( A ) MYC2 , PDF1 . 2A and NIMIN1 expression in ANAC032 transgenics compared to WT after spraying with Pst DC3000 ( 24 hpi ) normalised to their respective controls . FCh , fold change . Means ± SD ( n = 3 ; ' n ' represents independently performed experiments , each including the rosette leaves of at least three plants grown in individual pots ) .

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(A) MYC2, PDF1.2A and NIMIN1 expression in ANAC032 transgenics compared to WT after spraying with Pst DC3000 (24 hpi) normalised to their respective controls. FCh, fold change. Means ± SD (n = 3; 'n' represents independently performed experiments, each including the rosette leaves of at least three plants grown in individual pots).


( B ) EMSA showing binding of ANAC032 to MYC2 , PDF1 . 2A and NIMIN1 promoter regions ( in 5 ´ - DY682 - labelled double - stranded oligonucleotides ) harbouring ANAC032 binding sites . 1 , labelled probe only ; 2 , labelled probe plus GST protein 3 , labelled probe plus ANAC032 - GST protein ; 4 , labelled probe , ANAC032 - GST protein and 100 x competitor ( unlabelled oligonucleotide containing ANAC032 binding site ) .

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(B) EMSA showing binding of ANAC032 to MYC2, PDF1.2A and NIMIN1 promoter regions (in 5´-DY682-labelled double-stranded oligonucleotides) harbouring ANAC032 binding sites. 1, labelled probe only; 2, labelled probe plus GST protein 3, labelled probe plus ANAC032-GST protein; 4, labelled probe, ANAC032-GST protein and 100 x competitor (unlabelled oligonucleotide containing ANAC032 binding site).


( C ) Confocal microscopy image showing nuclear localization of ANAC032 - GFP fusion protein expressed from the ANAC032 promoter in ANAC032prom : ANAC032 - GFP / anac032 - 1 seedlings treated with Pst at 6 hpi . Left , bright field ; right , chlorophyll auto - fluorescence ( red ) and GFP fluorescence ( green ) under bright field .

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(C) Confocal microscopy image showing nuclear localization of ANAC032-GFP fusion protein expressed from the ANAC032 promoter in ANAC032prom:ANAC032-GFP/anac032-1 seedlings treated with Pst at 6 hpi. Left, bright field; right, chlorophyll auto-fluorescence (red) and GFP fluorescence (green) under bright field.


( D ) Expression of MYC2 , PDF1 . 2A , NIMIN1 and ANAC032 in two - week - old ANAC032prom : ANAC032 - GFP / anac032 - 1 seedlings compared to WT at 6 hpi with Pst , normalised to their respective controls . FCh , fold change . Means ± SD ( n = 3 ; ' n ' represents independently performed experiments , each including the rosette leaves of at least three plants grown in individual pots ) . Asterisks indicate a significant difference from WT , normalised to their respective controls ( p < 0 . 05 ; Student ´ s t - test ) .

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(D) Expression of MYC2, PDF1.2A, NIMIN1 and ANAC032 in two-week-old ANAC032prom:ANAC032-GFP/anac032-1 seedlings compared to WT at 6 hpi with Pst, normalised to their respective controls. FCh, fold change. Means ± SD (n = 3; 'n' represents independently performed experiments, each including the rosette leaves of at least three plants grown in individual pots). Asterisks indicate a significant difference from WT, normalised to their respective controls (p < 0.05; Student´s t-test).


( E ) ChIP - qPCR shows enrichment of MYC2 , PDF1 . 2Aand NIMIN1 promoter regions containing ANAC032 binding site compared to a promoter region lacking the ANAC032 binding site ( AT5G09810 ; Neg Cont ) . Means ± SD ( n = 3 ; ' n ' represents independently performed experiments , each including the rosette leaves of at least three plants grown in individual pots ) . Asterisks indicate a significant difference from negative control ( p < 0 . 01 ; Student ´ s t - test ) .

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(E) ChIP-qPCR shows enrichment of MYC2, PDF1.2Aand NIMIN1 promoter regions containing ANAC032 binding site compared to a promoter region lacking the ANAC032 binding site (AT5G09810; Neg Cont). Means ± SD (n = 3; 'n' represents independently performed experiments, each including the rosette leaves of at least three plants grown in individual pots). Asterisks indicate a significant difference from negative control (p < 0.01; Student´s t-test).


Transcript levels of ( A ) NIMIN1 and ( B ) PR1 in WT , anac032 - 1 and 35S : ANAC032 plants after 3 and 6 h of treatment with SA compared to their respective controls .

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Transcript levels of (A) NIMIN1 and (B) PR1 in WT, anac032-1 and 35S:ANAC032 plants after 3 and 6 h of treatment with SA compared to their respective controls.


( C ) , Expression of PDF1 . 2A in WT and anac032 - 1 plants after 3 and 6 h of treatment with SA compared to their respective controls . Transcript levels were measured using qRT - PCR and numbers on the y - axis indicate fold change ( FCh ; log2 basis ) . Means are shown ± SD ( n = 3 ; ' n ' represents independently performed experiments ) . Asterisks indicate a significant difference from wild type ( p < 0 . 05 ; Student ' s t test ) .

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(C), Expression of PDF1.2A in WT and anac032-1 plants after 3 and 6 h of treatment with SA compared to their respective controls. Transcript levels were measured using qRT-PCR and numbers on the y-axis indicate fold change (FCh; log2 basis). Means are shown ± SD (n = 3; 'n' represents independently performed experiments). Asterisks indicate a significant difference from wild type (p < 0.05; Student's t test).


Stomatal aperture 1 h ( A ) or 4 h ( B ) after spraying with Pst DC3000 or mock treatment .

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Stomatal aperture 1 h (A) or 4 h (B) after spraying with Pst DC3000 or mock treatment.


( C ) Microscope images of stomata at 1 and 4 hpi with Pst DC3000 ( Mock at 4 h ) .

(C) Microscope images of stomata at 1 and 4 hpi with Pst DC3000 (Mock at 4 h).


( D ) , Stomatal aperture 1 h after treatment with COR , ABA , or COR plus ABA , compared to mock . In ( A ) , ( B ) , and ( D ) , data are means ± SD of 12 to16 measurements ; in each measurement the rosette leaves from at least 6 - 8 plants were used ) . Asterisks indicate a significant difference from mock treatment ( p < 0 . 0001 ; Student ´ s t - test ) .

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(D), Stomatal aperture 1 h after treatment with COR, ABA, or COR plus ABA, compared to mock. In (A), (B), and (D), data are means ± SD of 12 to16 measurements; in each measurement the rosette leaves from at least 6 - 8 plants were used). Asterisks indicate a significant difference from mock treatment (p < 0.0001; Student´s t-test).


A / B . Hybrid Rab7 / Arl8b compartment . A . Representative confocal images of fixed HeLa cells expressing Arl8b - GFP ( green ) , immunolabeled against endogenous Rab7 ( eRab7 , magenta ) . Zoom insets ( 3x ) highlight select regions of colocalization ( white ) , white arrowheads point to vesicles positive for both GTPases . B . Colocalization ( Mander ' s overlap ) of endogenous Rab7 with Arl8b - GFP versus free GFP ( EV ) , n EV = 4 , n Arl8b = 9 images ( 3 ≥ cells per image ) analysed from 2 independent experiments . Significance : 2 - tailed student ' s t - test , * p < 0 . 05 .

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A/B. Hybrid Rab7/Arl8b compartment. A. Representative confocal images of fixed HeLa cells expressing Arl8b-GFP (green), immunolabeled against endogenous Rab7 (eRab7, magenta). Zoom insets (3x) highlight select regions of colocalization (white), white arrowheads point to vesicles positive for both GTPases. B. Colocalization (Mander's overlap) of endogenous Rab7 with Arl8b-GFP versus free GFP (EV), n EV=4, n Arl8b=9 images (3≥ cells per image) analysed from 2 independent experiments. Significance: 2-tailed student's t-test, * p<0.05.


C - F . Analysis of Arl8b compartment organization and dynamics as a function of Rab7 activity status . C . Left and middle panels : representative confocal images of live HeLa cells expressing mCherry - Rab7 or its mutants Q67L and T22N ( white ) , together with Arl8b - GFP ( green ) , taken at the start of time - lapse ( t0 ) . Right panels : tracks followed by Arl8b - positive null during the time - lapse lasting 50 s recorded at 0 . 5 s per frame , with highest displacement rates for each track depicted on a rainbow colour scale ( blue : immobile ; red : maximum mobility per time interval ) . Zoom insets ( 2 . 8x ) highlight select peripheral ( PP ) and perinuclear ( PN ) cell regions D . Plot of Arl8b - positive pixel distribution expressed as fractional distance along a straight line from center of nucleus ( 0 ) to the plasma membrane ( 1 . 0 ) , numbers of ( pixels ) plotted given above each scatter , n = 7 cells analysed per condition from 2 independent experiments . E / F . Quantification of mean Arl8b vesicle displacement and maximum speed , respectively , n Rab7 = 21 , n QL = 25 , n TN = 24 images ( 3 ≥ cells per image ) analysed from 2 independent experiments .

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C-F. Analysis of Arl8b compartment organization and dynamics as a function of Rab7 activity status. C. Left and middle panels: representative confocal images of live HeLa cells expressing mCherry-Rab7 or its mutants Q67L and T22N (white), together with Arl8b-GFP (green), taken at the start of time-lapse (t0). Right panels: tracks followed by Arl8b-positive null during the time-lapse lasting 50 s recorded at 0.5 s per frame, with highest displacement rates for each track depicted on a rainbow colour scale (blue: immobile; red: maximum mobility per time interval). Zoom insets (2.8x) highlight select peripheral (PP) and perinuclear (PN) cell regions D. Plot of Arl8b-positive pixel distribution expressed as fractional distance along a straight line from center of nucleus (0) to the plasma membrane (1.0), numbers of (pixels) plotted given above each scatter, n=7 cells analysed per condition from 2 independent experiments. E/F. Quantification of mean Arl8b vesicle displacement and maximum speed, respectively, n Rab7=21, n QL=25, n TN=24 images (3≥ cells per image) analysed from 2 independent experiments.


