Expression data dot plots of genes found differentially regulated in cluster C1 between day 23 BALO with ( C1 + ) without ( C1 - ) microinjected TR - Mac . circle illustrates number of expressing a specific gene

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Expression data dot plots of genes found differentially regulated in cluster C1 between day 23 BALO with (C1+) and without (C1-) microinjected TR-Mac. The circle size illustrates the number of cells expressing a specific gene


of terminally differentiated ( AEC I ciliated ) in day 23 BALO cultures with ( + ) without ( - ) microinjected TR ‑ Mac

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Percentage of terminally differentiated cells (AEC I and ciliated cells) in day 23 BALO cultures with (+) and without (-) microinjected TR‑Mac


Representative of E11 . 5 lung explants after treatment for 48 h with 100 μM Scra or mo142 - 3p reveals reduced branching morphogenesis after miR142 - 3p knockdown .

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Representative images of E11.5 lung explants after treatment for 48 h with 100 μM Scra or mo142-3p reveals reduced size and branching morphogenesis after miR142-3p knockdown.


Organoid diameter ( n = 30 - 50 per group ) in µm before addition of 4 μM Scra or mo142 - 3p at day 6 ( control ) 5 days after treatment ( at day 11 BALO culture ) in n = 3 biological

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Organoid diameter (n=30-50 per group) in µm before addition of 4 μM Scra or mo142-3p at day 6 (control) and 5 days after treatment (at day 11 BALO culture) in n=3 biological replicates


Representative of β - galactosidase in TOPGAL epithelium ( C ) BALO cultures before ( day 6 , control ) or 5 days after treatment with either 4 μM Scra or mo142 - 3p ( day 11 of co - culture ) . BASC rMC were isolated form lung homogenate of TOPGAL mice . β - galactosidase + rMC at day 11 of culture are indicated with arrows .

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

Representative images of β-galactosidase staining in TOPGAL epithelium (C) BALO cultures before (day 6, control) or 5 days after treatment with either 4 μM Scra or mo142-3p (day 11 of co-culture). BASC and rMC were isolated form the lung homogenate of TOPGAL mice. β-galactosidase+ rMC at day 11 of culture are indicated with arrows.


Representative of β - galactosidase in TOPGAL rMC ( D ) BALO cultures before ( day 6 , control ) or 5 days after treatment with either 4 μM Scra or mo142 - 3p ( day 11 of co - culture ) . BASC rMC were isolated form lung homogenate of TOPGAL mice . β - galactosidase + rMC at day 11 of culture are indicated with arrows .

Representative images of β-galactosidase staining in TOPGAL rMC (D) BALO cultures before (day 6, control) or 5 days after treatment with either 4 μM Scra or mo142-3p (day 11 of co-culture). BASC and rMC were isolated form the lung homogenate of TOPGAL mice. β-galactosidase+ rMC at day 11 of culture are indicated with arrows.


Representative transmission confocal after LysoTracker indicating branching number of branching points in n = 4 BALO 15 days after treatment with 4 μM Scra or mo142 - 3p ( day 21 of co - culture ) in n = 3 biological replicates .

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Representative transmission and confocal images after LysoTracker staining indicating branching and number of branching points in n=4 BALO 15 days after treatment with 4 μM Scra or mo142-3p (day 21 of co-culture) in n=3 biological replicates.


mRNA of epithelial mesenchymal miR142 - 3p expression in Scra mo142 - 3p - treated organoids 5 days after treatment ( n = 3 - 4 biological replicates with pooled from 4 cultures per replicate ) .

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mRNA levels of epithelial and mesenchymal miR142-3p expression in Scra and mo142-3p-treated organoids 5 days after treatment (n=3-4 biological replicates with pooled cells from 4 cultures per replicate).


Representative of day 21 BALO after microinjection of SC35M - GFP IAV ( green ) into central airway - like ( * ) after 0 12 h pi visualizes IVA spread to alveolar - like regions . Dotted lines illustrate BALO - borders . Scale bars represent 100 μm

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Representative images of day 21 BALO after microinjection of SC35M-GFP IAV (green) into the central airway-like (*) after 0 and 12 h pi visualizes IVA spread to the alveolar-like regions. Dotted lines illustrate BALO-borders. Scale bars represent 100 μm


of plaque forming units in supernatants from mock or SC35M IAV infected BALO 48 h after infection ( n = 5 biological replicates ) . N . D . : not - detectable

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Quantification of plaque forming units in supernatants from mock or SC35M IAV infected BALO 48 h after infection (n=5 biological replicates). N.D.: not-detectable


Relative NP expression in PR8 IAV infected ( HA + ) or non - infected ( HA - ) epithelial isolated from BALO 48 h pi , FACS - sorted according to EpCAM HA expression ( n = 3 biological replicates with pooled from 4 cultures per replicate )

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Relative NP expression in PR8 IAV infected (HA+) or non-infected (HA-) epithelial cells isolated from BALO 48 h pi, FACS-sorted according to EpCAM and HA expression (n=3 biological replicates with pooled cells from 4 cultures per replicate)


Representative flow cytometric data showing of EpCAM + NP + in mock - SC35M IAV ‑ infected BALO at 48 h pi in n = 3 biological replicates with pooled from 4 cultures per replicate

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Representative flow cytometric data showing the percentage of EpCAM+NP+ cells in mock- and SC35M IAV‑infected BALO at 48 h pi in n=3 biological replicates with pooled cells from 4 cultures per replicate


Representative of a day 21 distal region in BALO generated from mTmG reporter mouse infected by SC35M - Cre IAV after 0 , 10 , 14 25 h . Arrows indicate . Scale bars represent 25 μm 10 μm in insert

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Representative fluorescence images of a day 21 distal region in BALO generated from mTmG reporter mouse and infected by SC35M-Cre IAV after 0, 10, 14 and 25 h. Arrows indicate cell death. Scale bars represent 25 μm and 10 μm in the insert


mRNA expression of Ifnb in mock PR8 IAV - infected BALO at 48 h pi ( n = 3 biological replicates with pooled from 4 cultures per replicate )

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mRNA expression of Ifnb in mock and PR8 IAV-infected BALO at 48 h pi (n=3 biological replicates with pooled cells from 4 cultures per replicate)


Release of TNFα ( 6 h pi ) , IL - 6 ( 48 h pi ) , IL - 1β ( 48 h pi ) detected by Bio - Plex ® Multiplex Immunoassay in supernatant of mock IAV infected BALO cultures with without TR - Mac ( n = 3 biological replicates )

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Release of TNFα (6 h pi), IL-6 (48 h pi), IL-1β (48 h pi) detected by Bio-Plex® Multiplex Immunoassay in the supernatant of mock and IAV infected BALO cultures with and without TR-Mac (n=3 biological replicates)


Representative sections of WT MPSIIIA brains at 9 months of stained for astrocytes ( GFAP ) or microglia ( isolectin B4 , ILB4 ) . correspond to high power views covering cortical layers II - V . Scale bar , 100 μm . ( n = 4 mice per group ) .

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Representative sections of WT and MPSIIIA brains at 9 months of age stained for astrocytes (GFAP) or microglia (isolectin B4, ILB4). Images correspond to high power views covering cortical layers II-V. Scale bar, 100 μm. (n=4 mice per group).


Quantitative PCR for various inflammatory cytokines in whole brains from 9 month old WT MPSIIIA mice . Symbols above bars are versus WT . ( n = 6 mice per group ) . Data are expressed as mean ± STDEV , data for each cytokine were tested by unpaired t - ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . Symbols above bars are versus WT .

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Quantitative PCR for various inflammatory cytokines in whole brains from 9 month old WT and MPSIIIA mice. Symbols above bars are versus WT. (n=6 mice per group). Data are expressed as mean ± STDEV, data for each cytokine were tested by unpaired t-test; *P<0.05, **P<0.01, ***P<0.001. Symbols above bars are versus WT.


of IL - 1β IL - 1Ra in plasma from human healthy control MPSIII ( n = 6 control participants n = 21 MPSIII ) . Data are expressed as mean ± STDEV were tested by unpaired t - . C . IL - 1β , control vs . MPSIII P = 0 . 0407 ; D . IL - 1Ra , control vs . MPSIII P = 0 . 0001 . of IL - 1β IL - 1Ra in cerebrospinal fluid ( CSF ) from control MPSIII ( n = 3 control participants n = 21 MPSIII ) . Data are expressed as mean ± STDEV were tested by unpaired t - ; * P < 0 . 05 . F . IL - 1Ra , control vs . MPSIII P = 0 . 0167 .

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

Quantification of IL-1β and IL-1Ra levels in plasma from human healthy control and MPSIII patients (n=6 control participants and n=21 MPSIII patients). Data are expressed as mean ± STDEV and were tested by unpaired t-test. C. IL-1β, control vs. MPSIII P=0.0407; D. IL-1Ra, control vs. MPSIII P=0.0001. Quantification of IL-1β and IL-1Ra levels in cerebrospinal fluid (CSF) from control and MPSIII patients (n=3 control participants and n=21 MPSIII patients). Data are expressed as mean ± STDEV and were tested by unpaired t-test; *P<0.05. F. IL-1Ra, control vs. MPSIII P=0.0167.


expression of Il1b was measured in whole brain using quantitative PCR 1 hour , 2 hours 6 hours after I . P . challenge ( n = 6 mice per group ) . Data are expressed as mean ± STDEV were tested by two - way ANOVA with Bonferroni ' s post - ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . Symbols above bars are versus PBS at relevant time - point .

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The expression of Il1b was measured in whole brain using quantitative PCR 1 hour, 2 hours and 6 hours after I.P. challenge (n=6 mice per group). Data are expressed as mean ± STDEV and were tested by two-way ANOVA with Bonferroni's post-test; *P<0.05, **P<0.01, ***P<0.001. Symbols above bars are versus PBS at the relevant time-point.


of intracellular production of IL - 1β ( n = 3 independent experiments each with 3 inter - experimental replicates ) . Data are expressed as mean ± STDEV were tested by one - way ANOVA with Tukey ' s post - ; * * * P < 0 . 001 . Symbols above bars are versus PBS . of IL - 1β secreted into media ( n = 3 independent experiments each with 3 inter - experimental replicates ) . A horizontal dotted represents limit of detection of ELISA . Data are expressed as mean ± STDEV were tested by one - way ANOVA with Tukey ' s post - ; * * P < 0 . 01 , * * * P < 0 . 001 . Symbols above bars are versus PBS .

