A comparison of SILAC - derived abundance as a function of time , for several that either show differential behaviour between WT mutants ( Idh2 , Idp1 Aco2 ) that behave similarly between genetic backgrounds tested ( Hsp78 ) . Shown are mean ratios ( two replicates for mutants , three replicates for WT ) , error bars indicating peptide - relative s . d .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 comparison of the SILAC-derived abundance as a function of time, for several proteins that either show differential behaviour between WT and the mutants (Idh2, Idp1 and Aco2) and proteins that behave similarly between all the genetic backgrounds tested (Hsp78). Shown are mean protein ratios (two replicates for mutants, three replicates for WT), error bars indicating peptide-based relative s.d.


WT ( a ) , dnm1Δ ( b ) atg1Δ ( c ) atg32Δ ( d ) expressing chromosomally tagged GFP chimeras of Aco2 , Idh2 , Idp1 Hsp78 were incubated in SL medium for 7 days , 10 OD600 units were sampled at day 1 day 7 processed for immunoblotting with anti - GFP antibodies as described in Methods section . Positive control used in c d was from dnm1Δ expressing Idp1 - GFP from a plasmid at 4 - day incubation ( Fig . 1b , lane 4 ) . Release of free GFP in WT is reproducibly variable between these reporters , varies between 0 ( Idh2 ) 60 % ( Aco2 ) . ( e ) Densitometric of release of free GFP ( as % of total signal ) in four independent experiments , comparing results between WT dnm1Δ . Bars denote s . d . ( n = 4 ) .

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

WT (a), dnm1Δ (b) and atg1Δ cells (c) and atg32Δ cells (d) expressing chromosomally tagged GFP chimeras of Aco2, Idh2, Idp1 and Hsp78 were incubated in SL medium for 7 days, and 10 OD600 units were sampled at day 1 and day 7 and processed for immunoblotting with anti-GFP antibodies as described in the Methods section. Positive control used in c and d was from dnm1Δ cells expressing Idp1-GFP from a plasmid at 4-day incubation (Fig. 1b, lane 4). Release of free GFP in WT cells is reproducibly variable between these reporters, and varies between 0 (Idh2) and 60% (Aco2). (e) Densitometric quantification of the release of free GFP (as % of total signal) in four independent experiments, comparing the results between WT and dnm1Δ cells. Bars denote s.d. (n=4).


( a ) expressing chromosomally tagged GFP chimeras of Aco2 , Idh2 , Idp1 Hsp78 as well as a plasmid - borne ( pDJB12 ) , mitochondrially targeted RFP ( mtRFP ) were incubated in SL - leucine medium for 3 days imaged daily . Aco2 Idp1 show clear mitophagic targeting ( arrows point at specific examples ) by day 3 , whereas Idh2 Hsp78 show weak or no mitophagy . In addition , Idh2 Hsp78 show different degrees of segregation relative to mtRFP marker , whereas Idp1 - GFP Aco2 - GFP show a near - complete co - localization with mtRFP . Scale bar , 5 μm . ( b ) of frequency of mitophagy from a ; of showing mitophagic profiles in red ( magenta bars ; plasmid - borne mtRFP ) or green ( turquoise bars ; integrated C - terminal GFP chimerae ) was calculated for each GFP chimera . Idp - GFP , red N = 76 , green N = 82 ; Aco2 - GFP , red N = 79 , green N = 82 ; Idh2 - GFP , red N = 72 , green N = 72 ; Hsp78 - GFP , red N = 70 , green N = 70 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Cells expressing chromosomally tagged GFP chimeras of Aco2, Idh2, Idp1 and Hsp78 as well as a plasmid-borne (pDJB12), mitochondrially targeted RFP (mtRFP) were incubated in SL-leucine medium for 3 days and imaged daily. Aco2 and Idp1 show clear mitophagic targeting (white arrows point at specific examples) by day 3, whereas Idh2 and Hsp78 show weak or no mitophagy. In addition, Idh2 and Hsp78 show different degrees of segregation relative to the mtRFP marker, whereas Idp1-GFP and Aco2-GFP show a near-complete co-localization with mtRFP. Scale bar, 5 μm. (b) Quantification of the frequency of mitophagy from a; the percentage of cells showing mitophagic profiles in the red (magenta bars; plasmid-borne mtRFP) or green (turquoise bars; integrated C-terminal GFP chimerae) was calculated for each GFP chimera. Idp-GFP, red N=76, green N=82; Aco2-GFP, red N=79, green N=82; Idh2-GFP, red N=72, green N=72; Hsp78-GFP, red N=70, green N=70.


WT dnm1Δ expressing integrated Hsp78 - GFP plasmid - borne mtRFP were grown on lactate medium for 3 days . were imaged daily were analysed using ImageJ as described in Methods . Graphs illustrate distribution of pixels associated with specific values of RFP GFP channel intensities , respectively . Diagonal concentrations of dots indicate spatial correlation between signals . coloured photographic panels at bottom illustrate increased correlation of two channels at 3 - day time point in dnm1Δ mutant , relative to WT . Scale bar , 5 μm .

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

WT and dnm1Δ cells expressing integrated Hsp78-GFP and plasmid-borne mtRFP were grown on lactate medium for 3 days. Cells were imaged daily and the images were analysed using ImageJ as described in Methods. Graphs illustrate the distribution of pixels associated with specific values of RFP and GFP channel intensities, respectively. Diagonal concentrations of dots indicate spatial correlation between the signals. The coloured photographic panels at the bottom illustrate the increased intensity correlation of the two channels at the 3-day time point in the dnm1Δ mutant, relative to the WT cells. Scale bar, 5 μm.


B , C Serial transplantations of PR + / + PR − / − mammary epithelia . ( B ) stereo microscopy of third ‐ generation mammary outgrowths derived from 8 ‐ week ‐ old PR + / + ; EGFP PR − / − ; EGFP donor mice . ( C ) Table summarizing 3 independent serial transplant experiments with PR + / + ; EGFP PR − / − ; EGFP . Each engrafted is represented by a micrograph ; black sectors represent of fat pad filled by engrafted epithelium . Scale bar : 200 μm .

