( E , F ) Drop on HU showing a 10 - fold dilution series of from exponential SC cultures of indicated strains that were spotted on SC + / - 200mM HU . ( F ) Histogram presents of 2 independent HU sensitivity assays with mean data point values indicated for each yeast dilution .

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(E,F) Drop assay on HU showing a 10-fold dilution series of cells from exponential SC cultures of the indicated strains that were spotted on SC +/- 200mM HU. (F) Histogram presents quantification of 2 independent HU sensitivity assays with mean and individual data point values indicated for each yeast dilution.


( A ) Preparations of - bound were prepared in triplicate subjected to SDS - PAGE immunoblotting with indicated antibodies in a that either does or does not express Rpb3 - TAP of - bound Rpb1 - S2P Rpb1 - S5P is shown on right . Mcm2 is used as a loading control .

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(A) Preparations of chromatin-bound proteins were prepared in triplicate and subjected to SDS-PAGE and immunoblotting with the indicated antibodies in a strain that either does or does not express Rpb3-TAP Quantification of chromatin-bound Rpb1-S2P and Rpb1-S5P is shown on the right. Mcm2 is used as a loading control.


( F , G ) A 10 - fold dilution series of from exponential SC cultures of indicated strains were spotted on SC + / - 200mM of HU . A high level of HU was used to be able to demonstrate robust suppression of mec1 - S1991A phenotype . ( G ) Histogram presents of 2 independent HU sensitivity assays with mean data point values indicated for each yeast dilution .

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(F,G) A 10-fold dilution series of cells from exponential SC cultures of the indicated strains were spotted on SC +/- 200mM of HU. A high level of HU was used to be able to demonstrate robust suppression of the mec1-S1991A phenotype. (G) Histogram presents quantification of 2 independent HU sensitivity assays with mean and individual data point values indicated for each yeast dilution.


( B ) Amongst Mec1 - S1991 - dependent phosphopeptides on HU , factors involved in transcription are highlighted .

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(B) Amongst the Mec1-S1991-dependent phosphopeptides on HU, factors involved in transcription are highlighted.


( E ) Position relative to envelope ( Zone 1 of lacO - tagged GAL1 - GAL10 locus in indicated strains grown either 12h on glucose , or 90 min galactose or galactose + 0 . 2M HU . third panel shows relative retention at periphery ( Zone 1 ) on galactose + HU . number of is > 300 for each condition .

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(E) Position relative to the nuclear envelope (Zone 1 of the lacO-tagged GAL1-GAL10 locus in the indicated strains grown either 12h on glucose, or 90 min galactose or galactose + 0.2M HU. The third panel shows the relative retention at the nuclear periphery (Zone 1) on galactose + HU. The number of cells is >300 for each condition.


Total Rpb1 of exponentially growing that were synchronized in G1 with α - factor released into S phase at 25 ° C . Actin was used as a loading control .

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Total Rpb1 levels of exponentially growing cells that were synchronized in G1 with α-factor and released into S phase at 25°C. Actin was used as a loading control.


A stable inducible HEK - 293T expressing myc - PP2AC ( panel A were treated with DMSO , 20 μM DT - 061 or 2 μM iHAP1 for 2 hours myc - PP2AC affinity purified , respectively . binding of indicated was analysed by Western blotting A ) representative of 3 independent

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A stable inducible HEK-293T cell line expressing myc-PP2AC (panel A were treated with DMSO, 20 μM DT-061 or 2 μM iHAP1 for 2 hours and myc-PP2AC affinity purified, respectively. The binding of the indicated proteins was analysed by Western blotting A) representative of 3 independent experiments


A stable inducible HEK - 293T expressing myc - PP2AC ( panel B ) were treated with DMSO , 20 μM DT - 061 or 2 μM iHAP1 for 2 hours myc - PP2AC by quantitative mass spectrometry ( B ) . B ) of a biological triplicate .

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A stable inducible HEK-293T cell line expressing myc-PP2AC (panel B) were treated with DMSO, 20 μM DT-061 or 2 μM iHAP1 for 2 hours and myc-PP2AC by quantitative mass spectrometry (B). B) analysis of a biological triplicate.


a stable inducible HeLa expressing myc - PP2R1A ( C ) were treated with DMSO , 20 μM DT - 061 or 2 μM iHAP1 for 2 hours myc - PP2R1A affinity purified , respectively . binding of indicated was analysed by Western blotting C ) representative of 2 technical

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a stable inducible HeLa cell line expressing myc- PP2R1A (C) were treated with DMSO, 20 μM DT-061 or 2 μM iHAP1 for 2 hours and myc-PP2R1A affinity purified, respectively. The binding of the indicated proteins was analysed by Western blotting C) representative of 2 technical repeats


D ) HEK - 293T were treated with DMSO , 20 μM DT - 061 or 2 μM iHAP1 for 2 hours lysates were incubated with a microcystin affinity column to capture PPP complexes . Bound complexes were analysed by mass spectrometry to identify PP2A components . conducted on a biological triplicate .

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D) HEK-293T cells were treated with DMSO, 20 μM DT-061 or 2 μM iHAP1 for 2 hours and protein lysates were incubated with a microcystin affinity column to capture PPP complexes. Bound complexes were analysed by mass spectrometry to identify PP2A components. Analysis conducted on a biological triplicate.


E ) Doxycyclin - inducible CRISPR - Cas9 HeLa lines allow for depletion of specific B56 subunits . Growth assays in presence of indicated concentrations of DT - 061 or iHAP1 measured after 12 days of treatment . Mean SD of three independent experiments are shown .

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E) Doxycyclin-inducible CRISPR-Cas9 HeLa cell lines allow for depletion of specific B56 subunits. Growth assays in the presence of the indicated concentrations of DT-061 or iHAP1 measured after 12 days of treatment. Mean and SD of three independent experiments are shown.


A ) Schematic of experimental setup results of CRISPR screen . Genes were ranked on their normZ score . top hits which were further analysed are highlighted .

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A) Schematic of the experimental setup and results of CRISPR screen. Genes were ranked based on their normZ score. The top hits which were further analysed are highlighted.


B ) Validation of CRISPR screen results using RNAi depletion followed by SRB growth . were treated with DMSO ( Ctr ) or DT - 061 5 . 5 µM or iHAP1 0 . 5 µM . C ) Incucyte growth after RNAi depletion of indicated alone or in combination with DMSO or 5 . 5 μM DT - 061 or 0 . 5 μM iHAP1 treatment . Data information : B - C ) Mean SD of three independent experiments is shown .

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B) Validation of CRISPR screen results using RNAi depletion followed by SRB growth assay. Cells were treated with DMSO (Ctr) or DT-061 5.5 µM or iHAP1 0.5 µM. C) Incucyte growth assay after RNAi depletion of indicated proteins alone or in combination with DMSO or 5.5 μM DT-061 or 0.5 μM iHAP1 treatment. Data information: B-C) Mean and SD of three independent experiments is shown.


A ) In vitro tubulin polymerization showing dependent effect of iHAP1 on polymerization dynamics . Taxol Nocodazole served as controls for experimental setup . was performed in presence of indicated drug concentrations . Mean are shown from three to six independent experiments .

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A) In vitro tubulin polymerization assay showing concentration dependent effect of iHAP1 on polymerization dynamics. Taxol and Nocodazole served as controls for the experimental setup. The assay was performed in the presence of the indicated drug concentrations. Mean and SEM are shown from three to six independent experiments.


B ) Representative colour - coded temporal projection of U2OS EB1 - GFP following indicated treatment . Scale bar 10µm .

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B) Representative colour-coded temporal projection of U2OS EB1-GFP cells following indicated treatment. Scale bar 10µm.


C ) of microtubule dynamics acquired from manual tracking of EB1 - GFP comets as indicators for MT growth after drug treatment . mean SD are plotted from three independent experiments ( n = 10 per experiment ) . Data information : P - values were calculated using Student ' s t - or Mann - Whitney U - ( unpaired , two - ) . ns , not significant , * * * * p < 0 . 0001 .

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C) Quantification of microtubule dynamics acquired from manual tracking of EB1-GFP comets as indicators for MT growth after drug treatment. The mean and SD are plotted from three independent experiments (n = 10 cells per experiment). Data information: P-values were calculated using Student's t-test or Mann-Whitney U-test (unpaired, two-tailed). ns, not significant, ****p<0.0001.


D ) Representative temporal projection of mitotic spindles of U2OS EB1 - GFP following specified treatment . counterstained with SiR - shown in cyan in merged . Scale bar 10µm .

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D) Representative temporal projection of mitotic spindles of U2OS EB1-GFP cells following specified treatment. DNA counterstained with SiR-DNA shown in cyan in merged images. Scale bar 10µm.


E ) of number of EB1 - GFP comets per mitotic spindle . mean SD are plotted from three independent experiments ( Total number of = 26 ( DMSO ) , 20 ( 30µM DT - 061 ) , 27 ( DMSO + APC / C in . ) , 26 ( 2µM iHAP1 + APC / C in ) . Data information : P - values were calculated using Student ' s t - or Mann - Whitney U - ( unpaired , two - ) . ns , not significant , * * * * p < 0 . 0001 .

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E) Analysis of number of EB1-GFP comets per mitotic spindle. The mean and SD are plotted from three independent experiments (Total number of cells = 26 (DMSO), 20 (30µM DT-061), 27 (DMSO+APC/C in.) and, 26 (2µM iHAP1+APC/C in). Data information: P-values were calculated using Student's t-test or Mann-Whitney U-test (unpaired, two-tailed). ns, not significant, ****p<0.0001.


A ) Measurement of time spent in mitosis by time - lapse imaging taking into consideration whether exited mitosis or underwent during mitosis . n indicates number of analyzed from at least 2 independent experiments . Schematic of experimental protocol on top . were treated with iHAP1 when indicated at stated in figure . B ) Representative stills from time - lapse imaging experiments depicting two . Scale bar represents 20 µm .

