( G ) show representative immunoblots analyzed using antibodies against the HA tag ( Nrxn1α - HA ) , the 3G10 epitope ( for the HS stub ) and V5 ( CA10 - V5 ) , as indicated . CA10 in input samples is shown for demonstration of equal expression . H ) Quantification of the 3G10 epitope signal normalized to HA ( for Nrxn1α - HA ) signals , respectively . Data shown are mean ± SEM of 3 biological replicates . * , p < 0 . 05 ; * * * , p < 0 . 001 by 2 - way ANOVA and Holm - Sidak tests .

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(G) show representative immunoblots analyzed using antibodies against the HA tag (Nrxn1α-HA), the 3G10 epitope (for the HS stub) and V5 (CA10-V5), as indicated. CA10 in input samples is shown for demonstration of equal expression. H) Quantification of the 3G10 epitope signal normalized to HA (for Nrxn1α-HA) signals, respectively. Data shown are mean ±SEM of 3 biological replicates. *, p< 0.05; ***, p<0.001 by 2-way ANOVA and Holm-Sidak tests.


( D ) Comparison of 1H - 13C HSQC spectra of Nrxn1 stalk ( red ) and Nrxn1 stalk / CA10 ( black ) at a 2 : 1 molar ratio ( 50 mM phosphate buffer , pH 7 . 4 , 0 . 5 mM d10 - DTT , 10 % D2O ) . Nrxn1 residues with stronger chemical shift changes are indicated blue boxes . Asterisk ( * ) indicates an impurity from glycerol .

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(D) Comparison of 1H-13C HSQC spectra of Nrxn1 stalk (red) and Nrxn1 stalk/CA10 (black) at a 2:1 molar ratio (50 mM phosphate buffer, pH 7.4, 0.5 mM d10-DTT, 10% D2O). Nrxn1 residues with stronger chemical shift changes are indicated blue boxes. Asterisk (*) indicates an impurity from glycerol.


( B - C ) Extracted ion chromatograms ( EIC ) of DDILVASAECPSDDE peptides and its glycosylated forms , from Nrxn1β expressed in HEK293 cells .

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(B-C) Extracted ion chromatograms (EIC) of DDILVASAECPSDDE peptides and its glycosylated forms, from Nrxn1β expressed in HEK293 cells.


( D ) Quantification , expressed as % of total occupancy of each glycosylation form , upon control and CA10 expression conditions . Bar graph shows means ± SEM of 4 biological replicates ; * , p < 0 . 05 by Mann - Whitney tests comparing the occupancy of each glycosylation with and without CA10 ( n = 4 biological replicates ) .

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(D) Quantification, expressed as % of total occupancy of each glycosylation form, upon control and CA10 expression conditions. Bar graph shows means ±SEM of 4 biological replicates; *, p<0.05 by Mann-Whitney tests comparing the occupancy of each glycosylation with and without CA10 (n=4 biological replicates).


( A ) Immunoblot α - showing neurexin isoform distribution in cortex , hippocampus , midbrain and cerebellum dissected from 8 - week - old mice . ( B ) Quantification of lower molecular weight neurexin ( LMW ; white arrowhead ) in relation to total levels of α - neurexins [ upper band ( line ) + LMW ] . Bar graphs show mean ± SEM of 3 independent replicates * * * , p < 0 . 001 by 2 - way ANOVA and Holm - Sidak tests , comparing to levels in cerebellum .

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(A) Immunoblot α-showing neurexin isoform distribution in cortex, hippocampus , midbrain and cerebellum dissected from 8-week-old mice. (B) Quantification of lower molecular weight neurexin (LMW; white arrowhead) in relation to total levels of α-neurexins [upper band (line) + LMW]. Bar graphs show mean ± SEM of 3 independent replicates***, p<0.001 by 2-way ANOVA and Holm-Sidak tests, comparing to levels in cerebellum.


( C ) α - Neurexin immunoprecipitated from forebrain ( Fb ) and cerebellum ( Cb ) using an anti - pan - neurexin antibody was heparinase - treated ( ' Heps ' ) and analyzed by immunoblotting with antibodies against neurexin and the 3G10 monoclonal ( to detect the HS stub ) . Stars indicate non - neurexin bands ( see results ) . ( D ) Quantification of low molecular weight neurexin ( LMW ; white arrowhead ) in relation to total levels of α - neurexins [ upper band ( line ) + LMW ] . Bar graphs show mean ± SEMof 3 biological replicates . * * * , p < 0 . 001 by 2 - way ANOVA and Holm - Sidak tests comparing forebrain and cerebellum . ( E ) Quantification of the 3G10 signal normalized to the signal of the corresponding neurexin band . Bar graphs show mean ± SEM of 3 biological replicates . * * * , p < 0 . 001 by 2 - way ANOVA and Holm - Sidak tests comparing forebrain and cerebellum .

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(C) α-Neurexin immunoprecipitated from forebrain (Fb) and cerebellum (Cb) using an anti-pan-neurexin antibody was heparinase-treated ('Heps') and analyzed by immunoblotting with antibodies against neurexin and the 3G10 monoclonal (to detect the HS stub). Stars indicate non-neurexin bands (see results). (D) Quantification of low molecular weight neurexin (LMW; white arrowhead) in relation to total levels of α-neurexins [upper band (line) + LMW]. Bar graphs show mean ±SEMof 3 biological replicates. ***, p<0.001 by 2-way ANOVA and Holm-Sidak tests comparing forebrain and cerebellum. (E) Quantification of the 3G10 signal normalized to the signal of the corresponding neurexin band. Bar graphs show mean ±SEM of 3 biological replicates. ***, p<0.001 by 2-way ANOVA and Holm-Sidak tests comparing forebrain and cerebellum.


( A - B ) Cell surface binding to endogenous neurexins . ( A ) Recombinant Fc - tagged LRRTM2 was added to day 10 hippocampal neurons expressing CA10 or control ( empty vector ) by lentiviral transduction . Surface - bound protein was quantified as the Fc signal of and normalized to a staining for endogenous neurexin ( n = 4 cultures ; three fields imaged and averaged per well ) . Scatchard analysis for LRRTM2 revealed an apparent Kd of 29 . 5 nM and a Bmax of 0 . 49 nM for control and Kd 35 . 3 nM , Bmax 0 . 28 nM upon expression of CA10 ; significant ( p = 0 . 0006 ) by non - linear regression . Data shown as mean ± SEM of biological replicates ( n = 4 ) . ( B ) Increasing amounts of recombinant , Fc - tagged Nlgn1 was added to immature hippocampal neurons expressing CA10 or control ( empty vector ) by lentiviral transduction , and analyzed as in ( A ) . The apparent Kd for Nlgn1 was 186 nM and Bmax 1 . 65 nM for control and Kd 223 nM , Bmax 1 . 49 nM when CA10 was overexpressed . The difference was not significant ( p = 0 . 20 ) by non - linear regression . Data shown as mean ± SEM of biological replicates ( n = 4 ) .

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(A-B) Cell surface binding to endogenous neurexins. (A) Recombinant Fc-tagged LRRTM2 was added to day 10 hippocampal neurons expressing CA10 or control (empty vector) by lentiviral transduction. Surface-bound protein was quantified as the Fc signal of and normalized to a staining for endogenous neurexin (n=4 cultures; three fields imaged and averaged per well). Scatchard analysis for LRRTM2 revealed an apparent Kd of 29.5 nM and a Bmax of 0.49 nM for control and Kd 35.3 nM, Bmax 0.28 nM upon expression of CA10; significant (p=0.0006) by non-linear regression. Data shown as mean ± SEM of biological replicates (n=4). (B) Increasing amounts of recombinant, Fc-tagged Nlgn1 was added to immature hippocampal neurons expressing CA10 or control (empty vector) by lentiviral transduction, and analyzed as in (A). The apparent Kd for Nlgn1 was 186 nM and Bmax 1.65 nM for control and Kd 223 nM, Bmax 1.49 nM when CA10 was overexpressed. The difference was not significant (p=0.20) by non-linear regression. Data shown as mean ± SEM of biological replicates (n=4).


( C - D ) Artificial synapse formation assays . ( C ) Confocal images of hippocampal neurons transduced to express CA10 - FLAG or control ( empty vector ) and co - cultured with HEK293 cells expressing GFP , Nlgn1 - YFP , LRRTM2 - YFP or TrkC - YFP . Cells were stained with antibodies against the synaptic marker SV2 ( red ) and somatodendritic MAP2 ( blue ) ; GFP / YFP is shown in green . Scale bar 20 μm . ( D ) Quantifications of SV2 signal overlapping GFP / YFP - positive cells . Data shown as means ± SEM with the n , reflecting number of independent cover slips analyzed , indicated in each bar with the total number of cells imaged in parenthesis . Comparisons were made by one - way ANOVA with Holm - Sidak ' s posthoc test for multiple comparisons between the indicated groups ( GFP condition was omitted from analysis due to unequal variance ; * * * , p < 0 . 001 .

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(C-D) Artificial synapse formation assays. (C) Confocal images of hippocampal neurons transduced to express CA10-FLAG or control (empty vector) and co-cultured with HEK293 cells expressing GFP, Nlgn1-YFP, LRRTM2-YFP or TrkC-YFP. Cells were stained with antibodies against the synaptic marker SV2 (red) and somatodendritic MAP2 (blue); GFP/YFP is shown in green. Scale bar 20 μm. (D) Quantifications of SV2 signal overlapping GFP/YFP-positive cells. Data shown as means ±SEM with the n, reflecting number of independent cover slips analyzed, indicated in each bar with the total number of cells imaged in parenthesis. Comparisons were made by one-way ANOVA with Holm-Sidak's posthoc test for multiple comparisons between the indicated groups (GFP condition was omitted from analysis due to unequal variance; ***, p<0.001.


( E - F ) Analysis of cerebellin ( Cbln ) - induced artificial synapse formation . ( E ) Confocal images of hippocampal neurons transduced to express CA10 - FLAG or control ( empty vector ) and co - cultured exposed to anti - V5 - coupled beads pre - incubated with recombinant V5 - tagged Cbln1 or empty anti - V5 beads . Cells were stained with antibodies against the synaptic marker synapsin ( green ) , somatodendritic MAP2 ( blue ) ; beads are colored red and indicated by arrowheads . Scale bar 10 μm . ( F ) Quantifications of the synapsin signal overlapping red beads . Data shown as means ± SEM ( n = 3 independent experiments , each data point corresponding to 58 - 171 analyzed beads ) , with comparisons made by two - way ANOVA with Holm - Sidak ' s posthoc test for multiple comparisons between the indicated groups .

