F Histograms of unwinding rewinding rates of Sgs1 in presence of Top3 - Rmi1 RPA ( purple bars ) . mean rates for unwinding rewinding are 66 ± 2 bp / s ( N = 502 ) 70 ± 4 bp / s ( N = 369 ) , respectively . For comparison , velocity distributions for Sgs1 in presence of RPA are shown as

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F Histograms of unwinding and rewinding rates of Sgs1 in the presence of Top3-Rmi1 and RPA (purple bars). The mean rates for unwinding and rewinding are 66 ± 2 bp/s (N = 502) and 70 ± 4 bp/s (N = 369), respectively. For comparison, the velocity distributions for Sgs1 in the presence of RPA are shown as white bars


A Mean duration of bursts ( violet bars ) ( N > 40 ) pauses ( green bars ) ( N > 40 ) for different enzyme combinations . Error bars represent standard error of mean .

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A Mean duration of bursts (violet bars) (N > 40) and pauses (green bars) (N > 40) for the different enzyme combinations. Error bars represent standard error of the mean.


B Mean duration of a unidirectional unwinding event ( N > 350 ) . Error bars represent standard error of mean .

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B Mean duration of a unidirectional unwinding event (N > 350). Error bars represent standard error of the mean.


C Mean processivity of unidirectional unwinding ( N > 350 ) . Error bars represent standard error of mean .

C Mean processivity of unidirectional DNA unwinding (N > 350). Error bars represent standard error of the mean.


C : Time course of PIKfyve downregulation in brains of prion - infected mice ( 3 mice / time point ) . PIKfyve ( but not FIG4 , calnexin , NeuN synaptophysin ) was suppressed between 60 120 dpi . T : terminal disease stage .

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C: Time course of PIKfyve downregulation in brains of prion-infected mice (3 mice/time point). PIKfyve (but not FIG4, calnexin, NeuN and synaptophysin) was suppressed between 60 and 120 dpi. T: terminal disease stage.


E : Prion - infected Gt1 ( 75 dpi ) stained with Alexa - Fluor647 - succinimidyl ester which stains vacuoles negatively ( arrows ) .

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E: Prion-infected Gt1 cells (75 dpi) stained with Alexa-Fluor647-succinimidyl ester which stains vacuoles negatively (arrows).


G : tga20 COCS were treated with pooled IgG or POM1 ( 72 hours ) . POM1 suppressed PIKfyve but not FIG4 VAC14 . Right : of immunoblots . Each dot represents an independent experiment . * * : p < 0 . 01 ; * * * : p < 0 . 001 ; unpaired t - Error bars represent s . e . m .

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G: tga20 COCS were treated with pooled IgG or POM1 (72 hours). POM1 suppressed PIKfyve but not FIG4 and VAC14. Right: Quantification of immunoblots. Each dot represents an independent experiment. **: p<0.01; ***: p<0.001; unpaired t-test Error bars represent s.e.m.


H : Immunoblots of human CJD brains ( red : sampled region ) . Gt1Sc : prion - infected Gt1 . Loading controls : actin calnexin ( Cnx )

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H: Immunoblots of human CJD brains (red: sampled region). Gt1Sc: prion-infected Gt1 cells. Loading controls: actin and calnexin (Cnx)


C : Treatment of COCS with POM1 reduces of PIKfyve starting 48h post treatment . Each dot represents an experiment . Statistics : Unpaired t - . * * : p < 0 . 01 .

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C: Treatment of COCS with POM1 reduces the levels of PIKfyve starting 48h post treatment. Each dot represents an individual experiment. Statistics: Unpaired t-test. **: p<0.01.


G : Left : partially restored PIKfyve in prion - infected tga20 mice treated with LIN5044 ( LCP ) or vehicle . Right : of western blot . Each dot represents an biological replicate . * : p0 . 05 ; unpaired t - Error bars represent s . e . m .

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G: Left: partially restored PIKfyve levels in prion-infected tga20 mice treated with LIN5044 (LCP) or vehicle. Right: quantification of the western blot. Each dot represents an individual biological replicate. *: p0.05; unpaired t-test Error bars represent s.e.m.


H : Left , tga20 COCS were inoculated with RML or NBH , optionally treated with GSK2606414 starting at 21 dpi , lysed at 35 dpi . Depletion of PIKfyve in prion - infected samples was largely rescued by GSK2606414 ( GSK ) . Right , of western blot . Each dot represents an biological replicate . Statistics : Unpaired t - . * * : p < 0 . 01 ; unpaired t - ; Error bars represent s . e . m .

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H: Left, tga20 COCS were inoculated with RML or NBH, optionally treated with GSK2606414 starting at 21 dpi, and lysed at 35 dpi. Depletion of PIKfyve in prion-infected samples was largely rescued by GSK2606414 (GSK). Right, quantification of western blot. Each dot represents an individual biological replicate. Statistics: Unpaired t-test. **: p<0.01; unpaired t-test; Error bars represent s.e.m.


E , F : Acyl - rac of Gt1 subjected to siRNA against zDHHC9 , zDHHC21 or both ( 72 hours post transfection ) , showing synergistic decrease of PIKfyve acylation by suppression of zDHHC9 21 ( quantified in F ) . Each dot represents a separate experiment ( unpaired t - ) . * * : p < 0 . 01 . Error bars represent s . e . m .

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E, F: Acyl-rac of Gt1 cells subjected to siRNA against zDHHC9, zDHHC21 or both (72 hours post transfection), showing synergistic decrease of PIKfyve acylation by suppression of zDHHC9 and 21 (quantified in F). Each dot represents a separate experiment (unpaired t-test). **: p<0.01. Error bars represent s.e.m.


H - I : Flag - tagged zDHHC9 or zDHHC21 were expressed in Gt1 were transfected with plasmids encoding . After 72 hours , zDHHC9 / 21 ( magenta ) were localized to Golgi apparatus ( GM130 + , cyan ) in NBH - exposed but not in prion - infected . n = 3 biological replicates .

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H-I: Flag-tagged zDHHC9 or zDHHC21 were expressed in Gt1 cells were transfected with plasmids encoding. After 72 hours, zDHHC9/21 (magenta) were localized to the Golgi apparatus (GM130+, cyan) in NBH-exposed cells but not in prion-infected cells. n=3 individual biological replicates.


A : Upper row : prion - infected Gt1 ( 75 dpi ) immunostained for LAMP1 showing prominent vacuoles . Control : NBH - inoculated . Middle row : Gt1 transfected with shRNA targeting PIKfyve or luciferase ( control ) for 5 days immunostained for LAMP1 . Depletion of PIKfyve resulted in LAMP1 + vacuoles ( yellow ) . DAPI : stain . Lower row : prion - infected NBH treated ( 75 dpi ) contained LAMP2 - vacuoles . n = 3 biological replicates .

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A: Upper row: prion-infected Gt1 cells (75 dpi) immunostained for LAMP1 showing prominent vacuoles. Control: NBH-inoculated cells. Middle row: Gt1 cells transfected with shRNA targeting PIKfyve or luciferase (control) for 5 days and immunostained for LAMP1. Depletion of PIKfyve resulted in LAMP1+ vacuoles (yellow). DAPI: nuclear stain. Lower row: prion-infected and NBH treated cells (75 dpi) contained LAMP2- vacuoles. n=3 individual biological replicates.


D : tga20 COCS treated with POM1 or control IgG ( 10 days ) immunostained with NeuN LAMP1 . Nuclei : DAPI . POM1 - treated COCS showed increased LAMP1 expression ( : Appendix Fig . S4B ) . NeuN + exhibited vacuoles ( arrowheads ) .

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D: tga20 COCS treated with POM1 or control IgG (10 days) and immunostained with NeuN and LAMP1. Nuclei: DAPI. POM1-treated COCS showed increased LAMP1 expression (quantification: Appendix Fig. S4B). NeuN+ cells exhibited vacuoles (arrowheads).


G : Lysosomal enzymes , but not β - actin , were elevated in brains of terminally scrapie - sick mice . Panels show independent triplicates . Statistics : ANOVA . . * * : p < 0 . 01 . Error bars represent s . e . m .

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G: Lysosomal enzymes, but not β-actin, were elevated in brains of terminally scrapie-sick mice. Panels show independent triplicates. Statistics: ANOVA.. **: p<0.01. Error bars represent s.e.m.


A : Time course of TFEB phosphorylation in prion - infected C57BL / 6 mice . Total TFEB was unchanged but reduced TFEB phosphorylation was evident at ≥ 90 days . : Each dot represents an biological replicate . Statistics : Unpaired t - . * : p0 . 05 ; * * : p < 0 . 01 . Error bars represent s . e . m .

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A: Time course of TFEB phosphorylation in prion-infected C57BL/6 mice. Total TFEB was unchanged but reduced TFEB phosphorylation was evident at ≥90 days. Quantification: Each dot represents an individual biological replicate. Statistics: Unpaired t-test. *: p0.05; **: p<0.01. Error bars represent s.e.m.


B : TFEB - controlled transcripts in brains of terminally scrapie - sick C57BL / 6 mice were measured by qPCR . Prion infection led to upregulation of these genes , but not of STAT3 . Control : NBH - inoculated mice . ANOVA on independent triplicates . * * * : p < 0 . 001 . Error bars represent s . e . m .

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B: TFEB-controlled transcripts in brains of terminally scrapie-sick C57BL/6 mice were measured by qPCR. Prion infection led to upregulation of these genes, but not of STAT3. Control: NBH-inoculated mice. ANOVA on independent triplicates. ***: p<0.001. Error bars represent s.e.m.


E : Gt1 ( as in D ) were fixed stained with anti - TFEB antibody . Prion - infected showed translocation of TFEB . DAPI : Blue . : Each dot represents an biological replicate ( 30 were quantified per replicate ) . Statistics : Unpaired t - . * * : p < 0 . 01 . Error bars represent s . e . m .

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E: Gt1 cells (as in D) were fixed and stained with anti-TFEB antibody. Prion-infected cells showed nuclear translocation of TFEB. DAPI: Blue. Quantification: Each dot represents an individual biological replicate (30 images were quantified per replicate). Statistics: Unpaired t-test. **: p<0.01. Error bars represent s.e.m.