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A) Measurement of time spent in mitosis by time-lapse imaging taking into consideration whether cells exited mitosis or underwent cell death during mitosis. n indicates the number of cells analyzed from at least 2 independent experiments. Schematic of experimental protocol on the top. Cells were treated with iHAP1 when indicated at the concentration stated in the figure. B) Representative stills from time-lapse imaging experiments depicting the two phenotypes. Scale bar represents 20 µm.


A ) Time - lapse imaging of HeLa expressing a marker Golgi apparatus ( green ) ( n = 87 of from 2 experiments analysed showing dispersal of marker ) . At time 0 , DT - 061 was added . On right - side , co - localisation of DT - 061 ( red ) Golgi marker is shown . B ) As in A ) but with a marker for ER either with addition of DT - 061 alone or DT - 061 + okadaic acid ( 50 nM ) Data information : Experiments were repeated at least twice stills show a representative for each treatment condition . were treated with DT - 061 at stated in figure . A - B ) Scale bar indicates 20 µm .

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A) Time-lapse imaging of HeLa cells expressing a marker staining the Golgi apparatus (green) (n= 87 of cells from 2 experiments analysed all showing dispersal of marker). At time 0, DT-061 was added. On the right-hand side, the co-localisation of DT-061 (red) and the Golgi marker is shown. B) As in A) but with a marker for ER and either with the addition of DT-061 alone or DT-061 + okadaic acid (50 nM) Data information: Experiments were repeated at least twice and stills show a representative cell for each treatment condition. Cells were treated with DT-061 at the concentration stated in the figure. A-B) Scale bar indicates 20 µm.


C ) Time required for ER signal translocation into nucleus after drug treatment . Number of analysed per condition shown on top median indicated by red . Data information : Experiments were repeated at least

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C) Time required for ER signal translocation into the nucleus after drug treatment. Number of cells analysed per condition shown on top and median indicated by red line. Data information: Experiments were repeated at least twice


D ) of with ER signal translocation into nucleus after drug treatment . Number of analysed per condition shown on top . Data information : Experiments were repeated at least

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D) Percentage of cells with ER signal translocation into the nucleus after drug treatment. Number of cells analysed per condition shown on top. Data information: Experiments were repeated at least twice


A - B ) Time - lapse imaging of H358 expressing a marker for Golgi ( A ) or ER ( B ) . were treated with DT - 061 at time 0 . ( B , low panel ) . For A ) 177 were analysed from one experiment displayed dispersal of Golgi marker . Arrow indicates addition of compound . Data information : Experiments were repeated at least twice except filming of Golgi marker which was done once . Stills show a representative for each treatment condition . were treated with DT - 061 at stated in figure . A - B ) Scale bar indicates 20 µm .

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A-B) Time-lapse imaging of H358 cells expressing a marker for the Golgi (A) or the ER (B). Cells were treated with DT-061 at time 0. (B, low panel). For A) 177 cells were analysed from one experiment and all displayed dispersal of the Golgi marker. Arrow indicates addition of compound. Data information: Experiments were repeated at least twice except filming of the Golgi marker which was done once. Stills show a representative cell for each treatment condition. Cells were treated with DT-061 at the concentration stated in the figure. A-B) Scale bar indicates 20 µm.


C ) Time needed for ER translocation into nucleus after drug treatment . Median ( red ) : 160 min . Number of analysed per condition shown on top . Data information : Experiments were repeated at least

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C) Time needed for ER translocation into the nucleus after drug treatment. Median (red line): 160 min. Number of cells analysed per condition shown on top. Data information: Experiments were repeated at least twice


D ) of with ER translocation after drug treatment . Number of analysed per condition shown on top . Data information : Experiments were repeated at least

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D) Percentage of cells with ER nuclear translocation after drug treatment. Number of cells analysed per condition shown on top. Data information: Experiments were repeated at least twice


B ) Increase in molar quantity of sphingolipid classes assessed in MCF7 after feeding with precursor sphingosine . MCF7 were co - treated with 15 μM DT - 061 , 1 . 5 μM iHAP1 , 3 µg / mL Brefeldin A , or vehicle ( DMSO ) . determined molar quantities of sphingolipid classes were normalized to that of PC . presented values represent averages of five independent experiments statistical was performed using an independent t - . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . Cer : ceramide , HexCer : hexosylceramide , PC : phosphatidylcholine , SM : sphingomyelin .

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B) Increase in the molar quantity of sphingolipid classes assessed in MCF7 cells after feeding with the precursor sphingosine. MCF7 cells were co-treated with 15 μM DT-061, 1.5 μM iHAP1, 3 µg/mL Brefeldin A, or vehicle (DMSO). The determined molar quantities of sphingolipid classes were normalized to that of PC. The presented values represent averages of five independent experiments and statistical analysis was performed using an independent t-test. *p<0.05, **p<0.01, ***p<0.001. Cer: ceramide, HexCer: hexosylceramide, PC: phosphatidylcholine, and SM: sphingomyelin.


( A ) Bone Marrow Derived Macrophages ( BMDMs ) were treated with either AMPK activator 991 , Dex or a combination for 1 h before by western blot . ( B , C ) Phospho - AMPK ( B ) phospho - ACC ( C ) were quantified . Data information : results are means ± or z - score or displayed with boxes whiskers in which central band represents median . N = 6 ( B , C ) , biological replicates * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 statistical using student ' s t - ( B , C )

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(A) Bone Marrow Derived Macrophages (BMDMs) were treated with either the AMPK activator 991, Dex or a combination for 1 h before analysis by western blot. (B, C) Phospho-AMPK (B) and phospho-ACC (C) were quantified. Data information: results are means ± SEM or z-score or displayed with boxes and whiskers in which the central band represents the median. N= 6 (B,C), biological replicates * p<0.05, ** p<0.01, *** p<0.001 statistical analysis using student's t-test (B, C)


( D ) WT AMPKα1 - / - BMDMs were treated with Dex for 1 h before by western blot GR phosphorylation at S211 was quantified . Data information : results are means ± or z - score or displayed with boxes whiskers in which central band represents median . N = 3 ( D ) biological replicates * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 statistical using ANOVA ( D ,

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(D) WT and AMPKα1-/- BMDMs were treated with Dex for 1 h before analysis by western blot and GR phosphorylation at S211 was quantified. Data information: results are means ± SEM or z-score or displayed with boxes and whiskers in which the central band represents the median. N= 3 (D) biological replicates * p<0.05, ** p<0.01, *** p<0.001 statistical analysis using ANOVA tests (D,


( E ) WT AMPKα1 - / - BMDMs were treated with Dex for 24 h analyzed by - seq . Differentially expressed genes between any condition were clustered using c - means clustering .

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(E) WT and AMPKα1-/- BMDMs were treated with Dex for 24 h and analyzed by RNA-seq. Differentially expressed genes between any condition were clustered using c-means clustering.


( F ) WT AMPKα1 - / - BMDMs were treated with Dex for 24 h anti - / pro - inflammatory markers assessed using immunofluorescence . Data information : results are means ± or z - score or displayed with boxes whiskers in which central band represents median . N = 3 - 4 ( F ) biological

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(F) WT and AMPKα1-/- BMDMs were treated with Dex for 24 h and anti-/pro-inflammatory markers assessed using immunofluorescence. Data information: results are means ± SEM or z-score or displayed with boxes and whiskers in which the central band represents the median. N= 3-4 (F) biological replicates


( G WT AMPKα1 - / - BMDMs were treated with Dex for 72 h , washed conditioned medium was recovered after 24 h used to treat muscle . Proliferation ( Ki67 immunostaining ) ( G ) were assessed in muscle . Data information : results are means ± or z - score or displayed with boxes whiskers in which central band represents median . N = 4 biological replicates ( G * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 statistical using ANOVA Bar = 25 µm (

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(G WT and AMPKα1-/- BMDMs were treated with Dex for 72 h, washed and conditioned medium was recovered after 24 h used to treat muscle stem cells. Proliferation (Ki67 immunostaining) (G) were assessed in muscle stem cells. Data information: results are means ± SEM or z-score or displayed with boxes and whiskers in which the central band represents the median. N= 4 biological replicates (G * p<0.05, ** p<0.01, *** p<0.001 statistical analysis using ANOVA tests Bar=25 µm (G


H ) WT AMPKα1 - / - BMDMs were treated with Dex for 72 h , washed conditioned medium was recovered after 24 h used to treat muscle . differentiation ( desmin immunostaining counting of muscle with 2 nuclei or more ) ( H ) were assessed in muscle . Data information : results are means ± or z - score or displayed with boxes whiskers in which central band represents median . N = 4 biological replicates H ) . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 statistical using ANOVA , H ) . Bar = 25 µm H ) .

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H) WT and AMPKα1-/- BMDMs were treated with Dex for 72 h, washed and conditioned medium was recovered after 24 h used to treat muscle stem cells. differentiation (desmin immunostaining and counting the percentage of muscle cells with 2 nuclei or more) (H) were assessed in muscle stem cells. Data information: results are means ± SEM or z-score or displayed with boxes and whiskers in which the central band represents the median. N= 4 biological replicates H). * p<0.05, ** p<0.01, *** p<0.001 statistical analysis using ANOVA tests ,H). Bar=25 µm H).


