( a ) of mitochondria numbers from Atg18 control ( Atg18KG03090 / wild type ) mutant ( Atg18KG03090 / Df ( 3L ) 6112 ) or Atg7 control ( Atg7d30 / d14 ) mutant ( Atg7d77 / d14 ) . Mitochondria were quantified from 2 distinct 25 μm2 regions per from 2 per from at least 3 different per genotype .

O O O O B-SUBCELLULAR O O B-GENEPROD O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O B-GENEPROD O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O

(a) Quantification of mitochondria numbers from Atg18 control (Atg18KG03090/wild type) and mutant (Atg18KG03090/Df(3L)6112) or Atg7 control (Atg7d30/d14) and mutant (Atg7d77/d14) TEM images. Mitochondria were quantified from 2 distinct 25 μm2 regions per cell from 2 cells per animal from at least 3 different animals per genotype.


( b , c ) Midguts dissected from either early third instar larvae ( b ) or at puparium formation ( c ) that express GFP - labelled mitochondria in contain an Atg1Δ3D loss - of - function mutant clone ( lacking RFP ) . Wild - type control possess stronger RFP heterozygous have weaker RFP . Representative are shown . is shown as mean ± s . d . NS , not significant . Scale bars , 0 μm .

O O O O O B-TISSUE O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O O B-TISSUE O O O O O O B-GENEPROD O O B-SUBCELLULAR O O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O B-GENEPROD O O 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) Midguts dissected from either early third instar larvae (b) or at puparium formation (c) that express GFP-labelled mitochondria in all cells and contain an Atg1Δ3D loss-of-function mutant cell clone (lacking RFP). Wild-type control cells possess stronger RFP and heterozygous cells have weaker RFP. Representative images are shown. Quantification is shown as mean ± s.d. NS, not significant. Scale bars, 0 μm.


( a ) Midguts dissected from expressing Uba1IR specifically in GFP - marked clones of at puparium formation analysed by differential interference contrast ( DIC ) microscopy . Representative are shown . ( b ) ( μm2 ) from a ; n = 16 intestines per genotype with 1 - 5 measured per intestine .

O O O B-TISSUE O O O B-GENEPROD O O B-GENEPROD O O O O O B-TISSUE O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O B-TISSUE O O O O O O O O B-TISSUE O

(a) Midguts dissected from animals expressing Uba1IR specifically in GFP-marked clones of cells at puparium formation and analysed by fluorescence and differential interference contrast (DIC) microscopy. Representative images are shown. (b) Cell size quantification (μm2) from a; n = 16 animal intestines per genotype with 1-5 cells measured per intestine.


( c ) Midguts dissected from at puparium formation that contain Uba1H33 loss - of - function mutant clones ( lacking RFP ) analysed by differential interference contrast microscopy . Wild - type ( + / + ) control possess stronger RFP heterozygous Uba1H33 / wild - type ( H33 / + ) have weaker RFP . Representative are shown . ( d ) ( μm2 ) from c , n = 8 ( + / + ) , n = 11 ( H33 / + ) n = 11 ( H33 / H33 ) intestines per genotype with 1 - 5 measured per intestine .

O O O B-TISSUE O O O B-TISSUE O O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O 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-TISSUE O O O O O O O O B-TISSUE O

(c) Midguts dissected from animals at puparium formation that contain Uba1H33 loss-of-function mutant cell clones (lacking RFP) and analysed by fluorescence and differential interference contrast microscopy. Wild-type (+/+) control cells possess stronger RFP and heterozygous Uba1H33/wild-type (H33/+) cells have weaker RFP. Representative images are shown. (d) Quantification (μm2) from c, n = 8 (+/+), n = 11 (H33/+) and n = 11 (H33/H33) animal intestines per genotype with 1-5 cells measured per intestine.


( e ) Midguts dissected from at puparium formation that contain Uba1H33 loss - of - function MARCM mutant clones ( GFP - positive ) that also have mCherry - Atg8a expressed in analysed by microscopy . Control wild - type heterozygous have no GFP . Representative are shown .

O O O B-TISSUE O O O B-TISSUE O O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O O O O B-EXP_ASSAY O O O O O O O O B-GENEPROD O O O O O

(e) Midguts dissected from animals at puparium formation that contain Uba1H33 loss-of-function MARCM mutant cell clones (GFP-positive) that also have mCherry-Atg8a expressed in all cells and analysed by fluorescence microscopy. Control wild-type and heterozygous cells have no GFP. Representative images are shown.


( f ) Midguts expressing GFP - Atg5 in enterocytes ( larger nuclei ) , with an Uba1H33 loss - of - function clone ( lacking RFP ) at puparium formation analysed by microscopy . Representative are shown .

O O O B-TISSUE O B-GENEPROD O B-GENEPROD O B-CELL_TYPE O O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O B-TISSUE O O O B-EXP_ASSAY O O O O O

(f) Midguts expressing GFP-Atg5 in enterocytes (larger nuclei), and with an Uba1H33 loss-of-function clone cell (lacking RFP) at puparium formation analysed by fluorescence microscopy. Representative images are shown.


( g ) Midguts dissected from at puparium formation that contain Uba1H33 loss - of - function MARCM mutant clones ( GFP - positive ) that are stained with p62 antibody analysed by microscopy . Control wild - type heterozygous have no GFP . Representative are shown .

O O O B-TISSUE O O O B-TISSUE O O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O B-EXP_ASSAY O O O O O O O O B-GENEPROD O O O O O

(g) Midguts dissected from animals at puparium formation that contain Uba1H33 loss-of-function MARCM mutant cell clones (GFP-positive) that are stained with p62 antibody and analysed by fluorescence microscopy. Control wild-type and heterozygous cells have no GFP. Representative images are shown.


( h ) Midguts dissected from expressing Dts7 , a dominant - sensitive mutant of β2 subunit of proteasome , specifically in GFP - marked at puparium formation analysed by differential interference contrast microscopy . Representative are shown . ( i ) ( μm2 ) from h , n = 6 intestines per genotype with 1 - 5 measured per intestine . is shown as mean ± s . d . NS , not significant . Scale bars , 20 μm .

O O O B-TISSUE O O O B-GENEPROD O O O O O O O O O O O O O O B-GENEPROD O O O B-TISSUE O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O B-TISSUE O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O

(h) Midguts dissected from animals expressing Dts7, a dominant temperature-sensitive mutant of the β2 subunit of the proteasome, specifically in GFP-marked cells at puparium formation and analysed by fluorescence and differential interference contrast microscopy. Representative images are shown. (i) Cell size quantification (μm2) from h, n = 6 animal intestines per genotype with 1-5 cells measured per intestine. Quantification is shown as mean ± s.d. NS, not significant. Scale bars, 20 μm.


( a , b ) E1 - charging for Uba1 Atg7 with either Atg8a ( a ) or ubiquitin ( Ub ; b ) , n = 2 experiments with independently isolated analyses . Flag - tagged baculoviral - expressed E1s were incubated with either His - tagged Atg8a or Ub in presence or absence of β - mercaptoethanol , separated by electrophoresis blotted . Band shift was detected with anti - Flag antibody . Relative molecular mass ( Mr K ) ladders are indicated . Representative are shown . Relative molecular masses : Flag - Uba1 , 135K ; Flag - Atg7 , 81K ; His - Atg8a , 29K ; Ub , 9K .

O O O O O O O O O B-GENEPROD I-GENEPROD O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O B-SUBCELLULAR O O O O B-GENEPROD O O B-GENEPROD O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-EXP_ASSAY O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O O O

(a,b) E1-charging assay for Uba1 and Atg7 with either Atg8a (a) or ubiquitin (Ub; b), n = 2 experiments with independently isolated proteins and analyses. Flag-tagged baculoviral-expressed E1s were incubated with either His-tagged Atg8a or Ub in the presence or absence of β-mercaptoethanol, separated by electrophoresis and blotted. Band shift was detected with anti-Flag antibody. Relative molecular mass (Mr K) ladders are indicated. Representative images are shown. Relative molecular masses: Flag-Uba1, 135K; Flag-Atg7, 81K; His-Atg8a, 29K; Ub, 9K.


( c ) Midgut extracts at puparium formation from wild - type , Atg7 mutant ( Atg7d77 / d14 ) Atg8 ( Atg8KG07569 ) mutant blotted with anti - Atg8 anti - Tubulin , n = 3 independent biological experiments . Representative are shown .

O O O B-TISSUE O O B-TISSUE O O O O O O B-GENEPROD O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O

(c) Midgut protein extracts at puparium formation from wild-type, Atg7 mutant (Atg7d77/d14) and Atg8 (Atg8KG07569) mutant animals blotted with anti-Atg8 and anti-Tubulin, n = 3 independent biological experiments. Representative images are shown.


( d ) Midguts from control Atg310 / wild - type ( Atg3 / + ) Atg310 / Df ( 3L ) cat mutant ( Atg3 / Df ) at puparium formation analysed by differential interference contrast microscopy . Representative are shown . ( e ) ( μm2 ) from d , n = 11 ( Atg3 / + ) n = 9 ( Atg3 / Df ) intestines per genotype with 5 measured per intestine .

O O O B-TISSUE O O B-GENEPROD O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O B-TISSUE O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O B-TISSUE O O O O O O B-TISSUE O

(d) Midguts from control Atg310/wild-type (Atg3/+) and Atg310/Df(3L)cat mutant (Atg3/Df) animals at puparium formation analysed by differential interference contrast microscopy. Representative images are shown. (e) Cell size quantification (μm2) from d, n = 11 (Atg3/+) and n = 9 (Atg3/Df) animal intestines per genotype with 5 cells measured per intestine.


( f ) Midguts dissected from early third instar larvae that mis - express Atg1GS10797 ( Atg1 ) Atg3IR only in DsRed - marked clone analysed by differential interference contrast ( DIC ) microscopy . Representative are shown .

O O O B-TISSUE O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O

(f) Midguts dissected from early third instar larvae that mis-express Atg1GS10797 (Atg1) and Atg3IR only in the DsRed-marked cell clone and analysed by fluorescence and differential interference contrast (DIC) microscopy. Representative images are shown.


( g ) Midguts dissected from expressing Atg4DN specifically in DsRed - marked clones of at puparium formation analysed by differential interference contrast microscopy . Representative are shown . ( h ) ( μm2 ) from g , n = 10 intestines per genotype with 1 - 5 measured per intestine .

O O O B-TISSUE O O O B-GENEPROD O O B-GENEPROD O O O O O B-TISSUE O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O B-TISSUE O O O O O O O O B-TISSUE O

(g) Midguts dissected from animals expressing Atg4DN specifically in DsRed-marked clones of cells at puparium formation and analysed by fluorescence and differential interference contrast microscopy. Representative images are shown. (h) Cell size quantification (μm2) from g, n = 10 animal intestines per genotype with 1-5 cells measured per intestine.


( i ) Midguts dissected from expressing mCherry - Atg8a in , expressing UbIR specifically in GFP - marked clones of at puparium formation . Representative are shown .

