Confocal microscopy images of co - localization analysis of the BNIP3 - proficient ( shCntl , n = 27 cells ) or deficient ( shBNIP3 , n = 31 cells ) B16 - F10 cells transiently transfected with GFP - LC3 and immunostained for TOMM20 ( mitochondrial marker ) . Colocalization events of GFP - LC3 puncta ( green ) with TOMM20 ( red ) are highlighted with white arrows . The percentage of GFP - LC3 puncta colocalizing with TOMM20 was calculated in n cells from 3 biologically independent experiments and analyzed with Mann - Whitney ' s non - parametric test . Scale bars represent 10µm .

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Confocal microscopy images of co-localization analysis of the BNIP3-proficient (shCntl, n=27 cells) or deficient (shBNIP3, n=31 cells) B16-F10 cells transiently transfected with GFP-LC3 and immunostained for TOMM20 (mitochondrial marker). Colocalization events of GFP-LC3 puncta (green) with TOMM20 (red) are highlighted with white arrows. The percentage of GFP-LC3 puncta colocalizing with TOMM20 was calculated in n cells from 3 biologically independent experiments and analyzed with Mann-Whitney's non-parametric test. Scale bars represent 10µm.


Glut1 , Hk2 , Pkm1 , Pkm2 , Pdk1 , Mct1 , Ldha , Ldhb and Mct4 transcript levels measured by qPCR from the corresponding B16 - F10 cell lysates . Transcript expression is represented as the fold - change relative to their corresponding shCntl value . Heatmap shows the average values in each condition ( n = 4 ) and they were analyzed with a one - sample T - test only against shCntl , except for Glut1 shATG5 that was analyzed with a Wilcoxon rank test .

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Glut1, Hk2, Pkm1, Pkm2, Pdk1, Mct1, Ldha, Ldhb and Mct4 transcript levels measured by qPCR from the corresponding B16-F10 cell lysates. Transcript expression is represented as the fold-change relative to their corresponding shCntl value. Heatmap shows the average values in each condition (n=4) and they were analyzed with a one-sample T-test only against shCntl, except for Glut1 shATG5 that was analyzed with a Wilcoxon rank test.


Immunoblot detection of Hydroxylated HIF - 1α ( HIF - 1α - OH ) , total HIF - 1α , LDHA and ACTIN protein levels from lysates of B16 - F10 cells ( n = 4 ) or B16 - F10 tumors [ CntlKD ( n = 5 ) , BNIP3KD ( n = 5 ) and ATG5KD ( n = 7 ) ] ) . The HIF - 1α - OH / HIF - 1α ratio is shown in a bar graph below . Densitometric quantifications relative to ACTIN levels are shown below each corresponding band . Analysis was performed as indicated in the Data Information section , unless for HIF - 1α / Actin ratio ( in vivo ) , which was analyzed with Kruskal - Wallis non - parametric test with Dunn ' s multiple comparisons test , and LDHA / Actin ratio for shATG5 condition ( in vitro ) , which was analyzed with Wilcoxon ' s rank test .

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Immunoblot detection of Hydroxylated HIF-1α (HIF-1α-OH), total HIF-1α, LDHA and ACTIN protein levels from lysates of B16-F10 cells (n=4) or B16-F10 tumors [CntlKD (n=5), BNIP3KD (n=5) and ATG5KD (n=7)]). The HIF-1α-OH /HIF-1α ratio is shown in a bar graph below. Densitometric quantifications relative to ACTIN levels are shown below each corresponding band. Analysis was performed as indicated in the Data Information section, unless for HIF-1α/Actin ratio (in vivo), which was analyzed with Kruskal-Wallis non-parametric test with Dunn's multiple comparisons test, and LDHA/Actin ratio for shATG5 condition (in vitro), which was analyzed with Wilcoxon's rank test.


Immunohistochemical staining for HIF - 1α ( brown ) representative from CntlKD , BNIP3KD and ATG5KD , B16 - F10 tumor tissues . Higher magnification of the highlighted ( with a white square ) stained sections is shown below .