After cardiotoxin injection to damage muscle , AMPKα1fl / fl ( WT ) LysM - α1 - / - mice were treated with a single dose of Dexamethasone ( Dex ) intra - peritoneal ( i . p . ) ( 0 . 1 mg / kg ) at day 3 ( D3 ) Tibialis Anterior ( TA ) were harvested at day 8 ( D8 ) 14 ( D14 ) after injury . ( A ) Representative embryonic myosin heavy chain ( EmbMHC ) immunostaining on whole TA muscle section at D8 D14 after injury . ( B ) of ( A ) , results are expressed as of positive myofibers expressing EmbMHC on whole muscle section . Data information : results are displayed with box whiskers in which central band represents median ( B N = 7 - 12 ( B * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 using ANOVA . Bars = 500 µm ( A ) ,

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After cardiotoxin injection to damage the muscle, AMPKα1fl/fl (WT) and LysM-α1-/- mice were treated with a single dose of Dexamethasone (Dex) intra-peritoneal (i.p.) (0.1 mg/kg) at day 3 (D3) and Tibialis Anterior (TA) muscles were harvested at day 8 (D8) and 14 (D14) after injury. (A) Representative embryonic myosin heavy chain (EmbMHC) immunostaining on whole TA muscle section at D8 and D14 after injury. (B) Quantification of (A), results are expressed as percentage of positive myofibers expressing the EmbMHC on the whole muscle section. Data information: results are displayed with box and whiskers in which the central band represents the median (B N= 7-12 muscles (B * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001 using ANOVA tests. Bars = 500 µm (A),


( F ) Macrophage polarization was determined by immunofluorescence in vehicle , DAMPs DAMPs + Dex treated WT AMPKα1 - / - BMDMs . Data information : results are displayed with box whiskers in which central band represents means ± ( F ) . N = 4 - 5 biological replicates ( F ) . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 using ANOVA . Bars = 25 µm ( F ) .

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(F) Macrophage polarization was determined by immunofluorescence in vehicle, DAMPs and DAMPs+Dex treated WT and AMPKα1-/- BMDMs. Data information: results are displayed with box and whiskers in which the central band represents the means ± SEM (F). N= 4-5 biological replicates (F). * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001 using ANOVA tests. Bars = 25 µm (F).


( F ) Macrophage polarization was determined by immunofluorescence in vehicle , LPS LPS + Dex treated WT AMPKα1 - / - BMDMs , data are expressed as positive . Data information : Results are displayed with box whiskers in which central band represents means ± ( F ) . N = 3 ( F ) biological replicates . Statistical by ANOVA F ) * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . Bar = 25 µm ( F ) .

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(F) Macrophage polarization was determined by immunofluorescence in vehicle, LPS and LPS+Dex treated WT and AMPKα1-/- BMDMs, data are expressed as percentage positive cells. Data information: Results are displayed with box and whiskers in which the central band represents the means ± SEM (F). N= 3 (F) biological replicates. Statistical analysis by ANOVA F) * p<0.05, ** p<0.01, *** p<0.001. Bar = 25 µm (F).


( F ) WT AMPKα1 - / - Bone Marrow Derived Macrophages ( BMDMs ) were treated with either Dex or AMPK activator 991 phosphorylated on serine were immunoprecipitated . Westen - blot for FOXO3 , AMPKα1 , actin phosphorylated form of FOXO3 were done . of band is provided normalized to those of actin total FOXO3 , a representative example is shown . Data information : results are box whiskers in which central band represents median F , N = 4 - 5 ( F biological replicates . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 * * * * p < 0 . 0001 using ANOVA Kruskal - Wallis

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(F) WT and AMPKα1-/- Bone Marrow Derived Macrophages (BMDMs) were treated with either Dex or the AMPK activator 991 and protein phosphorylated on serine were immunoprecipitated. Westen-blot for FOXO3, AMPKα1, actin and the phosphorylated form of FOXO3 were done. Quantification of the band intensity is provided normalized to those of actin and total FOXO3, a representative example is shown. Data information: results are box and whiskers in which the central band represents the median F, N= 4-5 (F biological replicates. * p<0.05, ** p<0.01, *** p<0.001 **** p<0.0001 using ANOVA and Kruskal-Wallis tests


, C . siRNA - mediated knockdown of EXOSC10 ( EXO10 ) , but not of DIS3 DIS3L , significantly increased generation of poly - GA in HeLa expressing ( G4C2 ) 80 repeats . ctrl " indicates a control vector which lacks G4C2 repeat but still contains 5 ' flanking region 3xTAG .

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, C. siRNA-mediated knockdown of EXOSC10 (EXO10), but not of DIS3 and DIS3L, significantly increased generation of poly-GA in HeLa cells expressing (G4C2) 80 repeats. ctrl" indicates a control vector which lacks the G4C2 repeat but still contains the 5' flanking region and 3xTAG.


. Increased G4C2 repeat expression upon EXOSC10 knockdown on RT - qPCR .

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. Increased G4C2 repeat RNA expression upon EXOSC10 knockdown on RT-qPCR.


E , F . Knockdown of EXOSC10 increased foci in HeLa expressing ( G4C2 ) 80 repeats visualized by in situ hybridization targeting sense foci . In total 25 ( Ct ) 24 ( EXO10 # 5 ) were analyzed in 3 independent experiments . G . Knockdown of EXOSC10 increased foci in HeLa expressing ( G4C2 ) 80 repeats . Each dot represents average foci ( Arbitrary unit ) of foci - positive in each randomly taken image . In total 25 ( Ct ) 24 ( EXO10 # 5 ) were analyzed in 3 independent experiments .

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E, F. Knockdown of EXOSC10 increased RNA foci in HeLa cells expressing (G4C2) 80 repeats visualized by in situ hybridization targeting sense RNA foci. In total 25 (Ct) and 24 (EXO10#5) images were analyzed in 3 independent experiments. G. Knockdown of EXOSC10 increased RNA foci intensity in HeLa cells expressing (G4C2) 80 repeats. Each dot represents average cellular RNA foci intensity (Arbitrary unit) of RNA foci-positive cells in each randomly taken image. In total 25 (Ct) and 24 (EXO10#5) images were analyzed in 3 independent experiments.


A , B . Dose - dependent suppression of poly - GA by exogenous EXOSC10 upon EXOSC10 knockdown .

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A, B. Dose-dependent suppression of poly-GA by exogenous EXOSC10 upon EXOSC10 knockdown.


C . Suppression of G4C2 repeat by exogenous EXOSC10 on EXOSC10 knockdown . of transfected EXOSC10 plasmid was 0 . 2 ng / μl in 1 ml medium .

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C. Suppression of G4C2 repeat RNA by exogenous EXOSC10 on EXOSC10 knockdown. Concentration of transfected EXOSC10 plasmid was 0.2 ng/μl in 1 ml medium.


D . Overexpression of EXOSC10 decreases G4C2 repeat .

D. Overexpression of EXOSC10 decreases G4C2 repeat RNA.


A , B . Under transcription inhibition with Actinomycin D , with EXOSC10 knockdown ( EXO10 KD ) showed more stable expression of repeat compared to with control knockdown with non - targeting siRNA ( Ct KD ) .

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A, B. Under transcription inhibition with Actinomycin D, cells with EXOSC10 knockdown (EXO10 KD) showed more stable expression of the repeat RNA compared to cells with control knockdown with non-targeting siRNA (Ct KD).


C , D . Degradation of FAM - labeled synthetic ( G4C2 ) 8 repeat with recombinant human EXOSC10 ( hEXO10 ) ( Upper left ) . 3 ' blocked FAM - labeled G4C2 repeat resisting 3 ' exoribonuclease activity is not degraded by recombinant EXOSC10 ( Lower left ) . Lack of recombinant EXOSC10 ( Upper right ) or Mg2 + ion which is essential co - factor of EXOSC10 ( Lower right ) abolished degradation activity . 3 or 4 independent experiments . of top bands are shown in ( D ) .

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C, D. Degradation of FAM-labeled synthetic (G4C2)8 repeat RNA with recombinant human EXOSC10 (hEXO10) (Upper left). 3' blocked FAM-labeled G4C2 repeat RNA resisting 3' exoribonuclease activity is not degraded by recombinant EXOSC10 (Lower left). Lack of recombinant EXOSC10 (Upper right) or Mg2+ ion which is essential co-factor of EXOSC10 (Lower right) abolished the RNA degradation activity. 3 or 4 independent experiments. Quantification of the top bands are shown in (D).


E , F . Degradation of antisense C4G2 repeat substrate with recombinant human EXOSC10 ( Left ) . Without recombinant EXOSC10 , no substrate degradation was observed ( Right ) . 5 or 6 independent experiments . is shown in ( F ) .

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E, F. Degradation of antisense C4G2 repeat RNA substrate with recombinant human EXOSC10 (Left). Without recombinant EXOSC10, no substrate degradation was observed (Right). 5 or 6 independent experiments. Quantification is shown in (F).


A . In situ hybridization . G4C2 foci were detected in a C9 - derived fibroblast but not in a control - derived fibroblast with non - targeting siRNA treatment ( left panels , control / si Ct . middle panels , C9 / si Ct ) . Knockdown of EXSOC10 increased foci ( right panels , C9 / siEXOSC10 # 5 ) . Nuclei were stained with DAPI ( blue ) . arrows indicate foci . green arrows show foci .

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A. In situ hybridization. G4C2 RNA foci were detected in a C9 patient-derived fibroblast but not in a control-derived fibroblast with non-targeting siRNA treatment (left panels, control/si Ct. middle panels, C9/si Ct). Knockdown of EXSOC10 increased RNA foci (right panels, C9/siEXOSC10#5). Nuclei were stained with DAPI (blue). White arrows indicate nuclear RNA foci. The green arrows show cytoplasmic RNA foci.


B . of % with G4C2 foci . 3 independent knockdown experiments were performed . 10 DAPI positive per experiment were randomly selected z - stack were obtained . In total , z - stack from 90 per group ( control siRNA or EXOSC10 siRNA ) were counted for foci . C . of number of G4C2 foci / . 3 independent knockdown experiments were performed . 10 DAPI positive per experiment were randomly selected z - stack were obtained . In total , z - stack from 90 per group ( control siRNA or EXOSC10 siRNA ) were counted for foci . D . of % with C4G2 foci . 3 independent knockdown experiments were performed . 10 DAPI positive per experiment were randomly selected z - stack were obtained . In total , z - stack from 90 per group ( control siRNA or EXOSC10 siRNA ) were counted for foci . E . of number of C4G2 foci / . 3 independent knockdown experiments were performed . 10 DAPI positive per experiment were randomly selected z - stack were obtained . In total , z - stack from 90 per group ( control siRNA or EXOSC10 siRNA ) were counted for foci .