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

Quantification of intracellular production of IL-1β (n=3 independent experiments each with 3 inter-experimental replicates). Data are expressed as mean ± STDEV and were tested by one-way ANOVA with Tukey's post-test; ***P<0.001. Symbols above bars are versus PBS. Quantification of IL-1β secreted into the media (n=3 independent experiments each with 3 inter-experimental replicates). A horizontal dotted line represents the limit of detection of the ELISA. Data are expressed as mean ± STDEV and were tested by one-way ANOVA with Tukey's post-test; **P<0.01, ***P<0.001. Symbols above bars are versus PBS.


WT mixed glial cultures were co - treated with MPSIIIA GAG ( 4 μg / ml ) / or TLR4 inhibitor CLI - 095 ( 1 μg / ml ) for 24 hours . intracellular production of IL - 1β was measured ( n = 3 independent experiments each with 3 inter - experimental replicates ) . Data are expressed as mean ± STDEV were tested by one - way ANOVA with Tukey ' s post - ; * * * P < 0 . 001 . Symbols above bars are versus MPSIIIA GAG + vehicle .

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WT mixed glial cultures were co-treated with MPSIIIA GAG (4 μg/ml) and/or the TLR4 inhibitor CLI-095 (1 μg/ml) for 24 hours. The intracellular production of IL-1β was measured (n=3 independent experiments each with 3 inter-experimental replicates). Data are expressed as mean ± STDEV and were tested by one-way ANOVA with Tukey's post-test; ***P<0.001. Symbols above bars are versus MPSIIIA GAG + vehicle.


Multi - heparinase ( HepM ) / or chondroitinase ABC ( cABC ) digests were performed on MPSIIIA GAG , resulting oligosaccharides applied to a WT mixed glial culture for 24 hours alongside heparin , bovine HS ( bHS ) , porcine DS ( pDS ) WT GAG . intracellular production of IL - 1β was measured ( n = 3 independent experiments each with 3 inter - experimental replicates ) . Data are expressed as mean ± STDEV were tested by one - way ANOVA with Tukey ' s post - ; * P < 0 . 05 , * * * P < 0 . 001 . Symbols above bars are versus MPSIIIA GAG alone .

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Multi-heparinase (HepM) and/or chondroitinase ABC (cABC) digests were performed on MPSIIIA GAG, and the resulting oligosaccharides applied to a WT mixed glial culture for 24 hours alongside heparin, bovine HS (bHS), porcine DS (pDS) and WT GAG. The intracellular production of IL-1β was measured (n=3 independent experiments each with 3 inter-experimental replicates). Data are expressed as mean ± STDEV and were tested by one-way ANOVA with Tukey's post-test; *P<0.05, ***P<0.001. Symbols above bars are versus MPSIIIA GAG alone.


MPSIIIA GAG was 2 - O - desulphated ( hashed bars ) utilising 120 mM NaOH , compositional HS disaccharide performed via RP - HPLC against untreated MPSIIIA GAG ( black bars ) to validate complete 2 - O - desulphation . contribution of each modification were determined ( n = 3 GAG samples ) . Data are expressed as mean ± STDEV were tested by two - way ANOVA with Bonferroni ' s post - ; 2S MPSIIIA GAG vs . 2S 2 - ODS MPSIIIA GAG P < 0 . 0001 . NS , N - sulphated glucosamine ; 2S , 2 - O - sulphate group ; 6S , 6 - O sulphate group .

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MPSIIIA GAG was 2-O-desulphated (hashed bars) utilising 120 mM NaOH, and compositional HS disaccharide analysis performed via RP-HPLC against untreated MPSIIIA GAG (black bars) to validate complete 2-O-desulphation. Percentage contribution of each modification were determined (n=3 GAG samples). Data are expressed as mean ± STDEV and were tested by two-way ANOVA with Bonferroni's post-test; 2S MPSIIIA GAG vs. 2S 2-ODS MPSIIIA GAG P<0.0001. NS, N-sulphated glucosamine; 2S, 2-O-sulphate group; 6S, 6-O sulphate group.


MPSIIIA GAG was 2 - O - desulphated resulting oligosaccharides applied to a WT mixed glial culture for 24 hours . intracellular production of IL - 1β was measured ( n = 3 independent experiments each with 3 inter - experimental replicates ) . Data are expressed as mean ± STDEV were tested by one - way ANOVA with Tukey ' s post - ; MPSIIIA GAG vs . 2ODS MPSIIIA GAG P < 0 . 0001 . 2ODS , 2 - O - desulphated .

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MPSIIIA GAG was 2-O-desulphated and the resulting oligosaccharides applied to a WT mixed glial culture for 24 hours. The intracellular production of IL-1β was measured (n=3 independent experiments each with 3 inter-experimental replicates). Data are expressed as mean ± STDEV and were tested by one-way ANOVA with Tukey's post-test; MPSIIIA GAG vs. 2ODS MPSIIIA GAG P<0.0001. 2ODS, 2-O-desulphated.


Representative sections from 9 month old WT MPSIIIA brains showing secondary storage of GM2 ganglioside , cholesterol , amyloid beta peptide 1 - 40 1 - 42 within cortical layers II to V / VI , x20 , scale bar : 50 μm . Inserts , 100x , scale bar : 10μm . ( n = 4 mice per group )

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Representative sections from 9 month old WT and MPSIIIA brains showing secondary storage of GM2 ganglioside, cholesterol, protein aggregates and amyloid beta peptide 1-40 and 1-42 within cortical layers II to V/VI, x20, scale bar: 50 μm. Inserts, 100x, scale bar: 10μm. (n=4 mice per group)


ATP in whole brain homogenate from 9 month old WT MPSIIIA mice ( n = 6 mice per group ) . Data are expressed as mean ± STDEV were tested by unpaired t - . WT vs . MPSIIIA P < 0 .

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ATP levels in whole brain homogenate from 9 month old WT and MPSIIIA mice (n=6 mice per group). Data are expressed as mean ± STDEV and were tested by unpaired t-test. WT vs. MPSIIIA P<0.02


Brain transcription of mRNA species for various inflammasome components ( n = 6 mice per group ) . Data are expressed as mean ± STDEV , data for each cytokine were tested by unpaired t - ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . Symbols above bars are versus

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Brain transcription of mRNA species for various inflammasome components (n=6 mice per group). Data are expressed as mean ± STDEV, data for each cytokine were tested by unpaired t-test; *P<0.05, **P<0.01, ***P<0.001. Symbols above bars are versus WT


Caspase 1 activity in whole brain homogenate from WT MPSIIIA mice ( n = 6 mice per group ) . Data are expressed as mean ± STDEV were tested by unpaired t - . WT vs . MPSIIIA P < 0 .

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Caspase 1 activity levels in whole brain homogenate from WT and MPSIIIA mice (n=6 mice per group). Data are expressed as mean ± STDEV and were tested by unpaired t-test. WT vs. MPSIIIA P<0.0001


WT ( E - G ) Nlrp3 - / - ( H - J ) mixed glia were primed with MPSIIIA GAG or LPS . ATP ( 5 mM ) , cholesterol crystals ( 1 mg / ml ) or amyloid - β fibrils ( Aβ1 - 40 4 . 3 μM Aβ1 - 42 5 μM ) were added 4 hours post - priming stimulus , secretion of IL - 1β into media measured via ELISA after a further 20 hours ( n = 3 independent experiments each with 3 inter - experimental replicates ) . Data are expressed as mean ± STDEV were tested by one - way ANOVA with Tukey ' s post - ; * P < 0 . 05 , * * * P < 0 .

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WT (E-G) and Nlrp3-/- (H-J) mixed glia were primed with MPSIIIA GAG or LPS. ATP (5 mM), cholesterol crystals (1 mg/ml) or amyloid-β fibrils (Aβ1-40 4.3 μM and Aβ1-42 5 μM) were added 4 hours post-priming stimulus, and the secretion of IL-1β into the media measured via ELISA after a further 20 hours (n=3 independent experiments each with 3 inter-experimental replicates). Data are expressed as mean ± STDEV and were tested by one-way ANOVA with Tukey's post-test; *P<0.05, ***P<0.001


biological activity of LV - mediated IL - 1Ra was assessed in vitro . RAW264 . 7 macrophages were transduced with LV . IL1RN or LV . GFP vectors at a MOI of 10 . After 72 hours were stimulated with recombinant hIL - 1β expression of TNF - α assessed after a further 48 hours . ( n = 3 independent experiments each with 3 inter - experimental replicates ) . Data are expressed as mean ± STDEV were tested by two - way ANOVA with Bonferroni ' s post - ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . Symbols above bars are versus NTC within a treatment group .

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

The biological activity of LV-mediated IL-1Ra was assessed in vitro. RAW264.7 macrophages were transduced with LV.IL1RN or LV.GFP vectors at a MOI of 10. After 72 hours cells were stimulated with recombinant hIL-1β and the expression of TNF-α assessed after a further 48 hours. (n=3 independent experiments each with 3 inter-experimental replicates). Data are expressed as mean ± STDEV and were tested by two-way ANOVA with Bonferroni's post-test; *P<0.05, **P<0.01, ***P<0.001. Symbols above bars are versus NTC within a treatment group.


A portion of transduced HSCs were plated into CFU assays , incubated for 12 days CFU colonies analysed . Vector copy number were quantified ( n = 6 CFU assays ) .

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A portion of transduced HSCs were plated into CFU assays, incubated for 12 days and CFU colonies analysed. Vector copy number were quantified (n=6 CFU assays).


A portion of transduced HSCs were plated into CFU assays , incubated for 12 days CFU colonies analysed . production of secreted human IL - 1Ra were quantified ( n = 6 CFU assays ) .

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A portion of transduced HSCs were plated into CFU assays, incubated for 12 days and CFU colonies analysed. protein production of secreted human IL-1Ra were quantified (n=6 CFU assays).


A portion of transduced HSCs were plated into CFU assays , incubated for 12 days CFU colonies analysed . intracellular human IL - 1Ra were quantified ( n = 6 CFU assays ) .