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B, C Serial transplantations of PR+/+ and PR−/− mammary epithelia. (B) Fluorescence stereo microscopy of third‐generation mammary outgrowths derived from 8‐week‐old PR+/+; EGFP and PR−/−; EGFP donor mice. (C) Table summarizing 3 independent serial transplant experiments with PR+/+; EGFP and PR−/−; EGFP. Each engrafted gland is represented by a micrograph; black sectors represent area of fat pad filled by engrafted epithelium. Scale bar: 200 μm.


D , E Serial transplantation of RANKL + / + RANKL − / − mammary epithelia . ( D ) stereo microscopy of third ‐ generation mammary outgrowths derived from 5 ‐ week ‐ old RANKL + / + ; EGFP RANKL − / − ; EGFP donor mice . Insets : higher magnification showing side branches present in WT control ( arrowheads ) absent from RANKL − / − ; EGFP epithelium . ( E ) Table summarizing three independent serial transplant experiments with RANKL + / + ; EGFP RANKL − / − ; EGFP donor mice . Scale bar : 200 μm .

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D, E Serial transplantation of RANKL+/+ and RANKL−/− mammary epithelia. (D) Fluorescence stereo microscopy of third‐generation mammary outgrowths derived from 5‐week‐old RANKL+/+; EGFP and RANKL−/−; EGFP donor mice. Insets: higher magnification showing side branches present in the WT control (arrowheads) absent from RANKL−/−; EGFP epithelium. (E) Table summarizing three independent serial transplant experiments with RANKL+/+; EGFP and RANKL−/−; EGFP donor mice. Scale bar: 200 μm.


F , G Serial transplantation of Wnt4 + / + Wnt4 − / − mammary epithelia . ( F ) stereo microscopy of third ‐ generation mammary outgrowths derived from mammary buds of E12 . 5 E13 . 5 Wnt4 + / + ; EGFP Wnt4 − / − ; EGFP . Scale bar : 200 μm . ( G ) Table summarizing three independent serial transplant experiments with Wnt4 + / + ; EGFP donor mice . Scale bar : 200 μm .

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

F, G Serial transplantation of Wnt4+/+ and Wnt4−/− mammary epithelia. (F) Fluorescence stereo microscopy of third‐generation mammary outgrowths derived from mammary buds of E12.5 and E13.5 Wnt4+/+; EGFP and Wnt4−/−; EGFP embryos. Scale bar: 200 μm. (G) Table summarizing three independent serial transplant experiments with Wnt4+/+; EGFP donor mice. Scale bar: 200 μm.


H Box plot showing difference between of reconstitution between WT MT grafts in each transplant generation . P values were determined by Mann - Whitney U ‐ .

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H Box plot showing the difference between percentage of reconstitution between WT and MT contralateral grafts in each transplant generation. P values were determined by Mann-Whitney U‐test.


A Epifluorescence stereo microscopy of inguinal mammary from a 5 ‐ day ‐ old Wnt4 : : Cre ; mT / mG ( n = 7 ) . Scale bars : 0 . 5 mm 0 . 1 mm ( inset ) .

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A Epifluorescence stereo microscopy of inguinal mammary gland from a 5‐day‐old Wnt4::Cre; mT/mG female (n = 7). Scale bars: 0.5 mm and 0.1 mm (inset).


B , C Histology sections of mammary from a 5 ‐ day ‐ old ( B ; n = 7 ) a 10 ‐ day ‐ old ( C ; n = 5 ) Wnt4 : : Cre ; mT / mG stained by double immunofluorescence microscopy for EGFP ( green ) PR ( magenta , not detected ) , counterstained with DAPI ( blue ) . Scale bar : 50 mm .

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

B, C Histology sections of mammary glands from a 5‐day‐old (B; n = 7) and a 10‐day‐old (C; n = 5) Wnt4::Cre; mT/mG female stained by double immunofluorescence microscopy for EGFP (green) and PR (magenta, not detected), counterstained with DAPI (blue). Scale bar: 50 mm.


D - F EGFP ( green ) PR ( magenta ) co ‐ immunofluorescence microscopy counterstained with DAPI ( blue ) on histology sections from mT / mG ; Wnt4 : : Cre mammary at different developmental stages . ( D ) TEB of a 4 ‐ week ‐ old ( n = 4 ) ; scale bar : 30 μm . ( E ) of an 8 ‐ week ‐ old ( n = 3 ) ; scale bar : 100 μm ; inset , scale bar : 20 μm . ( F ) of a at day 10 . 5 of pregnancy ( n = 3 ) ; scale bar : 150 μm ; inset , scale bar : 30 μm .

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

D-F EGFP (green) and PR (magenta) co‐immunofluorescence microscopy counterstained with DAPI (blue) on histology sections from mT/mG; Wnt4::Cre mammary glands at different developmental stages. (D) TEB of a 4‐week‐old female (n = 4); scale bar: 30 μm. (E) Ducts of an 8‐week‐old female (n = 3); scale bar: 100 μm; inset, scale bar: 20 μm. (F) Duct of a female at day 10.5 of pregnancy (n = 3); scale bar: 150 μm; inset, scale bar: 30 μm.


G - I EGFP ( green ) , PR ( magenta ) , p63 ( ) triple co ‐ immunofluorescence microscopy counterstained with DAPI ( blue ) on histological sections from mT / mG ; Wnt4 : : Cre mammary at different developmental stages . ( G ) of 5 ‐ day ‐ old ( n = 3 ) . ( H ) TEB of a 4 ‐ week ‐ old ( n = 3 ) . ( I ) of an 8 ‐ week ‐ old ( n = 3 ) . Scale bars : 30 μm .

G-I EGFP (green), PR (magenta), and p63 (white) triple co‐immunofluorescence microscopy counterstained with DAPI (blue) on histological sections from mT/mG; Wnt4::Cre mammary glands at different developmental stages. (G) Ducts of 5‐day‐old female (n = 3). (H) TEB of a 4‐week‐old female (n = 3). (I) Duct of an 8‐week‐old female (n = 3). Scale bars: 30 μm.