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(E-F) Analysis of cerebellin(Cbln)-induced artificial synapse formation. (E) Confocal images of hippocampal neurons transduced to express CA10-FLAG or control (empty vector) and co-cultured exposed to anti-V5-coupled beads pre-incubated with recombinant V5-tagged Cbln1 or empty anti-V5 beads. Cells were stained with antibodies against the synaptic marker synapsin (green), somatodendritic MAP2 (blue); beads are colored red and indicated by arrowheads. Scale bar 10 μm. (F) Quantifications of the synapsin signal overlapping red beads. Data shown as means ±SEM (n=3 independent experiments, each data point corresponding to 58-171 analyzed beads), with comparisons made by two-way ANOVA with Holm-Sidak's posthoc test for multiple comparisons between the indicated groups.


( C ) Immunoblots analysis of TOMM34 expression in parental and metformin adaptive HCC cells .

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(C) Immunoblots analysis of TOMM34 expression in parental and metformin adaptive HCC cells.


( G ) The morphology ( Left ) , migration ( Middle ) and invasion ( Right ) of metformin adaptive Huh7 cells with ( shTOM ) or without ( shNC ) knockdown of TOMM34 are shown ( 5 × 104 cells for Transwell assay ) . Scale bars , ( Left ) 25 μm , ( Middle ) and ( Right ) 100 μm . ( n = 3 biological replicates , Two - way ANOVA ) . Data information : Data are presented as means ± S . D .

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(G) The morphology (Left), migration (Middle) and invasion (Right) of metformin adaptive Huh7 cells with (shTOM) or without (shNC) knockdown of TOMM34 are shown (5×104 cells for Transwell assay). Scale bars, (Left) 25 μm, (Middle) and (Right) 100 μm. (n=3 biological replicates, Two-way ANOVA). Data information: Data are presented as means ± S.D.


( I ) A tail vein injection model was established using adaptive Huh7 cells to assess cell migratory ability . Scale bars , ( Left ) 1000 μm , ( Right ) 25 μm . ( n = 6 mice for each group , Student ' s t - test ) . Data information : Data are presented as means ± S . D .

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(I) A tail vein injection model was established using adaptive Huh7 cells to assess cell migratory ability. Scale bars, (Left) 1000 μm, (Right) 25 μm. (n=6 mice for each group, Student's t-test). Data information: Data are presented as means ± S.D.


The effects of TOMM34 on HCC lung metastasis in the tail vein injection mouse model ( shTOM : shTOMM34 , TOMM34 knockdown cells ; Scale bars , ( Left ) 1000 μm , ( Right ) 25 μm . ( n = 5 mice for each group , Student ' s t - test ) . Data information : Data are presented as means ± S . D .

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The effects of TOMM34 on HCC lung metastasis in the tail vein injection mouse model (shTOM: shTOMM34, TOMM34 knockdown cells; Scale bars, (Left) 1000 μm, (Right) 25 μm. (n=5 mice for each group, Student's t-test). Data information: Data are presented as means ± S.D.


The effects of TOMM34 on HCC lung metastasis in the tail vein injection mouse model OE - TOM : OE - TOMM34 , TOMM34 overexpressed cells ) . Scale bars , ( Left ) 1000 μm , ( Right ) 25 μm . ( n = 5 mice for each group , Student ' s t - test ) . Data information : Data are presented as means ± S . D .

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The effects of TOMM34 on HCC lung metastasis in the tail vein injection mouse model OE-TOM: OE-TOMM34, TOMM34 overexpressed cells). Scale bars, (Left) 1000 μm, (Right) 25 μm. (n=5 mice for each group, Student's t-test). Data information: Data are presented as means ± S.D.


( L ) Western blot analysis showing the effects of TOMM34 on the expression of EMT markers in HCC cells ( shTOM : shTOMM34 , TOMM34 knockdown cells ; OE - TOM : OE - TOMM34 , TOMM34 overexpressed cells ) .

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(L) Western blot analysis showing the effects of TOMM34 on the expression of EMT markers in HCC cells (shTOM: shTOMM34, TOMM34 knockdown cells; OE-TOM: OE-TOMM34, TOMM34 overexpressed cells).


( M ) The protein expression of TOMM34 in clinical HCC samples with or without metastasis . Scale bars , ( Left ) 100 μm , ( Right ) 25 μm . ( n = 33 in metastasis present group , n = 53 in metastasis absent group , Student ' s t - test ) . Data information : Data are presented as means ± S . D .

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(M) The protein expression of TOMM34 in clinical HCC samples with or without metastasis. Scale bars, (Left) 100 μm, (Right) 25 μm. (n = 33 in metastasis present group, n = 53 in metastasis absent group, Student's t-test). Data information: Data are presented as means ± S.D.


( C ) Transmission electron microscopy ( TEM ) of Huh7 shNC and shTOM ( shTOMM34 ) cells . Yellow arrowheads indicate mitochondria , bule arrowheads indicate nucleus , red arrowheads indicate endoplasmic reticulum . Scale bars , ( Left ) 20 μm , ( Middle ) 4 μm , ( Right ) 1 μm . ( n = 5 biological replicates , One - way ANOVA ) . Data information : Data are presented as means ± S . D .

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(C) Transmission electron microscopy (TEM) of Huh7 shNC and shTOM (shTOMM34) cells. Yellow arrowheads indicate mitochondria, bule arrowheads indicate nucleus, red arrowheads indicate endoplasmic reticulum. Scale bars, (Left) 20 μm, (Middle) 4 μm, (Right) 1 μm. (n=5 biological replicates, One-way ANOVA). Data information: Data are presented as means ± S.D.


( G ) Western blot analysis showing the protein expression of the main mitochondrial respiratory chain complex subunit ( shTOM : shTOMM34 , TOMM34 knockdown cells ; OE - TOM : OE - TOMM34 , TOMM34 overexpressed cells ) .

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(G) Western blot analysis showing the protein expression of the main mitochondrial respiratory chain complex subunit (shTOM: shTOMM34, TOMM34 knockdown cells; OE-TOM: OE-TOMM34, TOMM34 overexpressed cells).


( B ) Co - IP assays indicate the interaction between exogenous TOMM34 and ATP5B in HEK293T cells .

(B) Co-IP assays indicate the interaction between exogenous TOMM34 and ATP5B in HEK293T cells.


( C ) Co - IP assays indicate the interaction between endogenous TOMM34 and ATP5B in Huh7 HCC cells .

(C) Co-IP assays indicate the interaction between endogenous TOMM34 and ATP5B in Huh7 HCC cells.


( E ) The mRNA and protein expression of ATP5B in indicated cells were determined by qPCR ( n = 3 technical replicates , Two - way ANOVA ) or immunoblotting ( shTOM : shTOMM34 , TOMM34 knockdown cells ; OE - TOM : OE - TOMM34 , TOMM34 - overexpressed cells ) . Data information : Data are presented as means ± S . D .

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(E) The mRNA and protein expression of ATP5B in indicated cells were determined by qPCR (n=3 technical replicates, Two-way ANOVA) or immunoblotting (shTOM: shTOMM34, TOMM34 knockdown cells; OE-TOM: OE-TOMM34, TOMM34-overexpressed cells). Data information: Data are presented as means ± S.D.


( F - G ) Western blot showing protein level of ATP5B in Huh7 ( F ) or PLC / PRF / 5 ( G ) cells treated with or without 10 mg / mL CHX for 12 hours or 25 μM MG132 for 8 hours . Relative grey value of ATP5B was calculated using Image J ( shTOM : shTOMM34 , TOMM34 knockdown cells ; OE - TOM : OE - TOMM34 , TOMM34 overexpressed cells ; CHX , cyclohexane ) . ( n = 3 technical replicates , Student ' s t - test ) . Data information : Data are presented as means ± S . D .

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(F-G) Western blot showing protein level of ATP5B in Huh7 (F) or PLC/PRF/5 (G) cells treated with or without 10 mg/mL CHX for 12 hours or 25 μM MG132 for 8 hours. Relative grey value of ATP5B was calculated using Image J (shTOM: shTOMM34, TOMM34 knockdown cells; OE-TOM: OE-TOMM34, TOMM34 overexpressed cells; CHX, cyclohexane). (n=3 technical replicates, Student's t-test). Data information: Data are presented as means ± S.D.


( H ) Tissue immunofluorescence assay showing the co - localization of TOMM34 and ATP5B at the invasive front of clinical HCC tissues . Scale bars , 20 μm .

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(H) Tissue immunofluorescence assay showing the co-localization of TOMM34 and ATP5B at the invasive front of clinical HCC tissues. Scale bars, 20 μm.


( I ) A positive correlation between TOMM34 and ATP5B levels in human HCC samples was shown by representative immunohistochemical images Scale bars , 50 μm . ( n = 72 ) .

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(I) A positive correlation between TOMM34 and ATP5B levels in human HCC samples was shown by representative immunohistochemical images Scale bars, 50 μm. (n=72).


( B - C ) Western blot showing the expression of indicated proteins in adaptive HCC cells with or without ATP5B silencing .

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(B-C) Western blot showing the expression of indicated proteins in adaptive HCC cells with or without ATP5B silencing.


( D - E ) Transwell assays showing the migration and invasion of adaptive HCC cells with or without the silence of ATP5B ( 5 × 104 adaptive Huh7 cells , 1 × 105 adaptive PLC / PRF / 5 cells ) , Scale bars , 100 μm . ( n = 3 biological replicates , Two - way ANOVA ) . Data information : Data are presented as means ± S . D .

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(D-E) Transwell assays showing the migration and invasion of adaptive HCC cells with or without the silence of ATP5B (5×104 adaptive Huh7 cells, 1×105 adaptive PLC/PRF/5 cells), Scale bars, 100 μm. (n=3 biological replicates, Two-way ANOVA). Data information: Data are presented as means ± S.D.


( G ) Western blot showing the expression of EMT markers in cells with ( shTOM ) or without ( shNC ) knockdown of TOMM34 or overexpression of ATP5B .

(G) Western blot showing the expression of EMT markers in cells with (shTOM) or without (shNC) knockdown of TOMM34 or overexpression of ATP5B.


