SPR sensorgram showing the binding kinetics for human ACE2 and immobilized 2019 - nCoV S . Data are shown as black lines and the best fit of the data to a 1 : 1 binding model is shown in red .

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SPR sensorgram showing the binding kinetics for human ACE2 and immobilized 2019-nCoV S. Data are shown as black lines and the best fit of the data to a 1:1 binding model is shown in red.


Negative - stain EM 2D class averages of 2019 - nCoV S bound by ACE2 . Averages have been rotated so that ACE2 is positioned above the 2019 - nCoV S protein with respect to the viral membrane . A cartoon depicting the ACE2 - bound 2019 - nCoV S protein is shown ( right ) with ACE2 in blue and S protein monomers colored tan , pink and green .

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Negative-stain EM 2D class averages of 2019-nCoV S bound by ACE2. Averages have been rotated so that ACE2 is positioned above the 2019-nCoV S protein with respect to the viral membrane. A cartoon depicting the ACE2-bound 2019-nCoV S protein is shown (right) with ACE2 in blue and S protein monomers colored tan, pink and green.


A biolayer interferometry sensorgram that shows binding to ACE2 by the 2019 - nCoV RBD - SD1 . Binding data are shown as a black line and the best fit of the data to a 1 : 1 binding model is shown in red .

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A biolayer interferometry sensorgram that shows binding to ACE2 by the 2019-nCoV RBD-SD1. Binding data are shown as a black line and the best fit of the data to a 1:1 binding model is shown in red.


Biolayer interferometry to measure cross - reactivity of the SARS - CoV RBD - directed antibodies S230 , m396 and 80R . Sensortips with immobilized antibodies were dipped into wells containing 2019 - nCoV RBD - SD1 and the resulting data are shown as a black line .

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Biolayer interferometry to measure cross-reactivity of the SARS-CoV RBD-directed antibodies S230, m396 and 80R. Sensortips with immobilized antibodies were dipped into wells containing 2019-nCoV RBD-SD1 and the resulting data are shown as a black line.


( E ) . Relative luciferase activity of the sensor ( + ) reporter or sensor ( - ) reporter induced by expression vectors of the top 5 genes identified in the screen or the GFP vector in HEK293 - FT cells . Error bars show SD ; n = 3 . Significance was assessed using 2 ‐ tailed Student ' s t ‐ test , < 0 . 05 * .

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(E). Relative luciferase activity of the sensor (+) reporter or sensor (-) reporter induced by expression vectors of the top 5 genes identified in the screen or the GFP vector in HEK293-FT cells. Error bars show SD; n=3. Significance was assessed using 2‐tailed Student's t‐test, < 0.05*.


( F ) . Heat map of relative miRNA expression of various miRNAs in HEK - 293FT cells transfected with expression vectors of the top 5 genes from the screen , or the ctrl ( GFP ) vector . Red color represents suppressed expression compared with ctrl ( GFP ) . n = 3 .

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(F). Heat map of relative miRNA expression of various miRNAs in HEK-293FT cells transfected with expression vectors of the top 5 genes from the screen, or the ctrl (GFP) vector. Red color represents suppressed expression compared with ctrl (GFP). n=3.


( A ) . Volcano plot of TaqMan array showing the mean average expression and p - value of miRNA expression profiles between TruB1 KD and ctrl ( scramble ) . N = 3 . Significance was assessed using 2 ‐ tailed Student ' s t ‐ test . MiRNAs suppressed ( Relative expression TruB1 KD / ctrl < 0 . 8 and p - value < 0 . 1 ) are enclosed by the red square .

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(A). Volcano plot of TaqMan array showing the mean average expression and p-value of miRNA expression profiles between TruB1 KD and ctrl (scramble). N=3. Significance was assessed using 2‐tailed Student's t‐test. MiRNAs suppressed (Relative expression TruB1 KD / ctrl <0.8 and p-value < 0.1) are enclosed by the red square.