F : TFEB responsive genes were upregulated in Gt1 chronically infected with RML prions ( 75 dpi ) . Panels depict independent triplicates . Statistics : ANOVA . * : p0 . 05 . Error bars represent s . e . m . G : TFEB responsive genes were upregulated in transfected with shPIKfyve for 5 days compared to shCtrl ( shRNA against luciferase ) . Panels depict independent triplicates . Statistics : ANOVA . * : p0 . 05 . Error bars represent s . e . m .

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F: TFEB responsive genes were upregulated in Gt1 cells chronically infected with RML prions (75 dpi). Panels depict independent triplicates. Statistics: ANOVA. *: p0.05. Error bars represent s.e.m. G: TFEB responsive genes were upregulated in cells transfected with shPIKfyve for 5 days compared to shCtrl (shRNA against luciferase). Panels depict independent triplicates. Statistics: ANOVA. *: p0.05. Error bars represent s.e.m.


C : Prion - infected Gt1 were lentivirally transduced with GADD34 at 75 dpi . Control : empty lentiviral vector . At 79 dpi , GADD34 overexpression had normalized eIF2α phosphorylation restored PIKfyve . Right : of western blots ( n = 3 ; unpaired t - ) . Each dot represents an experiment . * * : p < 0 . 01 . Error bars represent s . e . m .

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C: Prion-infected Gt1 cells were lentivirally transduced with GADD34 at 75 dpi. Control: empty lentiviral vector. At 79 dpi, GADD34 overexpression had normalized eIF2α phosphorylation and restored PIKfyve levels. Right: Quantification of western blots (n=3; unpaired t-test). Each dot represents an individual experiment. **: p<0.01. Error bars represent s.e.m.


E : tga20 COCS were infected with RML optionally treated with bPIP ( 5µg / ml ) . At 45 dpi , NeuN morphometry revealed ablation of cerebellar granule layer ( CGN ) in prion - infected slices its rescue by bPIP . Control : NBH treated COCS . Each dot represents an slice ( Statistics : ANOVA ) . * : p < 0 . 05 . * * * * p < 0 . 0001 .

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E: tga20 COCS were infected with RML and optionally treated with bPIP (5µg/ml). At 45 dpi, NeuN morphometry revealed ablation of cerebellar granule layer (CGN) in prion-infected slices and its rescue by bPIP. Control: NBH treated COCS. Each dot represents an individual slice (Statistics: ANOVA). *: p<0.05. **** p<0.0001.


F : tga20 COCS were treated with POM1 ( optionally pre - blocked with recPrP ) treated with bPIP ( 5µg / ml ) . At 14 dpi NeuN morphometry revealed POM1 - induced ablation of cerebellar granule layer ( CGL ) rescue by bPIP .

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F: tga20 COCS were treated with POM1 (optionally pre-blocked with recPrP) and treated with bPIP (5µg/ml). At 14 dpi NeuN morphometry revealed POM1-induced ablation of cerebellar granule layer (CGL) and rescue by bPIP.


( A ) HeLa stably expressing GFP ‐ LC3B ( left panel ) or GFP ‐ GATE ‐ 16 ( right panel ) were transfected with either non ‐ targeting siRNA ( control siRNA ) or a pool of LC3 siRNAs ( A , B , C ) , using DharmaFect reagent . After 72 h interval , were incubated for 2 h in EBSS medium in absence or presence of 0 . 1 μM Baf A subjected to western blot after lysis with RIPA extraction buffer .

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(A) HeLa cells stably expressing GFP‐LC3B (left panel) or GFP‐GATE‐16 (right panel) were transfected with either non‐targeting siRNA (control siRNA) or a pool of LC3 siRNAs (A, B, C), using DharmaFect reagent. After 72 h interval, the cells were incubated for 2 h in EBSS medium in the absence or presence of 0.1 μM Baf A and subjected to western blot analysis after lysis with RIPA extraction buffer.


( B ) of relative p62 GFP ‐ GATE ‐ 16 level ( upper panel ) degradation ( lower panel ) was performed as described in ' Materials methods ' . Mean ± s . d . of three independent experiments is presented .

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(B) Quantification of relative p62 and GFP‐GATE‐16 level (upper panel) and degradation (lower panel) was performed as described in 'Materials and methods'. Mean±s.d. of three independent experiments is presented.


( C ) HeLa stably expressing GFP ‐ GATE ‐ 16 were transfected with non ‐ targeting siRNA or a pool of LC3 siRNAs treated as in ( A ) . were subjected to immunostaining with anti ‐ p62 after fixation , analysed by confocal microscopy . GFP of three independent experiments was measured as described in ' Materials methods ' ( right panel ) . Scale bar : 20 μm .

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(C) HeLa cells stably expressing GFP‐GATE‐16 were transfected with non‐targeting siRNA or a pool of LC3 siRNAs and treated as in (A). The cells were subjected to immunostaining with anti‐p62 after fixation, and analysed by confocal microscopy. The GFP intensity of three independent experiments was measured as described in 'Materials and methods' (right panel). Scale bar: 20 μm.


( A ) HeLa stably expressing GFP ‐ GATE ‐ 16 ( left panel ) or GFP ‐ LC3B ( right panel ) were transfected with either non ‐ targeting siRNA ( control siRNA ) or a pool of GABARAP siRNAs ( GABARAP , GABARAPL1 , GATE ‐ 16 ) using DharmaFect reagent . After 72 h interval , were incubated for 2 h in EBSS medium in absence or presence of 0 . 1 μM Baf A subjected to western blot after lysis with RIPA extraction buffer .

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(A) HeLa cells stably expressing GFP‐GATE‐16 (left panel) or GFP‐LC3B (right panel) were transfected with either non‐targeting siRNA (control siRNA) or a pool of GABARAP siRNAs (GABARAP, GABARAPL1, and GATE‐16) using DharmaFect reagent. After 72 h interval, the cells were incubated for 2 h in EBSS medium in the absence or presence of 0.1 μM Baf A and subjected to western blot analysis after lysis with RIPA extraction buffer.


( B ) of relative p62 GFP ‐ LC3B level ( upper panel ) degradation ( lower panel ) were performed as described in ' Materials methods ' . Mean ± s . d . of three independent experiments is presented .

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(B) Quantification of relative p62 and GFP‐LC3B level (upper panel) and degradation (lower panel) were performed as described in 'Materials and methods'. Mean±s.d. of three independent experiments is presented.


( C ) HeLa stably expressing GFP ‐ LC3B were transfected with non ‐ targeting siRNA or a pool of GABARAP siRNAs treated as in ( A ) . were subjected to immunostaining with anti ‐ p62 after fixation , analysed by confocal microscopy . GFP of three independent experiments was measured as described in ' Materials methods ' ( right panel ) . Scale bar : 20 μm .

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(C) HeLa cells stably expressing GFP‐LC3B were transfected with non‐targeting siRNA or a pool of GABARAP siRNAs and treated as in (A). The cells were subjected to immunostaining with anti‐p62 after fixation, and analysed by confocal microscopy. The GFP intensity of three independent experiments was measured as described in 'Materials and methods' (right panel). Scale bar: 20 μm.


( A ) Bulk degradation was tested in HeLa that were transfected with non ‐ targeting siRNA ( control siRNA ) , a pool of GABARAP / GATE ‐ 16 siRNAs , or a pool of LC3 siRNAs . rate of long ‐ lived degradation was measured in incubated in either αMEM ( control ) or EBSS ( starvation ) medium , in presence or absence of 10 mM of 3 ‐ MA , 72 h after siRNA transfection . Values expressing degradation are represented as mean ± s . d . of three determinations .

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(A) Bulk protein degradation was tested in HeLa cells that were transfected with non‐targeting siRNA (control siRNA), a pool of GABARAP/GATE‐16 siRNAs, or a pool of LC3 siRNAs. The rate of long‐lived protein degradation was measured in cells incubated in either αMEM (control) or EBSS (starvation) medium, in the presence or absence of 10 mM of 3‐MA, 72 h after siRNA transfection. Values expressing the protein degradation percentage are represented as the mean±s.d. of three determinations.


( B ) HeLa stably expressing GFP ‐ LC3B ( left panel ) or GFP ‐ GATE ‐ 16 ( right panel ) were transfected with a pool of GABARAP / GATE ‐ 16 siRNAs or a pool of LC3 , respectively , with non ‐ targeting siRNA . Seventy ‐ two hours after transfection , were collected after 5 h of starvation relative level of GFP ‐ LC3B or GFP ‐ GATE ‐ 16 was measured using flow cytometry . Values represent mean ± s . d . of three experiments .

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(B) HeLa cells stably expressing GFP‐LC3B (left panel) or GFP‐GATE‐16 (right panel) were transfected with a pool of GABARAP/GATE‐16 siRNAs or a pool of LC3, respectively, and with non‐targeting siRNA. Seventy‐two hours after transfection, the cells were collected after 5 h of starvation and the relative level of GFP‐LC3B or GFP‐GATE‐16 was measured using flow cytometry. Values represent the mean±s.d. of three experiments.


( A ) HeLa stably expressing YFP ‐ Atg5 were transfected with non ‐ targeting siRNA ( control siRNA ) , a pool of LC3 siRNAs , or a pool of GABARAP / GATE ‐ 16 siRNAs using DharmaFect reagent . Seventy ‐ two hours after transfection , were incubated for 2 h in EBSS medium , fixed , immunostained with anti ‐ Atg16 antibodies . of Atg5 Atg16 ‐ labelled structures from three independent experiments is presented on right panel . Scale bar : 20 μm .

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(A) HeLa cells stably expressing YFP‐Atg5 were transfected with non‐targeting siRNA (control siRNA), a pool of LC3 siRNAs, or a pool of GABARAP/GATE‐16 siRNAs using DharmaFect reagent. Seventy‐two hours after transfection, the cells were incubated for 2 h in EBSS medium, fixed, and immunostained with anti‐Atg16 antibodies. Quantification of Atg5 and Atg16‐labelled puncta structures from three independent experiments is presented on the right panel. Scale bar: 20 μm.


( B ) HeLa stably expressing YFP ‐ Atg5 were treated as in ( A ) monitored by live microscopy . of lifespan of Atg5 ‐ labelled structures is presented . Mean ± s . d . of three independent experiments is presented . * P0 . 05 , * * P0 . 001 .

