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 .

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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 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 .

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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 aggregates with larger sizes . Representative Z - projections ( Maximum intensity ) of mutant aggregates . 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 ) 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 .

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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 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 ) .

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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 all ( n = 221 ) samples shows that gut communities cluster by mothers and 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 and 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 the interquartile range with median for each TC exposure class and time point grouping for infants and 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 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 ) .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 ) .

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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 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 .

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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 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 .

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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 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 .

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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 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 ) .

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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 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 .

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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 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 .

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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 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 ) .

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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 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 .

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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 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 .

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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 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 .

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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 ) 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 .

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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 muscles were co ‐ transfected with YFP ‐ LC3 and Bnip3 or Bnip3BH3mut expression plasmids and 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 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 .

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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 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 .

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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 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 ) .

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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 muscles were collected 12 and 21 days after transfection . Cross ‐ sectional area 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 and renilla ‐ luciferase activity was determined . Each condition represents the 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 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 .

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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 muscles were transfected with Bnip3 together with d . n . AMPK or mock vector and cross ‐ sectional area 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 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 .

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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 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 .

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

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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 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 .

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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 , 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 .

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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 , 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 .

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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 assay using H3N - GST . The components included in the assay and the 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 the histone H3 peptide corresponding to residues 10 - 17 . The observed y and b ions and 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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 ) .

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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 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 .

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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 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 .

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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 the effects of H3 lysine substitution mutants . Silencing at the 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 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 ) .

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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 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 .

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

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


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

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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 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 ) .

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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 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 .

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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 and clr4 mutant strains . Silencing at the 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 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 .

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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 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 .

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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 . 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 .

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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 ) 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 .

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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 protein interaction analysis using the indicated epitope tags and purification methods . Domain architecture of bait and 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 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 .

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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 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 ) .

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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 ) 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 .

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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 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 ) .

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

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 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 .

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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 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 .

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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 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 .

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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 . 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 .

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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 . 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 .

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

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 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 ) .

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

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 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 .

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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 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 .

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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.


A and E . Western blot analysis of genetically modified Drosophila S2 cells . Unmodified S2 cells ( wild - type , wt ) , or S2 cells depleted of dmDis3l2 ( dmDis3l2ko ) by CRISPR / Cas9 or expressing FLAG - tagged catalytic mutant dmDis3l2 ( dmDis3l2CM OE ) were analyzed . Antibodies are indicated . Note , that panels were assembled from two technical replicates of identical lysates ( see source data ) .

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

A and E. Western blot analysis of genetically modified Drosophila S2 cells. Unmodified S2 cells (wild-type, wt), or S2 cells depleted of dmDis3l2 (dmDis3l2ko) by CRISPR/Cas9 or expressing FLAG-tagged catalytic mutant dmDis3l2 (dmDis3l2CM OE) were analyzed. Antibodies are indicated. Note, that panels were assembled from two technical replicates of identical lysates (see source data).


A . Protein / RNA co - immunopurification experiments . FLAG - tagged versions of indicated proteins were expressed in S2 cells , followed by immunopurification and RNA isolation . RNA was visualized by CIP / PNK treatment using - 32P - ATP followed by denaturing polyacrylamide gel electrophoresis and phosphorimaging . Star indicates non - specific contaminant .

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A. Protein/RNA co-immunopurification experiments. FLAG-tagged versions of indicated proteins were expressed in S2 cells, followed by immunopurification and RNA isolation. RNA was visualized by CIP/PNK treatment using -32P-ATP followed by denaturing polyacrylamide gel electrophoresis and phosphorimaging. Star indicates non-specific contaminant.


B . Meta - analysis of 3 ′ end counts in dmDis3l2CM - IP mapping to the indicated region around the annotated 3 ′ end of 68 tRNA loci in the Drosophila melanogaster genome . Cumulative relative distribution of 3 ′ end signal , relative to the annotated 3 ′ end , is reported ( bottom ) . The average genome thymine ( T ) - content for the same regions is displayed ( top ) .