J - O Epifluorescence stereo microscopy of mammary harvested from 15 ‐ day ‐ old Wnt4 : : GFP either WT ( n = 18 ) ( J , M ) , ERα − / − ( n = 4 ) ( K , N ) , or PR − / − ( n = 3 ) ( L , O ) . dTomato expression ( J - L ) ; EGFP expression ( M - O ) is not abrogated in ERα − / − nor PR − / − epithelia . Arrowheads mark main originating from nipple . Scale bar : 50 μm .

J-O Epifluorescence stereo microscopy of mammary glands harvested from 15‐day‐old Wnt4::GFP females either WT (n = 18) (J, M), ERα−/− (n = 4) (K, N), or PR−/− (n = 3) (L, O). dTomato expression (J-L); EGFP expression (M-O) is not abrogated in ERα−/− nor PR−/− epithelia. Arrowheads mark the main duct originating from the nipple. Scale bar: 50 μm.


Q Bar plots showing relative PR Wnt4 mRNA expression normalized to CK18 mRNA in mammary organoids from 5 pubertal ( 6 old ) 3 adult ( 11 old ) mice exposed for 6 h to vehicle ( C ) , 17β ‐ estradiol ( 20 nmol ) ( E2 ) , or R5020 ( 20 nmol ) ( P ) . Bars represent mean ± SD of 3 independent experiments .

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

Q Bar plots showing relative PR and Wnt4 mRNA expression normalized to CK18 mRNA in mammary organoids from 5 pubertal (6 weeks old) and 3 adult (11 weeks old) mice exposed for 6 h to vehicle (C), 17β‐estradiol (20 nmol) (E2), or R5020 (20 nmol) (P). Bars represent the mean ± SD of 3 independent experiments.


R - X Epifluorescence stereo microscopy of mammary that were engrafted with Wnt4 : : GFP epithelium from 8 ‐ week ‐ old , either PRWT ( R , T , V , X ) or PR − / − ( S , U , W ) . dTomato expression ( R , S ) ; EGFP expression ( T , U ) double epifluorescence ( V , W , X ) on engrafted 3 after surgery when recipients were 6 old . Representative result from three independent experiments . Arrowheads point to TEBs ( V , X ) or to origin of growth ( W ) . Scale bar ( R - W ) : 5 mm , ( X ) : 1 mm .

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

R-X Epifluorescence stereo microscopy of contralateral mammary glands that were engrafted with Wnt4::GFP epithelium from 8‐week‐old females, either PRWT (R, T, V, X) or PR−/− (S, U, W). dTomato expression (R, S); EGFP expression (T, U) double epifluorescence (V, W, X) on contralateral engrafted glands 3 weeks after surgery when recipients were 6 weeks old. Representative result from three independent experiments. Arrowheads point to TEBs (V, X) or to origin of growth (W). Scale bar (R-W): 5 mm, (X): 1 mm.


A - D Representative epifluorescence stereo microscopy of inguinal mammary from a 10 ‐ day ‐ old mT / mG ( A , C ) MMTV : : Cre ; mT / mG ( B , D ) ( n = 7 ) . Scale bar : 1 mm .

O O O O O O O O O O O O O O O O O O O O O O O O O O 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-D Representative epifluorescence stereo microscopy of inguinal mammary glands from a 10‐day‐old mT/mG (A, C) and MMTV::Cre; mT/mG (B, D) female (n = 7). Scale bar: 1 mm.


E Higher magnification of inguinal mammary from a 10 ‐ day ‐ old MMTV : : Cre ; mT / mG . Scale bar : 0 . 2 mm . F Immunofluorescence microscopy for EGFP ( green ) on a mammary section from a 10 ‐ day ‐ old MMTV : : Cre ; mT / mG counterstained with DAPI ( blue ) ( n = 7 ) . Scale bar : 30 μm .

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

E Higher magnification of inguinal mammary gland from a 10‐day‐old MMTV::Cre; mT/mG female. Scale bar: 0.2 mm.F Immunofluorescence microscopy for EGFP (green) on a mammary gland section from a 10‐day‐old MMTV::Cre; mT/mG female counterstained with DAPI (blue) (n = 7). Scale bar: 30 μm.


G Epifluorescence stereo microscopy of mammary from 10 ‐ day ‐ old either MMTV : : Cre ; mT / mG Wnt4fl / + or MMTV : : Cre ; mT / mG ; Wnt4fl / fl . Scale bar : 1 mm . Arrowhead marks main originating from nipple . H Bar plot showing of branching points in prepubertal Cre + ; Wnt4fl / fl ( n = 12 ) relative to Cre + ; Wnt4fl / + ( n = 10 ) . Two ‐ , paired Student ' s t ‐ was used to calculate statistical significance .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Epifluorescence stereo microscopy of mammary glands from 10‐day‐old littermates either MMTV::Cre; mT/mG Wnt4fl/+ or MMTV::Cre; mT/mG; Wnt4fl/fl. Scale bar: 1 mm. Arrowhead marks the main duct originating from the nipple.H Bar plot showing ratio of branching points in prepubertal Cre+;Wnt4fl/fl (n = 12) relative to Cre+;Wnt4fl/+ littermates (n = 10). Two‐tailed, paired Student's t‐test was used to calculate statistical significance.


I stereo microscopy of mammary from 35 ‐ day ‐ old either MMTV : : Cre ; mT / m ; Wnt4fl / + or MMTV : : Cre ; mT / m ; Wnt4fl / fl . Note elongation is delayed in Wnt4 ‐ deficient mammary epithelium compared to Wnt4fl / + control . LN : lymph node . Scale bar : 4 mm . J , K Bar plots showing number of terminal end buds ( TEBs ) ( J ) of mammary fat pad filled by system ( K ) . Ctrl ( Wnt4fl / wt or Wnt4fl / fl ; Cre − ) : n = 6 , fl / + ; Cre + : n = 4 , fl / fl ; Cre + : n = 7 ) . Data are presented as mean ± SD . Two ‐ Student ' s t ‐ was used to calculate statistical significance .