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(B) HeLa cells stably expressing YFP‐Atg5 were treated as in (A) and monitored by live microscopy. Quantification of the lifespan of Atg5‐labelled puncta structures is presented. Mean±s.d. of three independent experiments is presented. *P0.05, **P0.001.


( C ) YFP ‐ Atg5 were transfected with siRNA pool starved as in ( A ) . Cryo sections of fixed were immunolabelled using anti ‐ GFP antibodies analysed by as described in ' Materials methods ' . of Atg5 ‐ labelled structures is presented at right panel . * P0 . 05 , * * P0 . 001 .

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(C) YFP‐Atg5 cells were transfected with siRNA pool and starved as in (A). Cryo sections of fixed cells were immunolabelled using anti‐GFP antibodies and analysed by TEM as described in 'Materials and methods'. Quantification of the Atg5‐labelled structures size is presented at the right panel. *P0.05, **P0.001.


( A ) Control HeLa HeLa stably expressing GFP ‐ LC3B or GFP ‐ GATE ‐ 16 were starved for 2 h in EBSS medium fixed immunostained with anti ‐ Atg16 antibodies . of Atg16 ‐ labelled structures from three independent experiments is presented at right panel . Arrowheads represent Atg ‐ 16 ‐ labelled . Scale bar : 20 μm .

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(A) Control HeLa cells and HeLa cells stably expressing GFP‐LC3B or GFP‐GATE‐16 were starved for 2 h in EBSS medium fixed and immunostained with anti‐Atg16 antibodies. Quantification of Atg16‐labelled puncta structures from three independent experiments is presented at the right panel. Arrowheads represent Atg‐16‐labelled puncta. Scale bar: 20 μm.


( B ) HeLa stably expressing GFP ‐ LC3B ( left panel ) or GFP ‐ GATE ‐ 16 ( middle panel ) were starved for 2 h in EBSS medium . were fixed their cryo sections were immunolabelled as in ( Figure 4B ) . of of GFP ‐ labelled structures ( right panel ) from three independent experiments is presented . * P0 . 05 , * * P0 . 001 .

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(B) HeLa cells stably expressing GFP‐LC3B (left panel) or GFP‐GATE‐16 (middle panel) were starved for 2 h in EBSS medium. The cells were fixed and their cryo sections were immunolabelled as in ( Figure 4B). Quantification of the size of GFP‐labelled structures (right panel) from three independent experiments is presented. *P0.05, **P0.001.


( A , B ) Control HeLa HeLa stably expressing GFP ‐ LC3B ( A ) or GFP ‐ GATE ‐ 16 ( B ) were transfected with reciprocal siRNA pools using DharmaFect reagent . After 72 h interval , were incubated for 2 h in EBSS , subjected to immunostaining with anti ‐ Atg16 after fixation , analysed by confocal microscopy . of containing Atg16 structures larger than 2 μm ( A ) or Atg16 ‐ labelled structures ( B ) from three independent experiments in comparison to control to G to A mutants ( D ) is presented at lower panel . Scale bar : 20 μm .

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

(A, B) Control HeLa cells and HeLa cells stably expressing GFP‐LC3B (A) or GFP‐GATE‐16 (B) were transfected with the reciprocal siRNA pools using DharmaFect reagent. After 72 h interval, the cells were incubated for 2 h in EBSS, subjected to immunostaining with anti‐Atg16 after fixation, and analysed by confocal microscopy. Quantification of cells containing Atg16 structures larger than 2 μm (A) or Atg16‐labelled puncta structures (B) from three independent experiments in comparison to control and to G to A mutants (D) is presented at the lower panel. Scale bar: 20 μm.


( C ) HeLa stably expressing silent GFP ‐ LC3B or GFP ‐ GATE ‐ 16 were transfected with their siRNA pools using DharmaFect reagent . After 72 ‐ h interval , were incubated for 2 h in EBSS medium subjected to immunostaining with anti ‐ Atg16 anti ‐ p62 antibodies followed by fixation . were analysed by confocal microscopy . Arrows represent , which do not express GFP whereas arrowheads represent , which express GFP ‐ . of Atg16 ‐ labelled structures from three independent experiments is presented at right panel . Scale bar : 20 μm .

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(C) HeLa cells stably expressing silent GFP‐LC3B or GFP‐GATE‐16 were transfected with their siRNA pools using DharmaFect reagent. After 72‐h interval, the cells were incubated for 2 h in EBSS medium and subjected to immunostaining with anti‐Atg16 and anti‐p62 antibodies followed by fixation. The cells were analysed by confocal microscopy. Arrows represent cells, which do not express GFP proteins whereas arrowheads represent cells, which express GFP‐proteins. Quantification of Atg16‐labelled puncta structures from three independent experiments is presented at the right panel. Scale bar: 20 μm.


( D ) HeLa stably expressing GFP ‐ LC3BG120A or GFP ‐ GATE ‐ 16G116Awere transfected with either pool of GABARAP / GATE ‐ 16 siRNAs or LC3 siRNAs , respectively , a non ‐ targeting siRNA . were starved for 2 h , fixed , immunostained with anti ‐ Atg16 anti ‐ p62 antibodies . is presented in ( A , B ) at lower panels . Scale bar : 20 μm .

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(D) HeLa cells stably expressing GFP‐LC3BG120A or GFP‐GATE‐16G116Awere transfected with either pool of GABARAP/GATE‐16 siRNAs or LC3 siRNAs, respectively, and a non‐targeting siRNA. The cells were starved for 2 h, fixed, and immunostained with anti‐Atg16 and anti‐p62 antibodies. Quantification is presented in (A, B) at the lower panels. Scale bar: 20 μm.


( E ) HeLa stably expressing GFP ‐ LC3B ( left panel ) or GFP ‐ GATE ‐ 16 ( right panel ) were transfected with reciprocal siRNA pool or with non ‐ targeting siRNA starved for 2 h in EBSS medium . were fixed their cryo sections were immuno ‐ labelled using anti ‐ GFP antibodies analysed by as described in ' Materials methods ' . of containing Atg16 structures larger than 2 μm ( left panel ) or of Atg16 ‐ labelled structures ( right panel ) is presented .

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(E) HeLa cells stably expressing GFP‐LC3B (left panel) or GFP‐GATE‐16 (right panel) were transfected with the reciprocal siRNA pool or with non‐targeting siRNA and starved for 2 h in EBSS medium. The cells were fixed and their cryo sections were immuno‐labelled using anti‐GFP antibodies and analysed by TEM as described in 'Materials and methods'. Quantification of cells containing Atg16 structures larger than 2 μm (left panel) or the size of Atg16‐labelled structures (right panel) is presented.


( A ) Control HeLa HeLa stably expressing GFP ‐ LC3B or GFP ‐ GATE ‐ 16 were transfected with HsAtg4AC77A ‐ Myc ‐ His6 . After 48 h interval , were incubated for 2 h in EBSS , subjected to immunostaining with anti ‐ Atg16 anti ‐ myc followed by confocal microscopy . of containing Atg16 structures larger than 2 μm from three independent experiments is presented at right panel . Scale bar : 20 μm .

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(A) Control HeLa cells and HeLa cells stably expressing GFP‐LC3B or GFP‐GATE‐16 were transfected with HsAtg4AC77A‐Myc‐His6. After 48 h interval, the cells were incubated for 2 h in EBSS, and subjected to immunostaining with anti‐Atg16 and anti‐myc followed by confocal microscopy analysis. Quantification of cells containing Atg16 structures larger than 2 μm from three independent experiments is presented at the right panel. Scale bar: 20 μm.


( B ) HeLa were transfected with HsAtg4AC77A ‐ Myc ‐ His6 or empty vector ( mock ) . After a 48 h interval , were incubated for 2 h in EBSS , fixed , their ultrathin sections were analysed by as described in ' Materials methods ' . * P0 . 05 .

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(B) HeLa cells were transfected with HsAtg4AC77A‐Myc‐His6 or empty vector (mock). After a 48 h interval, the cells were incubated for 2 h in EBSS, fixed, and their ultrathin sections were analysed by TEM as described in 'Materials and methods'. *P0.05.


A Range of example RAD51 expression score ( RAD51NES ) values with respective unmixed monochrome . EpCAM is used as a tumour marker an internal sample quality control . Scale bar is 50μm .

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A Range of example RAD51 nuclear expression score (RAD51NES) values with respective unmixed monochrome fluorescent IHC staining images. EpCAM is used as a tumour marker and an internal sample quality control. Scale bar is 50μm.


C Distribution of RAD51 expression score ( RAD51NES ) in BCC cohort . cohort is divided into RAD51 - Low expressing cases ( first quartile , Q1 - blue ) , intermediate cases ( interquartile range , IQR - grey ) RAD51 - High expressing cases ( fourth quartile , Q4 - brown ) . Dashed denotes median RAD51NES in this cohort .

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C Distribution of RAD51 nuclear expression score (RAD51NES) in the BCC cohort. The cohort is divided into RAD51-Low expressing cases (first quartile, Q1 - blue), intermediate cases (interquartile range, IQR - grey) and RAD51-High expressing cases (fourth quartile, Q4 - brown). Dashed line denotes the median RAD51NES in this cohort.


D Survival of BCC cohort . Kaplan - Meier plots for progression - free survival ( PFS ) ( left ) overall survival ( OS ) ( right ) stratified according to fourth quartile ( Q4 ) first quartile ( Q1 ) of RAD51NES . Log - rank , shading denotes 95 % confidence intervals .

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D Survival analysis of the BCC cohort. Kaplan-Meier plots for progression-free survival (PFS) (left) and overall survival (OS) (right) stratified according to fourth quartile (Q4) and first quartile (Q1) of RAD51NES. Log-rank test, shading denotes 95% confidence intervals.


E Number of cases with progression at 12 24 months . Chi - square .

E Number of cases with progression at 12 and 24 months. Chi-square test.


A Distribution of RAD51NES in SCOTROC4 cohort . Dashed denotes median RAD51NES in this cohort .

A Distribution of RAD51NES in the SCOTROC4 cohort. Dashed line denotes the median RAD51NES in this cohort.