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B. Meta-analysis of 3′ end counts in dmDis3l2CM-IP mapping to the indicated region around the annotated 3′ end of 68 tRNA loci in the Drosophila melanogaster genome. Cumulative relative distribution of 3′ end signal, relative to the annotated 3′ end, is reported (bottom). The average genome thymine (T)-content for the same regions is displayed (top).


C . Northern hybridization experiment using total RNA from adult whole male flies using probes against 3 ′ trailer or mature tRNAAla ( TGC ) . Wild - type flies ( w1118 ) , or flies bearing a homozygous frame - shift mutation in the first coding exon ( dmDis3l2 - / - ) or a homozygous amino acid - exchange mutation in the catalytic site ( dmDis3l2CM ) in the endogenous dmdis3l2 locus in the same genetic background were used . Probes against 2S rRNA served as loading control . D . Quantification of four independent biological replicates of experiment shown in C . P - Value determined by Student ' s t - test .

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C. Northern hybridization experiment using total RNA from adult whole male flies using probes against 3′ trailer or mature tRNAAla(TGC). Wild-type flies (w1118), or flies bearing a homozygous frame-shift mutation in the first coding exon (dmDis3l2-/-) or a homozygous amino acid-exchange mutation in the catalytic site (dmDis3l2CM) in the endogenous dmdis3l2 locus in the same genetic background were used. Probes against 2S rRNA served as loading control.D. Quantification of four independent biological replicates of experiment shown in C. P-Value determined by Student's t-test.


E . Exoribonuclease assay using immunopurified dmDis3l2 and in vitro transcribed , 5 ′ radiolabeled mature tRNA , containing a CCA modification or 3 ′ trailer - containing tRNA . Reactions were incubated for the indicated time and subjected to polyacrylamide gel electrophoresis and phosphorimaging .

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E. Exoribonuclease assay using immunopurified dmDis3l2 and in vitro transcribed, 5′ radiolabeled mature tRNA, containing a CCA modification or 3′ trailer-containing tRNA. Reactions were incubated for the indicated time and subjected to polyacrylamide gel electrophoresis and phosphorimaging.


F . Quantification of three independent replicates of experiment shown in E . Data represent mean SD . Decay rates ( kobs ) SEM of fit were determined upon fitting to single exponential decay kinetics .

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F. Quantification of three independent replicates of experiment shown in E. Data represent mean SD. Decay rates (kobs) SEM of fit were determined upon fitting to single exponential decay kinetics.


A . Impact of RNA secondary structures on RNA decay rates ( kobs ) of 256 different RNA substrates subjected to E . coli RNase R ( ecoRNaseR ) - or Drosophila melanogaster Dis3l2 ( dmDis3l2 ) - directed RNA decay assay as described in Fig . 2 . RNA secondary structures were determined by RNAfold ( Gruber et al , 2008 ) and reported as effective free energy ( EFE ) . Error bars represent SEM of curve fit . Grey boxes represent tukey boxplot of individual EFE groups . White line indicates median . P - Value determined by Mann - Whitney test comparing individual EFE groups against all substrates . Unstructured RNA used for subsequent analysis are indicated ( EFE groups 4 to 6 ) .

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

A. Impact of RNA secondary structures on RNA decay rates (kobs) of 256 different RNA substrates subjected to E.coli RNase R (ecoRNaseR)- or Drosophila melanogaster Dis3l2 (dmDis3l2)-directed RNA decay assay as described in Fig. 2. RNA secondary structures were determined by RNAfold (Gruber et al, 2008) and reported as effective free energy (EFE). Error bars represent SEM of curve fit. Grey boxes represent tukey boxplot of individual EFE groups. White line indicates median. P-Value determined by Mann-Whitney test comparing individual EFE groups against all substrates. Unstructured RNA used for subsequent analysis are indicated (EFE groups 4 to 6).


B . Nucleotide content of randomized 3 ′ end sequence among the indicated number of substrate RNAs , ranked according to decay kinetics from slow ( left ) to fast ( right ) . Fold - change in abundance for individual nucleotides is reported ( bottom ) . Only unstructured RNA substrates ( see A ) were considered .

