.e.m.; differences with Ctr (*) and between WT LRRK2 and G/S LRRK2 (§) were significant at P 0.05; unpaired t-test in a and ANOVA and Bonferroni test in b,c.


( a ) Immunoblots of rat liver lysosomes incubated with WT or G / S LRRK2 alone or with 1 mM GTP / or GAPDH . Duplicate samples are shown . Bottom : of lysosome - bound LRRK2 expressed as a multiple of amount bound without additions ( n = 6 independent experiments ) .

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(a) Immunoblots of rat liver lysosomes incubated with WT or G/S LRRK2 alone or with 1 mM GTP and/or GAPDH. Duplicate samples are shown. Bottom: quantification of lysosome-bound LRRK2 expressed as a multiple of the amount bound without additions (n = 6 independent experiments).


( b ) Effect of kinase inhibitor on WT , G / S , or kinase - dead mutant D / A LRRK2 incubated with rat liver lysosomes in presence or absence of GAPDH ( 5 μg ) . Right : expressed as a multiple of association without additions ( n = 3 - 5 independent experiments ) .

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(b) Effect of kinase inhibitor on WT, G/S, or the kinase-dead mutant D/A LRRK2 incubated with rat liver lysosomes in presence or absence of GAPDH (5 μg). Right: quantification expressed as a multiple of the association without additions (n = 3-5 independent experiments).


( c , d ) Transient transfection of HEK293 with myc - or GFP - tagged LRRK2 constructs indicated at top left in c . Full , full ; M , KFERQ - like motifs ( M1 - M8 ) . ( c ) Binding to GST - hsc70 . Left , representative immunoblots ; input lanes , one - tenth of input material . NT , non - transfected . Immunoblot for hsc70 is shown as loading control . Top right : of LRRK2 bound to hsc70 . Where indicated , samples were also incubated with RNase A to compete KFERQ - mediated binding of hsc70 ( n = 3 or 4 independent experiments ) . ( d ) Coimmunoprecipitation ( IP ) of hsc70 with anti - myc or anti - GFP in same . Inputs ( Inp ) , one - quarter of starting volume .

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(c,d) Transient transfection of HEK293 cells with the myc- or GFP-tagged LRRK2 constructs indicated at top left in c. Full, full length; M, KFERQ-like motifs (M1-M8). (c) Binding to GST-hsc70. Left, representative immunoblots; input lanes, one-tenth of input material. NT, non-transfected cells. Immunoblot for hsc70 is shown as loading control. Top right: quantification of LRRK2 bound to hsc70. Where indicated, samples were also incubated with RNase A to compete the KFERQ-mediated binding of hsc70 (n = 3 or 4 independent experiments). (d) Coimmunoprecipitation (IP) of hsc70 with anti-myc or anti-GFP in the same cells. Inputs (Inp), one-quarter of starting volume.


( e ) Binding or LRRK2 to rat liver lysosomes treated with indicated concentrations of trypsin . LAMP - 1 is shown to confirm integrity of lysosomes .

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(e) Binding or LRRK2 to rat liver lysosomes treated with the indicated concentrations of trypsin. LAMP-1 is shown to confirm the integrity of the lysosomes.


( f ) Co - IP of LAMP - 2A with anti - LRRK2 in lysosomes incubated with WT or G / S LRRK2 . Inp , one - quarter of starting sample ; IgG , immunoglobulin G . Values : LAMP - 2A recovered in IP ( IP L2A ) corrected for amount of LRRK2 pulled down expressed as a multiple of WT value .

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(f) Co-IP of LAMP-2A with anti-LRRK2 in lysosomes incubated with WT or G/S LRRK2. Inp, one-quarter of starting sample; IgG, immunoglobulin G. Values: LAMP-2A recovered in the IP (IP L2A) corrected for the amount of LRRK2 pulled down and expressed as a multiple of WT value.


( g , h ) Duplicate LAMP - 2A immunoblots of rat liver lysosomes incubated alone ( − ) or with WT or G / S LRRK2 ( g ) or with lysosomes from control ( Ctr ) or from WT - LRRK2 or G / S - LRRK2 transgenic mice ( h ) subjected to blue native electrophoresis . Arrow , 700 - kDa multimeric . values are mean + s . e . m . ; differences compared to none or untreated ( * ) or to WT or full - ( § ) were significant at P 0 . 05 ; unpaired t - in a ANOVA Bonferroni in b , c . Full - blots gels in Supplementary Figure 12 .