B Survival of SCOTROC4 cohort . Kaplan - Meier plots for PFS ( left ) OS ( right ) stratified according to quartiles of RAD51NES . Log - rank , shading denotes 95 % confidence intervals .

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B Survival analysis of the SCOTROC4 cohort. Kaplan-Meier plots for PFS (left) and OS (right) stratified according to quartiles of RAD51NES. Log-rank test, shading denotes 95% confidence intervals.


C Number of cases with progression at 12 24 months . Chi - square .

C Number of cases with progression at 12 and 24 months. Chi-square test.


D Survival of HRD positive according to quartile of RAD51NES . Log - rank , shading denotes 95 % confidence intervals . E , Survival of HRD negative . Log - rank , shading denotes 95 % confidence intervals .

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D Survival analysis of HRD positive patients according to quartile of RAD51NES. Log-rank test, shading denotes 95% confidence intervals. E, Survival analysis of HRD negative patients. Log-rank test, shading denotes 95% confidence intervals.


A In - vitro survival of HGSOC lines upon stable RAD51 overexpression . Mean with standard deviation is shown of at least three biological replicates per point . Extra - sum - of - squares F .

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A In-vitro cell survival assay of HGSOC cell lines upon stable RAD51 overexpression. Mean with standard deviation is shown of at least three biological replicates per point. Extra-sum-of-squares F test.


B In - vitro colony forming assays comparing RAD51 - overexpressing control HGSOC , Caov - 3 , after treatment with increasing doses of carboplatin . Mean SD of four biological replicates ( left ) a representative experiment ( right ) . P - value for a comparison between lines for each drug treatment condition is indicated above bars . T - .

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B In-vitro colony forming assays comparing RAD51-overexpressing and control HGSOC cell line, Caov-3, after treatment with increasing doses of carboplatin. Mean and SD of four biological replicates (left) a representative experiment (right). P-value for a comparison between cell lines for each drug treatment condition is indicated above the bars. T-test.


E Volcano plot for fold changes of immune genes enriched or depleted in RAD51 - High tumours vs RAD51 - Low tumours from TCGA . T - ; dashed denoted threshold of significance , Bonferroni corrected for multiple testing .

E Volcano plot for fold changes of immune genes enriched or depleted in RAD51-High tumours vs RAD51-Low tumours from TCGA. T-test; dashed line denoted threshold of significance, Bonferroni corrected for multiple testing.


F Immune genes enriched in RAD51 - High RAD51 - Low tumours across four EOC mRNA cohorts ( TCGA , AOCS , MGH , Duke ) .

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F Immune genes enriched in RAD51- High and RAD51-Low tumours across four EOC mRNA cohorts (TCGA, AOCS, MGH, Duke).


G Example unmixed demonstrating presence of CD8 + T in Tumour ( T ) Stroma ( S ) in a RAD51 - High a RAD51 - Low tumour from BCC cohort . Scale bar is 50μm .

G Example unmixed fluorescent IHC images demonstrating the presence of CD8+ T cells in the Tumour (T) and Stroma (S) in a RAD51-High and a RAD51-Low tumour from the BCC cohort. Scale bar is 50μm.


H CD8 + cytotoxic T - ( Tcyt ) infiltration in BCC cohort . Absolute tumour cytotoxic T - infiltration numbers tumour / stroma ( T / S ) cytotoxic T - number in RAD51 - High - Low cases ( left ) . Bar is median . Mann - Whitney .

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H CD8+ cytotoxic T-cell (Tcyt) infiltration analysis in the BCC cohort. Absolute tumour cytotoxic T-cell infiltration numbers and tumour/stroma (T/S) cytotoxic T-cell number ratio in RAD51-High and -Low cases (left). Bar is median. Mann-Whitney test.


( A ) MOG1 interacts genetically with H2B deubiquitination machinery . Ten - fold serial dilutions of indicated strains were spotted on YPD incubated for 2 days at indicated

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(A) MOG1 interacts genetically with the H2B deubiquitination machinery. Ten-fold serial dilutions of the indicated strains were spotted on YPD and incubated for 2 days at the indicated temperatures


( B ) Expression of total H2B H2Bub1 were analyzed in wild - type ( WT ) , mog1Δ , ubp8Δ ubp8Δmog1Δ whole lysates by western blotting using anti - total H2B anti - H2Bub1 antibodies . Bar graphs of H2Bub1 after H2B normalization show mean standard deviations of at least three independent experiments . P - value was calculated using Student ´ s t - . Error bars represent SD . P - value was calculated using Student ´ s t - ( * , P = 0 . 01 - 0 . 05 ; * * , P = 0 . 001 - 0 . 01 ; * * * , P < 0 . 001 )

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(B) Expression levels of total H2B and H2Bub1 were analyzed in wild-type (WT), mog1Δ, ubp8Δ and ubp8Δmog1Δ whole cell lysates by western blotting using anti-total H2B and anti-H2Bub1 antibodies. Bar graphs of H2Bub1 levels after H2B normalization show the mean and standard deviations of at least three independent experiments. The P-value was calculated using Student´s t-test. Error bars represent SD. The P-value was calculated using Student´s t-test (*, P = 0.01-0.05; **, P = 0.001-0.01; ***, P < 0.001)


Ten - fold serial dilutions of indicated strains were spotted on YPD incubated for 2 days at indicated temperatures ( C ) , ( D were then spotted on YPD or YPGal incubated for 2

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Ten-fold serial dilutions of the indicated strains were spotted on YPD and incubated for 2 days at the indicated temperatures (C), (D and were then spotted on YPD or YPGal and incubated for 2 days


Ten - fold serial dilutions of indicated strains were spotted on YPD incubated for 2 days at indicated temperatures ( E were then spotted on YP incubated for 2

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Ten-fold serial dilutions of the indicated strains were spotted on YPD and incubated for 2 days at the indicated temperatures (E and were then spotted on YP and incubated for 2 days


( A ) expression of H3K4me3 H3 in total extracts of wild - type ( WT ) , mog1Δ , set1Δ spp1Δ strains were analyzed by western blotting using indicated antibodies . Bar graphs of H3K4me3 after H3 normalization show mean standard deviations of at least three independent experiments . P - value was calculated using Student ´ s t - ( * , P = 0 . 01 - 0 . 05 ; * * , P = 0 . 001 - 0 . 01 ; * * * , P < 0 . 001 )

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(A) The expression levels of H3K4me3 and H3 in total extracts of wild-type (WT), mog1Δ, set1Δ and spp1Δ strains were analyzed by western blotting using the indicated antibodies. Bar graphs of H3K4me3 levels after H3 normalization show the mean and standard deviations of at least three independent experiments. The P-value was calculated using Student´s t-test (*, P = 0.01-0.05; **, P = 0.001-0.01; ***, P < 0.001)


( B ) ChIP of H3K4me3 presence at ADH1 , PMA1 YEF3 promoter ( P ) 5 ' ORF in wild - type ( WT ) or mog1Δ strains relative to total H3 . bar graphs indicate mean standard deviation for at least three independent experiments . significance of differences was calculated using Student ´ s t - ( * , P value = 0 . 01 - 0 . 05 ; * * , P value = 0 . 001 - 0 . 01 ; * * * , P < 0 . 001 )

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(B) ChIP analysis of H3K4me3 presence at ADH1, PMA1 and YEF3 promoter (P) and 5'ORF in wild-type (WT) or mog1Δ strains relative to total H3. The bar graphs indicate the mean and standard deviation for at least three independent experiments. The significance of the differences was calculated using Student´s t-test (*, P value = 0.01-0.05; **, P value = 0.001-0.01; ***, P < 0.001)


( C ) MOG1 interacts genetically with H3 methylases Set1 Set2 . Ten - fold serial dilutions of indicated strains were spotted on YPD incubated for 2 days at indicated temperatures . ( D ) histone H3K4A H3K4Amog1Δ strains were compared with their isogenic strains as in ( C )

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(C) MOG1 interacts genetically with the H3 methylases Set1 and Set2. Ten-fold serial dilutions of the indicated strains were spotted on YPD and incubated for 2 days at the indicated temperatures. (D) histone H3K4A and H3K4Amog1Δ strains were compared with their isogenic strains as in (C)


( A ) Ten - fold serial dilutions of indicated strains were spotted on YPD containing indicated concentrations of hydroxyurea ( HU ) were incubated for 2 - 3 days .

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(A) Ten-fold serial dilutions of the indicated strains were spotted on YPD containing the indicated concentrations of hydroxyurea (HU) and were incubated for 2-3 days.


( B ) mRNA synthesis rates ( SR ) as determined in Genomic Run - On ( C ) mRNA abundance ( RA ) , in mog1 ∆ compared with wild type ( WT ) . Bar graphs showing average level standard deviation of three experiments ( n = 3 ) for SR RA values obtained as median of whole gene dataset values in arbitrary units ( x 107 ) from image . Error bars represent SD . Significance of differences was obtained using Student ´ s t - statistical difference is considered as * , P value = 0 . 01 - 0 . 05 ; * * , P value = 0 . 001 - 0 . 01 ; * * * , P value < 0 . 001 . scatter plots ( right panels ) show variation of transcription ( B ) or mRNA ( C ) of genes . Pearson correlation values are : - 0 . 039 ( SR ) - 0 . 042 ( RA ) . This low correlation flat shape of clouds on scatter plots ( see red tendency ) indicates that there is no bias of transcriptional effect with regard to expression level . ( D ) Box plots of SR RA median of mog1Δ compared with WT for genes ( Total ) , SAGA TFIID - dominated genes . These data were obtained from three independent replicates ( n = 3 ) of RA SR data that were averaged as explained in main text . median standard deviation for whole data set ( Total ) for 351 ( for RA ) or 316 ( SR ) SAGA - dominated genes for 3903 ( RA ) or 3403 ( SR ) TFIID - dominated genes are shown . Significance of differences was calculated using Student ´ s t - . No significant difference between different groups was found . similar variations in SRs RAs indicate that there are no changes in mRNA half - lives upon MOG1 deletion ( shown in Fig EV4 ) . total number of genes with confident value in analyses was 3953 for RA 3989 for