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B. Quantification of %cell with G4C2 RNA foci. 3 independent knockdown experiments were performed. 10 DAPI positive cells per experiment were randomly selected and z-stack images were obtained. In total, z-stack images from 90 cells per group (control siRNA or EXOSC10 siRNA) were counted for RNA foci. C. Quantification of the number of G4C2 RNA foci/cell. 3 independent knockdown experiments were performed. 10 DAPI positive cells per experiment were randomly selected and z-stack images were obtained. In total, z-stack images from 90 cells per group (control siRNA or EXOSC10 siRNA) were counted for RNA foci. D. Quantification of %cell with C4G2 RNA foci. 3 independent knockdown experiments were performed. 10 DAPI positive cells per experiment were randomly selected and z-stack images were obtained. In total, z-stack images from 90 cells per group (control siRNA or EXOSC10 siRNA) were counted for RNA foci. E. Quantification of the number of C4G2 RNA foci/cell. 3 independent knockdown experiments were performed. 10 DAPI positive cells per experiment were randomly selected and z-stack images were obtained. In total, z-stack images from 90 cells per group (control siRNA or EXOSC10 siRNA) were counted for RNA foci.


F , G . Increased endogenous repeat expression upon EXOSC10 knockdown in strand specific RT - qPCR . 10 independent knockdown experiments ( Case1 N = 4 , Case2 N = 4 , Case3 N = 2 ) were performed .

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F, G. Increased endogenous repeat RNA expression upon EXOSC10 knockdown in strand specific RT-qPCR. 10 independent knockdown experiments (Case1 N=4, Case2 N=4, Case3 N=2) were performed.


A , B , C . Double knockdown of EXOSC10 DIS3 significantly increased poly - GA repeat expression compared with single knockdown of EXOSC10 in expressing ( G4C2 ) 80 repeats .

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A, B, C. Double knockdown of EXOSC10 and DIS3 significantly increased poly-GA and repeat RNA expression compared with single knockdown of EXOSC10 in cells expressing (G4C2) 80 repeats.


D . Overexpression of DIS3 compensated EXOSC10 knockdown - induced repeat accumulation .

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D. Overexpression of DIS3 compensated EXOSC10 knockdown-induced repeat RNA accumulation.


E , F . Catalytically dead mutant EXOSC10 rescued EXOSC10 knockdown - induced GA increase .

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E, F. Catalytically dead mutant EXOSC10 rescued EXOSC10 knockdown-induced GA increase.


C - F . HA - tagged poly - GA , poly - GP , poly - GR or poly - PR ( red ) EXOSC10 ( green ) were immunostained with anti - HA anti - EXOSC10 antibodies . Nuclei were stained with DAPI ( blue ) .

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C-F. HA-tagged poly-GA, poly-GP, poly-GR or poly-PR (red) and EXOSC10 (green) were immunostained with anti-HA and anti-EXOSC10 antibodies. Nuclei were stained with DAPI (blue).


G . EXOSC10 ( green ) were immunostained with anti - EXOSC10 antibody in transfected with control mock plasmid . Nuclei were stained with DAPI ( blue ) .

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G. EXOSC10 (green) were immunostained with anti-EXOSC10 antibody in cells transfected with control mock plasmid. Nuclei were stained with DAPI (blue).


H , I . of frequency of with mislocalized EXOSC10 . Only DPR - positive were quantified ( except mock transfected expressing no DPR . ( Total : ( figure H ) mock 300 , GA 112 , GP 69 , GR 53 , ( figure I ) mock 445 , PR 159 ) .

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H, I. Quantification of the frequency of cells with mislocalized EXOSC10. Only DPR-positive cells were quantified (except mock transfected cells expressing no DPR. (Total cell counts: (figure H) mock 300 cells, GA 112 cells, GP 69 cells, GR 53 cells, (figure I) mock 445 cells, PR 159 cells).


B . Immunoblot validation of successful fractionation using antibodies against HDAC1 ( marker ) GAPDH ( marker ) .

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B. Immunoblot validation of successful fractionation using antibodies against HDAC1 (nuclear marker) and GAPDH (cytoplasmic marker).


C . Co - immunoprecipitation of GFP - DPR interacting in fraction using GFP - trap beads . Immunoprecipitants were served for immunoblot with anti - EXOSC10 antibody . same was reprobed with anti - GFP antibody . D . of relative intensities of " EXOSC10 in co - IP fraction " / " EXOSC10 in input " . E . Co - immunoprecipitation of GFP - DPR interacting in fraction using GFP - trap beads followed by immunoblot using anti - EXOSC10 antibody . same was reprobed with anti - GFP antibody . F . of relative intensities of " EXOSC10 in co - IP fraction " / " EXOSC10 in input " .

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C. Co-immunoprecipitation of GFP-DPR interacting proteins in cytoplasmic fraction using GFP-trap beads. Immunoprecipitants were served for immunoblot with anti-EXOSC10 antibody. The same membrane was reprobed with anti-GFP antibody. D. Quantification of the relative intensities of "EXOSC10 in co-IP fraction" / "EXOSC10 in input". E. Co-immunoprecipitation of GFP-DPR interacting proteins in nuclear fraction using GFP-trap beads followed by immunoblot using anti-EXOSC10 antibody. The same membrane was reprobed with anti-GFP antibody. F. Quantification of the relative intensities of "EXOSC10 in co-IP fraction" / "EXOSC10 in input".


B , C . Increased expression of 3 ' extended snoRA48 precursor upon EXOSC10 knockdown ( B ) or GFP - poly - GR177 / GFP - poly - PR166 expression ( C ) on RT - qPCR . D , E . Increased expression of 3 ' extended snoRA68 precursor upon EXOSC10 knockdown ( D ) or GFP - poly - GR177 / GFP - poly - PR166 expressions ( E ) on RT - qPCR .

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B, C. Increased expression of 3'extended snoRA48 precursor upon EXOSC10 knockdown (B) or GFP-poly-GR177/ GFP-poly-PR166 expression (C) on RT-qPCR. D, E. Increased expression of 3'extended snoRA68 precursor upon EXOSC10 knockdown (D) or GFP-poly-GR177 /GFP-poly-PR166 expressions (E) on RT-qPCR.


F . RT - qPCR showed increased G4C2 repeat expression from co - transfected EF1 ( G4C2 ) 80 repeat construct in presence of GFP , GFP - poly - GR177 or GFP - poly - PR166 .

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F. RT-qPCR analysis showed increased cellular G4C2 repeat RNA expression from co-transfected EF1 (G4C2)80 repeat construct in the presence of GFP, GFP-poly-GR177 or GFP-poly-PR166.


Western blotting of Gadd45a expression in mouse intestine crypt lysates from WT G3Terc - / - mice ( A ) . of Gadd45a expression , β - actin level was used as loading control ( n = 7 mice per genotype ) , two independent experiments were carried out ( B ) .

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Western blotting analysis of Gadd45a protein expression in mouse intestine crypt lysates from WT and G3Terc-/- mice (A). Quantification of Gadd45a protein expression, β-actin protein level was used as the loading control (n=7 mice per genotype), two independent experiments were carried out (B).


of Gadd45a - ChIP values as of total telomeric ALU elements in WT G3Terc - / - MEF ( n = 3 for each genotype ) .

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Quantification of Gadd45a-ChIP values as percentage of total telomeric DNA and ALU elements in WT and G3Terc-/- MEF cells (n=3 for each genotype).


Dot - bot following Gadd45a - ChIP in WT G3Terc - / - MEF .

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Dot-bot assay following Gadd45a-ChIP in WT and G3Terc-/- MEF cells.


Kaplan - Meier survival curves of mice with indicated genotypes ( WT , n = 44 ; Gadd45a - / - , n = 48 ; G3Terc - / - , n = 37 ; G3 - dKO , n = 41 ) . P value comparing survival of G3Terc - / - with G3 - dKO mice is calculated with log - rank .

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Kaplan-Meier survival curves of the mice with indicated genotypes (WT, n=44; Gadd45a-/-, n=48; G3Terc-/-, n=37; G3-dKO, n=41). P value comparing the survival of G3Terc-/- with G3-dKO mice is calculated with the log-rank test.


average of mice with indicated genotypes at indicated time points ( G3 - dKO ( n = 19 ) vs G3Terc - / - ( n = 17 ) , P = 0 . 004 ; G3 - dKO ( n = 14 ) vs G3Terc - / - ( n = 14 ) , P = 0 . 0038 ) . P values are calculated with log - rank .

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The average body weight of mice with indicated genotypes at indicated time points (G3-dKO Female (n=19) vs G3Terc-/- Female (n=17), P=0.004; G3-dKO Male (n=14) vs G3Terc-/- Male (n=14), P=0.0038). P values are calculated with log-rank test.


Whole mount of colon samples from 7 - month old mice with indicated genotypes ( G ) . on density of colon crypts ( H ) ( n = 5 mice per group ) .

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Whole mount images of colon samples from 7-month old mice with indicated genotypes (G). Quantification on density of colon crypts (H) (n=5 mice per group).


H & E - stained small intestine sections from 7 - month old mice with indicated genotypes ( I ) . of number of small intestine basal crypts ( J ) ( n = 5 - 6 mice per group ) .

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H&E-stained small intestine sections from 7-month old mice with indicated genotypes (I). Quantification of the number of small intestine basal crypts (J) (n=5-6 mice per group).


Olfm4 - positive intestinal ( indicated by arrows ) in small intestine sections ( A ) . of intestinal ( Olfm4 + ) number per 100 μm ( n = 4 - 5 mice per group ) of small intestine sections ( B ) .

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Olfm4-positive intestinal stem cells (indicated by arrows) in small intestine sections (A). Quantification of intestinal stem cell (Olfm4+) number per 100 μm (n=4-5 mice per group) of small intestine sections (B).


