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 2Western blot for phosphorylated and total ERK in HEK293 cells transfected with native and ΔA781 _ N783 ( left ) or P824R ( right ) CSF - 1R and treated with CSF - 1 ( D , E ) or IL - 34 ( F , G ) for 10 minutes . Horizontal line indicates untreated cells , with increasing concentrations 10 , 50 and 100 ng / ml . Corresponding densitometry displays the ratio of phospho - ERK to total ERK , normalised to the ratio of the untreated control for the native receptor ( * * * * p < 0 . 0001 , * * * p < 0 . 0005 , * * p < 0 . 01 , * p < 0 . 05 , one - way ANOVA with Sidak ' s post - test for multiple comparison , n = 3 biological replicates , , error bars indicate SEM ) . Western blot for CSF - 1R in HEK293 cells transfected with native and ΔA781 _ N783 ( top ) or P824R ( bottom ) CSF - 1R and treated with rapamycin . Horizontal line indicates untreated cells , with concentrations 0 . 1 , 0 . 5 , 1 , 5 , 10μM . Corresponding densitometry ( below ) displays the fold change of CSF - 1R relative to the untreated control for each transfection ( * * * * p < 0 . 0001 , one - way ANOVA with Sidak ' s post - test for multiple comparison to untreated , n = 3 biological replicates , error bars indicate SEM ) . Western blot for CSF - 1R in HEK293 cells transfected with native and ΔA781 _ N783 ( top ) or P824R ( bottom ) CSF - 1R and treated with 3 - methyladenosine . Horizontal line indicates untreated cells , with increasing concentrations 0 . 1 , 0 . 2 , 0 . 5 , 1mM . Corresponding densitometry ( below ) displays the fold change of CSF - 1R relative to the untreated control for each transfection ( * * * * p < 0 . 0001 , * p = 0 . 0114 , one - way ANOVA with Sidak ' s post - test for multiple comparison to untreated , n = 3 biological replicates , error bars indicate SEM ) . Western blot for CSF - 1R and Actin in HEK293 cells transfected with native and ΔA781 _ N783 ( top ) or P824R ( bottom ) CSF - 1R and treated with MG132 or PLX3397 . Horizontal line indicates untreated cells , 20μM PLX3397 , with increasing concentrations 0 . 1 , 0 . 5 , 0 . 1 , 5μM . Corresponding densitometry ( below ) displays the fold change of CSF - 1R relative to the untreated control for each transfection ( * * * p = 0 . 0002 , * * p = 0 . 002 , one - way ANOVA with Sidak ' s post - test for multiple comparison to untreated , n = 3 biological replicates , error bars indicate 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 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 O O O O O O O O O O

Figure 2Western blot for phosphorylated and total ERK in HEK293 cells transfected with native and ΔA781_N783 (left) or P824R (right) CSF-1R and treated with CSF-1 (D, E) or IL-34 (F, G) for 10 minutes. Horizontal line indicates untreated cells, with increasing concentrations 10, 50 and 100 ng/ml. Corresponding densitometry displays the ratio of phospho-ERK to total ERK, normalised to the ratio of the untreated control for the native receptor (**** p < 0.0001, *** p < 0.0005, ** p < 0.01, * p < 0.05, one-way ANOVA with Sidak's post-test for multiple comparison, n = 3 biological replicates, , error bars indicate SEM).Western blot for CSF-1R in HEK293 cells transfected with native and ΔA781_N783 (top) or P824R (bottom) CSF-1R and treated with rapamycin. Horizontal line indicates untreated cells, with concentrations 0.1, 0.5, 1, 5, 10μM. Corresponding densitometry (below) displays the fold change of CSF-1R relative to the untreated control for each transfection (**** p < 0.0001, one-way ANOVA with Sidak's post-test for multiple comparison to untreated, n = 3 biological replicates, error bars indicate SEM). Western blot for CSF-1R in HEK293 cells transfected with native and ΔA781_N783 (top) or P824R (bottom) CSF-1R and treated with 3-methyladenosine. Horizontal line indicates untreated cells, with increasing concentrations 0.1, 0.2, 0.5, 1mM. Corresponding densitometry (below) displays the fold change of CSF-1R relative to the untreated control for each transfection (**** p < 0.0001, * p = 0.0114, one-way ANOVA with Sidak's post-test for multiple comparison to untreated, n = 3 biological replicates, error bars indicate SEM). Western blot for CSF-1R and Actin in HEK293 cells transfected with native and ΔA781_N783 (top) or P824R (bottom) CSF-1R and treated with MG132 or PLX3397. Horizontal line indicates untreated cells, 20μM PLX3397, with increasing concentrations 0.1, 0.5, 0.1, 5μM. Corresponding densitometry (below) displays the fold change of CSF-1R relative to the untreated control for each transfection (*** p = 0.0002, ** p = 0.002, one-way ANOVA with Sidak's post-test for multiple comparison to untreated, n = 3 biological replicates, error bars indicate SEM).


Figure 3Immunocytochemistry of microglia isolated from Csf - 1rflx / wt ; Cx3cr1 - Cre - and Csf - 1rflx / wt ; Cx3cr1 - Cre + P0 mouse pup brains . Microglia were stimulated with LPS and exposed to fluorescent opsonised latex beads for 1 hour before fixation and quantification of bead + cells via microscopy . Cells were stained using MitoTracker ( red ) . Arrowheads ( white ) indicate bead + cells . Quantification of mouse microglial phagocytic activity expressed as percentage bead + cells ( n = 4 assays , error bars indicate SEM ) . L ) Immunocytochemistry of macrophages differentiated in vitro from Csf - 1rflx / wt ; Cx3cr1 - Cre - and Csf - 1rflx / wt ; Cx3cr1 - Cre + mouse bone marrow . Macrophages were stimulated with LPS and exposed to fluorescent opsonised latex beads for 1 hour before fixation and quantification of bead + cells via microscopy . Cells were stained using MitoTracker ( red ) and DAPI ( blue ) . Arrowheads ( white ) indicate bead + cells . M ) Quantification of mouse macrophage phagocytic activity expressed as percentage bead + cells . ( * p = 0 . 024 , Student ' s t - test with Welch ' s correction , n = 4 assays , error bars indicate 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 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

Figure 3Immunocytochemistry of microglia isolated from Csf-1rflx/wt;Cx3cr1-Cre- and Csf-1rflx/wt;Cx3cr1-Cre+ P0 mouse pup brains. Microglia were stimulated with LPS and exposed to fluorescent opsonised latex beads for 1 hour before fixation and quantification of bead+ cells via microscopy. Cells were stained using MitoTracker(red). Arrowheads (white) indicate bead+ cells. Quantification of mouse microglial phagocytic activity expressed as percentage bead+ cells (n = 4 assays, error bars indicate SEM).L) Immunocytochemistry of macrophages differentiated in vitro from Csf-1rflx/wt;Cx3cr1-Cre- and Csf-1rflx/wt;Cx3cr1-Cre+ mouse bone marrow. Macrophages were stimulated with LPS and exposed to fluorescent opsonised latex beads for 1 hour before fixation and quantification of bead+ cells via microscopy. Cells were stained using MitoTracker (red) and DAPI (blue). Arrowheads (white) indicate bead+ cells. M) Quantification of mouse macrophage phagocytic activity expressed as percentage bead+ cells. (* p = 0.024, Student's t-test with Welch's correction, n = 4 assays, error bars indicate SEM).