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(B) mRNA synthesis rates (SR) as determined in Genomic Run-On analysis and (C) mRNA abundance (RA), in mog1∆ compared with wild type (WT) cells. Bar graphs showing the average level and standard deviation of three experiments (n=3) for SR and RA values obtained as the median of the whole gene dataset values in arbitrary units (x 107) from image analysis quantification. Error bars represent SD. Significance of the differences was obtained using Student´s t-test and statistical difference is considered as *, P value = 0.01-0.05; **, P value = 0.001-0.01; ***, P value < 0.001. The scatter plots (right panels) show the variation of the transcription (B) or mRNA (C) levels of individual genes. Pearson correlation values are: -0.039 (SR) and -0.042 (RA). This low correlation and the flat shape of the clouds on scatter plots (see red tendency line) indicates that there is no bias of the transcriptional effect with regard to expression level. (D) Box plots of SR and RA median levels of mog1Δ cells compared with WT for all genes (Total), SAGA and TFIID-dominated genes. These data were obtained from three independent replicates (n=3) of the RA and SR data that were averaged as explained in the main text. The median and standard deviation for the whole data set (Total) and for 351 (for RA) or 316 (SR) SAGA-dominated genes and for the 3903 (RA) or 3403 (SR) TFIID-dominated genes are shown. Significance of the differences was calculated using Student´s t-test. No significant difference between the different groups was found. The similar variations in SRs and RAs indicate that there are no changes in mRNA half-lives upon MOG1 deletion (shown in Fig EV4). The total number of genes with confident value in the analyses was 3953 for RA and 3989 for SR


( A ) ChIP of TAP - tagged Rad6 presence at ADH1 , PMA1 YEF3 coding regions ( 5 ' & 3 ' ) in WT or mog1Δ strains . Bar charts indicate mean standard deviation for at least three independent experiments . Significance of differences was obtained using Student ´ s t - ( * , P value = 0 . 01 - 0 . 05 ; * * , P value = 0 . 001 - 0 . 01 , * * * , P value = 0 . 0001 - 0 . 001 , * * * * , P value < 0 . 0001 ) . ( B ) , ( C ) ( D ) Bre1 - TAP , Rtf1 - TAP Rpb3 respectively , were analyzed as described in ( A ) . ( E ) Bar charts showing relative occupancy to 5 ' ORFs ( left panel ) or 3 ' ORFs ( right panel ) of each factor relative to Rpb3 occupancy at these gene positions .

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(A) ChIP analysis of TAP-tagged Rad6 presence at ADH1, PMA1 and YEF3 coding regions (5' & 3') in WT or mog1Δ strains. Bar charts indicate the mean and standard deviation for at least three independent experiments. Significance of the differences was obtained using Student´s t-test (*, P value = 0.01-0.05; **, P value = 0.001-0.01, ***, P value = 0.0001-0.001, ****, P value <0.0001). (B), (C) and (D) Bre1-TAP, Rtf1-TAP and Rpb3 respectively, were analyzed as described in (A). (E) Bar charts showing the relative chromatin occupancy to 5'ORFs (left panel) or 3'ORFs (right panel) of each factor relative to Rpb3 occupancy at these gene positions.


( A ) Co - purification of Rtf1 - PK in expressing Mog1 - TAP ( + , + ) . A non - tagged ( - , - ) expressing only Rtf1 - PK ( + , - ) were included as negative controls for co - purification . Inputs IPs are depicted for each .

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(A) Co-purification of Rtf1-PK in cells expressing Mog1-TAP (+,+). A non-tagged strain (-,-) and cells expressing only Rtf1-PK (+,-) were included as negative controls for co-purification. Inputs and IPs are depicted for each strain.


( B ) ChIP of presence of HA - tagged Mog1 compared to a non - tagged ( WT ) at PMA1 YEF3 promoter ( P ) coding regions ( 5 ' ORF ) . occupancy level was calculated as signal of IP samples in relation to Input signal normalized to an Intergenic region . Bar charts indicate mean standard deviation for at least three independent experiments . Significance of differences was obtained using Student ´ s t - is presented as a P - value ( * , P < 0 . 05 ) .

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(B) ChIP analysis of the presence of HA-tagged Mog1 compared to a non-tagged strain (WT) at PMA1 and YEF3 promoter (P) and coding regions (5'ORF). The occupancy level was calculated as the signal ratio of the IP samples in relation to the Input signal normalized to an Intergenic region. Bar charts indicate the mean and standard deviation for at least three independent experiments. Significance of the differences was obtained using Student´s t-test and is presented as a P-value (*, P<0.05).


( D ) Mog1 - HA immunoprecipitation in expressing Bre1 - PK ( upper panel ) Mog1 - TAP co - precipitation in expressing Ada2 - HA ( lower panel ) are indicate as in ( A ) Inputs IPs are depicted for each .

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(D) Mog1-HA immunoprecipitation in cells expressing Bre1-PK (upper panel) and Mog1-TAP co-precipitation in cells expressing Ada2-HA (lower panel) are indicate as in (A) Inputs and IPs are depicted for each strain.


Representative of of mRNA export are shown . localization of poly ( A ) + was assessed by in situ hybridization using Cy3 - labeled oligo ( dT ) probes ( red ) in indicated strains . In situ hybridization was monitored at 30 ºC after shifting of to 39 ºC for 3 hours . was stained by DAPI ( blue ) . In scale bar corresponds to 2 μM . are representative pictures from 2 independent experiments of approximately 600 . percentages indicate number of showing mRNA export defect from these experiments .

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Representative images of the analysis of nuclear mRNA export are shown. The cellular localization of poly(A)+RNA was assessed by in situ hybridization using Cy3-labeled oligo(dT) probes (red) in the indicated strains. In situ hybridization was monitored at 30 ºC and after shifting of the cells to 39 ºC for 3 hours. DNA was stained by DAPI (blue). In all images the scale bar corresponds to 2 μM. Images are representative pictures from 2 independent experiments of approximately 600 cells. The percentages indicate the number of cells showing mRNA export defect from these experiments.


( A ) Mog1E65K Ran - binding mutant complements slow growth of mog1ΔsetΔ mutants . Ten - fold serial dilutions of wild - type ( WT ) , mog1Δ , set1Δ , mog1ΔsetΔ strains transformed with wild - type MOG1 ( pMOG1 ) or with Ran - binding mutant ( pMOG1E65K ) as indicated were spotted on SC - LEU incubated for 2 days at indicated temperatures .

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(A) The Mog1E65K Ran-binding mutant complements the slow growth of mog1ΔsetΔ mutants. Ten-fold serial dilutions of wild-type (WT), mog1Δ, set1Δ, and mog1ΔsetΔ strains transformed with wild-type MOG1 (pMOG1) or with the Ran-binding mutant (pMOG1E65K) as indicated were spotted on SC-LEU and incubated for 2 days at the indicated temperatures.


( B ) Mog1E65K Ran - binding mutant complements low H3K4me3 H2Bub1 of mog1Δ . expression of H3K4me3 , total H3 , H2Bub1 , total H2B PGK1 in whole lysates from wild type ( WT ) or from mog1Δ transformed with an empty plasmid ( + pEmpty ) , wild type MOG1 ( pMOG1 ) or a Ran - binding mutant ( pMOG1E65K ) were analyzed by western blotting using corresponding antibodies .

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(B) The Mog1E65K Ran-binding mutant complements the low H3K4me3 and H2Bub1 levels of mog1Δ. The expression levels of H3K4me3, total H3, H2Bub1, total H2B and PGK1 in whole cell lysates from wild type (WT) or from mog1Δ cells transformed with an empty plasmid (+pEmpty), wild type MOG1 (pMOG1) or a Ran-binding mutant (pMOG1E65K) were analyzed by western blotting using the corresponding antibodies.


( C ) Gsp2 is not involved in H2Bub1 regulation . expression of total H2B of H2Bub1 were analyzed in whole lysates from wild type ( WT ) gsp2Δ strains by western blotting using anti - total H2B anti - H2Bub1 antibodies .

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(C) Gsp2 is not involved in H2Bub1 regulation. The expression levels of total H2B and of H2Bub1 were analyzed in whole cell lysates from wild type (WT) and gsp2Δ strains by western blotting using anti-total H2B and anti-H2Bub1 antibodies.


( a ) Nox2 - specific ROS production was assessed using Nox2 redox biosensor p47 - roGFP redox biosensor Cat : catalase , PEG - Cat : pegylated catalase . (

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(a) Nox2-specific ROS production was assessed using the Nox2 redox biosensor p47-roGFP redox biosensor Cat: catalase, PEG-Cat: pegylated catalase. (


b ) Measurement of intracellular glutathione redox potential with Grx1 - roGFP2 .

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b) Measurement of intracellular glutathione redox potential with Grx1-roGFP2.


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(c) Analysis of Rac1 and (d) Src.


. ( e ) Immunoblot of precipitated p47phox probed with an anti - phosphoserine or anti - p47phox antibody .

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. (e) Immunoblot of precipitated p47phox probed with an anti-phosphoserine or anti-p47phox antibody.


( f ) Nox2 - specific intracellular ROS production was measured using p47 - roGFP redox biosensor .

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(f) Nox2-specific intracellular ROS production was measured using p47-roGFP redox biosensor.


g ) Extracellular ROS production was assessed using Amplex - red dye .

g) Extracellular ROS production was assessed using Amplex-red dye.


( h ) Plasma calcium influx was measured by analysing Fura - 2 quench rate upon addition of extracellular Mn2 + .

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(h) Plasma membrane calcium influx was measured by analysing the Fura-2 fluorescence quench rate upon addition of extracellular Mn2+.


( i ) Intracellular RNS generation was measured using DAF - FM . Bars represent average ± s . e . m . from n = 15 fibres for each condition in ( a , b , f , g ,

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(i) Intracellular RNS generation was measured using DAF-FM. Bars represent average±s.e.m. from n=15 individual fibres for each condition in (a,b,f,g,i


, j ) . Markers of autophagy were analysed in isolated fibres ( incubated with or without PP2 ) from FDBs .

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,j). Markers of autophagy were analysed in isolated fibres (incubated with or without PP2) from FDBs.