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(g,h) Duplicate LAMP-2A immunoblots of rat liver lysosomes incubated alone (−) or with WT or G/S LRRK2 (g) or with lysosomes from control (Ctr) or from WT-LRRK2 or G/S-LRRK2 transgenic mice (h) and subjected to blue native electrophoresis. Arrow, 700-kDa multimeric complex. All values are mean + s.e.m.; differences compared to none or untreated (*) or to WT or full-length protein (§) were significant at P 0.05; unpaired t-test in a and ANOVA and Bonferroni test in b,c. Full-length blots and gels in Supplementary Figure 12.


( a ) Immunofluorescence for LAMP - 2A LAMP - 1 of sections from dorsal motor nuclei of vagus nerves of brains from two different unaffected control ( Ctr ) individuals two individuals with Parkinson ' s disease with G / S LRRK2 mutation . Left : from representative fields . Right : number of per . Black dots , values in ; red lines , mean .

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(a) Immunofluorescence for LAMP-2A and LAMP-1 of sections from the dorsal motor nuclei of the vagus nerves of brains from two different unaffected control (Ctr) individuals and two individuals with Parkinson's disease with the G/S LRRK2 mutation. Left: images from representative fields. Right: number of fluorescent puncta per cell. Black dots, values in individual cells; red lines, mean.


( b ) Immunohistochemistry for LAMP - 2A in same samples .

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(b) Immunohistochemistry for LAMP-2A in the same samples.


( c ) Immunofluorescence for LAMP - 2A ( red ) LRRK2 ( green ) in same samples . Merged from two fluorophore channels are shown ( for colocalization , see Supplementary Fig . 7b ) . Scale bars , 10 μm . Values are mean + s . e . m . ; * P 0 . 05 , ANOVA Bonferroni .

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(c) Immunofluorescence for LAMP-2A (red) and LRRK2 (green) in the same samples. Merged images from the two fluorophore channels are shown (for colocalization quantification, see Supplementary Fig. 7b). Scale bars, 10 μm. Values are mean + s.e.m.; *P 0.05, ANOVA and Bonferroni test.


( a ) Top : LRRK2 immunoblot of rat liver lysosomes incubated with WT , G / S , D / A ( kinase - dead ) , I / T or R / C mutant LRRK2 , alone or in presence of protease inhibitors ( PI ) . Input lanes , one - quarter of input material . Bottom : of binding uptake ( calculated as in Fig . 2b ; n = 4 or 5 independent experiments ) .

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(a) Top: LRRK2 immunoblot of rat liver lysosomes incubated with WT, G/S, D/A (kinase-dead), I/T or R/C mutant LRRK2, alone or in the presence of protease inhibitors (PI). Input lanes, one-quarter of input material. Bottom: quantification of binding and uptake (calculated as in Fig. 2b; n = 4 or 5 independent experiments).


( b ) Left : immunoblots of lysosomes incubated as in a , alone or in presence of increasing concentrations of RNase A ( left ) or GAPDH ( right ) . Right : of LRRK2 bound , as a multiple of binding when incubated with lysosomes alone ( n = 3 independent experiments ) .

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(b) Left: immunoblots of lysosomes incubated as in a, alone or in the presence of increasing concentrations of RNase A (left) or GAPDH (right). Right: quantification of LRRK2 bound, as a multiple of the binding when incubated with lysosomes alone (n = 3 independent experiments).


( c ) Top : coimmunoprecipitation ( co - IP ) of LAMP - 2A ( L2A ) with anti - LRRK2 from lysosomes incubated with LRRK2proteins as in a . Co - immunoprecipitated fractions were immunoblotted for LAMP - 2A LRRK2 . Input : immunoblot for LAMP - 2A before affinity purification . Bottom : amount of LAMP - 2A coimmunoprecipitated , expressed as a multiple of value with WT LRRK2 ( n = 3 or 4 independent experiments ) .