( H ) Transwell assays showing the migration and invasion of indicated cells ( 5 × 104 cells ; shTOM , shTOMM34 , TOMM34 knockdown cells ) . Scale bars , 100 μm . ( n = 4 biological replicates , two - way ANOVA ) . Data information : Data are presented as means ± S . D .

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(H) Transwell assays showing the migration and invasion of indicated cells (5×104 cells; shTOM, shTOMM34, TOMM34 knockdown cells). Scale bars, 100 μm. (n=4 biological replicates, two-way ANOVA). Data information: Data are presented as means ± S.D.


( A - B ) Co - IP assay was performed to detect the interaction between TOMM34 and ATP5B treated with or without Gboxin for 24 hours in parental or adaptive PLC / PRF / 5 cells .

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(A-B) Co-IP assay was performed to detect the interaction between TOMM34 and ATP5B treated with or without Gboxin for 24 hours in parental or adaptive PLC/PRF/5 cells.


( C - D ) Immunoblotting of EMT markers of HCC cells treated with or without 200 nM Gboxin for 24 hours ( shTOM : shTOMM34 , TOMM34 knockdown cells ; OE - TOM : OE - TOMM34 , TOMM34 overexpressed cells ) .

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(C-D) Immunoblotting of EMT markers of HCC cells treated with or without 200 nM Gboxin for 24 hours (shTOM: shTOMM34, TOMM34 knockdown cells; OE-TOM: OE-TOMM34, TOMM34 overexpressed cells).


( E - F ) Transwell assay showing the migration and invasion of HCC cells treated with or without 200 nM Gboxin for 36 hours ( 5 × 104 cells ; shTOM : shTOMM34 , TOMM34 knockdown cells ) . Scale bars , 100 μm . ( n = 3 biological replicates , two - way ANOVA ) . Data information : Data are presented as means ± S . D .

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

(E-F) Transwell assay showing the migration and invasion of HCC cells treated with or without 200 nM Gboxin for 36 hours (5×104 cells; shTOM: shTOMM34, TOMM34 knockdown cells). Scale bars, 100 μm. (n=3 biological replicates, two-way ANOVA). Data information: Data are presented as means ± S.D.


( A RNA sequencing of CD31 and CD34 double - positive retinal endothelial cells from P5 wild - type mice . ( A ) RNA levels ( FPKM ; fragments per kilobase million ) of marker genes of endothelial cells ( green ; CD31 ( gene name PECAM1 ) , CD34 , von Willebrand Factor ( vWF ) , tyrosine - protein kinase receptor Tie2 ( TEK ) , VE - cadherin ( CDH5 ) , endoglin ( ENG ) , CD146 ( MCAM ) and VEGFR2 ( KDR ) ) , astrocytes ( blue , GFAP ) , immune cells ( red ; T - cells ( CD3E ) , B - cells ( CD19 ) , all leucocytes ( CD45 , PTPRC ) , and monocytes / macrophages ( F4 / 80 , EMR1 ) ) , Müller glia cells ( orange ; aquaporin 4 ( AQP4 ) ) , neurons ( yellow ; retinal ganglion cells ( RNA binding fox - 1 homolog 3 , RBFOX3 ) , amacrine cells ( parvalbumin , PVALB ) , bipolar cells ( PKC - α , PRKCA ) , horizontal cell ( calbindin , CALB1 ) , photoreceptors ( rods , CD73 ( NT5E ) ; cones , transducing ( GNAT1 ) ) , and retinal pigment epithelial cells ( magenta ; retinal pigment epithelium - specific 65 kDa protein ( RPE65 ) .

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(A RNA sequencing of CD31 and CD34 double-positive retinal endothelial cells from P5 wild-type mice. (A) RNA levels (FPKM; fragments per kilobase million) of marker genes of endothelial cells (green; CD31 (gene name PECAM1), CD34, von Willebrand Factor (vWF), tyrosine-protein kinase receptor Tie2 (TEK), VE-cadherin (CDH5), endoglin (ENG), CD146 (MCAM) and VEGFR2 (KDR)), astrocytes (blue, GFAP), immune cells (red; T-cells (CD3E), B-cells (CD19), all leucocytes (CD45, PTPRC), and monocytes/macrophages (F4/80, EMR1)), Müller glia cells (orange; aquaporin 4 (AQP4)), neurons (yellow; retinal ganglion cells (RNA binding fox-1 homolog 3, RBFOX3), amacrine cells (parvalbumin, PVALB), bipolar cells (PKC-α, PRKCA), horizontal cell (calbindin, CALB1), photoreceptors (rods, CD73 (NT5E); cones, transducing (GNAT1)), and retinal pigment epithelial cells (magenta; retinal pigment epithelium-specific 65 kDa protein (RPE65).


B ) RNA sequencing of CD31 and CD34 double - positive retinal endothelial cells from P5 wild - type mice . ( B ) RNA levels of VASP ( red ) , EVL ( green ) and Mena ( blue ) in the CD31 and CD34 double - positive P5 retinal endothelial cells . Error bars represent SEM ; n = 18 animals ( 36 retinas ) from six independent litters ; four independent experiments . * * * p < 0 . 001 , one - way ANOVA with Bonferroni ' s multi comparison test .

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B) RNA sequencing of CD31 and CD34 double-positive retinal endothelial cells from P5 wild-type mice. (B) RNA levels of VASP (red), EVL (green) and Mena (blue) in the CD31 and CD34 double-positive P5 retinal endothelial cells. Error bars represent SEM; n = 18 animals (36 retinas) from six independent litters; four independent experiments. ***p<0.001, one-way ANOVA with Bonferroni's multi comparison test.


( C - E ) Staining of Ena / VASP proteins in blood vessels of P5 mouse retinas . P5 wild - type mouse retinas were fixed and stained with isolectin B4 ( IB4 , green ) to visualize endothelial cells and VASP - ( C ) , EVL - ( D ) , or Mena - specific ( E ) antibodies ( red ) . Yellow color in the merged images indicates the expression of VASP and EVL proteins in endothelial cells . No Mena protein expression was detected in P5 retinal endothelial cells ( E ) . Representative images from three independent experiments are shown . Scale bars , 10 µm .

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(C-E) Staining of Ena/VASP proteins in blood vessels of P5 mouse retinas. P5 wild-type mouse retinas were fixed and stained with isolectin B4 (IB4, green) to visualize endothelial cells and VASP - (C), EVL- (D), or Mena-specific (E) antibodies (red). Yellow color in the merged images indicates the expression of VASP and EVL proteins in endothelial cells. No Mena protein expression was detected in P5 retinal endothelial cells (E). Representative images from three independent experiments are shown. Scale bars, 10 µm.


( C - E ) Staining of Ena / VASP proteins in blood vessels of P5 mouse retinas . P5 wild - type mouse retinas were fixed and stained with isolectin B4 ( IB4 , green ) to visualize endothelial cells and

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(C-E) Staining of Ena/VASP proteins in blood vessels of P5 mouse retinas. P5 wild-type mouse retinas were fixed and stained with isolectin B4 (IB4, green) to visualize endothelial cells and VASP


( A ) Wild - type and EVL - / - P5 retinas were digested , labelled with CD31 - , CD34 - and EVL - specific antibodies and analyzed by flow cytometry . Endothelial cells were defined as CD31 / CD34 - double positive cells ( gate indicated in the left panel ) and analyzed for EVL expression ( middle panel ) . Mean fluorescence intensity ( MFI ) of EVL in wild - type ( magenta ) and EVL - / - ( black ) cells is shown in the right panel . WT : n = 3 , EVL - / - : n = 2 ; two retinas per animal . Error bars represent SEM .

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(A) Wild-type and EVL-/- P5 retinas were digested, labelled with CD31-, CD34- and EVL-specific antibodies and analyzed by flow cytometry. Endothelial cells were defined as CD31/CD34-double positive cells (gate indicated in the left panel) and analyzed for EVL expression (middle panel). Mean fluorescence intensity (MFI) of EVL in wild-type (magenta) and EVL-/- (black) cells is shown in the right panel. WT: n=3, EVL-/-: n=2; two retinas per animal. Error bars represent SEM.


( B - C ) EVL protein expression in sparse / migrating wild - type ( WT ) and EVL - / - mouse brain endothelial cells ( MBEC ) and mouse lung endothelial cells ( MLEC ) . Actin was used as loading control . Western blots are representative of three independent experiments .

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(B-C) EVL protein expression in sparse/migrating wild-type (WT) and EVL-/- mouse brain endothelial cells (MBEC) and mouse lung endothelial cells (MLEC). Actin was used as loading control. Western blots are representative of three independent experiments.


( D ) MLEC from wild - type ( upper panel ) and EVL - / - ( lower panel ) mice stimulated with 10 ng / ml VEGF were stained for actin ( cyan ) and EVL ( magenta ) . Asterisks indicate focal adhesions , white arrows indicate filopodia and white arrowheads indicate the leading edge of lamellipodia . Representative images from four independent experiments are shown . Scale bar , 20 µm .

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(D) MLEC from wild-type (upper panel) and EVL-/- (lower panel) mice stimulated with 10 ng/ml VEGF were stained for actin (cyan) and EVL (magenta). Asterisks indicate focal adhesions, white arrows indicate filopodia and white arrowheads indicate the leading edge of lamellipodia. Representative images from four independent experiments are shown. Scale bar, 20 µm.


( A ) Isolectin B4 stained vasculature in whole mount retinas of wild - type ( WT ) and global EVL - / - mice on postnatal days 3 , 5 and 7 ( P3 , P5 , P7 ) assessed by confocal microscopy . Scale bars 200 µm .

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(A) Isolectin B4 stained vasculature in whole mount retinas of wild-type (WT) and global EVL-/- mice on postnatal days 3, 5 and 7 (P3, P5, P7) assessed by confocal microscopy. Scale bars 200 µm.


( B ) Analysis of the radial vascular outgrowth relative to retinal radius and normalized to wild - type littermates . ( C ) Impact of individual and combined VASP / EVL deletion on sprouting angiogenesis in the postnatal mouse retina at P5 ( EVL - deficient animals ( E - / - ) , green ; VASP - deficient animals ( V - / - ) , red ; VASP / EVL - double deficient animals ( EV - / - ) , orange ) .

