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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


( g ) iPSC lines from controls and from patients with G / S LRRK2 - mutation Parkinson ' s disease , differentiated for 30 d and immunostained for LAMP - 2A , α - syn and TH . Merged channels ( left ) and magnified green and red channels from boxed region ( right ) . Arrows , colocalization LAMP - 2A and α - syn . ( h ) Percentage of LAMP - 2A + puncta colocalizing with α - syn in TH + cells ( n > 50 ) for two control and two LRRK2 iPSC lines treated as in g .

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(g) iPSC lines from controls and from patients with G/S LRRK2-mutation Parkinson's disease, differentiated for 30 d and immunostained for LAMP-2A, α-syn and TH. Merged channels (left) and magnified green and red channels from boxed region (right). Arrows, colocalization LAMP-2A and α-syn. (h) Percentage of LAMP-2A+ puncta colocalizing with α-syn in TH+ cells (n > 50) for two control and two LRRK2 iPSC lines treated as in g.


( i , j ) iPSC lines as in g , differentiated for 30 or 75 d and untreated or treated with RNAi for LAMP - 2A . ( i ) Immunofluorescence for LAMP - 2A , α - syn and GFP in LAMP - 2A - RNAi iPSC lines differentiated for 30 d . Bottom : higher magnifications of boxed areas . ( j ) Percentage of TH + cells showing cytoplasmic accumulation of α - syn . n ≥ 2 experiments using two control and two LRRK2 iPSC lines ( n > 70 and n > 150 TH + cells in Ctr and LRRK2 , respectively ) . Scale bars , 10 μm . All values are mean + s . e . m . Differences from WT or control ( * ) or between control or knockdown cells ( § ) are significant at P 0 . 05 ; unpaired t - test in a , h and ANOVA and Bonferroni test in f , j . Full - length blots and gels in Supplementary Figure 12 .

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

(i,j) iPSC lines as in g, differentiated for 30 or 75 d and untreated or treated with RNAi for LAMP-2A. (i) Immunofluorescence for LAMP-2A, α-syn and GFP in LAMP-2A-RNAi iPSC lines differentiated for 30 d. Bottom: higher magnifications of boxed areas. (j) Percentage of TH+ cells showing cytoplasmic accumulation of α-syn. n ≥ 2 experiments using two control and two LRRK2 iPSC lines (n > 70 and n > 150 TH+ cells in Ctr and LRRK2, respectively). Scale bars, 10 μm. All values are mean + s.e.m. Differences from WT or control (*) or between control or knockdown cells (§) are significant at P 0.05; unpaired t-test in a,h and ANOVA and Bonferroni test in f,j. Full-length blots and gels in Supplementary Figure 12.


A . Whole mount in situ hybridization ( WISH ) of evi1at 20 ( left ) and 32 ( middle ) hpf . evi1expression is visible in various structures of the brain , neuronal structures , the posterior pronephric duct ( ppnd ) and the branchial arches ( ba ) , as well as in the VDA ( ventral dorsal aorta ) region . Additionally , evi1co - localizes with the endothelial marker flk1 ( right ) .

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

A. Whole mount in situ hybridization (WISH) of evi1at 20 (left) and 32 (middle) hpf. evi1expression is visible in various structures of the brain, neuronal structures, the posterior pronephric duct (ppnd) and the branchial arches (ba), as well as in the VDA (ventral dorsal aorta) region. Additionally, evi1co-localizes with the endothelial marker flk1 (right).


B , C . WISH of runx1 / c - myb ; in HSPCs ( B ) , mpo in neutrophils ( left ) and l - plastin ( right ) in monocytes / macrophages ( C ) at 36 hpf in control ( upper ) and evi1 MO ( lower ) injected embryos . D - G . WISH of globinin erythrocytes of 6 dpf old embryos ( D ) , of rag - 1in the thymus of 5 dpf embryos ( E ; red asterisk ) , of cd41at 52 hpf ( F ) , and gata2bat 32 hpf ( G ) for both control ( upper ) and evi1 morpholino ( lower ) injected embryos . H . Quantitation of results is shown for each gene , displaying the percentages of embryos with normal vs . changed expression in each condition . A Fisher ´ s exact test was applied to calculate statistical significance . P < 0 . 001 ( * * * ) .