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B. Nucleotide content of randomized 3′ end sequence among the indicated number of substrate RNAs, ranked according to decay kinetics from slow (left) to fast (right). Fold-change in abundance for individual nucleotides is reported (bottom). Only unstructured RNA substrates (see A) were considered.


C . Decay rate of the indicated number of sequences , grouped according to individual nucleotide content in the randomized 3 ′ end sequence . Data is represented as boxplot , outliers are not shown . P - Value reports significant differences compared to all substrates as determined by Mann - Whitney test . Grey area represents the inner quartile range and white line the median of all substrates .

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C. Decay rate of the indicated number of sequences, grouped according to individual nucleotide content in the randomized 3′ end sequence. Data is represented as boxplot, outliers are not shown. P-Value reports significant differences compared to all substrates as determined by Mann-Whitney test. Grey area represents the inner quartile range and white line the median of all substrates.


( C ) HeLa GFP - LC3 cells were transfected with indicated siRNAs and infected with DsRed - labeled Salmonella . The percentage of GFP - LC3 + Salmonella at 1 hr post - infection is shown . Data are shown as means ± SD ; n = 75 cells per condition from three biological replicates . Data are representative of three independent experiments .

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(C) HeLa GFP-LC3 cells were transfected with indicated siRNAs and infected with DsRed-labeled Salmonella. The percentage of GFP-LC3+ Salmonella at 1 hr post-infection is shown. Data are shown as means ± SD; n = 75 cells per condition from three biological replicates. Data are representative of three independent experiments.


( D ) Representative epifluorescent microscopy images of HeLa cells used to generate data shown in ( C ) . The scale bars represent 10 μm .

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(D) Representative epifluorescent microscopy images of HeLa cells used to generate data shown in (C). The scale bars represent 10 μm.


( E ) Western blot of HeLa cells transfected with the indicated siRNAs and treated with Torin E64D / PepA . Data are representative of three independent experiments .

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(E) Western blot of HeLa cells transfected with the indicated siRNAs and treated with Torin E64D/PepA. Data are representative of three independent experiments.


( G ) HeLa GFP - LC3 cells were transfected with control siRNA , CLEC12A siRNA1 , or CLEC12A siRNA2 as well as CLEC12A rescue constructs ( R1 or R2 ) and infected for 1 hr with DsRed - labeled Salmonella . Data are shown as mean ± SD ; n = 75 per condition . Data are representative of three independent experiments .

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(G) HeLa GFP-LC3 cells were transfected with control siRNA, CLEC12A siRNA1, or CLEC12A siRNA2 as well as CLEC12A rescue constructs (R1 or R2) and infected for 1 hr with DsRed-labeled Salmonella. Data are shown as mean ± SD; n = 75 per condition. Data are representative of three independent experiments.


( H ) HeLa cells from ( G ) were lysed , and HA - CLEC12A expression levels were assessed by western blot . Data are representative of three independent experiments .

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(H) HeLa cells from (G) were lysed, and HA-CLEC12A expression levels were assessed by western blot. Data are representative of three independent experiments.


( A ) Representative epifluorescent images showing impaired antibacterial autophagy in ATG16L1 KO HeLa cells complemented with the indicated ATG16L1 protein in the presence of control siRNA or CLEC12A siRNA . The scale bars represent 10 μm . ( B ) Percentage of LC3 - Salmonella colocalization in WT HeLa cells , ATG16L1 KO HeLa cells , and ATG16L1 KO HeLa cells stably expressing ATG16L1 ∗ 300T or ∗ 300A alleles in the presence of control siRNA or CLEC12A siRNA . Data are shown as means ± SD ; n = 75 . Data shown are representative of three independent experiments .