Figure 4A ) Immunohistochemistry of Csf - 1rflx / wt ; Cx3cr1 - Cre - ( top ) and Csf - 1rflx / wt ; Cx3cr1 - Cre + ( bottom ) mice unilaterally injected with Aβ1 - 42 in the hippocampus and stained for IB4 ( green ) , F4 / 80 ( white ) and Aβ ( red ) . Scale bars indicate 50 μm . B ) Quantification of F4 / 80 and IB4 immunopositivity following intrahippocampal injection of Aβ1 - 42 . Quantification of immunopositivity in both injected and uninjected hippocampi of Csf - 1rflx / wt ; Cx3cr1 - Cre - and Csf - 1rflx / wt ; Cx3cr1 - Cre + mice . ( Two - way ANOVA with Sidak ' s test for multiple comparisons . Asterisks ( * ) indicate comparison to immunopositivity values of the uninjected hippocampus , obliques ( # ) indicate comparison between Csf - 1rflx / wt ; Cx3cr1 - Cre - and Csf - 1rflx / wt ; Cx3cr1 - Cre + mice , # # or * * p < 0 . 003 , # # # # or * * * * p < 0 . 0001 , n = 5 mice per group , error bars indicate SEM ) . C ) Immunohistochemistry of Csf - 1rflx / wt ; Tie2 - Cre - ( top ) and Csf - 1rflx / wt ; Tie2 - Cre + mice ( ottom ) mice unilaterally injected with Aβ1 - 42 in the hippocampus and stained for IB4 ( green ) , F4 / 80 ( white ) and Aβ ( red ) . D ) Quantification of F4 / 80 and IB4 immunopositivity following intrahippocampal injection of Aβ1 - 42 . Quantification of immunopositivity in both injected and uninjected hippocampi of Csf - 1rflx / wt ; Tie2 - Cre - and Csf - 1rflx / wt ; Tie2 - Cre + mice . ( n = 5 mice per group . Two - way ANOVA with Sidak ' s test for multiple comparisons . Asterisks ( * ) indicate comparison to immunopositivity values of the uninjected hippocampus , * p < 0 . 01 , * * * p < 0 . 0005 , error bars indicate 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 O O O O 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

Figure 4A) Immunohistochemistry of Csf-1rflx/wt;Cx3cr1-Cre- (top) and Csf-1rflx/wt;Cx3cr1-Cre+ (bottom) mice unilaterally injected with Aβ1-42 in the hippocampus and stained for IB4 (green), F4/80 (white) and Aβ (red). Scale bars indicate 50 μm. B) Quantification of F4/80 and IB4 immunopositivity following intrahippocampal injection of Aβ1-42. Quantification of immunopositivity in both injected and uninjected hippocampi of Csf-1rflx/wt;Cx3cr1-Cre- and Csf-1rflx/wt;Cx3cr1-Cre+ mice. (Two-way ANOVA with Sidak's test for multiple comparisons. Asterisks (*) indicate comparison to immunopositivity values of the uninjected hippocampus, obliques (#) indicate comparison between Csf-1rflx/wt;Cx3cr1-Cre- and Csf-1rflx/wt;Cx3cr1-Cre+ mice, ## or ** p < 0.003, #### or **** p < 0.0001, n = 5 mice per group,error bars indicate SEM).C) Immunohistochemistry of Csf-1rflx/wt;Tie2-Cre- (top) and Csf-1rflx/wt;Tie2-Cre+ mice (ottom) mice unilaterally injected with Aβ1-42 in the hippocampus and stained for IB4 (green), F4/80 (white) and Aβ (red). D) Quantification of F4/80 and IB4 immunopositivity following intrahippocampal injection of Aβ1-42. Quantification of immunopositivity in both injected and uninjected hippocampi of Csf-1rflx/wt;Tie2-Cre- and Csf-1rflx/wt;Tie2-Cre+ mice. (n = 5 mice per group. Two-way ANOVA with Sidak's test for multiple comparisons. Asterisks (*) indicate comparison to immunopositivity values of the uninjected hippocampus, *p < 0.01, *** p < 0.0005, error bars indicate SEM)).


Figure 5qPCR of wild type ( blue ) or Csf - 1r + / - ( red ) endothelial cells treated with control , Csf - 1r + / + MCM or Csf - 1r + / - MCM . Statistical analyses of inter - genotype changes . ( * * * * p < 0 . 0001 , * * p < 0 . 005 , Scatter plots represent technical replicates of n = 2 independent primary cell isolations and microglia conditionings , Two - way ANOVA with multiple comparisons and Sidak post - test , error bars indicate 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

Figure 5qPCR of wild type (blue) or Csf-1r+/- (red) endothelial cells treated with control, Csf-1r+/+ MCM or Csf-1r+/- MCM. Statistical analyses of inter-genotype changes. (**** p < 0.0001, ** p < 0.005, Scatter plots represent technical replicates of n = 2 independent primary cell isolations and microglia conditionings, Two-way ANOVA with multiple comparisons and Sidak post-test, error bars indicate SEM)).


Figure 6IHC of ALSP patient post mortem tissue for amyloid - β ( green ) and CLD5 ( red ) , ( B ) human IgG ( green ) and PDGFRβ ( red ) , ( C ) GFAP ( green ) and CLD5 ( red ) . ΔA781 _ N783 and P824R indicate the CSF - 1R variant present . ( D ) Iba1 ( green ) and GFAP ( red ) , ( E ) amyloid - β ( green ) and CD68 ( red ) , ( f ) CD163 ( green ) and CLD5 ( red ) , imaging of an ALSP patient with the ΔA781 _ N783 CSF - 1R variant . ΔA781 _ N783 and P824R indicate the CSF - 1R variant present . ( G ) Fibrinogen ( green ) and CLD5 ( red ) and ( H ) FLAIR imaging of an ALSP patient with the ΔA781 _ N783 CSF - 1R variant . ΔA781 _ N783 and P824R indicate the CSF - 1R variant present .