( D ) . Relative expression of let - 7 family miRNAs and other miRNAs in HEK293FT cells with TruB1 KD or ctrl ( siRNA ) determined by qRT - PCR . Significance was assessed using 2 ‐ tailed Student ' s t ‐ test , < 0 . 05 * . Data information : All experiments were performed in triplicate . Error bars show SD .

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(D). Relative expression of let-7 family miRNAs and other miRNAs in HEK293FT cells with TruB1 KD or ctrl (siRNA) determined by qRT-PCR. Significance was assessed using 2‐tailed Student's t‐test, < 0.05*. Data information: All experiments were performed in triplicate. Error bars show SD.


( E ) . Relative expression of primary - let - 7 family miRNAs as in ( D ) determined by qRT - PCR . Significance was assessed using 2 ‐ tailed Student ' s t ‐ test , < 0 . 05 * . Data information : All experiments were performed in triplicate . Error bars show SD .

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(E). Relative expression of primary-let-7 family miRNAs as in (D) determined by qRT-PCR. Significance was assessed using 2‐tailed Student's t‐test, < 0.05*. Data information: All experiments were performed in triplicate. Error bars show SD.


( C ) . Relative miRNA expression of let - 7a in HEK - 293 cells infected with tetracycline - inducible expressing lentiviruses for TruB1 , mt1 , mt2 or GFP 5 days after doxycycline treatment , as determined by qRT - PCR . Significance was assessed using 2 ‐ tailed Student ' s t ‐ test , < 0 . 05 * . Data information : All experiments were performed in triplicate . Error bars show SD .

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(C). Relative miRNA expression of let-7a in HEK-293 cells infected with tetracycline-inducible expressing lentiviruses for TruB1, mt1, mt2 or GFP 5 days after doxycycline treatment, as determined by qRT-PCR. Significance was assessed using 2‐tailed Student's t‐test, < 0.05*. Data information: All experiments were performed in triplicate. Error bars show SD.


( D ) . Northern blotting for let - 7a and RNU6B in HeLa cells infected with lentiviruses encoding tetracycline - inducible expression of TruB1 , mt1 , mt2 , or GFP , 5 days after doxycycline treatment .

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(D). Northern blotting for let-7a and RNU6B in HeLa cells infected with lentiviruses encoding tetracycline-inducible expression of TruB1, mt1, mt2, or GFP, 5 days after doxycycline treatment.


( G ) . Location of pseudouridine sites detected by the CMC primer extension method . Total RNA purified from HEK - 293FT cells were treated with CMC . CMC treated RNA were reverse - transcribed with RI - labelled specific primers for tRNAphe or pri - let - 7b . ddATP was used for sequence control . Pseudouridines are indicated by black arrows .

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(G). Location of pseudouridine sites detected by the CMC primer extension method. Total RNA purified from HEK-293FT cells were treated with CMC. CMC treated RNA were reverse-transcribed with RI-labelled specific primers for tRNAphe or pri-let-7b. ddATP was used for sequence control. Pseudouridines are indicated by black arrows.


( A ) . RIP analysis of pri - let - 7a1 and Flag - TruB1 from HEK - 293FT cells . RNA was extracted from IP material and analyzed by qRT - PCR . HEK - 293FT cells were infected with lentiviruses encoding tetracycline - inducible expression of TruB1 , mt1 , mt2 , or GFP and treated with doxycycline . Error bars show SD ; n = 3 . Significance was assessed using 2 ‐ tailed Student ' s t ‐ test , < 0 . 05 * .

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(A). RIP analysis of pri-let-7a1 and Flag-TruB1 from HEK-293FT cells. RNA was extracted from IP material and analyzed by qRT-PCR. HEK-293FT cells were infected with lentiviruses encoding tetracycline-inducible expression of TruB1, mt1, mt2, or GFP and treated with doxycycline. Error bars show SD; n=3. Significance was assessed using 2‐tailed Student's t‐test, < 0.05*.