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(A) Representative epifluorescent images showing impaired antibacterial autophagy in ATG16L1 KO HeLa cells complemented with the indicated ATG16L1 protein in the presence of control siRNA or CLEC12A siRNA. The scale bars represent 10 μm. (B) Percentage of LC3-Salmonella colocalization in WT HeLa cells, ATG16L1 KO HeLa cells, and ATG16L1 KO HeLa cells stably expressing ATG16L1∗300T or ∗300A alleles in the presence of control siRNA or CLEC12A siRNA. Data are shown as means ± SD; n = 75. Data shown are representative of three independent experiments.


( A ) Confocal micrographs of LC3 - Listeria colocalization in WT and Clec12a − / − BMDMs at 1 hr post - infection . The scale bars represent 10 μm . ( B ) Quantification of LC3 - Listeria colocalization in WT and Clec12a − / − BMDMs . Data shown represent mean ± SD of n = 3 independent experiments ; unpaired t test . ∗ ∗ p < 0 . 01 .

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(A) Confocal micrographs of LC3-Listeria colocalization in WT and Clec12a−/− BMDMs at 1 hr post-infection. The scale bars represent 10 μm. (B) Quantification of LC3-Listeria colocalization in WT and Clec12a−/− BMDMs. Data shown represent mean ± SD of n = 3 independent experiments; unpaired t test. ∗∗p < 0.01.


( C and D ) Salmonella cfus quantified per gram of stool ( C ) and per organ ( D ) at 4 days post - infection are displayed . Data are shown as mean ± SD ; ∗ ∗ p ≤ 0 . 01 . Data are representative of at least two independent experiments ( n = 9 for WT ; n = 7 or 8 for Clec12a − / − ) .

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(C and D) Salmonella cfus quantified per gram of stool (C) and per organ (D) at 4 days post-infection are displayed. Data are shown as mean ± SD; ∗∗p ≤ 0.01. Data are representative of at least two independent experiments (n = 9 for WT; n = 7 or 8 for Clec12a−/−).


( E ) Clinical score for infected mice at 4 days post - infection . Data are shown as mean ± SD . Data are representative of at least two independent experiments . ∗ ∗ ∗ ∗ p < 0 . 0001 ; unpaired t test . ( n = 14 for WT ; n = 13 for Clec12a − / − ) .

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(E) Clinical score for infected mice at 4 days post-infection. Data are shown as mean ± SD. Data are representative of at least two independent experiments. ∗∗∗∗p < 0.0001; unpaired t test. (n = 14 for WT; n = 13 for Clec12a−/−).


( F ) Survival curve for infected WT and Clec12a − / − mice ( n = 9 mice per genotype ) .

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(F) Survival curve for infected WT and Clec12a−/− mice (n = 9 mice per genotype).


( A ) Gentamicin protection assay in HeLa cells showing fold replication of Salmonella in cells treated with the indicated siRNAs . Data are shown as mean ± SD ; n = 6 . Data are representative of four independent experiments .

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(A) Gentamicin protection assay in HeLa cells showing fold replication of Salmonella in cells treated with the indicated siRNAs. Data are shown as mean ± SD; n = 6. Data are representative of four independent experiments.


( B ) Quantitation of CLEC12A - bacteria and LC3 - bacteria colocalization at indicated time points . Data are shown as mean ± SD ; n = 3 independent experiments . ( C ) Representative confocal micrographs of HeLa cells showing association of intracellular bacteria with GFP - CLEC12A at 20 , 40 , 60 , and 80 min post - infection . The scale bars represent 10 μm .

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(B) Quantitation of CLEC12A-bacteria and LC3-bacteria colocalization at indicated time points. Data are shown as mean ± SD; n = 3 independent experiments. (C) Representative confocal micrographs of HeLa cells showing association of intracellular bacteria with GFP-CLEC12A at 20, 40, 60, and 80 min post-infection. The scale bars represent 10 μm.


( A - C ) Representative epifluorescent images of HeLa cells showing colocalization of ( A ) CLEC12A ( green ) and galectin 8 or galectin 3 ( red ) around intracellular Salmonella ( blue ) , ( B ) GFP - CLEC12A ( green ) and NDP52 ( red ) around Salmonella ( blue ) , and ( C ) endogenous CLEC12A ( red ) and GFP - LC3 ( green ) around Salmonella ( blue ) .

