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 .

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 .

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 .

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 .

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

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 .

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


( C ) Expression profile of the indicated ion channel analyzed by RT - PCR in the WM266 . 4 and 451Lu metastatic melanoma cell lines ( middle and bottom panels , respectively ) . Expected amplicons sizes ( positive controls in top panel ) are in base pairs ( bp ) ; TRPV1 = 120 , TRPV2 = 199 , TRPV3 = 226 , TRPV4 = 190 / 370 , TRPV6 = 208 , TRPC1 = 201 , TRPC6 = 121 , TRPM2 = 303 , TRPM7 = 226 , TRPA1 = 140 , Orai1 = 161 , STIM1 = 109 , STIM2 = 114 ) .

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(C) Expression profile of the indicated ion channel analyzed by RT-PCR in the WM266.4 and 451Lu metastatic melanoma cell lines (middle and bottom panels, respectively). Expected amplicons sizes (positive controls in top panel) are in base pairs (bp); TRPV1=120, TRPV2=199, TRPV3=226, TRPV4=190/370, TRPV6=208, TRPC1=201, TRPC6=121, TRPM2=303, TRPM7=226, TRPA1=140, Orai1=161, STIM1=109, STIM2=114).


( F ) TRPV2 immunoblotting in 36 out of the 60 NCI - 60 cell lines . Tubulin was used as a loading control . Note that after longer exposure times , TRPV2 expression was also detected in some breast and prostate cancer cell lines , as it has been previously described ( Elbaz et al , 2018 ; Gambade et al , 2016 ; Monet et al , 2010 ) .

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(F) TRPV2 immunoblotting in 36 out of the 60 NCI-60 cell lines. Tubulin was used as a loading control. Note that after longer exposure times, TRPV2 expression was also detected in some breast and prostate cancer cell lines, as it has been previously described (Elbaz et al, 2018; Gambade et al, 2016; Monet et al, 2010).


( G ) TRPV2 protein expression in melanoma cell lines harboring either B - RAF ( black ) or N - Ras ( blue ) mutations . β - Actin was used as a loading control .

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(G) TRPV2 protein expression in melanoma cell lines harboring either B-RAF (black) or N-Ras (blue) mutations. β-Actin was used as a loading control.


( A ) Analysis for total and plasma membrane ( PM ) TRPV2 expression in melanoma cell lines . β - actin was used as loading and cell integrity control . PM fractions were isolated using the surface protein biotinylation technique . The higher molecular weight bands of TRPV2 correspond to the glycosylated and mature

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(A) Analysis for total and plasma membrane (PM) TRPV2 expression in melanoma cell lines. β-actin was used as loading and cell integrity control. PM fractions were isolated using the surface protein biotinylation technique. The higher molecular weight bands of TRPV2 correspond to the glycosylated and mature channel


( C ) TRPV2 overexpression in the non - invasive 501mel cell line ( GFP - TRPV2 ) or downregulation by lentiviral - delivery of TRPV2 specific shRNAs ( shRNA V2 - 1 and V2 - 2 ) in the WM266 . 4 and 451Lu metastatic melanoma cell lines assessed by western - blot . β - actin was used as a loading control .

(C) TRPV2 overexpression in the non-invasive 501mel cell line (GFP-TRPV2) or downregulation by lentiviral-delivery of TRPV2 specific shRNAs (shRNA V2-1 and V2-2) in the WM266.4 and 451Lu metastatic melanoma cell lines assessed by western-blot. β-actin was used as a loading control.


( E ) Average normalized quenching rates ( as in D ) presented as mean ± SEM ( n > 3 biological replicates ) . * * P = 0 . 0095 , the Mann - Whitney test ( left panel ) ; * P = 0 . 0399 - 0 . 0336 ( middle panel ) and * P = 0 . 0438 , * * P = 0 . 0033 ( right panel ) the Kruskal - Wallis test followed by Dunn ' s multiple comparisons test .

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(E) Average normalized quenching rates (as in D) presented as mean ± SEM (n>3 biological replicates). **P = 0.0095, the Mann-Whitney test (left panel); *P = 0.0399-0.0336 (middle panel) and *P = 0.0438, **P = 0.0033 (right panel) the Kruskal-Wallis test followed by Dunn's multiple comparisons test.


( A ) Impact of TRPV2 genetic manipulation in 501mel , WM266 . 4 and 451Lu on serum - induced migration and matrigel invasion . Representative pictures show crystal violet - stained cells that have migrated through 8 µm pore size membranes . Histograms illustrate the average numbers of migrating / invading cells normalized to control ( presented as mean ± SEM of n = 3 independent experiments ) . For 501mel the Mann - Whitney test was used for statistical analysis ( * * P = 0 . 0022 ) ; For WM266 . 4 & 451Lu one - way ANOVA followed by Dunnett ' s multiple comparisons tests was used ( * * P < 0 . 01 ; * * * P < 0 . 001 ; * * * * P < 0 . 0001 ; See appendix Table S2 for exact P values ) .

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(A) Impact of TRPV2 genetic manipulation in 501mel, WM266.4 and 451Lu on serum-induced migration and matrigel invasion. Representative pictures show crystal violet-stained cells that have migrated through 8 µm pore size membranes. Histograms illustrate the average numbers of migrating/invading cells normalized to control (presented as mean ± SEM of n=3 independent experiments). For 501mel the Mann-Whitney test was used for statistical analysis (**P = 0.0022); For WM266.4&451Lu one-way ANOVA followed by Dunnett's multiple comparisons tests was used (**P < 0.01; ***P < 0.001; ****P < 0.0001 ; See appendix Table S2 for exact P values).


( D ) TRPV2 - silencing effect on 3D invasion . Representative images were taken every day for 3 days after collagen embedding of spheroids from WM266 . 4 cells expressing either shRNA ctrl or TRPV2 - targeting shRNAs ( scale bar = 0 . 5 mm ) . ( E ) Quantification of collagen invasion . For each spheroid , the cell - covered area at day 2 was normalized to the starting area of the collagen - embedded spheroids . Histograms represent the invasion relative to control WM266 . 4 spheroids ( shRNA Ctrl ) from at least 12 spheroids from three independent experiments ( See also appendix Fig S3C ) . M . * * * * P < 0 . 0001 and * * P = 0 . 0012 the Kruskal - Wallis test followed by Dunn ' s multiple comparisons test .

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(D) TRPV2-silencing effect on 3D invasion. Representative images were taken every day for 3 days after collagen embedding of spheroids from WM266.4 cells expressing either shRNA ctrl or TRPV2-targeting shRNAs (scale bar=0.5 mm). (E) Quantification of collagen invasion. For each spheroid, the cell-covered area at day 2 was normalized to the starting area of the collagen-embedded spheroids. Histograms represent the invasion relative to control WM266.4 spheroids (shRNA Ctrl) from at least 12 spheroids from three independent experiments (See also appendix Fig S3C). M. ****P <0.0001 and **P = 0.0012 the Kruskal-Wallis test followed by Dunn's multiple comparisons test.


