Figure 5WT and Mfn1 / 2 double KO were transfected with : A ) mt - RFP , ER - GFP and plasmids expressing the indicated proteins . Mitochondria - ER colocalization was analyzed using Mander ' s coefficient ( n = 15 cells analyzed in 3 independent experiments ) . Data are presented as mean ± SEM . B ) GFP and ERMITO - RLuc and indicated expression vectors . After 24 h , the transfected cells were sorted and plated for another 24 h , after which RLuc was analyzed ( n = 3 independent experiments ) . Data are presented as mean ± SEM . C ) Data information : The red dashed line indicates the WT cells results . * p < 0 . 05 versus Mfn1 / 2 DKO cells , one - way ANOVA followed by Tukey ' s post hoc test . Note that experiments were performed at the same time , so the values of WT and Mfn2 KO transfected with control plasmid are the same but the figures have been split in two for the sake of linearity . WT and Mfn1 / 2 double KO were transfected with : C ) GFP and the indicated plasmids . After 48 h , mitochondrial Ca2 + was determined ( n = 30 cells from 3 independent experiments ) . Data are presented as mean ± SEM . Data information : The red dashed line indicates the WT cells results . * p < 0 . 05 versus Mfn1 / 2 DKO cells , one - way ANOVA followed by Tukey ' s post hoc test . Note that experiments were performed at the same time , so the values of WT and Mfn2 KO transfected with control plasmid are the same but the figures have been split in two for the sake of linearity . WT and Mfn1 / 2 double KO were transfected with : D ) LAR - GECO1 and the indicated plasmids . After 48 h ER Ca2 + was released with caffeine ( 20 mM ) as indicated ( n = 25 - 27 cells from 3 independent experiments ) . Data are presented as mean ± SEM . Data information : The red dashed line indicates the WT cells results . * p < 0 . 05 versus Mfn1 / 2 DKO cells , one - way ANOVA followed by Tukey ' s post hoc test . Note that experiments were performed at the same time , so the values of WT and Mfn2 KO transfected with control plasmid are the same but the figures have been split in two for the sake of linearity . WT and Mfn1 / 2 double KO were transfected with : E ) GFP and the indicated plasmid . After 48 h , the effect of caffeine - induced ( 20 mM ) ER Ca2 + release on mitochondrial Ca2 + was measured ( n = 25 cells from 3 independent experiments ) . Data are presented as mean ± SEM .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5WT and Mfn1/2 double KO were transfected with: A) mt-RFP, ER-GFP and plasmids expressing the indicated proteins. Mitochondria-ER colocalization was analyzed using Mander's coefficient (n= 15 cells analyzed in 3 independent experiments). Data are presented as mean ± SEM. B) GFP and ERMITO-RLuc and indicated expression vectors. After 24 h, the transfected cells were sorted and plated for another 24 h, after which RLuc was analyzed (n= 3 independent experiments). Data are presented as mean ± SEM. C) Data information: The red dashed line indicates the WT cells results. *p<0.05 versus Mfn1/2 DKO cells, one-way ANOVA followed by Tukey's post hoc test. Note that experiments were performed at the same time, so the values of WT and Mfn2 KO transfected with control plasmid are the same but the figures have been split in two for the sake of linearity.WT and Mfn1/2 double KO were transfected with: C) GFP and the indicated plasmids. After 48 h, mitochondrial Ca2+ was determined (n= 30 cells from 3 independent experiments). Data are presented as mean ± SEM. Data information: The red dashed line indicates the WT cells results. *p<0.05 versus Mfn1/2 DKO cells, one-way ANOVA followed by Tukey's post hoc test. Note that experiments were performed at the same time, so the values of WT and Mfn2 KO transfected with control plasmid are the same but the figures have been split in two for the sake of linearity.WT and Mfn1/2 double KO were transfected with: D) LAR-GECO1 and the indicated plasmids. After 48 h ER Ca2+ was released with caffeine (20 mM) as indicated (n= 25-27 cells from 3 independent experiments). Data are presented as mean ± SEM. Data information: The red dashed line indicates the WT cells results. *p<0.05 versus Mfn1/2 DKO cells, one-way ANOVA followed by Tukey's post hoc test. Note that experiments were performed at the same time, so the values of WT and Mfn2 KO transfected with control plasmid are the same but the figures have been split in two for the sake of linearity.WT and Mfn1/2 double KO were transfected with: E) GFP and the indicated plasmid. After 48 h, the effect of caffeine-induced (20 mM) ER Ca2+ release on mitochondrial Ca2+ was measured (n= 25 cells from 3 independent experiments). Data are presented as mean ± SEM.