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(B) Analysis of the radial vascular outgrowth relative to retinal radius and normalized to wild-type littermates. (C) Impact of individual and combined VASP/EVL deletion on sprouting angiogenesis in the postnatal mouse retina at P5 (EVL-deficient animals (E-/-), green; VASP-deficient animals (V-/-), red; VASP/EVL-double deficient animals (EV-/-), orange).


( B ) Analysis of the radial vascular outgrowth relative to retinal radius and normalized to wild - type littermates .

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(B) Analysis of the radial vascular outgrowth relative to retinal radius and normalized to wild-type littermates.


The retinal vasculature of endothelial cell specific EVL knockout mice ( EVLΔEC ) and littermate controls ( EVLfl / fl ) on P5 was analyzed by Isolectin B4 staining .

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The retinal vasculature of endothelial cell specific EVL knockout mice (EVLΔEC) and littermate controls (EVLfl/fl) on P5 was analyzed by Isolectin B4 staining.


Radial outgrowth of the retinal vasculature ; in global EVL - / - mice are shown for comparison ; 4 - 5 independent experiments .

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Radial outgrowth of the retinal vasculature; in global EVL-/- mice are shown for comparison; 4 - 5 independent experiments.


( C ) Quantification of the branch points in a field of 170 μm x 300 μm directly behind the vascular front . ( D ) Tipp cells numbers per field of view ( 387 , 5 x 387 , 5 µm ) .

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(C) Quantification of the branch points in a field of 170 μm x 300 μm directly behind the vascular front. (D) Tipp cells numbers per field of view (387,5 x 387,5 µm).


( C ) Quantification of the branch points in a field of 170 μm x 300 μm directly behind the vascular front .

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(C) Quantification of the branch points in a field of 170 μm x 300 μm directly behind the vascular front.


( E ) Filopodia numbers normalized to the length of the angiogenic front .

(E) Filopodia numbers normalized to the length of the angiogenic front.


( F ) Endothelial cell proliferation analyzed by BrdU incorporation ( red ) of ERG positive ( blue ) endothelial nuclei .

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(F) Endothelial cell proliferation analyzed by BrdU incorporation (red) of ERG positive (blue) endothelial nuclei.


CD31 and CD34 double positive endothelial cells were isolated from wild - type or EVL - / - P5 retinas by FACS and analyzed by RNA - sequencing . Two independent experiments ; in total 18 retinas from WT mice and 18 retinas from EVL - / - mice from three different litters , each . ( A ) RNA levels ( FPKM ) of VASP , EVL and Mena in endothelial cells from wild - type or EVL - / - retinas . Error bars represent SEM .

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CD31 and CD34 double positive endothelial cells were isolated from wild-type or EVL-/- P5 retinas by FACS and analyzed by RNA-sequencing. Two independent experiments; in total 18 retinas from WT mice and 18 retinas from EVL-/- mice from three different litters, each. (A) RNA levels (FPKM) of VASP, EVL and Mena in endothelial cells from wild-type or EVL-/- retinas. Error bars represent SEM.


CD31 and CD34 double positive endothelial cells were isolated from wild - type or EVL - / - P5 retinas by FACS and analyzed by RNA - sequencing . Two independent experiments ; in total 18 retinas from WT mice and 18 retinas from EVL - / - mice from three different litters , each .

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CD31 and CD34 double positive endothelial cells were isolated from wild-type or EVL-/- P5 retinas by FACS and analyzed by RNA-sequencing. Two independent experiments; in total 18 retinas from WT mice and 18 retinas from EVL-/- mice from three different litters, each.


( D ) Analysis of mRNA levels of Esm1 , Ptgs2 ( cyclooxygenase 2 ) , serpine 1 and paxillin in EVL - deficient MLEC relative to WT controls set to 100 . n = 3 - 4 independent experiments , error bars represent SEM , one sample t - test , * p < 0 . 05 ; * * p < 0 . 01 .

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(D) Analysis of mRNA levels of Esm1, Ptgs2 (cyclooxygenase 2), serpine 1 and paxillin in EVL-deficient MLEC relative to WT controls set to 100. n = 3-4 independent experiments, error bars represent SEM, one sample t-test, *p<0.05; **p<0.01.


( E ) ESM1 protein expression in P5 retinas of wild - type ( WT ) and global EVL - / - mice . Retinas were fixed and stained with isolectin B4 ( IB4 , green ) to visualize endothelial cells and antibodies directed against the tip cell marker ESM1 ( red ) . Representative images from three independent experiments are shown . Scale bars , 100 µm .

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(E) ESM1 protein expression in P5 retinas of wild-type (WT) and global EVL-/- mice. Retinas were fixed and stained with isolectin B4 (IB4, green) to visualize endothelial cells and antibodies directed against the tip cell marker ESM1 (red). Representative images from three independent experiments are shown. Scale bars, 100 µm.


( F ) Analysis of retinal ESM1 protein levels normalized to wild - type littermates . Error bars represent SEM ; * * * p < 0 . 001 , unpaired Student ' s t - test ; three different litters .

(F) Analysis of retinal ESM1 protein levels normalized to wild-type littermates. Error bars represent SEM; ***p<0.001, unpaired Student's t-test; three different litters.


( A ) Proliferation of WT and EVL - / - MLEC was assessed by BrdU incorporation . The number of BrdU positive cells ( red ) was normalized to the total number of cells ( white ) . Eight fields of view per condition and genotype for each of 5 independent WT and EVL - / - cell batches ; error bars represent SEM ; * * p < 0 . 01 , * * * p < 0 . 001 , # p = 0 . 171 non - significant vs . Basal EVL - / - , one - way ANOVA with Bonferroni ' s multi comparison test ; scale bar = 50 µm .

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(A) Proliferation of WT and EVL-/- MLEC was assessed by BrdU incorporation. The number of BrdU positive cells (red) was normalized to the total number of cells (white). Eight fields of view per condition and genotype for each of 5 independent WT and EVL-/- cell batches; error bars represent SEM; **p<0.01, ***p<0.001, #p=0.171 non-significant vs. Basal EVL-/-, one-way ANOVA with Bonferroni's multi comparison test; scale bar = 50 µm.


( C ) Sprouting of WT and EVL - / - primary MLEC spheres in the absence ( Basal ) or presence of VEGF ( 50 ng / ml ) ; asterisks indicate sprouts ; scale bars , 100 µm . ( D ) Solid bars represent proportion of WT and EVL - / - MLEC spheres forming sprouts in the absence ( − ) or presence ( + ) of VEGF . n - numbers are indicated ; n . s . non - significant ( p = 0 . 557 ) ; * * * p < 0 . 001 , Fisher ' s exact test . ( E ) Quantification of MLEC sphere sprout length without or with VEGF . Dots represent individual sprouts . Horizontal lines represent mean , error bars represent SEM . n = 20 - 30 beads ( from three independent experiments ) . * * * p < 0 . 001 , n . s . non - significant ( p = 0 . 543 ) , one - way ANOVA with Bonferroni ' s multi comparison test .

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(C) Sprouting of WT and EVL-/- primary MLEC spheres in the absence (Basal) or presence of VEGF (50 ng/ml); asterisks indicate sprouts; scale bars, 100 µm. (D) Solid bars represent proportion of WT and EVL-/- MLEC spheres forming sprouts in the absence (−) or presence (+) of VEGF. n-numbers are indicated; n.s. non-significant (p=0.557); ***p<0.001, Fisher's exact test. (E) Quantification of MLEC sphere sprout length without or with VEGF. Dots represent individual sprouts. Horizontal lines represent mean, error bars represent SEM. n = 20-30 beads (from three independent experiments). ***p<0.001, n.s. non-significant (p=0.543), one-way ANOVA with Bonferroni's multi comparison test.


( C ) Sprouting of WT and EVL - / - primary MLEC spheres in the absence ( Basal ) or presence of VEGF ( 50 ng / ml ) ; asterisks indicate sprouts ; scale bars , 100 µm .

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(C) Sprouting of WT and EVL-/- primary MLEC spheres in the absence (Basal) or presence of VEGF (50 ng/ml); asterisks indicate sprouts; scale bars, 100 µm.


( A ) VEGFR2 protein levels in wild - type ( WT ) and EVL - deficient ( EVL - / - ) endothelial cells . Actin was used as loading control . The lower panel shows quantification of VEGFR2 to actin ratios from five independent cell batches ; error bars represent SEM ; n . s . non - significant ( p = 0 . 388 ) , unpaired Student ' s t - test .

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(A) VEGFR2 protein levels in wild-type (WT) and EVL-deficient (EVL-/-) endothelial cells. Actin was used as loading control. The lower panel shows quantification of VEGFR2 to actin ratios from five independent cell batches; error bars represent SEM; n.s. non-significant (p=0.388), unpaired Student's t-test.


( B ) Antibody feeding assay to monitor VEGFR2 internalization after VEGF stimulation . Left panel : schematic diagram showing the two step staining procedure of surface and internalized VEGFR2 antibodies , which are displayed in yellow and green in the merged confocal images of WT and EVL - / - endothelial cells ( middle panel ) , respectively ; scale bar , 10 µm . The bar graphs show quantification of the number of VEGFR2 clusters and the ratio of internalized to total VEGFR2 ratios normalized to wild - type ; n = 4 - 6 independent cell batches , error bars represent SEM ; * * p < 0 . 01 , * * * p < 0 . 001 , unpaired Student ' s t - test .

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(B) Antibody feeding assay to monitor VEGFR2 internalization after VEGF stimulation. Left panel: schematic diagram showing the two step staining procedure of surface and internalized VEGFR2 antibodies, which are displayed in yellow and green in the merged confocal images of WT and EVL-/- endothelial cells (middle panel), respectively; scale bar, 10 µm. The bar graphs show quantification of the number of VEGFR2 clusters and the ratio of internalized to total VEGFR2 ratios normalized to wild-type; n = 4-6 independent cell batches, error bars represent SEM; **p<0.01, ***p<0.001, unpaired Student's t-test.


( A ) Western blots and quantification of VEGFR2 phosphorylation levels relative to total VEGFR2 levels in WT and EVL - / - endothelial cells under basal and VEGF - stimulated conditions ( 80 ng / ml ) ; n = 5 independent cell batches , error bars represent SEM ; * p < 0 . 05 , one - way ANOVA with Bonferroni ' s multi comparison test .

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(A) Western blots and quantification of VEGFR2 phosphorylation levels relative to total VEGFR2 levels in WT and EVL-/- endothelial cells under basal and VEGF-stimulated conditions (80 ng/ml); n = 5 independent cell batches, error bars represent SEM; *p<0.05, one-way ANOVA with Bonferroni's multi comparison test.