( B ) Representative confocal images of the in situ detection of endogenous TRPV2 interactions with paxillin and vinculin by proximity - ligation assays ( PLA ) . Low confluency WM266 . 4 and 451Lu metastatic melanoma cells plated on fibronectin - coated coverslips were stained for F - actin ( green ) , cell nuclei ( blue ) and PLA reaction using antibodies specific of the indicated proteins ( red ) ( scale bar = 20 µm ; identical for all pictures ) . Red fluorescent spots indicate the association of the two proteins of interest , close to 40 nm . Scatter plots represent the quantification of the number of PLA spots per cell ( bars indicate the medians ) between TRPV2 and a control antibody ( IgG ) , paxillin ( PAX ) or vinculin ( VCL ) . ( n = 29 - 51 cells from at least three independent experiments ) .

(B) Representative confocal images of the in situ detection of endogenous TRPV2 interactions with paxillin and vinculin by proximity-ligation assays (PLA). Low confluency WM266.4 and 451Lu metastatic melanoma cells plated on fibronectin-coated coverslips were stained for F-actin (green), cell nuclei (blue) and PLA reaction using antibodies specific of the indicated proteins (red) (scale bar = 20 µm ; identical for all pictures). Red fluorescent spots indicate the association of the two proteins of interest, close to 40 nm. Scatter plots represent the quantification of the number of PLA spots per cell (bars indicate the medians) between TRPV2 and a control antibody (IgG), paxillin (PAX) or vinculin (VCL). (n=29-51 cells from at least three independent experiments).


( C ) Super - resolution ( dSTORM ) imaging of the clustering of TRPV2 channels with paxillin , but not with vinculin , in migrating WM266 . 4 cells ( Scale bar = 5µm ) . a and b insets show expanded views of a region of the cell and arrows highlight TRPV2 channel and paxillin co - clusters .

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(C) Super-resolution (dSTORM) imaging of the clustering of TRPV2 channels with paxillin, but not with vinculin, in migrating WM266.4 cells (Scale bar = 5µm). a and b insets show expanded views of a region of the cell and arrows highlight TRPV2 channel and paxillin co-clusters.


( B ) Comparison of calpain activity in control ( GFP ) or overexpressing TRPV2 ( GFP - TRPV2 ) 501mel cells , and in control ( shRNA Ctrl ) or TRPV2 - silenced ( shRNA V2 - 1 , - 2 ) WM266 . 4 cells or 451Lu cells . Bar graphs show mean normalized calpain activity ± SEM ( n = 3 biological replicates ) . For 501mel the Mann - Whitney test was used for statistical analysis ( * P = 0 . 05 ) ; For WM266 . 4 & 451Lu one - way ANOVA followed by Dunnett ' s multiple comparisons tests was used ( * * P < 0 . 01 ; See appendix Table S2 for exact P values ) .

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(B) Comparison of calpain activity in control (GFP) or overexpressing TRPV2 (GFP-TRPV2) 501mel cells, and in control (shRNA Ctrl) or TRPV2-silenced (shRNA V2-1, -2) WM266.4 cells or 451Lu cells. Bar graphs show mean normalized calpain activity ± SEM (n=3 biological replicates). For 501mel the Mann-Whitney test was used for statistical analysis (*P = 0.05); For WM266.4&451Lu one-way ANOVA followed by Dunnett's multiple comparisons tests was used (**P < 0.01; See appendix Table S2 for exact P values).


( C ) Representative immunoblots showing the full - length ( 230 kDa ; Black Arrows ) and the calpain - mediated degradation product ( 190 kDa ; Grey Arrows ) of talin after overexpression or repression of TRPV2 , in the corresponding cell lines . Tubulin was used as a loading control .

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(C) Representative immunoblots showing the full-length (230 kDa ; Black Arrows) and the calpain-mediated degradation product (190 kDa ; Grey Arrows) of talin after overexpression or repression of TRPV2, in the corresponding cell lines. Tubulin was used as a loading control.


A - D Representative bioluminescence imaging ( BLI ) data of mice injected intravenously with the non - metastatic melanoma cell line 501mel - Luc transfected with either GFP - TRPV2 or GFP control ( A - B ) , or with the invasive 451Lu - Luc melanoma cell line expressing either control shRNA or TRPV2 targeting shRNA ( C - D ) . Tumor growth and metastasis formation were monitored for 64 days ( 501mel - Luc ) or 35 days ( 451Lu - Luc ) after injection . Graphs presented in A and C show in vivo normalized photon flux quantification at the end time point ( ns P = 0 . 0903 ( A ) and * * P = 0 . 0064 ( C ) , the Mann - Withney test ) . Graphs presented in B and D show the number of metastatic foci per animal ( B ) or per lungs ( D ) counted at necropsy ( * P = 0 . 0397 ( B ) and ns P = 0 . 0691 ( D ) , unpaired t - tests ) . For ( D ) Representative ex vivo BLI images of lung metastasis are

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A-D Representative bioluminescence imaging (BLI) data of mice injected intravenously with the non-metastatic melanoma cell line 501mel-Luc transfected with either GFP-TRPV2 or GFP control (A-B), or with the invasive 451Lu-Luc melanoma cell line expressing either control shRNA or TRPV2 targeting shRNA (C-D). Tumor growth and metastasis formation were monitored for 64 days (501mel-Luc) or 35 days (451Lu-Luc) after injection. Graphs presented in A and C show in vivo normalized photon flux quantification at the end time point (ns P = 0.0903 (A) and **P = 0.0064 (C), the Mann-Withney test). Graphs presented in B and D show the number of metastatic foci per animal (B) or per lungs (D) counted at necropsy (*P = 0.0397 (B) and ns P = 0.0691 (D), unpaired t-tests). For (D) Representative ex vivo BLI images of lung metastasis are shown


( A ) TRPV2 RNA expression levels ( microarray probe : 219282 _ s _ at ) measured in normal skin ( n = 7 biological replicates ) , nevi ( n = 9 biological replicates ) , primary ( n = 31 biological replicates ) and metastatic ( n = 73 biological replicates ) melanomas ( GEO : GSE46517 ) ( Data ref : Kwong LN , 2013 ) . TRPV2 levels are increased in malignant lesions compared to normal skin and nevi . Data is represented as a whisker - box plot with outliers plotted as individual points ( Boxes extend from the 25th to 75th percentiles , whiskers from the 10th to 90th percentiles , the horizontal line in each box is plotted at the median ; ns P = 0 , 1383 , * P = 0 , 0488 and * * P = 0 , 0016 , the Kruskal - Wallis test ) . The horizontal dotted line corresponds to TRPV2 median expression in nevi .

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(A) TRPV2 RNA expression levels (microarray probe: 219282_s_at) measured in normal skin (n=7 biological replicates), nevi (n=9 biological replicates) , primary (n=31 biological replicates) and metastatic (n=73 biological replicates) melanomas (GEO: GSE46517)(Data ref: Kwong LN, 2013). TRPV2 levels are increased in malignant lesions compared to normal skin and nevi. Data is represented as a whisker-box plot with outliers plotted as individual points (Boxes extend from the 25th to 75th percentiles, whiskers from the 10th to 90th percentiles, the horizontal line in each box is plotted at the median; ns P = 0,1383, *P = 0,0488 and **P = 0,0016, the Kruskal-Wallis test). The horizontal dotted line corresponds to TRPV2 median expression in nevi.