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 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

Figure 6IHC of ALSP patient post mortem tissue for amyloid-β (green) and CLD5 (red), (B) human IgG (green) and PDGFRβ (red), (C) GFAP (green) and CLD5 (red). ΔA781_N783 and P824R indicate the CSF-1R variant present.(D) Iba1 (green) and GFAP (red), (E) amyloid-β (green) and CD68 (red), (f) CD163 (green) and CLD5 (red), imaging of an ALSP patient with the ΔA781_N783 CSF-1R variant. ΔA781_N783 and P824R indicate the CSF-1R variant present.(G) Fibrinogen (green) and CLD5 (red) and (H) FLAIR imaging of an ALSP patient with the ΔA781_N783 CSF-1R variant. ΔA781_N783 and P824R indicate the CSF-1R variant present.


Figure 1Pooled clones ( A - D ) and single cell clones ( E - H ) of HEK293 / sw cells untransfected or stably transfected with the indicated WT and mutant PS1 constructs were analyzed for γ - secretase expression and APP processing . ( A ) PS1 , PS2 and NCT were analyzed in cell lysates by immunoblotting using antibodies PS1N ( PS1 ) , BI - HF5C ( PS2 ) and N1660 ( NCT ) , respectively . ( B ) Full length APP and APP CTFs were analyzed by immunoblotting using antibody 6687 . ( C ) Conditioned media were analyzed for secreted APPs by immunoblotting using antibody 22C11 and for total Aβ by combined immunoprecipitation / immunoblotting using antibodies 3552 / 2D8 . Pooled clones ( A - D ) and single cell clones ( E - H ) of HEK293 / sw cells untransfected or stably transfected with the indicated WT and mutant PS1 constructs were analyzed for γ - secretase expression and APP processing . ( D ) Conditioned media were analyzed for individual Aβ species on Tris - Bicine urea SDS - PAGE by combined immunoprecipitation / immunoblotting of Aβ using antibodies 3552 / 2D8 . Pooled clones of HEK293 / sw cells stably transfected with PS1 L166P were used as reference . Note that more sample was loaded for the PS1 L435F mutant to facilitate analysis . Pooled clones ( A - D ) and single cell clones ( E - H ) of HEK293 / sw cells untransfected or stably transfected with the indicated WT and mutant PS1 constructs were analyzed for γ - secretase expression and APP processing . ( E ) PS1 expression levels were analyzed in cell lysates of HEK293 / sw cells stably expressing PS1 WT or PS1 L435F by immunoblotting using antibodies PS1NT and 5E12 , respectively . Pooled clones ( A - D ) and single cell clones ( E - H ) of HEK293 / sw cells untransfected or stably transfected with the indicated WT and mutant PS1 constructs were analyzed for γ - secretase expression and APP processing . ( F ) Conditioned media were analyzed by ELISA specific for Aβ40 , Aβ42 and Aβ43 . Data represent mean ± s . e . m . ( n = 6 ) . Absolute levels and Aβ ratios are shown . Pooled clones ( A - D ) and single cell clones ( E - H ) of HEK293 / sw cells untransfected or stably transfected with the indicated WT and mutant PS1 constructs were analyzed for γ - secretase expression and APP processing . ( G ) Secreted Aβ was analyzed as in ( D ) . To verify individual Aβ species , Aβ standards were co - migrated . Conditioned media were analyzed for individual Aβ species on Tris - Bicine urea SDS - PAGE by combined immunoprecipitation / immunoblotting of Aβ using antibodies 3552 / 2D8 . Pooled clones ( A - D ) and single cell clones ( E - H ) of HEK293 / sw cells untransfected or stably transfected with the indicated WT and mutant PS1 constructs were analyzed for γ - secretase expression and APP processing . ( H ) Total Aβ in conditioned media was analyzed by MALDI - TOF MS following immunoprecipitation with antibody 4G8 . Observed ( Aβ42 , 4513 . 6 ; Aβ43 , 4615 . 3 ) and predicted molecular masses ( Aβ42 , 4514 . 1 ; Aβ43 , 4615 . 2 ) were in good agreement .

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 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 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 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 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 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

Figure 1Pooled clones (A-D) and single cell clones (E-H) of HEK293/sw cells untransfected or stably transfected with the indicated WT and mutant PS1 constructs were analyzed for γ-secretase expression and APP processing.(A) PS1, PS2 and NCT were analyzed in cell lysates by immunoblotting using antibodies PS1N (PS1), BI-HF5C (PS2) and N1660 (NCT), respectively.(B) Full length APP and APP CTFs were analyzed by immunoblotting using antibody 6687. (C) Conditioned media were analyzed for secreted APPs by immunoblotting using antibody 22C11 and for total Aβ by combined immunoprecipitation/immunoblotting using antibodies 3552/2D8.Pooled clones (A-D) and single cell clones (E-H) of HEK293/sw cells untransfected or stably transfected with the indicated WT and mutant PS1 constructs were analyzed for γ-secretase expression and APP processing. (D) Conditioned media were analyzed for individual Aβ species on Tris-Bicine urea SDS-PAGE by combined immunoprecipitation/immunoblotting of Aβ using antibodies 3552/2D8. Pooled clones of HEK293/sw cells stably transfected with PS1 L166P were used as reference. Note that more sample was loaded for the PS1 L435F mutant to facilitate analysis.Pooled clones (A-D) and single cell clones (E-H) of HEK293/sw cells untransfected or stably transfected with the indicated WT and mutant PS1 constructs were analyzed for γ-secretase expression and APP processing. (E) PS1 expression levels were analyzed in cell lysates of HEK293/sw cells stably expressing PS1 WT or PS1 L435F by immunoblotting using antibodies PS1NT and 5E12, respectively.Pooled clones (A-D) and single cell clones (E-H) of HEK293/sw cells untransfected or stably transfected with the indicated WT and mutant PS1 constructs were analyzed for γ-secretase expression and APP processing. (F) Conditioned media were analyzed by ELISA specific for Aβ40, Aβ42 and Aβ43. Data represent mean ± s.e.m. (n=6). Absolute levels and Aβ ratios are shown.Pooled clones (A-D) and single cell clones (E-H) of HEK293/sw cells untransfected or stably transfected with the indicated WT and mutant PS1 constructs were analyzed for γ-secretase expression and APP processing.(G) Secreted Aβ was analyzed as in (D). To verify individual Aβ species, Aβ standards were co-migrated. Conditioned media were analyzed for individual Aβ species on Tris-Bicine urea SDS-PAGE by combined immunoprecipitation/immunoblotting of Aβ using antibodies 3552/2D8.Pooled clones (A-D) and single cell clones (E-H) of HEK293/sw cells untransfected or stably transfected with the indicated WT and mutant PS1 constructs were analyzed for γ-secretase expression and APP processing.(H) Total Aβ in conditioned media was analyzed by MALDI-TOF MS following immunoprecipitation with antibody 4G8. Observed (Aβ42, 4513.6; Aβ43, 4615.3) and predicted molecular masses (Aβ42, 4514.1; Aβ43, 4615.2) were in good agreement.