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A portion of transduced HSCs were plated into CFU assays, incubated for 12 days and CFU colonies analysed. intracellular human IL-1Ra were quantified (n=6 CFU assays).


Donor chimerism was determined by flow cytometry at 4 months of ( 2 months post - transplant ) ( n = 10 mice ) .

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Donor chimerism was determined by flow cytometry at 4 months of age (2 months post-transplant) (n=10 mice).


on peripheral blood composition was determined by flow cytometry at 4 months of ( 2 months post - transplant ) ( n = 10 mice ) .

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based on peripheral blood composition was determined by flow cytometry at 4 months of age (2 months post-transplant) (n=10 mice).


Human IL - 1Ra expression was quantified in plasma of mice at 6 months of ( 4 months post - transplant ) ( n = 10 mice per group ) . Data are expressed as mean ± STDEV were tested by one - way ANOVA with Tukey ' s post - ; WT vs . LV . IL1RN P < 0 . 0001 ; MPSIIIA vs . LV . IL1RN P < 0 . 0001 . Symbols above bars are versus WT .

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 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

Human IL-1Ra expression was quantified in the plasma of mice at 6 months of age (4 months post-transplant) (n=10 mice per group). Data are expressed as mean ± STDEV and were tested by one-way ANOVA with Tukey's post-test; WT vs. LV.IL1RN P<0.0001; MPSIIIA vs. LV.IL1RN P<0.0001. Symbols above bars are versus WT.


Vector copy number was evaluated in brain , spleen , liver , blood ( WBC ) bone marrow ( BM ) of mice at 6 months of ( 4 months post - transplant ) ( n = 10 mice per group ) .

Vector copy number was evaluated in brain, spleen, liver, white blood cells (WBC) and bone marrow (BM) of mice at 6 months of age (4 months post-transplant) (n=10 mice per group).


of human IL - 1Ra in brain of transplanted MPSIIIA mice ( n = 10 mice per group ) . Data are expressed as mean ± STDEV were tested by one - way ANOVA with Tukey ' s post - ; WT vs . LV . IL1RN P < 0 . 0001 ; MPSIIIA vs . LV . IL1RN P < 0 . 0001 . Symbols above bars are versus WT .

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Quantification of human IL-1Ra levels in the brain of transplanted MPSIIIA mice (n=10 mice per group). Data are expressed as mean ± STDEV and were tested by one-way ANOVA with Tukey's post-test; WT vs. LV.IL1RN P<0.0001; MPSIIIA vs. LV.IL1RN P<0.0001. Symbols above bars are versus WT.


Representative sections from 6 - month old MPSIIIA LV . IL1RN transplanted MPSIIIA mice . Sections show CA3 region of hippocampus . expressing high of human IL - 1Ra are indicated with black arrows . 40x , scale bar , 50 μm ( n = 4 mice per group ) .

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Representative sections from 6-month old MPSIIIA and LV.IL1RN transplanted MPSIIIA mice. Sections show the CA3 region of the hippocampus. Cells expressing high levels of human IL-1Ra are indicated with black arrows. 40x, scale bar, 50 μm (n=4 mice per group).


Mice were placed in centre of Y - maze allowed to explore freely for 10 minutes . of spontaneous alternations in Y - maze were measured to assess working memory ( n = 10 mice per group ) . Data are expressed as mean ± STDEV were tested by one - way ANOVA with Tukey ' s post - ; * P < 0 . 05 , * * P < 0 .

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Mice were placed in the centre of the Y-maze and allowed to explore freely for 10 minutes. The percentage of spontaneous alternations in the Y-maze were measured to assess working memory (n=10 mice per group). Data are expressed as mean ± STDEV and were tested by one-way ANOVA with Tukey's post-test; *P<0.05, **P<0.01


A one - hour open field was performed path measured as an indicator of hyperactivity ( n = 10 mice per group ) . Data are expressed as mean ± STDEV were tested by one - way ANOVA with Tukey ' s post - ; * * * P < 0 . 001 . Symbols above bars are versus

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A one-hour open field test was performed and path length measured as an indicator of hyperactivity (n=10 mice per group). Data are expressed as mean ± STDEV and were tested by one-way ANOVA with Tukey's post-test; ***P<0.001. Symbols above bars are versus WT


Representative of hippocampus cortex ( layers II - V ) from control treated mice stained with anti - mouse glial fibrillary acidic ( GFAP ; red ) / DAPI ( , blue ) to identify activated astrocytes , 20x , scale bar : 50μm Representative of hippocampus cortex ( layers II - V ) from control treated mice stained with isolectin B4 ( ILB4 ) to identify activated microglia , 20x , scale bar :

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Representative images of the hippocampus and cortex (layers II-V) from control and treated mice stained with anti-mouse glial fibrillary acidic protein (GFAP; red)/DAPI (nuclear, blue) to identify activated astrocytes, 20x, scale bar: 50μm Representative images of the hippocampus and cortex (layers II-V) from control and treated mice stained with isolectin B4 (ILB4) to identify activated microglia, 20x, scale bar: 50μ


average number of GFAP positive astrocyte over three fields of view per mouse of hippocampus ( C ) cortex ( D ) were counted ( n = 4 mice per group , average of 3 fields of view per mouse ) . Data are expressed as mean ± STDEV were tested by one - way ANOVA with Tukey ' s post - ; * * P < 0 . 01 , * * * P < 0 . 001 . Symbols above bars are versus

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

The average number of GFAP positive astrocyte cells over three fields of view per mouse of the hippocampus (C) and the cortex (D) were counted (n=4 mice per group, average of 3 fields of view per mouse). Data are expressed as mean ± STDEV and were tested by one-way ANOVA with Tukey's post-test; **P<0.01, ***P<0.001. Symbols above bars are versus WT


average number of ILB4 + positive microglial over three fields of view per mouse of hippocampus ( C ) cortex ( D ) were counted ( n = 4 mice per group , average of 3 fields of view per mouse ) . Data are expressed as mean ± STDEV were tested by one - way ANOVA with Tukey ' s post - ; * * * P < 0 . 001 . Symbols above bars are versus

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

The average number of ILB4+ positive microglial cells over three fields of view per mouse of the hippocampus (C) and the cortex (D) were counted (n=4 mice per group, average of 3 fields of view per mouse). Data are expressed as mean ± STDEV and were tested by one-way ANOVA with Tukey's post-test; ***P<0.001. Symbols above bars are versus WT


Colocalization of AcGFP - Siwi , AcGFP - BmAgo3 mCherry - BmVasa in BmN4 . scans ( in enlarged region ) show that Siwi - D670A mutant but not BmAgo3 - D697A mutant dissociates from BmVasa foci . scans of were normalized to highest value depicted at right panel . Scale bar , 8 μm .

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Colocalization of AcGFP-Siwi, AcGFP-BmAgo3 and mCherry-BmVasa in BmN4 cells. Line scans (white line in the enlarged region) show that Siwi-D670A mutant but not BmAgo3-D697A mutant dissociates from BmVasa foci. Line scans of fluorescence intensity were normalized to the highest value and depicted at the right panel. Scale bar, 8 μm.


Colocalization of AcGFP - Siwi mCherry - BmDcp2 in BmN4 . scans ( in enlarged region ) show that Siwi - D670A mutant but not BmAgo3 - D697A mutant overlaps with BmDcp2 foci . scans of were normalized to highest value depicted at right panel . Scale bar , 8 μm .

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Colocalization of AcGFP-Siwi and mCherry-BmDcp2 in BmN4 cells. Line scans (white line in the enlarged region) show that Siwi-D670A mutant but not BmAgo3-D697A mutant overlaps with BmDcp2 foci. Line scans of fluorescence intensity were normalized to the highest value and depicted at the right panel. Scale bar, 8 μm.


( Left ) Effect of 1 , 6 - Hexanediol treatment on AcGFP - Siwi AcGFP - Siwi - D670A foci . Z - stacks were taken prior to addition of 1 , 6 - Hexanediol or medium after 30 minutes incubation at RT . Z - projections of middle 6 μm stacks are then normalized pseudo - colored with Fire LUT to visualize Siwi foci . Scale bar 10 μm . Scale bars represent pixel in arbitrary units ( A . U . ) ( Right ) High granule / of Siwi - D670A persists despite 1 , 6 - Hexanediol treatment . Average from each of independent Z - stacks ( n = 30 per set ) is quantified ( see Materials Methods ) depicted as box plots . Representative data from N = 3 independent experiments are shown . P - values were calculated by Asymptotic Wilcoxon rank sum .

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

(Left) Effect of 1,6-Hexanediol treatment on AcGFP-Siwi and AcGFP-Siwi-D670A foci. Z-stacks were taken prior to the addition of 1,6-Hexanediol or medium and after 30 minutes incubation at RT. Z-projections of the middle 6 μm stacks are then normalized and pseudo-colored with Fire LUT to visualize the Siwi foci. Scale bar 10 μm. Scale bars represent pixel intensity in arbitrary units (A.U.) (Right) High granule/cell intensity ratio of Siwi-D670A persists despite 1,6-Hexanediol treatment. Average intensity ratio from each of the independent Z-stacks (n=30 per set) is quantified (see Materials and Methods) and depicted as box plots. Representative data from N = 3 independent experiments are shown. P-values were calculated by Asymptotic Wilcoxon rank sum test.


Co - expression of Siwi - D670A increases granule - to - of BmSpnE BmQin foci . ( Upper ) Representative Z - projections ( maximum ) of mCherry - BmSpnE mCherry - BmQin foci . AcGFP signal from FITC channel is not shown . Scale bar 8 μm . ( Bottom ) Average from each of independent Z - stacks ( n = 6 per set ) is quantified ( see Materials Methods ) depicted as box plots . Representative data from N ≥ 3 independent experiments are shown . P - values were calculated by Asymptotic Wilcoxon rank sum .

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

Co-expression of Siwi-D670A increases granule-to-cell intensity ratio of BmSpnE and BmQin foci. (Upper) Representative Z-projections (maximum intensity) of mCherry-BmSpnE and mCherry-BmQin foci. AcGFP signal from FITC channel is not shown. Scale bar 8 μm. (Bottom) Average intensity ratio from each of the independent Z-stacks (n=6 per set) is quantified (see Materials and Methods) and depicted as box plots. Representative data from N ≥ 3 independent experiments are shown. P-values were calculated by Asymptotic Wilcoxon rank sum test.