( B ) Representative melanoma tissue lesions from a tissue microarray ( TMA ) comparing TRPV2 staining between a benign nevus and a malignant melanoma ( See also appendix Fig S10 ) ( Scale bar = 200µm ) . Top right insets show the same images stained with an anti - melanoma triple cocktail ( HMB45 + A103 + T311 ) . Regions depicted with a black line represent the interface between the diffusely stained tumor ( T ) and the surrounding normal stroma ( S ) . Right panel shows the scatter plot of the complete quantification of TRPV2 staining in 77 patients with malignant melanoma compared to 16 nevi tissues ( dot , single patient sample ) with lines for mean ± SEM ( * P = 0 . 0473 , the Mann - Whitney test ) .

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(B) Representative melanoma tissue lesions from a tissue microarray (TMA) comparing TRPV2 staining between a benign nevus and a malignant melanoma (See also appendix Fig S10) (Scale bar = 200µm). Top right insets show the same images stained with an anti-melanoma triple cocktail (HMB45+A103+T311). Regions depicted with a black line represent the interface between the diffusely stained tumor (T) and the surrounding normal stroma (S). Right panel shows the scatter plot of the complete quantification of TRPV2 staining in 77 patients with malignant melanoma compared to 16 nevi tissues (dot, single patient sample) with lines for mean ± SEM (*P = 0.0473, the Mann-Whitney test).


( F ) Analysis of TRPV2 expression by quantitative RT - PCR ( left panel ) and western - blot ( right panel ) in the WM115 / WM266 . 4 pair of isogenic melanoma cell lines . The WM115 cell line was derived from an in situ tumor while the WM266 . 4 was established from a skin metastasis of the same patient . Relative transcript levels are presented as mean ± SEM from n = 4 - 9 biological replicates ( * * P = 0 . 0028 , the Mann - Whitney test ) .

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(F) Analysis of TRPV2 expression by quantitative RT-PCR (left panel) and western-blot (right panel) in the WM115/WM266.4 pair of isogenic melanoma cell lines. The WM115 cell line was derived from an in situ tumor while the WM266.4 was established from a skin metastasis of the same patient. Relative transcript levels are presented as mean ± SEM from n=4-9 biological replicates (**P = 0.0028, the Mann-Whitney test).


( G ) Kaplan - Meier plot showing the association of TRPV2 expression ( 10 % highest versus 10 % lowest expressers in the skin cutaneous melanoma cohort TCGA data set ) with melanoma patient survival ( ( * ) P = 0 , 0144 , the Log - rank ( Mantel - Cox ) test ) .

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(G) Kaplan-Meier plot showing the association of TRPV2 expression (10% highest versus 10% lowest expressers in the skin cutaneous melanoma cohort TCGA data set) with melanoma patient survival ((*)P = 0,0144, the Log-rank (Mantel-Cox) test).


( A ) ROS burst response in wild - type ( WT ) and map4k4 lines in response to 1 μM flg22 . Values are means ± SEM , n = 16 ( biological replicates ) . Results are representative of three independent experiments . Statistical analysis was performed using a one - way ANOVA with Tukey post - test , p < 0 . 01 . Data information Different letters indicate statistical significant base on one - way ANOVA with Tukey post - test , samples sharing letters are not significantly different .

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(A) ROS burst response in wild-type (WT) and map4k4 lines in response to 1 μM flg22. Values are means ± SEM, n = 16 (biological replicates). Results are representative of three independent experiments. Statistical analysis was performed using a one-way ANOVA with Tukey post-test, p < 0.01. Data information Different letters indicate statistical significant base on one-way ANOVA with Tukey post-test, samples sharing letters are not significantly different.


( B ) qRT - PCR analysis of the expression of PTI marker gene FRK1 in wild - type and map4k4 - 1 seedlings 1 h after treatment with 1 μM flg22 . Bars represent means ± SD based on three biological replicates , p < 0 . 01 . Data information : Different letters indicate statistical significant base on one - way ANOVA with Tukey post - test , samples sharing letters are not significantly different .

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(B) qRT-PCR analysis of the expression of PTI marker gene FRK1 in wild-type and map4k4-1 seedlings 1 h after treatment with 1 μM flg22. Bars represent means ± SD based on three biological replicates, p < 0.01. Data information: Different letters indicate statistical significant base on one-way ANOVA with Tukey post-test, samples sharing letters are not significantly different.


( D ) Heat map of gene expression response to flg22 in wild - type and map4k4 - 1 , red and blue indicate high and low expression levels .

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(D) Heat map of gene expression response to flg22 in wild-type and map4k4-1, red and blue indicate high and low expression levels.


( F ) qRT - PCR analysis of 14 days old seedlings treated with H2O or 1 µM flg22 for 1 hour . Values are the means of three biological replicates ± SD , p < 0 . 01 . Data information : Different letters indicate statistical significant base on one - way ANOVA with Tukey post - test , samples sharing letters are not significantly different .

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(F) qRT-PCR analysis of 14 days old seedlings treated with H2O or 1 µM flg22 for 1 hour. Values are the means of three biological replicates ± SD, p < 0.01. Data information: Different letters indicate statistical significant base on one-way ANOVA with Tukey post-test, samples sharing letters are not significantly different.


( A ) left panel , map4k4 - 1 shows enhanced resistance to Pseudomonas syringae pv . tomato DC3000 . The growth of Pst DC3000 after inoculation in the indicated genotypes ( Col - 0 , white bars , map4k4 - 1 , green bars ) . Bacterial titers were determined 2 hours ( day 0 ) and 3 days after inoculation . Right panel , disease symptoms of wild - type and map4k4 - 1 at 3 days after inoculation . Values are the means ± SD , n = 6 ( biological replicates ) , p < 0 . 01 . Data information : Statistical analysis were performed using a one - way ANOVA with Newman - Keuls multiple comparison tests or t - tests . n . s . indicate non - significant .

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(A) left panel, map4k4-1 shows enhanced resistance to Pseudomonas syringae pv. tomato DC3000. The growth of Pst DC3000 after inoculation in the indicated genotypes (Col-0, white bars, map4k4-1, green bars). Bacterial titers were determined 2 hours (day 0) and 3 days after inoculation. Right panel, disease symptoms of wild-type and map4k4-1 at 3 days after inoculation. Values are the means ± SD, n = 6 (biological replicates), p < 0.01. Data information: Statistical analysis were performed using a one-way ANOVA with Newman-Keuls multiple comparison tests or t-tests. n.s. indicate non-significant.


( B ) map4k4 - 1 disease resistance phenotype was recovered to wild - type levels in two independent complementation lines , c867 and c872 . Bacterial titers were determined as described above . Values are the means ± SD , n = 6 ( biological replicates ) , p < 0 . 01 . Data information : Statistical analysis were performed using a one - way ANOVA with Newman - Keuls multiple comparison tests or t - tests . n . s . indicate non - significant .