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(A-C) Representative epifluorescent images of HeLa cells showing colocalization of (A) CLEC12A (green) and galectin 8 or galectin 3 (red) around intracellular Salmonella (blue), (B) GFP-CLEC12A (green) and NDP52 (red) around Salmonella (blue), and (C) endogenous CLEC12A (red) and GFP-LC3 (green) around Salmonella (blue).


( D ) CLEC12A or GFP - CLEC12A ( green ) and GABARAPL2 or ATG16L1 ( red ) around intracellular Salmonella ( blue ) . Images are representative of three or more independent experiments .

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(D) CLEC12A or GFP-CLEC12A (green) and GABARAPL2 or ATG16L1 (red) around intracellular Salmonella (blue). Images are representative of three or more independent experiments.


( E ) Representative epifluorescent images of HeLa cells transfected with a control siRNA or a siRNA against CLEC12A and infected with DsRed - labeled Salmonella for 1 hr . ( F ) Quantitation of ubiquitin - bacteria colocalization at 1 hr post - infection in cells shown in ( E ) . Data are shown as means ± SEM from three independent experiments .

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(E) Representative epifluorescent images of HeLa cells transfected with a control siRNA or a siRNA against CLEC12A and infected with DsRed-labeled Salmonella for 1 hr. (F) Quantitation of ubiquitin-bacteria colocalization at 1 hr post-infection in cells shown in (E). Data are shown as means ± SEM from three independent experiments.


( G ) Representative epifluorescent images of HeLa cells transfected and infected as in ( E ) . ( H ) Quantitation of NDP52 - bacteria colocalization at 1 hr post - infection in cells shown in ( G ) . Data are shown as means ± SEM from three independent experiments .

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(G) Representative epifluorescent images of HeLa cells transfected and infected as in (E). (H) Quantitation of NDP52-bacteria colocalization at 1 hr post-infection in cells shown in (G). Data are shown as means ± SEM from three independent experiments.


( I ) Representative confocal micrographs of HeLa cells expressing GFP - CLEC12A . Cells were untreated or exposed to hypertonic media containing PEG 1000 , followed by hypotonic shock .

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(I) Representative confocal micrographs of HeLa cells expressing GFP-CLEC12A. Cells were untreated or exposed to hypertonic media containing PEG 1000, followed by hypotonic shock.


( E ) HeLa GFP - LC3 cells were transfected with indicated siRNAs for 48 hr and then infected with DsRed - labeled Salmonella for 1 hr . The fraction of GFP - positive intracellular Salmonella is shown . Data are mean ± SD and are representative of three independent experiments .

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(E) HeLa GFP-LC3 cells were transfected with indicated siRNAs for 48 hr and then infected with DsRed-labeled Salmonella for 1 hr. The fraction of GFP-positive intracellular Salmonella is shown. Data are mean ± SD and are representative of three independent experiments.


( F ) Gentamicin protection assay in HeLa cells showing increased intracellular bacterial replication after 48 hr of knockdown of ATG16L1 , KLHL9 , NEDD8 , or KLHL13 . Data shown as mean ± SD ; n = 4 . Data are representative of three independent experiments .

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(F) Gentamicin protection assay in HeLa cells showing increased intracellular bacterial replication after 48 hr of knockdown of ATG16L1, KLHL9, NEDD8, or KLHL13. Data shown as mean ± SD; n = 4. Data are representative of three independent experiments.


B Forest plots display the hazard ratios of genes at the 17q12 amplicon according to the DFS ( top right ) and OS ( bottom right ) of breast cancer patients in the METABRIC dataset . Genes located at the HER2 amplicon were nominated according to hazard - ratio levels ( all P - values < 0 . 01 ) . The HER2 co - amplification percentage of the indicated genes is represented as bar graphs ( left ) .

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B Forest plots display the hazard ratios of genes at the 17q12 amplicon according to the DFS (top right) and OS (bottom right) of breast cancer patients in the METABRIC dataset. Genes located at the HER2 amplicon were nominated according to hazard-ratio levels (all P-values < 0.01). The HER2 co-amplification percentage of the indicated genes is represented as bar graphs (left).