Depletion of BmSpnE or BmQin results in segregation of Siwi - D670A from BmDcp2 - containing P - bodies . ( Top - left ) Representative Z - projections ( Maximum ) . Scale bar 8 μm . ( Top - right ) Enlarged from box . Scale bar 2 μm . ( Bottom ) Box plot showing that depletion of BmQin ( n = 32 ) or BmSpnE ( n = 32 ) reduces colocalization between Siwi - D670A BmDcp2 , compared to control ( dsRLuc , n = 32 ) . Representative data from N = 3 independent experiments are shown . Bonferroni - corrected P - values were calculated by Asymptotic Wilcoxon rank sum . See also Figure EV3 .

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Depletion of BmSpnE or BmQin results in segregation of Siwi-D670A from BmDcp2-containing P-bodies. (Top-left) Representative Z-projections (Maximum intensity). Scale bar 8 μm. (Top-right) Enlarged area from the white box. Scale bar 2 μm. (Bottom) Box plot showing that depletion of BmQin (n=32 cells) or BmSpnE (n=32 cells) reduces colocalization ratio between Siwi-D670A and BmDcp2, compared to control (dsRLuc, n=32 cells). Representative data from N = 3 independent experiments are shown. Bonferroni-corrected P-values were calculated by Asymptotic Wilcoxon rank sum test. See also Figure EV3.


Depletion of Siwi results in segregation of BmQin from BmDcp2 - containing P - bodies . Box plot showing that depletion of Siwi ( dsSiwi , n = 47 ) reduces colocalization between BmQin BmDcp2 , compared to control ( dsRLuc , n = 55 ) . Representative data from N = 4 independent experiments are shown . Depletion of Siwi results in segregation of BmSpnE from BmDcp2 - containing P - bodies . Box plot showing that depletion of Siwi ( dsSiwi , n = 55 ) reduces colocalization between BmSpnE BmDcp2 , compared to control ( dsRLuc , n = 55 ) . Representative data from N = 6 independent experiments are shown . Depletion of Siwi results in colocalization of BmSpnE BmVasa . Box plot showing that depletion of Siwi ( dsSiwi , n = 18 ) significantly up - regulates colocalization between BmSpnE BmVasa , compared to control ( dsRLuc , n = 18 ) . Representative data from N = 3 independent experiments are shown . Depletion of BmVasa results in segregation of Siwi from BmAgo3 foci . Box plot showing that depletion of BmVasa ( dsBmVasa , n = 12 ) down - regulates colocalization between Siwi BmAgo3 , compared to control ( dsRLuc , n = 12 ) . Data points collected from N = 2 independent experiments are shown ( statistical is not performed for N < 3 data ) . Depletion of BmVasa results in entanglement of Siwi at piP - bodies . Interaction interfaces are indicated by arrows . Box plot showing that depletion of BmVasa ( dsBmVasa , n = 45 ) mildly up - regulates colocalization between Siwi BmDcp2 , compared to control ( dsRLuc , n = 49 ) . Representative data from N = 5 independent experiments are shown .

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

Depletion of Siwi results in segregation of BmQin from BmDcp2-containing P-bodies. Box plot showing that depletion of Siwi (dsSiwi, n=47 cells) reduces colocalization ratio between BmQin and BmDcp2, compared to control (dsRLuc, n=55 cells). Representative data from N = 4 independent experiments are shown. Depletion of Siwi results in segregation of BmSpnE from BmDcp2-containing P-bodies. Box plot showing that depletion of Siwi (dsSiwi, n=55 cells) reduces colocalization ratio between BmSpnE and BmDcp2, compared to control (dsRLuc, n=55 cells). Representative data from N = 6 independent experiments are shown. Depletion of Siwi results in colocalization of BmSpnE and BmVasa. Box plot showing that depletion of Siwi (dsSiwi, n=18 cells) significantly up-regulates colocalization ratio between BmSpnE and BmVasa, compared to control (dsRLuc, n=18 cells). Representative data from N = 3 independent experiments are shown. Depletion of BmVasa results in segregation of Siwi from BmAgo3 foci. Box plot showing that depletion of BmVasa (dsBmVasa, n=12 cells) down-regulates colocalization ratio between Siwi and BmAgo3, compared to control (dsRLuc, n=12 cells). Data points collected from N = 2 independent experiments are shown (statistical test is not performed for N < 3 data). Depletion of BmVasa results in entanglement of Siwi at piP-bodies. Interaction interfaces are indicated by white arrows. Box plot showing that depletion of BmVasa (dsBmVasa, n=45 cells) mildly up-regulates colocalization ratio between Siwi and BmDcp2, compared to control (dsRLuc, n=49 cells). Representative data from N = 5 independent experiments are shown.


Co - expression of BmVasa - E339Q / Siwi - D670A results in with larger sizes . Representative Z - projections ( Maximum ) of mutant . Scale bar 1 μm .

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Co-expression of BmVasa-E339Q/Siwi-D670A results in aggregates with larger sizes. Representative Z-projections (Maximum intensity) of mutant aggregates. Scale bar 1 μm.


Split violin plots of piRNA expression fold change between AcGFP ( control ) piRNA factor ( s ) overexpressed libraries . Overexpression of Siwi - D670A , BmVasa - E339Q both mutants increase non - TE - derived piRNA ( red , 626 genes ) while TE - derived piRNA ( blue , 818 genes ) does not change . Bonferroni - corrected P - values effect sizes ( r ) were calculated by Asymptotic Wilcoxon rank sum .

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Split violin plots of piRNA expression fold change between AcGFP (control) and piRNA factor(s) overexpressed libraries. Overexpression of Siwi-D670A, BmVasa-E339Q and both mutants increase non-TE-derived piRNA (red, 626 genes) while TE-derived piRNA (blue, 818 genes) does not change. Bonferroni-corrected P-values and the effect sizes (r) were calculated by Asymptotic Wilcoxon rank sum test.


Fig 1 : Urinary triclosan are elevated in TC mothers following 6 months of exposure . Summaries are provided as interquartile range with median of triclosan molecules in a picogram / microliter ( pg / L ) . Urinary triclosan measurements are available for 38 mothers ( 17 TC , 21 nTC ) 33 infants ( 15 TC , 18 nTC ) . p - value for mothers in Figure 1 ( represented by * * * ) is p = 5 . 07e - 5 ( Mann - Whitney U ) .

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Fig 1: Urinary triclosan levels are elevated in TC mothers following 6 months of exposure. Summaries are provided as the interquartile range with median of triclosan molecules in a picogram/microliter (pg/L). Urinary triclosan measurements are available for 38 mothers (17 TC, 21 nTC) and 33 infants (15 TC, 18 nTC). The p-value for the mothers in Figure 1 (represented by ***) is p=5.07e-5 (Mann-Whitney U test).


( A ) PCoA of Bray - Curtis dissimilarity for ( n = 221 ) samples shows that gut communities cluster by mothers infants .

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(A) PCoA of Bray-Curtis dissimilarity for all (n=221) samples shows that gut communities cluster by mothers and infants.


( B ) PCoA separated by time treatment .

(B) PCoA separated by time and treatment.


Fig 3 : TC randomization does not decrease gut microbial diversity in infants or mothers . Shannon diversity measures are plotted as interquartile range with median for each TC exposure class time point grouping for infants mothers .

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Fig 3: TC randomization does not decrease gut microbial diversity in infants or mothers. Shannon diversity measures are plotted as the interquartile range with median for each TC exposure class and time point grouping for infants and mothers.


( A ) Differentially abundant taxa between nTC TC households . Values left of grey indicate an enrichment in nTC household values to right indicate an enrichment in TC households . ( A ) Analyses are separated by mothers infants for samples across 3 time points ( FDR adjusted p - value < 0 . 01 ) .

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(A) Differentially abundant taxa between nTC and TC households. Values left of the grey line indicate an enrichment in nTC household and values to the right indicate an enrichment in TC households. (A) Analyses are separated by mothers and infants for all samples across the 3 time points (FDR adjusted p-value < 0.01).


Differentially abundant taxa are displayed for ( B ) infantsat per visit ( FDR adjusted p - value < 0 . 05 ) .

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Differentially abundant taxa are displayed for (B) infantsat per visit (FDR adjusted p-value < 0.05).


Differentially abundant taxa are displayed for ( C ) mothers at per visit ( FDR adjusted p - value < 0 . 05 ) .

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Differentially abundant taxa are displayed for(C) mothers at per visit (FDR adjusted p-value < 0.05).


( A ) Adult were transfected with YFP ‐ LC3 with pDsRed2 ‐ Mito . were starved for 24 h or denervated for 7 days . were exposed observed in situ using confocal in vivo microscopy . At least five per condition have been studied . Scale bar , 20 μm .

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(A) Adult muscles were transfected with YFP‐LC3 and with pDsRed2‐Mito. Animals were starved for 24 h or denervated for 7 days. Muscles were exposed and observed in situ using confocal in vivo microscopy. At least five animals per condition have been studied. Scale bar, 20 μm.


( B ) Adult were transfected with YFP ‐ LC3 with pDsRed2 ‐ Mito . Lysosomes were inhibited by treating mice with 50 mg / kg of chloroquine for 7 days . Chloroquine treatment started at day of denervation lasted for 7 days , whereas for fasting experiments at 6th day of chloroquine injection food was removed for 24 h . were exposed observed in situ using confocal in vivo microscopy . At least five per condition have been studied . Scale bar , 20 μm .

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(B) Adult muscles were transfected with YFP‐LC3 and with pDsRed2‐Mito. Lysosomes were inhibited by treating mice with 50 mg/kg of chloroquine for 7 days. Chloroquine treatment started at the day of denervation and lasted for 7 days, whereas for fasting experiments at the 6th day of chloroquine injection food was removed for 24 h. Muscles were exposed and observed in situ using confocal in vivo microscopy. At least five animals per condition have been studied. Scale bar, 20 μm.


( A ) Imaging of FoxO3 ‐ mediated modification of mitochondrial network in soleus muscle of living mice . Adult soleus were transfected with pDsRed2 ‐ Mito either c . a . FoxO3 or mock vector ( control ) . Two later , were exposed observed in situ using two ‐ photon microscopy . At least five per condition were studied .