Effects of select transport route activation on the spatial organization of late compartments . A . Representative confocal images of fixed HeLa cells ectopically expressing Arl8b - GFP or Arl8b - Myc ( green ) , in combination with HA - RILP ( cyan ) and / or Myc - SKIP , PLEKHM1 - FLAG , or GFP - FYCO1 ( red ) , immunolabeled against endogenous Rab7 ( eRab7 , magenta ) and the indicated epitope tags . Cell and nuclear boundaries are demarcated with solid and dashed lines respectively , zoom insets ( 3 . 5x ) highlight select peripheral ( PP ) and perinuclear ( PN ) cell regions , scale bar : 10µm . B . Colocalization ( Mander ' s overlap ) between the indicated pairs of proteins , n ≥ 3 images ( 3 ≥ cells per image ) analysed per condition from 2 independent experiments .

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Effects of select transport route activation on the spatial organization of late compartments. A. Representative confocal images of fixed HeLa cells ectopically expressing Arl8b-GFP or Arl8b-Myc (green), in combination with HA-RILP (cyan) and/or Myc-SKIP, PLEKHM1-FLAG, or GFP-FYCO1 (red), immunolabeled against endogenous Rab7 (eRab7, magenta) and the indicated epitope tags. Cell and nuclear boundaries are demarcated with solid and dashed lines respectively, zoom insets (3.5x) highlight select peripheral (PP) and perinuclear (PN) cell regions, scale bar: 10µm. B. Colocalization (Mander's overlap) between the indicated pairs of proteins, n≥3 images (3≥ cells per image) analysed per condition from 2 independent experiments.


C . Plot of eRab7 - or Arl8b - GFP / Myc - Arl8b - positive pixel distribution in response to the indicated effector perturbations , expressed as fractional distance along a straight line from center of nucleus ( 0 ) to the plasma membrane ( 1 . 0 ) , number of ( pixels ) plotted given above each scatter , n ≥ 6 cells per condition analysed from 2 independent experiments .

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C. Plot of eRab7- or Arl8b-GFP / Myc-Arl8b-positive pixel distribution in response to the indicated effector perturbations, expressed as fractional distance along a straight line from center of nucleus (0) to the plasma membrane (1.0), number of (pixels) plotted given above each scatter, n≥6 cells per condition analysed from 2 independent experiments.


D . Colocalization ( Mander ' s overlap ) between Arl8b and Rab7 as a function of the indicated effector perturbations , n ≥ 5 images ( 3 ≥ cells per image ) analysed per condition from 2 independent experiments .

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D. Colocalization (Mander's overlap) between Arl8b and Rab7 as a function of the indicated effector perturbations, n≥5 images (3≥ cells per image) analysed per condition from 2 independent experiments.


A - D . Correlative light and electron microscopy ( CLEM ) on A . untransfected ( WT ) HeLa cells harbouring endogenous CD63 labeled with GFP as compared to those ectopically expressing B . HA - RILP , C . HA - SKIP or D . both HA - RILP and HA - SKIP . Left panels : wide field fluorescence images of fixed cells showing endogenous GFP - CD63 ( green ) and SiR - lysosome - stained endosomes and / or lysosomes ( red ) . Cell and nuclear boundaries are demarcated with solid and dashed lines respectively , zoom insets highlight regions selected for EM imaging . Middle panels : overview electron micrographs of perinuclear ( PN ) and peripheral ( PP ) cell regions selected for further analysis . Various endolysosomal subtypes are designated by arrowheads : MVBs ( white ) , lysosomes ( black ) , endolysosomes ( yellow ) and abnormal / enlarged endolysosomes ( red ) . Right panels : graphical representations of endosomal distribution under the indicated conditions based on ultrastructural characterization .

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A-D. Correlative light and electron microscopy (CLEM) on A. untransfected (WT) HeLa cells harbouring endogenous CD63 labeled with GFP as compared to those ectopically expressing B. HA-RILP, C. HA-SKIP or D. both HA-RILP and HA-SKIP. Left panels: wide field fluorescence images of fixed cells showing endogenous GFP-CD63 (green) and SiR-lysosome-stained endosomes and/or lysosomes (red). Cell and nuclear boundaries are demarcated with solid and dashed lines respectively, zoom insets highlight regions selected for EM imaging. Middle panels: overview electron micrographs of perinuclear (PN) and peripheral (PP) cell regions selected for further analysis. Various endolysosomal subtypes are designated by arrowheads: MVBs (white), lysosomes (black), endolysosomes (yellow) and abnormal/enlarged endolysosomes (red). Right panels: graphical representations of endosomal distribution under the indicated conditions based on ultrastructural characterization.


A . Electron micrograph of sections immunolabelled against RFP - SKIP ( 15nm gold ) and GFP - Rab7 ( 10nm gold ) . Arrowheads and zoom inset ( 1 . 75x ) highlight presence of RFP - SKIP and GFP - Rab7 on the same endosomal membrane , scale bar : 200nm .

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A. Electron micrograph of sections immunolabelled against RFP-SKIP (15nm gold) and GFP-Rab7 (10nm gold). Arrowheads and zoom inset (1.75x) highlight presence of RFP-SKIP and GFP-Rab7 on the same endosomal membrane, scale bar: 200nm.


B . Co - immunoprecipitations ( Co - IP ) of HA - RILP and RFP - SKIP with GFP - Rab7 ( R7 ) versus its mutants Q67L ( QL ) and T22N ( TN ) from HEK293T cells using GFP - trap beads . Representative immunoblots against GFP , HA and RFP are shown , EV : empty vector , IP : immunoprecipitation , TL : total lysate ( see also Fig EV1E ) . C . Quantification of interaction between SKIP and Rab7 mutants expressed as fraction Co - IP relative to wild type Rab7 , n = 3 independent experiments .

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B. Co-immunoprecipitations (Co-IP) of HA-RILP and RFP-SKIP with GFP-Rab7 (R7) versus its mutants Q67L (QL) and T22N (TN) from HEK293T cells using GFP-trap beads. Representative immunoblots against GFP, HA and RFP are shown, EV: empty vector, IP: immunoprecipitation, TL: total lysate (see also Fig EV1E). C. Quantification of interaction between SKIP and Rab7 mutants expressed as fraction Co-IP relative to wild type Rab7, n=3 independent experiments.


B . Co - immunoprecipitations ( Co - IP ) of HA - RILP and RFP - SKIP with GFP - Rab7 ( R7 ) versus its mutants Q67L ( QL ) and T22N ( TN ) from HEK293T cells using GFP - trap beads . Representative immunoblots against GFP , HA and RFP are shown , EV : empty vector , IP : immunoprecipitation , TL : total lysate ( see also Fig EV1E ) .

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B. Co-immunoprecipitations (Co-IP) of HA-RILP and RFP-SKIP with GFP-Rab7 (R7) versus its mutants Q67L (QL) and T22N (TN) from HEK293T cells using GFP-trap beads. Representative immunoblots against GFP, HA and RFP are shown, EV: empty vector, IP: immunoprecipitation, TL: total lysate (see also Fig EV1E).


D . Pull - down ( PD ) of RFP - SKIP or RFP - RILP from HEK293T cell lysates using recombinant GST - Rab7 versus GST - Arl8b and free GST . Representative immunoblots against RFP and GST are shown ( see also EV1F ) .

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D. Pull-down (PD) of RFP-SKIP or RFP-RILP from HEK293T cell lysates using recombinant GST-Rab7 versus GST-Arl8b and free GST. Representative immunoblots against RFP and GST are shown (see also EV1F).


E . In vitro glutathione precipitation assays . E . SKIP truncation analysis by PD against GST - Rab7 . Top panels : representative immunoblots against Myc and GST ( see also Fig EV1G ) . Bottom panels : schematic representation of SKIP domain organization . Regions of SKIP capable of interacting with Arl8b versus Rab7 are demarcated with solid black lines . An alignment of human ( h ) and murine ( m ) SKIP sequences to known effectors of Rab7 surrounding the conserved KML / I effector motif at residues 610 - 612 of SKIP is provided . H . Graphical summary of SKIP as a dual effector of Arl8b and Rab7 .

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E. In vitro glutathione precipitation assays. E. SKIP truncation analysis by PD against GST-Rab7. Top panels: representative immunoblots against Myc and GST (see also Fig EV1G). Bottom panels: schematic representation of SKIP domain organization. Regions of SKIP capable of interacting with Arl8b versus Rab7 are demarcated with solid black lines. An alignment of human (h) and murine (m) SKIP sequences to known effectors of Rab7 surrounding the conserved KML/I effector motif at residues 610-612 of SKIP is provided. H. Graphical summary of SKIP as a dual effector of Arl8b and Rab7.


F / G . Co - IP of RFP - SKIP versus its KMI motif mutants AAI and AAA with constitutively active GFP - Rab7 Q67L using RFP - trap beads ( see also Fig EV1H ) . F . Representative immunoblots against GFP and RFP . G . Quantification of interaction between SKIP mutants with Rab7 expressed as fraction Co - IP relative to wild type SKIP , n = 3 independent experiments .

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F/G. Co-IP of RFP-SKIP versus its KMI motif mutants AAI and AAA with constitutively active GFP-Rab7 Q67L using RFP-trap beads (see also Fig EV1H). F. Representative immunoblots against GFP and RFP. G. Quantification of interaction between SKIP mutants with Rab7 expressed as fraction Co-IP relative to wild type SKIP, n=3 independent experiments.


F / G . Co - IP of RFP - SKIP versus its KMI motif mutants AAI and AAA with constitutively active GFP - Rab7 Q67L using RFP - trap beads ( see also Fig EV1H ) . F . Representative immunoblots against GFP and RFP .

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F/G. Co-IP of RFP-SKIP versus its KMI motif mutants AAI and AAA with constitutively active GFP-Rab7 Q67L using RFP-trap beads (see also Fig EV1H). F. Representative immunoblots against GFP and RFP.