( B ) . EMSA of 32p - ATP - labelled pri - let - 7a1 or pri - let - 7a1 loop mt mixed with recombinant TruB1 , mt1 or mt2 at several doses . RNP : Ribonucleoprotein complexes .

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(B). EMSA of 32p-ATP-labelled pri-let-7a1 or pri-let-7a1 loop mt mixed with recombinant TruB1, mt1 or mt2 at several doses. RNP: Ribonucleoprotein complexes.


( E ) . Tag densities represented as dots . Density of miRNA clusters in HITS - CLIP were normalized by miRNA expression as determined by TaqMan array .

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(E). Tag densities represented as dots. Density of miRNA clusters in HITS-CLIP were normalized by miRNA expression as determined by TaqMan array.


( A ) . In vitro processing assay for RI - labelled pri - let - 7a1 . Autoradiographs of gels showing pri - let - 7a1 treated with whole cell lysate ( WCL ) from HEK - 293FT cells transfected with GFP , TruB1 , mt1 , or mt2 ( overexpression , left ) , and TruB1 KD or ctrl ( siRNA , right ) . Dicer KD was used to show the height of pre - let - 7a1 . ( B ) RI intensities of ( A ) were quantified and normalized to ctrl . Relative processing rate of pri - let - 7a1 into pre - and mature - are shown . Significance was assessed using 2 ‐ tailed Student ' s t ‐ test , < 0 . 05 * . Data information : All experiments were performed in triplicate . Error bars show SD .

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(A). In vitro processing assay for RI-labelled pri-let-7a1. Autoradiographs of gels showing pri-let-7a1 treated with whole cell lysate (WCL) from HEK-293FT cells transfected with GFP, TruB1, mt1, or mt2 (overexpression, left), and TruB1 KD or ctrl (siRNA, right). Dicer KD was used to show the height of pre-let-7a1. (B) RI intensities of (A) were quantified and normalized to ctrl. Relative processing rate of pri-let-7a1 into pre- and mature- are shown. Significance was assessed using 2‐tailed Student's t‐test, < 0.05*. Data information: All experiments were performed in triplicate. Error bars show SD.


( C ) . RIP assay of pri - let - 7a1 and DGCR8 from HEK - 293FT cells . Western blotting for input or immunoprecipitate ( IP ) using anti - DGCR8 antibody and anti - actin antibody are shown on top . RNA was extracted from IP material and analyzed by qRT - PCR ( bottom ) . Significance was assessed using 2 ‐ tailed Student ' s t ‐ test , < 0 . 05 * . ( D ) . RIP assay of pri - let - 7a1 and Flag - tagged TruB1 from HEK - 293FT cells with Lin28B KD or ctrl ( siRNA ) . Western blotting for input or IP material using anti - Flag antibody , anti - Lin28B antibody , and anti - actin antibody are shown on top . RNA was extracted from IP material and analyzed by qRT - PCR ( bottom ) . Significance was assessed using 2 ‐ tailed Student ' s t ‐ test , < 0 . 05 * . Data information : All experiments were performed in triplicate . Error bars

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(C). RIP assay of pri-let-7a1 and DGCR8 from HEK-293FT cells. Western blotting for input or immunoprecipitate (IP) using anti-DGCR8 antibody and anti-actin antibody are shown on top. RNA was extracted from IP material and analyzed by qRT-PCR (bottom). Significance was assessed using 2‐tailed Student's t‐test, < 0.05*. (D). RIP assay of pri-let-7a1 and Flag-tagged TruB1 from HEK-293FT cells with Lin28B KD or ctrl (siRNA). Western blotting for input or IP material using anti-Flag antibody, anti-Lin28B antibody, and anti-actin antibody are shown on top. RNA was extracted from IP material and analyzed by qRT-PCR (bottom). Significance was assessed using 2‐tailed Student's t‐test, < 0.05*. Data information: All experiments were performed in triplicate. Error bars show


( B ) . Relative luciferase activity of KRAS reporter or Ctrl ( empty ) reporter in HEK293FT cells infected with lentiviruses expressing tetracycline - inducible TruB1 , mt1 , mt2 , or GFP 5 days after doxycycline treatment . Significance was assessed using 2 ‐ tailed Student ' s t ‐ test , < 0 . 05 * . Data information : All experiments were performed in triplicate . Error bars show SD .