Representative of small intestine crypt culture ( day 2 ) . Arrows mark surviving cysts ( C ) . of numbers of cysts per culture well ( D ) ( n = 4 - 5 mice / genotype ) .

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Representative images of small intestine crypt culture (day 2). Arrows mark the surviving cysts (C). Quantification of numbers of cysts per culture well (D)(n=4-5 mice/genotype).


In vitro crypt culture ( day 8 ) from small intestine crypts isolated from mice with indicated genotypes . Arrows mark crypt - like structure ( E ) . number of organoids per culture wells ( n = 4 - 5 mice per group ) . Two hundred crypts are seeded per well ( F ) .

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In vitro crypt culture (day 8) from small intestine crypts isolated from mice with indicated genotypes. Arrows mark the crypt-like structure (E). The number of organoids per culture wells (n=4-5 mice per group). Two hundred crypts are seeded per well (F).


Representative of TUNEL in small intestine sections . Arrows indicate TUNEL positive ( G ) . of TUNEL positive per crypt ( n = 5 mice per group ) ( H ) .

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Representative images of TUNEL staining in small intestine sections. Arrows indicate TUNEL positive cells (G). Quantification of TUNEL positive cells per crypt (n=5 mice per group) (H).


Representative of phosphorylated histone 3 ( pH3 ) antibody in mouse small intestine . Arrows indicate pH3 positive ( I ) . of pH3 positive crypt per 100 μm of small intestines ( n = 4 - 6 mice per group ) ( J ) .

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Representative images of phosphorylated histone 3 (pH3) antibody staining in mouse small intestine. Arrows indicate pH3 positive cells (I). Quantification of pH3 positive crypt cells per 100 μm of small intestines (n=4-6 mice per group) (J).


Representative of immunostaining for p53 on small intestine sections . Arrows indicate p53 - positive ( A ) . of of p53 positive per crypt ( n = 5 - 6 mice per group ) ( B ) .

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Representative images of immunostaining for p53 on small intestine sections. Arrows indicate p53-positive cells (A). Quantification of the percentage of p53 positive cells per crypt (n=5-6 mice per group) (B).


Representative of γH2AX antibody on small intestine paraffin sections from WT , Gadd45a - / - , G3Terc - / - , G3 - dKO mice ( 7 months old ) . Arrows indicate γH2AX positive nuclei ( C ) . of of γH2AX positive per small intestine crypt ( n = 4 - 6 mice per group ) ( D ) .

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Representative images of γH2AX antibody staining on small intestine paraffin sections from WT, Gadd45a-/-, G3Terc-/-, and G3-dKO mice (7 months old). Arrows indicate γH2AX positive nuclei (C). Quantification of the percentage of γH2AX positive cells per small intestine crypt (n=4-6 mice per group) (D).


Representative of small intestine crypt sections stained with antibody against pATM ( Ser - 1981 ) ( E ) . of frequencies of pATM ( Ser - 1981 ) positive crypt ( n = 4 - 6 of 7 - month old mice per genotype ) ( F ) .

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Representative images of small intestine crypt sections stained with antibody against pATM (Ser-1981)(E). Quantification of frequencies of pATM (Ser-1981) positive crypt cells (n=4-6 of 7-month old mice per genotype) (F).


Representative image of anaphase bridge ( indicated with arrows ) in basal crypt of G3Terc - / - mice ( G ) . of with anaphase bridges in pH3 positive basal crypt population ( n = 4 - 6 mice per genotype ) ( H ) .

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Representative image of the anaphase bridge (indicated with arrows) in the basal crypt of G3Terc-/- mice (G). The percentage of cells with anaphase bridges in pH3 positive basal crypt cell population (n=4-6 mice per genotype) (H).


Heat map indicating relative expression of DDR cycle - related gene transcripts in MEFs from indicated genotypes ( Data are extracted from RNAseq datasets ) .

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Heat map indicating the relative expression levels of DDR and cell cycle-related gene transcripts in MEFs from indicated genotypes (Data are extracted from RNAseq datasets).


CpG island methylation status of subtelomeric regions on chromosome 1q . gDNAs from MEF with indicated genotypes were PCR amplified analysed by bisulfite sequencing . Yellow blue bars denote frequencies of methylated unmethylated CpG islands , respectively , at each position ( A ) . of subtelomeric CpG island methylation ( B ) .

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CpG island methylation status of subtelomeric regions on chromosome 1q. gDNAs from MEF with indicated genotypes were PCR amplified and analysed by bisulfite sequencing. Yellow and blue bars denote the frequencies of methylated and unmethylated CpG islands, respectively, at each position (A). Quantification of subtelomeric CpG island methylation (B).


Telomeric dot - bot of tri - methyl H3K9 ( H3K9me3 ) - ChIP in indicated

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Telomeric quantification and dot-bot assay of tri-methyl H3K9 (H3K9me3)-ChIP in indicated genotypes


Telomeric dot - bot of acetylated H3K9 ( H3K9ac ) - ChIP in indicated

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Telomeric quantification and dot-bot assay of acetylated H3K9 (H3K9ac)-ChIP in indicated genotypes


Telomeric dot - bot of HP1a - ChIP in indicated

Telomeric quantification and dot-bot assay of HP1a-ChIP in indicated genotypes


Representative of small intestine organoids from WT G3Terc - / - mice incubated with or without APE1 inhibitor ( CRT0044876 , 10μM ) treatment . Arrows indicate crypt - like structure in organoid cultures ( A ) . of organoid number ( per well ) ( in 104 μm2 ) ( n = 3 - 4 mice per genotype / treatment ) ( B - C ) .

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Representative images of small intestine organoids from WT and G3Terc-/- mice incubated with or without APE1 inhibitor (CRT0044876, 10μM) treatment. Arrows indicate the crypt-like structure in organoid cultures (A). Quantification of organoid number (per well) and size (in 104 μm2) (n=3-4 mice per genotype/treatment) (B-C).


Representative of γH2AX antibody on dissociated organoid from WT G3Terc - / - organoid cultures with or without APE1 inhibitor ( CRT0044876 , 10μM ) ( DNAs are stained with DAPI ) ( D ) . of γH2AX positive ( n = 3 - 4 mice per group ) ( E ) .

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Representative images of γH2AX antibody staining on dissociated organoid cells from WT and G3Terc-/- organoid cultures with or without APE1 inhibitor (CRT0044876, 10μM) (DNAs are stained with DAPI) (D). Quantification of γH2AX positive cells (n=3-4 mice per group) (E).


Telomeric DNA quantification and dot-blot assay of H3K9me3-ChIP


Telomeric DNA quantification and dot-blot assay of H3K9ac-ChIP


Telomeric DNA quantification and dot-blot assay of HP1a-ChIP


Representative of Olfm4 in small intestine sections from mice treated with or without APE1 inhibitor ( CRT0044876 , CRT ) . Arrows indicate Olfm4 positive intestine ( I ) . of Olfm4 positive intestine ( n = 4 mice per group ) ( J ) .

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Representative images of Olfm4 staining in small intestine sections from mice treated with or without APE1 inhibitor (CRT0044876, CRT). Arrows indicate Olfm4 positive intestine stem cells (I). Quantification of Olfm4 positive intestine stem cells (n=4 mice per group) (J).


knockdown efficiency of shRNA - GADD45A in human WI38 fibroblasts .

The knockdown efficiency of shRNA-GADD45A in human WI38 fibroblasts.


proliferation curves of human WI38 fibroblasts treated with control shRNA or shRNA against GADD45A .

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The proliferation curves of human WI38 fibroblasts treated with control shRNA or shRNA against GADD45A.


Representative of β - gal in human fibroblast cultures with control shRNA - GADD45A treatments at passage 54 ( C ) . of of β - gal positive in human fibroblasts with control shRNA - GADD45A treatments ( duplicates per group ) ( D ) .

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Representative images of β-gal staining in human fibroblast cultures with control and shRNA-GADD45A treatments at passage 54 (C). Quantification of the percentage of β-gal positive cells in human fibroblasts with control and shRNA-GADD45A treatments (duplicates per group) (D).


Representative of GADD45A γH2AX in colon sections from young ( 35 - 50 y / o ) old ( 75 - 85 y / o ) people ( E ) . of of GADD45A positive in young old human colon tissues ( n = 8 per group ) ( F ) . of of γH2AX positive in young old human colon tissues ( n = 6 per group ) ( G ) .

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Representative images of GADD45A and γH2AX staining in colon sections from young (35-50 y/o) and old (75-85 y/o) people (E). Quantification of the percentage of GADD45A positive cells in young and old human colon tissues (n=8 per age group) (F). Quantification of the percentage of γH2AX positive cells in young and old human colon tissues (n=6 per age group) (G).


( a ) Kinetics of appearance of P14 in spleens of C57BL / 6 mice given adoptive transfer of 1 × 104 P14 , monitored after infection of recipients with LCMV Armstrong .

(a) Kinetics of the appearance of P14 cells in spleens of C57BL/6 mice given adoptive transfer of 1 × 104 P14 cells, monitored after infection of recipients with LCMV Armstrong strain.


( b ) Flow cytometry of at various stages of P14 differentiation , assessed by forward scatter ( FSC ) .

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(b) Flow cytometry of cell size at various stages of P14 differentiation, assessed by forward scatter (FSC).


( c ) Immunoblot of LC3b p62 at various stages of P14 differentiation . Data are representative of two independent experiments with samples pooled from 5 - 20 mice for each time point .

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(c) Immunoblot analysis of LC3b and p62 at various stages of P14 differentiation. Data are representative of two independent experiments with samples pooled from 5-20 mice for each time point.


( b ) Flow cytometry of P14 from spleens of recipient mice given adoptive transfer of P14 transduced with retrovirus as in a , assessed on days 5 , 8 30 after infection of recipients with LCMV . Numbers in blue outlined areas indicate GFP − among transduced ( Thy - 1 . 1 + ) P14 . ( c ) Summary of results in b , showing autophagy activity as GFP − . * P ≤ 0 . 0005 ( unpaired t - ) .