I - K . Time - lapse of SKIP - mediated transport of late endosomes . I . Schematic representation of tamoxifen - induced activation of SKIP onto endosomal membranes . J / K . Live HeLa cells co - expressing GFP - ER - SKIP ( green ) and mCherry - Rab7 ( magenta ) together with HA - RILP ( unstained ) expressed at low levels ( cells transfected at 1 : 5 RILP : SKIP ratio ) were imaged in the J . absence or K . presence of tamoxifen , allowing on - demand association of SKIP with endosomal membranes . Confocal frames from time - lapses taken at the indicated time - points following treatment are shown . Cell and nuclear boundaries are demarcated with solid and dashed lines respectively , zoom insets ( 3x ) highlight select peripheral ( PP ) and perinuclear ( PN ) cell regions , scale bars :

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I-K. Time-lapse of SKIP-mediated transport of late endosomes. I. Schematic representation of tamoxifen-induced activation of SKIP onto endosomal membranes. J/K. Live HeLa cells co-expressing GFP-ER-SKIP (green) and mCherry-Rab7 (magenta) together with HA-RILP (unstained) expressed at low levels (cells transfected at 1:5 RILP:SKIP ratio) were imaged in the J. absence or K. presence of tamoxifen, allowing on-demand association of SKIP with endosomal membranes. Confocal frames from time-lapses taken at the indicated time-points following treatment are shown. Cell and nuclear boundaries are demarcated with solid and dashed lines respectively, zoom insets (3x) highlight select peripheral (PP) and perinuclear (PN) cell regions, scale bars: 10µm


A - C . Consequences of effector depletion on the endogenous Arl8b / Rab7 hybrid compartment . A . Representative confocal images of fixed HeLa cells harbouring GFP - tagged endogenous Arl8b ( G - eArl8b , green ) , transfected with the indicated siRNAs and immunolabeled against endogenous Rab7 ( eRab7 , magenta ) . B . Colocalization ( Mander ' s overlap ) between endogenous Arl8b and Rab7 in response to effector depletion , n siC = 10 , n siRILP = 9 , n siSKIP = 9 images ( 4 ≥ cells per image ) analysed from 2 independent experiments . C . Immunoblot analysis for depletion efficiency of SKIP and RILP , with actin as loading control .

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A-C. Consequences of effector depletion on the endogenous Arl8b/Rab7 hybrid compartment. A. Representative confocal images of fixed HeLa cells harbouring GFP-tagged endogenous Arl8b (G-eArl8b, green), transfected with the indicated siRNAs and immunolabeled against endogenous Rab7 (eRab7, magenta). B. Colocalization (Mander's overlap) between endogenous Arl8b and Rab7 in response to effector depletion, n siC=10, n siRILP=9, n siSKIP=9 images (4≥ cells per image) analysed from 2 independent experiments. C. Immunoblot analysis for depletion efficiency of SKIP and RILP, with actin as loading control.


D / E . Effect of Rab7 GTPase activity status on its association with the peripheral SKIP compartment . D . Left panels : Representative confocal images of fixed HeLa cells expressing GFP - Rab7 or its mutants Q67L or T22N ( green ) together with HA - RILP ( red ) and Myc - SKIP ( blue ) , immunolabeled against the indicated epitope tags . Right panels : Schematic overview per condition . E . Colocalization ( Mander ' s overlap ) between the indicated protein pairs , n Rab7 = 5 , n QL = 5 , n TN = 7 images ( 2 ≥ cells per image ) analysed from 2 independent experiments . F . Graphical summary of Rab7 removal from the SKIP compartment .

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D/E. Effect of Rab7 GTPase activity status on its association with the peripheral SKIP compartment. D. Left panels: Representative confocal images of fixed HeLa cells expressing GFP-Rab7 or its mutants Q67L or T22N (green) together with HA-RILP (red) and Myc-SKIP (blue), immunolabeled against the indicated epitope tags. Right panels: Schematic overview per condition. E. Colocalization (Mander's overlap) between the indicated protein pairs, n Rab7=5, n QL=5, n TN=7 images (2≥ cells per image) analysed from 2 independent experiments. F. Graphical summary of Rab7 removal from the SKIP compartment.


B . Representative confocal images of fixed HeLa cells depleted of TBC1D2 , 1D5 , or 1D15 using siRNA oligo pools and immunolabeled against CD63 ( white ) , scale bar : 10µm . C . Plot of CD63 pixel distribution as a function of TBC1D2 / 1D5 / 1D15 depletion expressed as fractional distance along a straight line from center of nucleus ( 0 ) to the plasma membrane ( 1 . 0 ) , number of ( pixels ) plotted given above each scatter , n ≥ 8 cells per condition analysed from 2 independent experiments .

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B. Representative confocal images of fixed HeLa cells depleted of TBC1D2, 1D5, or 1D15 using siRNA oligo pools and immunolabeled against CD63 (white), scale bar: 10µm. C. Plot of CD63 pixel distribution as a function of TBC1D2/1D5/1D15 depletion expressed as fractional distance along a straight line from center of nucleus (0) to the plasma membrane (1.0), number of (pixels) plotted given above each scatter, n≥8 cells per condition analysed from 2 independent experiments.


D / E . Rescue of TBC1D15 depletion phenotype . D . Representative confocal images of fixed HeLa cells transfected with either control siRNA ( siC ) or oligo # 3 targeting TBC1D15 ( siTBC1D15 ) and ectopically expressing either GFP - EV , GFP - TBC1D5 or siRNA - resistant GFP - TBC1D15res ( green ) , immunolabeled for CD63 ( magenta ) , scale bar : 10µm . E . Quantification of rescue expressed as % cells ( average ) in the population exhibiting one of 3 phenotypes : clustered , dispersed or intermediate ; total numbers of cells analysed per condition appear above each bar , n = 3 independent experiments .

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D/E. Rescue of TBC1D15 depletion phenotype. D. Representative confocal images of fixed HeLa cells transfected with either control siRNA (siC) or oligo #3 targeting TBC1D15 (siTBC1D15) and ectopically expressing either GFP-EV, GFP-TBC1D5 or siRNA-resistant GFP-TBC1D15res (green), immunolabeled for CD63 (magenta), scale bar: 10µm. E. Quantification of rescue expressed as % cells (average) in the population exhibiting one of 3 phenotypes: clustered, dispersed or intermediate; total numbers of cells analysed per condition appear above each bar, n=3 independent experiments.


Effects of TBC1D15 depletion on the morphology and dynamics of late organelles . F . Representative electron micrographs of fixed HeLa cells transfected with either control siRNA ( siC ) or a pool of oligos targeting TBC1D15 ( siTBC1D15 ) are shown , scale bars as indicated .

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Effects of TBC1D15 depletion on the morphology and dynamics of late organelles. F. Representative electron micrographs of fixed HeLa cells transfected with either control siRNA (siC) or a pool of oligos targeting TBC1D15 (siTBC1D15) are shown, scale bars as indicated.


G . Analysis of late compartment dynamics as a function of TBC1D15 depletion . Tracks followed by SiR - lysosome - positive vesicles during a time - lapse lasting 255 sec ( 5 s per frame ) , with highest displacement rates for each track depicted on a rainbow colour scale ( blue : immobile ; red : maximum mobility per time interval ) . Zoom insets ( 3x ) highlight select peripheral ( PP ) and perinuclear ( PN ) regions , scale bar : 10µm EV10 ) . H . Quantification of high motility fraction ( % tracks with displacement rates above 0 . 9µm / sec ) , n siC = 27 , n si1D15 = 37 images ( 2 ≥ cells per image ) analysed from 2 independent experiments . Effectiveness of TBC1D15 ( si1D15 ) depletion is confirmed by immunoblot against endogenous TBC1D15 ( e1D15 ) following its immunoprecipitation ( IP ) , TL : total lysate .

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G. Analysis of late compartment dynamics as a function of TBC1D15 depletion. Tracks followed by SiR-lysosome-positive vesicles during a time-lapse lasting 255 sec (5 s per frame), with highest displacement rates for each track depicted on a rainbow colour scale (blue: immobile; red: maximum mobility per time interval). Zoom insets (3x) highlight select peripheral (PP) and perinuclear (PN) regions, scale bar: 10µm EV10). H. Quantification of high motility fraction (% tracks with displacement rates above 0.9µm/sec), n siC=27, n si1D15=37 images (2≥ cells per image) analysed from 2 independent experiments. Effectiveness of TBC1D15 (si1D15) depletion is confirmed by immunoblot against endogenous TBC1D15 (e1D15) following its immunoprecipitation (IP), TL: total lysate.


I . FRAP of GFP - Rab7 in live HeLa cells transfected with either control siRNA ( siC , black line ) or a pool of oligos targeting TBC1D15 ( si1D15 , red line ) . Plotted is average GFP - Rab7 signal recovery during 200 s following bleaching , expressed as % of pre - bleach signal , n = 3 bleach regions per sample .

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I. FRAP of GFP-Rab7 in live HeLa cells transfected with either control siRNA (siC, black line) or a pool of oligos targeting TBC1D15 (si1D15, red line). Plotted is average GFP-Rab7 signal recovery during 200 s following bleaching, expressed as % of pre-bleach signal, n=3 bleach regions per sample.


Effects of depleting known GAPs for Rab7 on late compartment segregation mediated by RILP and SKIP . A . Representative confocal images of fixed HeLa cells transfected with either control siRNA ( siC ) , two different siRNA oligos targeting TBC1D15 ( # 1 and # 3 ) or oligo pool targeting TBC1D17 and ectopically expressing GFP - Arl8b ( green ) in combination with HA - RILP ( cyan ) and Myc - SKIP ( red ) , immunolabeled against endogenous Rab7 ( eRab7 , magenta ) and the indicated epitope tags . Cell and nuclear boundaries are demarcated with solid and dashed lines respectively , zoom insets ( 3 . 5x ) highlight select peripheral ( PP ) and perinuclear ( PN ) cell regions , scale bar : 10µm .