( B ) Western blots and quantification of ERK1 / 2 phosphorylation levels relative to total ERK levels in WT and EVL - / - endothelial cells under basal and VEGF - stimulated conditions ( 80 ng / ml ) ; n = 6 independent cell batches , error bars represent SEM ; * p < 0 . 05 , one - way ANOVA with Bonferroni ' s multi comparison test .

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(B) Western blots and quantification of ERK1/2 phosphorylation levels relative to total ERK levels in WT and EVL-/- endothelial cells under basal and VEGF-stimulated conditions (80 ng/ml); n = 6 independent cell batches, error bars represent SEM; *p<0.05, one-way ANOVA with Bonferroni's multi comparison test.


( C ) ERK1 / 2 phosphorylation in P5 retinas of wild - type ( WT ) and global EVL - / - mice . Retinas were fixed and stained with isolectin B4 ( IB4 , green ) to visualize endothelial cells and antibodies directed against phospho - ERK1 / 2 ( red ) . Representative images from three independent experiments are shown . Scale bars , 100 µm . ( D ) Analysis of endothelial ERK1 / 2 - phosphorylation normalized to wild - type littermates . Error bars represent SEM ; * * * p < 0 . 001 , unpaired Student ' s t - test ; two different litters .

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(C) ERK1/2 phosphorylation in P5 retinas of wild-type (WT) and global EVL-/- mice. Retinas were fixed and stained with isolectin B4 (IB4, green) to visualize endothelial cells and antibodies directed against phospho-ERK1/2 (red). Representative images from three independent experiments are shown. Scale bars, 100 µm. (D) Analysis of endothelial ERK1/2-phosphorylation normalized to wild-type littermates. Error bars represent SEM; ***p<0.001, unpaired Student's t-test; two different litters.


( C ) ERK1 / 2 phosphorylation in P5 retinas of wild - type ( WT ) and global EVL - / - mice . Retinas were fixed and stained with isolectin B4 ( IB4 , green ) to visualize endothelial cells and antibodies directed against phospho - ERK1 / 2 ( red ) . Representative images from three independent experiments are shown . Scale bars , 100 µm .

(C) ERK1/2 phosphorylation in P5 retinas of wild-type (WT) and global EVL-/- mice. Retinas were fixed and stained with isolectin B4 (IB4, green) to visualize endothelial cells and antibodies directed against phospho-ERK1/2 (red). Representative images from three independent experiments are shown. Scale bars, 100 µm.


( A - C ) MITOL - HA formed small dot - like structures following CCCP treatment . HeLa cells transiently expressing Flag - Parkin and MITOL - HA were treated with 15 µM CCCP for 3 hours , and then subjected to immunocytochemistry . MITOL and Tom20 ( mitochondrial marker ; A ) signals co - localized well without CCCP treatment , whereas the MITOL - positive small dots were not coincident with Tom20 , Sec61β ( ER marker ; B ) , or LAMP1 ( lysosomal marker ; C ) after CCCP treatment . Scale bars , 10 µm . ( D ) MITOL - HA co - localized with catalase ( peroxisome marker ) following CCCP treatment . Higher magnification images of the boxed regions are shown in the small panel . Scale bars , 10 µm . Arrowheads indicate representative examples of MITOL - HA co - localization with catalase . ( E ) Correlation statistics for the localization of MITOL - HA and Tom20 , Sec61β , LAMP1 , or catalase . Dots indicate individual Pearson correlation coefficient data points . In the box - plots , the center lines indicate the medians , the box limits indicate the 25th and 75th percentiles as determined in the R software package , and the whiskers extend 1 . 5 times the interquartile range from the 25th and 75th percentiles . Means and the number of samples are shown on the box and X - axis , respectively . Statistical significance was calculated using a one - tailed Welch ' s t - test . ( F ) The line - graph shows a line scan of fluorescence through three MITOL - positive peroxisomes ( red bar in Fig 1D ) that clearly indicates co - localization of MITOL ( green line ) and catalase ( magenta line ) .

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

(A-C) MITOL-HA formed small dot-like structures following CCCP treatment. HeLa cells transiently expressing Flag-Parkin and MITOL-HA were treated with 15 µM CCCP for 3 hours, and then subjected to immunocytochemistry. MITOL and Tom20 (mitochondrial marker; A) signals co-localized well without CCCP treatment, whereas the MITOL-positive small dots were not coincident with Tom20, Sec61β (ER marker; B), or LAMP1 (lysosomal marker; C) after CCCP treatment. Scale bars, 10 µm. (D) MITOL-HA co-localized with catalase (peroxisome marker) following CCCP treatment. Higher magnification images of the boxed regions are shown in the small panel. Scale bars, 10 µm. Arrowheads indicate representative examples of MITOL-HA co-localization with catalase. (E) Correlation statistics for the localization of MITOL-HA and Tom20, Sec61β, LAMP1, or catalase. Dots indicate individual Pearson correlation coefficient data points. In the box-plots, the center lines indicate the medians, the box limits indicate the 25th and 75th percentiles as determined in the R software package, and the whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles. Means and the number of samples are shown on the box and X-axis, respectively. Statistical significance was calculated using a one-tailed Welch's t-test. (F) The line-graph shows a line scan of fluorescence through three MITOL-positive peroxisomes (red bar in Fig 1D) that clearly indicates co-localization of MITOL (green line) and catalase (magenta line).


( G ) Peroxisomal membrane protein ( PMP ) 34 - FusionRed also co - localized with MITOL - GFP in Parkin - expressing HeLa cells after 3 hours of CCCP treatment . Higher magnification images of the boxed regions are shown in the small panel . Scale bars , 10 µm . ( H ) Correlation statistics for the localization of MITOL - GFP and PMP34 - FusionRed . Dots indicate individual Pearson correlation coefficient data points . In the box - plots , the center lines indicate the medians , box limits indicate the 25th and 75th percentiles as determined in the R software package , and the whiskers extend 1 . 5 times the interquartile range from the 25th and 75th percentiles . Means and the number of samples are shown on the box and X - axis , respectively . Statistical significance was calculated using a one - tailed Welch ' s t - test .

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

(G) Peroxisomal membrane protein (PMP) 34-FusionRed also co-localized with MITOL-GFP in Parkin-expressing HeLa cells after 3 hours of CCCP treatment. Higher magnification images of the boxed regions are shown in the small panel. Scale bars, 10 µm. (H) Correlation statistics for the localization of MITOL-GFP and PMP34-FusionRed. Dots indicate individual Pearson correlation coefficient data points. In the box-plots, the center lines indicate the medians, box limits indicate the 25th and 75th percentiles as determined in the R software package, and the whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles. Means and the number of samples are shown on the box and X-axis, respectively. Statistical significance was calculated using a one-tailed Welch's t-test.


( A ) MITOL - HA did not move to peroxisomes , but was rather retained on mitochondria even after CCCP treatment in HeLa cells lacking endogenous Parkin . Wild - type HeLa cells or HeLa cells stably expressing GFP - Parkin were transfected with MITOL - HA , treated with 15 µM CCCP for 3 hours , and then subjected to immunocytochemistry with anti - HA and anti - Tom20 antibodies . Higher magnification images of the boxed regions are shown in the small panel . Scale bars , 10 µm .

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 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

(A) MITOL-HA did not move to peroxisomes, but was rather retained on mitochondria even after CCCP treatment in HeLa cells lacking endogenous Parkin. Wild-type HeLa cells or HeLa cells stably expressing GFP-Parkin were transfected with MITOL-HA, treated with 15 µM CCCP for 3 hours, and then subjected to immunocytochemistry with anti-HA and anti-Tom20 antibodies. Higher magnification images of the boxed regions are shown in the small panel. Scale bars, 10 µm.


( B ) Correlation statistics for the localization of MITOL - HA and Tom20 in the absence or presence of GFP - Parkin . Dots indicate individual Pearson correlation coefficient data points . In the box - plots , the center lines indicate the medians , the box limits indicate the 25th and 75th percentiles as determined in the R software package , and the whiskers extend 1 . 5 times the interquartile range from the 25th and 75th percentiles . Means and the number of samples are shown on the box and X - axis , respectively . Statistical significance was calculated using a one - tailed Welch ' s t - test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Correlation statistics for the localization of MITOL-HA and Tom20 in the absence or presence of GFP-Parkin. Dots indicate individual Pearson correlation coefficient data points. In the box-plots, the center lines indicate the medians, the box limits indicate the 25th and 75th percentiles as determined in the R software package, and the whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles. Means and the number of samples are shown on the box and X-axis, respectively. Statistical significance was calculated using a one-tailed Welch's t-test.


( C ) MITOL was not degraded following mitochondrial depolarization . HeLa cells stably expressing 3Flag - MITOL were transfected with a GFP - Parkin plasmid or the pEGFP - C1 vector , treated with 15 µM CCCP + / - 10 µM MG132 for 3 hours , and then immunoblotted with anti - Flag , anti - ubiquitin , and anti - tubulin antibodies . Black arrowheads indicate 3Flag - MITOL in the upper panel and mono - ubiquitin in the middle panel , respectively .

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

(C) MITOL was not degraded following mitochondrial depolarization. HeLa cells stably expressing 3Flag-MITOL were transfected with a GFP-Parkin plasmid or the pEGFP-C1 vector, treated with 15 µM CCCP +/- 10 µM MG132 for 3 hours, and then immunoblotted with anti-Flag, anti-ubiquitin, and anti-tubulin antibodies. Black arrowheads indicate 3Flag-MITOL in the upper panel and mono-ubiquitin in the middle panel, respectively.


( D ) The total cellular amount of MITOL was not dramatically reduced following extended valinomycin treatment . Immunoblotting combined with fractionation analysis showed that Mitofusin2 ( MFN2 ) underwent rapid degradation within 3 hours of valinomycin treatment , in particular in the 3 , 000 g pellet ( mitochondria - rich fractions ) . Cytochrome c oxidase subunit 2 ( MTCO2 , inner mitochondrial protein ) was significantly reduced at 24 hours 10 μM valinomycin treatment . In contrast to those two proteins , MITOL degradation was minimal . Note that the chemical apoptosis inhibitor ZVAD - FMK ( 10 μM ) was added to cells along with valinomycin to prevent cell death .