Figure 2 ( A ) PS1 / 2 - / - MEF cells stably transduced with PS1 WT or PS1 L435F were analyzed for PS1 expression and APP processing by immunoblotting as in Fig 1A . Antibody 5E12 was used for the detection of the PS1 CTF . ( B ) Stably transduced PS1 / 2 - / - MEF cells of A were transiently transfected with APPsw - 6myc and conditioned media were analyzed for Aβ species by Tris - Bicine urea SDS - PAGE . Individual Aβ species were verified by co - migration with Aβ standards ( left panel ) and quantified ( right panel ) . Data represent mean ± s . e . m . of n = 3 independently performed transfections . Note that more sample was loaded for the PS1 L435F mutant to facilitate immunoblot analysis . ( C ) Secreted Aβ from cells in B were quantified by ELISA . Data represent mean ± s . e . m . of n = 5 ( PS1 WT ) or 6 ( PS1 L435F ) independently performed transfections .

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Figure 2(A) PS1/2-/- MEF cells stably transduced with PS1 WT or PS1 L435F were analyzed for PS1 expression and APP processing by immunoblotting as in Fig 1A. Antibody 5E12 was used for the detection of the PS1 CTF.(B) Stably transduced PS1/2-/- MEF cells of A were transiently transfected with APPsw-6myc and conditioned media were analyzed for Aβ species by Tris-Bicine urea SDS-PAGE. Individual Aβ species were verified by co-migration with Aβ standards (left panel) and quantified (right panel). Data represent mean ± s.e.m. of n=3 independently performed transfections. Note that more sample was loaded for the PS1 L435F mutant to facilitate immunoblot analysis.(C) Secreted Aβ from cells in B were quantified by ELISA. Data represent mean ± s.e.m. of n=5 (PS1 WT) or 6 (PS1 L435F) independently performed transfections.


Figure 3Immunohistochemical detection of Aβ40 ( left column ) , Aβ42 ( medium column ) and Aβ43 ( right column ) in consecutive frontal cortex paraffin sections of two FAD cases with PS1 L435F mutation ( cases # 1 and # 2 , 1st and 2nd rows ) , another FAD case with different PS1 mutation ( case # 3 , 3rd row ) and one SAD case ( case # 4 , forth row ) . In both PS1 L435F mutation cases the numerous Aβ plaques mainly contain Aβ42 and Aβ43 but less Aβ40 . This is in contrast to Aβ plaques of the control FAD and SAD cases , in which solely Aβ42 predominates and Aβ43 is sparse ; even levels of Aβ40 seem to be lower than in the PS1 L435F cases . Note that in both PS1 L435F cases several plaques are larger than those seen in cases # 3 and # 4 , representing cotton wool plaques . Scale bar = 500 µm . Magnification is identical in all pictures .

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Figure 3Immunohistochemical detection of Aβ40 (left column), Aβ42 (medium column) and Aβ43 (right column) in consecutive frontal cortex paraffin sections of two FAD cases with PS1 L435F mutation (cases #1 and #2, 1st and 2nd rows), another FAD case with different PS1 mutation (case #3, 3rd row) and one SAD case (case #4, forth row). In both PS1 L435F mutation cases the numerous Aβ plaques mainly contain Aβ42 and Aβ43 but less Aβ40. This is in contrast to Aβ plaques of the control FAD and SAD cases, in which solely Aβ42 predominates and Aβ43 is sparse; even levels of Aβ40 seem to be lower than in the PS1 L435F cases. Note that in both PS1 L435F cases several plaques are larger than those seen in cases #3 and #4, representing cotton wool plaques. Scale bar = 500 µm. Magnification is identical in all pictures.


Figure 2a ) Kaplan - Meier to first fever ( ≥ 38 ° C ) episode within 24 hours of receiving vaccines administered at 4 months of age ; control n = 88 and 4CMenB n = 89 . b ) Principal component ( PC ) analysis of RNA - sequencing data ( 14837 genes , n = 253 ) from all study time points . The lines connect participant ' s pre - and post - vaccination samples . The ellipses are the two - dimensional ( PC1 and PC2 ) 95 % confidence intervals for each study time point . A contribution plot ( top right ) displays the genes contributing most to PC1 / PC2 , i . e . 0 . 15 implies 0 . 15 % contribution of that variable to the principal components displayed . c ) Plotted are the CIBERSORTx neutrophil fractions from whole blood RNA - sequencing data , with median and interquartile range . P - values were determined from a two sample Wilcoxon rank sum test . The number of individuals in each group is display in the x - axis . d ) Spearman ' s rank correlation between neutrophil counts measure by full blood counts ( n = 142 ) and those estimated by CIBERSORTx , using the LM22 signature matrix .

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Figure 2a) Kaplan-Meier to first fever (≥38°C) episode within 24 hours of receiving vaccines administered at 4 months of age; control n=88 and 4CMenB n=89.b) Principal component (PC) analysis of RNA-sequencing data (14837 genes, n= 253) from all study time points. The lines connect participant's pre- and post-vaccination samples. The ellipses are the two-dimensional (PC1 and PC2) 95% confidence intervals for each study time point. A contribution plot (top right) displays the genes contributing most to PC1/PC2, i.e. 0.15 implies 0.15% contribution of that variable to the principal components displayed.c) Plotted are the CIBERSORTx neutrophil fractions from whole blood RNA-sequencing data, with median and interquartile range. P-values were determined from a two sample Wilcoxon rank sum test. The number of individuals in each group is display in the x-axis.d) Spearman's rank correlation between neutrophil counts measure by full blood counts (n= 142) and those estimated by CIBERSORTx, using the LM22 signature matrix.