O O O B-TISSUE O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O B-GENEPROD O O O O O B-TISSUE O O O O O O

(i) Midguts dissected from animals expressing mCherry-Atg8a in all cells, and expressing UbIR specifically in GFP-marked clones of cells at puparium formation. Representative images are shown.


( j ) Midguts dissected from expressing UbIR specifically in GFP - marked clones of at puparium formation analysed by differential interference contrast microscopy . Representative are shown . ( k ) ( μm2 ) from ( j ) , n = 12 intestines per genotype with 1 - 5 measured per intestine .

O O O B-TISSUE O O O B-GENEPROD O O B-GENEPROD O O O O O B-TISSUE O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O B-TISSUE O O O O O O O O B-TISSUE O

(j) Midguts dissected from animals expressing UbIR specifically in GFP-marked clones of cells at puparium formation and analysed by fluorescence and differential interference contrast microscopy. Representative images are shown. (k) Cell size quantification (μm2) from (j), n = 12 animal intestines per genotype with 1-5 cells measured per intestine.


( l , m ) Midguts dissected from early third instar larvae that exhibit Atg1GS10797 ( Atg1 ) mis - expression in either a wild - type GFP - positive clone ( l ) or a Uba1H33 loss - of - function MARCM mutant clone ( GFP - positive ; m ) analysed by differential interference contrast microscopy . Control wild - type heterozygous Uba1H33 / wild - type have no GFP . Representative are shown . ( n ) ( μm2 ) from l m , n = 22 ( control ) , n = 16 ( Atg1 ) n = 6 ( Atg1 + Uba1H33 ) intestines per genotype with 1 - 5 measured per intestine . data for control ( l , m ) were pooled because they are genetically identical . is shown as mean ± s . d . NS , not significant . Scale bars , 20 μm .

O O O O O B-TISSUE O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O 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-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O B-TISSUE O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(l,m) Midguts dissected from early third instar larvae that exhibit Atg1GS10797 (Atg1) mis-expression in either a wild-type GFP-positive cell clone (l) or a Uba1H33 loss-of-function MARCM mutant cell clone (GFP-positive; m) and analysed by fluorescence and differential interference contrast microscopy. Control wild-type and heterozygous Uba1H33/wild-type cells have no GFP. Representative images are shown. (n) Quantification (μm2) from l and m, n = 22 (control), n = 16 (Atg1) and n = 6 (Atg1 +Uba1H33) animal intestines per genotype with 1-5 cells measured per intestine. Quantification data for control cells (l,m) were pooled because they are genetically identical. Quantification is shown as mean ± s.d. NS, not significant. Scale bars, 20 μm.


( a , b ) Midguts dissected from either early third instar larvae ( a ) or at puparium formation ( b ) that express GFP - labelled mitochondria in enterocytes ( larger nuclei ) contain Uba1H33 loss - of - function mutant clones ( lacking RFP ) . Wild - type control possess stronger RFP heterozygous have weaker RFP . Representative are shown .

O O O O O B-TISSUE O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O O B-TISSUE O O O O O O B-GENEPROD O O B-SUBCELLULAR O B-CELL_TYPE O O O O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O

(a,b) Midguts dissected from either early third instar larvae (a) or at puparium formation (b) that express GFP-labelled mitochondria in enterocytes (larger nuclei) and contain Uba1H33 loss-of-function mutant cell clones (lacking RFP). Wild-type control cells possess stronger RFP and heterozygous cells have weaker RFP. Representative images are shown.


( c ) Midguts dissected at puparium formation that express Uba1IR ( GFP in nucleus cytoplasm ) stained with ATP synthase V ( ATPV ) to detect mitochondria in . Representative are shown .

O O O B-TISSUE O O B-TISSUE O O O B-GENEPROD O B-GENEPROD O B-SUBCELLULAR I-SUBCELLULAR O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O O B-SUBCELLULAR O O O O O O

(c) Midguts dissected at puparium formation that express Uba1IR (GFP in nucleus and cytoplasm) and stained with ATP synthase complex V (ATPV) to detect mitochondria in all cells. Representative images are shown.


( d - f ) Representative immuno - of Uba1IRclone at puparium formation . Uba1IR - expressing possess gold particles ; control lack gold particles ( d ) . Control possess numerous autolysosomes in cytoplasm ( arrowheads ) few mitochondria ( asterisks ) ; Uba1IR - expressing possess numerous mitochondria few autophagic structures . Scale bars , 20 μm ( a - c ) 1 μm ( d - f ) .

O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-TISSUE O O B-GENEPROD O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O O O O B-SUBCELLULAR O B-SUBCELLULAR O O O O B-SUBCELLULAR O O O O B-GENEPROD O O O O B-SUBCELLULAR O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O

(d-f) Representative immuno-TEM images of Uba1IRclone cells at puparium formation. Uba1IR-expressing cells possess gold particles; control cells lack gold particles (d). Control cells possess numerous autolysosomes in the cytoplasm (arrowheads) and few mitochondria (asterisks); Uba1IR-expressing cells possess numerous mitochondria and few autophagic structures. Scale bars, 20 μm (a-c) and 1 μm (d-f).


( C - I ) BMDMs were primed with LPS for 4 h then treated with LicoB for 1 h , prior to stimulation with nigericin for 45 min or ATP for 1 h . Western blot analyses of pro - caspase - 1 ( p45 ) , pro - IL - 1β , NLRP3 , ASC ( arrow indicates ASC in whole lysate ( WCL ) ; activated caspase - 1 ( p20 ) IL - 1β ( p17 ) in culture supernatants ( SN ) of BMDMs ( C , G ) . Caspase - 1 activity ( D , H ) , IL - 1β secretion ( E , I ) , LDH release ( F , J ) in SN were measured . Coomassie Blue was used as supernatant loading control , while lamin B was used as lysate loading control .

O O O O O B-CELL_TYPE O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O B-CELL_TYPE O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O B-GENEPROD I-GENEPROD O O O O O O O

(C-I) BMDMs were primed with LPS for 4 h and then treated with LicoB for 1 h, prior to stimulation with nigericin for 45 min or ATP for 1 h. Western blot analyses of pro-caspase-1 (p45), pro-IL-1β, NLRP3, and ASC (the arrow indicates ASC in the whole cell lysate (WCL); activated caspase-1 (p20) and cleaved IL-1β (p17) in the culture supernatants (SN) of BMDMs (C, G). Caspase-1 activity (D, H), IL-1β secretion (E, I), and LDH release (F, J) in the SN were measured. Coomassie Blue staining was used as the supernatant loading control, while lamin B was used as the lysate loading control.


( A - C ) BMDMs were primed with LPS then treated with LicoB ( 20 μΜ ) for 1 h , followed by stimulation with nigericin , ATP , poly ( I : C ) , or MSU . Pam3CSK4 - primed BMDMs were treated with LicoB ( 20 μM ) then transfected with LPS . Western blot analyses of pro - caspase - 1 ( p45 ) , pro - IL - 1β , NLRP3 , ASC in whole lysate ( WCL ) ; activated caspase - 1 ( p20 ) IL - 1β ( p17 ) in culture supernatants ( SN ) of BMDMs . ( A ) . Caspase - 1 activity ( B ) IL - 1β secretion ( C ) in SN were measured .

O O O O O B-CELL_TYPE O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-CELL_TYPE O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O B-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O B-CELL_TYPE O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O

(A-C) BMDMs were primed with LPS and then treated with LicoB (20 μΜ) for 1 h, followed by stimulation with nigericin, ATP, poly (I:C), or MSU. Pam3CSK4-primed BMDMs were treated with LicoB (20 μM) and then transfected with LPS. Western blot analyses of pro-caspase-1 (p45), pro-IL-1β, NLRP3, and ASC in the whole cell lysate (WCL); and activated caspase-1 (p20) and cleaved IL-1β (p17) in the culture supernatants (SN) of BMDMs. (A). Caspase-1 activity (B) and IL-1β secretion (C) in the SN were measured.


( D - F ) LPS - primed BMDMs were treated with LicoB ( 20 μM ) for 1 h then stimulated with nigericin for 45 min , or poly ( dA : dT ) / Salmonella for 6 h . Western blot analyses of pro - caspase - 1 ( p45 ) , pro - IL - 1β , NLRP3 , ASC in whole lysate ( WCL ) ; activated caspase - 1 ( p20 ) IL - 1β ( p17 ) in culture supernatants ( SN ) of BMDMs ( D ) . Caspase - 1 activity ( E ) IL - 1β secretion ( F ) in SN were measured .

O O O O O B-SMALL_MOLECULE O O B-CELL_TYPE O O O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-ORGANISM O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O B-CELL_TYPE O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O

(D-F) LPS-primed BMDMs were treated with LicoB (20 μM) for 1 h and then stimulated with nigericin for 45 min, or poly(dA:dT)/Salmonella for 6 h. Western blot analyses of pro-caspase-1 (p45), pro-IL-1β, NLRP3, and ASC in the whole cell lysate (WCL); and activated caspase-1 (p20) and cleaved IL-1β (p17) in the culture supernatants (SN) of BMDMs (D). Caspase-1 activity (E) and IL-1β secretion (F) in the SN were measured.


( A - C ) LPS - primed BMDMs were pre - treated with indicated dose of LicoB for 1 h then stimulated with ATP for 1 h . Western blot was used to detect cross - linked ASC in Triton X - insoluble pellet , arrow indicates ASC ( A ) . Caspase - 1 activity ( B ) IL - 1β secretion ( C ) in SN were measured again to verify NLRP3 activation .

O O O O O B-SMALL_MOLECULE O O B-CELL_TYPE O O O O O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-GENEPROD O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O B-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O B-GENEPROD O O

(A-C) LPS-primed BMDMs were pre-treated with the indicated dose of LicoB for 1 h and then stimulated with ATP for 1 h. Western blot analysis was used to detect cross-linked ASC in the Triton X-insoluble pellet, the arrow indicates ASC (A). Caspase-1 activity (B) and IL-1β secretion (C) in the SN were measured again to verify NLRP3 activation.


( E ) Western blot of cross - linked ASC in Triton X - insoluble pellet from LPS - primed BMDMs pre - treated with LicoB ( 20 μM ) or vehicle then stimulated with nigericin , poly ( dA : dT ) , or Salmonella .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O B-CELL_TYPE O O O O B-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O B-ORGANISM O

(E) Western blot analysis of cross-linked ASC in the Triton X-insoluble pellet from LPS-primed BMDMs pre-treated with LicoB (20 μM) or vehicle and then stimulated with nigericin, poly (dA:dT), or Salmonella.


( A ) Western blot of in whole lysates ( WCL ) from BMDMs stimulated with LPS for 3 h then treated with LicoB for 1 h ( LicoB after LPS ) , or BMDMs first treated with LicoB for 1 h then stimulated with LPS for 3 h ( LicoB before LPS ) .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-CELL_TYPE O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O B-CELL_TYPE O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O

(A) Western blot analysis of protein in whole cell lysates (WCL) from BMDMs stimulated with LPS for 3 h and then treated with LicoB for 1 h (LicoB after LPS), or BMDMs first treated with LicoB for 1 h and then stimulated with LPS for 3 h (LicoB before LPS).