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

(D) The total cellular amount of MITOL was not dramatically reduced following extended valinomycin treatment. Immunoblotting combined with fractionation analysis showed that Mitofusin2 (MFN2) underwent rapid degradation within 3 hours of valinomycin treatment, in particular in the 3,000 g pellet (mitochondria-rich fractions). Cytochrome c oxidase subunit 2 (MTCO2, inner mitochondrial protein) was significantly reduced at 24 hours 10 μM valinomycin treatment. In contrast to those two proteins, MITOL degradation was minimal. Note that the chemical apoptosis inhibitor ZVAD-FMK (10 μM) was added to cells along with valinomycin to prevent cell death.


( E ) Quantification of 3Flag - MITOL , MFN2 , and MTCO2 protein levels in the PNS and 3 , 000 g pellet fraction following 10 µM valinomycin + ZVAD - FMK treatment at the indicated times . Data represent the mean fold change ± s . e . m relative to untreated samples in three biological replicates .

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

(E) Quantification of 3Flag-MITOL, MFN2, and MTCO2 protein levels in the PNS and 3,000 g pellet fraction following 10 µM valinomycin + ZVAD-FMK treatment at the indicated times. Data represent the mean fold change ± s.e.m relative to untreated samples in three biological replicates.


( F ) Pre - existing MITOL on mitochondria moves to peroxisomes following CCCP treatment . Following doxycycline treatment for 3 hours to induce MITOL expression , cells were washed with fresh medium to stop the synthesis of new MITOL . After treatment with or without CCCP for more than 3 hours , cells were immunostained using anti - Flag , anti - Pex14 ( peroxisomal membrane protein ) , and anti - Hsp60 antibodies . Higher magnification images of the boxed regions are shown in the bottom panel . Scale bars , 10 µm .

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 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

(F) Pre-existing MITOL on mitochondria moves to peroxisomes following CCCP treatment. Following doxycycline treatment for 3 hours to induce MITOL expression, cells were washed with fresh medium to stop the synthesis of new MITOL. After treatment with or without CCCP for more than 3 hours, cells were immunostained using anti-Flag, anti-Pex14 (peroxisomal membrane protein), and anti-Hsp60 antibodies. Higher magnification images of the boxed regions are shown in the bottom panel. Scale bars, 10 µm.


( A ) Wild - type or PINK1 knock out ( KO ) HeLa cells were transfected with GFP - Parkin wild - type or the C431S mutant , treated with 15 µM CCCP , and then immuno - stained with anti - Flag and anti - catalase antibodies . Wild - type Parkin , but not the catalytically inactive Parkin ( C431S ) mutant , mediated 3Flag - MITOL translocation to peroxisomes following mitophagy stimulation . In PINK1 KO HeLa cells , the redistribution of MITOL to peroxisomes was not observed even in the presence of wild - type Parkin when mitochondria were damaged . Higher magnification images of the boxed regions are shown in the small panel . Scale bars , 10 µm . Arrowheads indicate representative examples of MITOL - peroxisome co - localization observed only in the presence of wild - type Parkin .

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

(A) Wild-type or PINK1 knock out (KO) HeLa cells were transfected with GFP-Parkin wild-type or the C431S mutant, treated with 15 µM CCCP, and then immuno-stained with anti-Flag and anti-catalase antibodies. Wild-type Parkin, but not the catalytically inactive Parkin (C431S) mutant, mediated 3Flag-MITOL translocation to peroxisomes following mitophagy stimulation. In PINK1 KO HeLa cells, the redistribution of MITOL to peroxisomes was not observed even in the presence of wild-type Parkin when mitochondria were damaged. Higher magnification images of the boxed regions are shown in the small panel. Scale bars, 10 µm. Arrowheads indicate representative examples of MITOL-peroxisome co-localization observed only in the presence of wild-type Parkin.


( B ) The redistribution of MITOL from mitochondria to peroxisomes does not require its own E3 activity . The E3 - inactive MITOL Cys65Ser / Cys68Ser ( CS ) and H43W mutants were transfected into HeLa cells stably expressing HA - Parkin , treated with 15 µM CCCP , and then subjected to immunocytochemistry with anti - Flag and anti - catalase antibodies . After 3 hours of CCCP treatment , both the CS and H43W mutants co - localized with catalase . Higher magnification images of the boxed regions are shown in the small panel . Scale bars , 10 µm . Arrowheads indicate representative examples of 3Flag - MITOL co - localization with catalase .

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

(B) The redistribution of MITOL from mitochondria to peroxisomes does not require its own E3 activity. The E3-inactive MITOL Cys65Ser/Cys68Ser (CS) and H43W mutants were transfected into HeLa cells stably expressing HA-Parkin, treated with 15 µM CCCP, and then subjected to immunocytochemistry with anti-Flag and anti-catalase antibodies. After 3 hours of CCCP treatment, both the CS and H43W mutants co-localized with catalase. Higher magnification images of the boxed regions are shown in the small panel. Scale bars, 10 µm. Arrowheads indicate representative examples of 3Flag-MITOL co-localization with catalase.


( C ) Correlation statistics for the localization of 3Flag - MITOL and catalase in the presence of GFP - Parkin wild - type or an inactive C431S mutant . Dots indicate individual Pearson correlation coefficient data points . In the box - plots , the center lines indicate the medians , the box limits indicate the 25th and 75th percentiles as determined in the R software package , and the whiskers extend 1 . 5 times the interquartile range from the 25th and 75th percentiles . Means and the number of samples are shown on the box and X - axis , respectively . Statistical significance was calculated using a one - tailed Welch ' s t - test . MITOL overlapped with catalase only in the presence of wild - type Parkin in wild - type HeLa cells . In PINK1 knockout ( KO ) HeLa cells , wild - type Parkin was unable to induce the peroxisomal translocation of MITOL .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Correlation statistics for the localization of 3Flag-MITOL and catalase in the presence of GFP-Parkin wild-type or an inactive C431S mutant. Dots indicate individual Pearson correlation coefficient data points. In the box-plots, the center lines indicate the medians, the box limits indicate the 25th and 75th percentiles as determined in the R software package, and the whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles. Means and the number of samples are shown on the box and X-axis, respectively. Statistical significance was calculated using a one-tailed Welch's t-test. MITOL overlapped with catalase only in the presence of wild-type Parkin in wild-type HeLa cells. In PINK1 knockout (KO) HeLa cells, wild-type Parkin was unable to induce the peroxisomal translocation of MITOL.


( D ) Correlation statistics for the localization of 3Flag - MITOL wild - type or inactive mutants with catalase . Dots indicate individual Pearson correlation coefficient data points . In the box - plots , the center lines indicate the medians , the box limits indicate the 25th and 75th percentiles as determined in the R software package , and the whiskers extend 1 . 5 times the interquartile range from the 25th and 75th percentiles . Means and the number of samples are shown on the box and X - axis , respectively . Statistical significance was calculated using a one - tailed Welch ' s t - test .

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

(D) Correlation statistics for the localization of 3Flag-MITOL wild-type or inactive mutants with catalase. Dots indicate individual Pearson correlation coefficient data points. In the box-plots, the center lines indicate the medians, the box limits indicate the 25th and 75th percentiles as determined in the R software package, and the whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles. Means and the number of samples are shown on the box and X-axis, respectively. Statistical significance was calculated using a one-tailed Welch's t-test.


( B ) Peroxisomal translocation of other mitochondrial proteins such as MitoNEET / CISD1 , Fis1 , Miro1 / 2 , and Tom70 ( mitochondrial outer membrane proteins ) , and Hsp60 ( mitochondrial matrix protein ) was not observed in CCCP - treated cells . HeLa cells stably expressing HA - Parkin were treated with 15 µM CCCP for 3 hours , and then immuno - stained with anti - MitoNEET / CISD1 , anti - Fis1 , anti - Miro1 / 2 , anti - Tom70 , anti - Hsp60 , and anti - catalase antibodies . Scale bars , 10 µm .

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

(B) Peroxisomal translocation of other mitochondrial proteins such as MitoNEET/CISD1, Fis1, Miro1/2, and Tom70 (mitochondrial outer membrane proteins), and Hsp60 (mitochondrial matrix protein) was not observed in CCCP-treated cells. HeLa cells stably expressing HA-Parkin were treated with 15 µM CCCP for 3 hours, and then immuno-stained with anti-MitoNEET/CISD1, anti-Fis1, anti-Miro1/2, anti-Tom70, anti-Hsp60, and anti-catalase antibodies. Scale bars, 10 µm.


( C ) A multi - spanning outer membrane protein PBR ( Peripheral Benzodiazepine Receptor ) did not co - localize with catalase , but did with Tom20 during mitophagy . HeLa cells stably expressing HA - Parkin were treated with 15 µM CCCP for 3 hours , and then immuno - stained with anti - Flag , anti - Tom20 , and anti - catalase antibodies . Scale bars , 10 µm .

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

(C) A multi-spanning outer membrane protein PBR (Peripheral Benzodiazepine Receptor) did not co-localize with catalase, but did with Tom20 during mitophagy. HeLa cells stably expressing HA-Parkin were treated with 15 µM CCCP for 3 hours, and then immuno-stained with anti-Flag, anti-Tom20, and anti-catalase antibodies. Scale bars, 10 µm.


( A ) siRNA - based knockdown of endogenous Tom70 , Tom20 , Tom40 , and Sam50 . HeLa cells treated with the corresponding siRNAs and immunoblotted with the indicated antibodies . AIF , Apoptosis - inducing factor .

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

(A) siRNA-based knockdown of endogenous Tom70, Tom20, Tom40, and Sam50. HeLa cells treated with the corresponding siRNAs and immunoblotted with the indicated antibodies. AIF, Apoptosis-inducing factor.


( B ) HeLa cells were immuno - stained with anti - Flag and anti - catalase antibodies after treatment of each siRNA . Tom20 - and Tom40 - siRNA treatment inhibited import of Su9 - GFP ( the targeting sequence of the FoF1 ATPase subunit 9 ) into the mitochondria , leading to an accumulation of Su9 - GFP precursor proteins in the cytosol and cell nucleus . 3Flag - MITOL still localized to mitochondria in Tom20 , Tom40 , and Sam50 siRNA - treated cells . In contrast , 3Flag - MITOL dispersed into the cytosol when Tom70 was knocked down . Scale bars , 10 µm .