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Immunohistochemical staining for HIF-1α (brown) representative from CntlKD, BNIP3KD and ATG5KD, B16-F10 tumor tissues. Higher magnification of the highlighted (with a white square) stained sections is shown below.


Immunoblot detection of Hydroxylated HIF - 1α ( HIF - 1α - OH ) , total HIF - 1α , LDHA , PHD2 and ACTIN protein levels from normoxic lysates of B16 - F10 cells transfected in presence of non - targeting siRNA sequences ( Scr ) or siRNA against PHD2 .

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Immunoblot detection of Hydroxylated HIF-1α (HIF-1α-OH), total HIF-1α, LDHA, PHD2 and ACTIN protein levels from normoxic lysates of B16-F10 cells transfected in presence of non-targeting siRNA sequences (Scr) or siRNA against PHD2.


Relative intracellular Fe2 + levels in normoxic B16 - F10 cells ( n = 4 ) . Representative pictures of FerroOrange intensity are shown on the left and analyzed using a RM one - way ANOVA ( Geisser - Greenhouse correction ) with Holm - Sidak ' s multiple comparisons test . Scale bars represent 50µm .

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Relative intracellular Fe2+ levels in normoxic B16-F10 cells (n=4). Representative pictures of FerroOrange intensity are shown on the left and analyzed using a RM one-way ANOVA (Geisser-Greenhouse correction) with Holm-Sidak's multiple comparisons test. Scale bars represent 50µm.


Immunoblot detection of Hydroxylated HIF - 1α ( HIF - 1α - OH ) , total HIF - 1α , LDHA , NCOA4 and ACTIN levels from normoxic lysates of B16 - F10 cells transfected in presence of non - targeting siRNA sequences ( Scr ) or siRNA against NCOA4 ( n = 4 ) .

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Immunoblot detection of Hydroxylated HIF-1α (HIF-1α-OH), total HIF-1α, LDHA, NCOA4 and ACTIN levels from normoxic lysates of B16-F10 cells transfected in presence of non-targeting siRNA sequences (Scr) or siRNA against NCOA4 (n=4).


Growth curves of B16 - F10 tumors generated by shCntl or shBNIP3 cells expressing Luciferase ( Luc ) or an undegradable HIF - 1α ( HIF - 1α - AA ) construct ( n = 7 for CntlKD Luc / BNIP3KD HIF - 1α - AA , n = 8 CntlKD HIF - 1α - AA and n = 5 for BNIP3KD Luc ) and analyzed with a two - way ANOVA with Tukey ' s multiple comparisons test . Representative double immunostaining for CD31 and αSMA in B16 - F10 tumors ( n = 5 per condition ) . Scale bars represent 50µm .

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Growth curves of B16-F10 tumors generated by shCntl or shBNIP3 cells expressing Luciferase (Luc) or an undegradable HIF-1α (HIF-1α-AA) construct (n=7 for CntlKD Luc/BNIP3KD HIF-1α-AA, n=8 CntlKD HIF-1α-AA and n=5 for BNIP3KD Luc) and analyzed with a two-way ANOVA with Tukey's multiple comparisons test. Representative double immunostaining for CD31 and αSMA in B16-F10 tumors (n=5 per condition). Scale bars represent 50µm.


B Observed dsDNA processing patterns of Sgs1 , including an overview over consecutive bursts and pauses ( upper panel ) as well as detailed views into individual bursts containing multiple unwinding events ( lower panels ) . A typical unwinding event of Sgs1 starts with slow , gradual unwinding of the dsDNA followed by DNA rehybridization , that can be almost instant ( 76 % of events , see blue sections in lower right panel ) or contain slow rewinding sections ( 24 % of events , see orange sections in lower right panel ) .

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B Observed dsDNA processing patterns of Sgs1, including an overview over consecutive bursts and pauses (upper panel) as well as detailed views into individual bursts containing multiple unwinding events (lower panels). A typical unwinding event of Sgs1 starts with slow, gradual unwinding of the dsDNA followed by DNA rehybridization, that can be almost instant (76% of events, see blue sections in lower right panel) or contain slow rewinding sections (24% of events, see orange sections in lower right panel).