Figure 6B ) Representative Western blots of the indicated proteins during the neuronal maturation in vitro ( n = 3 independent experiments ) . C ) Representative Western blots of the indicated proteins of WT and Mfn2 KO neurons at DIV7 ( n = 3 independent experiments ) . D D ) Primary neurons of tamoxifen - inducible Mfn2 KO mice were transfected at DIV3 with ER - GFP , mt - RFP plus the indicated plasmids . Tamoxifen - treated ( for 72 h ) and vehicle - treated neurons ( WT ; red dashed line ) were fixed and mitochondria - ER co - localization was analyzed using Mander ' s coefficient ( n = 15 neurons analyzed in 3 independent experiments ) . Data are presented as mean ± SEM . Data information : * p < 0 . 05 versus WT ; # p < 0 . 05 versus Mfn2 KO neurons , one - way ANOVA followed by Tukey ' s post - hoc test . Scale bar : 500 μm . ( F ) neurite length measurement and representative tracings ( G ) of primary cortical neurons of WT and Mfn2 KO mice co - transfected at DIV4 with GFP and the indicated plasmids . After 72 h , the neurons were fixed , immunofluorescence with anti - GFP antibodies was performed and neurite length was measured ( n = 30 neurons from 3 independent experiments ) . Data are presented as mean ± SEM . Data information : * p < 0 . 05 versus WT ; # p < 0 . 05 versus Mfn2 KO neurons , one - way ANOVA followed by Tukey ' s post - hoc test . Scale bar : 500 μm . E

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Figure 6B) Representative Western blots of the indicated proteins during the neuronal maturation in vitro (n= 3 independent experiments). C) Representative Western blots of the indicated proteins of WT and Mfn2 KO neurons at DIV7 (n= 3 independent experiments). D D) Primary neurons of tamoxifen-inducible Mfn2 KO mice were transfected at DIV3 with ER-GFP, mt-RFP plus the indicated plasmids. Tamoxifen-treated (for 72 h) and vehicle-treated neurons (WT; red dashed line) were fixed and mitochondria-ER co-localization was analyzed using Mander's coefficient (n= 15 neurons analyzed in 3 independent experiments). Data are presented as mean ± SEM. Data information: *p<0.05 versus WT; #p<0.05 versus Mfn2 KO neurons, one-way ANOVA followed by Tukey's post-hoc test. Scale bar: 500 μm.(F) neurite length measurement and representative tracings (G) of primary cortical neurons of WT and Mfn2 KO mice co-transfected at DIV4 with GFP and the indicated plasmids. After 72 h, the neurons were fixed, immunofluorescence with anti-GFP antibodies was performed and neurite length was measured (n= 30 neurons from 3 independent experiments). Data are presented as mean ± SEM. Data information: *p<0.05 versus WT; #p<0.05 versus Mfn2 KO neurons, one-way ANOVA followed by Tukey's post-hoc test. Scale bar: 500 μm. E