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(b) Flow cytometry of P14 cells from the spleens of recipient mice given adoptive transfer of P14 cells transduced with retrovirus as in a, assessed on days 5, 8 and 30 after infection of recipients with LCMV. Numbers in blue outlined areas indicate percent GFP− cells among transduced (Thy-1.1+) P14 cells. (c) Summary of results in b, showing autophagy activity as percent GFP− cells. *P ≤ 0.0005 (unpaired t-test).


( d ) Multispectral - imaging flow cytometry of P14 from spleens of recipient mice given adoptive transfer of P14 transduced with retrovirus as in a , assessed on day 8 after infection , showing GFP signals from GFP + transduced ( CD45 . 1 + Thy - 1 . 1 + ) P14 . Original magnification , × 60 . ( e ) Summary of results in d , presented as frequency of with more than one GFP + punctum among vector transduced Thy - 1 . 1 + P14 . Each symbol represents an mouse ; small horizontal lines indicate mean ( ± s . e . m . ) . Data are representative of three independent experiments ( b , d ) or three independent experiments with three or more mice per group ( e ; error bars , s . e . m . ) or six mice per group ( c ; error bars , s . e . m . ) .

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(d) Multispectral-imaging flow cytometry of P14 cells from the spleens of recipient mice given adoptive transfer of P14 cells transduced with retrovirus as in a, assessed on day 8 after infection, showing GFP signals from GFP+ transduced (CD45.1+ Thy-1.1+) P14 cells. Original magnification, ×60. (e) Summary of results in d, presented as the frequency of cells with more than one GFP+ punctum among vector transduced Thy-1.1+ P14 cells. Each symbol represents an individual mouse; small horizontal lines indicate the mean (±s.e.m.). Data are representative of three independent experiments (b,d) or three independent experiments with three or more mice per group (e; error bars, s.e.m.) or six mice per group (c; error bars, s.e.m.).


( b ) Flow cytometry of splenocytes from recipient mice ( n = 6 ) given adoptive transfer of P14 transduced with retrovirus as in a , assessed on days 5 8 after infection of recipients with LCMV ( left ) , of P14 per spleen ( right ) . Numbers adjacent to outlined areas ( left ) indicate P14 ( CD45 . 1 + ) . * P ≤ 0 . 0005 ( unpaired t - ) .

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(b) Flow cytometry of splenocytes from recipient mice (n = 6) given adoptive transfer of P14 cells transduced with retrovirus as in a, assessed on days 5 and 8 after infection of recipients with LCMV (left), and quantification of P14 cells per spleen (right). Numbers adjacent to outlined areas (left) indicate percent P14 (CD45.1+) cells. *P ≤ 0.0005 (unpaired t-test).


( c ) Flow cytometry of of P14 transduced with wild - type mCherry - GFP - LC3b retrovirus ( assessed as in Fig . 1b ) in mice treated as in b , assessed at days 5 8 after infection .

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(c) Flow cytometry of the size of P14 cells transduced with wild-type mCherry-GFP-LC3b retrovirus (assessed as in Fig. 1b) in mice treated as in b, assessed at days 5 and 8 after infection.


( d ) Efficiency of retroviral transduction ( left ) expression of GFP mCherry ( right ) in retrovirus - transduced P14 , assessed by flow cytometry gated on CD45 . 1 + P14 ( left ) or CD45 . 1 + Thy - 1 . 1 + P14 ( right ) . Numbers in quadrants indicate CD45 . 1 + Thy - 1 . 1 − ( untransduced ) P14 ( top left ) or CD45 . 1 + Thy - 1 . 1 + ( transduced ) P14 ( top right ; magenta ) ( left ) or in each ( right ) . ( e ) of geometric mean ( gMFI ) of GFP to that of mCherry ( GFP / mCherry ) for in d ; each symbol represents an mouse ; numbers above lines connecting groups indicate difference in . Data are representative of two independent experiments with three or more mice per group ( error bars ( b ) , s . e . m . ) .

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(d) Efficiency of retroviral transduction (left) and expression of GFP and mCherry (right) in retrovirus-transduced P14 cells, assessed by flow cytometry gated on CD45.1+ P14 cells (left) or CD45.1+Thy-1.1+ P14 cells (right). Numbers in quadrants indicate percent CD45.1+Thy-1.1− (untransduced) P14 cells (top left) or CD45.1+Thy-1.1+ (transduced) P14 cells (top right; magenta) (left) or percent cells in each (right). (e) Ratio of the geometric mean fluorescence intensity (gMFI) of GFP to that of mCherry (GFP/mCherry) for the cells in d; each symbol represents an individual mouse; numbers above lines connecting groups indicate difference in ratio. Data are representative of two independent experiments with three or more mice per group (error bars (b), s.e.m.).


( a ) Genomic deletion of Atg7 in H - 2Db - gp33 + T in total activated ( CD44hiCD62Llo ) CD8 + T from Atg7fl / fl mice ( Cre − ) Atg7fl / flGzmb - Cremice ( Cre + ) at day 8 after infection with LCMV Armstrong ; results are presented relative to those of Atg7fl / fl mice , set as 100 % . Each symbol represents an mouse ; small horizontal lines indicate mean ( ± s . e . m . ) .

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(a) Genomic deletion of Atg7 in H-2Db-gp33+ T cells and in total activated (CD44hiCD62Llo) CD8+ T cells from Atg7fl/fl mice (Cre −) and Atg7fl/flGzmb-Cremice (Cre +) at day 8 after infection with LCMV Armstrong strain; results are presented relative to those of Atg7fl/fl mice, set as 100%. Each symbol represents an individual mouse; small horizontal lines indicate the mean (±s.e.m.).


( b ) Immunoblot of Atg7 products of its enzymatic activity ( left margin ) in CD44hiCD62Llo CD8 + T isolated at day 8 after infection as in a ; β - actin serves as a loading control .

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(b) Immunoblot analysis of Atg7 and the products of its enzymatic activity (left margin) in CD44hiCD62Llo CD8+ T cells isolated at day 8 after infection as in a; β-actin serves as a loading control.


( c ) Flow cytometry of T in peripheral blood at day 8 after infection as in a , gated on CD8 + T ( left ) , of H - 2Db - gp33 + T identified at left , identifying terminal effector ( KLRG1hiCD127lo ) memory precursor ( KLRG1loCD127hi ) populations ( right ) . Numbers adjacent to outlined areas ( left ) indicate H - 2Db - gp33 + T ; numbers in quadrants ( right ) indicate in each .

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(c) Flow cytometry of T cells in the peripheral blood at day 8 after infection as in a, gated on CD8+ T cells (left), and of the H-2Db-gp33+ T cells identified at left, identifying terminal effector (KLRG1hiCD127lo) and memory precursor (KLRG1loCD127hi) populations (right). Numbers adjacent to outlined areas (left) indicate percent H-2Db-gp33+ T cells; numbers in quadrants (right) indicate percent cells in each.


( d ) Total antigen - specific CD8 + T in spleen , liver lungs at day 8 after infection as in a .

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(d) Total antigen-specific CD8+ T cells in the spleen, liver and lungs at day 8 after infection as in a.


( e ) Production of IFN - γ TNF in obtained 8 d after infection stimulated ex vivo with various peptides ( above plots ) , gated on total CD8 + T . Numbers in quadrants indicate in each .

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(e) Production of IFN-γ and TNF in cells obtained 8 d after infection and stimulated ex vivo with various peptides (above plots), gated on total CD8+ T cells. Numbers in quadrants indicate percent cells in each.


( f ) Flow cytometry of CD8 + T in peripheral blood of mice at day 15 after infection as in a ( numbers in plots as in c , left ) .

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(f) Flow cytometry of CD8+ T cells in the peripheral blood of mice at day 15 after infection as in a (numbers in plots as in c, left).


( g ) of tetramer - positive in spleens at day 15 after infection as in a .

(g) Quantification of tetramer-positive cells in spleens at day 15 after infection as in a.


( h ) Kinetics of appearance H - 2Db - gp33 + CD8 + T among peripheral blood mononuclear ( PBMCs ) after infection as in a ( left ) ; each represents one mouse . Right , frequency of H - 2Db - gp33 + CD8 + T remaining on day 15 relative to that on day 8 , set as 100 % .

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(h) Kinetics of the appearance H-2Db-gp33+ CD8+ T cells among peripheral blood mononuclear cells (PBMCs) after infection as in a (left); each line represents one mouse. Right, frequency of H-2Db-gp33+ CD8+ T cells remaining on day 15 relative to that on day 8, set as 100%.


( i ) of tetramer - positive at day 30 after infection as in a . Horizontal dashed lines ( g , h ) indicate limit of detection . * P ≤ 0 . 05 , * * P ≤ 0 . 005 * * * P ≤ 0 . 0005 ( unpaired t - ) . Data were pooled from two independent experiments ( a ) or at least two independent experiments ( c - i ) with three to seven mice per group ( error bars , s . e . m . ) or are from one experiment with samples obtained from two independent experiments ( b ) .

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(i) Quantification of tetramer-positive cells at day 30 after infection as in a. Horizontal dashed lines (g,h) indicate the limit of detection. *P ≤ 0.05, **P ≤ 0.005 and ***P ≤ 0.0005 (unpaired t-test). Data were pooled from two independent experiments (a) or at least two independent experiments (c-i) with three to seven mice per group (error bars, s.e.m.) or are from one experiment with samples obtained from two independent experiments (b).


( a ) Flow cytometry of circulating LCMV - specific CD8 + T among peripheral blood mononuclear from Atg5fl / fl Atg5fl / flGzmb - Cremice on days 8 , 15 50 after infection with LCMV Armstrong . Numbers adjacent to outlined areas indicate H - 2Db - gp33 + T , gated on CD8 + T . ( b ) Longitudinal kinetics of appearance of H - 2Db - gp33 + T in peripheral blood of mice as in a ( left ) , H - 2Db - gp33 + CD8 + T remaining on day 15 ( right ) ( presented as in Fig . 4h ) .