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(B). Relative luciferase activity of KRAS reporter or Ctrl (empty) reporter in HEK293FT cells infected with lentiviruses expressing tetracycline-inducible TruB1, mt1, mt2, or GFP 5 days after doxycycline treatment. Significance was assessed using 2‐tailed Student's t‐test, < 0.05*. Data information: All experiments were performed in triplicate. Error bars show SD.


( D ) . Relative expression of let - 7 families determined by qRT - PCR in HEK - 293FT cells with KD of let - 7 family members or ctrl ( using 2 ' - O - methylated antisense inhibitor ) . Significance was assessed using 2 ‐ tailed Student ' s t ‐ test , < 0 . 05 * . Data information : All experiments were performed in triplicate . Error bars show SD .

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(D). Relative expression of let-7 families determined by qRT-PCR in HEK-293FT cells with KD of let-7 family members or ctrl (using 2'-O-methylated antisense inhibitor). Significance was assessed using 2‐tailed Student's t‐test, < 0.05*. Data information: All experiments were performed in triplicate. Error bars show SD.


( E ) . Real - time glo assay for HEK293FT cells infected with lentiviruses expressing tetracycline - inducible TruB1 or GFP , 5 days after doxycycline treatment , with or without KD of let - 7 family members . GFP : GFP without let - 7 KD . WT : overexpression of wild type TruB1 without let - 7 KD . WT + let7 KD : WT : overexpression of wild type TruB1 with let - 7 KD . GFP + let - 7KD : GFP with let - 7 KD . Significance was assessed using 2 ‐ tailed Student ' s t ‐ test , < 0 . 05 * .

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(E). Real-time glo assay for HEK293FT cells infected with lentiviruses expressing tetracycline-inducible TruB1 or GFP, 5 days after doxycycline treatment, with or without KD of let-7 family members. GFP: GFP without let-7 KD. WT: overexpression of wild type TruB1 without let-7 KD. WT + let7 KD: WT: overexpression of wild type TruB1 with let-7 KD. GFP + let-7KD: GFP with let-7 KD. Significance was assessed using 2‐tailed Student's t‐test, < 0.05*.


Gating strategy for sorting of EpCAMhighCD24lowSca ‑ 1 + cells and rMC from lung homogenate of adult mice .

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Gating strategy for sorting of EpCAMhighCD24lowSca‑1+ cells and rMC from lung homogenate of adult mice.


Time - course of epithelial stem / progenitor cell proliferation and differentiation within BALO at days 8 , 11 and 21 of co - culture with rMC .

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Time-course of epithelial stem/progenitor cell proliferation and differentiation within BALO at days 8, 11 and 21 of co-culture with rMC.


Clonal expansion of EpCAMhighCD24lowSca ‑ 1 + cells derived from either GFP - expressing mice or tdTomato - expressing ROSAmT / mG mice at day 21 of co ‑ culture .

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Clonal expansion of EpCAMhighCD24lowSca‑1+ cells derived from either GFP-expressing mice or tdTomato-expressing ROSAmT/mG mice at day 21 of co‑culture.


Percentages of SCGB1A1 + SFTPC + ( BALO ) , SCGB1A1 + SFTPC - ( bronchiolospheres ) and SCGB1A1 - SFTPC + ( alveolospheres ) organoids per well at day 21 of culture derived from EpCAMhighCD24lowSca - 1 + cells isolated from Scgb1a1mCherrySftpcYFP reporter mice ( n = 4 biological replicates ) .

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Percentages of SCGB1A1+SFTPC+ (BALO), SCGB1A1+SFTPC- (bronchiolospheres) and SCGB1A1-SFTPC+ (alveolospheres) organoids per well at day 21 of culture derived from EpCAMhighCD24lowSca-1+ cells isolated from Scgb1a1mCherrySftpcYFP reporter mice (n=4 biological replicates).