( I ) lysates of LPS - primed BMDMs treated with / without nigericin were incubated with sepharose or LicoB - sepharose . pull - down samples input were analysed using Western blot . ( J ) lysates of LPS - primed BMDMs were incubated with sepharose or LicoB - sepharose in presence of different concentrations of free LicoB ( 0 . 5 mM 1 mM ) . pull - down samples input were analysed using Western blot .

O O O O O B-SMALL_MOLECULE O O B-CELL_TYPE O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-SMALL_MOLECULE O O B-CELL_TYPE O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O B-EXP_ASSAY I-EXP_ASSAY O

(I) Cell lysates of LPS-primed BMDMs treated with/without nigericin were incubated with sepharose or LicoB-sepharose. The pull-down samples and input were analysed using Western blot analysis. (J) Cell lysates of LPS-primed BMDMs were incubated with sepharose or LicoB-sepharose in the presence of different concentrations of free LicoB (0.5 mM and 1 mM). The pull-down samples and input were analysed using Western blot.


( K ) HEK - 293T were transfected with Flag - NLRP3 or Flag - vector then treated with LicoB ( 40 μM ) . Immunoprecipitation was performed with anti - DYKDDDDK ( Flag ) affinity gel agarose beads ; Western blot has been shown .

O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O B-SMALL_MOLECULE O O O O O B-EXP_ASSAY O O O O O O O B-GENEPROD O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O

(K) HEK-293T cells were transfected with Flag-NLRP3 or Flag-vector and then treated with LicoB (40 μM). Immunoprecipitation was performed with anti-DYKDDDDK (Flag) affinity gel agarose beads; Western blot analysis has been shown.


( A ) Survival of WT mice pre - treated with vehicle , LicoB ( 20 mg / kg ) , LicoB ( 40 mg / kg ) , MCC950 ( 40 mg / kg ) , or LicoB ( 40 mg / kg ) + MCC950 ( 40 mg / kg ) , followed by i . p . injection with LPS ( 20 mg / kg ) ; mice survival was monitored for 72 h ( n = 10 mice per group ) .

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(A) Survival of WT mice pre-treated with vehicle, LicoB (20 mg/kg), LicoB (40 mg/kg), MCC950 (40 mg/kg), or LicoB (40 mg/kg) + MCC950 (40 mg/kg), followed by i.p. injection with LPS (20 mg/kg); mice survival was monitored for 72 h (n=10 mice per group).


C57BL / 6 mice received vehicle or LicoB ( 40 mg / kg ) for 17 consecutive doses , once every two days for 34 days . Following that treatment , serum of ALT ( H ) ( n = 8 mice per group ) .

O O O B-ORGANISM O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O B-TISSUE O B-GENEPROD O O O O O O O B-ORGANISM O O O O

C57BL/6 mice received vehicle or LicoB (40 mg/kg) for 17 consecutive doses, once every two days for 34 days. Following that treatment, the serum levels of ALT (H) (n=8 mice per group).


C57BL / 6 mice received vehicle or LicoB ( 40 mg / kg ) for 17 consecutive doses , once every two days for 34 days . Following that treatment , serum of AST ( I ) , CRE ( J ) were measured , ( n = 8 mice per group ) .

O O O B-ORGANISM O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O B-TISSUE O B-GENEPROD O O O O B-SMALL_MOLECULE O O O O O O O O O O B-ORGANISM O O O O

C57BL/6 mice received vehicle or LicoB (40 mg/kg) for 17 consecutive doses, once every two days for 34 days. Following that treatment, the serum levels of AST (I), and CRE (J) were measured, (n=8 mice per group).


( A - D ) Mice were pre - treated with LicoB or MCC950 for 1 h , then i . p . injected with MSU ( 50 mg / kg ) treated for 6 h ( n = 6 mice per group ) . of IL - 1β in peritoneal lavage fluid ( A ) serum ( B ) were measured using ELISA . of peritoneal exudate ( PECs ) ( C ) neutrophils ( Ly6G CD11b ) ( D ) using flow cytometry .

O O O O O B-ORGANISM O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-ORGANISM O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-TISSUE I-TISSUE I-TISSUE O O O B-TISSUE O O O O O O B-EXP_ASSAY O O O O O O O O O O B-CELL_TYPE O B-GENEPROD I-GENEPROD O O O O O B-EXP_ASSAY I-EXP_ASSAY O

(A-D) Mice were pre-treated with LicoB or MCC950 for 1 h, then i.p. injected with MSU (50 mg/kg) and treated for 6 h (n=6 mice per group). The levels of IL-1β in the peritoneal lavage fluid (A) and serum (B) were measured using ELISA. Quantification of peritoneal exudate cells (PECs) (C) and neutrophils (Ly6G and CD11b) (D) using flow cytometry.


Under same growth conditions ( permitted water , free food , a 12 h / 12 h / cycle at 20 ± 2 ° C ) , eight - week - old C57BL / 6 mice were continuously fed with methionine - choline - supplemented ( MCS ) or methionine - choline - deficient ( MCD ) diets for 6 , at same time , gavaged with LicoB , MCC950 , or a combination of LicoB MCC950 ( n = 8 mice per group ( B , C ( A ) Representative liver section stained with H & E , Sirius Red , Masson ' s trichrome stains . Scale bar : 0 . 5 cm ( top row ) 200 μm ( 3 bottom ows ) . ( B , C ) serum of ALT AST ( n = 8 mice per group ) were measured using ELISA .

O O O O O O O O O O O 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-ORGANISM O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-ORGANISM O O O O O O O O O O B-TISSUE O B-EXP_ASSAY O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O 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-TISSUE O B-GENEPROD I-GENEPROD O O O O B-ORGANISM O O O O O O B-EXP_ASSAY O

Under the same growth conditions (permitted water, free food, and a 12 h/12 h dark/light cycle at 20±2°C), eight-week-old male C57BL/6 mice were continuously fed with methionine- and choline-supplemented (MCS) or methionine- and choline-deficient (MCD) diets for 6 weeks, and at the same time, gavaged with LicoB, MCC950, or a combination of LicoB and MCC950 (n=8 mice per group (B, C (A) Representative liver section images stained with H&E, Sirius Red, and Masson's trichrome stains. Scale bar: 0.5 cm (top row) and 200 μm (3 bottom ows). (B, C) The serum levels of ALT and AST (n=8 mice per group) were measured using ELISA.


Under same growth conditions ( permitted water , free food , a 12 h / 12 h / cycle at 20 ± 2 ° C ) , eight - week - old C57BL / 6 mice were continuously fed with methionine - choline - supplemented ( MCS ) or methionine - choline - deficient ( MCD ) diets for 6 , at same time , gavaged with LicoB , MCC950 n = 6 mice per group Real - time quantitative PCR was used to detect mRNA of Col1a1 ( E ) , TNF - α ( F ) in mice livers , as described in ( A ) ( n = 6 mice per group ) .

O O O O O O O O O O O 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-ORGANISM O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O B-ORGANISM O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O B-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-ORGANISM O O O O O O O O O O O O B-ORGANISM O O O O

Under the same growth conditions (permitted water, free food, and a 12 h/12 h dark/light cycle at 20±2°C), eight-week-old male C57BL/6 mice were continuously fed with methionine- and choline-supplemented (MCS) or methionine- and choline-deficient (MCD) diets for 6 weeks, and at the same time, gavaged with LicoB, MCC950 n=6 mice per group Real-time quantitative PCR was used to detect the mRNA levels of Col1a1 (E), TNF-α (F) in the mice livers, as described in (A) (n=6 mice per group).


Under same growth conditions ( permitted water , free food , a 12 h / 12 h / cycle at 20 ± 2 ° C ) , eight - week - old C57BL / 6 mice were continuously fed with methionine - choline - supplemented ( MCS ) or methionine - choline - deficient ( MCD ) diets for 6 , at same time , gavaged with LicoB , MCC950 n = 6 mice per group Real - time quantitative PCR was used to detect mRNA of IL - 1β ( G ) , IL - 18 ( H ) in mice livers , as described in ( A ) ( n = 6 mice per group ) .

O O O O O O O O O O O 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-ORGANISM O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O B-ORGANISM O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-ORGANISM O O O O O O O O O O O O B-ORGANISM O O O O

Under the same growth conditions (permitted water, free food, and a 12 h/12 h dark/light cycle at 20±2°C), eight-week-old male C57BL/6 mice were continuously fed with methionine- and choline-supplemented (MCS) or methionine- and choline-deficient (MCD) diets for 6 weeks, and at the same time, gavaged with LicoB, MCC950 n=6 mice per group Real-time quantitative PCR was used to detect the mRNA levels of IL-1β (G), and IL-18 (H) in the mice livers, as described in (A) (n=6 mice per group).


Release of IL - 6 / or TNF release ( ELISA ) from ( C ) human MoMacs ( n = 6 - 8 ) Data information represent data ( mean + ) combined from ' n ' ( given in brackets for each panel ) technical or biological replicates ( human donor * p < 0 . 05 according to Wilcoxon signed rank sum (

B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O O B-EXP_ASSAY O O O O O B-ORGANISM B-CELL_TYPE O O O O O O O O O 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-ORGANISM O O O O O O O O O O O O O O

Release of IL-6 and/or TNF release (ELISA) from (C) human MoMacs (n=6-8) Data information represent data (mean+SEM) combined from 'n' (given in brackets for each panel) technical or biological replicates (human donor * p<0.05 according to Wilcoxon signed rank sum (


Release of IL - 6 / or TNF release ( ELISA ) fro ( D ) murine BMDMs ( n = 6 Data information represent data ( mean + ) combined from ' n ' ( given in brackets for each panel ) technical or biological replicate mic * p < 0 . 05 according to Wilcoxon signed rank sum ( C ,

B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O O B-EXP_ASSAY O O O O O B-ORGANISM B-CELL_TYPE O O O O O 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-ORGANISM O O O O O O O O O O O O O O O

Release of IL-6 and/or TNF release (ELISA) fro (D) murine BMDMs (n=6 Data information represent data (mean+SEM) combined from 'n' (given in brackets for each panel) technical or biological replicate mic * p<0.05 according to Wilcoxon signed rank sum (C, D


Release of IL - ( ELISA ) fro THP - 1 ( n = 3 ) Data information I one representative of ' n ' ( given in brackets for each panel ) independent experiments is shown ( mean + SD ) Student ' s t -

B-EXP_ASSAY O B-GENEPROD I-GENEPROD O B-EXP_ASSAY O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Release of IL- (ELISA) fro THP-1 cells (n=3) Data information I one representative of 'n' (given in brackets for each panel) independent experiments is shown (mean+SD) Student's t-tes


( f ) GFP in vivo post intradermal injection of chitin oligomers into LysMEGFP / + C57BL / 6 mice ( n = 3 / group ) Data information represent data ( mean + ) combined from ' n ' ( given in brackets for each panel ) technical or biological replicate