figf1 ( a ) Representative differential interference contrast microscopy images of midgut cells from wild - type animals at the early third instar larval ( Early 3rd ) , late third instar larval ( Late 3rd ) and at puparium formation ( white prepupal , WPP ) stages . ( b ) Autophagy detected by formation of mCherry - Atg8a punctate spots in midgut cells from wild - type animals at indicated stages . Representative images are shown . ( c ) Midguts from control Atg18KG03090 / wild - type ( Atg18 / + ) , n = 14 , and Atg18KG03090 / Df ( 3L ) 6112 mutant ( Atg18 / Df ) , n = 11 , animals at puparium formation analysed by differential interference contrast microscopy . Representative images are shown . ( d ) Wild - type , control ( Atg18KG03090 / + ) and Atg18 mutant ( Atg18 / Df ) midgut cell size quantification ( μm2 ) at indicated stages ; n = 10 animal intestines per genotype with 5 cells measured per intestine per stage . ( e ) Differential interference contrast images of midgut cells from Atg2EP3697 / wild - type control ( Atg2 / + ) and Atg2EP3697 / Df ( 3L ) 6091 mutant ( Atg2 / Df ) animals at puparium formation . Representative images are shown . ( f ) Cell size quantification ( μm2 ) from e , n = 12 ( Atg2 / + ) and n = 7 ( Atg2 / Df ) animal intestines per genotype with 5 cells measured per intestine . ( g - j ) Representative TEM images of intestine cells 2 h after puparium formation . ( g , i ) Control Atg18KG03090 / wild - type ( Atg18 / + , g ) and control Atg2EP3697 / wild - type ( Atg2 / + , i ) cells with an enlarged image showing a double - membrane structure ( arrowhead ) that surrounds a mitochondrion and endoplasmic reticulum . Arrows indicate autolysosomes . ( h , j ) Atg18KG03090 / Df ( 3L ) 6112mutant ( Atg18 / Df , h ) and Atg2EP3697 / Df ( Atg2 / Df , j ) mutant cells lacking autophagic structures . Quantification is shown as mean ± s . d . Scale bars , 20 μm ( a - c and e ) and 1 μm ( g - j ) .

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figf1(a) Representative differential interference contrast microscopy images of midgut cells from wild-type animals at the early third instar larval (Early 3rd), late third instar larval (Late 3rd) and at puparium formation (white prepupal, WPP) stages.(b) Autophagy detected by formation of mCherry-Atg8a punctate spots in midgut cells from wild-type animals at indicated stages. Representative images are shown.(c) Midguts from control Atg18KG03090/wild-type (Atg18/+), n = 14, and Atg18KG03090/Df(3L)6112 mutant (Atg18/Df), n = 11, animals at puparium formation analysed by differential interference contrast microscopy. Representative images are shown. (d) Wild-type, control (Atg18KG03090/+) and Atg18 mutant (Atg18/Df) midgut cell size quantification (μm2) at indicated stages; n = 10 animal intestines per genotype with 5 cells measured per intestine per stage.(e) Differential interference contrast images of midgut cells from Atg2EP3697/wild-type control (Atg2/+) and Atg2EP3697/Df(3L)6091 mutant (Atg2/Df) animals at puparium formation. Representative images are shown. (f) Cell size quantification (μm2) from e, n = 12 (Atg2/+) and n = 7 (Atg2/Df) animal intestines per genotype with 5 cells measured per intestine.(g-j) Representative TEM images of intestine cells 2 h after puparium formation. (g,i) Control Atg18KG03090/wild-type (Atg18/+, g) and control Atg2EP3697/wild-type (Atg2/+, i) cells with an enlarged image showing a double-membrane structure (arrowhead) that surrounds a mitochondrion and endoplasmic reticulum. Arrows indicate autolysosomes. (h,j) Atg18KG03090/Df(3L)6112mutant (Atg18/Df, h) and Atg2EP3697/Df (Atg2/Df, j) mutant cells lacking autophagic structures. Quantification is shown as mean ± s.d. Scale bars, 20 μm (a-c and e) and 1 μm (g-j).