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(c) Top: coimmunoprecipitation (co-IP) of LAMP-2A (L2A) with anti-LRRK2 from lysosomes incubated with LRRK2proteins as in a. Co-immunoprecipitated fractions were immunoblotted for LAMP-2A and LRRK2. Input: immunoblot for LAMP-2A before the affinity purification. Bottom: amount of LAMP-2A coimmunoprecipitated, expressed as a multiple of the value with WT LRRK2 (n = 3 or 4 independent experiments).


( d ) Co - IP of LAMP - 2A with anti - GFP in tet - on HEK293 expressing GFP - LRRK2 pulsed for 24 h with doxycycline . Inputs ( Inp ) , one - quarter of material added .

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(d) Co-IP of LAMP-2A with anti-GFP in tet-on HEK293 cells expressing GFP-LRRK2 proteins and pulsed for 24 h with doxycycline. Inputs (Inp), one-quarter of material added.


( e ) same as in d control transduced with KFERQ - PA - mCherry1 . Left : representative . Nuclei are highlighted with DAPI in blue . Right : after photoactivation 16 h of serum deprivation ( n > 150 ) . values are mean + s . e . m . Differences with WT ( * ) or with indicated mutants ( § ) were significant at P 0 . 05 ; unpaired t - in a - c ANOVA Bonferroni in e . Scale bars , 10 μm . Full - blots gels in Supplementary Figure 12 .

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(e) The same cells as in d and control cells transduced with KFERQ-PA-mCherry1. Left: representative images. Nuclei are highlighted with DAPI in blue. Right: Quantification after photoactivation and 16 h of serum deprivation (n > 150 cells). All values are mean + s.e.m. Differences with WT (*) or with the indicated mutants (§) were significant at P 0.05; unpaired t-test in a-c and ANOVA and Bonferroni test in e. Scale bars, 10 μm. Full-length blots and gels in Supplementary Figure 12.


( a ) α - syn immunoblot of rat liver lysosomes incubated with WT or mutant ( A53T ) α - syn alone ( − ) or with WT LRRK2 or G / S LRRK2 . Inp , input ; Mon , monomers . Right : of α - syn oligomers , expressed as a multiple of amounts in samples without LRRK2 ( n = 4 or 5 independent experiments ) .

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(a) α-syn immunoblot of rat liver lysosomes incubated with WT or mutant (A53T) α-syn alone (−) or with WT LRRK2 or G/S LRRK2. Inp, input; Mon, monomers. Right: quantification of α-syn oligomers, expressed as a multiple of amounts in samples without LRRK2 (n = 4 or 5 independent experiments).


( b - d ) Degradation of radiolabeled ( b ) , binding of GST - α - syn ( c ) of endogenous α - syn ( d ) in intact brain lysosomes ( Lysos ) from non - transgenic ( Ctr ) G / S LRRK2 transgenic mice . Bottom panel in c shows lower exposure to visualize monomers of α - syn . Homog , homogenates ; olig , oligomers ; I , input ( n = 3 independent experiments ) .

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(b-d) Degradation of radiolabeled proteins (b), binding of GST-α-syn (c) and levels of endogenous α-syn (d) in intact brain lysosomes (Lysos) from non-transgenic (Ctr) and G/S LRRK2 transgenic mice. Bottom panel in c shows lower exposure to visualize monomers of α-syn. Homog, homogenates; olig, oligomers; I, input (n = 3 independent experiments).


( e ) Ventral midbraindopaminergic neuronal cultures from control ( Ctr ) or WT - LRRK2 or G / S - LRRK2 transgenic mice untreated or treated with RNAi for LAMP - 2A ( L2A ( − ) ) . ( f ) Colocalization between α - syn total LAMP - 2 from in e ( n > 50 tyrosine hydroxylase ( TH ) - positive neurons ) . Total LAMP - 2 is not reduced in L2A knockdown because LAMP - 2B is upregulated .

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(e) Ventral midbraindopaminergic neuronal cultures from control (Ctr) or WT-LRRK2 or G/S-LRRK2 transgenic mice untreated or treated with RNAi for LAMP-2A (L2A(−)). (f) Colocalization between α-syn and total LAMP-2 from cells in e (n > 50 tyrosine hydroxylase (TH)-positive neurons). Total LAMP-2 is not reduced in L2A knockdown cells because LAMP-2B is upregulated.