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I Fluorescence stereo microscopy of mammary glands from 35‐day‐old littermates either MMTV::Cre; mT/m; Wnt4fl/+ or MMTV::Cre; mT/m;Wnt4fl/fl. Note ductal elongation is delayed in Wnt4‐deficient mammary epithelium compared to Wnt4fl/+ control. LN: lymph node. Scale bar: 4 mm.J, K Bar plots showing number of terminal end buds (TEBs) (J) and area of mammary fat pad filled by the ductal system (K). Ctrl (Wnt4fl/wt or Wnt4fl/fl; Cre−): n = 6, fl/+; Cre+: n = 4, and fl/fl; Cre+: n = 7). Data are presented as the mean ± SD. Two‐tailed Student's t‐test was used to calculate statistical significance.


L , M Bar plots showing BrdU incorporation index in inguinal mammary of 4 ‐ week ‐ old Wnt4fl / + ; Cre + ( n = 3 ) or Wnt4fl / fl ; Cre + ( n = 4 ) in ( L ) in TEBs ( M ) . Data are presented as mean ± SD . Two ‐ Student ' s t ‐ was used to calculate statistical significance .

L, M Bar plots showing BrdU incorporation index in inguinal mammary glands of 4‐week‐old Wnt4fl/+; Cre+ (n = 3) or Wnt4fl/fl; Cre+ (n = 4) in the ducts (L) and in the TEBs (M). Data are presented as the mean ± SD. Two‐tailed Student's t‐test was used to calculate statistical significance.


A Whole ‐ mount microscopy of X ‐ gal ‐ stained Axin2 + / lacZ in E12 . 5 showing β ‐ galactosidase expression in mammary buds ( arrows ) ( n = 8 ) . Scale bar : 1 mm . Arrowheads mark mammary buds .

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A Whole‐mount microscopy of X‐gal‐stained Axin2+/lacZ in E12.5 embryo showing β‐galactosidase expression in the mammary buds (arrows) (n = 8). Scale bar: 1 mm. Arrowheads mark mammary buds.


B - F Whole ‐ mount microscopy of X ‐ gal ( blue ) ‐ carmine alum ( red ) ‐ stained mammary harvested from Axin2 + / lacZ mice at distinct developmental stages . ( B ) At postnatal day 1 , β ‐ galactosidase activity detected in nipple ( n = 6 ) . Scale bar : 1 mm . ( C , D ) In 5 ‐ week ‐ old mammary , reporter activity was detected around ( small arrows ) in neck region of terminal end buds ( TEBs ) ( large arrows ) ( C ) ( n = 8 ) . Scale bars : 400 μm ( C ) 100 μm ( D ) . ( E ) At 8 . 5 day of pregnancy , reporter expression was detected in . Higher magnification ( inset ) suggests myoepithelial expression ( n = 10 ) . Scale bar : 1 mm . ( F ) Whole ‐ mount at day 14 . 5 of pregnancy : reporter activity is limited to ( n = 5 ) . Scale bar : 200 μm .

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B-F Whole‐mount microscopy of X‐gal (blue)‐ and carmine alum (red)‐stained mammary glands harvested from Axin2+/lacZ mice at distinct developmental stages. (B) At postnatal day 1, β‐galactosidase activity detected in the nipple area (n = 6). Scale bar: 1 mm. (C, D) In 5‐week‐old mammary glands, reporter activity was detected around the ducts (small arrows) and in the neck region of the terminal end buds (TEBs) (large arrows) (C) (n = 8). Scale bars: 400 μm (C) and 100 μm (D). (E) At 8.5 day of pregnancy, reporter expression was detected in the ducts. Higher magnification (inset) suggests myoepithelial expression (n = 10). Scale bar: 1 mm. (F) Whole‐mount at day 14.5 of pregnancy: reporter activity is limited to ducts (n = 5). Scale bar: 200 μm.


G Histology section of Axin2 : : LacZ mammary at day 8 . 5 of pregnancy counterstained with red ; luminal epithelial show no detectable β ‐ galactosidase activity but myoepithelial do . Scale bar : 200 μm .

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G Histology section of Axin2::LacZ mammary gland at day 8.5 of pregnancy counterstained with nuclear red; luminal epithelial cells show no detectable β‐galactosidase activity but myoepithelial cells do. Scale bar: 200 μm.


H β ‐ galactosidase activity ( blue ) colocalizes with myoepithelial marker p63 ( green ) detected by immunofluorescence microscopy . Arrows point to myoepithelial . L , lumen . Scale bar : 100 μm .

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H β‐galactosidase activity (blue) colocalizes with the myoepithelial marker p63 (green) detected by immunofluorescence microscopy. Arrows point to myoepithelial cells. L, lumen. Scale bar: 100 μm.


I Representative whole ‐ mount stereo microscopy of X ‐ gal ( blue ) ‐ carmine alum ( magenta ) ‐ stained mammary biopsies taken from 14 ‐ week ‐ old Axin2 : : LacZ collected at diestrus estrus , respectively ( n = 3 ) . Scale bar : 200 μm .

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I Representative whole‐mount stereo microscopy of X‐gal (blue)‐ and carmine alum (magenta)‐stained mammary gland biopsies taken from 14‐week‐old Axin2::LacZ females collected at diestrus and estrus, respectively (n = 3). Scale bar: 200 μm.


J Relative Wnt4 Axin2 mRNA expression in mammary from three mice in estrus versus diestrus assessed by semiquantitative qRT - PCR normalized to 18S rRNA . Two ‐ , paired Student ' s t ‐ was used to calculate statistical significance .

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J Relative Wnt4 and Axin2 mRNA expression in mammary glands from three mice in estrus versus diestrus assessed by semiquantitative qRT-PCR normalized to 18S rRNA. Two‐tailed, paired Student's t‐test was used to calculate statistical significance.