figf2 ( a ) Midguts dissected from animals expressing Atg18IR specifically in DsRed - marked clones of cells at puparium formation and analysed by fluorescence and differential interference contrast ( DIC ) microscopy . Representative images are shown . ( b ) Quantification ( μm2 ) from a , n = 12 animal intestines per genotype with 1 - 5 cells measured per intestine . ( c ) Midguts expressing mCherry - Atg8a in all cells , and expressing Atg18IR specifically in GFP - marked cell clones dissected from animals at 2 h after puparium formation . Representative images are shown . ( d ) Midguts dissected from animals at puparium formation that contain an Atg1Δ3D loss - of - function mutant cell clone ( lacking GFP ) and analysed by fluorescence and differential interference contrast microscopy . Wild - type ( + / + ) control cell possesses stronger GFP and heterozygous Atg1Δ3D / wild - type ( Δ3D / + ) cells have weaker GFP . Representative images are shown . ( e ) Quantification ( μm2 ) from d , n = 7 ( + / + ) , n = 8 ( Δ3D / + ) and n = 8 ( Δ3D / Δ3D ) animal intestines per genotype with 1 - 5 cells measured per intestine . ( f ) Midguts dissected from animals expressing mCherry - Atg8a in all cells , and expressing Atg1IR specifically in GFP - marked clones of cells at 2 h after puparium formation . Representative images are shown . ( g - j ) Representative cut views from three - dimensional reconstitution images from wild - type ( g , h ) and Atg1 knockdown ( i , j ) cells at early third instar larval stage ( g , i ) and at puparium formation ( h , j ) . ( k ) Quantification of cell volume ( μm3 ) from g - j , n = 5 animal intestines per genotype with 1 - 5 cells measured per intestine per stage . Black columns : early third instar larval stage . White columns : puparium formation stage . Quantification is shown as mean ± s . d . NS , not significant . Scale bars , 20 μm .

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figf2(a) Midguts dissected from animals expressing Atg18IR specifically in DsRed-marked clones of cells at puparium formation and analysed by fluorescence and differential interference contrast (DIC) microscopy. Representative images are shown. (b) Quantification (μm2) from a, n = 12 animal intestines per genotype with 1-5 cells measured per intestine.(c) Midguts expressing mCherry-Atg8a in all cells, and expressing Atg18IR specifically in GFP-marked cell clones dissected from animals at 2 h after puparium formation. Representative images are shown.(d) Midguts dissected from animals at puparium formation that contain an Atg1Δ3D loss-of-function mutant cell clone (lacking GFP) and analysed by fluorescence and differential interference contrast microscopy. Wild-type (+/+) control cell possesses stronger GFP and heterozygous Atg1Δ3D/wild-type (Δ3D/+) cells have weaker GFP. Representative images are shown. (e) Quantification (μm2) from d, n = 7 (+/+), n = 8 (Δ3D/+) and n = 8 (Δ3D/Δ3D) animal intestines per genotype with 1-5 cells measured per intestine.(f) Midguts dissected from animals expressing mCherry-Atg8a in all cells, and expressing Atg1IR specifically in GFP-marked clones of cells at 2 h after puparium formation. Representative images are shown.(g-j) Representative cut views from three-dimensional reconstitution images from wild-type (g,h) and Atg1 knockdown (i,j) cells at early third instar larval stage (g,i) and at puparium formation (h,j). (k) Quantification of cell volume (μm3) from g-j, n = 5 animal intestines per genotype with 1-5 cells measured per intestine per stage. Black columns: early third instar larval stage. White columns: puparium formation stage. Quantification is shown as mean ± s.d. NS, not significant. Scale bars, 20 μm.