C The frequency of CDK12 amplification in HER2 - amplified - and HER2 - nonamplified breast cancer . The bar graphs indicate the frequency of HER2 and CDK12 co - amplified cases among the patients with HER2 gene amplification ( left ) . Tables show the percentage of CDK12 amplification in the indicated cohorts ( right ) . Amp , amplification ; non - amp , non - amplification .

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C The frequency of CDK12 amplification in HER2-amplified- and HER2-nonamplified breast cancer. The bar graphs indicate the frequency of HER2 and CDK12 co-amplified cases among the patients with HER2 gene amplification (left). Tables show the percentage of CDK12 amplification in the indicated cohorts (right). Amp, amplification; non-amp, non-amplification.


D Scatter plot showing the correlation between CDK12 expression and its copy number alteration ( CNA ) in METABRIC ( left ) . The r value was calculated as the Pearson ' s correlation coefficient . Box plots showing the mRNA levels of CDK12 in the indicated subtypes from METABRIC ( right ) . P - values were calculated using one - way ANOVA , followed by post - hoc LSD test .

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D Scatter plot showing the correlation between CDK12 expression and its copy number alteration (CNA) in METABRIC (left). The r value was calculated as the Pearson's correlation coefficient. Box plots showing the mRNA levels of CDK12 in the indicated subtypes from METABRIC (right). P-values were calculated using one-way ANOVA, followed by post-hoc LSD test.


E Survival analysis of breast cancer patients according to the expression of CDK12 in METABRIC ( top ) and KM plotter ( bottom ) using the Kaplan - Meier method with the log - rank test .

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E Survival analysis of breast cancer patients according to the expression of CDK12 in METABRIC (top) and KM plotter (bottom) using the Kaplan-Meier method with the log-rank test.


A FACS analysis of the percentages of CD44 + / CD24 − / ESA + breast CSC - like populations in CDK12 - overexpressing ZR - 75 - 30 and HCC - 1419 cells and CDK12 - siRNA - transfected BT474 and HCC - 1954 cells . Top , representative dot plot of CD44 - APC versus CD24 - PE expression for viable cells ( red ) and ESA - positive cells ( blue ) from the viable cells . Bottom , the percentage of CD44 + / CD24 - / ESA + cell population ( blue dots at lower right quadrant in the plots ) was quantified in the indicated cell lines . Mean ± s . d . of three independent samples . P - values were based on two - tailed Student ' s t - test ( ZR - 75 - 30 and HCC - 1419 cells ) and one - way ANOVA with a post - hoc LSD test ( BT474 and HCC - 1954 cells ) . Amp , amplification ; non - amp , non - amplification ; Trz , trastuzumab .

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A FACS analysis of the percentages of CD44+/CD24−/ESA+ breast CSC-like populations in CDK12-overexpressing ZR-75-30 and HCC-1419 cells and CDK12-siRNA-transfected BT474 and HCC-1954 cells. Top, representative dot plot of CD44-APC versus CD24-PE expression for viable cells (red) and ESA-positive cells (blue) from the viable cells. Bottom, the percentage of CD44+/CD24-/ESA+ cell population (blue dots at lower right quadrant in the plots) was quantified in the indicated cell lines. Mean ± s.d. of three independent samples. P-values were based on two-tailed Student's t-test (ZR-75-30 and HCC-1419 cells) and one-way ANOVA with a post-hoc LSD test (BT474 and HCC-1954 cells). Amp, amplification; non-amp, non-amplification; Trz, trastuzumab.


B ALDEFLUOR assay for analysing cell population with a high ALDH activity in the indicated cell lines . Cells either treated with ALDH inhibitor DEAB ( with DEAB ; negative control ) or untreated ( without DEAB ) were incubated with ALDEFLURO substrate ( BAAA ) . In the FACS analysis , the percentage of ALDEFLUOR - positive population was determined based on the shift of fluorescent cells seen in the dot plots in the absence of DEAB . Mean ± s . d . . P - values were calculated based on a two - tailed Student ' s t - test cell line information was indicated as follows : a / a , CDK12 - amplified / HER2 - amplified cells ; n / a , CDK12 non - amplified / HER2 - amplified cells S , trastuzumab - sensitive .