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Effects of depleting known GAPs for Rab7 on late compartment segregation mediated by RILP and SKIP. A. Representative confocal images of fixed HeLa cells transfected with either control siRNA (siC), two different siRNA oligos targeting TBC1D15 (#1 and #3) or oligo pool targeting TBC1D17 and ectopically expressing GFP-Arl8b (green) in combination with HA-RILP (cyan) and Myc-SKIP (red), immunolabeled against endogenous Rab7 (eRab7, magenta) and the indicated epitope tags. Cell and nuclear boundaries are demarcated with solid and dashed lines respectively, zoom insets (3.5x) highlight select peripheral (PP) and perinuclear (PN) cell regions, scale bar: 10µm.


B . Colocalization of SKIP with endogenous Rab7 ( Mander ' s overlap ) in response to depletion of TBC - containing proteins , n ≥ 4 images ( 3 ≥ cells per image ) analysed per condition from 2 or more independent

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B. Colocalization of SKIP with endogenous Rab7 (Mander's overlap) in response to depletion of TBC-containing proteins, n≥4 images (3≥ cells per image) analysed per condition from 2 or more independent experiments


C . Plot of Rab7 - positive pixel distribution expressed as fractional distance along a straight line from center of nucleus ( 0 ) to the plasma membrane ( 1 . 0 ) , number of ( pixels ) plotted given above each scatter , n ≥ 8 cells analysed per condition from 2 independent experiments .

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C. Plot of Rab7-positive pixel distribution expressed as fractional distance along a straight line from center of nucleus (0) to the plasma membrane (1.0), number of (pixels) plotted given above each scatter, n≥8 cells analysed per condition from 2 independent experiments.


D . Validation of depletion efficiencies expressed as fraction of the indicated mRNA remaining relative to siC assayed by qPCR , n = 3 independent experiments .

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D. Validation of depletion efficiencies expressed as fraction of the indicated mRNA remaining relative to siC assayed by qPCR, n=3 independent experiments.


E . Effect of TBC1D15 depletion on Rab7 - dependent MHC - II receptor trafficking to the SKIP - positive compartment , n siC = 21 , n si1D15 = 21 cells analysed from 3 independent

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E. Effect of TBC1D15 depletion on Rab7-dependent MHC-II receptor trafficking to the SKIP-positive compartment, n siC=21, n si1D15=21 cells analysed from 3 independent experiments


Representative confocal images of either control ( siC ) Hela cells harbouring endogenous TBC1D15 tagged with GFP ( Ge1D15 , green ) or those depleted of FIS1 using a pool of siRNA oligos , fixed and immunolabeled for the mitochondrial marker TOM20 ( blue ) in combination with either endogenous A . Rab7 Zoom insets ( 2 . 5x ) highlight select cell regions , arrowheads point to vesicles positive for TBC1D15 and negative for TOM20 ( yellow ) .

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Representative confocal images of either control (siC) Hela cells harbouring endogenous TBC1D15 tagged with GFP (Ge1D15, green) or those depleted of FIS1 using a pool of siRNA oligos, fixed and immunolabeled for the mitochondrial marker TOM20 (blue) in combination with either endogenous A. Rab7 Zoom insets (2.5x) highlight select cell regions, arrowheads point to vesicles positive for TBC1D15 and negative for TOM20 (yellow).


Representative confocal images of either control ( siC ) Hela cells harbouring endogenous TBC1D15 tagged with GFP ( Ge1D15 , green ) or those depleted of FIS1 using a pool of siRNA oligos , fixed and immunolabeled for the mitochondrial marker TOM20 ( blue ) in combination with either endogenous B . CD63 ( red ) . Zoom insets ( 2 . 5x ) highlight select cell regions , arrowheads point to vesicles positive for TBC1D15 and negative for TOM20 ( yellow ) .

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Representative confocal images of either control (siC) Hela cells harbouring endogenous TBC1D15 tagged with GFP (Ge1D15, green) or those depleted of FIS1 using a pool of siRNA oligos, fixed and immunolabeled for the mitochondrial marker TOM20 (blue) in combination with either endogenous B. CD63 (red). Zoom insets (2.5x) highlight select cell regions, arrowheads point to vesicles positive for TBC1D15 and negative for TOM20 (yellow).


Effect of FIS1 on late compartment distribution . C . Representative confocal images of fixed HeLa cells depleted of FIS1 using a siRNA oligo pool and immunolabeled against CD63 ( white ) .

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Effect of FIS1 on late compartment distribution. C. Representative confocal images of fixed HeLa cells depleted of FIS1 using a siRNA oligo pool and immunolabeled against CD63 (white).


D . Plot of CD63 pixel distribution as a function of FIS1 depletion expressed as fractional distance along a straight line from center of nucleus ( 0 ) to the plasma membrane ( 1 . 0 ) , number of ( pixels ) plotted given above each scatter , n siC = 14 , n siFis1 = 17 cells analysed from 2 independent experiments .

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D. Plot of CD63 pixel distribution as a function of FIS1 depletion expressed as fractional distance along a straight line from center of nucleus (0) to the plasma membrane (1.0), number of (pixels) plotted given above each scatter, n siC=14, n siFis1=17 cells analysed from 2 independent experiments.


E . Colocalization of SKIP with endogenous Rab7 ( Mander ' s overlap ) in response to FIS1 depletion , n siC = 13 , n siFis1 = 9 images ( 3 ≥ cells per image ) analysed from 2 independent

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E. Colocalization of SKIP with endogenous Rab7 (Mander's overlap) in response to FIS1 depletion, n siC=13, n siFis1=9 images (3≥ cells per image) analysed from 2 independent experiments


F . Validation of FIS1 depletion efficiency assayed by qPCR and expressed as fraction FIS1 mRNA remaining relative to siC , n = 3 independent experiments .

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F. Validation of FIS1 depletion efficiency assayed by qPCR and expressed as fraction FIS1 mRNA remaining relative to siC, n=3 independent experiments.


G - J . Recruitment of endogenous TBC1D15 to the SKIP compartment . G . Left panels : representative confocal images of fixed HeLa cells harbouring endogenous TBC1D15 tagged with GFP ( G - e1D15 , green ) and ectopically expressing either empty vector , HA - SKIP or PLEKHM1 - HA ( red ) , immunolabeled against HA . Zoom insets ( 2 . 7x ) highlight select peripheral ( PP ) and perinuclear ( PN ) cell regions . Right panels : pixel plots of endogenous GFP - TBC1D15 ( green line ) and HA signals ( red line ) corresponding to the dashed white lines in G . H - J . Colocalization ( Mander ' s overlap ) between the indicated pairs of proteins , n ≥ 5 images , 3 ≥ cells per image , analysed per condition from 2 independent

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G-J. Recruitment of endogenous TBC1D15 to the SKIP compartment. G. Left panels: representative confocal images of fixed HeLa cells harbouring endogenous TBC1D15 tagged with GFP (G-e1D15, green) and ectopically expressing either empty vector, HA-SKIP or PLEKHM1-HA (red), immunolabeled against HA. Zoom insets (2.7x) highlight select peripheral (PP) and perinuclear (PN) cell regions. Right panels: pixel plots of endogenous GFP-TBC1D15 (green line) and HA signals (red line) corresponding to the dashed white lines in G. H-J. Colocalization (Mander's overlap) between the indicated pairs of proteins, n≥5 images, 3≥ cells per image, analysed per condition from 2 independent experiments


Effects of SKIP KMI motif on HOPS complex recruitment . A . Analysis of SKIP / HOPS interactions by co - IP of RFP / mCherry - HOPS subunits with Myc - SKIP versus the KMI motif mutant AAA from HEK293T cells using RFP - trap beads . Representative immunoblots against RFP and Myc are shown , IP : immunoprecipitation , TL : total lysate . B . Quantification of SKIP / HOPS interactions , n = 3 independent experiments . Top graph : fold change in binding of HOPS subunits to AAA mutant relative to wild type ( WT ) SKIP ( 1 . 0 ) , significance : one - way ANOVA test ( relative to VPS41 ) . Bottom graph : Co - IP per HOPS subunit relative to VPS39 , significance : one - way ANOVA test ( relative to VPS39 ) , * p < 0 . 05 , * * * p < 0 . 001 , ns : not significant .

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Effects of SKIP KMI motif on HOPS complex recruitment. A. Analysis of SKIP/HOPS interactions by co-IP of RFP/mCherry-HOPS subunits with Myc-SKIP versus the KMI motif mutant AAA from HEK293T cells using RFP-trap beads. Representative immunoblots against RFP and Myc are shown, IP: immunoprecipitation, TL: total lysate. B. Quantification of SKIP/HOPS interactions, n=3 independent experiments. Top graph: fold change in binding of HOPS subunits to AAA mutant relative to wild type (WT) SKIP (1.0), significance: one-way ANOVA test (relative to VPS41). Bottom graph: Co-IP per HOPS subunit relative to VPS39, significance: one-way ANOVA test (relative to VPS39), * p<0.05, *** p<0.001, ns: not significant.


C . Colocalization ( Mander ' s overlap ) between SKIP and RFP - VPS39 ( images or mCherry - VPS41 ( images , n ≥ 10 images ( 2 ≥ cells per image ) analysed per condition from 2 independent experiments .

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C. Colocalization (Mander's overlap) between SKIP and RFP-VPS39 (images or mCherry-VPS41 (images , n≥10 images (2≥ cells per image) analysed per condition from 2 independent experiments.


D . Representative confocal images of fixed HeLa cells ectopically expressing Myc - SKIP , KMI motif mutant ( AAA ) or RUN domain truncation mutant ( ∆ RUN ) ( red ) together with RFP - VPS39 ( green ) , immunolabeled for endogenous VPS11 ( eVPS11 , blue ) .

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D. Representative confocal images of fixed HeLa cells ectopically expressing Myc-SKIP, KMI motif mutant (AAA) or RUN domain truncation mutant (∆RUN) (red) together with RFP-VPS39 (green), immunolabeled for endogenous VPS11 (eVPS11, blue).