K Stereo microscopy of X ‐ gal ‐ carmine alum ‐ stained mammary from ovariectomized Axin2 : : LacZ treated for 72 h with vehicle ( n = 4 ) ( left ) , 17 ‐ β ‐ estradiol ( E2 ) ( n = 6 ) ( center ) , 17 ‐ β ‐ estradiol progesterone ( E2 P ) ( n = 8 ) ( right ) . Scale bar : 200 μm .

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K Stereo microscopy of X‐gal‐ and carmine alum‐stained mammary glands from ovariectomized Axin2::LacZ females treated for 72 h with vehicle (n = 4) (left), 17‐β‐estradiol (E2) (n = 6) (center), 17‐β‐estradiol and progesterone (E2 and P) (n = 8) (right). Scale bar: 200 μm.


L , M Stereo microscopy of whole ‐ mounted X ‐ gal stained after engraftment with mammary buds from Axin2 : : LacZ transgenic Wnt4 + / + or Wnt4 − / − E12 . 5 E13 . 5 ( L ) or Axin2 : : LacZ transgenic either PR + / − or PR − / − 8 ‐ week ‐ old ( M ) , at day 8 . 5 of pregnancy . β ‐ galactosidase expression reflecting Axin2 transcription is readily detected in PR + / − as well as Wnt4 + / + mammary epithelia but not in PR − / − ( n = 6 ) Wnt4 − / − counterparts ( n = 6 ) . Blue : X ‐ gal ; magenta : carmine alum counterstain . Scale bars : 200 μm .

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L, M Stereo microscopy of contralateral glands whole‐mounted and X‐gal stained after engraftment with mammary buds from Axin2::LacZ transgenic and Wnt4+/+ or Wnt4−/− female E12.5 and E13.5 embryos (L) or Axin2::LacZ transgenic and either PR+/− or PR−/− 8‐week‐old females (M), at day 8.5 of pregnancy. β‐galactosidase expression reflecting Axin2 transcription is readily detected in PR+/− as well as Wnt4+/+ mammary epithelia but not in the PR−/− (n = 6) and Wnt4−/− counterparts (n = 6). Blue: X‐gal staining; magenta: carmine alum counterstain. Scale bars: 200 μm.


A X ‐ gal ‐ stained whole ‐ mounts of Axin2 : : LacZ at E14 . 5 either Wnt4 + / + or Wnt4 − / − ( n = 5 or 4 , respectively ) . Arrows mark mammary buds . Scale bar : 5 mm .

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A X‐gal‐stained whole‐mounts of Axin2::LacZ embryos at E14.5 either Wnt4+/+or Wnt4−/− (n = 5 or 4, respectively). Arrows mark mammary buds. Scale bar: 5 mm.


B Whole ‐ mount of X ‐ gal ‐ carmine alum ‐ stained mammary harvested from 6 ‐ week ‐ old either Wnt4fl / wt ; MMTV : : Cre + ; Axin2 + / lacZ ( n = 5 ) or Wnt4fl / flMMTV : : Cre + ; Axin2 + / lacZ ( n = 4 ) . Left insets : higher magnification ; right insets : histology section of same . Note expression of reporter detected around neck of TEBs in Wnt4 ‐ deficient littermate control is comparable . LN , lymph node . Scale bars : 1 mm , inset scale bar : 100 μm .

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B Whole‐mount analysis of X‐gal‐ and carmine alum‐stained mammary glands harvested from 6‐week‐old littermates either Wnt4fl/wt;MMTV::Cre+;Axin2+/lacZ (n = 5) or Wnt4fl/flMMTV::Cre+;Axin2+/lacZ (n = 4). Left insets: higher magnification; right insets: histology section of the same gland. Note expression of reporter detected around the neck of TEBs in Wnt4‐deficient and littermate control is comparable. LN, lymph node. Scale bars: 1 mm, and inset scale bar: 100 μm.


( A - D ) Serum NfL concentrations of preataxic ( green ) ataxic ( red ) SCA3 subjects controls ( blue ) were measured in two independent cohorts , each with a different Simoa approach : cohort # 1 , recruited by ESMI consortium ( A , C ) , cohort # 2 , recruited by EuroSCA / RiSCA consortium ( B , D ) . Boxes show ranges between lower upper quartiles , central bands show medians , whiskers show data within 1 . 5 ∙ IQR of median , with dots representing outliers . Groups were compared with Mann Whitney U ( * * * p < . 001 , * * p < . 01 , ns p ≥ . 05 , two - , Bonferroni - corrected ; In scatter plots , NfL values were plotted as a function of subjects ' . dashed grey lines visualise optimal cut - offs for differentiating ataxic SCA3 subjects from controls in each cohort ( cohort # 1 : 20 . 0 pg / ml , 98 . 7 % sensitivity , 92 . 2 % specificity ; cohort # 2 : 50 . 9 pg / ml , 92 . 6 % sensitivity , 100 % specificity ; cut - offs were derived by maximising Youden ' s index irrespective of ) . Note logarithmic scale of y - axes .

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(A-D) Serum NfL concentrations of preataxic (green) and ataxic (red) SCA3 subjects and controls (blue) were measured in two independent cohorts, each with a different Simoa approach: cohort #1, recruited by the ESMI consortium (A, C), and cohort #2, recruited by the EuroSCA/RiSCA consortium (B, D). Boxes show the ranges between lower and upper quartiles, the central bands show the medians, and the whiskers show data within 1.5∙IQR of the median, with dots representing outliers. Groups were compared with Mann Whitney U tests (*** p<.001, ** p<.01, ns p≥.05, two-tailed, Bonferroni-corrected; In the scatter plots, the individual NfL values were plotted as a function of subjects' age. The dashed grey lines visualise the optimal cut-offs for differentiating ataxic SCA3 subjects from controls in each cohort (cohort #1: 20.0 pg/ml, 98.7% sensitivity, 92.2% specificity; cohort #2: 50.9 pg/ml, 92.6% sensitivity, 100% specificity; cut-offs were derived by maximising Youden's index irrespective of age). Note the logarithmic scale of the y-axes.