figf3 ( a ) Midguts dissected from animals at puparium formation that contain Vps34Δm22 loss - of - function mutant cell clones ( lacking GFP ) and analysed by fluorescence and differential interference contrast ( DIC ) microscopy . Wild - type ( + / + ) control cells possess stronger GFP and heterozygous Vps34Δm22 / wild - type ( Δm22 / + ) cells have weaker GFP . Representative images are shown . ( b ) Quantification ( μm2 ) from a , n = 14 animal intestines per genotype with 1 - 5 cells measured per intestine . ( c ) Midguts dissected from animals expressing Atg8aIR specifically in GFP - marked clones of cells at puparium formation and analysed by fluorescence and differential interference contrast microscopy . Representative images are shown . ( d ) Cell size quantification ( μm2 ) from c , n = 10 animal intestines per genotype with 1 - 5 cells measured per intestine . ( e ) Midguts dissected from animals expressing Atg12IR specifically in DsRed - marked clones of cells at puparium formation and analysed by fluorescence and differential interference contrast microscopy . Representative images are shown . ( f ) Cell size quantification ( μm2 ) from e , n = 14 animal intestines per genotype with 1 - 5 cells measured per intestine . ( g - i ) Midguts dissected from early third instar larvae that mis - express Atg1GS10797 ( Atg1 ; g , GFP in nucleus and cytoplasm ) , both Atg1 + Atg8aIR ( h ) , or both Atg1 + Atg7IR ( i ) and stained to detect Discs large ( green ) on the cortex of all cells . Representative images are shown . ( g ' and i ' ) DIC images of midguts expressing mCherry - Atg8a in all cells , Atg1 ( g ' ) , and Atg1 + Atg7IR ( i ' ) in GFP - marked clones of cells . mCherry - Atg8a puncta are shown in insets . Representative images are shown . Quantification is shown as mean ± s . d . Scale bars , 20 μm .

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figf3(a) Midguts dissected from animals at puparium formation that contain Vps34Δm22 loss-of-function mutant cell clones (lacking GFP) and analysed by fluorescence and differential interference contrast (DIC) microscopy. Wild-type (+/+) control cells possess stronger GFP and heterozygous Vps34Δm22/wild-type (Δm22/+) cells have weaker GFP. Representative images are shown. (b) Quantification (μm2) from a, n = 14 animal intestines per genotype with 1-5 cells measured per intestine.(c) Midguts dissected from animals expressing Atg8aIR specifically in GFP-marked clones of cells at puparium formation and analysed by fluorescence and differential interference contrast microscopy. Representative images are shown. (d) Cell size quantification (μm2) from c, n = 10 animal intestines per genotype with 1-5 cells measured per intestine.(e) Midguts dissected from animals expressing Atg12IR specifically in DsRed-marked clones of cells at puparium formation and analysed by fluorescence and differential interference contrast microscopy. Representative images are shown. (f) Cell size quantification (μm2) from e, n = 14 animal intestines per genotype with 1-5 cells measured per intestine.(g-i) Midguts dissected from early third instar larvae that mis-express Atg1GS10797 (Atg1; g, GFP in nucleus and cytoplasm), both Atg1 +Atg8aIR (h), or both Atg1 +Atg7IR (i) and stained to detect Discs large (green) on the cortex of all cells. Representative images are shown. (g' and i') DIC images of midguts expressing mCherry-Atg8a in all cells, Atg1 (g'), and Atg1+Atg7IR (i') in GFP-marked clones of cells. mCherry-Atg8a puncta are shown in insets. Representative images are shown. Quantification is shown as mean ± s.d. Scale bars, 20 μm.