Effect of HOPS over - expression on recruitment of TBC1D15 to the SKIP compartment . E . Representative confocal images of fixed HeLa cells harbouring endogenous GFP - TBC1D15 ( Ge1D15 , green ) and ectopically expressing HA - SKIP ( red ) together with mCh - VPS18 or RFP - VPS39 ( white ) , immunolabeled for endogenous VPS18 or VPS11 ( blue ) , as

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Effect of HOPS over-expression on recruitment of TBC1D15 to the SKIP compartment. E. Representative confocal images of fixed HeLa cells harbouring endogenous GFP-TBC1D15 (Ge1D15, green) and ectopically expressing HA-SKIP (red) together with mCh-VPS18 or RFP-VPS39 (white), immunolabeled for endogenous VPS18 or VPS11 (blue), as indicated


F - I . Colocalization of SKIP and HOPS complex with TBC1D15 as a function of HOPS subunit over - expression . F / H . Mander ' s overlap between F . SKIP or H . eVPS18 and endogenous TBC1D15 , n ≥ 5 images ( 2 ≥ cells per image ) analysed per condition from 2 independent experiments , significance : one - way ANOVA test ( relative to EV ) , * * p < 0 . 01 , * * * p < 0 . 001 , ns : not significant . G / I . Mander ' s overlap between G . SKIP or I . eVPS11 and endogenous TBC1D15 as a function VPS18 overabundance , with or without FIS1 depletion ( siFIS1 ) , n ≥ 3 images ( 2 ≥ cells per image ) analysed per condition from 2 or more independent experiments .

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F-I. Colocalization of SKIP and HOPS complex with TBC1D15 as a function of HOPS subunit over-expression. F/H. Mander's overlap between F. SKIP or H. eVPS18 and endogenous TBC1D15, n≥5 images (2≥ cells per image) analysed per condition from 2 independent experiments, significance: one-way ANOVA test (relative to EV), ** p<0.01, *** p<0.001, ns: not significant. G/I. Mander's overlap between G. SKIP or I. eVPS11 and endogenous TBC1D15 as a function VPS18 overabundance, with or without FIS1 depletion (siFIS1), n≥3 images (2≥ cells per image) analysed per condition from 2 or more independent experiments.


A / B . In situ SKIP / TBC1D15 complex formation assayed using proximity - based biotin ligation ( BioID ) . A . Neutravidin precipitates ( PD ) from biotin - treated HEK293T cells ectopically expressing GFP - TBC1D15 ( GFP - 1D15 ) together with HA - BioID - SKIP or HA - BioID - EV . Representative immunoblots against GFP , HA and VPS18 , TL : total lysate . B . Quantification of biotinylation of GFP - TBC1D15 ( top graph ) and endogenous VPS18 ( eVPS18 , bottom graph ) by BioID - SKIP , expressed relative to BioID - EV ( 1 . 0 ) , n = 4 independent experiments .

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A/B. In situ SKIP/TBC1D15 complex formation assayed using proximity-based biotin ligation (BioID). A. Neutravidin precipitates (PD) from biotin-treated HEK293T cells ectopically expressing GFP-TBC1D15 (GFP-1D15) together with HA-BioID-SKIP or HA-BioID-EV. Representative immunoblots against GFP, HA and VPS18 , TL: total lysate. B. Quantification of biotinylation of GFP-TBC1D15 (top graph) and endogenous VPS18 (eVPS18, bottom graph) by BioID-SKIP, expressed relative to BioID-EV (1.0), n=4 independent experiments.


C . Analysis of TBC1D15 / HOPS interactions by co - IP of RFP / mCherry - HOPS subunits with GFP - TBC1D15 from HEK293T cells using GFP - trap beads . Left panel : schematic of HOPS complex composition , with subunits tested marked in yellow . Right panel : representative immunoblots against GFP and RFP ; IP : immunoprecipitation , TL : total lysate .

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C. Analysis of TBC1D15/HOPS interactions by co-IP of RFP/mCherry-HOPS subunits with GFP-TBC1D15 from HEK293T cells using GFP-trap beads. Left panel: schematic of HOPS complex composition, with subunits tested marked in yellow. Right panel: representative immunoblots against GFP and RFP; IP: immunoprecipitation, TL: total lysate.


Effects of VPS18 depletion on the recruitment of TBC1D15 to the SKIP complex . D . Representative confocal images of fixed HeLa cells harbouring endogenous GFP - TBC1D15 ( G - e1D15 , green ) , transfected with either control siRNA ( siC ) or oligo pool targeting VPS18 ( siVPS18 ) and ectopically expressing HA - SKIP ( red ) , immunolabeled against endogenous VPS18 ( eVPS18 , blue ) and HA , scale bar : 10µm . Cell and nuclear boundaries are demarcated with solid and dashed lines respectively , zoom insets ( 3 . 5x ) highlight select peripheral ( PP ) and perinuclear ( PN ) cell regions . E . Colocalization ( Mander ' s overlap ) between SKIP and endogenous TBC1D15 as a function of VPS18 depletion , n siC = 9 , n siVPS18 = 11 images ( 2 ≥ cells per image ) analysed from 2 independent experiments .

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Effects of VPS18 depletion on the recruitment of TBC1D15 to the SKIP complex. D. Representative confocal images of fixed HeLa cells harbouring endogenous GFP-TBC1D15 (G-e1D15, green), transfected with either control siRNA (siC) or oligo pool targeting VPS18 (siVPS18) and ectopically expressing HA-SKIP (red), immunolabeled against endogenous VPS18 (eVPS18, blue) and HA, scale bar: 10µm. Cell and nuclear boundaries are demarcated with solid and dashed lines respectively, zoom insets (3.5x) highlight select peripheral (PP) and perinuclear (PN) cell regions. E. Colocalization (Mander's overlap) between SKIP and endogenous TBC1D15 as a function of VPS18 depletion, n siC=9, n siVPS18=11 images (2≥ cells per image) analysed from 2 independent experiments.


F . Neutravidin precipitates ( PD ) from biotin - treated HeLa cells ectopically expressing GFP - TBC1D15 ( GFP - 1D15 ) together with BioID - SKIP or BioID - EV . Representative immunoblots against GFP , HA and VPS18 are shown , TL : total lysate . G . Quantification of GFP - TBC1D15 biotinylation by BioID - SKIP over BioID - EV in response to VPS18 depletion expressed as % of control ( siC ) , n = 4 independent experiments performed in HeLa or HEK293T cells . H . Quantification of VPS18 protein abundance from experiments in G , n = 4 .

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F. Neutravidin precipitates (PD) from biotin-treated HeLa cells ectopically expressing GFP-TBC1D15 (GFP-1D15) together with BioID-SKIP or BioID-EV. Representative immunoblots against GFP, HA and VPS18 are shown, TL: total lysate. G. Quantification of GFP-TBC1D15 biotinylation by BioID-SKIP over BioID-EV in response to VPS18 depletion expressed as % of control (siC), n=4 independent experiments performed in HeLa or HEK293T cells. H. Quantification of VPS18 protein abundance from experiments in G, n=4.


. Effect of TBC1D15 activity on Rab7 displacement from the SKIP compartment . Colocalization ( Mander ' s overlap ) between Rab7 and TBC1D15 ( WT or R417A mutant ) in the presence of either SKIP or PLEKHM1 combined with the indicated VPS proteins , n ≥ 5 images ( 2 ≥ cells per image ) analysed from 2 independent

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. Effect of TBC1D15 activity on Rab7 displacement from the SKIP compartment. Colocalization (Mander's overlap) between Rab7 and TBC1D15 (WT or R417A mutant) in the presence of either SKIP or PLEKHM1 combined with the indicated VPS proteins, n≥ 5 images (2≥ cells per image) analysed from 2 independent experiments


A DNA content of mCFU - E and BaF3 - EpoR cells in response to different Epo concentrations . [ 3H ] - Thymidine incorporation was measured after 14 h ( mCFU - E ) or 38 h ( BaF3 - EpoR ) . Data represented as mean ± standard deviation , N = 3 . Lines represent sigmoidal regression . EC50 values are given .

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A DNA content of mCFU-E and BaF3-EpoR cells in response to different Epo concentrations. [3H]-Thymidine incorporation was measured after 14 h (mCFU-E) or 38 h (BaF3-EpoR). Data represented as mean ± standard deviation, N=3. Lines represent sigmoidal regression. EC50 values are given.


B Cell doubling with time in response to 50 U / ml Epo . Cell numbers were determined by manual counting using trypan blue exclusion assay . Data represented as mean ± standard deviation , N = 3 . Lines represent exponential regression . Doubling time is indicated .

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B Cell doubling with time in response to 50 U/ml Epo. Cell numbers were determined by manual counting using trypan blue exclusion assay. Data represented as mean ± standard deviation, N=3. Lines represent exponential regression. Doubling time is indicated.


C Size determinations of mCFU - E and BaF3 - EpoR cells . Exemplary fluorescence microscopy pictures upon Hoechst staining for nucleus visualization with 60x objective . The bar respresents 10 µm distance ( upper panel ) . Cell diameter was measured by Imaging flow cytometry . Cytoplasm was stained with Calcein , nuclei were stained with DRAQ5 . Probability density function of size distribution with indicated mean diameter of mCFU - E and BaF3EpoR cells . All cells were growth - factor deprived and unstimulated .

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C Size determinations of mCFU-E and BaF3-EpoR cells. Exemplary fluorescence microscopy pictures upon Hoechst staining for nucleus visualization with 60x objective. The bar respresents 10 µm distance (upper panel). Cell diameter was measured by Imaging flow cytometry. Cytoplasm was stained with Calcein, nuclei were stained with DRAQ5. Probability density function of size distribution with indicated mean diameter of mCFU-E and BaF3EpoR cells. All cells were growth-factor deprived and unstimulated.