C Histograms of the observed unwinding and rewinding velocities for Sgs1 . The mean unwinding rate was 65 ± 2 bp / s ( N = 899 ) . The mean rewinding rate was 115 ± 6 bp / s ( N = 287 ) . We attribute the large rewinding rate in the absence of RPA to an increased error in inferring the rewinding rate from the short rewinding sections .

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C Histograms of the observed unwinding and rewinding velocities for Sgs1. The mean unwinding rate was 65 ± 2 bp/s (N = 899). The mean rewinding rate was 115 ± 6 bp/s (N = 287). We attribute the large rewinding rate in the absence of RPA to an increased error in inferring the rewinding rate from the short rewinding sections.


B Observed dsDNA processing pattern of Sgs1 in the presence of RPA including an overview over successive bursts and pauses ( upper panel ) and detailed views of single bursts ( lower panels ) . Unwinding events are always followed by slow rewinding events ( orange sections ) .

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B Observed dsDNA processing pattern of Sgs1 in the presence of RPA including an overview over successive bursts and pauses (upper panel) and detailed views of single bursts (lower panels). Unwinding events are always followed by slow rewinding events (orange sections).


C Histograms of the observed unwinding and rewinding velocities for Sgs1 in the presence of RPA ( brown bars ) . The mean unwinding rate was 51 ± 3 bp / s ( N = 2139 ) . The mean rewinding rate was 66 ± 3 bp / s ( N = 2091 ) . White bars show for comparison the distributions of unwinding and rewinding velocities for Sgs1

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C Histograms of the observed unwinding and rewinding velocities for Sgs1 in the presence of RPA (brown bars). The mean unwinding rate was 51 ± 3 bp/s (N = 2139). The mean rewinding rate was 66 ± 3 bp/s (N = 2091). White bars show for comparison the distributions of unwinding and rewinding velocities for Sgs1 alone


D Force - dependence of the Sgs1 unwinding ( black squares ) ( N = 2139 ) and rewinding rates ( black diamonds ) ( N = 2091 ) in the presence of RPA ( errors given as standard deviations ) . Red circles represent the force - dependent DNA opening and closure rates measured for RPA

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D Force-dependence of the Sgs1 unwinding (black squares) (N = 2139) and rewinding rates (black diamonds) (N = 2091) in the presence of RPA (errors given as standard deviations). Red circles represent the force-dependent DNA opening and closure rates measured for RPA alone


B Typical dsDNA processing event . The dashed red line connects the minima at the end of the rewinding sections The inset shows a DNA unwinding event in the presence of nuclease - dead Dna2 E675A and RPA .

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B Typical dsDNA processing event. The dashed red line connects the minima at the end of the rewinding sections The inset shows a DNA unwinding event in the presence of nuclease-dead Dna2 E675A and RPA.


C Histograms of the observed unwinding and rewinding rates ( green bars ) with mean values of 69 ± 3 bp / s ( N = 365 ) and 74 ± 5 bp / s ( N = 282 ) , respectively . White bars show for comparison the unwinding and rewinding velocities of Sgs1 in the presence of

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C Histograms of the observed unwinding and rewinding rates (green bars) with mean values of 69 ± 3 bp/s (N = 365) and 74 ± 5 bp/s (N = 282), respectively. White bars show for comparison the unwinding and rewinding velocities of Sgs1 in the presence of RPA


B dsDNA processing events observed for Sgs1 in the presence of Top3 - Rmi1 . Typical events consist of periods with slow gradual unwinding typically followed by instant DNA rezipping as seen also for Sgs1 alone .

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B dsDNA processing events observed for Sgs1 in the presence of Top3-Rmi1. Typical events consist of periods with slow gradual unwinding typically followed by instant DNA rezipping as seen also for Sgs1 alone.