O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O O B-SMALL_MOLECULE O O B-GENEPROD O O O O O B-ORGANISM O O O O O O O O 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) GFP in vivo fluorescence post intradermal injection of chitin oligomers into LysMEGFP/+ C57BL/6 mice (n=3/group) Data information represent data (mean+SEM) combined from 'n' (given in brackets for each panel) technical or biological replicate mic


( G ) Representative in vivo imaging results I one representative of ' n ' ( given in brackets for each panel ) independent experiments is shown ( mean + SD )

O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O 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) Representative in vivo imaging results I one representative of 'n' (given in brackets for each panel) independent experiments is shown (mean+SD)


Leukocyte infiltration in vivo after intratracheal administration assessed by ( H ) BALF flow cytometry ( n = 3 - 4 / group Data information represent data ( mean + ) combined from ' n ' ( given in brackets for each panel ) technical or biological replicate mic ) , one - way ANOVA (

B-CELL_TYPE B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O 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-ORGANISM O O O O O O O

Leukocyte infiltration in vivo after intratracheal administration assessed by (H) BALF flow cytometry (n=3-4/group Data information represent data (mean+SEM) combined from 'n' (given in brackets for each panel) technical or biological replicate mic ), one-way ANOVA (H


Leukocyte infiltration in vivo after intratracheal administration assessed b ( I ) histology ( n = 2 ) , scale bar = 200 μm , arrows show infiltrate I one representative of ' n ' ( given in brackets for each panel ) independent experiments is shown ( mean + SD )

B-CELL_TYPE B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O

Leukocyte infiltration in vivo after intratracheal administration assessed b (I) histology analysis (n=2), scale bar = 200 μm, arrows show cellular infiltrate I one representative of 'n' (given in brackets for each panel) independent experiments is shown (mean+SD)


A . thaliana seedling ( J ) target gene mRNA induction ( n = 3 I one representative of ' n ' ( given in brackets for each panel ) independent experiments is shown ( mean + SD ) Student ' s t -

B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O O O O O O 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. thaliana seedling (J) target gene mRNA induction (n=3 I one representative of 'n' (given in brackets for each panel) independent experiments is shown (mean+SD) Student's t-tes


A . thalian leaf piece ( K ) ROS production ( 0 - 45 min , n = 6 ) Data information represent data ( mean + ) combined from ' n ' ( given in brackets for each panel ) technical or biological replicate

B-ORGANISM I-ORGANISM I-ORGANISM B-TISSUE O O O O B-SMALL_MOLECULE O O O O O O O O O O O O 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. thalian leaf piece (K) ROS production (0-45 min, n=6) Data information represent data (mean+SEM) combined from 'n' (given in brackets for each panel) technical or biological replicate plant leave


TNF released from WT or Myd88 ( A KO BMDM ( total n = 6 - 9 / group combined from 3 experiments ) Data information : I represent data ( mean + ) combined from ' n ' technical or biological replicate mic Student ' t -

B-GENEPROD B-EXP_ASSAY O O O B-GENEPROD O O O B-CELL_TYPE O O O O O O O O O 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-ORGANISM O O O O

TNF released from WT or Myd88 (A KO BMDM (total n=6-9/group combined from 3 experiments) Data information: I represent data (mean+SEM) combined from 'n' technical or biological replicate mic Student' t-test


TNF released from W or Tlr KO BMDM ( total n = 6 - 9 / group combined from 3 experiments ) Student ' s t -

B-GENEPROD B-EXP_ASSAY O O O B-GENEPROD O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O

TNF released from W or Tlr KO BMDM (total n=6-9/group combined from 3 experiments) Student's t-tes


( C ) IL - 6 released from MoMacs ( n = 5 ) treated with non - targeting ( NT ) , TLR2 - or MyD88 - specific siRNA relative to NT condition one - sample t -

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(C) IL-6 released from primary MoMacs (n=5) treated with non-targeting (NT), TLR2- or MyD88-specific siRNA relative to NT condition one-sample t-tes


( D ) CD62L shedding from human PMNs ( n = 4 - 8 ) with or without isotype control or anti - TLR2 blocking antibody Data information : I represent data ( mean + ) combined from ' n ' technical or biological replicate ( human donor * p < 0 . 05 according to Mann -

O O O B-GENEPROD O O O B-CELL_TYPE O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O

(D) CD62L shedding from human PMNs (n=4-8) with or without isotype control or anti-TLR2 blocking antibody Data information: I represent data (mean+SEM) combined from 'n' technical or biological replicate (human donor * p<0.05 according to Mann-Whitney


( E ) IL - 6 released from mock or TLR2 Cas9 - CRISPR - edited THP - 1 ( n = 3 ) I one representative of ' n ' independent experiments is shown ( mean + SD ) ) , Student ' s t -

O O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O O O B-GENEPROD I-GENEPROD O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O 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) IL-6 protein released from mock or TLR2 Cas9-CRISPR-edited THP-1 cells (n=3) I one representative of 'n' independent experiments is shown (mean+SD) ), Student's t-tes


( F ) NF - κB activity in empty vector ( EV ) or TLR2 - transfected HEK293T ( n = 3 ) I one representative of ' n ' independent experiments is shown ( mean + SD )

O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR B-EXP_ASSAY O O O O O O O B-GENEPROD O O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O O

(F) NF-κB activity in empty vector (EV) or TLR2-transfected HEK293T cells (n=3) I one representative of 'n' independent experiments is shown (mean+SD)


( G ) MPO release from intradermally injected WT or Tlr2 KO mice ( n = 5 - 15 biopsies each ) Data information I represent data ( mean + ) combined from ' n ' technical or biological replicate mic ) , Student ' s t -

O O O B-GENEPROD B-EXP_ASSAY O O O O O B-GENEPROD O B-ORGANISM O O O 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-ORGANISM O O O O O O O

(G) MPO release from intradermally injected WT or Tlr2 KO mice (n=5-15 biopsies each) Data information I represent data (mean+SEM) combined from 'n' technical or biological replicate mic ), Student's t-tes


( H ) BALF IL - 6 TNF from intratracheally treated WT or Tlr2 KO mice ( n = 4 - 5 mice each ) one - way ANOVA with Dunnett ' s multiple compariso

O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD O B-ORGANISM O O O O O O B-ORGANISM O O O O O O O O O O O O

(H) BALF IL-6 and TNF from intratracheally treated WT or Tlr2 KO mice (n=4-5 mice each) one-way ANOVA with Dunnett's multiple compariso


( I ) Relative IL10 mRNA in whole blood of WT or heterozygous TLR2 R753Q carriers ( n = 5 each ) Data information : I represent data ( mean + ) combined from ' n ' technical or biological replicate ( human donor * p < 0 . 05 according to Mann -

O O O O B-GENEPROD O O B-TISSUE I-TISSUE O O O O B-GENEPROD O O O O 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-ORGANISM O O O O O O O O O O O

(I) Relative IL10 mRNA in whole blood of WT or heterozygous TLR2 R753Q carriers (n=5 each) Data information: I represent data (mean+SEM) combined from 'n' technical or biological replicate (human donor * p<0.05 according to Mann-Whitney


( A ) NF - κB activity in TLR2 - transfected HEK293T ( n = 3 ) Data information I one representative of ' n ' independent experiments is shown ( mean + SD ) Student ' s t -

O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR B-EXP_ASSAY O B-GENEPROD O O B-CELL_LINE O O O O 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) NF-κB activity in TLR2-transfected HEK293T cells (n=3) Data information I one representative of 'n' independent experiments is shown (mean+SD) Student's t-tes


( B ) IL - 8 release ROS production from PMNs ( n = 7 ) Data information represent data ( mean + ) from ' n ' technical or biological replicates ( donors ) * p < 0 . 05 according to Wilcoxon signed rank

O O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY B-SMALL_MOLECULE O O B-CELL_TYPE O O O O O O O O O O O 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) IL-8 release and ROS production from primary PMNs (n=7) Data information represent data (mean+SEM) from 'n' technical or biological replicates (donors) * p<0.05 according to Wilcoxon signed rank su


( D ) IRAK4 S186 T345 phospho - site containing IRAK4 peptides quantified in proteome - wide phospho - screen in THP - 1 ( n = 1 ) Data informatio I one representative of ' n ' independent experiments is shown ( mean + SD ) for details on data acquisition for panel see Expanded View

O O O B-GENEPROD O O B-EXP_ASSAY O O O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O 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) IRAK4 S186 and T345 phospho-site containing IRAK4 peptides quantified in proteome-wide phospho-screen in THP-1 cells (n=1) Data informatio I one representative of 'n' independent experiments is shown (mean+SD) for details on data acquisition and analysis for panel see Expanded View Information


( F ) Microarray intensities ( excerpt ) of chitin - specific signature genes ( n = 5 ) Data information represent data ( mean + ) from ' n ' technical or biological replicates ( donors ) for details on data acquisition for panels ( D - F ) see Expanded View

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O 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) Microarray intensities (excerpt) of chitin-specific signature genes (n=5) Data information represent data (mean+SEM) from 'n' technical or biological replicates (donors) for details on data acquisition and analysis for panels (D-F) see Expanded View Information


( G ) Relative cytokine mRNAs in whole blood stimulation ( n = 5 - 13 ) Data information represent data ( mean + ) from ' n ' technical or biological replicates ( donors ) * p < 0 . 05 according to Wilcoxon signed rank

O O O O B-GENEPROD O O B-TISSUE I-TISSUE O O O O O O O O O O O O O O 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) Relative cytokine mRNAs in whole blood stimulation (n=5-13) Data information represent data (mean+SEM) from 'n' technical or biological replicates (donors) * p<0.05 according to Wilcoxon signed rank su


Flow cytometric quantificatio of Alexa647 - labeled C10 - 15 interaction with mTLR2 - Fc ( n = 4 each ) Data information : sub - panel represent data ( mean + SE combined from ' n ' biological replicate mic I one representative of ' n ' independent experiments is show p < 0 . 05 according to Mann - Whitney U (

B-EXP_ASSAY I-EXP_ASSAY O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O B-GENEPROD O O O 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-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O

Flow cytometric quantificatio of Alexa647-labeled C10-15 interaction with mTLR2-Fc Protein (n=4 each) Data information: Quantification sub-panel represent data (mean+SE combined from 'n' biological replicate mic I one representative of 'n' independent experiments is show p<0.05 according to Mann-Whitney U (A


microscale thermophoresis ( MST ) ( B ) of Alexa647 - labeled C10 - 15 interaction with mTLR2 - Fc ( n = 4 each ) I one representative of ' n ' independent experiments is

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O B-GENEPROD O O O O O O O O O O O O O O O O O O

microscale thermophoresis (MST) analysis (B) of Alexa647-labeled C10-15 interaction with mTLR2-Fc Protein (n=4 each) I one representative of 'n' independent experiments is show


( C ) Flow cytometr of Candida albicans stained with control IgG or mTLR2 - Fc anti - Fc - Alexa594 , together with ConA - Alexa488 CFW ( n = 4 each ) . Scale bar = 5 μm Data information : sub - panel represent data ( mean + SE combined from ' n ' biological replicate mic I one representative of ' n ' independent experiments is show one - sample t - (