D Epo - induced phosphorylation of EpoR , AKT and ERK of mCFU - E and BaF3 - EpoR cells . Above each panel the number of growth - factor deprived cells examined per time point is indicated as well as the concentration of Epo applied for stimulation . To the left the position of the molecular weight marker is indicated in kDa and arrowheads indicate the position of the protein of interest . For the detection of the EpoR , 5x106 mCFU - E cells were stimulated with 2 . 5 U / ml Epo and 5x106 BaF3 - EpoR cells were stimulated with 5 U / ml Epo . Cells were lysed , subjected to immunoprecipitation with anti - EpoR and were analyzed by immunoblotting using either anti - pTyr ( pEpoR ) or anti - EpoR ( EpoR ) antibodies . For the detection of AKT , mCFU - E cells were stimulated with 2 . 5 U / ml Epo and BaF3 - EpoR cells were stimulated with 5 U / ml Epo . Per time point cellular lysates equivalent to 5x105 mCFU - E cells and 1x106 BaF3 - EpoR cells were analyzed by immunoblotting using anti - pAKT and anti - AKT antibodies . For the detection of ERK , cells were stimulated with 50 U / ml Epo . Per time point cellular lysates equivalent to 8x105 cells were analyzed by immunoblotting . Immunoblot detection was performed with chemiluminescence utilizing a CCD camera device ( ImageQuant ) .

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D Epo-induced phosphorylation of EpoR, AKT and ERK of mCFU-E and BaF3-EpoR cells. Above each panel the number of growth-factor deprived cells examined per time point is indicated as well as the concentration of Epo applied for stimulation. To the left the position of the molecular weight marker is indicated in kDa and arrowheads indicate the position of the protein of interest. For the detection of the EpoR, 5x106 mCFU-E cells were stimulated with 2.5 U/ml Epo and 5x106 BaF3-EpoR cells were stimulated with 5 U/ml Epo. Cells were lysed, subjected to immunoprecipitation with anti-EpoR and were analyzed by immunoblotting using either anti-pTyr (pEpoR) or anti-EpoR (EpoR) antibodies. For the detection of AKT, mCFU-E cells were stimulated with 2.5 U/ml Epo and BaF3-EpoR cells were stimulated with 5 U/ml Epo. Per time point cellular lysates equivalent to 5x105 mCFU-E cells and 1x106 BaF3-EpoR cells were analyzed by immunoblotting using anti-pAKT and anti-AKT antibodies. For the detection of ERK, cells were stimulated with 50 U/ml Epo. Per time point cellular lysates equivalent to 8x105 cells were analyzed by immunoblotting. Immunoblot detection was performed with chemiluminescence utilizing a CCD camera device (ImageQuant).


E Absolute concentrations of pEpoR , pAKT and ppERK in mCFU - E and BaF3 - EpoR cells with time in response to 50 U / ml Epo . Experimental data of a representative experiment is depicted with filled circles , dashed lines represent splines . Error bars indicate standard deviation estimated by an error model .

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E Absolute concentrations of pEpoR, pAKT and ppERK in mCFU-E and BaF3-EpoR cells with time in response to 50 U/ml Epo. Experimental data of a representative experiment is depicted with filled circles, dashed lines represent splines. Error bars indicate standard deviation estimated by an error model.


F Expression of cell - cycle indicator genes of mCFU - E and BaF3 - EpoR cells in response to 5 U / ml Epo . Genes were selected based on microarray analysis . Experimental data is shown as fold change to unstimulated cells with mean ± standard deviation , N = 3 . Welch Modified Two - Sample t - Test , * p < 0 . 05 .

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F Expression of cell-cycle indicator genes of mCFU-E and BaF3-EpoR cells in response to 5 U/ml Epo. Genes were selected based on microarray analysis. Experimental data is shown as fold change to unstimulated cells with mean ± standard deviation, N=3. Welch Modified Two-Sample t-Test, * p<0.05.


G Fold change of fractions of cells in S / G2 / M phase of the cell cycle with respect to 0 U / ml Epo . Growth - factor deprived cells were stimulated with indicated Epo doses for given time . Fractions in sub - G1 , G1 , S , G2 / M were determined by propidium iodide ( PI ) staining for DNA content . N = 3 . Welch Modified Two - Sample t - Test , n . s . = not significant , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 005 . The percentage of cells in S / G2 / M phase in unstimulated cells is additionally indicated .

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G Fold change of fractions of cells in S/G2/M phase of the cell cycle with respect to 0 U/ml Epo. Growth-factor deprived cells were stimulated with indicated Epo doses for given time. Fractions in sub-G1, G1, S, G2/M were determined by propidium iodide (PI) staining for DNA content. N=3. Welch Modified Two-Sample t-Test, n.s. = not significant, * p < 0.05 , ** p < 0.01 , *** p < 0.005. The percentage of cells in S/G2/M phase in unstimulated cells is additionally indicated.


B - G Model calibration with time - resolved quantitative immunoblot data of mCFU - E cells in blue and BaF3 - EpoR cells in black . Growth - factor deprived mCFU - E cells ( 5x106 cells per condition ) and BaF3 - EpoR cells ( 1x107 cells per condition ) were stimulated with different Epo doses and absolute concentrations were determined for pEpoR ( B ) , pAKT ( C ) , ppERK ( D ) . The scale for pS6 ( E ) was estimated in arbitrary units .

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B-G Model calibration with time-resolved quantitative immunoblot data of mCFU-E cells in blue and BaF3-EpoR cells in black. Growth-factor deprived mCFU-E cells (5x106 cells per condition) and BaF3-EpoR cells (1x107 cells per condition) were stimulated with different Epo doses and absolute concentrations were determined for pEpoR (B), pAKT (C), ppERK (D). The scale for pS6 (E) was estimated in arbitrary units.


GTP - Ras ( F ) and ppERK were determined upon stimulation with indicated , color - coded Epo doses . pEpoR was analyzed by immunoprecipitation followed by immunoblotting , GTP - Ras was analyzed after pulldown using a fusion protein harboring GST fused to the Ras binding domain of Raf1 followed by detection by quantitative immunoblotting . For pAkt and ppERK , cellular lysates were subjected to quantitative immunoblotting . Calibrator proteins were used for EpoR , AKT , GTP - Ras and ERK to facilitate the conversion to nM concentrations . Experimental data represented by filled circles . Error bars represent standard deviation estimated by an error model . Solid lines represent model trajectories .

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GTP-Ras (F) and ppERK were determined upon stimulation with indicated, color-coded Epo doses. pEpoR was analyzed by immunoprecipitation followed by immunoblotting, GTP-Ras was analyzed after pulldown using a fusion protein harboring GST fused to the Ras binding domain of Raf1 followed by detection by quantitative immunoblotting. For pAkt and ppERK, cellular lysates were subjected to quantitative immunoblotting. Calibrator proteins were used for EpoR, AKT, GTP-Ras and ERK to facilitate the conversion to nM concentrations. Experimental data represented by filled circles. Error bars represent standard deviation estimated by an error model. Solid lines represent model trajectories.


A Model calibration with mCFU - E wild - type cells , and mCFU - E cells overexpressing SHIP1 or PTEN , and BaF3 - EpoR wild - type cells . Experimental data represented by filled circles . Error bars represent standard deviation estimated by an error model . Solid lines represent model trajectories .

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A Model calibration with mCFU-E wild-type cells, and mCFU-E cells overexpressing SHIP1 or PTEN, and BaF3-EpoR wild-type cells. Experimental data represented by filled circles. Error bars represent standard deviation estimated by an error model. Solid lines represent model trajectories.


B Model prediction of pAKT dynamics in BaF3 - EpoR cells overexpressing PTEN or SHIP1 . Model predictions are represented by solid lines . Experimental validation data obtained by quantitative immunoblotting represented by filled circles . Error bars represent standard deviation estimated by an error model .

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B Model prediction of pAKT dynamics in BaF3-EpoR cells overexpressing PTEN or SHIP1. Model predictions are represented by solid lines. Experimental validation data obtained by quantitative immunoblotting represented by filled circles. Error bars represent standard deviation estimated by an error model.


D Experimental validation of basal DUSP expression in mCFU - E and BaF3 - EpoR . Quantitative RT - PCR was performed with all samples on the same plate , allowing a direct comparison of mRNA levels between mCFU - E and BaF3 - EpoR cells . Data is normalized to the Rpl32 gene . Ratios of expression in BaF3 - EpoR cells compared to mCFU - E cells are shown as box plots . N = 10 . For details , see Appendix K .

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D Experimental validation of basal DUSP expression in mCFU-E and BaF3-EpoR. Quantitative RT-PCR was performed with all samples on the same plate, allowing a direct comparison of mRNA levels between mCFU-E and BaF3-EpoR cells. Data is normalized to the Rpl32 gene. Ratios of expression in BaF3-EpoR cells compared to mCFU-E cells are shown as box plots. N=10. For details, see Appendix K.


A Model prediction and experimental validation for pAKT , ppERK and pS6 dynamics of 32D - EpoR cells in response to 50 U / ml Epo . Simulations are based on measured , cell - type - specific protein abundance of 32D - EpoR cells and global kinetic rates estimated from mCFUE and BaF3 - EpoR cells . Model predictions are represented by solid lines . Experimental validation data obtained by quantitative immunoblotting represented by filled circles . Error bars represent standard deviation estimated by an error model .

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A Model prediction and experimental validation for pAKT, ppERK and pS6 dynamics of 32D-EpoR cells in response to 50 U/ml Epo. Simulations are based on measured, cell-type-specific protein abundance of 32D-EpoR cells and global kinetic rates estimated from mCFUE and BaF3-EpoR cells. Model predictions are represented by solid lines. Experimental validation data obtained by quantitative immunoblotting represented by filled circles. Error bars represent standard deviation estimated by an error model.


C Model prediction and experimental validation for RSK , AKT , ERK and S6 activation upon RSK overexpression ( oe ) and 5 U / ml Epo stimulation in 32DEpoR cells . Simulations are based on measured , cell - type - specific protein abundance of 32D - EpoR cells and global kinetic rates estimated from mCFUE and BaF3 - EpoR cells . Model predictions are represented by solid lines . Experimental validation data obtained by quantitative immunoblotting represented by filled circles . Error bars represent standard deviation estimated by an error model . Integrated pS6 was significantly higher upon RSK overexpression as compared to 32D - EpoR wild - type ( wt ) cells , N = 4 ( lower right panel ) . Two - Sample t - Test , * * * p < 0 . 005 .