Representative confocal images of days 8 to 21 of culture showing endogenous SCGB1A1 and SFTPC expression during BALO formation derived from EpCAMhighCD24lowSca - 1 + SCGB1A1 + SFTPC + cells isolated from Scgb1a1mCherrySftpcYFP reporter mice .

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Representative confocal images of days 8 to 21 of culture showing endogenous SCGB1A1 and SFTPC expression during BALO formation derived from EpCAMhighCD24lowSca-1+SCGB1A1+SFTPC+ cells isolated from Scgb1a1mCherrySftpcYFP reporter mice.


Heat map ( left ) and tSNE plot ( right ) ( F ) of digested day 21 BALO cultures depicting four distinct clusters ( C1 , airway , blue ; C2 , alveolar , purple ; C3 , MYO , orange , and C4 , LIF , green ) .

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Heat map (left) and tSNE plot (right) (F) of digested day 21 BALO cultures depicting four distinct clusters (C1, airway, blue; C2, alveolar, purple; C3, MYO, orange, and C4, LIF, green).


Violin plots of selected genes representing airway associated - genes ( G ) ( Itgb4 , Trp63 , Krt7 , and Sox2 ) and alveoli associated ‑ genes ( H ) ( Cxcl15 , Lyz2 , Sftpc , and Hopx ) . Each violin plot shows the frequency distribution of the mean transcript level ( log2 ) .

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Violin plots of selected genes representing airway associated-genes (G) (Itgb4, Trp63, Krt7, and Sox2) and alveoli associated‑genes (H) (Cxcl15, Lyz2, Sftpc, and Hopx). Each violin plot shows the frequency distribution of the mean transcript level (log2).


mRNA expression analysis of epithelial cell differentiation markers Sftpc ( AEC II ) , Hopx ( AEC I ) , Foxj1 ( ciliated cells ) , Muc5ac ( secretory cells ) and p63 ( basal cells ) in BALO at days 0 , 10 , and 21 of culture ( n = 3 biological replicates with pooled cells from 4 cultures per replicate )

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mRNA expression analysis of epithelial cell differentiation markers Sftpc (AEC II), Hopx (AEC I), Foxj1 (ciliated cells), Muc5ac (secretory cells) and p63 (basal cells) in BALO at days 0, 10, and 21 of culture (n=3 biological replicates with pooled cells from 4 cultures per replicate)


Electron microscopy of BALO alveoli showing two cuboidal epithelial cells ( AEC II ) connected by tight junctions ( red square ) with numerous mitochondria ( M ) and lamellar bodies ( LB ) . A LIF with numerous lipid droplets ( * ) and a MYO with cisterns of rough ER ( red arrow ) are located at the basal side ( left ) . The alveolar lumen is filled with lamellar surfactant including tubular myelin ( white arrow ) ( lower right ) . Exocytosis of a lamellar body ( * ) from an AEC II ( upper right ) . Scale bars indicate 500 nm .

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Electron microscopy of BALO alveoli showing two cuboidal epithelial cells (AEC II) connected by tight junctions (red square) with numerous mitochondria (M) and lamellar bodies (LB). A LIF with numerous lipid droplets (*) and a MYO with cisterns of rough ER (red arrow) are located at the basal side (left). The alveolar lumen is filled with lamellar surfactant including tubular myelin (white arrow) (lower right). Exocytosis of a lamellar body (*) from an AEC II (upper right). Scale bars indicate 500 nm.


Electron microscopy of bronchiolar - like airway depicting columnar ciliated cells ( red arrowhead ) with basal bodies ( black arrowhead ) at the left side of the longitudinally sectioned lumen . The boxed area indicates an apical junctional complex between two ciliated cells . 1 = tight junction , 2 = adherens junction . Scale bar indicates 500 nm ( in insert : 100 nm ) .