Figure 3a ) Volcano plot highlighting differentially expressed genes ( DEGs , false - discovery rate [ FDR ] < 0 . 01 ; red upregulated and blue downregulated ) at each study time point versus pre - vaccination ( 4 months of age ) 4 hours post - vaccination ( 719 DEGs , n = 28 ) . P - values were obtained from the moderated t - statistic , after adjustment for multiple testing ( Benjamini and Hochberg ' s method ) . The top 10 genes , ranked by FDR , are labelled . Volcano plot highlighting differentially expressed genes ( DEGs , false - discovery rate [ FDR ] < 0 . 01 ; red upregulated and blue downregulated ) at each study time point versus pre - vaccination b ) Same as ( a ) but 24 hours post - vaccination ( 5553 DEGs , n = 31 ) Volcano plot highlighting differentially expressed genes ( DEGs , false - discovery rate [ FDR ] < 0 . 01 ; red upregulated and blue downregulated ) at each study time point versus pre - vaccination c ) Same as ( a ) but 3 days post - vaccination ( 159 DEGs , n = 30 ) Volcano plot highlighting differentially expressed genes ( DEGs , false - discovery rate [ FDR ] < 0 . 01 ; red upregulated and blue downregulated ) at each study time point versus pre - vaccination d ) Same as ( a ) but 7 days post - vaccination ( 6 DEGs , n = 36 ) .

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Figure 3a) Volcano plot highlighting differentially expressed genes (DEGs, false-discovery rate [FDR] <0.01; red upregulated and blue downregulated) at each study time point versus pre-vaccination (4 months of age) 4 hours post-vaccination (719 DEGs, n=28). P-values were obtained from the moderated t-statistic, after adjustment for multiple testing (Benjamini and Hochberg's method). The top 10 genes, ranked by FDR, are labelled.Volcano plot highlighting differentially expressed genes (DEGs, false-discovery rate [FDR] <0.01; red upregulated and blue downregulated) at each study time point versus pre-vaccination b) Same as (a) but 24 hours post-vaccination (5553 DEGs, n=31)Volcano plot highlighting differentially expressed genes (DEGs, false-discovery rate [FDR] <0.01; red upregulated and blue downregulated) at each study time point versus pre-vaccination c) Same as (a) but 3 days post-vaccination (159 DEGs, n=30)Volcano plot highlighting differentially expressed genes (DEGs, false-discovery rate [FDR] <0.01; red upregulated and blue downregulated) at each study time point versus pre-vaccination d) Same as (a) but 7 days post-vaccination (6 DEGs, n=36).


Figure 4 a ) Top genes differentially regulated between vaccine groups 4 hours post - vaccination . Note , only genes differentially expressed in either study group ( concomitant 4CMenB or control vaccines alone ) were included in intergroup analysis . The plotted lines are the LOESS ( locally estimated scatterplot smoothing ) regression curves with the 95 % confidence intervals in grey . The FDR was derived by comparing fold - changes in gene expression in the control group with the test group , from baseline to the time point designated , and is reported if statistically significant ( FDR < 0 . 05 ) . P - values were obtained from the moderated t - statistic , after adjustment for multiple testing ( Benjamini and Hochberg ' s method ) . Pre - vaccination samples n = 125 , 4 hour samples n = 28 , 24 hour samples n = 31 , 3 day sample n = 30 , 7 day samples n = 36 . The expression E value , are the gene expression value derived from the voom - limma workflow ( Law et al . , 2014 ) . Note , only genes differentially expressed in either study group ( concomitant 4CMenB or control vaccines alone ) were included in intergroup analysis . The plotted lines are the LOESS ( locally estimated scatterplot smoothing ) regression curves with the 95 % confidence intervals in grey . The FDR was derived by comparing fold - changes in gene expression in the control group with the test group , from baseline to the time point designated , and is reported if statistically significant ( FDR < 0 . 05 ) . b ) Same as ( a ) but top genes differentially regulated between vaccine groups 24 hours post - vaccination . Note , only genes differentially expressed in either study group ( concomitant 4CMenB or control vaccines alone ) were included in intergroup analysis . The plotted lines are the LOESS ( locally estimated scatterplot smoothing ) regression curves with the 95 % confidence intervals in grey . The FDR was derived by comparing fold - changes in gene expression in the control group with the test group , from baseline to the time point designated , and is reported if statistically significant ( FDR < 0 . 05 ) . P - values were obtained from the moderated t - statistic , after adjustment for multiple testing ( Benjamini and Hochberg ' s method ) . c ) Same as ( a ) but top genes differentially regulated between vaccine groups 3 days post - vaccination . Note , only genes differentially expressed in either study group ( concomitant 4CMenB or control vaccines alone ) were included in intergroup analysis . The plotted lines are the LOESS ( locally estimated scatterplot smoothing ) regression curves with the 95 % confidence intervals in grey . The FDR was derived by comparing fold - changes in gene expression in the control group with the test group , from baseline to the time point designated , and is reported if statistically significant ( FDR < 0 . 05 ) . P - values were obtained from the moderated t - statistic , after adjustment for multiple testing ( Benjamini and Hochberg ' s method ) . d ) Same as ( a ) but top genes differentially regulated between vaccine groups 7 days post - vaccination .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Top genes differentially regulated between vaccine groups 4 hours post-vaccination. Note, only genes differentially expressed in either study group (concomitant 4CMenB or control vaccines alone) were included in intergroup analysis. The plotted lines are the LOESS (locally estimated scatterplot smoothing) regression curves with the 95% confidence intervals in grey. The FDR was derived by comparing fold-changes in gene expression in the control group with the test group, from baseline to the time point designated, and is reported if statistically significant (FDR <0.05). P-values were obtained from the moderated t-statistic, after adjustment for multiple testing (Benjamini and Hochberg's method). Pre-vaccination samples n= 125, 4 hour samples n= 28, 24 hour samples n = 31, 3 day sample n= 30, 7 day samples n= 36. The expression E value, are the gene expression value derived from the voom-limma workflow (Law et al., 2014).  Note, only genes differentially expressed in either study group (concomitant 4CMenB or control vaccines alone) were included in intergroup analysis. The plotted lines are the LOESS (locally estimated scatterplot smoothing) regression curves with the 95% confidence intervals in grey. The FDR was derived by comparing fold-changes in gene expression in the control group with the test group, from baseline to the time point designated, and is reported if statistically significant (FDR <0.05). b) Same as (a) but top genes differentially regulated between vaccine groups 24 hours post-vaccination.  Note, only genes differentially expressed in either study group (concomitant 4CMenB or control vaccines alone) were included in intergroup analysis. The plotted lines are the LOESS (locally estimated scatterplot smoothing) regression curves with the 95% confidence intervals in grey. The FDR was derived by comparing fold-changes in gene expression in the control group with the test group, from baseline to the time point designated, and is reported if statistically significant (FDR <0.05). P-values were obtained from the moderated t-statistic, after adjustment for multiple testing (Benjamini and Hochberg's method). c) Same as (a) but top genes differentially regulated between vaccine groups 3 days post-vaccination.  Note, only genes differentially expressed in either study group (concomitant 4CMenB or control vaccines alone) were included in intergroup analysis. The plotted lines are the LOESS (locally estimated scatterplot smoothing) regression curves with the 95% confidence intervals in grey. The FDR was derived by comparing fold-changes in gene expression in the control group with the test group, from baseline to the time point designated, and is reported if statistically significant (FDR <0.05). P-values were obtained from the moderated t-statistic, after adjustment for multiple testing (Benjamini and Hochberg's method). d) Same as (a) but top genes differentially regulated between vaccine groups 7 days post-vaccination.