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C Model prediction and experimental validation for RSK, AKT, ERK and S6 activation upon RSK overexpression (oe) and 5 U/ml Epo stimulation in 32DEpoR cells. Simulations are based on measured, cell-type-specific protein abundance of 32D-EpoR cells and global kinetic rates estimated from mCFUE and BaF3-EpoR cells. Model predictions are represented by solid lines. Experimental validation data obtained by quantitative immunoblotting represented by filled circles. Error bars represent standard deviation estimated by an error model. Integrated pS6 was significantly higher upon RSK overexpression as compared to 32D-EpoR wild-type (wt) cells, N=4 (lower right panel). Two-Sample t-Test, *** p<0.005.


D Impact of AKT , Ras or PTEN overexpression on BaF3 - induced pS6 dynamics in EpoR and BaF3 - PTEN cells . Quantitative AKT upon overexpression of PTEN , Ras , a constitutive active BaF3 protein , or the empty vector control in EpoR cells or BaF3 - mCFU - E cells . BaF3 - factor deprived mCFU - E cells ( 5x106 cells per condition ) and Epo - immunoblotting cells ( 1x107 cells per condition ) were stimulated with 5 U / ml Epo for indicated time points . Cellular lysates were analyzed by S6 employing sequential reprobing anti - pAKT , anti - ppERK , anti - pS6 , anti - Epo and to ensure equal loading with anti - beta - actin antibodies . Detection was performed with chemiluminescence using a CCD camera device ( ImageQuant ) . Quantification of pS6 on the right is depicted as fold change to wild - type samples at 30 minutes after Epo stimulation . Error bars represent standard deviation . oe : overexpression . N = 3 . Welch Modified Two - Sample t - Test , n . s . = not significant , * p < 0 .

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

D Impact of AKT, Ras or PTEN overexpression on BaF3-induced pS6 dynamics in EpoR and BaF3-PTEN cells. Quantitative AKT upon overexpression of PTEN, Ras, a constitutive active BaF3 protein, or the empty vector control in EpoR cells or BaF3-mCFU-E cells. BaF3-factor deprived mCFU-E cells (5x106 cells per condition) and Epo-immunoblotting cells (1x107 cells per condition) were stimulated with 5 U/ml Epo for indicated time points. Cellular lysates were analyzed by S6 employing sequential reprobing anti-pAKT, anti-ppERK, anti-pS6, anti-Epo and to ensure equal loading with anti-beta-actin antibodies. Detection was performed with chemiluminescence using a CCD camera device (ImageQuant). Quantification of pS6 on the right is depicted as fold change to wild-type samples at 30 minutes after Epo stimulation. Error bars represent standard deviation. oe: overexpression. N=3. Welch Modified Two-Sample t-Test, n.s. = not significant, * p < 0.05


A Epo - induced signaling upon AKT or MEK inhibitor treatment . Growth - factor deprived cells were pretreated for half an hour with AKT VIII or U0126 , respectively , and subsequently stimulated with 5 U / ml Epo . PDI served as loading control .

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A Epo-induced signaling upon AKT or MEK inhibitor treatment. Growth-factor deprived cells were pretreated for half an hour with AKT VIII or U0126, respectively, and subsequently stimulated with 5 U/ml Epo. PDI served as loading control.


B Model calibration with integrated pAKT , ppERK and pS6 data upon inhibitor treatment and 5 U / ml Epo stimulation of mCFU - E , 32D - EpoR and BaF3 - EpoR cells . Area under curve from time - resolved , quantitative immunoblotting data was calculated and is represented by filled circles . Error bars represent standard deviation estimated by an error model . Solid lines represent model trajectories .

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B Model calibration with integrated pAKT, ppERK and pS6 data upon inhibitor treatment and 5 U/ml Epo stimulation of mCFU-E, 32D-EpoR and BaF3-EpoR cells. Area under curve from time-resolved, quantitative immunoblotting data was calculated and is represented by filled circles. Error bars represent standard deviation estimated by an error model. Solid lines represent model trajectories.


C Analysis of cell - cycle indicator genes upon AKT VIII and U0126 treatment . Growth - factor deprived cells were pretreated for half an hour indicated doses of a single inhibitor , followed by stimulation with 5 U / ml Epo for 0 h and 3 h . The expression of cyclinD2 , cyclinG2 and p27 was measured by quantitative RT - PCR and normalized to the Rpl32 gene . Genes were selected based on microarray analysis . Experimental data is shown as fold change to unstimulated cells with mean ± standard deviation , N = 3 . Welch Modified Two - Sample t - Test , n . s . not significant , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 005 .

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C Analysis of cell-cycle indicator genes upon AKT VIII and U0126 treatment. Growth-factor deprived cells were pretreated for half an hour indicated doses of a single inhibitor, followed by stimulation with 5 U/ml Epo for 0 h and 3 h. The expression of cyclinD2, cyclinG2 and p27 was measured by quantitative RT-PCR and normalized to the Rpl32 gene. Genes were selected based on microarray analysis. Experimental data is shown as fold change to unstimulated cells with mean ± standard deviation, N=3. Welch Modified Two-Sample t-Test, n.s. not significant, * p<0.05, ** p<0.01, *** p<0.005.


B Linear regression fit for the respective cell - cycle indicator of experimental data . Data points represent mean ± standard deviation , N = 3 . Solid lines represent the linear regression fit .

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B Linear regression fit for the respective cell-cycle indicator of experimental data. Data points represent mean ± standard deviation, N=3. Solid lines represent the linear regression fit.


Lower panel : Experimental validation of PTEN and SHIP1 overexpression effects on Epo - dependent proliferation . Proliferation was assessed using [ 3H ] - Thymidine incorporation 14 h ( mCFU - E ) or 38 h ( BaF3 - EpoR ) after retroviral transduction with PTEN or SHIP1 construct for overexpression . Data represented as mean ± standard deviation , N = 3 . EC50 values are given .

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Lower panel: Experimental validation of PTEN and SHIP1 overexpression effects on Epo-dependent proliferation. Proliferation was assessed using [3H]-Thymidine incorporation 14 h (mCFU-E) or 38 h (BaF3-EpoR) after retroviral transduction with PTEN or SHIP1 construct for overexpression. Data represented as mean ± standard deviation, N=3. EC50 values are given.


Lower panel : Experimental validation of proliferation upon combined inhibitor treatment and 5 U / ml Epo stimulation . Proliferation was measured as cell numbers after 14 h ( mCFU - E ) or 38 h ( BaF3 - EpoR , 32D - EpoR ) with Coulter Counter . Maximum was scaled to

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Lower panel: Experimental validation of proliferation upon combined inhibitor treatment and 5 U/ml Epo stimulation. Proliferation was measured as cell numbers after 14 h (mCFU-E) or 38 h (BaF3-EpoR, 32D-EpoR) with Coulter Counter. Maximum was scaled to 1


A Venn diagramm representing overlap of hCFU - E proteome from three independent healthy donors . In total 6 , 925 proteins were

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A Venn diagramm representing overlap of hCFU-E proteome from three independent healthy donors. In total 6,925 proteins were quantified


B Size determinations of hCFU - E compared to mCFU - E cells . Cell diameter was measured by Imaging flow cytometry . Cytoplasm was stained with Calcein , nuclei were stained with DRAQ5 . Probability density function of size distribution with indicated mean diameter of hCFU - E and mCFU - E cells . All cells were growth - factor deprived and unstimulated .

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B Size determinations of hCFU-E compared to mCFU-E cells. Cell diameter was measured by Imaging flow cytometry. Cytoplasm was stained with Calcein, nuclei were stained with DRAQ5. Probability density function of size distribution with indicated mean diameter of hCFU-E and mCFU-E cells. All cells were growth-factor deprived and unstimulated.


C Cytoplasmic volumes of BaF3 - EpoR , 32D - EpoR , mCFU - E and the hCFU - E cells . All volumes were determined using imaging flow cytometry as described in ( B ) .

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C Cytoplasmic volumes of BaF3-EpoR, 32D-EpoR, mCFU-E and the hCFU-E cells. All volumes were determined using imaging flow cytometry as described in (B).


D Correlation between protein molecules per cell for key signaling components determined by quantiative immunoblotting or quantitative mass spectrometry and the " proteomic ruler " method ( Wiśniewski et al , 2014 ) in BaF3 - EpoR and mCFU - E cells . Diagonal line as guide to the eye . Spearman ' s rank - based coefficient of correlation ρ = 0 . 88 .

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D Correlation between protein molecules per cell for key signaling components determined by quantiative immunoblotting or quantitative mass spectrometry and the "proteomic ruler" method (Wiśniewski et al, 2014) in BaF3-EpoR and mCFU-E cells. Diagonal line as guide to the eye. Spearman's rank-based coefficient of correlation ρ=0.88.


E Protein abundance of key signaling components were determined by mass spectrometry of whole - cell lysates of hCFU - E cells . Copy numbers of proteins per cell were obtained by the " proteomic ruler " method and converted to cytoplasmic concentrations with the volumes calculated from ( B ) and shown in ( C ) .

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E Protein abundance of key signaling components were determined by mass spectrometry of whole-cell lysates of hCFU-E cells. Copy numbers of proteins per cell were obtained by the "proteomic ruler" method and converted to cytoplasmic concentrations with the volumes calculated from (B) and shown in (C).


F Model prediction and experimental validation of proliferation upon single or combined inhibitor treatment with AKT VIII and U0126 upon 5 U / ml Epo stimulation . Maximum proliferation was scaled to 1 . Proliferation was measured as cell numbers , counted with a hemocytometer after 96 hours by trypan blue exclusion assay . Solid lines represent model trajectories . Experimental data is represented as mean ± standard deviation . N =

F Model prediction and experimental validation of proliferation upon single or combined inhibitor treatment with AKT VIII and U0126 upon 5 U/ml Epo stimulation. Maximum proliferation was scaled to 1. Proliferation was measured as cell numbers, counted with a hemocytometer after 96 hours by trypan blue exclusion assay. Solid lines represent model trajectories. Experimental data is represented as mean ± standard deviation. N=3


( D ) Immunofluorescence staining of the spinal cord at GW7 , GW8 , GW10 , GW11 , and GW23 for RBFOX3 ( red ) , OLIG2 ( green ) , and GFAP ( white ) . The appearance of astrocytes and oligodendrocytes was observed from GW8 onward . Scale bars : 100 μm .