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Electron microscopy of bronchiolar-like airway depicting columnar ciliated cells (red arrowhead) with basal bodies (black arrowhead) at the left side of the longitudinally sectioned lumen. The boxed area indicates an apical junctional complex between two ciliated cells. 1 = tight junction, 2 = adherens junction. Scale bar indicates 500 nm (in insert: 100 nm).


Staining of lamellar bodies in BALO with RFP LysoTracker . Scale bars represent 100 μm .

Staining of lamellar bodies in BALO with RFP LysoTracker. Scale bars represent 100 μm.


Western blot analysis of the surfactant proteins : pro - SPC , mature SPC , pro - SPB , mature SPB , and SPA in lung homogenate ( LH ) , AEC and day 21 BALO ( n = 3 biological replicates ) .

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Western blot analysis of the surfactant proteins: pro-SPC, mature SPC, pro-SPB, mature SPB, and SPA in lung homogenate (LH), AEC and day 21 BALO (n=3 biological replicates).


F - G . Alveolar diameter ( F ) and number of alveoli ( G ) in BALO at days 15 , 21 , 30 and 40 of culture was measured from n = 5 BALO in n = 3 biological replicates with tdTomato + rMC .

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F-G. Alveolar diameter (F) and number of alveoli (G) in BALO at days 15, 21, 30 and 40 of culture was measured from n=5 BALO in n=3 biological replicates with tdTomato+ rMC.


H - I . Representative scheme and images of day 40 BALO alveoli ( H ) and airway ( I ) . BALO alveolar - like structures ( H ) are shown ( h ' ) ( red arrowheads ) in semithin section ( 0 . 5 µm ) stained with Toluidine blue . Scale bar indicates 100 μm ( left ) . Electron microscopy showing AEC I ( h ' ' ) ultrastructure within BALO . Scale bars indicate 2500 nm ( center ) and 1000 nm ( right ) . An airway - like structure ( i ' ) is shown with secretory and ciliated cells ( red arrowheads ) in semithin sections ( 0 . 5 µm ) longitudinally cut and stained with Toluidine blue . Scale bar indicates 50 μm ( far left ) . Electron microscopy of a bronchiolar - like airway ( I ) depicting pseudostratified epithelium ( i ' ' ) with a basal - like cell ( BC ) , not reaching the lumen in which cilia ( C ) are seen , located between a secretory ( SC ) and a ciliated cell ( Ci ) . Scale bars indicate 1000 nm ( left and right ) . Mature cilia ( i ' ' ' ) in BALO at higher magnification depicting the 9x2 + 2 structure with central doubled microtubules ( insert , red arrowhead ) , a characteristic for motile cilia . Scale bar indicates 100 nm ( far right ) .

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H-I. Representative scheme and images of day 40 BALO alveoli (H) and airway (I). BALO alveolar-like structures (H) are shown (h') (red arrowheads) in semithin section (0.5 µm) stained with Toluidine blue. Scale bar indicates 100 μm (left). Electron microscopy showing AEC I (h'') ultrastructure within BALO. Scale bars indicate 2500 nm (center) and 1000 nm (right). An airway-like structure (i') is shown with secretory and ciliated cells (red arrowheads) in semithin sections (0.5 µm) longitudinally cut and stained with Toluidine blue. Scale bar indicates 50 μm (far left). Electron microscopy of a bronchiolar-like airway (I) depicting pseudostratified epithelium (i'') with a basal-like cell (BC), not reaching the lumen in which cilia (C) are seen, located between a secretory (SC) and a ciliated cell (Ci). Scale bars indicate 1000 nm (left and right). Mature cilia (i''') in BALO at higher magnification depicting the 9x2+2 structure with central doubled microtubules (insert, red arrowhead), a characteristic for motile cilia. Scale bar indicates 100 nm (far right).


Representative images of day 21 BALO and tdTomato + rMC stained with LipidTOX ( green ) . Scale bar represents 100 μm .

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Representative images of day 21 BALO and tdTomato+ rMC stained with LipidTOX (green). Scale bar represents 100 μm.