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(B) map4k4-1 disease resistance phenotype was recovered to wild-type levels in two independent complementation lines, c867 and c872. Bacterial titers were determined as described above. Values are the means ± SD, n = 6 (biological replicates), p < 0.01. Data information: Statistical analysis were performed using a one-way ANOVA with Newman-Keuls multiple comparison tests or t-tests. n.s. indicate non-significant.


( C ) qRT - PCR analysis of PR1 expression levels in Col - 0 , map4k4 - 1 and c867 after mock ( MgCl2 ) or Pst DC3000 inoculation , values are the means ± SD , n = 3 ( biological replicates ) , p < 0 . 01 . Data information : Statistical analysis were performed using a one - way ANOVA with Newman - Keuls multiple comparison tests or t - tests . n . s . indicate non - significant .

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(C) qRT-PCR analysis of PR1 expression levels in Col-0, map4k4-1 and c867 after mock (MgCl2) or Pst DC3000 inoculation, values are the means ± SD, n = 3 (biological replicates), p < 0.01. Data information: Statistical analysis were performed using a one-way ANOVA with Newman-Keuls multiple comparison tests or t-tests. n.s. indicate non-significant.


( D ) Flg22 - protection assay . Col - 0 , map4k4 - 1 and c867 were pretreated with H2O or flg22 1 day before Pst DC3000 inoculation . The bacterial population was measured 3 days after infection . Values are the means ± SD , n = 6 . * * p < 0 . 01 , Results are representative of three independent experiments . Data information : Statistical analysis were performed using a one - way ANOVA with Newman - Keuls multiple comparison tests or t - tests . n . s . indicate non - significant .

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(D) Flg22-protection assay. Col-0, map4k4-1 and c867 were pretreated with H2O or flg22 1 day before Pst DC3000 inoculation. The bacterial population was measured 3 days after infection. Values are the means ± SD, n = 6. **p < 0.01, Results are representative of three independent experiments. Data information: Statistical analysis were performed using a one-way ANOVA with Newman-Keuls multiple comparison tests or t-tests. n.s. indicate non-significant.


( A ) Subcellular localization of MAP4K4 - GFP in Arabidopsis mesophyll protoplasts . MAP4K4 - GFP ( green ) partially co - localized with the plasma membrane dye FM4 - 64 ( red ) . 14 hours after transformation , photographs were taken when the plasma membrane of mesophyll protoplasts was stained by FM4 - 64 ( < 3 min ) . Scale bars = 10 µm .

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(A) Subcellular localization of MAP4K4-GFP in Arabidopsis mesophyll protoplasts. MAP4K4-GFP (green) partially co-localized with the plasma membrane dye FM4-64 (red). 14 hours after transformation, photographs were taken when the plasma membrane of mesophyll protoplasts was stained by FM4-64 (< 3 min). Scale bars = 10 µm.


( B ) Auto - phosphorylation of recombinant HisMBP - MAP4K4 wild - type , no auto - phosphorylation in the kinase - inactive HisMBP - MAP4K4 - D139A protein . The phosphorylation level was determined by Pro - Q Diamond phosphoprotein gel stain ( upper panel ) , protein loading amounts were visualized by Coomassie G - 250 staining ( lower panel ) . The predicted molecular weight of HisMBP - MAP4K4 is 118 . 4 kDa .

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(B) Auto-phosphorylation of recombinant HisMBP-MAP4K4 wild-type, no auto-phosphorylation in the kinase-inactive HisMBP-MAP4K4-D139A protein. The phosphorylation level was determined by Pro-Q Diamond phosphoprotein gel stain (upper panel), protein loading amounts were visualized by Coomassie G-250 staining (lower panel). The predicted molecular weight of HisMBP-MAP4K4 is 118.4 kDa.


( C ) BiFC visualization of interaction between MAP4K4 and BIK1 in Arabidopsis mesophyll protoplasts . BIK1 - nYFP and MAP4K4 - cYFP were co - expressed in protoplasts to show fluorescence of complemented YFP signal ( green ) , 14 h after transformation , the results were photographed by confocal laser scanning microscopy , scale bars = 10 µm . Chl indicates Chloroplast , BF indicate Bright field . nYFP and cYFP constructs were used as empty controls . MAP3K5 - K375M and MKK4 were used as control .

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(C) BiFC visualization of interaction between MAP4K4 and BIK1 in Arabidopsis mesophyll protoplasts. BIK1-nYFP and MAP4K4-cYFP were co-expressed in protoplasts to show fluorescence of complemented YFP signal (green), 14 h after transformation, the results were photographed by confocal laser scanning microscopy, scale bars = 10 µm. Chl indicates Chloroplast, BF indicate Bright field. nYFP and cYFP constructs were used as empty controls. MAP3K5-K375M and MKK4 were used as control.


( D ) MAP4K4 associates with BIK1 in vivo by Co - IP . BIK1 - HA and MAP4K4 - GFP were co - expressed in plants by crossing of 35S : : BIK1 - HA and 35S : : MAP4K4 - GFP transgenic lines . 14 days old plants were treated ( + ) with 1 µM flg22 or H2O ( - ) for 10 min . GFP trap beads were used for immunoprecipitation , and target proteins were detected by western blots using anti - GFP or anti - HA antibodies . The predicted molecular weight of BIK1 - HA , MAP4K4 - GFP is 49 . 3 kDa and 103 . 8 kDa respectively .

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(D) MAP4K4 associates with BIK1 in vivo by Co-IP. BIK1-HA and MAP4K4-GFP were co-expressed in plants by crossing of 35S::BIK1-HA and 35S::MAP4K4-GFP transgenic lines. 14 days old plants were treated (+) with 1 µM flg22 or H2O (-) for 10 min. GFP trap beads were used for immunoprecipitation, and target proteins were detected by western blots using anti-GFP or anti-HA antibodies. The predicted molecular weight of BIK1-HA, MAP4K4-GFP is 49.3 kDa and 103.8 kDa respectively.


( A ) Decreased BIK1 abundance in map4k4 - 1 . 35S : : BIK1 - HA was introduced into map4k4 - 1 by crossing , sibling lines carrying either map4k4 - 1 or MAP4K4 genotype were isolated from F3 generation . BIK1 levels were detected by western blot analysis using anti - HA antibodies .

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(A) Decreased BIK1 abundance in map4k4-1. 35S::BIK1-HA was introduced into map4k4-1 by crossing, sibling lines carrying either map4k4-1 or MAP4K4 genotype were isolated from F3 generation. BIK1 levels were detected by western blot analysis using anti-HA antibodies.


( B ) MAP4K4 regulates BIK1 accumulation through the proteasome pathway . 10 days old 35S : : BIK1 - HA seedlings of WT or map4k4 - 1 were treated with DMSO ( - ) or the proteasome inhibitor MG132 ( 50 µM ) for 5h , then the plants were collected for immunoblot analysis .

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(B) MAP4K4 regulates BIK1 accumulation through the proteasome pathway. 10 days old 35S::BIK1-HA seedlings of WT or map4k4-1 were treated with DMSO (-) or the proteasome inhibitor MG132 (50 µM) for 5h, then the plants were collected for immunoblot analysis.