( E , F ) Serum pNfH of preataxic ataxic SCA3 subjects controls were also measured in both cohorts , each with a different Simoa approach ( two - Mann Whitney U , Bonferroni - corrected ;

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(E, F) Serum pNfH levels of preataxic and ataxic SCA3 subjects and controls were also measured in both cohorts, each with a different Simoa approach (two-tailed Mann Whitney U tests, Bonferroni-corrected;


( A ) Serum NfL of ataxic SCA3 subjects significantly correlated with disease severity , as quantified by Scale for Rating Assessment of Ataxia ( SARA ) score ( r = 0 . 43 , p < . 001 , Pearson ' s correlation , two - ) .

(A) Serum NfL levels of ataxic SCA3 subjects significantly correlated with disease severity, as quantified by the Scale for the Rating and Assessment of Ataxia (SARA) score (r=0.43, p<.001, Pearson's correlation, two-tailed test).


( B ) cross - sectional NfL also reflected longitudinal disease progression , as quantified by annual SARA score change ( available for 35 subjects ) . Boxes show ranges between lower upper quartiles , central bands show medians , whiskers show data within 1 . 5 ∙ IQR of median , with dots representing outliers . Subjects with high disease progression ( annual SARA score increase ≥ 0 . 71 points / year , n = 18 , median split ) had significantly higher serum of NfL than subjects with low disease progression

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(B) The cross-sectional NfL levels also reflected longitudinal disease progression, as quantified by the annual SARA score change (available for 35 subjects). Boxes show the ranges between lower and upper quartiles, the central bands show the medians, and the whiskers show data within 1.5∙IQR of the median, with dots representing outliers. Subjects with high disease progression (annual SARA score increase ≥0.71 points/year, n=18, median split) had significantly higher serum levels of NfL than subjects with low disease progression


( B ) cross - sectional NfL also reflected longitudinal disease progression , as quantified by annual SARA score change ( available for 35 subjects ) . Boxes show ranges between lower upper quartiles , central bands show medians , whiskers show data within 1 . 5 ∙ IQR of median , with dots representing outliers . Subjects with high disease progression ( annual SARA score increase ≥ 0 . 71 points / year , n = 18 , median split ) had significantly higher serum of NfL than subjects with low disease

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( C ) We modelled serum NfL ( log - transformed ) in SCA3 carriers ( n = 123 ) with predictors ATXN3 CAG repeat , their squares possible interactions ( for details , see Results Methods ) . highly significant predictors ( , its square , repeat , intercept , p < . 001 , explained variance : R2 = 0 . 37 ) were used to generate diagram . For a given , each increase in CAG repeat was associated with higher NfL concentrations . steepness of slopes declined with increasing . In controls ( black , n = 125 ) , relation between NfL level ( log - transformed ) was linear .

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(C) We modelled serum NfL levels (log-transformed) in SCA3 carriers (n=123) with the predictors age and ATXN3 CAG repeat length, their squares and all possible interactions (for details, see Results and Methods). The highly significant predictors (age, its square, and repeat length, and the intercept, all p<.001, explained variance: R2=0.37) were used to generate the diagram. For a given age, each increase in CAG repeat count was associated with higher NfL concentrations. The steepness of the slopes declined with increasing age. In controls (black, n=125), the relation between NfL level (log-transformed) and age was linear.


( A ) Serum NfL in preataxic SCA3 subjects ( cross - sectional data ) were plotted over time from individually estimated ataxia onset . NfL increased significantly with proximity to estimated onset ( F ( 1 , 21 ) = 39 . 68 , p < . 001 , R2 = 0 . 64 ) . For benchmarking of preataxic subjects , blue dashed visualises optimal cut - off for differentiating ataxic SCA3 subjects from controls in pooled cohort ( 20 . 3 pg / ml , 99 . 0 % sensitivity , 95 . 2 % specificity ) .

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(A) Serum NfL levels in preataxic SCA3 subjects (cross-sectional data) were plotted over the time from the individually estimated ataxia onset. NfL levels increased significantly with proximity to the estimated onset (F(1,21)=39.68, p<.001, R2=0.64). For benchmarking the levels of preataxic subjects, the blue dashed line visualises the optimal cut-off for differentiating ataxic SCA3 subjects from controls in the pooled cohort (20.3 pg/ml, 99.0% sensitivity, 95.2% specificity).


( B ) To determine time at which NfL become significantly increased in preataxic stage of SCA3 , NfL of preataxic carriers need to be related to NfL of controls at same , as NfL physiologically increase with . For each preataxic subject , difference between measured NfL value ( green dot ) NfL value predicted for control subjects of same ( solid blue ) was visualised by of vertical green . Standardisation of this difference relative to NfL distribution in controls

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(B) To determine the time at which NfL levels become significantly increased in the preataxic stage of SCA3, NfL levels of preataxic carriers need to be related to NfL levels of controls at the same age, as NfL levels physiologically increase with age. For each preataxic subject, the difference between the measured NfL value (green dot) and the NfL value predicted for control subjects of the same age (solid blue line) was visualised by the length of the vertical green line. Standardisation of this difference relative to the NfL distribution in controls


( B ) To determine time at which NfL become significantly increased in preataxic stage of SCA3 , NfL of preataxic carriers need to be related to NfL of controls at same , as NfL physiologically increase with . For each preataxic subject , difference between measured NfL value ( green dot ) NfL value predicted for control subjects of same ( solid blue ) was visualised by of vertical green . Standardisation of this difference relative to NfL distribution in

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( C ) In preataxic SCA3 subjects , NfL z - scores increased significantly ( F ( 1 , 21 ) = 30 . 78 , p < . 001 , R2 = 0 . 58 ) with subjects approaching expected of onset . NfL of preataxic subjects were significantly increased compared to controls ( i . e . z - score > 1 . 96 ) 7 . 5 years before expected onset , indicated by non - overlapping 95 % confidence intervals of SCA3 subjects ( black solid ) controls ( blue dashed ) .