O O O B-EXP_ASSAY I-EXP_ASSAY O B-ORGANISM I-ORGANISM B-EXP_ASSAY O O O O B-GENEPROD O O O O O O B-SMALL_MOLECULE O O O B-GENEPROD O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O 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-ORGANISM O O O O O O O O O O O O O O O O O

(C) Flow cytometr of Candida albicans stained with control IgG or mTLR2-Fc anti-Fc-Alexa594, together with ConA-Alexa488 and CFW (n=4 each). Scale bar = 5 μm Data information: Quantification sub-panel represent data (mean+SE combined from 'n' biological replicate mic I one representative of 'n' independent experiments is show one-sample t-test (C


( D ) microscopy of Candida albicans stained with control IgG or mTLR2 - Fc anti - Fc - Alexa594 , together with ConA - Alexa488 CFW ( n = 4 each ) . Scale bar = 5 μm I one representative of ' n ' independent experiments is

O O O B-EXP_ASSAY O B-ORGANISM I-ORGANISM B-EXP_ASSAY O O O O B-GENEPROD O O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O

(D) fluorescence microscopy of Candida albicans stained with control IgG or mTLR2-Fc anti-Fc-Alexa594, together with ConA-Alexa488 and CFW (n=4 each). Scale bar = 5 μm I one representative of 'n' independent experiments is show


( g ) MST of Alexa647 - labeled C10 - 15 mTLR2 - Fc with SSL3 titration ( n = 2 ) Data information I one representative of ' n ' independent experiments is

O O O B-EXP_ASSAY O B-SMALL_MOLECULE O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE B-GENEPROD O O O B-GENEPROD B-EXP_ASSAY O O O O O O O O O O O O O O O O O

(g) MST analysis of Alexa647-labeled C10-15 and mTLR2-Fc protein with SSL3 titration (n=2) Data information I one representative of 'n' independent experiments is show


( H ) NF - κB activation in TLR2 - transfected HEK293T ( n = 3 Data information I one representative of ' n ' independent experiments is shown ( for asmean + SD ) ) , Student ' s t -

O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR B-EXP_ASSAY O B-GENEPROD O O B-CELL_LINE O O O O O O 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) NF-κB activation in TLR2-transfected HEK293T cells (n=3 Data information I one representative of 'n' independent experiments is shown (for asmean+SD) ), Student's t-tes


IL - 8 release from PMNs ( n = 2 ) without or with SSL3 Data information I one representative of ' n ' independent experiments is show asmean + SD ) Student ' s t -

B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O B-CELL_TYPE O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O

IL-8 release from primary PMNs (n=2) without or with SSL3 Data information I one representative of 'n' independent experiments is show asmean+SD) Student's t-tes


( J ) BALF TNF in C57BL / 6 mice ( n = 5 / group ) upon C10 - 15 administration without or with SSL3 Data information : Quantificatio - panel represent data ( mean + SE combined from ' n ' biological replicate mic one - way ANOVA with Dunnett ' s multiple comparison ( J )

O O O O B-GENEPROD O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O

(J) BALF TNF in C57BL/6 mice (n=5/group) upon C10-15 administration without or with SSL3 Data information: Quantificatio -panel represent data (mean+SE combined from 'n' biological replicate mic one-way ANOVA with Dunnett's multiple comparison (J)


( K ) ROS production between 0 45 min post chitin application in A . thaliana leaf pieces ( n = 6 ) Data information : Quantificatio panels ( represent dat mean ± SD in K combined from ' n ' biological replicate

O O O B-SMALL_MOLECULE O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O

(K) ROS production between 0 and 45 min post chitin application in A. thaliana leaf pieces (n=6) Data information: Quantificatio panels ( represent dat mean±SD in K combined from 'n' biological replicate plant leave


( L ) MST of Alexa647 - labeled C10 - 15 mTLR2 - Fc with C5 titration ( n = 2 ) Data informatio I one representative of ' n ' independent experiments is

O O O B-EXP_ASSAY O B-SMALL_MOLECULE O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE B-GENEPROD O O O B-SMALL_MOLECULE B-EXP_ASSAY O O O O O O O O O O O O O O O O O

(L) MST analysis of Alexa647-labeled C10-15 and mTLR2-Fc Protein with C5 titration (n=2) Data informatio I one representative of 'n' independent experiments is show


B . Screening results of compounds competing Dvl - CXXC5 interaction . Each 2 μM of compounds was used for competing as described in Fig 1A . [ n = 3 ]

O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O

B . Screening results of compounds competing Dvl-CXXC5 interaction. Each 2 μM of the compounds was used for competing as described in Fig 1A. [n=3]


C , D . calvaria were treated with 2 μM of each compound for 2 days . were subjected to immunofluorescence analyses to visualize β - catenin ( C ; green ) . relative numbers of β - catenin - positive nuclei were counted ( D ) . [ n = 3 ]

O O O O B-TISSUE O O O O O O O O O O O O O O O B-EXP_ASSAY O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-SUBCELLULAR O O O O O O O O O O O

C, D. Primary calvaria cells were treated with 2 μM of each compound for 2 days. The cells were subjected to immunofluorescence analyses to visualize β-catenin (C; green). The relative numbers of β-catenin-positive nuclei were counted (D). [n=3]


E , F . calvariae from 4 - day - old mice were cultured ex vivo for 7 days with 2 µM of each compound . Representative calvaria sections were visualized by H & amp ; amp ; E ( E ) . calvaria thicknesses were measured from using Image Pro software ( F ) . [ n = 3 ]

O O O O B-TISSUE O O O O O O B-ORGANISM O O O O O O O O O O O O O O O B-TISSUE O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-TISSUE O O O O O O O O O O O O O O O O O

E, F . The calvariae from 4-day-old mice were cultured ex vivo for 7 days with 2 µM of each compound. Representative calvaria sections were visualized by H&amp;E staining (E). The calvaria thicknesses were measured from the images using Image Pro software (F). [n=3]


B . A competition curve for Dvl - CXXC5 interaction by KY - 02061 .

O O O O O O B-GENEPROD O B-GENEPROD O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O

B . A competition curve for the Dvl-CXXC5 interaction by KY-02061.


C - E . NMR titration analyses for Dvl PZD domain with KY - 02061 . 1H - 15N - HSQC analyses were performed to analyze interaction of 15N - labelled Dvl - PDZ domain with KY - 02061 . 1H - 15N - HSQC spectrum of different molar ratios ( Dvl PDZ domain : KY - 02061 ) are displayed as red ( 1 : 0 ) , orange ( 1 : 10 ) , purple ( 1 : 20 ) , cyan ( 1 : 40 ) , green ( 1 : 60 ) blue ( 1 : 80 ) ( C , residues with meaningful chemical shift change are indicated by arrows ) . Plot of chemical shift changes ( Δδ ) as a function of residue number in molecular 1 : 80 ( D , a red - colored indicates for Δδ = 0 . 05 ) . residues with Δδ greater than 0 . 05 are visualized as a stick model on ribbon representation of Dvl PDZ domain structure ( E ) .

O O O O B-EXP_ASSAY O O O B-GENEPROD O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-SMALL_MOLECULE O O B-GENEPROD O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O B-GENEPROD O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O O O O O O O

C-E. NMR titration analyses for Dvl PZD domain with KY-02061. 1H-15N-HSQC analyses were performed to analyze the interaction of 15N-labelled Dvl-PDZ domain with KY-02061. The 1H-15N-HSQC spectrum of different molar ratios (Dvl PDZ domain:KY-02061) are displayed as red (1:0), orange (1:10), purple (1:20), cyan (1:40), green (1:60) and blue (1:80) (C, residues with meaningful chemical shift change are indicated by arrows). Plot of chemical shift changes (Δδ) as a function of residue number in molecular ratio 1:80 (D, a red-colored line indicates the line for Δδ=0.05). The residues with Δδ greater than 0.05 are visualized as a stick model on the ribbon representation of the Dvl PDZ domain structure (E).


F . Molecular docking of Dvl binding motif ( DBM ) or KY - 02061 to Dvl PDZ domain were analyzed by in silico experiments . superimposed structure of DBM ( green ) KY - 02061 ( yellow ) on surface of Dvl PDZ domain ( gray ) was visualized .

O O O O O B-GENEPROD O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-GENEPROD O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O B-GENEPROD O O O O O O O O

F. Molecular docking of Dvl binding motif (DBM) or KY-02061 to Dvl PDZ domain were analyzed by in silico experiments. The superimposed structure of DBM (green) and KY-02061 (yellow) on the surface of Dvl PDZ domain (gray) was visualized.


A . MC3T3E1 were transfected with pTOPFLASH together with pCMV - β - gal . After 24 h , were treated with DMSO , KY - 02061 in DMSO , or 5 µM PolyR - DBM for 2 days . luciferase activities of whole lysates were measured normalized with β - galactosidase activities . [ n = 3 ]

O O B-CELL_LINE O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O

A . MC3T3E1 cells were transfected with pTOPFLASH together with pCMV-β-gal. After 24 h, the cells were treated with DMSO, KY-02061 in DMSO, or 5 µM PolyR-DBM for 2 days. The luciferase activities of whole cell lysates were measured and normalized with β-galactosidase activities. [n=3]


B . calvaria were isolated from calvariae of 4 - day - old mice ( Manton et al , 2007 ) , treated with DMSO or KY - 02061 in DMSO for 4 days . ALP activity were visualized by ALP .

O O B-TISSUE O O O B-TISSUE O O O O O O B-ORGANISM O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O B-EXP_ASSAY O O O O B-GENEPROD O

B . Primary calvaria cells were isolated from the calvariae of 4-day-old mice (Manton et al, 2007), and treated with DMSO or KY-02061 in DMSO for 4 days. ALP activity levels were visualized by ALP staining.


C . calvaria were treated with DMSO or 5 µM KY - 02061 in DMSO for 14 days . were subjected to immunofluorescence analyses to visualize Runx2 ( green ) β - catenin ( red ) . nuclei were counterstained with DAPI ( blue ) .

O O B-TISSUE O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O B-EXP_ASSAY O O O B-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-SUBCELLULAR O O O B-EXP_ASSAY O O O O

C. Primary calvaria cells were treated with DMSO or 5 µM KY-02061 in DMSO for 14 days. The cells were subjected to immunofluorescence analyses to visualize Runx2 (green) and β-catenin (red). The cell nuclei were counterstained with DAPI (blue).


D , E . Calvariae from 4 - day - old mice were cultured ex vivo for 7 days with KY - 02061 in DMSO ( D ) . calvaria thicknesses were measured from stained sections using Image Pro software ( E ) . [ n = 3 ]

O O O O B-TISSUE O O O O O O B-ORGANISM O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O

D, E. Calvariae from 4-day-old mice were cultured ex vivo for 7 days with KY-02061 in DMSO (D). The calvaria thicknesses were measured from the stained sections using Image Pro software (E). [n=3]


B . A competition curve for Dvl - CXXC5 interaction by KY - 02327 .