Fluorescence images of αSMA ( green ) and neutral lipids ( LipidTOX red ) staining in WT ‑ derived rMC and BALO ( dotted lines indicate single BALO or insert ) at day 21 of culture . Scale bars represent 50 μm ( left ) and 25 μm ( right ) .

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Fluorescence images of αSMA (green) and neutral lipids (LipidTOX red) staining in WT‑derived rMC and BALO (dotted lines indicate single BALO or insert) at day 21 of culture. Scale bars represent 50 μm (left) and 25 μm (right).


Representative flow cytometric dot plots and histograms of PDGFRα expression in rMC ( EpCAM - CD45 - CD31 - Sca - 1 + ) isolated from the lung homogenate of PdgfraGFP reporter mice .

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Representative flow cytometric dot plots and histograms of PDGFRα expression in rMC (EpCAM-CD45-CD31-Sca-1+) isolated from the lung homogenate of PdgfraGFP reporter mice.


Representative images of BALO formation at day 8 , 15 and 21 of co - culture . WT BASC were co ‑ cultivated with sorted rMC ( total PDGFRα + population ) or rMC expressing either low or high levels of PDGFRα ‑ GFP . Scale bars represent 100 μm .

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Representative images of BALO formation at day 8, 15 and 21 of co-culture. WT BASC were co‑cultivated with sorted rMC (total PDGFRα+ population) or rMC expressing either low or high levels of PDGFRα‑GFP. Scale bars represent 100 μm.


tSNE plots and violin plots depicting selected genes representing MYO ( Pdgfrα , Tagln , Acta2 , Eln , and Axin2 ) ( top panels ) and LIF associated - genes ( Fgf10 , Apoe , Serpina3n , Gsn , and Gas6 ) ( bottom panels ) . Each violin plot shows the frequency distribution of the mean transcript level ( log2 ) . C3 ( MYO , orange ) and C4 ( LIF , green ) refer to the scRNA - Seq experiment in Fig . 1G .

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tSNE plots and violin plots depicting selected genes representing MYO (Pdgfrα, Tagln, Acta2, Eln, and Axin2) (top panels) and LIF associated-genes (Fgf10, Apoe, Serpina3n, Gsn, and Gas6) (bottom panels). Each violin plot shows the frequency distribution of the mean transcript level (log2). C3 (MYO, orange) and C4 (LIF, green) refer to the scRNA-Seq experiment in Fig. 1G.


Fluorescence image of αSMA + PDGFRαhigh MYO ( yellow arrows ) and LipidTOX + PDGFRαlow LIF ( red arrows ) from sorted PDGFRα - GFP rMC after 21 days of BALO culture . Scale bars represent 50 μm ( left ) and 25 μm ( right ) .

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Fluorescence image of αSMA+PDGFRαhigh MYO (yellow arrows) and LipidTOX+PDGFRαlow LIF (red arrows) from sorted PDGFRα-GFP rMC after 21 days of BALO culture. Scale bars represent 50 μm (left) and 25 μm (right).


Fluorescence image containing PDGFRαhigh MYO from sorted PDGFRα - GFP rMC within BALO derived from tdTomato + mice at day 21 of culture . Scale bars represent 100 μm ( left ) and 50 μm ( right ) .

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Fluorescence image containing PDGFRαhigh MYO from sorted PDGFRα-GFP rMC within BALO derived from tdTomato+ mice at day 21 of culture. Scale bars represent 100 μm (left) and 50 μm (right).