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

(B) HeLa cells were immuno-stained with anti-Flag and anti-catalase antibodies after treatment of each siRNA. Tom20- and Tom40-siRNA treatment inhibited import of Su9-GFP (the targeting sequence of the FoF1 ATPase subunit 9) into the mitochondria, leading to an accumulation of Su9-GFP precursor proteins in the cytosol and cell nucleus. 3Flag-MITOL still localized to mitochondria in Tom20, Tom40, and Sam50 siRNA-treated cells. In contrast, 3Flag-MITOL dispersed into the cytosol when Tom70 was knocked down. Scale bars, 10 µm.


( C ) Statistical analysis of the MITOL subcellular localization following 15 µM CCCP treatment for 3 hours in cells treated with control , Tom70 , Tom20 , Tom40 , or Sam50 siRNAs . Su9 - GFP was not imported into the mitochondria , but rather localized to the cytosol and nucleus following Tom20 and Tom40 knockdown . The number of HeLa cells with cytosol - localized Su9 - GFP or 3Flag - MITOL in each siRNA experiment were determined . Data represent the mean ± s . e . m from > 100 cells in three biological replicates . ( In case of Tom40 , and Sam50 siRNAs , data represent the mean ± s . e . m from > 60 cells in three biological replicates . ) Statistical significance was calculated using a one - tailed Welch ' s t - test .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Statistical analysis of the MITOL subcellular localization following 15 µM CCCP treatment for 3 hours in cells treated with control, Tom70, Tom20, Tom40, or Sam50 siRNAs. Su9-GFP was not imported into the mitochondria, but rather localized to the cytosol and nucleus following Tom20 and Tom40 knockdown. The number of HeLa cells with cytosol-localized Su9-GFP or 3Flag-MITOL in each siRNA experiment were determined. Data represent the mean ± s.e.m from > 100 cells in three biological replicates. (In case of Tom40, and Sam50 siRNAs, data represent the mean ± s.e.m from > 60 cells in three biological replicates.) Statistical significance was calculated using a one-tailed Welch's t-test.


( D ) HeLa cells treated with siControl or siTom70 were fractionated into cytosolic and mitochondrial fractions and then immunoblotted with anti - lactate dehydrogenase ( LDH ) , anti - Tom20 , and anti - Flag antibodies . LDH was used as a cytosolic marker . Although some MITOL detached from mitochondria during fractionation , sufficient amounts of MITOL were collected in the mitochondria - enriched fraction in control cells . In contrast , almost all of the MITOL was collected in the cytosolic fraction in siTom70 - treated cells .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) HeLa cells treated with siControl or siTom70 were fractionated into cytosolic and mitochondrial fractions and then immunoblotted with anti-lactate dehydrogenase (LDH), anti-Tom20, and anti-Flag antibodies. LDH was used as a cytosolic marker. Although some MITOL detached from mitochondria during fractionation, sufficient amounts of MITOL were collected in the mitochondria-enriched fraction in control cells. In contrast, almost all of the MITOL was collected in the cytosolic fraction in siTom70-treated cells.


( A ) CRISPR / Cas9 gene editing was used to generate peroxisome - null cell lines by knocking out PEX19 . In wild - type HCT116 cells , catalase localized to peroxisomes ( dot - like structures ) , whereas in PEX19 - / - cells catalase was diffusely localized throughout the cytosol , indicating loss of peroxisomes . Scale bars , 10 µm .

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

(A) CRISPR/Cas9 gene editing was used to generate peroxisome-null cell lines by knocking out PEX19. In wild-type HCT116 cells, catalase localized to peroxisomes (dot-like structures), whereas in PEX19 -/- cells catalase was diffusely localized throughout the cytosol, indicating loss of peroxisomes. Scale bars, 10 µm.


Wild - type and PEX19 - / - HCT116 cells stably expressing HA - Parkin and 3Flag - MITOL were treated with 10 µM valinomycin for 3 hours , and then subjected to immunocytochemistry with anti - Flag , anti - catalase , and anti - Hsp60 ( B ) antibodies . In wild - type HCT116 cells , MITOL translocated to peroxisomes , whereas MITOL remained associated with mitochondria in PEX19 - / - cells . Arrowheads indicate representative examples of 3Flag - MITOL co - localization with catalase rather than Hsp60 in wild - type HCT116 cells . Higher magnification images of the boxed regions are shown in the small panel . Scale bars , 10 µm .

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

Wild-type and PEX19 -/- HCT116 cells stably expressing HA-Parkin and 3Flag-MITOL were treated with 10 µM valinomycin for 3 hours, and then subjected to immunocytochemistry with anti-Flag, anti-catalase, and anti-Hsp60 (B) antibodies. In wild-type HCT116 cells, MITOL translocated to peroxisomes, whereas MITOL remained associated with mitochondria in PEX19 -/- cells. Arrowheads indicate representative examples of 3Flag-MITOL co-localization with catalase rather than Hsp60 in wild-type HCT116 cells. Higher magnification images of the boxed regions are shown in the small panel. Scale bars, 10 µm.


Wild - type and PEX19 - / - HCT116 cells stably expressing HA - Parkin and 3Flag - MITOL were treated with 10 µM valinomycin for 3 hours , and then subjected to immunocytochemistry with anti - Flag anti - Tom20 ( C ) antibodies . In wild - type HCT116 cells , MITOL translocated to peroxisomes , whereas MITOL remained associated with mitochondria in PEX19 - / - cells . Higher magnification images of the boxed regions are shown in the small panel . Scale bars , 10 µm .

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

Wild-type and PEX19 -/- HCT116 cells stably expressing HA-Parkin and 3Flag-MITOL were treated with 10 µM valinomycin for 3 hours, and then subjected to immunocytochemistry with anti-Flag anti-Tom20 (C) antibodies. In wild-type HCT116 cells, MITOL translocated to peroxisomes, whereas MITOL remained associated with mitochondria in PEX19 -/- cells. Higher magnification images of the boxed regions are shown in the small panel. Scale bars, 10 µm.


( A ) siRNA - based knockdown of Pex3 , Pex16 , and Pex19 . HeLa cell lysates treated with the indicated plasmids and siRNAs were immunoblotted with the indicated antibodies .

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(A) siRNA-based knockdown of Pex3, Pex16, and Pex19. HeLa cell lysates treated with the indicated plasmids and siRNAs were immunoblotted with the indicated antibodies.


( B ) A comparable amount of MITOL was detected in control , PEX3 - , PEX16 - , and PEX19 - knockdown cells . HeLa cell lysates stably expressing HA - Parkin and 3Flag - MITOL treated with the corresponding siRNAs were immunoblotted with the indicated antibodies .

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(B) A comparable amount of MITOL was detected in control, PEX3-, PEX16-, and PEX19-knockdown cells. HeLa cell lysates stably expressing HA-Parkin and 3Flag-MITOL treated with the corresponding siRNAs were immunoblotted with the indicated antibodies.


( C ) MITOL translocation from the mitochondria to peroxisomes is highly dependent on Pex3 . HeLa cells stably expressing 3Flag - MITOL and HA - Parkin were transfected with control , PEX3 , PEX16 , or PEX19 siRNA , treated with 15 µM CCCP for 3 hours , and then subjected to immunocytochemistry with anti - Flag and anti - catalase antibodies . Scale bars , 10 µm .

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

(C) MITOL translocation from the mitochondria to peroxisomes is highly dependent on Pex3. HeLa cells stably expressing 3Flag-MITOL and HA-Parkin were transfected with control, PEX3, PEX16, or PEX19 siRNA, treated with 15 µM CCCP for 3 hours, and then subjected to immunocytochemistry with anti-Flag and anti-catalase antibodies. Scale bars, 10 µm.


( D ) Correlation statistics for the localization of 3Flag - MITOL and catalase in cells transfected with the indicated siRNAs . Dots indicate individual Pearson correlation coefficient data points . In the box - plots , the center lines indicate the medians , the box limits indicate the 25th and 75th percentiles as determined in the R software package , and the whiskers extend 1 . 5 times the interquartile range from the 25th and 75th percentiles . Means and the number of samples are shown on the box and X - axis , respectively . Statistical significance was calculated using a one - tailed Welch ' s t - test .

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(D) Correlation statistics for the localization of 3Flag-MITOL and catalase in cells transfected with the indicated siRNAs. Dots indicate individual Pearson correlation coefficient data points. In the box-plots, the center lines indicate the medians, the box limits indicate the 25th and 75th percentiles as determined in the R software package, and the whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles. Means and the number of samples are shown on the box and X-axis, respectively. Statistical significance was calculated using a one-tailed Welch's t-test.


( A ) MITOL did not merge with Hsp60 in control siRNA - treated cells , whereas most MITOL co - localized with Hsp60 in p97 / VCP knockdown cells in response to mitophagy stimuli . HeLa cells stably expressing 3Flag - MITOL and HA - Parkin were transfected with control or p97 / VCP siRNA , treated with 15 µM CCCP for 3 hours , and then subjected to immunocytochemistry with anti - Flag , anti - catalase , and anti - Hsp60 antibodies . Scale bars , 10 µm .

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

(A) MITOL did not merge with Hsp60 in control siRNA-treated cells, whereas most MITOL co-localized with Hsp60 in p97/VCP knockdown cells in response to mitophagy stimuli. HeLa cells stably expressing 3Flag-MITOL and HA-Parkin were transfected with control or p97/VCP siRNA, treated with 15 µM CCCP for 3 hours, and then subjected to immunocytochemistry with anti-Flag, anti-catalase, and anti-Hsp60 antibodies. Scale bars, 10 µm.


( B ) HeLa cell lysates treated with control or p97 / VCP siRNA were immunoblotted with anti - p97 / VCP and anti - tubulin antibodies .

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(B) HeLa cell lysates treated with control or p97/VCP siRNA were immunoblotted with anti-p97/VCP and anti-tubulin antibodies.


( C ) Overexpression of an p97 / VCP ATP hydrolysis - defective mutant , E305Q / E578Q ( p97QQ ) , blocked MITOL redistribution from mitochondria to peroxisomes , while overexpression of wild - type p97 / VCP had no effect on MITOL redistribution . HeLa cells stably expressing GFP - Parkin were transfected with MITOL - HA and Flag - p97 / VCP wild - type or p97QQ , treated with 15 µM CCCP for 3 hours , and then subjected to immunocytochemistry with anti - HA and anti - catalase antibodies . Higher magnification images of the boxed regions are shown in the small panel . Scale bars , 10 µm . Arrowheads indicate representative examples of MITOL - peroxisome co - localization that was only observed in the presence of a functional VCP .