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(A) Imaging of FoxO3‐mediated modification of the mitochondrial network in soleus muscle of living mice. Adult soleus muscles were transfected with pDsRed2‐Mito and either c.a.FoxO3 or mock vector (control). Two weeks later, muscles were exposed and observed in situ using two‐photon microscopy. At least five animals per condition were studied.


( B ) Overexpression of c . a . FoxO3 induces mitochondrial changes removal through autophagy . Muscle fibres were transfected by electroporation with YFP ‐ LC3 , pDsRed2 ‐ Mito either c . a . FoxO3 or mock vector . Two later , fibres were analysed for mitochondrial distribution by confocal microscopy . A higher magnification of square is depicted on right panels . arrows point autophagosomes engulfing mitochondria .

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(B) Overexpression of c.a.FoxO3 induces mitochondrial changes and removal through autophagy. Muscle fibres were transfected by electroporation with YFP‐LC3, pDsRed2‐Mito and either c.a.FoxO3 or mock vector. Two weeks later, fluorescent fibres were analysed for mitochondrial distribution by confocal microscopy. A higher magnification of the square is depicted on the right panels. White arrows point autophagosomes engulfing the mitochondria.


( C ) Overexpression of c . a . FoxO3 induces reduction in myofibre decrease in SDH . Left image : immunostaining for FoxO3 shows atrophic fibres , which over ‐ expressed FoxO3 ( red ) are labelled by asterisks . Nuclei are stained by Hoechst ( blue ) . Right image : histochemistry for succinate dehydrogenase ( SDH ) on serial section . Note that fibres positive for FoxO3 are negative for SDH ( asterisks ) .

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(C) Overexpression of c.a.FoxO3 induces reduction in myofibre size and decrease in SDH staining. Left image: immunostaining for FoxO3 shows atrophic fibres, which over‐expressed FoxO3 (red) and are labelled by asterisks. Nuclei are stained by Hoechst (blue). Right image: histochemistry for succinate dehydrogenase (SDH) on serial section. Note that fibres positive for FoxO3 are negative for SDH staining (asterisks).


( D ) Electron micrograph of a control c . a . FoxO3 ‐ expressing fibres . were processed with standard fixation ‐ embedding procedure . Black arrows indicate subsarcolemmal intermyofibrillar mitochondria , respectively . Note that subsarcolemmal mitochondria are reduced in FoxO3 overexpressing fibres , whereas intermyofibrillar mitochondria are smaller paler than control ones . Red arrowheads show a vesicle containing mitochondria . image is representative of FoxO3 ‐ transfected fibres .

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(D) Electron micrograph of a control and c.a.FoxO3‐expressing fibres. Muscles were processed with standard fixation‐embedding procedure. Black and white arrows indicate subsarcolemmal and intermyofibrillar mitochondria, respectively. Note that subsarcolemmal mitochondria are reduced in FoxO3 overexpressing fibres, whereas intermyofibrillar mitochondria are smaller and paler than control ones. Red arrowheads show a vesicle containing mitochondria. The image is representative of the FoxO3‐transfected fibres.


( A ) RNAi ‐ mediated knockdown of Fis1 showed by western blot . MEF were transfected with vectors expressing four different Fis shRNAs . For in vivo experiments Fis ( 4 ) was used . Efficiency of knockdown is indicated in Supplementary Table S2 .

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(A) RNAi‐mediated knockdown of Fis1 showed by western blot assay. MEF cells were transfected with vectors expressing four different Fis shRNAs. For in vivo experiments Fis (4) was used. Efficiency of protein knockdown is indicated in Supplementary Table S2.


( B ) Blockade of Fis1 of Bnip3 by RNAi reduces muscle loss during fasting . Adult skeletal were transfected with specific shRNAs for mouse Fis1 ( oligo 4 ) mouse Bnip3 or co ‐ transfected 8 days later mice were starved for 48 h . Cross ‐ sectional ( CSA ) of transfected fibres , identified by GFP , was measured as described earlier ( * P0 . 01 ) n = 450 .

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(B) Blockade of Fis1 and of Bnip3 by RNAi reduces muscle loss during fasting. Adult skeletal muscles were transfected with specific shRNAs for mouse Fis1 (oligo 4) and mouse Bnip3 or co‐transfected and 8 days later mice were starved for 48 h. Cross‐sectional area (CSA) of transfected fibres, identified by GFP, was measured as described earlier (*P0.01) n=450.


( C ) RNAi of Fis1 Bnip3 significantly prevents MuRF ‐ 1 atrogin ‐ 1 promoters activation during starvation . TA were transfected with either 5 kb of MuRF ‐ 1 promoter ( left panel ) or 3 . 5 kb of atrogin ‐ 1 promoter ( right panel ) with or without shRNAs vectors against Fis Bnip3 . In fasting experiments , after 7 days from transfection mice were starved for 24 h . A renilla ‐ luciferase construct ( pRL ‐ TK ) was co ‐ transfected to normalized for transfection efficiency . activation of MuRF ‐ 1 atrogin ‐ 1 promoters was determined by firefly activity normalize for renilla . Each condition represents average of at least three independent experiments ± s . e . m . ( * P0 . 05 ) .

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(C) RNAi of Fis1 and Bnip3 significantly prevents MuRF‐1 and atrogin‐1 promoters activation during starvation. TA muscles were transfected with either 5 kb of the MuRF‐1 promoter (left panel) or 3.5 kb of atrogin‐1 promoter (right panel) with or without shRNAs vectors against Fis and Bnip3. In fasting experiments, after 7 days from transfection mice were starved for 24 h. A renilla‐luciferase construct (pRL‐TK) was co‐transfected to normalized for transfection efficiency. The activation of MuRF‐1 and atrogin‐1 promoters was determined by firefly activity and normalize for the renilla. Each condition represents the average of at least three independent experiments±s.e.m. (*P0.05).


( D ) Muscle atrophy induced by FoxO3 is reduced by d . n . DRP1 . Adult skeletal were transfected with HA ‐ tagged c . a . FoxO3 with or without d . n . DRP1 examined after 2 . CSA of transfected fibres , identified by anti ‐ HA immunofluorescence , was measured as described earlier ( * P0 . 001 ) n = 400 .

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(D) Muscle atrophy induced by FoxO3 is reduced by d.n.DRP1. Adult skeletal muscles were transfected with HA‐tagged c.a.FoxO3 with or without d.n.DRP1 and examined after 2 weeks. CSA of transfected fibres, identified by anti‐HA immunofluorescence, was measured as described earlier (*P0.001) n=400.


( E ) Blockade of fission machinery by d . n . DRP1 or Bnip3 inhibition by RNAi reduces FoxO3 ‐ mediated autophagosome formation . were transfected with YFP ‐ LC3 , c . a . FoxO3 in presence or absence of d . n . DRP1 or shRNAs against Bnip3 . Twelve days later , were collected analysed for vesicles formation ( * P0 . 001 ) n = 450 .

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(E) Blockade of the fission machinery by d.n.DRP1 or Bnip3 inhibition by RNAi reduces FoxO3‐mediated autophagosome formation. Animals were transfected with YFP‐LC3, c.a.FoxO3 in presence or absence of d.n.DRP1 or shRNAs against Bnip3. Twelve days later, muscles were collected and analysed for fluorescent vesicles formation (*P0.001) n=450.


( F ) FoxO3 ‐ dependent activation of MuRF ‐ 1 atrogin ‐ 1 is significantly reduced by inhibition of fission machinery . MuRF ‐ 1 promoter ( left panel ) or atrogin ‐ 1 promoter ( right panel ) was co ‐ transfected into adult TA muscle with or without c . a . FoxO3 in presence or absence of d . n . DRP1 . A renilla ‐ luciferase vector was co ‐ transfected to normalize for transfection efficiency . Eight days later , firefly renilla ‐ luciferase activity was determined . Each condition represents average of at least three independent experiments ± s . e . m . ( * P0 . 05 ) .

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(F) FoxO3‐dependent activation of MuRF‐1 and atrogin‐1 is significantly reduced by inhibition of the fission machinery. MuRF‐1 promoter (left panel) or atrogin‐1 promoter (right panel) was co‐transfected into adult TA muscle with or without c.a.FoxO3 and in the presence or absence of d.n.DRP1. A renilla‐luciferase vector was co‐transfected to normalize for transfection efficiency. Eight days later, firefly and renilla‐luciferase activity was determined. Each condition represents the average of at least three independent experiments±s.e.m. (*P0.05).


Overexpression of DRP1 / Fis1 Bnip3 induces changes in mitochondrial network in vivo . In vivo imaging of mitochondrial network was performed in expressing Bnip3 , an atrophy ‐ related gene under FoxO3 control , DRP1 / Fis1 . Adult were transfected with plasmids encoding mitochondrially targeted yellow ( mtM13 ‐ YFP ) either mock vector , Bnip3 , HA ‐ DRP1 , Fis1 or DRP1 / Fis1 . Seven days later ( for Bnip3 ) or 12 days later ( for DRP1 Fis1 ) , were observed in vivo with confocal microscopy . TMRM was injected into to monitor mitochondria , which retain potential . transfected with mtM13 ‐ YFP mock vector showed orderly pattern of intermyofibrillar subsarcolemmal mitochondria ( upper panel ‐ IMF ) . Scale bar : 20 μm .

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Overexpression of DRP1/Fis1 and Bnip3 proteins induces changes in the mitochondrial network in vivo. In vivo imaging of the mitochondrial network was performed in muscles expressing Bnip3, an atrophy‐related gene under FoxO3 control, and DRP1/Fis1 proteins. Adult muscles were transfected with plasmids encoding mitochondrially targeted yellow fluorescent protein (mtM13‐YFP) and either mock vector, Bnip3, HA‐DRP1, Fis1 or DRP1/Fis1. Seven days later (for Bnip3) or 12 days later (for DRP1 and Fis1), muscles were observed in vivo with confocal microscopy. TMRM was injected into the muscles to monitor mitochondria, which retain membrane potential. Muscles transfected with mtM13‐YFP and mock vector showed the orderly pattern of intermyofibrillar and subsarcolemmal mitochondria (upper panel‐IMF). Scale bar: 20 μm.