figf4 ( a ) Midguts dissected from animals at puparium formation that contain Atg7d4 loss - of - function mutant cell clones ( lacking GFP ) and analysed by fluorescence and differential interference contrast ( DIC ) microscopy . Wild - type ( + / + ) control cells possess stronger GFP and heterozygous Atg7d4 / wild - type ( d4 / + ) cells have weaker GFP . Representative images are shown . ( b ) Cell size quantification ( μm2 ) from a , n = 8 animal intestines per genotype with 1 - 5 cells measured per intestine . ( c ) Midguts expressing mCherry - Atg8a in all cells , and with Atg7d4 loss - of - function clone cells ( lacking GFP ) at 2 h after puparium formation . Representative images are shown . ( e ) Midguts dissected from animals expressing Atg12IR specifically in DsRed - marked clones of cells at puparium formation and analysed by fluorescence and differential interference contrast microscopy . Representative images are shown . ( f ) Cell size quantification ( μm2 ) from e , n = 14 animal intestines per genotype with 1 - 5 cells measured per intestine . ( f ) Midguts expressing GFP - Atg5 in enterocytes ( larger nuclei ) with an Atg7d4 loss - of - function clone cell ( lacking RFP ) at puparium formation . Representative images are shown . ( g , h ) Representative TEM images of intestine cells 2 h after puparium formation . Arrows indicate autolysosomes . ( g , h ) Control ( Atg7d30 / d14 ; g ) and Atg7 mutant ( Atg7d77 / d14 ; h ) cells both contain autophagic structures . ( h , right ) Enlarged Atg7 mutant cell image of a double - membrane autophagosome ( arrowhead ) surrounding a mitochondrion . Quantification is shown as mean ± s . d . NS , not significant . Scale bars , 20 μm ( a , c , d and f ) and 1 μm ( g , h ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

figf4(a) Midguts dissected from animals at puparium formation that contain Atg7d4 loss-of-function mutant cell clones (lacking GFP) and analysed by fluorescence and differential interference contrast (DIC) microscopy. Wild-type (+/+) control cells possess stronger GFP and heterozygous Atg7d4/wild-type (d4/+) cells have weaker GFP. Representative images are shown. (b) Cell size quantification (μm2) from a, n = 8 animal intestines per genotype with 1-5 cells measured per intestine.(c) Midguts expressing mCherry-Atg8a in all cells, and with Atg7d4 loss-of-function clone cells (lacking GFP) at 2 h after puparium formation. Representative images are shown.(e) Midguts dissected from animals expressing Atg12IR specifically in DsRed-marked clones of cells at puparium formation and analysed by fluorescence and differential interference contrast microscopy. Representative images are shown. (f) Cell size quantification (μm2) from e, n = 14 animal intestines per genotype with 1-5 cells measured per intestine.(f) Midguts expressing GFP-Atg5 in enterocytes (larger nuclei) with an Atg7d4 loss-of-function clone cell (lacking RFP) at puparium formation. Representative images are shown.(g,h) Representative TEM images of intestine cells 2 h after puparium formation. Arrows indicate autolysosomes. (g,h) Control (Atg7d30/d14; g) and Atg7 mutant (Atg7d77/d14; h) cells both contain autophagic structures. (h, right) Enlarged Atg7 mutant cell image of a double-membrane autophagosome (arrowhead) surrounding a mitochondrion. Quantification is shown as mean ± s.d. NS, not significant. Scale bars, 20 μm (a,c,d and f) and 1 μm (g,h).


figf5 ( 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 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

figf5(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 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.


figf6 ( 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 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 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 ) , 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 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 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

figf6(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 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 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), 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 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 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.


figf7 ( 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 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 / + ) 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 ) 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 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 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 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 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

figf7(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 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/+) 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) 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 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 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 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 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.


figf8 ( 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 and cytoplasm ) and stained with ATP synthase complex V ( ATPV ) to detect mitochondria in all cells . Representative images are shown . ( 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 ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

figf8(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 and cytoplasm) and stained with ATP synthase complex V (ATPV) to detect mitochondria in all cells. Representative images are shown.(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).


Figure 1 ( 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 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(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.


Figure 2 ( 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 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 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(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 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.


Figure 3 ( 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 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 .

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Figure 3(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 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.


Figure 4 ( 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 ) 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 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 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(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) 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 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.