B. A competition curve for the Dvl-CXXC5 interaction by KY-02327.


O O O O O B-GENEPROD O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O B-GENEPROD O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Fluorescence quenching plot of Dvl PDZ domain upon addition of different amounts of KY-02327. From top to bottom final moral ratio between Dvl PDZ and KY-02327 was, 1:0, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:15, 1:20 and 1:25, respectively.


D - F . 1H - 15N - HSQC analyses were performed to analyze interaction of 15N - labelled Dvl - PDZ domain with KY - 02327 . 1H - 15N - HSQC spectrum of different molar ratios ( Dvl PDZ domain : KY - 02327 ) are displayed as red ( 1 : 0 ) , yellow ( 1 : 5 ) , green ( 1 : 10 ) , magenta ( 1 : 20 ) ( D , residues with meaningful chemical shift change are indicated by arrows ) . Plot of chemical shift changes ( Δδ ) as a function of residue number in molecular 1 : 20 ( E , a red - colored indicates for Δδ = 0 . 05 ) . residues with Δδ greater than 0 . 05 are visualized as a stick model on ribbon representation of Dvl PDZ domain structure ( F ) .

O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-SMALL_MOLECULE O O B-GENEPROD O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O B-GENEPROD O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O O O O O O O

D-F. 1H-15N-HSQC analyses were performed to analyze the interaction of 15N-labelled Dvl-PDZ domain with KY-02327. The 1H-15N-HSQC spectrum of different molar ratios (Dvl PDZ domain:KY-02327) are displayed as red (1:0), yellow (1:5), green (1:10), and magenta (1:20) (D, residues with meaningful chemical shift change are indicated by arrows). Plot of chemical shift changes (Δδ) as a function of residue number in molecular ratio 1:20 (E, a red-colored line indicates the line for Δδ=0.05). The residues with Δδ greater than 0.05 are visualized as a stick model on the ribbon representation of the Dvl PDZ domain structure (F).


G . Molecular docking of KY - 02061 or KY - 02327 to Dvl PDZ domain were analyzed by in silico experiments . superimposed structure of KY - 02061 ( yellow ) KY - 02327 ( cyan ) on surface of Dvl PDZ domain ( gray ) was visualized .

O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-GENEPROD O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O B-GENEPROD O O O O O O O O

G. Molecular docking of KY-02061 or KY-02327 to Dvl PDZ domain were analyzed by in silico experiments. The superimposed structure of KY-02061 (yellow) and KY-02327 (cyan) on the surface of Dvl PDZ domain (gray) was visualized.


A . MC3T3E1 were transfected with pTOPFLASH together with pCMV - β - gal . After 24 h , were treated with indicated dose of KY - 02327 for 2 days . luciferase activities of whole lysates were measured normalized with β - galactosidase activities . [ n = 3 ]

O O B-CELL_LINE O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-EXP_ASSAY O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O

A . MC3T3E1 cells were transfected with pTOPFLASH together with pCMV-β-gal. After 24 h, the cells were treated with indicated dose of KY-02327 for 2 days. The luciferase activities of whole cell lysates were measured and normalized with β-galactosidase activities. [n=3]


B - C . MC3T3E1 were treated with indicated concentrations of KY - 02327 for 2 days . β - Catenin α - tubulin were detected by immunoblot .

O O O O B-CELL_LINE O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O B-EXP_ASSAY O

B-C . MC3T3E1 cells were treated with indicated concentrations of KY-02327 for 2 days. β-Catenin and α-tubulin were detected by immunoblot.


B - C . MC3T3E1 were treated with indicated concentrations of KY - 02327 for 2 days . β - Catenin α - tubulin were detected by immunoblot ( B ) or immunofluorescence ( C ) analyses . Nuclei were counterstained with DAPI ( C , blue ) .

O O O O B-CELL_LINE O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O B-EXP_ASSAY O O O O O B-SUBCELLULAR O O O B-EXP_ASSAY O O O O O O

B-C . MC3T3E1 cells were treated with indicated concentrations of KY-02327 for 2 days. β-Catenin and α-tubulin were detected by immunoblot (B) or immunofluorescence (C) analyses. Nuclei were counterstained with DAPI (C, blue).


D . MC3T3E1 were treated with indicated concentrations of KY - 02327 for 14 days . mRNA level of collagen 1a ( Col1a ) was measured by quantitative real - time qRT - PCR . [ n = 3 ]

O O B-CELL_LINE O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O B-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O

D. MC3T3E1 cells were treated with indicated concentrations of KY-02327 for 14 days. The mRNA level of collagen 1a (Col1a) was measured by quantitative real-time qRT-PCR. [n=3]


E . MC3T3E1 were treated with indicated concentrations of KY - 02327 for 21 days . mRNA level of osteocalcin ( OCN ) was measured by qRT - PCR . [ n = 3 ]

O O B-CELL_LINE O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O B-GENEPROD O B-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O

E. MC3T3E1 cells were treated with indicated concentrations of KY-02327 for 21 days. The mRNA level of osteocalcin (OCN) was measured by qRT-PCR. [n=3]


A - F . Vehicle or 20 mg of KY - 02327 per kg of ( mpk ) was orally administered or 0 . 06 mpk of N - terminal fragment of PTH ( 1 - 34 ) was subcutaneously injected into Sham - operated ( Sham ) or ovariectomized ( OVX ) mice on 5 sequential days per week for 4 [ n = 4 ] . After calcein injection , prepared femoral sections of OVX mice were examined under microscope to visualize integrated calcein ( A , green , arrows ) . Nuclei were counterstained with DAPI ( A , blue ) . Osteoblast numbers ( B ) calcein double labelled surfaces ( C ) were measured in femoral sections .

O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O B-TISSUE O O O B-ORGANISM O O O B-EXP_ASSAY O O O B-SMALL_MOLECULE O O O O O O O O B-SUBCELLULAR O O O B-EXP_ASSAY O O O O O O B-CELL_TYPE O O O O B-SMALL_MOLECULE O O O O O O O O O B-TISSUE O O

A-F. Vehicle or 20 mg of KY-02327 per kg of animal body weight (mpk) was orally administered or 0.06 mpk of the N-terminal fragment of PTH (amino acids 1-34) was subcutaneously injected into the Sham-operated (Sham) or ovariectomized (OVX) mice on 5 sequential days per week for 4 weeks [n=4]. After calcein injection, the prepared femoral sections of the OVX mice were examined under fluorescence microscope to visualize integrated calcein (A, green, arrows). Nuclei were counterstained with DAPI (A, blue). Osteoblast numbers (B) and calcein double labelled surfaces (C) were measured in the femoral sections.


A - F . Vehicle or 20 mg of KY - 02327 per kg of ( mpk ) was orally administered or 0 . 06 mpk of N - terminal fragment of PTH ( 1 - 34 ) was subcutaneously injected into Sham - operated ( Sham ) or ovariectomized ( OVX ) mice on 5 sequential days per week for 4 [ n = 4 ] . mouse femurs were analyzed using micro - CT BMD ( D ) BV / TV ( E ) were calculated from micro - CT data .

O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O B-ORGANISM B-TISSUE O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O

A-F. Vehicle or 20 mg of KY-02327 per kg of animal body weight (mpk) was orally administered or 0.06 mpk of the N-terminal fragment of PTH (amino acids 1-34) was subcutaneously injected into the Sham-operated (Sham) or ovariectomized (OVX) mice on 5 sequential days per week for 4 weeks [n=4]. The mouse femurs were analyzed using micro-CT and BMD (D) and BV/TV (E) were calculated from the micro-CT data.


A - F . Vehicle or 20 mg of KY - 02327 per kg of ( mpk ) was orally administered or 0 . 06 mpk of N - terminal fragment of PTH ( 1 - 34 ) was subcutaneously injected into Sham - operated ( Sham ) or ovariectomized ( OVX ) mice on 5 sequential days per week for 4 [ n = 4 ] . three - dimensional of femoral trabecular bone were reconstructed ( F ) , trabecular number ( G ) trabecular separation ( H ) were calculated .

O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O B-TISSUE I-TISSUE I-TISSUE O O O O O O B-TISSUE O O O O B-TISSUE O O O O O O O

A-F. Vehicle or 20 mg of KY-02327 per kg of animal body weight (mpk) was orally administered or 0.06 mpk of the N-terminal fragment of PTH (amino acids 1-34) was subcutaneously injected into the Sham-operated (Sham) or ovariectomized (OVX) mice on 5 sequential days per week for 4 weeks [n=4]. The three-dimensional images of femoral trabecular bone were reconstructed (F), and trabecular number (G) and trabecular separation (H) were calculated.


B , C Kinetic change of mRNA expression of Top1 , Btbd1 , Tdp1 , Srsf1 in microglia following LPS / IFNγ stimulation ( n = 6 , technical replicates ) . Data information : Data are presented as mean ± s . e . m . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; Statistical analyses were performed using two - way ANOVA with Dunnett ' s Multiple Comparison for ( B C ) ,

O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-CELL_TYPE O B-SMALL_MOLECULE O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Kinetic change of mRNA expression of Top1, Btbd1, Tdp1, and Srsf1 in microglia following LPS/IFNγ stimulation (n = 6, technical replicates). Data information: Data are presented as the mean ± s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001; Statistical analyses were performed using two-way ANOVA with Dunnett's Multiple Comparison test for (B and C),


D , E expression of TOP1 was detected by Western blotting ( n = 3 , biological replicates ) . Data information : Data are presented as mean ± s . e . m . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 Statistical analyses were performed using student ' s unpaired two - t - .

O O O B-EXP_ASSAY O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D, E The protein expression of TOP1 was detected by Western blotting (n = 3, biological replicates). Data information: Data are presented as the mean ± s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001 Statistical analyses were performed using the student's unpaired two-tailed t-test.


F - H Immunostaining of TOP1 ( red ) in stimulated microglia ( 4 h after LPS / IFNγ stimulation ) or resting microglia , co - stained with Hoechst ( blue ) ( bar = 20 μm ) ; for lower - left panel in ( H ) , n = 3 technical replicates per group ( 2 to 5 random fields per well were analyzed ) ; for lower right panel , 201 from unstimulated microglia 256 from stimulated microglia were analyzed . Data information : Data are presented as mean ± s . e . m . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; Statistical analyses were performed using student ' s unpaired two - t - .

O O O B-EXP_ASSAY O B-GENEPROD O O O O O B-CELL_TYPE O O O O B-SMALL_MOLECULE O B-GENEPROD O O O O B-CELL_TYPE O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O 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-CELL_TYPE O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-H Immunostaining of TOP1 (red) in stimulated microglia (4 h after LPS/IFNγ stimulation) or resting microglia, co-stained with Hoechst (blue) (bar = 20 μm); for the lower-left panel in (H), n = 3 technical replicates per group (2 to 5 random fields per well were analyzed); for the lower right panel, 201 cells from unstimulated microglia and 256 cells from stimulated microglia were analyzed. Data information: Data are presented as the mean ± s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001; Statistical analyses were performed using the student's unpaired two-tailed t-test.


G Immunofluorescent of spinal cord sections from mice at EAE peak control mice with anti - F4 / 80 ( green ) , TOP1 ( magenta ) , nuclei with Hoechst ( blue ) . For left panels , bar = 400 μm ; for magnified panels , bar = 100 μm . yellow arrowhead indicates a representative F4 / 80 + with high TOP1 expression .