( C ) Flg22 - induced BIK1 phosphorylation assay . 10 days old 35S : : BIK1 - HA seedlings of WT or map4k4 - 1 were treated with H2O ( - ) or 1 µM flg22 for 15 min , BIK1 phosphorylation levels were detected by mobility - shift assay using anti - HA immunoblot .

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(C) Flg22-induced BIK1 phosphorylation assay. 10 days old 35S::BIK1-HA seedlings of WT or map4k4-1 were treated with H2O (-) or 1 µM flg22 for 15 min, BIK1 phosphorylation levels were detected by mobility-shift assay using anti-HA immunoblot.


( D , E and F ) Identification of phosphorylation sites by LC - MS / MS analysis . Five residues in GST - BIK1 - K105E were phosphorylated in vitro by HisMBP - MAP4K4 . The peptide sequences are shown above the spectrum with the PH designating phosphorylated residue . In the spectrum , the matched fragment ions shows in red line for terminal series ions and in orange for immonium ions and modified ions . ( G ) S233 and T242 were required for BIK1 hyper - phosphorylation . BIK1 - HA , BIK1S233 - HA and BIK1S242 - HA mutants were expressed in Col - 0 protoplasts and treated with 100 nM flg22 for 15 min , BIK1 phosphorylation levels were detected by mobility - shift assay using anti - HA immunoblot . Each experiment was performed at least three times with similar results .

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(D, E and F) Identification of phosphorylation sites by LC-MS/MS analysis. Five residues in GST-BIK1-K105E were phosphorylated in vitro by HisMBP-MAP4K4. The peptide sequences are shown above the spectrum with the PH designating phosphorylated residue. In the spectrum, the matched fragment ions shows in red line for terminal series ions and in orange for immonium ions and modified ions. (G) S233 and T242 were required for BIK1 hyper-phosphorylation. BIK1-HA, BIK1S233-HA and BIK1S242-HA mutants were expressed in Col-0 protoplasts and treated with 100 nM flg22 for 15 min, BIK1 phosphorylation levels were detected by mobility-shift assay using anti-HA immunoblot. Each experiment was performed at least three times with similar results.


( A ) MAP4K4 associates with PP2C38 in vivo by Co - IP . MAP4K4 - GFP and PP2C38 - HA were co - expressed in protoplasts . GFP trap beads were used for immunoprecipitation , and target proteins were detected by western blots using anti - GFP or anti - HA antibodies . The predicted molecular weight of MAP4K4 - GFP and PP2C38 - HA is 103 . 8kDa and 48 . 1 kDa respectively .

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(A) MAP4K4 associates with PP2C38 in vivo by Co-IP. MAP4K4-GFP and PP2C38-HA were co-expressed in protoplasts. GFP trap beads were used for immunoprecipitation, and target proteins were detected by western blots using anti-GFP or anti-HA antibodies. The predicted molecular weight of MAP4K4-GFP and PP2C38-HA is 103.8kDa and 48.1 kDa respectively.


( B ) Identification of phosphorylation sites by LC - MS / MS analysis . In vitro phosphorylation site of HisMBP - PP2C38 by HisMBP - MAP4K4 . The peptide sequence is shown above the spectrum with the PH designating phosphorylated residue . In the spectrum , the matched fragment ions shows in red line for terminal series ions and in orange for immonium ions and modified ions .

(B) Identification of phosphorylation sites by LC-MS/MS analysis. In vitro phosphorylation site of HisMBP-PP2C38 by HisMBP-MAP4K4. The peptide sequence is shown above the spectrum with the PH designating phosphorylated residue. In the spectrum, the matched fragment ions shows in red line for terminal series ions and in orange for immonium ions and modified ions.


( C ) S77 is required for flg22 - induced PP2C38 band shift . PP2C38 - HA and PP2C38 - S77A - HA were expressed in Col - 0 and map4k4 protoplasts and treated with 1µM flg22 for 30 min . The phosphorylated PP2C38 was detected by western blot mobility shift assay .

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(C) S77 is required for flg22-induced PP2C38 band shift. PP2C38-HA and PP2C38-S77A-HA were expressed in Col-0 and map4k4 protoplasts and treated with 1µM flg22 for 30 min. The phosphorylated PP2C38 was detected by western blot mobility shift assay.


( D ) flg22 - induced PP2C38 band shift was compromised in map4k4 mutants . PP2C38 - HA were expressed in Col - 0 and map4k4 protoplasts and treated with 1µM flg22 for 30 min .

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(D) flg22-induced PP2C38 band shift was compromised in map4k4 mutants. PP2C38-HA were expressed in Col-0 and map4k4 protoplasts and treated with 1µM flg22 for 30 min.


( E ) PP2C38 dephosphorylates MAP4K4 by its phosphatase activity . HisMBP - MAP4K4 was incubated with HisMBP - PP2C38 or HisMBP - PP2C38D88N D289N ( phosphatase - inactive ) or alone , and the phosphorylation level of MAP4K4 was determined by Pro - Q Diamond phosphoprotein gel stain . All the experiments were performed three times with similar results .

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(E) PP2C38 dephosphorylates MAP4K4 by its phosphatase activity. HisMBP-MAP4K4 was incubated with HisMBP-PP2C38 or HisMBP-PP2C38D88N D289N (phosphatase-inactive) or alone, and the phosphorylation level of MAP4K4 was determined by Pro-Q Diamond phosphoprotein gel stain. All the experiments were performed three times with similar results.


( A ) BIK1 transgene restores flg22 - induced ROS burst in map4k4 - 1 . 35S : : BIK1 - HA WT or 35S : : BIK1 - HA map4k4 - 1 were generated by crossing as described before . ROS burst response in the indicated genotypes in response to 1 μM flg22 . Values are means ± SEM , n = 16 . Results are representative of three independent experiments . Data information : Different letters indicate statistical significant base on one - way ANOVA with Tukey post - test , samples sharing letters are not significantly different , p < 0 . 05 .

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(A) BIK1 transgene restores flg22-induced ROS burst in map4k4-1. 35S::BIK1-HA WT or 35S::BIK1-HA map4k4-1 were generated by crossing as described before. ROS burst response in the indicated genotypes in response to 1 μM flg22. Values are means ± SEM, n = 16. Results are representative of three independent experiments. Data information: Different letters indicate statistical significant base on one-way ANOVA with Tukey post-test, samples sharing letters are not significantly different, p < 0.05.


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(B) Morphological phenotype of 6-week-old of Col-0, map4k4-1, bik1 and map4k4-1 bik1 mutants.


( C ) ROS burst response in Col - 0 , map4k4 - 1 , bik1 and map4k4 - 1 bik1 mutants . Values are means ± SEM , n = 16 . Results are representative of three independent experiments . Data information : Different letters indicate statistical significant base on one - way ANOVA with Tukey post - test , samples sharing letters are not significantly different , p < 0 . 05 .