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

(C) Overexpression of an p97/VCP ATP hydrolysis-defective mutant, E305Q/E578Q (p97QQ), blocked MITOL redistribution from mitochondria to peroxisomes, while overexpression of wild-type p97/VCP had no effect on MITOL redistribution. HeLa cells stably expressing GFP-Parkin were transfected with MITOL-HA and Flag-p97/VCP wild-type or p97QQ, treated with 15 µM CCCP for 3 hours, and then subjected to immunocytochemistry with anti-HA and anti-catalase antibodies. Higher magnification images of the boxed regions are shown in the small panel. Scale bars, 10 µm. Arrowheads indicate representative examples of MITOL-peroxisome co-localization that was only observed in the presence of a functional VCP.


( D ) NMS - 873 , a specific inhibitor of p97 / VCP , prevented MITOL translocation following CCCP - treatment . HeLa cells stably expressing HA - Parkin were transfected with 3Flag - MITOL , treated with 15 µM CCCP in the presence or absence of 10 µM NMS - 873 for 3 hours , and then subjected to immuno - staining with anti - Flag , anti - catalase , and anti - HA antibodies . Higher magnification images of the boxed regions are shown in the small panel . Scale bars , 10 µm . Arrowheads indicate representative examples of MITOL - peroxisome co - localization that was only observed in the presence of a functional VCP .

O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR 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 B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) NMS-873, a specific inhibitor of p97/VCP, prevented MITOL translocation following CCCP-treatment. HeLa cells stably expressing HA-Parkin were transfected with 3Flag-MITOL, treated with 15 µM CCCP in the presence or absence of 10 µM NMS-873 for 3 hours, and then subjected to immuno-staining with anti-Flag, anti-catalase, and anti-HA antibodies. Higher magnification images of the boxed regions are shown in the small panel. Scale bars, 10 µm. Arrowheads indicate representative examples of MITOL-peroxisome co-localization that was only observed in the presence of a functional VCP.


( E ) Correlation statistics for the localization of 3Flag - MITOL and catalase in the presence of NMS - 873 . Dots indicate individual Pearson correlation coefficient data points . In the box - plots , the center lines indicate the medians , the box limits indicate the 25th and 75th percentiles as determined in the R software package , and the whiskers extend 1 . 5 times the interquartile range from the 25th and 75th percentiles . Means and the number of samples are shown on the box and X - axis , respectively . Statistical significance was calculated using a one - tailed Welch ' s t - test .

O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Correlation statistics for the localization of 3Flag-MITOL and catalase in the presence of NMS-873. Dots indicate individual Pearson correlation coefficient data points. In the box-plots, the center lines indicate the medians, the box limits indicate the 25th and 75th percentiles as determined in the R software package, and the whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles. Means and the number of samples are shown on the box and X-axis, respectively. Statistical significance was calculated using a one-tailed Welch's t-test.


( A ) MITOL was ubiquitylated only in the presence of Parkin when the mitochondrial membrane was decreased . After 15 µM CCCP treatment for 3 hours , HeLa cells stably expressing HA - Parkin and 3Flag - MITOL were immunoprecipitated with anti - Flag magnetic beads , and then immunoblotted with the indicated antibodies . Red bars indicate ubiquitylation ; the black arrowhead indicates 3Flag - MITOL .

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

(A) MITOL was ubiquitylated only in the presence of Parkin when the mitochondrial membrane was decreased. After 15 µM CCCP treatment for 3 hours, HeLa cells stably expressing HA-Parkin and 3Flag-MITOL were immunoprecipitated with anti-Flag magnetic beads, and then immunoblotted with the indicated antibodies. Red bars indicate ubiquitylation; the black arrowhead indicates 3Flag-MITOL.


( B ) The characteristic ubiquitylation ladder was observed for wild - type MITOL , but was absent in the K268A and K54A / K268A mutants . Black arrowhead indicates 3Flag - MITOL .

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(B) The characteristic ubiquitylation ladder was observed for wild-type MITOL, but was absent in the K268A and K54A/K268A mutants. Black arrowhead indicates 3Flag-MITOL.


( C ) The MITOL K268A and K268R mutants were targeted to mitochondria under steady - state conditions , whereas peroxisomal localization following CCCP treatment was considerably disrupted . HeLa cells stably expressing HA - Parkin were transfected with 3Flag - MITOL wild - type , K268A , or K268R mutants , treated with 15 µM CCCP for 3 hours , and then subjected to immuno - staining with anti - Flag and anti - catalase antibodies . Scale bars , 10 µm .

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

(C) The MITOL K268A and K268R mutants were targeted to mitochondria under steady-state conditions, whereas peroxisomal localization following CCCP treatment was considerably disrupted. HeLa cells stably expressing HA-Parkin were transfected with 3Flag-MITOL wild-type, K268A, or K268R mutants, treated with 15 µM CCCP for 3 hours, and then subjected to immuno-staining with anti-Flag and anti-catalase antibodies. Scale bars, 10 µm.


( D ) The double K54 / K268 mutation did not change the MITOL localization pattern . The subcellular localization of the MITOL K268A or K268R mutants was not drastically changed by inclusion of the K54A or K54R mutations . Scale bars , 10 µm .

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(D) The double K54/K268 mutation did not change the MITOL localization pattern. The subcellular localization of the MITOL K268A or K268R mutants was not drastically changed by inclusion of the K54A or K54R mutations. Scale bars, 10 µm.


( E ) Correlation statistics for the localization of 3Flag - MITOL wild - type , K268A , or K268R mutants with catalase . Dots indicate individual Pearson correlation coefficient data points . In the box - plots , the center lines indicate the medians , the box limits indicate the 25th and 75th percentiles as determined in the R software package , and the whiskers extend 1 . 5 times the interquartile range from the 25th and 75th percentiles . Means and the number of samples are shown on the box and X - axis , respectively . Statistical significance was calculated using a one - tailed Welch ' s t - test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Correlation statistics for the localization of 3Flag-MITOL wild-type, K268A, or K268R mutants with catalase. Dots indicate individual Pearson correlation coefficient data points. In the box-plots, the center lines indicate the medians, the box limits indicate the 25th and 75th percentiles as determined in the R software package, and the whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles. Means and the number of samples are shown on the box and X-axis, respectively. Statistical significance was calculated using a one-tailed Welch's t-test.


( F ) The fold change in ubiquitylation of MITOL K268 , K40 , and K54 in valinomycin - treated samples versus untreated samples . After 3 hours of valinomycin treatment , PEX19 - / - HCT116 cells stably expressing HA - Parkin and 3Flag - MITOL were immunoprecipitated with anti - Flag magnetic beads , and then subjected to LC - MS / MS analysis for label - free quantification of ubiquitylated peptides . Error bars represent the mean ± s . e . m in three biological replicates .

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

(F) The fold change in ubiquitylation of MITOL K268, K40, and K54 in valinomycin-treated samples versus untreated samples. After 3 hours of valinomycin treatment, PEX19 -/- HCT116 cells stably expressing HA-Parkin and 3Flag-MITOL were immunoprecipitated with anti-Flag magnetic beads, and then subjected to LC-MS/MS analysis for label-free quantification of ubiquitylated peptides. Error bars represent the mean ± s.e.m in three biological replicates.


( A ) To generate MITOL - 3Flag knock - in ( KI ) HCT116 cell lines , 3xFlag gene cassettes were inserted upstream of the MITOL stop codon using CRISPR / Cas9 - based gene editing . Insertion of the 3Flag - tag was verified by immunoblotting with an anti - Flag antibody . Asterisk indicates a cross - reacting band .

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(A) To generate MITOL-3Flag knock-in (KI) HCT116 cell lines, 3xFlag gene cassettes were inserted upstream of the MITOL stop codon using CRISPR/Cas9-based gene editing. Insertion of the 3Flag-tag was verified by immunoblotting with an anti-Flag antibody. Asterisk indicates a cross-reacting band.


( B ) The MITOL subcellular localization was observed in MITOL - 3Flag KI HCT116 cells stably expressing HA - Parkin with anti - Flag , anti - catalase , and anti - Hsp60 antibodies . Endogenous MITOL ( detectable with an anti - Flag antibody ) overlapped with Hsp60 under steady - state conditions , whereas 3 hours of valinomycin ( 10 µM ) treatment induced translocation of endogenous MITOL from mitochondria to peroxisomes . Higher magnification images of the boxed regions are shown in the bottom panel . Scale bars , 10 µm .

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

(B) The MITOL subcellular localization was observed in MITOL-3Flag KI HCT116 cells stably expressing HA-Parkin with anti-Flag, anti-catalase, and anti-Hsp60 antibodies. Endogenous MITOL (detectable with an anti-Flag antibody) overlapped with Hsp60 under steady-state conditions, whereas 3 hours of valinomycin (10 µM) treatment induced translocation of endogenous MITOL from mitochondria to peroxisomes. Higher magnification images of the boxed regions are shown in the bottom panel. Scale bars, 10 µm.


( C ) Correlation statistics for the localization of endogenous MITOL - 3Flag with catalase . Dots indicate individual Pearson correlation coefficient data points . In the box - plots , the center lines show the medians , box limits indicate the 25th and 75th percentiles as determined by the R software package , and whiskers extend 1 . 5 times the interquartile range from the 25th and 75th percentiles . Means and the number of samples are shown on the box and the X - axis , respectively . Statistical significance was calculated using a one - tailed Welch ' s t - test .

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

(C) Correlation statistics for the localization of endogenous MITOL-3Flag with catalase. Dots indicate individual Pearson correlation coefficient data points. In the box-plots, the center lines show the medians, box limits indicate the 25th and 75th percentiles as determined by the R software package, and whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles. Means and the number of samples are shown on the box and the X-axis, respectively. Statistical significance was calculated using a one-tailed Welch's t-test.


( D ) The extraction of endogenous MITOL from depolarized mitochondria is blocked by the p97 / VCP inhibitor NMS - 873 . Higher magnification images of the boxed regions are shown in the lower panel . Scale bars , 10 µm .

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

(D) The extraction of endogenous MITOL from depolarized mitochondria is blocked by the p97/VCP inhibitor NMS-873. Higher magnification images of the boxed regions are shown in the lower panel. Scale bars, 10 µm.