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G Immunofluorescent staining of spinal cord sections from mice at EAE peak and control mice with anti-F4/80 (green), TOP1 (magenta), and nuclei with Hoechst (blue). For the left panels, bar = 400 μm; for the magnified panels, bar = 100 μm. The yellow arrowhead indicates a representative F4/80+ cell with high TOP1 expression.


I A magnified image of selected region ( box ) in ( G ) ; bar = 50 F4 / 80 + with increased TOP1 expression in nucleus in ( G ) . J mean of TOP1 expression in matter in ( G ) was graphed ; n = 3 to 4 mice per group with 3 - 5 sections from each mouse . Data information : Data are presented as mean ± s . e . m . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 Statistical analyses were performed using student ' s unpaired two - t - .

O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD B-EXP_ASSAY O B-SUBCELLULAR O O O O O O O O B-GENEPROD B-EXP_ASSAY O B-TISSUE O O O O O O O O O O O O B-ORGANISM O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

I A magnified image of the selected region (white box) in (G); bar = 50 F4/80+ cell with increased TOP1 expression in the nucleus in (G). J The mean fluorescent intensity of TOP1 expression in the white matter in (G) was graphed; n = 3 to 4 mice per group with 3-5 sections from each mouse. Data information: Data are presented as the mean ± s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001 Statistical analyses were performed using the student's unpaired two-tailed t-test.


K TOP1 expression in spinal cord homogenates of EAE mice dissected at disease peak ( scored 3 ) or control mice were examined by Western blotting . L of TOP1 expression of Western blot bands in ( K ) with normalization to GAPDH β - actin ( n = 3 biological samples ) . Data information : Data are presented as mean ± s . e . m . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 Statistical analyses were performed using student ' s unpaired two - t - .

O B-GENEPROD O O B-TISSUE I-TISSUE O O B-DISEASE B-ORGANISM O O O O O O O O O O B-ORGANISM O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

K TOP1 expression in the spinal cord homogenates of EAE mice dissected at disease peak (scored 3) or control mice were examined by Western blotting. L The quantification of TOP1 protein expression of Western blot bands in (K) with normalization to GAPDH and β-actin (n = 3 biological samples). Data information: Data are presented as the mean ± s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001 Statistical analyses were performed using the student's unpaired two-tailed t-test.


M , Western blotting of TOP1 expression from brain homogenates of mice receiving intracisternal LPS injection or PBS after 8 h ( n = 4 or 5 biological samples ) .

O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O B-TISSUE O O B-ORGANISM O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O O O O O O O O

M, Western blotting of TOP1 expression from brain homogenates of mice receiving intracisternal LPS injection or PBS after 8 h (n = 4 or 5 biological samples).


TOP1 expression from brain homogenates of mice receiving intracisternal LPS injection or PBS after 8 h ( n = 4 or 5 biological samples ) . Data information : Data are presented as mean ± s . e . m . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 Statistical analyses were performed using student ' s unpaired two - t - .

B-GENEPROD B-EXP_ASSAY O B-TISSUE O O B-ORGANISM O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

TOP1 expression from brain homogenates of mice receiving intracisternal LPS injection or PBS after 8 h (n = 4 or 5 biological samples). Data information: Data are presented as the mean ± s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001 Statistical analyses were performed using the student's unpaired two-tailed t-test.


O Microglia were sorted from LPS challenged mice ( i . p ) after 4 h or naive mice , mRNA expression of TOP1 was graphed ( n = 5 or 6 biological samples ) . Data information : Data are presented as mean ± s . e . m . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 Statistical analyses were performed using student ' s unpaired two - t - .

O B-CELL_TYPE O O O B-SMALL_MOLECULE O B-ORGANISM O O O O O O O O O O B-ORGANISM O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

O Microglia were sorted from LPS challenged mice (i.p) after 4 h or naive mice, and the mRNA expression of TOP1 was graphed (n = 5 or 6 biological samples). Data information: Data are presented as the mean ± s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001 Statistical analyses were performed using the student's unpaired two-tailed t-test.


P Brain sections from healthy control MS brain tissues , including active MS lesion , chronic active lesion , normal - appearing matter ( NAWM ) , were stained with anti - HLA - DR , anti - TOP1 , co - stained with Hoechst ( bar = 50 μm ) .

O B-TISSUE O O O O B-DISEASE B-TISSUE O O O O B-DISEASE O O O O O O O O O B-TISSUE O O O O O B-EXP_ASSAY O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O

P Brain sections from healthy control and MS brain tissues, including the active MS lesion, chronic active lesion, and the normal-appearing white matter (NAWM), were stained with anti-HLA-DR, anti-TOP1, and co-stained with Hoechst (bar = 50 μm).


C , D Immunostaining of iNOS ( red ) Phalloidin ( green ) in microglia ( n = 3 technical replicates with more than 5 random fields per well ) ( bar = 50 μm ) . Data information : Data shown are mean ± s . e . m . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; n . s . = no statistical significance . Statistical analyses were performed using one - way ANOVA with Dunnett ' s Multiple Comparison for ( D )

O O O B-EXP_ASSAY O B-GENEPROD O O O B-SMALL_MOLECULE O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, D Immunostaining of iNOS (red) and Phalloidin (green) in microglia (n = 3 technical replicates with more than 5 random fields per well) (bar = 50 μm). Data information: Data shown are mean ± s.e.m. *P<0.05; **P<0.01; ***P<0.001; n.s. = no statistical significance. Statistical analyses were performed using one-way ANOVA with Dunnett's Multiple Comparison Test for (D)


B Microglia in hippocampus thalamus were stained with Iba1 ( red ) ; bar = 50 μm ; microglia in yellow boxes were presented in ( E ) .

O B-CELL_TYPE O B-TISSUE I-TISSUE O B-EXP_ASSAY O B-GENEPROD O O O O O O O O O B-CELL_TYPE O O O O O O O O O O

B Microglia in the hippocampus and thalamus were stained with Iba1 (red); bar = 50 μm; microglia in the yellow boxes were presented in (E).


C , D of Iba1 in hippocampus thalamus in ( B ) ; n = 2 , 4 , 5 mice for naive , LPS , LPS + TPT group , respectively ( 2 - 6 sections per mouse were analyzed ) . Data information : Data shown are mean ± s . e . m . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; n . s . = no statistical significance . Statistical analyses comparing LPS _ PBS group with LPS _ TPT group were performed using Student ' s unpaired two - t -

O O O O B-GENEPROD O B-TISSUE I-TISSUE O O O O O O O O O O O O B-ORGANISM O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O

C, D Fluorescent intensity of Iba1 in hippocampus and thalamus in (B); n = 2, 4, 5 mice for naive, LPS, and LPS + TPT group, respectively (2-6 sections per mouse were analyzed). Data information: Data shown are mean ± s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001; n.s. = no statistical significance. Statistical analyses comparing the LPS_PBS group with the LPS_TPT group were performed using the Student's unpaired two-tailed t-test


L , M Cytokine array on brain homogenates after 24 h of LPS challenge ( n = 2 mice for naïve group ; n = 3 mice for LPS LPS + TPT groups ) .

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L, M Cytokine array on brain homogenates after 24 h of LPS challenge (n = 2 mice for the naïve group; n = 3 mice for the LPS and LPS + TPT groups).


K Spinal cord sections dissected from Day 30 from EAE batch in ( H ) were stained with CD11b ( red ) , Fluoromyelin ( green ) , Hoechst ( blue ) ( bar = 200 μm ) . L CD11b immunofluorescent in ( K ) was graphed ( n = 2 mice for control group ; n = 4 mice for EAE - PBS EAE - TPT groups ) . M of demyelinated in matter of spinal cord was graphed ( n = 2 mice for control group , n = 4 mice for EAE - PBS EAE - TPT groups ) . Data information : Data are presented as mean ± s . e . m . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; Student ' s unpaired two - t - was used for comparing EAE - PBS group with EAE - TPT group in L , M .

O B-TISSUE I-TISSUE O O O O O O B-DISEASE O O O O O O B-EXP_ASSAY O B-GENEPROD O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-GENEPROD B-EXP_ASSAY O O O O O O O O O O B-ORGANISM O O O O O O O B-ORGANISM O B-DISEASE O B-SMALL_MOLECULE B-DISEASE O B-SMALL_MOLECULE O O O O O O O B-TISSUE O B-TISSUE I-TISSUE O O O O O O B-ORGANISM O O O O O O O B-ORGANISM O B-DISEASE O B-SMALL_MOLECULE B-DISEASE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-DISEASE O B-SMALL_MOLECULE O O B-DISEASE O B-SMALL_MOLECULE O O O O O O

K Spinal cord sections dissected from Day 30 from the EAE batch in (H) were stained with CD11b (red), Fluoromyelin (green), and Hoechst (blue) (bar = 200 μm). L The CD11b immunofluorescent intensity in (K) was graphed (n = 2 mice for the control group; n = 4 mice for the EAE-PBS and EAE-TPT groups). M The percentage of demyelinated area in the white matter of the spinal cord was graphed (n = 2 mice for the control group, n = 4 mice for the EAE-PBS and EAE-TPT groups). Data information: Data are presented as the mean ± s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001; the Student's unpaired two-tailed t-test was used for comparing the EAE-PBS group with the EAE-TPT group in L, and M.


A - seq . of freshly isolated , proliferating differentiating satellite reveals an increased expression of linc - MYH in proliferating differentiating MuSCs , but no expression is found in in freshly isolated MuSCs .

O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-EXP_ASSAY O O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O B-EXP_ASSAY O B-CELL_TYPE O O O B-EXP_ASSAY O O O O O O B-CELL_TYPE O

A RNA-seq. of freshly isolated, proliferating and differentiating satellite cells reveals an increased expression of linc-MYH in proliferating and differentiating MuSCs, but no expression is found in in freshly isolated MuSCs.


Expression of linc - MYH is confined to skeletal muscle in mouse ( B ) tissues .

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Expression of linc-MYH is confined to skeletal muscle in mouse (B) tissues.


Expression of linc - MYH is confined to skeletal muscle in human ( C ) tissues .

Expression of linc-MYH is confined to skeletal muscle in human (C) tissues.


D Expression of linc - MYH is not detected in limb buds at E10 . 5 ( n = 3 ) , E13 . 5 ( n = 3 ) E15 . 5 ( n = 3 ) . Expression of linc - MYH in hindlimb muscle of E18 . 5 , newborn 7 day old ( n = 3 / 3 / 3 ) . Expression of linc - MYH in m . soleus ( soleus ) , m . tibialis anterior ( TA ) m . extensor digitorum longus ( EDL ) muscle at 3 ( n = 3 ) 8 of ( n = 5 ) . Biological replicates were used for PCR experiments , data are mean ± S . E . M .