Figure 5 ( 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 , 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 , the serum levels of AST ( I ) , and CRE ( J ) were measured , ( n = 8 mice per group ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(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, 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, the serum levels of AST (I), and CRE (J) were measured, (n=8 mice per group).


Figure 6 ( 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 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6(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.


Figure 7Under 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 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 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 ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7Under 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 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 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).


Figure 1Release 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 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 , DRelease 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 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 ) 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 ( HLeukocyte 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 - tesA . 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

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1Release 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 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, DRelease 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 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)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 (HLeukocyte 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-tesA. 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


Figure 2TNF 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 - testTNF 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 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 + SEM ) combined from ' n ' technical or biological replicate ( human donor * p < 0 . 05 according to Mann - Whitney ( 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 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 + SEM ) combined from ' n ' technical or biological replicate mic ) , Student ' s t - tes ( 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 + SEM ) combined from ' n ' technical or biological replicate ( human donor * p < 0 . 05 according to Mann -

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2TNF 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-testTNF 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 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+SEM) combined from 'n' technical or biological replicate (human donor * p<0.05 according to Mann-Whitney (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 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+SEM) combined from 'n' technical or biological replicate mic ), Student's t-tes (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+SEM) combined from 'n' technical or biological replicate (human donor * p<0.05 according to Mann-Whitney


Figure 3 ( 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 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 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 + 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 + SEM ) from ' n ' technical or biological replicates ( donors ) * p < 0 . 05 according to Wilcoxon signed rank

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

Figure 3 (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 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 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+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+SEM) from 'n' technical or biological replicates (donors) * p<0.05 according to Wilcoxon signed rank su


Figure 4Flow 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 ( Amicroscale 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 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 ) 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 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 cells ( n = 3 Data information I one representative of ' n ' independent experiments is shown ( for asmean + SD ) ) , Student ' s t - tesIL - 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 ) ( 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 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

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4Flow 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 (Amicroscale 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 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) 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 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 cells (n=3 Data information I one representative of 'n' independent experiments is shown (for asmean+SD) ), Student's t-tesIL-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) (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 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


Figure 1B . 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 . 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 . 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 ; amp ; E staining ( E ) . The calvaria thicknesses were measured from the images using Image Pro software ( F ) . [ n = 3 ]

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Figure 1B . 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. 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 . 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]


Figure 2B . 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 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 . The superimposed structure of DBM ( green ) and KY - 02061 ( yellow ) on the surface of Dvl PDZ domain ( gray ) was visualized .

O B-PANEL_START O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2B . 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 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. The superimposed structure of DBM (green) and KY-02061 (yellow) on the surface of Dvl PDZ domain (gray) was visualized.


Figure 3A . 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 . 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 . 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 ) . The calvaria thicknesses were measured from the stained sections using Image Pro software ( E ) . [ n = 3 ]

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3A . 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 . 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. 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). The calvaria thicknesses were measured from the stained sections using Image Pro software (E). [n=3]


Figure 4B . A competition curve for the Dvl - CXXC5 interaction by KY - 02327 . 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 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 . The superimposed structure of KY - 02061 ( yellow ) and KY - 02327 ( cyan ) on the surface of Dvl PDZ domain ( gray ) was visualized .

O B-PANEL_START O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4B. A competition curve for the Dvl-CXXC5 interaction by KY-02327.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 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. The superimposed structure of KY-02061 (yellow) and KY-02327 (cyan) on the surface of Dvl PDZ domain (gray) was visualized.


Figure 5A . 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 cells were treated with indicated concentrations of KY - 02327 for 2 days . β - Catenin and α - tubulin were detected by immunoblot . 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 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 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 ]

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5A . 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 cells were treated with indicated concentrations of KY-02327 for 2 days. β-Catenin and α-tubulin were detected by immunoblot.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 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 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]


Figure 6A - 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 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 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 .

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Figure 6A-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 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 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.