C Histogram of the unwinding rate of Sgs1 in the presence of Top3 - Rmi1 ( violet bars ) with a mean rate of 86 ± 3 bp / s ( N = 1101 ) . For comparison , the distribution of unwinding rates for Sgs1 alone is depicted with white

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C Histogram of the unwinding rate of Sgs1 in the presence of Top3-Rmi1 (violet bars) with a mean rate of 86 ± 3 bp/s (N = 1101). For comparison, the distribution of unwinding rates for Sgs1 alone is depicted with white bars


E dsDNA processing by Sgs1 in the presence of Top3 - Rmi1 and RPA . In contrast to the absence of RPA , DNA closure is seen as a slow rewinding .

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E dsDNA processing by Sgs1 in the presence of Top3-Rmi1 and RPA. In contrast to the absence of RPA, DNA closure is seen as a slow rewinding.


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

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

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

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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 and synaptophysin ) was suppressed between 60 and 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 cells ( 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 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 .

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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 cells . Loading controls : actin and 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 the levels of PIKfyve starting 48h post treatment . Each dot represents an individual experiment . Statistics : Unpaired t - test . * * : 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 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 .

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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 , 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 .

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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 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 .

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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 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 .

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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 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 .

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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 ) and immunostained with NeuN and LAMP1 . Nuclei : DAPI . POM1 - treated COCS showed increased LAMP1 expression ( quantification : Appendix Fig . S4B ) . NeuN + cells 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 . Quantification : Each dot represents an individual biological replicate . Statistics : Unpaired t - test . * : 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 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 .

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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 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 .

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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 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 .

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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 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 .

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 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: 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 ) and treated with bPIP ( 5µg / ml ) . At 14 dpi NeuN morphometry revealed POM1 - induced ablation of cerebellar granule layer ( CGL ) and rescue by bPIP .

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 B-CONTROLLED_VAR O

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 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 .

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

(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 ) 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 .

O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 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 .

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

(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 ) 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 .

O O O O O O O O O O O O O O O O O O O O O O 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) 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 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 .

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

(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 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 .

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

(B) HeLa cells 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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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(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 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 .

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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 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 .

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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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 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 .

(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 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 .

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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 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 .

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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 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 .

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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 and 24 months . Chi - square test .

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


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

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


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 .

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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 and 24 months . Chi - square test .

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


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 .

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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 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 .

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 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 .

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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 - test ; dashed line denoted threshold of significance , Bonferroni corrected for multiple testing .

O O O O O O O O O O O O O O O O O O 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 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 and RAD51 - Low tumours across four EOC mRNA cohorts ( TCGA , AOCS , MGH , Duke ) .

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


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 .

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 - 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 .

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 the H2B deubiquitination machinery . Ten - fold serial dilutions of the indicated strains were spotted on YPD and incubated for 2 days at the indicated

O O O O O O O O O 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) 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 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 )

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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 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

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 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

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

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 ) 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 )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 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 )

(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 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 )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 the indicated strains were spotted on YPD containing the indicated concentrations of hydroxyurea ( HU ) and were incubated for 2 - 3 days .

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 O O O O O O O O O

(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 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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 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 .

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

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 ) 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 ) 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 .

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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 . 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 the Nox2 redox biosensor p47 - roGFP redox biosensor Cat : catalase , PEG - Cat : pegylated catalase . (

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

(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 .

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


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


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

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

. (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 .

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

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

(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 individual fibres for each condition in ( a , b , f , g ,

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

(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 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 .

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

(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 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 ) .

(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 and anti - p62 antibodies .

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


( 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 ) .

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

(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 . 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 .

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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 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 .

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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 analysis of Src protein with anti - P - Src or anti - Src antibodies .

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


( 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 .

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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 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 ) .

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

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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 and eosin staining of cross - sections from Dia showing central nuclei ( arrow head ) and 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 analysis of macrophage content ( CD68 ) .

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


( 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 ) .

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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 ) 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 ) 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 .

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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 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 ) .

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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 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 .

(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 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 .

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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 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 .

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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.