( k ) Autophagosome formation was analysed using microscopy ( scale bar = 100 μm ) illustrated LC3 localization autophagosome formation . ( l ) Confocal microscopy detected p62 - LC3 localization in single fibres from FDBs ( scale bar = 140 50 μm for box areas ) . immunoblots were performed with isolated from FDBs probed with antibodies as indicated . Glyceraldehyde - 3 - phosphate dehydrogenase ( GAPDH ) was detected as a loading control . Representative are shown . Bars represent average ± s . e . m . from n = 3 independent biological experiments . Statistical differences between groups were determined using of variance ( ANOVA ) with Tukey ' s post hoc . * P0 . 05 * * P0 . 01 .

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(k) Autophagosome formation was analysed using fluorescence microscopy (scale bar=100 μm) and illustrated LC3 localization and autophagosome formation. (l) Confocal microscopy detected p62-LC3 localization in single fibres from FDBs (scale bar=140 and 50 μm for white box areas). All immunoblots were performed with isolated proteins from FDBs and probed with antibodies as indicated. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was detected as a loading control. Representative images are shown. Bars represent average ±s.e.m. from n=3 independent biological experiments. Statistical differences between groups were determined using analysis of variance (ANOVA) with Tukey's post hoc test. *P0.05 and **P0.01.


( a ) FDB lysates were analysed using immunoblotting with antibodies as indicated .

(a) FDB lysates were analysed using immunoblotting with antibodies as indicated.


( b ) Confocal microscopy shows effect of Nox2 inhibition on p62 - LC3 localization in single FDB fibres . Representative of independent biological experiments ( n = 3 ) are shown ( scale bar = 100 35 μm for box areas ) .

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(b) Confocal microscopy shows the effect of Nox2 inhibition on p62-LC3 localization in single FDB fibres. Representative images of independent biological experiments (n=3) are shown (scale bar=100 and 35 μm for white box areas).


( c ) Autophagic flux was analysed by immunoblotting with anti - LC3B anti - p62 antibodies .

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(c) Autophagic flux was analysed by immunoblotting with anti-LC3B and anti-p62 antibodies.


( d ) Effect of gp91 ds PP2 on LC3 - LAMP1 colocalization was analysed using confocal microscopy . Representative from independent biological experiments ( n = 3 ) are shown ( scale bar = 140 40 μm for red box areas ) .

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(d) Effect of gp91 ds and PP2 on LC3-LAMP1 colocalization was analysed using confocal microscopy. Representative images from independent biological experiments (n=3) are shown (scale bar=140 and 40 μm for red box areas).


(e) Immunoblot analysis of LAMP1,


( g ) Immunoblot of Caspase3 activity . immunoblots were performed with isolated from FDBs probed with antibodies as indicated . GAPDH was detected as a loading control . Representative are shown . Bars represent average ± s . e . m . from n = 3 independent biological experiments . Statistical differences between groups were determined using ANOVA with Tukey ' s post hoc . * P0 . 05 * * P0 . 01 .

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(g) Immunoblot of Caspase3 activity. All immunoblots were performed with isolated proteins from FDBs and probed with antibodies as indicated. GAPDH was detected as a loading control. Representative images are shown. Bars represent average ±s.e.m. from n=3 independent biological experiments. Statistical differences between groups were determined using ANOVA with Tukey's post hoc test. *P0.05 and **P0.01.


( a ) Intracellular ROS production was assessed using DCFH - DA . ( b ) Plasma calcium influx was measured by analysing quench of Fura - 2 upon addition of extracellular Mn2 + . ( a , b ) Were conducted in enzymatically digested single FDBs from WT , mdx p47 − / − - mdx mice . Bars represent average ± s . e . m . from n = 15 fibres for each condition .

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(a) Intracellular ROS production was assessed using DCFH-DA. (b) Plasma membrane calcium influx was measured by analysing the quench of Fura-2 fluorescence upon addition of extracellular Mn2+. (a,b) Were conducted in enzymatically digested single FDBs from WT, mdx and p47−/−-mdx mice. Bars represent average±s.e.m. from n=15 individual fibres for each condition.


( c ) Immunoblot of Src with anti - P - Src or anti - Src antibodies .

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(c) Immunoblot analysis of Src protein with anti-P-Src or anti-Src antibodies.


( d ) Autophagy marker were analysed by immunoblotting with antibodies as indicated ( c ) ( d ) were conducted in enzymatically isolated fibres from FDBs of WT , mdx p47 − / − - mdx mice . Representative are shown . GAPDH was detected as a loading control . Bars represent average ± s . e . m . from n = 3 independent biological experiments .

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(d) Autophagy marker proteins were analysed by immunoblotting with antibodies as indicated (c) and (d) were conducted in enzymatically isolated fibres from FDBs of WT, mdx and p47−/−-mdx mice. Representative images are shown. GAPDH was detected as a loading control. Bars represent average±s.e.m. from n=3 independent biological experiments.


( e ) LC3 - LAMP1 colocalization in single FDBs from mdx p47 − / − - mdx mice was analysed using confocal microscopy . Representative from n = 3 independent biological experiments are shown ( scale bar = 100 40 μm for red box areas ) .

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(e) LC3-LAMP1 colocalization in single FDBs from mdx and p47−/−-mdx mice was analysed using confocal microscopy. Representative images from n=3 independent biological experiments are shown (scale bar=100 and 40 μm for red box areas).


f ) Immunofluorescence to detect lysosomes ( LAMP1 ) in TA muscle from WT , mdx p47 − / − - mdx mice . arrows indicate lysosome yellow arrows indicate nucleus .

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f) Immunofluorescence assay to detect lysosomes (LAMP1) in TA muscle from WT, mdx and p47−/−-mdx mice. White arrows indicate lysosome and yellow arrows indicate nucleus.


( g ) Immunohistochemistry to detect lysosome ( LAMP1 ) in TA muscle from WT , mdx p47 − / − - mdx mice . Black arrows indicate LAMP1 positive ( brown ) structures . Histogram plot quantifying number of immunopositive LAMP1 structures per fibre . Representative from n = 3 independent biological experiments are shown . Scale bar represents 100 μm for f 140 μm for g . Statistical differences between groups were determined using ANOVA with Tukey ' s post hoc . * P0 . 05 * * P0 . 01 .

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(g) Immunohistochemistry to detect lysosome (LAMP1) in TA muscle from WT, mdx and p47−/−-mdx mice. Black arrows indicate LAMP1 positive (brown) structures. Histogram plot quantifying the number of immunopositive LAMP1 structures per fibre. Representative images from n=3 independent biological experiments are shown. Scale bar represents 100 μm for f and 140 μm for g. Statistical differences between groups were determined using ANOVA with Tukey's post hoc test. *P0.05 and **P0.01.


( a ) Haematoxylin eosin of cross - sections from Dia showing central nuclei ( arrow head ) smaller fibres ( arrow ) .

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(a) Haematoxylin and eosin staining of cross-sections from Dia showing central nuclei (arrow head) and smaller fibres (arrow).


( b ) Immunoblot of macrophage content ( CD68 ) .

(b) Immunoblot analysis of macrophage content (CD68).


( c ) Immunofluorescent bright field of Dia cross - sections showing fibre - type distribution . Type I ( red ) , IIA ( green ) , IIB / IIX ( x , unstained viewed from bright field overlay ) .

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(c) Immunofluorescent and bright field images of Dia cross-sections showing fibre-type distribution. Type I (red), IIA (green), IIB/IIX ( white x, unstained and viewed from bright field overlay).


( d ) Serum creatine kinase activity . ( e ) Force - frequency relationship in Dia muscle strips from WT ( black ) , mdx ( red ) p47 − / − - mdx ( blue ) mice . Scale bars represent 55 μm . For e , # P0 . 01 p47 − / − - mdx versus mdx . # # P0 . 01 p47 − / − - mdx versus WT mdx . Mdx was statistically different than WT at frequencies of stimulation . Statistical differences between groups were determined using ANOVA with Tukey ' s post hoc . * P0 . 05 * * P0 . 01 .

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(d) Serum creatine kinase activity. (e) Force-frequency relationship in Dia muscle strips from WT (black), mdx (red) and p47−/−-mdx (blue) mice. Scale bars represent 55 μm. For e, #P0.01 p47−/−-mdx versus mdx.##P0.01 p47−/−-mdx versus WT and mdx. Mdx was statistically different than WT at all frequencies of stimulation. Statistical differences between groups were determined using ANOVA with Tukey's post hoc test. *P0.05 and **P0.01.


( B ) Representative immunohistochemical with anti - pan keratin antibodies of a pair of lesions formed by parallel injections of versus Black HKC strains together with of pan - keratin positive areas ( mm2 ) of lesions ( dots ) formed by 6 HKCs strains of two ancestries . n ( tumors per ancestry ) = 8 . * p < 0 . 05 . Horizontal : median . Two - unpaired t - . Scale bar : 500μm .

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(B) Representative immunohistochemical analysis with anti-pan keratin antibodies of a pair of tumor lesions formed by parallel injections of White versus Black HKC strains together with quantification of the pan-keratin positive areas (mm2) of individual lesions (dots) formed by 6 HKCs strains of the two ancestries. n(tumors per ancestry) = 8. *p<0.05. Horizontal line: median. Two-tailed unpaired t-test. Scale bar: 500μm.


Double immunofluorescence of tumors formed by six HKC strains , as in previous panels , with antibodies against Ki67 ( C ) together with anti - pan - keratin antibodies for identification . Shown are representative of results of lesions ( dots ) . Digitally acquired were used to determine % of Ki67 positive ( C ) in pan - keratin positive areas , examining 3 - 5 fields in each case . For Ki67 , K10 K1 : n ( tumors per ancestry ) = 24 , 22 6 , respectively . * * * * p < 0 . 0001 ; * * p < 0 . 005 , horizontal : median , two - unpaired t . Scale bars : 100μm .

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Double immunofluorescence analysis of tumors formed by six HKC strains, as in the previous panels, with antibodies against Ki67 (C) together with anti-pan-keratin antibodies for tumor cells identification. Shown are representative images and quantification of results of individual lesions (dots). Digitally acquired images were used to determine the % of Ki67 positive cells (C) in pan-keratin positive areas, examining 3-5 fields in each case. For Ki67, K10 and K1 staining: n(tumors per ancestry) = 24, 22 and 6, respectively. ****p<0.0001; **p<0.005, horizontal line: median, two-tailed unpaired t test. Scale bars: 100μm.