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B ALDEFLUOR assay for analysing cell population with a high ALDH activity in the indicated cell lines. Cells either treated with ALDH inhibitor DEAB (with DEAB; negative control) or untreated (without DEAB) were incubated with ALDEFLURO substrate (BAAA). In the FACS analysis, the percentage of ALDEFLUOR-positive population was determined based on the shift of fluorescent cells seen in the dot plots in the absence of DEAB. Mean ± s.d. . P-values were calculated based on a two-tailed Student's t-test cell line information was indicated as follows: a/a, CDK12-amplified/HER2-amplified cells; n/a, CDK12 non-amplified/HER2-amplified cells S, trastuzumab-sensitive.


C The self - renewal of CSCs in the indicated stable cell lines was analysed using a tumorsphere - formation assay . The number of tumorsphere cells ( > 100 μm in diameter ) was counted after 3 , 5 , and 7 days . Data represent the mean ± s . d . ( n = 3 ) . P - value was calculated based on a two - tailed Student ' s t - test ( ZR - 75 - 30 and HCC - 1419 cells ) or ANOVA with a post - hoc LSD test ( BT474 and HCC - 1954 cells ) . cell line information was indicated as follows : a / a , CDK12 - amplified / HER2 - amplified cells ; n / a , CDK12 non - amplified / HER2 - amplified cells ; R , trastuzumab - resistant ; PR , trastuzumab - partial responsive ; S , trastuzumab - sensitive .

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C The self-renewal of CSCs in the indicated stable cell lines was analysed using a tumorsphere-formation assay. The number of tumorsphere cells (> 100 μm in diameter) was counted after 3, 5, and 7 days. Data represent the mean ± s.d. (n = 3). P-value was calculated based on a two-tailed Student's t-test (ZR-75-30 and HCC-1419 cells) or ANOVA with a post-hoc LSD test (BT474 and HCC-1954 cells). cell line information was indicated as follows: a/a, CDK12-amplified/HER2-amplified cells; n/a, CDK12 non-amplified/HER2-amplified cells; R, trastuzumab-resistant; PR, trastuzumab-partial responsive; S, trastuzumab-sensitive.


D Cell growth of the indicated stable cell lines treated with trastuzumab ( Trz ) or vehicle ( veh ) as measured by SRB assay . Trastuzumab - sensitive HER2 + breast cancer cell lines ( S ) and trastuzumab - insensitive HER2 + breast cancer cell lines ( partial responsive , PR ; resistant , R ) were treated with 1 µg / mL or 100 µg / mL trastuzumab , respectively . Data represent the mean ± s . d . P - values were calculated using RM ANOVA with a post - hoc LSD test . cell line information was indicated as follows : a / a , CDK12 - amplified / HER2 - amplified cells ; n / a , CDK12 non - amplified / HER2 - amplified cells ; R , trastuzumab - resistant ; PR , trastuzumab - partial responsive ; S , trastuzumab - sensitive .

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D Cell growth of the indicated stable cell lines treated with trastuzumab (Trz) or vehicle (veh) as measured by SRB assay. Trastuzumab-sensitive HER2+ breast cancer cell lines (S) and trastuzumab-insensitive HER2+ breast cancer cell lines (partial responsive, PR; resistant, R) were treated with 1 µg/mL or 100 µg/mL trastuzumab, respectively. Data represent the mean ± s.d. P-values were calculated using RM ANOVA with a post-hoc LSD test. cell line information was indicated as follows: a/a, CDK12-amplified/HER2-amplified cells; n/a, CDK12 non-amplified/HER2-amplified cells; R, trastuzumab-resistant; PR, trastuzumab-partial responsive; S, trastuzumab-sensitive.