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

B, C. WISH of runx1/c-myb ;in HSPCs (B), mpo in neutrophils (left) and l-plastin (right) in monocytes/macrophages (C) at 36 hpf in control (upper) and evi1 MO (lower) injected embryos.D-G. WISH of globinin erythrocytes of 6 dpf old embryos (D), of rag-1in the thymus of 5 dpf embryos (E; red asterisk), of cd41at 52 hpf (F), and gata2bat 32 hpf (G) for both control (upper) and evi1 morpholino (lower) injected embryos.H. Quantitation of results is shown for each gene, displaying the percentages of embryos with normal vs. changed expression in each condition. A Fisher´s exact test was applied to calculate statistical significance. P < 0.001 (***).


I . WISH of runx1 / c - myb in HSPCs of uninjected control and UAS : mEvi1 plasmid DNA injected Tg ( - 1 . 5hsp70l : Gal4 ) embryos with heat - shock induction performed at 14 hpf . J . WISH of runx1 / c - myb in HSPCs of uninjected control and UAS : mEvi1 mRNA in Tg ( fli . 1 : Gal4FF ; UAS : RFP ) embryos , leading to endothelial - specific evi1 overexpression . K . Graph displays quantitation of results from I and J using the same method as above .

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

I. WISH of runx1/c-myb in HSPCs of uninjected control and UAS:mEvi1 plasmid DNA injected Tg(-1.5hsp70l:Gal4) embryos with heat-shock induction performed at 14 hpf.J. WISH of runx1/c-myb in HSPCs of uninjected control and UAS:mEvi1 mRNA in Tg(fli.1:Gal4FF;UAS:RFP) embryos, leading to endothelial-specific evi1 overexpression.K. Graph displays quantitation of results from I and J using the same method as above.


A . WISH of notch1b ( upper ) and dll4 ( lower ) in both control ( left ) and evi1 MO injected embryos ( right ) .

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

A. WISH of notch1b(upper) and dll4(lower) in both control (left) and evi1 MO injected embryos (right).


B . WISH of notch1b in uninjected ( left ) and UAS : mEvi1 plasmid DNA injected ( right ) Tg ( fli . 1 : Gal4FF ; UAS : RFP ) embryos resulting in endothelial - specific evi1 induction .

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

B. WISH of notch1b in uninjected (left) and UAS:mEvi1 plasmid DNA injected (right) Tg(fli.1:Gal4FF;UAS:RFP) embryos resulting in endothelial-specific evi1 induction.


A . Confocal time - lapse live imaging was performed in Tg ( c - myb : GFP ; kdrl : mKate - CAAX ) embryos from 28 to 42 hpf ( Movie EV 1 ) . Shown are three representative time - points in which the transformation from hemogenic endothelial cells to the hematopoietic fate is visible , indicated by the white arrowheads . For each time - point merged images are shown . White arrowheads denote double - positive cells . B . Cell counts of total numbers of kdrl + cmyb1 + cells " in view " , dividing and respectively emerging HSPCs . n = 10 movies from n = 3 biological replicates were analyzed . A non - parametric Mann - Whitney test was used to test for statistical significance and error bars are shown as ± s . d .

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A. Confocal time-lapse live imaging was performed in Tg(c-myb:GFP;kdrl:mKate-CAAX) embryos from 28 to 42 hpf (Movie EV 1). Shown are three representative time-points in which the transformation from hemogenic endothelial cells to the hematopoietic fate is visible, indicated by the white arrowheads. For each time-point merged images are shown. White arrowheads denote double-positive cells.B. Cell counts of total numbers of kdrl+cmyb1+ cells "in view", dividing and respectively emerging HSPCs. n=10 movies from n=3 biological replicates were analyzed. A non-parametric Mann-Whitney test was used to test for statistical significance and error bars are shown as ± s.d.


C . Gene expression analysis of endothelial ( kdrl , flt1 , dab2 , cdh5 ) and blood specific genes ( c - myb , CD45 , CD41 , runx1 ) in sorted kdrl + c - myb + cells from 34 hpf Tg ( c - myb : GFP ; kdrl : mKate - CAAX ) embryos . Cells were isolated from 15 - 25 pooled embryos per sample . Three biological experiments were performed with one representative shown . Error bars indicate s . d . of three technical replicates for each representative experiment .