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(a) Flow cytometry of circulating LCMV-specific CD8+ T cells among peripheral blood mononuclear cells from Atg5fl/fl and Atg5fl/flGzmb-Cremice on days 8, 15 and 50 after infection with LCMV Armstrong strain. Numbers adjacent to outlined areas indicate percent H-2Db-gp33+ T cells, gated on CD8+ T cells. (b) Longitudinal kinetics of the appearance of H-2Db-gp33+ T cells in the peripheral blood of mice as in a (left), and percent H-2Db-gp33+ CD8+ T cells remaining on day 15 (right) (all presented as in Fig. 4h).


( c ) Flow cytometry of T in tissues on day 279 after infection as in a ( numbers in plots as in a , among total gated CD8 + T ) . ( d ) Total T specific for H - 2Db - gp33 ( left ) or H - 2Db - NP396 ( right ) in spleen , liver lungs of mice as in a . * P ≤ 0 . 05 , * * P ≤ 0 . 005 * * * P ≤ 0 . 0005 ( unpaired t - ) . Data are representative of two independent experiments with three to seven mice per group ( error bars ( d ) , s . e . m . ) .

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

(c) Flow cytometry of T cells in tissues on day 279 after infection as in a (numbers in plots as in a, among total gated CD8+ T cells). (d) Total T cells specific for H-2Db-gp33 (left) or H-2Db-NP396 (right) in the spleen, liver and lungs of mice as in a. *P ≤ 0.05, **P ≤ 0.005 and ***P ≤ 0.0005 (unpaired t-test). Data are representative of two independent experiments with three to seven mice per group (error bars (d), s.e.m.).


( a , b ) Flow cytometry of CD8 + T obtained from chimeras generated ( as in Supplementary Fig . 5a ) by reconstitution of wild - type mice with a mixture of bone marrow from Atg7fl / fl C57BL / 6 wild - type C57BL / 6C57BL / 6 ( Atg7fl / fl vs C57BL / 6 ) or from Atg7fl / flGzmb - Cremice wild - type C57BL / 6mice ( Atg7fl / flGzmb - Cre vs C57BL / 6 ) , gated ( far left ) as populations specific for H - 2Db - gp33 ( a ) or H - 2Db - NP396 ( b ) then assessed , on days 8 , 15 30 after infection of recipients with LCMV Armstrong infection , as CD45 . 2 + ( Atg7fl / fl or Atg7fl / flGzmb - Cre ) donor or CD45 . 1 + ( C57BL / 6 ) donor ( middle ) . Far right , appearance of tetramer - positive T ( key , donor source ) in peripheral blood of chimeras from day 8 to day 30 after infection , presented relative to that at day 8 after infection , set as 100 % .

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 B-CONTROLLED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR 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 B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Flow cytometry of CD8+ T cell obtained from chimeras generated (as in Supplementary Fig. 5a) by reconstitution of wild-type mice with a mixture of bone marrow cells from Atg7fl/fl C57BL/6 and wild-type C57BL/6C57BL/6 (Atg7fl/fl vs C57BL/6) or from Atg7fl/flGzmb-Cremice and wild-type C57BL/6mice and (Atg7fl/flGzmb-Cre vs C57BL/6), gated (far left) as populations specific for H-2Db-gp33 (a) or H-2Db-NP396 (b) and then assessed, on days 8, 15 and 30 after infection of recipients with LCMV Armstrong infection, as CD45.2+ (Atg7fl/fl or Atg7fl/flGzmb-Cre) donor cells or CD45.1+ (C57BL/6) donor cells (middle). Far right, appearance of tetramer-positive T cells (key, donor source) in the peripheral blood of chimeras from day 8 to day 30 after infection, presented relative to that at day 8 after infection, set as 100%.


( c ) Frequency of CD45 . 2 + antigen - specific CD8 + T in spleen , liver lungs on day 30 after infectionwith LCMV Armstrongas in a , b . Data are representative of two independent experiments with three to four mice per group ( error bars , s . e . m . ) .

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

(c) Frequency of CD45.2+ antigen-specific CD8+ T cells in the spleen, liver and lungs on day 30 after infectionwith LCMV Armstrongas in a,b. Data are representative of two independent experiments with three to four mice per group (error bars, s.e.m.).


( a ) Kinetics of appearance of H - 2Db - gp276 + T in peripheral blood of Atg7fl / fl Atg7fl / flGzmb - Cre mice at various times ( horizontal axis ) after infection with LCMV clone 13 .

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

(a) Kinetics of the appearance of H-2Db-gp276+ T cells in the peripheral blood of Atg7fl/fl and Atg7fl/flGzmb-Cre mice at various times (horizontal axis) after infection with LCMV clone 13.


( b ) Total IFN - γ - secreting in spleens obtained from mice at day 8 or day 15 after infection as in a , assessed after 5 h of stimulation ex vivo with various peptides ( horizontal axes ) .

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(b) Total IFN-γ-secreting cells in spleens obtained from mice at day 8 or day 15 after infection as in a, assessed after 5 h of stimulation ex vivo with various peptides (horizontal axes).


( c ) Viral titers in serum of mice at various times ( horizontal axis ) after infection with LCMV clone 13 as in a . Each symbol represents an mouse ; small horizontal lines indicate mean ( ± s . e . m . ) . * P ≤ 0 . 05 , * * P ≤ 0 . 005 * * * P ≤ 0 . 0005 ( unpaired t - ) . Data are representative of two independent experiments with three to five mice per group ( error bars , s . e . m . ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Viral titers in serum of mice at various times (horizontal axis) after infection with LCMV clone 13 as in a. Each symbol represents an individual mouse; small horizontal lines indicate the mean (±s.e.m.). *P ≤ 0.05, **P ≤ 0.005 and ***P ≤ 0.0005 (unpaired t-test). Data are representative of two independent experiments with three to five mice per group (error bars, s.e.m.).


intestine - specific RNAi against irx - 1 reduces Smurf phenotype at day 8 of adulthood . Mean ± SD . Scale bar : 100 μm . Unpaired t - , two - . More than 30 worms analysed in each of three biological replicates .

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

The intestine-specific RNAi against irx-1 reduces the Smurf phenotype at day 8 of adulthood. Mean ± SD. Scale bar: 100 μm. Unpaired t-test, two-tailed. More than 30 worms analysed in each of the three biological replicates.


, C intestine - specific RNAi against irx - 1 reduces Smurf phenotype at day 8 of adulthood . Mean ± SD . Scale bar : 100 μm . Unpaired t - , two - . More than 30 worms analysed in each of three biological replicates .

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

, C The intestine-specific RNAi against irx-1 reduces the Smurf phenotype at day 8 of adulthood. Mean ± SD. Scale bar: 100 μm. Unpaired t-test, two-tailed. More than 30 worms analysed in each of the three biological replicates.


B , C BWM - specific RNAi against myrf - 2 improves sarcomere in worms at day 8 of adulthood . Arrowheads arrows respectively denote gaps of myofilaments disorganised myofilaments with GFP aggregations . Scale Bar : 10 μm . Mean ± SD . Unpaired t - , two - . n > = 50 .

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

B, C The BWM-specific RNAi against myrf-2 improves sarcomere in the worms at day 8 of adulthood. Arrowheads and arrows respectively denote the gaps of myofilaments and the disorganised myofilaments with GFP aggregations. Scale Bar: 10 μm. Mean ± SD. Unpaired t-test, two-tailed. n>=50.


D , E Suppressing myrf - 2 in BWM increases worms ' motility at day 5 of adulthood . tracks were from worms ' movement in 30 sec ( D ) . speed of worm movement was measured by of track versus ( E ) . Mean ± SD . n > = 120 . Unpaired t - , two - .

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

D, E Suppressing myrf-2 in BWM increases worms' motility at day 5 of adulthood. The tracks were from worms' movement in 30 sec (D). The speed of worm movement was measured by the ratio of track length versus body length (E). Mean ± SD. n>=120. Unpaired t-test, two-tailed.


( A ) survival curves of MyD88 flies injected with different doses of A . fumigatus conidia ( c / f : conidia injected per fly ; error bars represent mean ± SD of survival of biological triplicates of 20 flies each ) .

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

(A) survival curves of MyD88 flies injected with different doses of A. fumigatus conidia (c/f: conidia injected per fly; error bars represent mean ± SD of the survival of biological triplicates of 20 flies each).


( B ) fungal loads of single MyD88 mutant wild - type flies ( 50 conidia injected per fly ) .

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(B) fungal loads of single MyD88 mutant and wild-type flies (50 conidia injected per fly).


( C - D ) GFP - labeled A . fumigatus ( 50 conidia per fly ) injected in wild - type ( C ) or MyD88 mutant ( D ) flies form hyphae in thorax of flies ( arrows ) . Scale bars 50 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 B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O

(C-D) GFP-labeled A. fumigatus (50 conidia per fly) injected in wild-type (C) or MyD88 mutant (D) flies form hyphae in the thorax of the flies (arrows). Scale bars 50 μm.


( E ) expression level of Drosomycin induced by different doses of injected A . fumigatus conidia measured by RT - qPCR ; M . luteus ( OD = 10 ) represents positive control ( pooled data of n = 3 experiments , biological replicates ) .

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

(E) expression level of Drosomycin induced by different doses of injected A. fumigatus conidia measured by RT-qPCR; M. luteus (OD=10) represents the positive control (pooled data of n=3 experiments, biological replicates).


Hayan flies are susceptible to A . fumigatus . Survival ( F ) , of Hayan mutant flies ; Hayan 0h vs . 120h , ( error bars represent mean ± SD of survival of biological triplicates of 20 flies each ) .