( A ) Adult TA were transfected with either DRP1 , Fis1 , DRP1 Fis1 or mock vector . were collected 12 or 21 days after transfection cross ‐ sectional of transfected fibres , identified by immunofluorescence , was measured as described earlier ( * P0 . 001 ) n = 400 .

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(A) Adult TA muscles were transfected with either DRP1, Fis1, DRP1 and Fis1 or mock vector. Muscles were collected 12 or 21 days after transfection and cross‐sectional area of transfected fibres, identified by immunofluorescence, was measured as described earlier (*P0.001) n=400.


( B ) Bnip3 or Bnip3 l expression for 7 days causes a 20 % reduction of cross ‐ sectional . TA were transfected with Bnip3 , Bnip3l or mock vector . Cross ‐ sectional of transfected fibres , identified by immunofluorescence , was analysed ( * P0 . 001 ) n = 500 .

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(B) Bnip3 or Bnip3 l expression for 7 days causes a 20% reduction of the cross‐sectional area. TA muscles were transfected with Bnip3, Bnip3l or mock vector. Cross‐sectional area of transfected fibres, identified by immunofluorescence, was analysed (*P0.001) n=500.


( C ) of LC3 ‐ positive autophagic vesicles in adult co ‐ tranfected with YFP ‐ LC3 DRP1 , Fis1 or DRP1 / Fis1 expression plasmids or mock vector .

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(C) Quantification of LC3‐positive autophagic vesicles in adult muscles co‐tranfected with YFP‐LC3 and DRP1, Fis1 or DRP1/Fis1 expression plasmids or mock vector.


( D ) Mutations in BH3 domain of Bnip3 ( Bnip3BH3mut ) do not affect BNIP3 ‐ mediated autophagy . Adult were co ‐ transfected with YFP ‐ LC3 Bnip3 or Bnip3BH3mut expression plasmids LC3 ‐ positive vesicles were quantified .

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(D) Mutations in BH3 domain of Bnip3 (Bnip3BH3mut) do not affect BNIP3‐mediated autophagy. Adult muscles were co‐transfected with YFP‐LC3 and Bnip3 or Bnip3BH3mut expression plasmids and LC3‐positive vesicles were quantified.


( E ) Bnip3 ‐ mediated atrophy is not modified by mutating BH3 domain ( Bnip3BH3mut ) . Adult TA were transfected with Bnip3 or Bnip3BH3mut expression plasmids cross ‐ sectional was quantified 7 days later plotted as % of controls ( * P0 . 001 ) n = 440 .

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(E) Bnip3‐mediated atrophy is not modified by mutating the BH3 domain (Bnip3BH3mut). Adult TA muscles were transfected with Bnip3 or Bnip3BH3mut expression plasmids and cross‐sectional area was quantified 7 days later and plotted as % of controls (*P0.001) n=440.


( A ) In vivo imaging of mitochondrial network was performed in expressing Fis1K148R . Adult were transfected with plasmids encoding mitochondrially targeted yellow ( mtM13 ‐ YFP ) Fis1K148R . Twelve days later were observed in vivo with confocal microscopy . TMRM was injected into to monitor mitochondria , which retain potential . Mitochondrial network was greatly altered but mitochondrial potential was conserved .

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(A)In vivo imaging of the mitochondrial network was performed in muscles expressing Fis1K148R. Adult muscles were transfected with plasmids encoding mitochondrially targeted yellow fluorescent protein (mtM13‐YFP) and Fis1K148R. Twelve days later muscles were observed in vivo with confocal microscopy. TMRM was injected into the muscles to monitor mitochondria, which retain membrane potential. Mitochondrial network was greatly altered but mitochondrial membrane potential was conserved.


( B ) FDB muscle fibres were transfected by electroporation in vivo . Eight days later adult fibres were isolated placed in culture . Adult fibres were loaded with TMRM ( 5 nM ) for 30 min at 37 ° C . TMRM accumulates in mitochondria that maintain mitochondrial potential . Oligomycin ( Olm , 5 μM ) protonophore FCCP ( 4 μM ) were added at indicated time points . TMRM were monitored in at least 10 fibres per construct ( * P0 . 001 ) .

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(B) FDB muscle fibres were transfected by electroporation in vivo. Eight days later adult fibres were isolated and placed in cell culture. Adult fibres were loaded with TMRM (5 nM) for 30 min at 37°C. TMRM accumulates in the mitochondria that maintain mitochondrial membrane potential. Oligomycin (Olm, 5 μM) and the protonophore FCCP (4 μM) were added at the indicated time points. TMRM staining were monitored in at least 10 fibres per construct (*P0.001).


( C ) hFisK148R ‐ transfected were collected 12 21 days after transfection . Cross ‐ sectional of transfected fibres , identified by anti ‐ myc immunofluorescence , was quantified ( n = 550 ) .

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(C) hFisK148R‐transfected muscles were collected 12 and 21 days after transfection. Cross‐sectional area of transfected fibres, identified by anti‐myc immunofluorescence, was quantified (n=550).


( D ) Fis1 ‐ dependent activation of MuRF ‐ 1 . MuRF ‐ 1 promoter was co ‐ transfected into adult TA muscle with or without Fis1 or Fis1K148R . A renilla ‐ luciferase vector was co ‐ transfected to normalize for transfection efficiency . Eight days later , firefly renilla ‐ luciferase activity was determined . Each condition represents average of at least five independent experiments ± s . e . m . ( * P0 . 05 ) .

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(D) Fis1‐dependent activation of MuRF‐1. MuRF‐1 promoter was co‐transfected into adult TA muscle with or without Fis1 or Fis1K148R. A renilla‐luciferase vector was co‐transfected to normalize for transfection efficiency. Eight days later, firefly and renilla‐luciferase activity was determined. Each condition represents the average of at least five independent experiments±s.e.m. (*P0.05).


( A ) Left panel : mitochondrial fission activates AMPK . HEK293 were transfected with a mock vector ( control ) , Bnip3 , DRP1 , Fis1 or co ‐ transfected with DRP1 Fis1 . were collected 24 h later lysates were analysed by immunoblotting . Right panel : immunoblotting shows AMPK phosphorylation in EDL soleus during muscle atrophy . P ‐ AMPK / AMPK analysed by densitometric is indicated below panels . ( B - E ) Inhibition of AMPK blocks Bnip3 ‐ DRP1 / Fis1 ‐ mediated atrophy .

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(A) Left panel: mitochondrial fission activates AMPK. HEK293 cells were transfected with a mock vector (control), Bnip3, DRP1, Fis1 or co‐transfected with DRP1 and Fis1. Cells were collected 24 h later and lysates were analysed by immunoblotting. Right panel: immunoblotting analysis shows AMPK phosphorylation in EDL and soleus muscles during muscle atrophy. P‐AMPK/AMPK ratio analysed by densitometric analysis is indicated below the panels. (B-E) Inhibition of AMPK blocks Bnip3‐ and DRP1/Fis1‐mediated atrophy.


( B ) Adult TA were transfected with Bnip3 together with d . n . AMPK or mock vector cross ‐ sectional was measured ( * P0 . 001 ) n = 480 .

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(B) Adult TA muscles were transfected with Bnip3 together with d.n.AMPK or mock vector and cross‐sectional area was measured (*P0.001) n=480.


( C ) Adult TA were transfected with DRP1 Fis1 together with d . n . AMPK or mock vector . Twelve days later were collected cross ‐ sectional was quantified ( * P0 . 001 ) n = 400 .

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(C) Adult TA muscles were transfected with DRP1 and Fis1 together with d.n.AMPK or mock vector. Twelve days later muscles were collected and cross‐sectional area was quantified (*P0.001) n=400.


( D , E ) RNAi ‐ mediated knockdown of AMPK α 1 α 2 inhibits Bnip3 ‐ DRP1 / Fis1 ‐ dependent atrophy . Mouse TA were co ‐ transfected with Bnip3 or DRP1 Fis1 pSuper vectors expressing shRNAs against lacZ ( control ) or AMPK . Twelve days later , TA were collected cross ‐ sectional was quantified ( * P0 . 001 ) n = 450 .

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(D, E) RNAi‐mediated knockdown of AMPK α 1 and α 2 inhibits Bnip3‐ and DRP1/Fis1‐dependent atrophy. Mouse TA muscles were co‐transfected with Bnip3 or DRP1 and Fis1 and pSuper vectors expressing shRNAs against lacZ (control) or AMPK. Twelve days later, TA muscles were collected and the cross‐sectional area was quantified (*P0.001) n=450.


( A ) Quantitative PCR was performed in triplicates using specific oligonucleotides ( see Supplementary Table 1 ) ( * P0 . 01 ) .

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(A) Quantitative PCR analysis was performed in triplicates using specific oligonucleotides (see Supplementary Table 1) (*P0.01).


( B ) Immunoblotting shows that AICAR treatment causes phosphorylation of AMPK downstream target ACC but does not affect phosphorylation of AKT FoxO3 .

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(B) Immunoblotting analysis shows that AICAR treatment causes phosphorylation of AMPK and the downstream target ACC but does not affect phosphorylation of AKT and FoxO3.


( C ) FoxO3 binding to atrogin ‐ 1 MuRF ‐ 1 promoters , as determined by ChIP , is induced by AICAR treatment .

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(C) FoxO3 binding to atrogin‐1 and MuRF‐1 promoters, as determined by ChIP, is induced by AICAR treatment.


( D ) RNAi ‐ mediated knockdown of FoxO3 inhibits DRP1 / Fis1 ‐ dependent atrophy . Mouse TA were co ‐ transfected with DRP1 Fis1 pSuper vectors expressing shRNAs against either lacZ ( control ) or FoxO3 . Twelve days later , were collected cross ‐ sectional was quantified ( * P0 . 001 ) n = 450 .

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(D) RNAi‐mediated knockdown of FoxO3 inhibits DRP1/Fis1‐dependent atrophy. Mouse TA muscles were co‐transfected with DRP1 and Fis1 and pSuper vectors expressing shRNAs against either lacZ (control) or FoxO3. Twelve days later, muscles were collected and the cross‐sectional area was quantified (*P0.001) n=450.


A Purified TAP - tagged Rik1 - containing complexes analyzed by SDS - PAGE silver . mock : a mock purification from an untagged . identified by LC - MS / MS are indicated at right , with number of identified unique peptides in parentheses .