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(C) ROS burst response in Col-0, map4k4-1, bik1 and map4k4-1 bik1 mutants. Values are means ± SEM, n = 16. Results are representative of three independent experiments. Data information: Different letters indicate statistical significant base on one-way ANOVA with Tukey post-test, samples sharing letters are not significantly different, p < 0.05.


( D ) Relative expression level of PR1 in Col - 0 , map4k4 - 1 , bik1 and map4k4 - 1 bik1mutants by qRT - PCR analysis . Values are means ± SD , n = 3 ( biological replicates ) . Data information : Different letters indicate statistical significant base on one - way ANOVA with Tukey post - test , samples sharing letters are not significantly different , p < 0 . 05 .

(D) Relative expression level of PR1 in Col-0, map4k4-1, bik1 and map4k4-1 bik1mutants by qRT-PCR analysis. Values are means ± SD, n = 3 (biological replicates). Data information: Different letters indicate statistical significant base on one-way ANOVA with Tukey post-test, samples sharing letters are not significantly different, p < 0.05.


(E) Morphological phenotype of 6-week-old of Col-0, map4k4-1, map4k3 and map4k4-1 map4k3 mutants.


( F ) Relative expression level of PR1 in Col - 0 , map4k4 - 1 , map4k3 and map4k4 - 1 map4k3 mutants by qRT - PCR analysis . Values are means ± SD , n = 3 ( biological replicates ) . Data information : Different letters indicate statistical significant base on one - way ANOVA with Tukey post - test , samples sharing letters are not significantly different , p < 0 . 05 .

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(F) Relative expression level of PR1 in Col-0, map4k4-1, map4k3 and map4k4-1 map4k3 mutants by qRT-PCR analysis. Values are means ± SD, n = 3 (biological replicates). Data information: Different letters indicate statistical significant base on one-way ANOVA with Tukey post-test, samples sharing letters are not significantly different, p < 0.05.


( G ) ROS burst response in Col - 0 , map4k4 - 1 , map4k3 and map4k4 - 1 map4k3 mutants . Values are means ± SEM , n = 16 ( biological replicates ) . Data information : Different letters indicate statistical significant base on one - way ANOVA with Tukey post - test , samples sharing letters are not significantly different , p < 0 . 05 .

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(G) ROS burst response in Col-0, map4k4-1, map4k3 and map4k4-1 map4k3 mutants. Values are means ± SEM, n = 16 (biological replicates). Data information: Different letters indicate statistical significant base on one-way ANOVA with Tukey post-test, samples sharing letters are not significantly different, p < 0.05.


B ) Western blots showing absence of mitochondrial proteins from the different mitochondrial complexes : NDUFB8 ( complex I ) , SDHA ( complex II ) , UQCRC2 ( complex III ) and MTCO - 1 ( complex IV ) , in senescent ( 10 days after 20Gy X - ray ) Control and Parkin - expressing MRC5 fibroblasts . Data is representative of 3 independent experiments ;

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B) Western blots showing absence of mitochondrial proteins from the different mitochondrial complexes: NDUFB8 (complex I), SDHA (complex II), UQCRC2 (complex III) and MTCO-1 (complex IV), in senescent (10 days after 20Gy X-ray) Control and Parkin-expressing MRC5 fibroblasts. Data is representative of 3 independent experiments;


C ) Cellular oxygen consumption rates ( OCR ) in senescent ( 10 days after 20Gy X - ray ) Control and Parkin - expressing MRC5 fibroblasts . Data were obtained using Seahorse XF24 analyzer and shows mean ± S . D . n = 4 technical repeats ( representative of 2 independent experiments ) ;

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C) Cellular oxygen consumption rates (OCR) in senescent (10 days after 20Gy X-ray) Control and Parkin-expressing MRC5 fibroblasts. Data were obtained using Seahorse XF24 analyzer and shows mean±S.D. n=4 technical repeats (representative of 2 independent experiments);


D ) Representative 3D EM pictures of senescent ( 20 days after 20Gy X - ray ) Parkin - expressing MRC5 fibroblasts with or without CCCP . Mitochondria are in red , the nucleus in blue ;

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D) Representative 3D EM pictures of senescent (20 days after 20Gy X-ray) Parkin-expressing MRC5 fibroblasts with or without CCCP. Mitochondria are in red, the nucleus in blue;


E ) Representative images of cell size , Sen - β - Gal activity ( blue cytoplasmic staining ) , macro - H2A foci and SDHA immunofluorescence in proliferating and senescent ( 10 days after 20Gy X - ray ) Parkin - expressing MRC5 fibroblasts ;

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E) Representative images of cell size, Sen-β-Gal activity (blue cytoplasmic staining), macro-H2A foci and SDHA immunofluorescence in proliferating and senescent (10 days after 20Gy X-ray) Parkin- expressing MRC5 fibroblasts;


F ) Quantification of ROS levels ( DHE fluorescence ) , Sen - β - Gal positive cells and Senescence - associated heterochromatin foci ( SAHF ) observed by DAPI in proliferating and senescent ( 10 days after 20Gy X - ray ) Control and Parkin - expressing MRC5 fibroblasts . Data are mean ± S . E . M of n = 3 independent experiments ; Asterisks denote statistical significant P < 0 . 05 One - way ANOVA .

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 O

F) Quantification of ROS levels (DHE fluorescence), Sen-β-Gal positive cells and Senescence-associated heterochromatin foci (SAHF) observed by DAPI in proliferating and senescent (10 days after 20Gy X-ray) Control and Parkin-expressing MRC5 fibroblasts. Data are mean±S.E.M of n=3 independent experiments; Asterisks denote statistical significant P<0.05 One-way ANOVA.


G ) Representative western blots showing p21 and p16 expression in proliferating and senescent ( 10 days after 20Gy X - ray ) Control and Parkin - expressing MRC5 fibroblasts . Data are representative of 3 independent experiments ;

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

G) Representative western blots showing p21 and p16 expression in proliferating and senescent (10 days after 20Gy X-ray) Control and Parkin-expressing MRC5 fibroblasts. Data are representative of 3 independent experiments;


H ) Secreted protein measured in a cytokine array ( RayBiotech ) of a variety of inflammatory proteins in proliferating and senescent ( 10 days after 20Gy X - ray ) Parkin - expressing MRC5 fibroblasts . Data are mean ± S . E . M of n = 3 independent experiments . Asterisks denote statistical significant P < 0 . 05 One - way ANOVA .

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

H) Secreted protein measured in a cytokine array (RayBiotech) of a variety of inflammatory proteins in proliferating and senescent (10 days after 20Gy X-ray) Parkin-expressing MRC5 fibroblasts. Data are mean±S.E.M of n=3 independent experiments. Asterisks denote statistical significant P<0.05 One-way ANOVA.