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

Hayan flies are susceptible to A. fumigatus. Survival (F), of Hayan mutant flies; Hayan 0h vs. 120h, (error bars represent mean ± SD of the survival of biological triplicates of 20 flies each).


time course of fungal loads of single Hayan mutant flies ( 500 conidia per fly ) ( G ) , fungal load upon ( 500 conidia per fly ) ( H ) of Hayan mutant flies ; Hayan 0h vs . 120h , ( error bars represent mean ± SD of survival of biological triplicates of 20 flies each ) .

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time course of fungal loads of single Hayan mutant flies (500 conidia per fly) (G), and fungal load upon death (500 conidia per fly) (H) of Hayan mutant flies; Hayan 0h vs. 120h, (error bars represent mean ± SD of the survival of biological triplicates of 20 flies each).


( A ) survival of MyD88 flies injected with ∆ pptA wild - type ( Af ) A . fumigatus conidia ( error bars represent mean ± SD of survival of biological triplicates of 20 flies each ) .

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(A) survival of MyD88 flies injected with ∆pptA and wild-type (Af) A. fumigatus conidia (error bars represent mean ± SD of the survival of biological triplicates of 20 flies each).


( B ) fungal loads of single flies after injection of 500 ∆ pptA conidia .

(B) fungal loads of single flies after the injection of 500 ∆pptA conidia.


( E ) dose response of MyD88 flies after ∆ gliP ( gliotoxin ) mutant or wild - type [ ∆ akuB ] A . fumigatus infection ; error bars represent mean ± SD of survival of biological triplicates of 20 flies each ; wild - type flies are used as a control for dose of 250 conidia . ( F - H ) dose response of MyD88 wild - type flies after gliotoxin ( F ) , fumagillin ( G ) , helvolic acid ( H ) injection at indicated concentrations ( 20 flies per condition ) .

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

(E) dose response of MyD88 flies after ∆gliP (gliotoxin) mutant or wild-type [∆akuB] A. fumigatus infection; error bars represent mean ± SD of the survival of biological triplicates of 20 flies each; wild-type flies are used as a control for the dose of 250 conidia. (F-H) dose response of MyD88 and wild-type flies after gliotoxin (F), fumagillin (G), and helvolic acid (H) injection at the indicated concentrations (20 flies per condition).


( A ) survival of MyD88 or wild - type flies to 250 injected ∆ aspf1 ( restrictocin mutant ) or wild - type [ ∆ akuB ] A . fumigatus conidia ( 20 flies per condition ) ; MyD88 : ∆ aspf1 vs . ∆ akuB ( * * * p = 0 . 0007 ) . ( B ) survival of MyD88 flies after injection of different concentrations of restrictocin ( R ) ( 20 flies per condition ) . ( C - D ) survival of - treated ( C ) axenic ( D ) MyD88 mutant flies after restrictocin injection ( 20 flies per condition ) .

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

(A) survival of MyD88 or wild-type flies to 250 injected ∆aspf1 (restrictocin mutant) or wild-type [∆akuB] A. fumigatus conidia (20 flies per condition); MyD88: ∆aspf1 vs. ∆akuB (***p=0.0007). (B) survival of MyD88 flies after the injection of different concentrations of restrictocin (R) (20 flies per condition ). (C-D) survival of antibiotics-treated (C) and axenic (D) MyD88 mutant flies after restrictocin injection (20 flies per condition).


( E - F ) ribosomal cleavage measurement after restrictocin or PBS injection in wild - type ( E ) MyD88 ( F ) flies ; arrowheads show position of 28S - derived α - sarcin fragments whereas arrows on right show its electrophoretic band position .

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

(E-F) ribosomal RNA cleavage measurement after restrictocin or PBS injection in wild-type (E) and MyD88 (F) flies; the arrowheads show the position of the 28S RNA-derived α-sarcin fragments whereas arrows on the right show its electrophoretic band position.


( H ) SDS - PAGE of 35S - labelled translated produced in a rabbit reticulocytes lysate from a m7G - capped reporter containing 5 ' UTR of β - globin followed by Renilla luciferase coding ( arrow ) , in presence of increasing concentrations ( 0 . 125 to 25 nM ) of restrictocin .

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(H) SDS-PAGE analysis of 35S-labelled translated proteins produced in a rabbit reticulocytes lysate from a m7G-capped reporter RNA containing the 5'UTR of β-globin followed by the Renilla luciferase coding sequence (arrow), in the presence of increasing concentrations (0.125 to 25 nM) of restrictocin.


( A - B ) survival ( A ) fungal load ( B ) of Bom ∆ 55C ( 55C ) deficient flies compared to wild - type MyD88 after injection of 250 conidia ( error bars represent mean ± SD of survival of biological triplicates of 20 flies each ) ; * * * * p < 0 . 0001 . ( B ) : fungal burden does not increase in Bom ∆ 55C - deficient flies ; wt 0h vs . 48h , * * p = 0 . 001 ; 55C 0h vs . 48h , * * p = 0 . 007 ; pooled data of n = 3 experiments , biological replicates .

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

(A-B) survival (A) and fungal load (B) of Bom∆55C (55C) deficient flies compared to wild-type and MyD88 after injection of 250 conidia (error bars represent mean ± SD of the survival of biological triplicates of 20 flies each); ****p < 0.0001. (B): the fungal burden does not increase in Bom∆55C-deficient flies; wt 0h vs. 48h, **p=0.001; 55C 0h vs. 48h, **p=0.007; pooled data of n=3 experiments, biological replicates.


( C - D ) rescue of sensitivity of Bom ∆ 55C flies to verruculogen ( C ) or to restrictocin ( D ) by transgenic expression of 55C locus genes ( caption in D also applies to ( C ) ) . 55C flies vs . BomS1 , * p = 0 . 0495 , vs . BomS6 * p = 0 . 011 for verruculogen ( C ) ; 55C flies vs . BomBc1 or BomS3 , * * * * p < 0 . 0001 , 55C flies vs . BomS6 , * * p = 0 . 0028 for restrictocin ( D ) ( 20 flies per condition ) .

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

(C-D) rescue of the sensitivity of Bom∆55C flies to verruculogen (C) or to restrictocin (D) by the transgenic expression of individual 55C locus genes (caption in D also applies to (C)). 55C flies vs. BomS1, *p=0.0495, vs. BomS6 *p=0.011 for verruculogen assay (C); 55C flies vs. BomBc1 or BomS3, ****p<0.0001, 55C flies vs. BomS6, **p=0.0028 for restrictocin assay (D) (20 flies per condition).


( E - F ) expression of BomBc1 , BomS3 measured by RT - digital PCR 48 hours after challenge ; BomBc1 PBST vs . Af , * p = 0 . 015 , PBST vs . restrictocin ( R ) , * p = 0 . 015 ; BomS3 : PBST vs . Af , * p = 0 . 02 , PBST vs . R , * p = 0 . 03 ( pooled data of n = 3 experiments , biological replicates ) .

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

(E-F) expression levels of BomBc1, and BomS3 measured by RT-digital PCR 48 hours after challenge; BomBc1 PBST vs. Af, *p=0.015, PBST vs. restrictocin (R), *p=0.015; BomS3: PBST vs. Af, *p=0.02, PBST vs. R, *p=0.03 (pooled data of n=3 experiments, biological replicates).


( A - B ) tremor rate ( A ) survival ( B ) of flies ( 20 flies per condition ) overexpressing Tl [ 10B ] in neurons compared to wild - type after injection of verruculogen . ( A ) Each dot corresponds to tremor rate measured in a batch of 20 flies ; tremor rate wt vs . elav > UAS - Toll10B , * * p = 0 . 002 . ( C - D ) tremor rate ( C ) survival ( D ) of flies ( 20 flies per condition ) overexpressing Tl [ 10B ] in glia compared to wild - type after injection of verruculogen . ( C ) Each dot corresponds to tremor rate measured in a batch of 20 flies ; tremor rate wt vs . repo > UAS - Toll10B , * * p = 0 . 002 . ( D ) Survival wt V vs . repo > UAS - Toll10B V , * * p = 0 . 005 .

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

(A-B) tremor rate (A) and survival (B) of flies (20 flies per condition) overexpressing Tl[10B] in neurons compared to wild-type after injection of verruculogen. (A) Each dot corresponds to the tremor rate measured in a batch of 20 flies; tremor rate wt vs. elav> UAS-Toll10B, **p=0.002. (C-D) tremor rate (C) and survival (D) of flies (20 flies per condition) overexpressing Tl[10B] in glia compared to wild-type after injection of verruculogen. (C) Each dot corresponds to the tremor rate measured in a batch of 20 flies; tremor rate wt vs. repo> UAS-Toll10B, **p=0.002. (D) Survival wt V vs. repo> UAS-Toll10B V, **p=0.005.


( E - G ) recovery time from tremor ( E , F ) survival ( G ) of single flies overexpressing BomS6 ubiquitously ( E , G ) or in neurons ( F - G ) ( biological replicates ) compared to wild - type after injection of verruculogen ; in ( G ) inset represents survival of vehicle control groups .

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(E-G) recovery time from tremor (E,F) and survival (G) of single flies overexpressing BomS6 ubiquitously (E,G) or in neurons (F-G) (biological replicates) compared to wild-type after injection of verruculogen; in (G) the inset represents the survival of vehicle control groups.


( H - I ) recovery time from tremor ( H ) survival ( I ) of single flies overexpressing BomS6 in glia ( pooled data from n = 3 experiments , biological replicates ) compared to wild - type after injection of verruculogen ; in ( I ) inset represents survival of vehicle control groups . Recovery time ( H ) repo > mCherry vs . repo > BomS6 , p = 0 . 058 , survival ( I ) repo > mCherry V vs . repo > BomS6 V , * p = 0 . 016 .

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

(H-I) recovery time from tremor (H) and survival (I) of single flies overexpressing BomS6 in glia (pooled data from n=3 experiments, biological replicates) compared to wild-type after injection of verruculogen; in (I) the inset represents the survival of vehicle control groups. Recovery time (H) repo>mCherry vs. repo>BomS6, p=0.058, survival (I) repo>mCherry V vs. repo>BomS6 V, *p=0.016.