Figure 5 False - discovery rate ( FDR ) are displayed above and were derived from a two sample Wilcoxon rank sum test ( Benjamini - Hochberg correction ) . Vertical lines represent the interquartile range around the median . The red dashed horizontal line is upper limit of the normal CRP reference interval ( 0 - 5mg / l ) . The number of individuals in each group is display in the x - axis .

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Figure 5 False-discovery rate (FDR) are displayed above and were derived from a two sample Wilcoxon rank sum test (Benjamini-Hochberg correction). Vertical lines represent the interquartile range around the median. The red dashed horizontal line is upper limit of the normal CRP reference interval (0-5mg/l). The number of individuals in each group is display in the x-axis.


Figure 6a , b ) Agreement plot . Red = differentially expressed ( DE ) in febrile infants , blue = DE in afebrile infants , purple = DE in febrile and afebrile infants . c ) Exemplar genes differentially expressed at 4 hours and 24 hours , respectively , between infants who experienced a fever within 24 hours of vaccination and afebrile infants . The plotted lines are the LOESS ( locally estimated scatterplot smoothing ) regression curves with the 95 % confidence intervals in grey . The false - discovery rate ( FDR ) was derived by comparing fold - changes in gene expression between infants who experienced post - vaccination fever and those who remained afebrile . P - values were obtained from the moderated t - statistic , after adjustment for multiple testing ( Benjamini and Hochberg ' s method ) . Pre - vaccination samples n = 125 , 4 hour samples n = 28 , 24 hour samples n = 31 , 3 day sample n = 30 , 7 day samples n = 36 .

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Figure 6a, b) Agreement plot. Red = differentially expressed (DE) in febrile infants, blue = DE in afebrile infants, purple =DE in febrile and afebrile infants.c) Exemplar genes differentially expressed at 4 hours and 24 hours, respectively, between infants who experienced a fever within 24 hours of vaccination and afebrile infants. The plotted lines are the LOESS (locally estimated scatterplot smoothing) regression curves with the 95% confidence intervals in grey. The false-discovery rate (FDR) was derived by comparing fold-changes in gene expression between infants who experienced post-vaccination fever and those who remained afebrile. P-values were obtained from the moderated t-statistic, after adjustment for multiple testing (Benjamini and Hochberg's method). Pre-vaccination samples n= 125, 4 hour samples n= 28, 24 hour samples n = 31, 3 day sample n= 30, 7 day samples n= 36.


Figure 7Boxplots represent the median with interquartile ranges . False - discovery rate ( FDR ) are displayed above and were derived from a two sample Wilcoxon rank sum test ( Benjamini - Hochberg correction ) . N = 6 mice per vaccine group .

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Figure 7Boxplots represent the median with interquartile ranges. False-discovery rate (FDR) are displayed above and were derived from a two sample Wilcoxon rank sum test (Benjamini-Hochberg correction). N = 6 mice per vaccine group.


Figure 8Boxplots depicting the change in temperature from baseline to 24 hours after the second 1μg dose of each of a variety of Escherichia coli lipopolysaccharide ( LPS ) and peptidoglycan ( PGN ) preparations , with and without alum , and combinations compared with 1 / 5 of the human dose of 4CMenB . Boxplots represent the median with interquartile ranges . Vaccine group medians at each timepoint were compared with baseline for that vaccine using a Wilcoxon signed - rank test . * < 0 . 05 , * * < 0 . 01 , * * * * < 0 . 0001 . N = 6 mice per vaccine group .

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Figure 8Boxplots depicting the change in temperature from baseline to 24 hours after the second 1μg dose of each of a variety of Escherichia coli lipopolysaccharide (LPS) and peptidoglycan (PGN) preparations, with and without alum, and combinations compared with 1/5 of the human dose of 4CMenB. Boxplots represent the median with interquartile ranges. Vaccine group medians at each timepoint were compared with baseline for that vaccine using a Wilcoxon signed-rank test. * < 0.05, ** < 0.01, **** < 0.0001. N= 6 mice per vaccine group.


Figure 1 ( A , B ) CMH2DCFDA staining ( A ) and data quantification ( B ) showing H2O2 level in different aged fly brains with and without treatment with the indicated RET inhibitors ( n = 5 per group ) . ( I ) Dose - dependent effect of CPT on RET - ROS in mitochondria isolated from 45 days old flies and respiring under RET condition ( n = 4 ) . ( K ) CMH2DCFDA staining showing H2O2 level in young flies treated with DES or DES + CPT ( n = 5 per group ) .

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Figure 1(A, B) CMH2DCFDA staining (A) and data quantification (B) showing H2O2 level in different aged fly brains with and without treatment with the indicated RET inhibitors (n=5 per group).(I) Dose-dependent effect of CPT on RET-ROS in mitochondria isolated from 45 days old flies and respiring under RET condition (n=4).(K) CMH2DCFDA staining showing H2O2 level in young flies treated with DES or DES + CPT (n=5 per group).


Figure 2 ( E - H ) Survival curves comparing control flies and flies fed with food containing 5 µM CPT ( E ) , 10 µM CPT ( * * p < 0 . 01 logrank , * * p < 0 . 01 Wang Allison ) ( F ) , or 20 µM CPT ( * * p < 0 . 01 logrank , * * p < 0 . 01 Wang Allison ) ( G ) from 35 days onwards . Bar graphs ( H ) show quantification of mean and maximal lifespan ( n = 3 groups , 20 - 25 flies per group ) , using single factor ANOVA with Scheffe ' s analysis as a post hoc test . : Data are representative of at least three repeats . H ) Data are shown as mean ± Survival curves were analyzed with logrank test along with Wang Allison test for maximum lifespan . Asterisks indicate statistical significance ( * * p < 0 . 01 , * p < 0 . 05 ) . Dashed lines mark time points of 50 % survival .