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(C) In preataxic SCA3 subjects, NfL z-scores increased significantly (F(1,21)=30.78, p<.001, R2=0.58) with subjects approaching the expected age of onset. NfL levels of preataxic subjects were significantly increased compared to controls (i.e. z-score >1.96) 7.5 years before the expected onset, indicated by the non-overlapping 95% confidence intervals of SCA3 subjects (black solid line) and controls (blue dashed line).


( A , B ) Longitudinal intraindividual stability of NfL ( A ) pNfH ( B ) serum over 6 , assessed in both ataxic ( red ) preataxic ( green ) SCA3 control ( blue ) subjects ( n = 21 ) . Data of same individuals are linked by lines . ICC , intraclass correlation coefficient ( estimate 95 % CI ) .

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(A, B) Longitudinal intraindividual stability of NfL (A) and pNfH (B) serum levels over 6 weeks, assessed in both ataxic (red) and preataxic (green) SCA3 and control (blue) subjects (n=21). Data of the same individuals are linked by lines. ICC, intraclass correlation coefficient (estimate and 95% CI).


( C ) Sample estimations were performed for future intervention trials using reduction of Nf towards control as outcome measure . estimated number of subjects per study arm is plotted over assumed therapeutic effect for lowering Nf level in SCA3 subjects to observed in healthy controls ( NfL : dashed , pNfH : dotted ) .

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(C) Sample size estimations were performed for future intervention trials using the reduction of Nf levels towards control levels as outcome measure. The estimated number of subjects per study arm is plotted over the assumed therapeutic effect for lowering the Nf level in SCA3 subjects to levels observed in healthy controls (NfL: dashed line, pNfH: dotted line).


( A - B ) Plasma concentrations of NfL ( A ) pNfH ( B ) were measured in 304Q knock - in SCA3 mouse model . Heterozygous become symptomatic at ≈ 8 months of .

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(A-B) Plasma concentrations of NfL (A) and pNfH (B) were measured in the 304Q knock-in SCA3 mouse model. Heterozygous animals become symptomatic at ≈8 months of age.


( C - F ) Tissue of soluble aggregated mutant ataxin - 3 were measured in cerebellum ( C , E ) frontal lobe ( D , F ) .

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(C-F) Tissue levels of soluble and aggregated mutant ataxin-3 were measured in cerebellum (C, E) and frontal lobe (D, F).


figure shows degree to which each variable differs between heterozygous SCA3 mice wildtype controls for a given . For each variable , we quantified deviation of hetero zygous mice from controls of same by calculating effect r of group comparison ( independent t - ) . An effect of 0 indicates that value in heterozygous mice is not different from controls , while an effect of 1 would indicate strong abnormality . Note that drop in effect of Nf from 12 months to 18 months thus does not imply a putative reduction of Nf , but rather loss of effect in mutants relative to controls , as controls show strong - related Nf increases at 18 months of . Negative effect values were set to 0 .

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The figure shows the degree to which each variable differs between heterozygous SCA3 mice and wildtype controls for a given age. For each variable, we quantified the deviation of heterozygous mice from controls of the same age by calculating the effect size r of the group comparison (independent t-tests). An effect size of 0 indicates that the value in heterozygous mice is not different from controls, while an effect size of 1 would indicate strong abnormality. Note that the drop in the effect size of Nf levels from 12 months to 18 months thus does not imply a putative reduction of Nf levels, but rather the loss of the effect in mutants relative to controls, as controls show strong age-related Nf increases at 18 months of age. Negative effect size values were set to 0.


( A ) We assessed samples of cerebellar cortex , deep cerebellar nuclei , frontal cortex , pons pyramidal tract by immunohistochemistry with ataxin - 3 , comparing heterozygous 304Q SCA3 mice at 2 , 6 , 12 18 months of with wildtype at 18 months . ML : molecular layer , PC : Purkinje , GL : granular layer , scale bars : 20 µm .

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(A) We assessed samples of cerebellar cortex, deep cerebellar nuclei, frontal cortex, pons and pyramidal tract by immunohistochemistry with ataxin-3 staining, comparing heterozygous 304Q SCA3 mice at 2, 6, 12 and 18 months of age with wildtype animals at 18 months. ML: molecular layer, PC: Purkinje cells, GL: granular layer, scale bars: 20 µm.


( B ) We histologically assessed number integrity of Purkinje in cerebellar cortex by NfL , pNfH , Nissl calbindin , comparing heterozygous 304Q SCA3 mice at 2 , 6 , 12 18 months of with wildtype . ML : molecular layer , PC : Purkinje , GL : granular layer , scale bars : 20 µm .

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(B) We histologically assessed the number and integrity of Purkinje cells in the cerebellar cortex by NfL, pNfH, Nissl and calbindin staining, comparing heterozygous 304Q SCA3 mice at 2, 6, 12 and 18 months of age with wildtype animals. ML: molecular layer, PC: Purkinje cells, GL: granular layer, scale bars: 20 µm.


( B ) We histologically assessed number integrity of Purkinje in cerebellar cortex by NfL , pNfH , Nissl calbindin , comparing heterozygous 304Q SCA3 mice at 2 , 6 , 12 18 months of with wildtype .

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(B) We histologically assessed the number and integrity of Purkinje cells in the cerebellar cortex by NfL, pNfH, Nissl and calbindin staining, comparing heterozygous 304Q SCA3 mice at 2, 6, 12 and 18 months of age with wildtype animals.


Select 2D class averages of particles that were used to calculate 2019 - nCoV S reconstruction ( left ) . Side top views of prefusion structure of 2019 - nCoV S with a single RBD in " up " conformation ( right ) . two RBD " down " protomers are shown as cryo - EM density in either or gray RBD " up " protomer is shown in ribbons , colored corresponding to schematic in Fig 1A .