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(D) Immunofluorescence staining of the spinal cord at GW7, GW8, GW10, GW11, and GW23 for RBFOX3 (red), OLIG2 (green), and GFAP (white). The appearance of astrocytes and oligodendrocytes was observed from GW8 onward. Scale bars: 100 μm.


( C ) Immunofluorescence staining of the spinal cord from GW7 , GW8 , and GW10 for a dorsal marker ( PAX7 , green ) , neuronal precursor marker ( ASCL1 , red ) , and nuclei marker ( DAPI , blue ) . Images show the neuronal differentiation process on the dorsal side of the spinal cord . Scale bar : 100 μm .

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(C) Immunofluorescence staining of the spinal cord from GW7, GW8, and GW10 for a dorsal marker (PAX7, green), neuronal precursor marker (ASCL1, red), and nuclei marker (DAPI, blue). Images show the neuronal differentiation process on the dorsal side of the spinal cord. Scale bar: 100 μm.


( C ) Violin plot showing the expression of selected genes . EGFR + Cluster 14 is circled with a red dotted line .

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(C) Violin plot showing the expression of selected genes. EGFR+ Cluster 14 is circled with a red dotted line.


( D ) Violin plots produced by VlnPlot function of Seurat package representing changes in expression of EGFR , DLL1 , MIAT , and FAM189A2 in cluster 14 with development based on snRNA counts data .

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(D) Violin plots produced by VlnPlot function of Seurat package representing changes in expression of EGFR, DLL1, MIAT, and FAM189A2 in cluster 14 with development based on snRNA counts data.


( B ) Immunofluorescence staining of spinal cord sections at GW7 , GW11 , GW17 , and GW23 for EGFR ( green ) and nuclei ( DAPI , blue ) . EGFR expression on the ventral side was observed in GW7 and GW11 spinal cord . EGFR expression on the dorsal side was observed in GW11 , GW17 , and GW23 spinal cord . Dorsal EGFR expression was mainly concentrated within the dorsal horn area . Scale bar : 100 μm . ( Top ) Higher - magnification view of boxes at bottom . White dashed lines indicate dorsal horn borders .

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(B) Immunofluorescence staining of spinal cord sections at GW7, GW11, GW17, and GW23 for EGFR (green) and nuclei (DAPI, blue). EGFR expression on the ventral side was observed in GW7 and GW11 spinal cord. EGFR expression on the dorsal side was observed in GW11, GW17, and GW23 spinal cord. Dorsal EGFR expression was mainly concentrated within the dorsal horn area. Scale bar: 100 μm. (Top) Higher-magnification view of boxes at bottom. White dashed lines indicate dorsal horn borders.


( D ) Representative immunofluorescence staining of a GW11 spinal cord section . Most dorsal EGFR - expressing cells at GW11 co - expressed GFAP and SOX9 , but not OLIG2 . Scale bars : 100 μm .

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(D) Representative immunofluorescence staining of a GW11 spinal cord section. Most dorsal EGFR-expressing cells at GW11 co-expressed GFAP and SOX9, but not OLIG2. Scale bars: 100 μm.


GW11 EGFR - expressing cells exhibited radial glia morphology and most co - expressed SOX9 ( A ) but not OLIG2 ( B ) . However , EGFR co - expression with SOX9 or OLIG2 was found in GW17 EGFR - expressing cells ( C , D ) . White dashed lines marked dorsal horn borders . Higher - magnification view of boxes on left . Scale bars : 100 μm .

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GW11 EGFR-expressing cells exhibited radial glia morphology and most co-expressed SOX9 (A) but not OLIG2 (B). However, EGFR co-expression with SOX9 or OLIG2 was found in GW17 EGFR-expressing cells (C, D). White dashed lines marked dorsal horn borders. Higher-magnification view of boxes on left. Scale bars: 100 μm.


( A , B ) ( A ) Representative IHC images and ( B ) semi quantification of CRMP4 protein in human spinal cords ( SC ) cross sections from 2 control patients and 3 ALS patients . We analyzed total of 7 SC sections of controls and 14 SC sections of ALS patients , Data presented as mean ± SE . DAB : labeled CRMP4 . Scale bar : left images 20 µm , right insets 10 µm . Mann - Whitney test * * * p = 0 . 0003 .

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(A, B) (A) Representative IHC images and (B) semi quantification of CRMP4 protein in human spinal cords (SC) cross sections from 2 control patients and 3 ALS patients. We analyzed total of 7 SC sections of controls and 14 SC sections of ALS patients, Data presented as mean ±SE. DAB: labeled CRMP4. Scale bar: left images 20 µm, right insets 10 µm. Mann-Whitney test ***p = 0.0003.


( C ) Representative images of P90 SC cross sections of SOD1G93A and WT mice . Red : denotes NeuN , Green : denotes CRMP4 . Scale bar : 10 μm . ( D ) Quantification of the percentage of CRMP4 positive SC neurons in 3 WT VS . 3 SOD1G93A mice . We monitored CRMP4 expression in total of 108 cells in WT condition and 123 cells in SOD1G93A , an average of 36 or 41 cells in each repeat respectively . Student ' s t - test , n = 3 , Data presented as mean ± SE , * p = 0 . 0161 .

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(C) Representative images of P90 SC cross sections of SOD1G93A and WT mice. Red: denotes NeuN, Green: denotes CRMP4. Scale bar: 10 μm. (D) Quantification of the percentage of CRMP4 positive SC neurons in 3 WT VS. 3 SOD1G93A mice. We monitored CRMP4 expression in total of 108 cells in WT condition and 123 cells in SOD1G93A, an average of 36 or 41 cells in each repeat respectively. Student's t-test, n = 3, Data presented as mean ±SE, *p = 0.0161.


( A ) Representative images of ALS patient or non - ALS human control intra - muscular nerves . Red : denotes NFH , Green : denotes CRMP4 , White : denotes co - localization area using Imaris software . Scale bar : 20 μm . ( B ) Quantification of CRMP4 intensity levels in NFH positive intra - muscular distal nerves from 5 non - ALS controls and 4 sALS patients . We analyzed 40 terminal axons from the healthy samples ( ~ 8 axons per sample ) and 36 terminal axons from sALS samples ( ~ 8 axons per sample ) . Data presented as mean ± SE . Student ' s t - test , * p = 0 . 0475 .

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(A) Representative images of ALS patient or non-ALS human control intra-muscular nerves. Red: denotes NFH, Green: denotes CRMP4, White: denotes co-localization area using Imaris software. Scale bar: 20 μm. (B) Quantification of CRMP4 intensity levels in NFH positive intra-muscular distal nerves from 5 non-ALS controls and 4 sALS patients. We analyzed 40 terminal axons from the healthy samples (~8 axons per sample) and 36 terminal axons from sALS samples (~8 axons per sample). Data presented as mean ±SE. Student's t-test, *p = 0.0475.


( C ) Representative images of SOD1G93A / ChAT : : tdTomato or WTChAT : : tdTomato neuromuscular junctions at P90 . White : denotes BTX , Red : denotes direct ChAT , Green : denotes CRMP4 , Yellow : denotes Z projection of 3D Imaris co - localization of CRMP4 and ChAT . Scale bar : 10 µm . ( D ) Quantification of CRMP4 positive NMJs in gastrocnemius muscles from 3 WT or 3 SOD1G93A P90 mice . Total of 44 NMJ ' s in WT condition and 60 NMJ ' s in SOD1G93A condition . Student ' s t - test , n = 3 , Data presented as mean ± SE , * p = 0 . 0157 . ( E ) Quantification of the percent of partially denervated NMJ ' s in the presence or absence of CRMP4 immunostaining in 3 different SOD1G93A mice . We counted 24 NMJ ' s in SOD1G93A CRMP4 negatives and 67 NMJ ' s in SOD1G93A CRMP4 positives . Student ' s t - test , n = 3 , Data presented as mean ± SE , * p = 0 . 0352 .

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(C) Representative images of SOD1G93A/ChAT::tdTomato or WTChAT::tdTomato neuromuscular junctions at P90. White: denotes BTX, Red: denotes direct ChAT, Green: denotes CRMP4, Yellow: denotes Z projection of 3D Imaris co-localization of CRMP4 and ChAT. Scale bar: 10 µm. (D) Quantification of CRMP4 positive NMJs in gastrocnemius muscles from 3 WT or 3 SOD1G93A P90 mice. Total of 44 NMJ's in WT condition and 60 NMJ's in SOD1G93A condition. Student's t-test, n=3, Data presented as mean ±SE, *p = 0.0157. (E) Quantification of the percent of partially denervated NMJ's in the presence or absence of CRMP4 immunostaining in 3 different SOD1G93A mice. We counted 24 NMJ's in SOD1G93A CRMP4 negatives and 67 NMJ's in SOD1G93A CRMP4 positives. Student's t-test, n=3, Data presented as mean ±SE, *p = 0.0352.


( F ) Representative images of P90 SOD1G93A and WT sciatic nerves . Red : denotes NFH , Blue : denotes GFAP and green denotes CRMP4 , Yellow : denotes the Z projection of 3D Imaris co - localization of CRMP4 and NFH . Scale bar : 5 μm . ( G ) Quantification of the co - localization area of CRMP4 with NFH in the sciatic nerve in 3 SOD1G93A mice compared to 3 WT mice using Imaris analysis . 14 WT sciatic nerve sections and 11 SOD1G93A sections were monitored . Data presented as mean ± SE . Student ' s t - test , n = 3 , * * * * p < 0 . 0001 .

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(F) Representative images of P90 SOD1G93A and WT sciatic nerves. Red: denotes NFH, Blue: denotes GFAP and green denotes CRMP4, Yellow: denotes the Z projection of 3D Imaris co-localization of CRMP4 and NFH. Scale bar: 5 μm. (G) Quantification of the co-localization area of CRMP4 with NFH in the sciatic nerve in 3 SOD1G93A mice compared to 3 WT mice using Imaris analysis. 14 WT sciatic nerve sections and 11 SOD1G93A sections were monitored. Data presented as mean ±SE. Student's t-test, n=3, ****p<0.0001.