Double immunofluorescence of tumors formed by six HKC strains , as in previous panels , with antibodies against keratin 10 1 ( K10 , K1 ) ( D ) together with anti - pan - keratin antibodies for identification . Shown are representative of results of lesions ( dots ) . Digitally acquired were used to determine % of K1 / K10 positivity ( D ) in pan - keratin positive areas , examining 3 - 5 fields in each case . For Ki67 , K10 K1 : n ( tumors per ancestry ) = 24 , 22 6 , respectively . * * * * p < 0 . 0001 ; * * p < 0 . 005 , horizontal : median , two - unpaired t . Scale bars : 100μm .

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Double immunofluorescence analysis of tumors formed by six HKC strains, as in the previous panels, with antibodies against keratin 10 and 1 (K10, K1) (D) together with anti-pan-keratin antibodies for tumor cells identification. Shown are representative images and quantification of results of individual lesions (dots). Digitally acquired images were used to determine the % of K1/K10 positivity (D) in pan-keratin positive areas, examining 3-5 fields in each case. For Ki67, K10 and K1 staining: n(tumors per ancestry) = 24, 22 and 6, respectively. ****p<0.0001; **p<0.005, horizontal line: median, two-tailed unpaired t test. Scale bars: 100μm.


( B ) Kaplan Meier plots of long - term overall survival of H / NSCC from TCGA dataset divided on basis of high versus low HSD17B7 expression relative to its median expression value among ( darkgreen lightgreen lines ) ( red orange lines ) , showing statistically significant association of elevated HSD17B7 expression with poorer survival . Statistical significance was calculated by log - rank between groups . High _ : n = 57patients . Low _ : n = 79 . High _ : n = 203 . Low _ : n = 181 .

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(B) Kaplan Meier plots of long-term overall survival of H/NSCC patients from the TCGA dataset divided on the basis of high versus low HSD17B7 expression levels relative to its median expression value among males (darkgreen and lightgreen lines) and females (red and orange lines), showing statistically significant association of elevated HSD17B7 expression with poorer patient survival. Statistical significance was calculated by the log-rank test between the groups. High_female: n=57patients. Low_female: n= 79 patients. High_male: n= 203 patients. Low_male: n= 181 patients.


( G ) Immunofluorescence ( IF ) of HSD17B7 expression in Black HKCs . Shown are representative as well as IF signal at level of ( dots ) for indicated strains . 19 - 46 were counted in each case . n ( HKC strains per ancestry ) = 5 , * * * p < 0 . 0005 , horizontal : median , 2 - unpaired t - . Unit of Y - axis : pixels . Scale bar : 20 μm .

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(G) Immunofluorescence (IF) analysis of HSD17B7 protein expression in White and Black HKCs. Shown are representative images as well as IF signal quantification at the level of individual cells (dots) for the indicated strains. 19-46 cells were counted in each case. n(HKC strains per ancestry) = 5, ***p<0.0005, horizontal line: median, 2-tailed unpaired t-test. Unit of Y-axis: pixels. Scale bar: 20 μm.


( D - F ) Intradermal tumorigenicity assays with HKCs ( 269W ) stably infected with an HSD17B7 overexpressing lentivirus ( HSD17B7 - oe ) versus control ( Ctrl ) . Experimental conditions were same as in Fig 2 . Mice were sacrificed 15 days after injection . Shown are representative of density assessed by immunohistochemical with anti - pan keratin antibodies ( D , ) as well as % of Ki67 positive ( E ) K1 positivity ( F ) of pan - keratin positive areas . For ( D ) , n ( tumors per condition ) = 9 ; for ( E F ) n ( 3 fields per , 5 tumors per condition ) = 15 . * p < 0 . 05 , * * * * p < 0 . 0001 . Horizontal : median . 2 - tail unpaired t - . Scale bar : 500μm ( D ) , 100μm ( F ) .

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(D-F) Intradermal tumorigenicity assays with HKCs (269W) stably infected with an HSD17B7 overexpressing lentivirus (HSD17B7-oe) versus control (Ctrl). Experimental conditions were the same as in Fig 2. Mice were sacrificed 15 days after injection. Shown are representative images and quantification of tumor cells density assessed by immunohistochemical staining with anti-pan keratin antibodies (D,) as well as % of Ki67 positive cells (E) and K1 positivity (F) of pan-keratin positive areas. For (D), n(tumors per condition) = 9; for (E and F) n(3 fields per tumor, 5 tumors per condition) = 15. *p<0.05, ****p<0.0001. Horizontal line: median. 2-tail unpaired t-test. Scale bar: 500μm (D), 100μm (F).


( G , H ) Intradermal tumorigenicity assays with SCC13 overexpressing HSD17B7 ( HSD17B7 - oe ) versus control ( Ctrl ) . Mice were sacrificed 15 days after injection . Shown are representative of density , assessed by immunohistochemical with anti - pan keratin antibodies ( G ) as well as % of Ki67 positive in pan - keratin positive areas ( H ) . For ( G ) , n ( tumors per condition ) = 4 ; for ( H ) , n ( 3 - 5 fields per , 4 tumors per condition ) 15 , 19 . * p < 0 . 05 ; Horizontal : median . One - paired t - . Scale bar in ( G , H ) : 100μm .

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(G, H) Intradermal tumorigenicity assays with SCC13 cells overexpressing HSD17B7 (HSD17B7-oe) versus control (Ctrl). Mice were sacrificed 15 days after injection. Shown are representative images and quantification of tumor cells density, assessed by immunohistochemical staining with anti-pan keratin antibodies (G) as well as % of Ki67 positive cells in pan-keratin positive areas (H). For (G), n(tumors per condition) = 4; for (H), n(3-5 fields per tumor, 4 tumors per condition) 15, 19. *p<0.05; Horizontal line: median. One-tailed paired t-test. Scale bar in (G, H): 100μm.


( D ) Colony forming assays of indicated SCC lines plus / minus HSD17B7 silencing as in previous panels . plated in triplicate dishes 5 days after lentiviral vector infection selection were tested for clonogenic capability as in ( A ) . Data are represented as number of colonies + / - SD . n ( dishes per condition ) = 3 * p < 0 . 05 , * * p < 0 . 01 ; p * * * * < 0 . 0001 , 2 - unpaired t - .

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(D) Colony forming assays of the indicated SCC cell lines plus/minus HSD17B7 silencing as in the previous panels. Cells plated in triplicate dishes 5 days after lentiviral vector infection and selection were tested for clonogenic capability as in (A). Data are represented as number of colonies +/- SD. n(dishes per condition)= 3 *p<0.05, **p<0.01; p****<0.0001, 2-tailed unpaired t-test.


( C , D ) SCC13 stably infected with an HSD17B7 - expressing lentivirus ( HSD - oe ) versus empty vector control ( Ctrl ) ( C ) or two HSD17B7 - silencing lentivirus ( shHSD2 , shHSD4 ) versus vector control ( shCtrl ) ( D ) were analyzed as in previous panels . Shown are values for 3 parallel cultures per condition together with mean + / - SD . * * p < 0 . 005 ; * * * p < 0 . 0005 ; * * * * p < 0 . 0001 . 2 - unpaired t - . Additional metabolic measurement experiments of SCC plus / minus HSD17B7 overexpression silencing , in context of additional manipulations are shown in Fig 9 .

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(C, D) SCC13 cells stably infected with an HSD17B7-expressing lentivirus (HSD-oe) versus empty vector control (Ctrl) (C) or two HSD17B7-silencing lentivirus (shHSD2, shHSD4) versus vector control (shCtrl) (D) were analyzed as in the previous panels. Shown are individual values for 3 parallel cultures per condition together with mean +/- SD. **p<0.005; ***p<0.0005; ****p<0.0001. 2-tailed unpaired t-test. Additional metabolic measurement experiments of SCC cells plus/minus HSD17B7 overexpression and silencing, in the context of additional manipulations are shown in Fig 9.


( C ) Purified mitochondrial preparation from SCC13 infected with lentiviruses expressing wild type ( HSD - wt ) Y193G K197G mutated ( HSD - mut ) forms versus empty vector control ( Ctrl ) as in ( B ) were assayed for of Electron Transport Chain activity , ATP mtROS production as in Fig 8A . Data are displayed as average values of triplicate measurements together with mean + / - SD . n ( dishes per condition ) = 3 . * * p < 0 . 005 ; * * * p < 0 . 0005 . 2 - unpaired t - . experimental values are provided in Dataset EV3 .

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(C) Purified mitochondrial preparation from SCC13 cells infected with lentiviruses expressing wild type (HSD-wt) and Y193G and K197G mutated (HSD-mut) forms versus empty vector control (Ctrl) as in (B) were assayed for levels of Electron Transport Chain activity, ATP and mtROS production as in Fig 8A. Data are displayed as average values of triplicate measurements together with mean +/- SD. n(dishes per condition)= 3. **p<0.005; ***p<0.0005. 2-tailed unpaired t-test. Individual experimental values are provided in Dataset EV3.


( D ) SCC13 were infected with two HSD17B7 silencing lentiviruses ( shHSD2 , shHSD4 ) versus vector control ( shCtrl ) followed , 3 days after infection antibiotic selection , by treatment with Zymosterol ( 2 . 5uM ) or ethanol vehicle alone for 48hours . Purified mitochondrial preparation were assayed for of Electron Transport Chain activity , ATP mtROS production as in Fig 8A . Data are displayed as average values of triplicate measurements together with mean + / - SD . n ( dishes per condition ) = 3 . * * * * p < 0 . 0001 , 2 - unpaired t - . experimental values are provided in Dataset EV3 .

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(D) SCC13 cells were infected with two HSD17B7 silencing lentiviruses (shHSD2, shHSD4) versus vector control (shCtrl) followed, 3 days after infection and antibiotic selection, by treatment with Zymosterol (2.5uM) or ethanol vehicle alone for 48hours. Purified mitochondrial preparation were assayed for levels of Electron Transport Chain activity, ATP and mtROS production as in Fig 8A. Data are displayed as average values of triplicate measurements together with mean +/- SD. n(dishes per condition)= 3. ****p<0.0001, 2-tailed unpaired t-test. Individual experimental values are provided in Dataset EV3.