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Select 2D class averages of the particles that were used to calculate the 2019-nCoV S reconstruction (left). Side and top views of the prefusion structure of the 2019-nCoV S protein with a single RBD in the "up" conformation (right). The two RBD "down" protomers are shown as cryo-EM density in either white or gray and the RBD "up" protomer is shown in ribbons, colored corresponding to the schematic in Fig 1A.


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

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 2019 - nCoV S with respect to viral . A cartoon depicting ACE2 - bound 2019 - nCoV S is shown ( right ) with ACE2 in blue S monomers colored tan , pink 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 2019 - nCoV RBD - SD1 . Binding data are shown as a black best fit of 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 SARS - CoV RBD - directed antibodies S230 , m396 80R . Sensortips with immobilized antibodies were dipped into wells containing 2019 - nCoV RBD - SD1 resulting data are shown as a black .

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

(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 transfected with expression vectors of top 5 genes from screen , or 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 mean average expression p - value of miRNA expression profiles between TruB1 KD ctrl ( scramble ) . N = 3 . Significance was assessed using 2 ‐ Student ' s t ‐ . MiRNAs suppressed ( Relative expression TruB1 KD / ctrl < 0 . 8 p - value < 0 . 1 ) are enclosed by 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 other miRNAs in HEK293FT with TruB1 KD or ctrl ( siRNA ) determined by qRT - PCR . Significance was assessed using 2 ‐ Student ' s t ‐ , < 0 . 05 * . Data information : experiments were performed in triplicate . Error bars show SD .

(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 - let - 7 family miRNAs as in ( D ) determined by qRT - PCR . Significance was assessed using 2 ‐ Student ' s t ‐ , < 0 . 05 * . Data information : 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.


( C ) . Relative miRNA expression of let - 7a in HEK - 293 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 ‐ Student ' s t ‐ , < 0 . 05 * . Data information : experiments were performed in triplicate . Error bars show SD .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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). 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 RNU6B in HeLa infected with lentiviruses encoding tetracycline - inducible expression of TruB1 , mt1 , mt2 , or GFP , 5 days after doxycycline treatment .

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

(D). 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 CMC primer extension method . Total purified from HEK - 293FT were treated with CMC . CMC treated were reverse - transcribed with RI - labelled specific primers for tRNAphe or pri - let - 7b . ddATP was used for control . Pseudouridines are indicated by black arrows .

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

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

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

(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 .

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

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

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

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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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). 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 infected with lentiviruses expressing tetracycline - inducible TruB1 , mt1 , mt2 , or GFP 5 days after doxycycline treatment . Significance was assessed using 2 ‐ Student ' s t ‐ , < 0 . 05 * . Data information : experiments were performed in triplicate . Error bars show SD .

(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 with KD of let - 7 family members or ctrl ( using 2 ' - O - methylated antisense inhibitor ) . Significance was assessed using 2 ‐ Student ' s t ‐ , < 0 . 05 * . Data information : experiments were performed in triplicate . Error bars show SD .

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

(D). 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 for HEK293FT 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 ‐ Student ' s t ‐ , < 0 . 05 * .

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

(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 + rMC from lung homogenate of adult mice .

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


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

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

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

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

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

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

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

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

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

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 , Sox2 ) alveoli associated ‑ genes ( H ) ( Cxcl15 , Lyz2 , Sftpc , Hopx ) . Each violin plot shows frequency distribution of mean transcript level ( log2 ) .

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

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

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

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 ( AEC II ) connected by tight junctions ( red square ) with numerous mitochondria ( M ) lamellar bodies ( LB ) . A LIF with numerous lipid droplets ( * ) a MYO with cisterns of rough ER ( red arrow ) are located at basal side ( left ) . alveolar lumen is filled with lamellar surfactant including tubular myelin ( arrow ) ( lower right ) . Exocytosis of a lamellar ( * ) from an AEC II ( upper right ) . Scale bars indicate 500 nm .

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

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 ( red arrowhead ) with basal bodies ( black arrowhead ) at left side of longitudinally sectioned lumen . boxed indicates an apical junctional between two ciliated . 1 = tight junction , 2 = adherens junction . Scale bar indicates 500 nm ( in insert : 100 nm ) .

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

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).


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

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

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

O O O O O O O O O O O O O O O O O O O O O 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-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 of day 40 BALO alveoli ( H ) 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 ) 1000 nm ( right ) . An airway - like structure ( i ' ) is shown with secretory ciliated ( red arrowheads ) in semithin sections ( 0 . 5 µm ) longitudinally cut 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 ( BC ) , not reaching lumen in which cilia ( C ) are seen , located between a secretory ( SC ) a ciliated ( Ci ) . Scale bars indicate 1000 nm ( left right ) . Mature cilia ( i ' ' ' ) in BALO at higher magnification depicting 9x2 + 2 structure with central doubled microtubules ( insert , red arrowhead ) , a characteristic for motile cilia . Scale bar indicates 100 nm ( far right ) .

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

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 of day 21 BALO 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.


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

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

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 histograms of PDGFRα expression in rMC ( EpCAM - CD45 - CD31 - Sca - 1 + ) isolated from lung homogenate of PdgfraGFP reporter mice .

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

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

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

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

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

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.


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

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).


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 ) 50 μm ( right ) .

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

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 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 ) 50 μm ( right )

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

Representative images of 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)


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

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

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 ( 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 ) 500 nm ( right , in insert : 250 nm )

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

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

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

Representative 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 ) tSNE plot ( right ) of comparative of digested day 23 BALO cultures with ( + ) without ( - ) microinjected TR ‑ Mac depicting six distinct clusters ( C1 , club / secretory , red ; C2 , basal , yellow ; C3 , rMC , green ; C4 , AEC II , blue ; C5 , AEC I , purple ; C6 , ciliated , 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 + ) without ( C1 - ) microinjected TR - Mac . circle illustrates number of expressing a specific gene

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


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

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


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

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


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

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


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

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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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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.


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

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


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

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

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

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


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

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


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

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

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


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

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


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

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

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


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

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


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

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


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

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


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

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

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