A ) Representative images of colony assays of wild - type and PGC - 1β - / - MEFs grown at 3 % or 21 % O2 ( 10 days after seeding 5 , 000 cells / well ) . Data are representative of 3 independent experiments ;

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

A) Representative images of colony assays of wild-type and PGC-1β -/- MEFs grown at 3% or 21% O2 (10 days after seeding 5,000 cells/well). Data are representative of 3 independent experiments;


B ) Effect of 3 % or 21 % O2 and X - ray irradiation ( at 3 % O2 ) on the percentage of Ki67 ( at day 6 ) and Sen - β - Gal positive cells ( at day 10 ) and number ( N ) of 53BP1 foci ( at day 6 ) in wild - type and PGC - 1β - / - MEFs . Data are mean ± S . E . M of n = 3 independent experiments ; Asterisks denote statistical significant P < 0 . 05 One - way ANOVA .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Effect of 3% or 21% O2 and X-ray irradiation (at 3% O2) on the percentage of Ki67 (at day 6) and Sen-β-Gal positive cells (at day 10) and number (N) of 53BP1 foci (at day 6) in wild-type and PGC-1β -/- MEFs. Data are mean±S.E.M of n=3 independent experiments; Asterisks denote statistical significant P<0.05 One-way ANOVA.


C ) Representative images of Sen - β - Gal activity ( blue cytoplasmatic staining ) , Ki - 67 and 53BP1 foci in proliferating and senescent wild - type and PGC - 1β - / - MEFs ( scale bar = 10µm ) ;

O O O O O O O O O O O O O O O O O O O O O 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) Representative images of Sen-β-Gal activity (blue cytoplasmatic staining), Ki-67 and 53BP1 foci in proliferating and senescent wild-type and PGC-1β-/- MEFs (scale bar=10µm);


D ) mRNA expression of PGC - 1β CXCL - 1 and p16 in proliferating and senescent ( 10 days after 10Gy X - ray ) wild type and PGC - 1β - / - MEFs . Data are mean ± S . E . M . of n = 3 independent experiments ; Asterisks denote statistical significant P < 0 . 05 One - way ANOVA .

D) mRNA expression of PGC-1β CXCL-1 and p16 in proliferating and senescent (10 days after 10Gy X-ray) wild type and PGC-1β-/- MEFs. Data are mean±S.E.M. of n=3 independent experiments; Asterisks denote statistical significant P<0.05 One-way ANOVA.


E ) Effects of overexpression of PGC - 1β on the percentage of Ki67 and Sen - β - Gal positive cells , number ( N ) of 53BP1 foci and percentage change of mitochondrial mass ( NAO intensity ) in proliferating and senescent ( 2 days after 10Gy X - ray ) MEFs cultured at 3 % O2 . Data are mean ± S . E . M of n = 3 independent experiments . Asterisks denote statistical significant P < 0 . 05 One - way ANOVA .

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

E) Effects of overexpression of PGC-1β on the percentage of Ki67 and Sen-β-Gal positive cells, number (N) of 53BP1 foci and percentage change of mitochondrial mass (NAO intensity) in proliferating and senescent (2 days after 10Gy X-ray) MEFs cultured at 3% O2. Data are mean±S.E.M of n=3 independent experiments. Asterisks denote statistical significant P<0.05 One-way ANOVA.


A ) Representative western blot of mTORC1 activity measured by phosphorylated p70S6K ( T389 ) from 6 to 72 hours after 20Gy in MRC5 fibroblasts . Data are representative of 3 independent experiments ;

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

A) Representative western blot of mTORC1 activity measured by phosphorylated p70S6K (T389) from 6 to 72 hours after 20Gy in MRC5 fibroblasts. Data are representative of 3 independent experiments;


B ) Representative western blots of the mitochondrialproteins TOMM20 , NDUFB8 ( complex I ) , SDHA ( complex II ) , UQCRC2 ( complex III ) and MT - CO1 ( complex IV ) following 20Gy irradiation with or without 100nM Rapamycin treatment in MRC5fibroblasts . Data are representative of 4 independent experiments ;

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

B) Representative western blots of the mitochondrialproteins TOMM20, NDUFB8 (complex I), SDHA (complex II), UQCRC2 (complex III) and MT-CO1 (complex IV) following 20Gy irradiation with or without 100nM Rapamycin treatment in MRC5fibroblasts. Data are representative of 4 independent experiments;


C ) Effect of 100nM rapamycin on mitochondrial mass ( measured by NAO fluorescence ) 2 - 4 days following replication exhaustion ( RS ) or genotoxic stress ( generated by X - ray irradiation , Etoposide , Neocarcinostatin ( NCS ) , H2O2 or telomere dysfunction ( TRF2ΔBΔM ) ) in a variety of cell lines . Data are mean from 3 independent experiments per cell line or treatment ;

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

C) Effect of 100nM rapamycin on mitochondrial mass (measured by NAO fluorescence) 2-4 days following replication exhaustion (RS) or genotoxic stress (generated by X-ray irradiation, Etoposide, Neocarcinostatin (NCS), H2O2 or telomere dysfunction (TRF2ΔBΔM)) in a variety of cell lines. Data are mean from 3 independent experiments per cell line or treatment;


D ) ( top ) Representative transmission electron microscopy ( T . E . M . ) micrographs of proliferating and senescent ( 3 days after 20Gy X - ray ) MRC5 fibroblasts treated with or without 100nM rapamycin . Mitochondria are labeled in pink . Scale bar = 2µm ; ( bottom left and middle ) Quantification of mitochondrial volume fraction ( % Vv ) and mitochondrial number per cross section in proliferating and senescent ( 3 days after 20Gy X - ray ) MRC5 fibroblasts treated with or without 100nM rapamycin . T . E . M . mitochondrial analysis are mean ± S . E . M of 24 electron micrographs per condition ; ( bottom right ) mtDNA copy number analysis by q - PCR in proliferating and senescent ( 3 days after 20Gy X - ray ) MRC5 fibroblasts treated with or without 100nM Rapamycin . Data are mean ± S . E . M of n = 3 independent experiments ; Asterisks denote statistical significant P < 0 . 05 One - way ANOVA .

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

D) (top) Representative transmission electron microscopy (T.E.M.) micrographs of proliferating and senescent (3 days after 20Gy X-ray) MRC5 fibroblasts treated with or without 100nM rapamycin. Mitochondria are labeled in pink. Scale bar=2µm; (bottom left and middle) Quantification of mitochondrial volume fraction (%Vv) and mitochondrial number per cross section in proliferating and senescent (3 days after 20Gy X-ray) MRC5 fibroblasts treated with or without 100nM rapamycin. T.E.M. mitochondrial analysis are mean±S.E.M of 24 electron micrographs per condition; (bottom right) mtDNA copy number analysis by q-PCR in proliferating and senescent (3 days after 20Gy X-ray) MRC5 fibroblasts treated with or without 100nM Rapamycin. Data are mean±S.E.M of n=3 independent experiments; Asterisks denote statistical significant P<0.05 One-way ANOVA.