A , B methylation level of NTN1 ( A ) DAPK1 ( B ) 5 ' regions ( Illumina ' s HumanMethylation450K Array ( HM450 ) from Cancer Genome Atlas breast cohort ) in paired breast tissues ( normal : green circles , : red circles ) , n = 92 , Wilcoxon matched - pairs signed rank , P = 0 . 004 P = 0 . 0008 .

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A, B DNA methylation level of NTN1 (A) and DAPK1 (B) 5' regions (Illumina's HumanMethylation450K Array (HM450) from The Cancer Genome Atlas breast cohort) in paired breast tissues (normal: green circles, tumor: red circles), n = 92, Wilcoxon matched-pairs signed rank test, P = 0.004 and P = 0.0008.


C , D NTN1 ( C ) DAPK1 ( D ) gene expression in paired breast tissues ( normal : green bars , : red bars , RNAseq from TCGA breast cohort , n = 112 114 , respectively .

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C, D NTN1 (C) and DAPK1 (D) gene expression in paired breast tissues (normal: green bars, tumor: red bars, RNAseq from TCGA breast cohort, n = 112 and 114, respectively.


E , F Correlation between NTN1 ( E ) DAPK1 ( F ) gene expression methylation in breast cancer cohort ( TCGA , n = 807 ) . Pearson correlation , P = 6 . 7 . 10 - 5 , r = - 0 . 14 for NTN1 ( A ) P < 10 - 16 , r = - 0 . 32 for DAPK1 ( B ) , respectively . G , H / Normal methylation of NTN1 ( G ) DAPK1 ( H ) in humanbreasttumors ( data extracted from TCGA cohort , paired samples ) according to gene expression ( downregulated FC ≤ 0 . 5 , down , n = 33 , or upregulated FC ≥ 1 . 3 , up , n = 16 ) . P = 3 . 10 - 2 P = 3 . 10 - 4 two - sided Mann Whitney , for NTN1 DAPK1 , respectively .

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E, F Correlation between NTN1 (E) and DAPK1 (F) gene expression and DNA methylation in the breast cancer cohort (TCGA, n = 807). Pearson correlation, P = 6.7.10-5, r = -0.14 for NTN1 (A) and P<10-16, r = -0.32 for DAPK1 (B), respectively.G, H Tumor/Normal DNA methylation ratio of NTN1 (G) and DAPK1 (H) in humanbreasttumors (data extracted from TCGA cohort, paired samples) according to gene expression (downregulated FC ≤ 0.5, down, n = 33, or upregulated FC ≥ 1.3, up, n = 16). P = 3.10-2 and P = 3.10-4 two-sided Mann Whitney test, for NTN1 and DAPK1, respectively.


A Methyl - Cap - seq read density profiles of 5 ' end of DAPK1 , UNC5B , NTN1in MDA - MB - 231 ( blue ) HMLER ( green ) . Red boxes represent CpG islands ( CGis ) ; gray boxes regions analyzed by bisulfite - PCR - sequencing ; gray boxes regions analyzed by parallel sequencing of amplicons from bisulfite modified ; black boxes exons UTR . Chromosome coordinates of each gene are given ( black lines ) .

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A Methyl-Cap-seq read density profiles of the 5' end of DAPK1, UNC5B, and NTN1in MDA-MB-231 (blue) and HMLER (green) cells. Red boxes represent the CpG islands (CGis); light gray boxes the regions analyzed by bisulfite-PCR-sequencing; dark gray boxes the regions analyzed by parallel sequencing of amplicons from bisulfite modified DNA; and black boxes the exons and UTR. Chromosome coordinates of each gene are given (black lines).


B , C Gene expression was measured by Q - RT - PCR after 72 h for MDA - MB - 231 ( B ) HMLER ( C ) treated daily with DAC ( 10 µM ) . PBGD expression level was used as an internal control . Data are expressed as mean ± s . e . m . of at least 3 independent experiments . * * * * P < 0 . 0001 , two - unpaired Student ' s t - .

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B, C Gene expression was measured by Q-RT-PCR after 72 h for MDA-MB-231 (B) and HMLER cells (C) treated daily with DAC (10 µM). PBGD expression level was used as an internal control. Data are expressed as mean ± s.e.m. of at least 3 independent experiments. **** P<0.0001, two-tailed unpaired Student's t-test.


D , E Measurement of hypomethylation of DAPK1 ( D ) NTN1 ( E ) promoters after decitabine treatment of MDA - MB - 231 HMLER . Over 1960 sequences were analyzed per group in 2 independent experiments . * * * * P < 0 . 0001 , two - way ANOVA post - hoc Tukey - .

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D, E Measurement of DNA hypomethylation of the DAPK1 (D) and NTN1 (E) promoters after decitabine treatment of MDA-MB-231 and HMLER cells. Over 1960 sequences were analyzed per group in 2 independent experiments. **** P< 0.0001, two-way ANOVA and post-hoc Tukey-test.


A , B were treated with DAC ( 10 μM , 72 h ) , / or net1 - mAb ( 10 μg / ml , 48 h ) / or recombinant netrin - 1 ( 5 μg / ml , 48 h ) . TUNEL assays from 3 independent experiments . * * * P < 0 . 0001 one - way ANOVA .

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A, B Cells were treated with DAC (10 μM, 72 h), and/or net1-mAb (10 μg/ml, 48 h) and/or recombinant netrin-1 (5 μg/ml, 48 h). TUNEL assays from 3 independent experiments. *** P<0.0001 one-way ANOVA.


C mortality was assessed using toxilight . Data represent mean ± s . e . m . from 3 independent experiments . * * * * P < 0 . 0001 , one - way ANOVA .

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C Cellular mortality was assessed using the toxilight assay. Data represent mean ± s.e.m. from 3 independent experiments. **** P<0.0001, one-way ANOVA.


D Loss of methylation of NTN1 DAPK1 promoters in decitabine treated tumors , compared with PBS treated tumors . > 1700 sequences were analyzed per group in 2 independent experiments . * * * * P < 0 . 0001 , two - way ANOVA post - hoc Tukey - .

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D Loss of DNA methylation of the NTN1 and DAPK1 promoters in decitabine treated tumors, compared with PBS treated tumors. >1700 sequences were analyzed per group in 2 independent experiments. **** P< 0.0001, two-way ANOVA and post-hoc Tukey-test.


E , F After one week of treatment , tumors from xenografted mice were fixed in formalin , embedded in paraffin , sliced into 4 μm sections . ( E ) of DAPK1 , UNC5B , netrin - 1 were measured in 4 independent tumors per treatment group by immunohistochemistry , expressed as a of total surface normalized against control PBS - group . Data represented as means ± s . e . m . * * * * P < 0 . 0001 , two - way ANOVA post - hoc Tukey - . ( F ) Representative sections corresponding to MDA - MB - 231xenografts , scale bars = 50 μm .

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E, F After one week of treatment, tumors from xenografted mice were fixed in formalin, embedded in paraffin, and sliced into 4 μm sections. (E) Levels of DAPK1, UNC5B, and netrin-1 were measured in 4 independent tumors per treatment group by immunohistochemistry staining, and expressed as a percentage of total tumor surface normalized against the control PBS-group. Data represented as means ± s.e.m. **** P<0.0001, two-way ANOVA and post-hoc Tukey-test. (F) Representative tumor sections corresponding to MDA-MB-231xenografts, scale bars = 50 μm.


G , H When tumors reached 100 mm3 , mice were injected subcutaneously with decitabine ( 0 . 4 mg / kg ) or PBS / or intraperitoneally with net1 - mAb ( 10 mg / kg ) . ( G ) volumes were measured twice a week . statistical significance of differences obtained between control PBS - group treated ( DAC + net1 - mAb ) group was determined by two - way ANOVA post - hoc Tukey - . * * * P < 0 . 0001 . ( H ) weights were measured 3 days after end of experiment . statistical significance of differences obtained between groups was determined by one - way ANOVA , * * * P < 0 . 0001 . Error bars = s . e . m .

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G, H When tumors reached 100 mm3, mice were injected subcutaneously with decitabine (0.4 mg/kg) or PBS and/or intraperitoneally with net1-mAb (10 mg/kg). (G) Tumor volumes were measured twice a week. The statistical significance of the differences obtained between the control PBS-group and treated (DAC + net1-mAb) group was determined by two-way ANOVA and post-hoc Tukey-test. *** P<0.0001. (H) Tumor weights were measured 3 days after the end of the experiment. The statistical significance of the differences obtained between the groups was determined by one-way ANOVA, *** P<0.0001. Error bars = s.e.m.


A Loss of methylation of NTN1 DAPK1 promoters in decitabine treated PDX tumors , compared with control PBS - group . of mean methylation of 11 DAPK1 - CpGs was 94 % ( 550 amplicons analyzed ) , while that of 7 NTN1 - CpGs was 64 % ( 213 amplicons analyzed ) . Two - way ANOVA post - hoc Tukey - .

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A Loss of DNA methylation of the NTN1 and DAPK1 promoters in decitabine treated PDX tumors, compared with the control PBS-group. The percentage of mean DNA methylation of the 11 DAPK1-CpGs was 94% (550 amplicons analyzed), while that of the 7 NTN1-CpGs was 64% (213 amplicons analyzed). Two-way ANOVA and post-hoc Tukey-test.


B Expression of DAPK1 , UNC5B , NTN1 was measured by Q - RT - PCR in PDX tumors after 7 days of in vivo DAC treatment ( 0 . 4 mg / kg ) . level of PBGD expression was used as an internal control . Data are expressed as mean ± s . e . m . for at least 3 grafts per group . * * * * P < 0 . 0001 , , two - way ANOVA post - hoc Tukey - .

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

B Expression of DAPK1, UNC5B, and NTN1 was measured by Q-RT-PCR in PDX tumors after 7 days of in vivo DAC treatment (0.4 mg/kg). The level of PBGD expression was used as an internal control. Data are expressed as mean ± s.e.m. for at least 3 grafts per group. **** P<0.0001,, two-way ANOVA and post-hoc Tukey-test.