O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O 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. Gene expression analysis of endothelial (kdrl, flt1, dab2, cdh5) and blood specific genes (c-myb, CD45, CD41, runx1) in sorted kdrl+c-myb+ cells from 34 hpf Tg(c-myb:GFP;kdrl:mKate-CAAX) embryos. Cells were isolated from 15-25 pooled embryos per sample. Three biological experiments were performed with one representative shown. Error bars indicate s.d.
of three technical replicates for each representative experiment.


A . WISH for runx1 / c - mybin 36 hpf evi1 morphant ( left ) and evi1 morphant transgenic Tg ( 5xUAS - E1b : 6xMYC - notch1a ; - 1 . 5hsp70l : Gal4 ) embryos ( right ) with global heat - shock ( HS ) induction at 20 hpf . B . WISH for runx1 / c - myb in 36 hpf evi1 morphant ( left ) and evi1 morphant transgenic Tg ( 5xUAS - E1b : 6xMYC - notch1a ; cdh5 : gal4ff ) embryos with endothelial - specific NICD induction ( right ) . C . WISH for runx1 / c - myb in 36 hpf evi1 morphant ( left ) and evi1 morphant transgenic Tg ( Hsp70l : vegfaa ; myl7 : eGFP ) embryos ( right ) with global heat - shock induction at 20 hpf . D . WISH for runx1 / c - mybin 32 hpf evi1 MO injected embryos ( left ) and embryos injected with both evi1 MO and capped gata2 mRNA ( right ) . E . Quantitation of all rescue experiments . Shown are the percentages of embryos displaying normal or changed runx1 / c - myb expression . A Fisher ´ s exact test was applied to calculate statistical significance . ( n . s . , not significant ; p < 0 . 001 ( * * * ) ; p < 0 . 01 ( * * ) ) .

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A. WISH for runx1/c-mybin 36 hpf evi1 morphant (left) and evi1 morphant transgenic Tg(5xUAS-E1b:6xMYC-notch1a;-1.5hsp70l:Gal4) embryos (right) with global heat-shock (HS) induction at 20 hpf.B. WISH for runx1/c-myb in 36 hpf evi1 morphant (left) and evi1 morphant transgenic Tg(5xUAS-E1b:6xMYC-notch1a;cdh5:gal4ff) embryos with endothelial-specific NICD induction (right).C. WISH for runx1/c-myb in 36 hpf evi1 morphant (left) and evi1 morphant transgenic Tg(Hsp70l:vegfaa;myl7:eGFP) embryos (right) with global heat-shock induction at 20 hpf.D. WISH for runx1/c-mybin 32 hpf evi1 MO injected embryos (left) and embryos injected with both evi1 MO and capped gata2 mRNA (right).E. Quantitation of all rescue experiments. Shown are the percentages of embryos displaying normal or changed runx1/c-myb expression. A Fisher´s exact test was applied to calculate statistical significance. (n.s., not significant; p < 0.001 (***); p<0.01 (**)).


A . Confocal time - lapse live imaging was performed in Tg ( c - myb : GFP ; fli . 1 : UAS ; Gal4 : RFP ) evi1 morphant embryos ( top , Movie EV 2 ) or in Tg ( c - myb : GFP ; fli . 1 : UAS ; Gal4 : RFP ) crossed to Tg ( 5xUAS - E1b : 6xMYC - notch1a ) embryos prior to MO injection ( bottom , Movie EV 3 ) from 28 to 42 hpf . Shown are three representative time - points , in which the transformation from hemogenic endothelial cells to the hematopoietic fate is visible . For each time - point merged images are shown . NICD induction controlled by endothelial fli . 1 ( lower panel ) can rescue the evi1 morphant phenotype and increases the number of HSPCs cells emerging from the VDA ( arrows ) . B . Cell counts of emerging HSPCs in control and evi1 morphant fish with or without endothelial NICD induction . At least n = 5 movies from n = 3 biological replicates were analyzed . A non - parametric Mann - Whitney test was used to test for statistical significance and error bars are shown as ± s . d . ( n . s . , not significant , p < 0 . 05 ( * ) ) .