E ) Representative western blots showing expression of phosphorylated p70S6K ( T389 ) and AKT ( S473 ) , the mitochondrial protein NDUFB8 and the DDR downstream target p21 in MRC5 fibroblasts treated with or without 10µM of the ATM inhibitor KU55933 and in fibroblasts from a patient with Ataxia Telangiectasis ( AT ) at different time points after 20Gy X - ray . Data are representative of 3 independent experiments ( ATM inhibitor ) and 1 experiment ( AT patient ) ;

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

E) Representative western blots showing expression of phosphorylated p70S6K (T389) and AKT (S473), the mitochondrial protein NDUFB8 and the DDR downstream target p21 in MRC5 fibroblasts treated with or without 10µM of the ATM inhibitor KU55933 and in fibroblasts from a patient with Ataxia Telangiectasis (AT) at different time points after 20Gy X-ray. Data are representative of 3 independent experiments (ATM inhibitor) and 1 experiment (AT patient);


F ) Western blots showing the effect of the ATM inhibitor KU55933 on γH2A . X , AKT phosphorylation and p21 expression in MRC5fibroblasts after 20Gy X - ray . Data is from one experiment ;

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

F) Western blots showing the effect of the ATM inhibitor KU55933 on γH2A.X, AKT phosphorylation and p21 expression in MRC5fibroblasts after 20Gy X-ray. Data is from one experiment;


G ) Effect of rapamycin and / or ATM inhibitor ( KU55933 ) on mitochondrial mass ( NAO intensity ) in proliferating and senescent ( 3 days after 20Gy X - ray ) MRC5 fibroblasts . Data are mean ± S . E . M of n = 3 independent experiments . Asterisks denote statistical significant P < 0 . 05 One - way ANOVA .

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

G) Effect of rapamycin and/or ATM inhibitor (KU55933) on mitochondrial mass (NAO intensity) in proliferating and senescent (3 days after 20Gy X-ray) MRC5 fibroblasts. Data are mean±S.E.M of n=3 independent experiments. Asterisks denote statistical significant P<0.05 One-way ANOVA.


A ) ROS levels ( DHE intensity ) and mean number ( N ) of yH2A . X foci after mTOR knockdown ( 72 hours ) in proliferating and senescent ( 2 days after 20Gy X - ray ) MRC5 fibroblasts . Data are mean ± S . E . M of n = 3 independent experiments ; Asterisks denote statistical significant P < 0 . 05 One - way ANOVA .

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

A) ROS levels (DHE intensity) and mean number (N) of yH2A.X foci after mTOR knockdown (72 hours) in proliferating and senescent (2 days after 20Gy X-ray) MRC5 fibroblasts. Data are mean±S.E.M of n=3 independent experiments; Asterisks denote statistical significant P<0.05 One-way ANOVA.


B ) ROS levels ( DHE intensity ) and mean number ( N ) of yH2A . X foci in proliferating and senescent ( 3 days after 20Gy X - ray ) MRC5 fibroblasts treated with or without 100nM Rapamycin and / or 2 . 5mM of the antioxidant NAC . Data are mean ± S . E . M of n = 3 independent experiments ; Asterisks denote statistical significant P < 0 . 05 One - way ANOVA .

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 B-CONTROLLED_VAR 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

B) ROS levels (DHE intensity) and mean number (N) of yH2A.X foci in proliferating and senescent (3 days after 20Gy X-ray) MRC5 fibroblasts treated with or without 100nM Rapamycin and/or 2.5mM of the antioxidant NAC. Data are mean±S.E.M of n=3 independent experiments; Asterisks denote statistical significant P<0.05 One-way ANOVA.


C ) ( top ) Scheme illustrating the experimental design : human MRC5 fibroblasts were irradiated with 20Gy X - ray and treated at day 3 after IR with 10µM of the ATM inhibitor ( ATMi ) KU55933 and / or 100nM Rapamycin ( Rap ) ; ( bottom ) Effect of single or combined inhibition of ATM and mTORC1 on the mean number ( N ) of γH2A . X foci , Sen - β - Gal activity and p21 expression in proliferating and senescent ( 10 days after 20Gy X - ray ) MRC5fibroblasts . Data are mean ± S . E . M of n = 3 independent experiments . Western blots are representative of 3 independent experiments ; Asterisks denote statistical significant P < 0 . 05 One - way ANOVA .

O O O O O O O O O O O O O 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) (top) Scheme illustrating the experimental design: human MRC5 fibroblasts were irradiated with 20Gy X-ray and treated at day 3 after IR with 10µM of the ATM inhibitor (ATMi) KU55933 and/or 100nM Rapamycin (Rap); (bottom) Effect of single or combined inhibition of ATM and mTORC1 on the mean number (N) of γH2A.X foci, Sen-β-Gal activity and p21 expression in proliferating and senescent (10 days after 20Gy X-ray) MRC5fibroblasts. Data are mean±S.E.M of n=3 independent experiments. Western blots are representative of 3 independent experiments; Asterisks denote statistical significant P<0.05 One-way ANOVA.


D ) ROS levels ( DHE intensity ) in proliferating and senescent ( 3 days after 10Gy X - ray ) wild - type and PGC - 1β - / - MEFs ( top ) and PGC - 1β overexpressing MEFs ( bottom ) . Data are mean ± S . E . M . of n = 3 independent experiments ; Asterisks denote statistical significant P < 0 . 05 One - way ANOVA .

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

D) ROS levels (DHE intensity) in proliferating and senescent (3 days after 10Gy X-ray) wild-type and PGC-1β-/- MEFs (top) and PGC-1β overexpressing MEFs (bottom). Data are mean±S.E.M. of n=3 independent experiments; Asterisks denote statistical significant P<0.05 One-way ANOVA.


E ) ( top ) Representative images and quantification of immunofluorescence staining against FLAG - PGC - 1β ( red ) and 53BP1 ( green ) in proliferating and senescent ( 2 days after 10Gy X - ray ) PGC - 1β overexpressing MEFs cultured at 3 % O2 , with or without 250µM of the antioxidant Trolox ( scale bar = 10µm ) . Data are mean ± S . E . M , n = 3 independent experiments ; Asterisks denote statistical significant P < 0 . 05 One - way ANOVA .

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

E) (top) Representative images and quantification of immunofluorescence staining against FLAG-PGC-1β (red) and 53BP1 (green) in proliferating and senescent (2 days after 10Gy X-ray) PGC-1β overexpressing MEFs cultured at 3% O2, with or without 250µM of the antioxidant Trolox (scale bar=10µm). Data are mean±S.E.M, n=3 independent experiments; Asterisks denote statistical significant P<0.05 One-way ANOVA.


F ) ( top ) Effect of overexpression of mutated Rheb ( N153T ) and ( bottom ) effect of 100nM rapamycin on the mean number ( N ) of 53BP1 foci in proliferating and senescent ( 3 days after 10Gy X - ray ) wild - type and PGC - 1β - / - MEFs . Data are mean ± S . E . M of n = 3 independent experiments ( at least 125 cells were analyzed per condition ) . Asterisks denote statistical significant P < 0 . 05 One - way ANOVA .

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

F) (top) Effect of overexpression of mutated Rheb (N153T) and (bottom) effect of 100nM rapamycin on the mean number (N) of 53BP1 foci in proliferating and senescent (3 days after 10Gy X-ray) wild-type and PGC-1β -/- MEFs. Data are mean±S.E.M of n=3 independent experiments (at least 125 cells were analyzed per condition). Asterisks denote statistical significant P<0.05 One-way ANOVA.