Gating strategy to define TR ‑ Mac from BAL of adult tdTomato + mice

Gating strategy to define TR‑Mac from BAL of adult tdTomato+ mice


Representative images of BALO after microinjection of tdTomato + TR - Mac at day 14 . TR - Mac are preferentially found in alveoli ( right ) . Scale bar represent 100 μm ( left ) and 50 μm ( right )

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Representative images of BALO after microinjection of tdTomato+ TR-Mac at day 14. TR-Mac are preferentially found in alveoli (right). Scale bar represent 100 μm (left) and 50 μm (right)


Fluorescence confocal images of CD206 , Siglec - F , and isotype control staining 14 days after tdTomato + TR ‑ Mac microinjection in a day 28 BALO . Scale bars represent 100 μm ( left ) and 25 μm ( right )

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Fluorescence confocal images of CD206, Siglec-F, and isotype control staining 14 days after tdTomato+ TR‑Mac microinjection in a day 28 BALO. Scale bars represent 100 μm (left) and 25 μm (right)


Electron microscopy depicting filopodium of TR ‑ Mac ( white dashed lines ) with a characteristic actin filament bundle ( yellow background ) ( left panel ) in contact with AEC I within BALO alveolar - like structures . Uptake of surfactant by TR - mac is depicted by a ( * ) . Scale bar indicates 1000nm ( left , in insert : 500 nm ) and 500 nm ( right , in insert : 250 nm )

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Electron microscopy depicting filopodium of TR‑Mac (white dashed lines) with a characteristic actin filament bundle (yellow background) (left panel) in contact with AEC I within BALO alveolar-like structures. Uptake of surfactant by TR-mac is depicted by a (*). Scale bar indicates 1000nm (left, in insert: 500 nm) and 500 nm (right, in insert: 250 nm)


Representative confocal images of Cx43 staining in tdTomato + TR - Mac monoculture in Matrigel and tdTomato + TR ‑ Mac microinjected at day 14 and analyzed in a mature day 21 BALO . Scale bars represent 50 μm ( overview ) and 5 μm ( close up )

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Representative confocal images of Cx43 staining in tdTomato+ TR-Mac monoculture in Matrigel and tdTomato+ TR‑Mac microinjected at day 14 and analyzed in a mature day 21 BALO. Scale bars represent 50 μm (overview) and 5 μm (close up)


Heat map ( left ) and tSNE plot ( right ) of the comparative analysis of digested day 23 BALO cultures with ( + ) and without ( - ) microinjected TR ‑ Mac depicting six distinct clusters ( C1 , club / secretory cells , red ; C2 , basal cells , yellow ; C3 , rMC , green ; C4 , AEC II , blue ; C5 , AEC I , purple ; and C6 , ciliated cells , pink )

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Heat map (left) and tSNE plot (right) of the comparative analysis of digested day 23 BALO cultures with (+) and without (-) microinjected TR‑Mac depicting six distinct clusters (C1, club/secretory cells, red; C2, basal cells, yellow; C3, rMC, green; C4, AEC II, blue; C5, AEC I, purple; and C6, ciliated cells, pink)


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

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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


Percentage of terminally differentiated cells ( AEC I and ciliated cells ) in day 23 BALO cultures with ( + ) and 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 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 .

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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 ) and 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 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 .

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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 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 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 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 .

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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 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 ) .

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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 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

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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


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

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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 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 )

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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 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

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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 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

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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 and PR8 IAV - infected BALO at 48 h pi ( n = 3 biological replicates with pooled cells 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 the supernatant of mock and IAV infected BALO cultures with and 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 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 ) .

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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 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 .

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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.


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 .

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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.


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 .

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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.


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 .

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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 ) 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 .

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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 ) 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 .

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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 , 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 .

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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 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 .

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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 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 )

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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 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 .

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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 - test ; * 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 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 .

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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 ) 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 .

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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


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 .

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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 and 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 and CFU colonies analysed . protein 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 and 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 age ( 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).


based on peripheral blood composition was determined by flow cytometry at 4 months of age ( 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 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 .

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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 , white blood cells ( WBC ) and bone marrow ( BM ) of mice at 6 months of age ( 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).


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 .

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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 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 ) .

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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 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 .

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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 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

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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 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 :

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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


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

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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


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

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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 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 .

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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 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 .

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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 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 .

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(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 - 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 .

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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 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 .

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

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 .

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

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 .

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

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 ) .

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

(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 ) .

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

(F) 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).