O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-TISSUE I-TISSUE O O O 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-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O B-TISSUE O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-TISSUE O B-TISSUE O O O O B-TISSUE I-TISSUE O B-TISSUE O O O B-TISSUE I-TISSUE I-TISSUE O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O

D Expression of linc-MYH is not detected in limb buds at E10.5 (n = 3), E13.5 (n = 3) and E15.5 (n = 3). Expression of linc-MYH in hindlimb muscle of E18.5, newborn and 7 day old animals (n = 3/3/3). Expression of linc-MYH in m. soleus (soleus), m. tibialis anterior (TA) and and m. extensor digitorum longus (EDL) muscle at 3 weeks (n = 3) and 8 weeks of age (n = 5). Biological replicates were used for all PCR experiments, data are mean ± S.E.M.


E Murine linc - Myh is localized in extracts of C2C12 . Xist ( ) Gapdh ( mainly ) were used as controls ( n = 2 / 3 biological replicates . ) Data are mean ± S . E . M .

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E Murine linc-Myh is localized in nuclear extracts of C2C12 cells. Xist (nuclear) and Gapdh (mainly cytoplasmic) were used as controls (n = 2 nuclear / 3 cytoplasmic biological replicates.) Data are mean ± S.E.M.


F - H - FISH identifies linc - Myh in nuclei ( DAPI , blue ) of C2C12 myoblasts . ( G ) Adipor ( ) ( H ) Xist ( ) were used as controls .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O B-SUBCELLULAR O B-SMALL_MOLECULE O O O O B-CELL_LINE B-CELL_TYPE O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O

F-H RNA-FISH identifies linc-Myh in nuclei (DAPI, blue) of C2C12 myoblasts. (G) Adipor (cytoplasmic) and (H) Xist (nuclear) were used as controls.


( J ) Heat map of pulled down in same experiments as in I , demonstrating that INO80 remodeler exclusively interacts with linc - MYH . Blue color indicates pull - down of respective , color indicates that was not detected in respective sample .

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(J) Heat map of proteins pulled down in the same experiments as in I, demonstrating that INO80 chromatin remodeler complex exclusively interacts with linc-MYH. Blue color indicates pull-down of the respective protein, white color indicates that the protein was not detected in the respective sample.


- IP experiments using an anti - INO80 antibody to isolate INO80 from murine C2C12 ( K ) INO80 in L refer to replicate INO80 precipitation experiments . Precipitated RNAs were detected by RT - PCR .

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RNA-IP experiments using an anti-INO80 antibody to isolate the INO80 complex from murine C2C12 cells (K) INO80 in L refer to replicate INO80 precipitation experiments. Precipitated RNAs were detected by RT-PCR.


- IP experiments using an anti - INO80 antibody to isolate INO80 from human HSMM ( L ) . INO80 II in L refer to replicate INO80 precipitation experiments . Precipitated RNAs were detected by RT - PCR .

B-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD O O O B-SUBCELLULAR O B-ORGANISM B-CELL_TYPE O O O O B-GENEPROD O O O O O O B-GENEPROD B-EXP_ASSAY O O B-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O

RNA-IP experiments using an anti-INO80 antibody to isolate the INO80 complex from human HSMM cells (L). INO80 II in L refer to replicate INO80 precipitation experiments. Precipitated RNAs were detected by RT-PCR.


( A ) of linc - MYH KO ctrl mice at 10 ( n = 24 KO / 17 WT ; Student ' s t , two - , * * p < 0 . 01 )

O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O

Body weight (A) of male linc-MYH KO and ctrl mice at 10 weeks (n = 24 KO/17 WT; Student's t test, two-tailed, **p < 0.01)


tibia ( B ) of linc - MYH KO ctrl mice of 10 ( n = 9 KO / 13 WT ; Student ' s t , two - , not significant ) . Data are mean ± S . E . M .

B-TISSUE O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

tibia length (B) of male linc-MYH KO and ctrl mice of 10 weeks (n = 9 KO/13 WT; Student's t test, two-tailed, not significant). Data are mean ± S.E.M.


C , D of m . tibialis anterior ( TA ) m . digitorum longus ( EDL ) of linc - MYH KO ctrl mice at 10 ( n = 9 KO / 10 WT for TA , n = 10 KO / 11 WT for EDL ; Student ' s t , two - , * * p < 0 . 01 , * * * p < 0 . 001 ) . Data are mean ± S . E . M .

O O O O O O B-TISSUE I-TISSUE O B-TISSUE O O O B-TISSUE I-TISSUE O B-TISSUE O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O 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, D Weight of m. tibialis anterior (TA) and m. digitorum longus (EDL) of male linc-MYH KO and ctrl mice at 10 weeks (n = 9 KO/10 WT for TA, n = 10 KO/11 WT for EDL; Student's t test, two-tailed, **p < 0.01, ***p<0.001). Data are mean ± S.E.M.


distribution of in cross sections of TA ( E , F ) EDL ( G , H ) muscle in linc - MYH KO ( blue ) ctrl ( grey ) at 10 . A significant increase of fiber was observed for TA EDL of linc - MYH KO mice ( n = 5 KO / 4 WT for TA , n = 3 KO / 3 WT for EDL muscle , two - way ANOVA with Fisher ' s multiple comparisons ; * p < 0 . 05 , * * p < 0 . 01 ; > 250 per were counted ) . Data are mean ± S . E . M .

B-EXP_ASSAY O O O O O B-TISSUE O O O O O B-TISSUE O O O O O B-TISSUE O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O B-SUBCELLULAR O O O B-TISSUE I-TISSUE O B-GENEPROD I-GENEPROD I-GENEPROD O B-ORGANISM O O O O O O O O O O O O O O O O O O O B-TISSUE I-TISSUE O O O O O O O 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-EXP_ASSAY O O O O O O O O O O O O O O

Size distribution of fibers in cross sections of TA (E, F) and EDL (G, H) muscle in male linc-MYH KO (blue) and ctrl (grey) animals at 10 weeks. A significant increase of fiber size was observed for TA and EDL muscles of linc-MYH KO mice (n = 5 KO/4 WT for TA, n = 3 KO/3 WT for EDL muscle, two-way ANOVA with Fisher's multiple comparisons; *p < 0.05, **p < 0.01; > 250 fibers per animal were counted). Data are mean ± S.E.M.


distribution of in cross sections of TA EDL muscle in linc - MYH KO ( blue ) ctrl ( grey ) at 10 . A significant increase of fiber was observed for TA EDL of linc - MYH KO mice ( n = 5 KO / 4 WT for TA , n = 3 KO / 3 WT for EDL muscle , two - way ANOVA with Fisher ' s multiple comparisons ; * p < 0 . 05 , * * p < 0 . 01 ; > 250 per were counted ) . Data are mean ± S . E . M .

B-EXP_ASSAY O O O O O B-TISSUE I-TISSUE I-TISSUE O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O B-SUBCELLULAR O O O B-TISSUE I-TISSUE O B-GENEPROD I-GENEPROD I-GENEPROD O B-ORGANISM O O O O O O O O O O O O O O O O O O O B-TISSUE I-TISSUE O O O O O O O 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-EXP_ASSAY O O O O O O O O O O O O O O

Size distribution of fibers in cross sections of TA and EDL muscle in male linc-MYH KO (blue) and ctrl (grey) animals at 10 weeks. A significant increase of fiber size was observed for TA and EDL muscles of linc-MYH KO mice (n = 5 KO/4 WT for TA, n = 3 KO/3 WT for EDL muscle, two-way ANOVA with Fisher's multiple comparisons; *p < 0.05, **p < 0.01; > 250 fibers per animal were counted). Data are mean ± S.E.M.


K Numbers of myonuclei in cross sections of TA EDL muscle ( TA : n = 6 KO / 6 WT , EDL n = 3 KO / 3 WT , Mann - Whitney one - , * * p < 0 . 01 , * p = 0 . 05 ) Data are mean ± S . E . M .

O O O B-SUBCELLULAR O O O O B-TISSUE I-TISSUE I-TISSUE O B-TISSUE O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

K Numbers of myonuclei in cross sections of TA and EDL muscle (TA: n = 6 KO/6 WT, EDL n = 3 KO/3 WT animals, Mann-Whitney test one-tailed, **p<0.01, *p = 0.05) Data are mean ± S.E.M.


L - O Number of myonuclei / fiber of Pax7pos ( red ) MuSCs ( red arrows ) on myofibers isolated from flexor digitorum brevis muscle . Nuclei were stained using DAPI ( blue ) . Scale bar in L indicates 50 µm for L M . ( N ) number of myonuclei / isolated fiber is increased in linc - MYH KO compared to WT myofibers ( n = 4 KO / 4 WT , > 26 / ; Mann - Whitney two - , * p < 0 . 05 ) . ( O ) Number of MuSCs on isolated from linc - MYH KO WT mice ( n = 4 KO / 4 WT , Mann - Whitney two - ; * * * * p < 0 . 0001 ) . Data are mean ± S . E . M .

O O O O O B-SUBCELLULAR O B-SUBCELLULAR O B-GENEPROD O O O B-CELL_TYPE O O O O O O O O B-TISSUE I-TISSUE I-TISSUE I-TISSUE O B-SUBCELLULAR O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O B-SUBCELLULAR O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O 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-CELL_TYPE O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

L-O Number of myonuclei / fiber and of Pax7pos (red) MuSCs (red arrows) on myofibers isolated from flexor digitorum brevis muscle. Nuclei were stained using DAPI (blue). Scale bar in L indicates 50 µm for L and M. (N) The number of myonuclei / isolated fiber is increased in linc-MYH KO compared to WT myofibers (n = 4 KO/4 WT, > 26 fibers/animal; Mann-Whitney test two-tailed, * p < 0.05). (O) Number of MuSCs on isolated fibers from linc-MYH KO and WT mice (n = 4 KO/4 WT animals, Mann-Whitney test two-tailed; ****p < 0.0001). Data are mean ± S.E.M.


P - S Number of Pax7 ( red ) positive MuSCs in cross sections of tibialis anterior muscle ( P - R ) ; relative amount of quiescent MuSCs identified by double ( yellow arrows ) for Pax7 CalcR ( green ) does not significantly differ between WT linc - MYH KO ( S ) . Nuclei were stained with DAPI ( blue ) . Scale bar in P indicates 25 µm for P Q . ( n = 3 KO / 3 WT , Mann - Whitney one - ; * p = 0 . 05 , ns : not significant ) . data are mean ± S . E . M .

O O O O O B-GENEPROD O O O O B-CELL_TYPE O O O O B-TISSUE I-TISSUE I-TISSUE O O O O O O O O O O B-CELL_TYPE O O B-EXP_ASSAY O O O O O B-GENEPROD I-GENEPROD O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-SUBCELLULAR O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

P-S Number of Pax7 (red) positive MuSCs in cross sections of tibialis anterior muscle (P-R); the relative amount of quiescent MuSCs identified by double staining (yellow arrows) for Pax7 and CalcR (green) does not significantly differ between WT and linc-MYH KO animals (S). Nuclei were stained with DAPI (blue). Scale bar in P indicates 25 µm for P and Q. (n = 3 KO/3 WT animals, Mann-Whitney test one-tailed; *p = 0.05, ns: not significant). All data are mean ± S.E.M.