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

A. Confocal time-lapse live imaging was performed in Tg(c-myb:GFP;fli.1:UAS;Gal4:RFP) evi1 morphant embryos (top, Movie EV 2) or in Tg(c-myb:GFP;fli.1:UAS;Gal4:RFP) crossed to Tg(5xUAS-E1b:6xMYC-notch1a) embryos prior to MO injection (bottom, Movie EV 3) from 28 to 42 hpf. Shown are three representative time-points, in which the transformation from hemogenic endothelial cells to the hematopoietic fate is visible. For each time-point merged images are shown. NICD induction controlled by endothelial fli.1 (lower panel) can rescue the evi1 morphant phenotype and increases the number of HSPCs cells emerging from the VDA (arrows).B. Cell counts of emerging HSPCs in control and evi1 morphant fish with or without endothelial NICD induction. At least n=5 movies from n=3 biological replicates were analyzed. A non-parametric Mann-Whitney test was used to test for statistical significance and error bars are shown as ± s.d. (n.s., not significant, p<0.05 (*)).


A . WISH of dll4 ( upper ) and notch1b ( lower ) in control injected embryos ( left ) , evi1 morphants ( middle ) and evi1 morphant transgenic Tg ( Hsp70l : vegfaa ; myl7 : eGFP ) embryos at 32 hpf after heat - shock induction at 20 hpf ( right ) .

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A. WISH of dll4(upper) and notch1b(lower) in control injected embryos (left), evi1 morphants (middle) and evi1 morphant transgenic Tg(Hsp70l:vegfaa;myl7:eGFP) embryos at 32 hpf after heat-shock induction at 20 hpf (right).


B , C . WISH of runx1 / c - myb ( B ) or notch1b ( C ) after treatment with DMSO ( left ) , the VEGF receptor inhibitor SU5461 ( middle ) and respectively SU5461 after injection of UAS : mEvi1 ( inducing endothelial evi1 expression ) in Tg ( fli . 1 : Gal4FF ; UAS : RFP ) embryos ( right ) .

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B, C. WISH of runx1/c-myb(B) or notch1b(C) after treatment with DMSO (left), the VEGF receptor inhibitor SU5461 (middle) and respectively SU5461 after injection of UAS:mEvi1 (inducing endothelial evi1 expression) in Tg(fli.1:Gal4FF;UAS:RFP) embryos (right).


A . Expression of runx1 / c - myb in the VDA after treatment with DMSO vehicle control ( left ) , the PI3K / AKT inhibitor Wortmannin ( WM , middle ) and after joined treatment with WM and enforced NICD expression via HS at 14 hpf in Tg ( 5xUAS - E1b : 6xMYC - notch1a ; - 1 . 5hsp70l : Gal4 ) embryos ( right ) . Graph displays quantitation of results . Shown are the percentages of embryos with normal or decreased runx1 / c - mybexpression for each condition .

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

A. Expression of runx1/c-myb in the VDA after treatment with DMSO vehicle control (left), the PI3K/AKT inhibitor Wortmannin (WM, middle) and after joined treatment with WM and enforced NICD expression via HS at 14 hpf in Tg(5xUAS-E1b:6xMYC-notch1a;-1.5hsp70l:Gal4) embryos (right). Graph displays quantitation of results. Shown are the percentages of embryos with normal or decreased runx1/c-mybexpression for each condition.


B . Expression of notch1b after treatment with DMSO vehicle control ( left ) or Wortmannin ( WM , middle ) . Graph displays quantitation of results . Shown are the percentages of embryos with normal or decreased notch1b expression for each condition .

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

B. Expression of notch1b after treatment with DMSO vehicle control (left) or Wortmannin (WM, middle). Graph displays quantitation of results. Shown are the percentages of embryos with normal or decreased notch1b expression for each condition.


C . WISH for runx1 / c - myb in control injected embryos ( upper left ) , evi1 morphants ( upper right ) or respectively evi1 MO with myr - AKT injected embryos with HS at 14 hpf ( lower right ) and evi1 MO with UAS - myr - AKT injected transgenic Tg ( fli . 1 : Gal4FFubs3 ; UAS : RFP ) rk embryos . D . Quantitation of results from ( C ) . Shown are the percentages of embryos with normal or decreased runx1 / c - myb expression for each condition .

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C. WISH for runx1/c-myb in control injected embryos (upper left), evi1 morphants (upper right) or respectively evi1 MO with myr-AKT injected embryos with HS at 14 hpf (lower right) and evi1 MO with UAS-myr-AKT injected transgenic Tg(fli.1:Gal4FFubs3; UAS:RFP)rk embryos.D. Quantitation of results from (C). Shown are the percentages of embryos with normal or decreased runx1/c-myb expression for each condition.