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A Purified TAP-tagged Rik1-containing complexes analyzed by SDS-PAGE and silver staining. mock: a mock purification from an untagged strain. Proteins identified by LC-MS/MS are indicated at right, with the number of identified unique peptides in parentheses.


B In vitro ubiquitylation assays using biotinylated ubiquitin , purified CLRC , recombinant S . pombe histone H3 as substrate . were analyzed by Western blotting ( WB ) using indicated antibodies . Asterisks indicate ubiquitylated histone H3 species .

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B In vitro ubiquitylation assays using biotinylated ubiquitin, purified CLRC, and recombinant S. pombe histone H3 as the substrate. Proteins were analyzed by Western blotting (WB) using the indicated antibodies. Asterisks indicate ubiquitylated histone H3 species.


C Ubiquitylation assays using recombinant histone H3 , N - terminal tail of histone H3 ( residues 1 - 36 ) fused with GST ( H3N - GST ) , or GST alone as substrate . Both fission yeast ( sp ) human ( hs ) full - H3 H3N - GST fusion were examined . were analyzed by SDS - PAGE followed by either silver or Western blotting with an anti - biotin antibody . Asterisks indicate ubiquitylated histone H3 species .

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C Ubiquitylation assays using recombinant histone H3, the N-terminal tail of histone H3 (residues 1-36) fused with GST (H3N-GST), or GST alone as the substrate. Both fission yeast (sp) and human (hs) full-length H3 proteins and H3N-GST fusion proteins were examined. The proteins were analyzed by SDS-PAGE followed by either silver staining or Western blotting with an anti-biotin antibody. Asterisks indicate ubiquitylated histone H3 species.


A Ubiquitylation using H3N - GST . components included in ubiquitylated H3N - GST species are indicated at right .

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A Ubiquitylation assay using H3N-GST. The components included in the assay and the ubiquitylated H3N-GST species are indicated at right.


B MS / MS spectrum of histone H3 peptide corresponding to residues 10 - 17 . observed y b ions fragment map are shown .

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B MS/MS spectrum of the histone H3 peptide corresponding to residues 10-17. The observed y and b ions and fragment map are shown.


C Ubiquitylation using biotinylated ubiquitin recombinant wild - type H3N - GST ( WT ) arginine - substituted H3N - GST mutants as substrates . were analyzed by Western blotting silver . mock : reaction without substrate . Asterisks indicate ubiquitylated H3N - GST .

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C Ubiquitylation assay using biotinylated ubiquitin and recombinant wild-type H3N-GST (WT) and arginine-substituted H3N-GST mutants as substrates. Proteins were analyzed by Western blotting and silver staining. mock: reaction without substrate. Asterisks indicate ubiquitylated H3N-GST proteins.


A precipitated with anti - H3K4me2 or anti - H3K9me2 antibodies was analyzed by SDS - PAGE silver . input sample mock precipitation using total mouse IgG are also shown . gel slices indicated by boxes were excised subjected to LC - MS / MS .

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A Chromatin precipitated with anti-H3K4me2 or anti-H3K9me2 antibodies was analyzed by SDS-PAGE and silver staining. The input sample and mock precipitation using total mouse IgG are also shown. The gel slices indicated by boxes were excised and subjected to LC-MS/MS analysis.


MS / MS spectrum of histone H3 peptide corresponding to residues 9 - 17 . observed y b ions fragment map are shown . C Summary of ubiquitylated peptides identified by LC - MS / MS .

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MS/MS spectrum of the histone H3 peptide corresponding to residues 9-17. The observed y and b ions and fragment map are shown. C Summary of the ubiquitylated peptides identified by LC-MS/MS.


D Relative abundance of ubiquitylated lysine residues in - associated histone H3 fractions . precipitated with anti - H3K4me2 or anti - H3K9me2 antibodies was subjected to LC - MS / MS , abundance of ubiquitylated lysine residues in each fraction is shown Since short peptides at unmodified lysine residues could not be efficiently detected in LC - MS / MS , these data do not accurately reflect abundance including unmodified lysines .

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D Relative abundance of ubiquitylated lysine residues in chromatin-associated histone H3 fractions. Chromatin precipitated with anti-H3K4me2 or anti-H3K9me2 antibodies was subjected to LC-MS/MS analysis, and the abundance of ubiquitylated lysine residues in each fraction is shown Since short peptides cleaved at unmodified lysine residues could not be efficiently detected in the LC-MS/MS analysis, these data do not accurately reflect the abundance including unmodified lysines.


B Heterochromatic silencing assays of wild - type mutant Hht1 - FH strains . Silencing at mating - type Kint2 : : ura4 + was evaluated . Ten - fold serial dilutions of indicated strains were spotted onto non - selective medium ( N / S ) , medium lacking uracil ( - Ura ) , medium containing 5 - FOA ( 5 - FOA ) .

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B Heterochromatic silencing assays of wild-type and mutant Hht1-FH strains. Silencing at the mating-type Kint2::ura4+ was evaluated. Ten-fold serial dilutions of the indicated strains were spotted onto non-selective medium (N/S), medium lacking uracil (-Ura), and medium containing 5-FOA (5-FOA).


C ChIP of H3K9me2 associated with mating - type loci , relative to control act1 + locus . Error bars indicate standard errors from three technical replicates .

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C ChIP analysis of H3K9me2 levels associated with the mating-type loci, relative to the control act1+ locus. Error bars indicate standard errors from three technical replicates.


D ChIP of Rik1 - myc associated with mating - type cenH centromeric dg loci , relative to control act1 + locus . Error bars indicate standard errors from three technical replicates .

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D ChIP analysis of Rik1-myc levels associated with the mating-type cenH and centromeric dg loci, relative to the control act1+ locus. Error bars indicate standard errors from three technical replicates.


E Heterochromatic silencing assays comparing effects of H3 lysine substitution mutants . Silencing at mating - type kint2 : : ura4 + was

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E Heterochromatic silencing assays comparing the effects of H3 lysine substitution mutants. Silencing at the mating-type kint2::ura4+ was evaluated


In vitro HMTase assays . Recombinant H3N - GST ( WT or K9R ) pre - ubiquitylated by CLRC was purified used in HMTase with 6 × His - tagged recombinant Clr4 ( His - Clr4 ) . were analyzed by SDS - PAGE Coomassie Brilliant Blue ( CBB ) ( left ) autoradiography ( right ) .

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In vitro HMTase assays. Recombinant H3N-GST (WT or K9R) pre-ubiquitylated by the CLRC was purified and used in the HMTase assay with 6×His-tagged recombinant Clr4 (His-Clr4). Proteins were analyzed by SDS-PAGE and Coomassie Brilliant Blue (CBB) staining (left) and autoradiography (right).


C , D In vitro HMTase using disulfide - linked ubiquitylated histone H3 . H3K14CUb control histone H3 ( H3C110A ) were used as substrates in HMTase , analyzed 3H - methyl group incorporation was measured using a liquid scintillation counter ( D ) . Error bars indicate standard deviations calculated from three independent experiments .

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C,D In vitro HMTase assay using disulfide-linked ubiquitylated histone H3. H3K14CUb and control histone H3 (H3C110A) were used as substrates in the HMTase assay, and analyzed The 3H-methyl group incorporation was measured using a liquid scintillation counter (D). Error bars indicate the standard deviations calculated from three independent experiments.


B Recombinant were resolved by 10 % SDS - PAGE visualized by CBB .

B Recombinant proteins were resolved by 10% SDS-PAGE and visualized by CBB staining.


C In vitro HMTase using disulfide - linked ubiquitylated histone H3 . H3K14CUb control histone H3 ( H3C110A ) were used as substrates in HMTase ,

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C In vitro HMTase assay using disulfide-linked ubiquitylated histone H3. H3K14CUb and control histone H3 (H3C110A) were used as substrates in the HMTase assay, and analyzed


D In vitro pulldown assays of GST - Clr4 with biotinylated ubiquitin . Bound ubiquitin was analyzed by Western blotting with HRP - conjugated streptavidin ( left ) . Precipitated GST were analyzed by SDS - PAGE CBB ( right ) .

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D In vitro pulldown assays of GST-Clr4 proteins with biotinylated ubiquitin. Bound ubiquitin was analyzed by Western blotting with HRP-conjugated streptavidin (left). Precipitated GST proteins were analyzed by SDS-PAGE and CBB staining (right).


F Whole lysates prepared from control , expressing FLAG - tagged wild - type Clr4 ( F - clr4 ) or mutant Clr4 ( F - clr4 ∆ 63 - 127 ) , or clr4 ∆ were subjected to immunoblotting using indicated antibodies .

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F Whole cell lysates prepared from control cells, cells expressing FLAG-tagged wild-type Clr4 (F-clr4) or mutant Clr4 (F-clr4∆63-127), or clr4∆ cells were subjected to immunoblotting using the indicated antibodies.


G Heterochromatic silencing assays of wild - type clr4 mutant strains . Silencing at mating - type Kint2 : : ura4 + was evaluated .

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G Heterochromatic silencing assays of wild-type and clr4 mutant strains. Silencing at the mating-type Kint2::ura4+ was evaluated.


H ChIP of H3K9me2 associated with silencing marker gene inserted in mating - type loci ( Kint2 : : ura4 + ) mating - type cenH centromeric dg loci , relative to control act1 + locus . Error bars indicate standard errors from three technical replicates .

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H ChIP analysis of H3K9me2 levels associated with the silencing marker gene inserted in the mating-type loci (Kint2::ura4+) and the mating-type cenH and centromeric dg loci, relative to the control act1+ locus. Error bars indicate standard errors from three technical replicates.


I of Kint2 : : ura4 + , mating - type cenH , centromeric dg transcripts were quantified by RT - qPCR calculated as fold change to wild - type expression . Error bars indicate standard errors from three technical replicates .

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I The levels of Kint2::ura4+, mating-type cenH, and centromeric dg transcripts were quantified by RT-qPCR and calculated as fold change to the wild-type expression. Error bars indicate standard errors from three technical replicates.


B . interaction by Nano LC - MS . Unique peptide coverage , as well as signal intensities ( PSI ) for Tailor ( bait ) CG16940 / dmDis3l2 in control IP ( FLAG - GFP ) FLAG - TailorIP are indicated . A . u . , arbitrary units .