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Figure 2(E-H) Survival curves comparing control flies and flies fed with food containing 5 µM CPT (E), 10 µM CPT (**p<0.01 logrank, **p<0.01 Wang Allison) (F), or 20 µM CPT (**p<0.01 logrank, **p<0.01 Wang Allison) (G) from 35 days onwards. Bar graphs (H) show quantification of mean and maximal lifespan (n=3 groups, 20-25 flies per group), using single factor ANOVA with Scheffe's analysis as a post hoc test. : Data are representative of at least three repeats. H) Data are shown as mean ± Survival curves were analyzed with logrank test along with Wang Allison test for maximum lifespan. Asterisks indicate statistical significance (**p< 0.01, *p< 0.05). Dashed lines mark time points of 50% survival.


Figure 3 ( C ) Images and data quantification showing accumulation of protein aggregates in the brains of aged flies with or without CPT treatment ( n = 5 ) . ( D ) Images and data quantification showing DA neuron mitochondrial morphology in the brains of aged flies with or without CPT treatment ( n = 5 ) . Images ( E ) showing 4 - HNE levels within or near TH - positive DA neurons in young flies , old flies , and old flies treated with CPT ( n = 4 sets , 5 samples per set ) . Arrows point to HNE signals co - labelled with TH - positive DA neurons .

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Figure 3(C) Images and data quantification showing accumulation of protein aggregates in the brains of aged flies with or without CPT treatment (n=5).(D) Images and data quantification showing DA neuron mitochondrial morphology in the brains of aged flies with or without CPT treatment (n=5).Images (E) showing 4-HNE levels within or near TH-positive DA neurons in young flies, old flies, and old flies treated with CPT (n=4 sets, 5 samples per set). Arrows point to HNE signals co-labelled with TH- positive DA neurons.


Figure 4 ( D ) CMH2DCFDA staining of H2O2 and quantification of signal intensity in the muscle tissues of 50 - day old control flies and flies with NDUFS2 or NDUFS3 knockdown ( n = 5 per group ) . ( F ) Survival curves of wild type flies with or without NMN treatment from 35 days onwards ( * * p < 0 . 01 logrank , * * P < 0 . 01 Wang Allison ) ( n = 3 groups , 20 - 25 flies per group ) . ( H ) Images and data quantification showing accumulation of protein aggregates in the muscle of young flies , old flies , and old flies treated with CPT ( n = 5 per group ) .

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Figure 4(D) CMH2DCFDA staining of H2O2 and quantification of signal intensity in the muscle tissues of 50-day old control flies and flies with NDUFS2 or NDUFS3 knockdown (n=5 per group).(F) Survival curves of wild type flies with or without NMN treatment from 35 days onwards (**p<0.01 logrank, **P<0.01 Wang Allison) (n=3 groups, 20-25 flies per group).(H) Images and data quantification showing accumulation of protein aggregates in the muscle of young flies, old flies, and old flies treated with CPT (n=5 per group).


Figure 5 ( A , B ) Survival curves showing effect of neuronal dSirt2 knockdown on fly lifespan under normal and CPT - treatment conditions ( n = 3 groups , 20 - 25 flies per group ) . ( C , D ) Survival curves showing effect of muscle dFoxo knockdown on fly lifespan under normal and CPT - treatment conditions ( n = 3 groups , 20 - 25 flies per group ) . ( E , F ) Survival curves showing effect of neuronal ATG1 knockdown on fly lifespan under normal and CPT - treatment conditions ( n = 3 groups , 20 - 25 flies per group ) . ( H ) Images and data quantification in single factor ANOVA with Scheffe ' s analysis as a post hoc test showing lysotracker staining in the muscle of young flies , old flies , and old flies treated with CPT ( n = 5 per group ) .

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Figure 5(A, B) Survival curves showing effect of neuronal dSirt2 knockdown on fly lifespan under normal and CPT-treatment conditions (n=3 groups, 20-25 flies per group). (C, D) Survival curves showing effect of muscle dFoxo knockdown on fly lifespan under normal and CPT-treatment conditions (n=3 groups, 20-25 flies per group). (E, F) Survival curves showing effect of neuronal ATG1 knockdown on fly lifespan under normal and CPT-treatment conditions (n=3 groups, 20-25 flies per group).(H) Images and data quantification in single factor ANOVA with Scheffe's analysis as a post hoc test showing lysotracker staining in the muscle of young flies, old flies, and old flies treated with CPT (n=5 per group).


Figure 6 ( A , B ) Survival curves showing effect of CPT treatment on the lifespan of Mhc > APP . C99 flies ( * * p < 0 . 01 logrank , * * p < 0 . 01 Wang Allison ) ( A ) and elav > APP . BACE flies ( * * p < 0 . 01 logrank , * * p < 0 . 01 Wang Allison ) ( B ) ( n = 3 groups , 20 - 25 flies per group ) . ( E ) Immunofluorescence images and data quantification showing effect of CPT on the formation of ubiquitin - and 6E10 - positive amyloid aggregates in the brain of elav > APP . BACE flies ( n = 5 ) . ( G ) Quantification of the effect of CPT on DA neuron number in the PPL1 cluster of elav > APP / BACE fly brain ( n = 4 sets , 5 brains per set ) .

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Figure 6(A, B) Survival curves showing effect of CPT treatment on the lifespan of Mhc>APP.C99 flies (**p<0.01 logrank, **p<0.01 Wang Allison) (A) and elav>APP.BACE flies (**p<0.01 logrank, **p<0.01 Wang Allison) (B) (n=3 groups, 20-25 flies per group).(E) Immunofluorescence images and data quantification showing effect of CPT on the formation of ubiquitin- and 6E10-positive amyloid aggregates in the brain of elav>APP.BACE flies (n=5).(G) Quantification of the effect of CPT on DA neuron number in the PPL1 cluster of elav>APP/BACE fly brain (n=4 sets, 5 brains per set).


Figure 7 ( A , B ) Quantification of effect of CPT on ROS level as detected by staining with the H2O2 dye CMH2DCFDA ( A ) , and on NAD + / NADH ratio ( B ) in control , FAD , and DS iPSC - derived neurons . ( D ) Survival curves showing effect of CPT on lifespan of PS19 tauopathy mice ( * p < 0 . 05 logrank ) ( n = 9 in vehicle group , 8 in CPT group ) .