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B. Protein interaction analysis by Nano LC-MS. Unique peptide counts and protein sequence coverage, as well as signal intensities (PSI) for Tailor (bait) and CG16940/dmDis3l2 in control IP (FLAG-GFP) and FLAG-TailorIP are indicated. A.u., arbitrary units.


D . Co - immunoprecipitation of FLAG - dmDis3l2 ( bait ) GFP - Tailor full - truncations expressed in Drosophila S2cells . GFP - Nibbler ( Nbr ) served as negative control . Domain architecture of GFP - Tailor truncations are indicated .

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D. Co-immunoprecipitation of FLAG-dmDis3l2 (bait) and GFP-Tailor full-length and truncations expressed in Drosophila S2cells. GFP-Nibbler (Nbr) served as negative control. Domain architecture of GFP-Tailor truncations are indicated.


E - G . Recombinant interaction using indicated epitope tags purification methods . Domain architecture of bait prey are indicated .

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E- G. Recombinant protein interaction analysis using the indicated epitope tags and purification methods. Domain architecture of bait and prey are indicated.


H I . Immunostaining imaging of Myc - Tailor GFP - dmDis3l2 ( H ) or endogenous dmDis3l2 ( I ) in S2 . Single color channel show total inversions . Scale bar = 5 µm .

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H and I. Immunostaining and imaging of Myc-Tailor and GFP-dmDis3l2 (H) or endogenous dmDis3l2 (I) in S2 cells. Single color channel images show total inversions. Scale bar = 5 µm.


B . In vitro exoribonuclease using 5 ′ radiolabeled substrate immunopurified wild - type ( WT ) or catalytic mutant ( CM ) FLAG - dmDis3l2 ( mutation indicated in Figure 1C ) , incubated for indicated time separated on a 15 % polyacrylamide gel followed by phosphorimaging . of degraded substrate in is indicated . C . Change in abundance of 256 different substrate RNAs as determined by high - throughput sequencing of substrates in experiment shown in ( B ) .

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B. In vitro exoribonuclease assay using 5′ radiolabeled RNA substrate and immunopurified wild-type (WT) or catalytic mutant (CM) FLAG-dmDis3l2 (mutation indicated in Figure 1C), incubated for the indicated time and separated on a 15 % polyacrylamide gel followed by phosphorimaging. Quantification of degraded substrate in percent is indicated.C. Change in abundance of 256 different substrate RNAs as determined by high-throughput sequencing of substrates in experiment shown in (B).


D . Decay rates of 256 different substrate RNAs . Data shown in ( C ) was normalized to overall decrease in substrate abundance as determined by phosphorimaging ( as shown in B ) fit to indicated model for exponential decay . Apparent decay rate of substrates , as determined by phosphorimaging is indicated in red . E . Selected examples for substrates shown in ( D ) . Values report observed decay rate ( kobs ) in min - 1 . F . Overview of decay rates ( kobs ) of 256 different substrate RNAs , as determined in ( D ) . Error represents of curve fit .

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D. Decay rates of 256 different substrate RNAs. Data shown in (C) was normalized to overall decrease in substrate abundance as determined by phosphorimaging (as shown in B) and fit to the indicated model for exponential decay. Apparent decay rate of all substrates, as determined by phosphorimaging is indicated in red.E. Selected examples for individual substrates shown in (D). Values report the observed decay rate (kobs) in min-1.F. Overview of decay rates (kobs) of all 256 different substrate RNAs, as determined in (D). Error represents SEM of curve fit.


G . Impact of secondary structures , as determined by RNAfold ( Gruber et al , 2008 ) , reported as effective free energy ( EFE , kcal / mol ) , on decay rates ( kobs ) are shown . Error bars represent of curve fit . Grey boxes represent tukey boxplot of EFE groups . indicates median . P - Value was determined by Mann - Whitney comparing EFE groups against substrates . Unstructured substrates used for subsequent are indicated ( EFE groups 4 to 6 ) .

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G. Impact of RNA secondary structures, as determined by RNAfold (Gruber et al, 2008), and reported as effective free energy (EFE, kcal/mol), on decay rates (kobs) are shown. Error bars represent SEM of curve fit. Grey boxes represent tukey boxplot of individual EFE groups. White line indicates median. P-Value was determined by Mann-Whitney test comparing individual EFE groups against all substrates. Unstructured substrates used for subsequent analysis are indicated (EFE groups 4 to 6).


H . Nucleotide content of single - stranded substrates , ranked from slow ( top ) to fast ( bottom ) decay by dmDis3l2 . I . Cumulative distribution of decay rates for substrates with indicated nucleotide at each one of four randomized position at 3 ′ end of substrate . P - Value was determined by Mann - Whitney comparing nts against .

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H. Nucleotide content of single-stranded RNA substrates, ranked from slow (top) to fast (bottom) decay by dmDis3l2.I. Cumulative distribution of decay rates for RNA substrates with the indicated nucleotide at each one of four randomized position at the 3′ end of the substrate RNA. P-Value was determined by Mann-Whitney test comparing individual nts against all.


J . Functional coupling between two base positions within randomized 3 ′ end of substrate . Red squares show average increase in decay rate compared to substrates . Black squares show no or negative effect .

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J. Functional coupling between two base positions within the randomized 3′ end of substrate RNA. Red squares show average increase in decay rate compared to all substrates. Black squares show no or negative effect.


K . Boxplot decay rates of substrates containing no , one , two , or three uridine ( s ) within randomized 3 ′ end of substrate . Median decay rates of fit are indicated . L . Decay rate of substrates , or substrates containing no , one , two , or three uridine ( s ) within randomized 3 ′ end of substrate . Median quartile ranges are indicated . P - values were determined by Mann - Whitney . Substrate containing 4 uridines ( 4U ) is indicated .

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K. Boxplot decay rates of substrates containing no, one, two, or three uridine(s) within the randomized 3′ end of the substrate RNA. Median decay rates SEM of fit are indicated.L. Decay rate of all substrates, or substrates containing no, one, two, or three uridine(s) within the randomized 3′ end of the substrate RNA. Median and quartile ranges are indicated. P-values were determined by Mann-Whitney test. Substrate containing 4 uridines (4U) is indicated.


A . decay using synthetic , 5 ′ radiolabeled pre - miR - 1003 , immunopurified dmDis3l2 wild - type ( WT ) or catalytic mutant ( CM ) Tailor . Reactions were incubated for indicated time separated by 15 % denaturing polyacrylamide gel electrophoresis subjected to phosphorimaging . B . of at least three independent replicates of experiment shown in A . Data represent mean SD . Half - life was determined by fitting data to single exponential ( dmDis3l2 dmDis3l2 / TailorCM ) or sigmoidal ( dmDis3l2 / TailorWT ) reaction kinetics .

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A. RNA decay assay using synthetic, 5′ radiolabeled pre-miR-1003, immunopurified dmDis3l2 and wild-type (WT) or catalytic mutant (CM) Tailor. Reactions were incubated for the indicated time and separated by 15% denaturing polyacrylamide gel electrophoresis and subjected to phosphorimaging.B. Quantification of at least three independent replicates of experiment shown in A. Data represent mean SD. Half-life was determined by fitting data to single exponential (dmDis3l2 and dmDis3l2/TailorCM) or sigmoidal (dmDis3l2/TailorWT) reaction kinetics.


C . decay using synthetic , 5 ′ radiolabeled pre - miR - 1003 containing indicated number or uridine residues at 3 ′ end immunopurified dmDis3l2 . D . of three independent replicates of experiment shown in C . Data represent mean SD . Data were fit to single exponential reaction kinetics . E . Reaction kinetics ( kobs ) determined in D plotted against number of 3 ′ terminal uridine residues . Error of fit is shown as .

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C. RNA decay assay using synthetic, 5′ radiolabeled pre-miR-1003 containing the indicated number or uridine residues at the 3′ end and immunopurified dmDis3l2.D. Quantification of three independent replicates of experiment shown in C. Data represent mean SD. Data were fit to single exponential reaction kinetics.E. Reaction kinetics (kobs) determined in D plotted against number of 3′ terminal uridine residues. Error of fit is shown as SEM.


A E . Western blot of genetically modified Drosophila S2 . Unmodified S2 ( wild - type , wt ) , or S2 depleted of Tailor ( tailorko ) by CRISPR / Cas9 or expressing FLAG - tagged Tailor ( TailorOE ) were analyzed . Antibodies are indicated . Note , that panels were assembled from two technical replicates of identical lysates ( see source data ) .

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A and E. Western blot analysis of genetically modified Drosophila S2 cells. Unmodified S2 cells (wild-type, wt), or S2 cells depleted of Tailor (tailorko) by CRISPR/Cas9 or expressing FLAG-tagged Tailor (TailorOE) were analyzed. Antibodies are indicated. Note, that panels were assembled from two technical replicates of identical lysates (see source data).


B , C , F G . Lysate of S2 ( as characterized in A E ) were incubated with - 32P - UTP ( B F ) or - 32P - ATP ( C G ) for indicated time followed by extraction , denaturing polyacrylamide gel electrophoresis phosphorimaging .

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B, C, F and G. Lysate of S2 cells (as characterized in A and E) were incubated with -32P-UTP (B and F) or -32P-ATP (C and G) for the indicated time followed by RNA extraction, denaturing polyacrylamide gel electrophoresis and phosphorimaging.


D H . of at least five ( D ) or seven ( H ) independent replicates of experiment shown in B / C F / G , respectively . Uridylation signal for substrate S1 ( indicated in B F ) was corrected for adenylation activity normalized to wild - type 2 min time point . Data represent mean . P - values were determined using Student ' s t - . * p < 0 . 05 , * * p < 10 - 2 , * * * p < 10 - 3 , * * * * p < 10 - 4 .

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D and H. Quantification of at least five (D) or seven (H) independent replicates of experiment shown in B/C and F/G, respectively. Uridylation signal intensity for substrate S1 (indicated in B and F) was corrected for adenylation activity and normalized to wild-type 2 min time point. Data represent mean SEM. P-values were determined using Student's t-test. * p<0.05, ** p<10-2, *** p<10-3, **** p<10-4.