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Figure 7(A, B) Quantification of effect of CPT on ROS level as detected by staining with the H2O2 dye CMH2DCFDA (A), and on NAD+/NADH ratio (B) in control, FAD, and DS iPSC-derived neurons.(D) Survival curves showing effect of CPT on lifespan of PS19 tauopathy mice (*p<0.05 logrank) (n=9 in vehicle group, 8 in CPT group).


Figure 1 ( f ) Percent survival of 15 d old plants with and without SA187 under NHS , HS and LAT conditions . Data information : percent survival measuremts after recovery at 22 ° C for 4 days . For percent survival , plants with green leaves were scored as live plant out of 12 plants for each biological repeat in different temperature treatments . All treatments are compared with direct 44 ° C heat stressed plants . Plots represent the mean of 3 biological replicates bars representSE Asterisks indicate a statistical difference based on the Mann - Whitney test ( * P < 0 . 05 )

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Figure 1(f) Percent survival of 15 d old plants with and without SA187 under NHS, HS and LAT conditions. Data information: percent survival measuremts after recovery at 22°C for 4 days. For percent survival, plants with green leaves were scored as live plant out of 12 plants for each biological repeat in different temperature treatments. All treatments are compared with direct 44°C heat stressed plants. Plots represent the mean of 3 biological replicates bars representSE Asterisks indicate a statistical difference based on the Mann-Whitney test (* P < 0.05 )


Figure 3 ( a , b ) Dynamics of HSFA2 and HSP101 transcript levels in control ( NHS , NHS + 187 ) , 44 ° C heat - stressed non - colonized and SA187 - colonized ( HS , HS + 187 ) , thermoprimed ( LAT ) as well as thermoprimed and SA187 - colonized plants ( LAT + 187 ) at 1 h , 24 h , 48 h , 72 h and 96 h of recovery at 22 ° C . SA187 - colonized ( HS + 187 ) and thermoprimed ( LAT ) plants showed higher transcript levels in comparison to plants exposed at 44 ° C HS for 30 min ( HS ) after 1 h of recovery at 22 ° C . Data information : All plots represent the means of 3 biological replicates . Error bars represent SE . Asterisks indicate a statistical difference based on Student ' s t - test ( * P ≤ 0 . 05 ( c , d ) . Transcript levels of heat stress memory genes HSP18 . 1 and APX2 in control plants ( NHS , NHS + 187 ) , 44 ° C heat - stressed non - colonized and SA187 - colonized ( HS , HS + 187 ) , thermoprimed ( LAT ) as well as thermoprimed SA187 - colonized plants ( LAT + 187 ) at 1 h , 24 h , 48 h , 72 h and 96 h of recovery at 22 ° C . Transcript levels were normalized to tubulin as reference gene and the respective 22 ° C NHS plants were harvested at the same time points . All treatments are compared with direct 44 ° C HS treatment for statistical significance . Data information : All plots represent the means of 3 biological replicates . Error bars represent SE . Asterisks indicate a statistical difference based on Student ' s t - test ( * P ≤ 0 . 05 ; * * P ≤ 0 . 01 ; * * * P ≤ 0 . 001 ( g ) Relative enrichment of H3K4me3 at APX2 and HSP18 . 2 in control non - primed ( NP ) , SA187 - colonized non - primed plants ( NP + 187 ) , 37 ° C - primed ( P ) and 37 ° C - primed SA187 - colonized plants ( P + 187 ) at 24 and 72 h after priming as determined by chromatin immunoprecipitation - qPCR for the indicated regions of APX2 and HSP18 . 2 . Amplification values were normalized to input and H3 and region 1 of non - primed ( NP ) plants . Data information : All plots represent the means of 3 biological replicates . Error bars represent SE . Asterisks indicate a statistical difference based on Student ' s t - test ( * P ≤ 0 . 05 ; * * P ≤ 0 . 01 ; * * * P ≤ 0 . 001 for differences between NP in comparison to NP + 187 , P and P + 187 treatments ) .

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 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 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

Figure 3(a,b) Dynamics of HSFA2 and HSP101 transcript levels in control (NHS, NHS+187), 44°C heat-stressed non-colonized and SA187-colonized (HS, HS+187), thermoprimed (LAT) as well as thermoprimed and SA187-colonized plants (LAT+187) at 1 h, 24 h, 48 h, 72 h and 96 h of recovery at 22°C. SA187-colonized (HS+187) and thermoprimed (LAT) plants showed higher transcript levels in comparison to plants exposed at 44°C HS for 30 min (HS) after 1 h of recovery at 22°C. Data information: All plots represent the means of 3 biological replicates. Error bars represent SE. Asterisks indicate a statistical difference based on Student's t-test (*P ≤ 0.05(c,d). Transcript levels of heat stress memory genes HSP18.1 and APX2 in control plants (NHS, NHS+187), 44°C heat-stressed non-colonized and SA187-colonized (HS, HS+187), thermoprimed (LAT) as well as thermoprimed SA187-colonized plants (LAT+187) at 1 h, 24 h, 48 h, 72 h and 96 h of recovery at 22°C . Transcript levels were normalized to tubulin as reference gene and the respective 22°C NHS plants were harvested at the same time points. All treatments are compared with direct 44°C HS treatment for statistical significance. Data information: All plots represent the means of 3 biological replicates. Error bars represent SE. Asterisks indicate a statistical difference based on Student's t-test (*P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001(g) Relative enrichment of H3K4me3 at APX2 and HSP18.2 in control non-primed (NP), SA187-colonized non-primed plants (NP+187), 37°C-primed (P) and 37°C-primed SA187-colonized plants (P+187) at 24 and 72 h after priming as determined by chromatin immunoprecipitation-qPCR for the indicated regions of APX2 and HSP18.2. Amplification values were normalized to input and H3 and region 1 of non-primed (NP) plants. Data information: All plots represent the means of 3 biological replicates. Error bars represent SE. Asterisks indicate a statistical difference based on Student's t-test (*P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001 for differences between NP in comparison to NP+187, P and P+187 treatments).


Figure 4 ( b , c ) Fresh weight and percent survival of Col - 0 , hsfa2 , hsfa1q and ein3 - 1 plants in HS , HS + 187 , LAT and LAT + 187 treatments . Due to the dwarf size of hsfa1q mutants , LAT treatment was performed on d 18 and HS at d 20 . All treatments are compared with plants upon 44 ° C HS . Data information : All plots represent the means of 3 biological replicates ( n = 36 , 12 plants per biological repeat ) . Error bars represent SD of three biological repeats . Asterisks indicate a statistical difference based on the Student ' s t - test ( * P ≤ 0 . 05 ; * * P ≤ 0 . 01 ; * * * P ≤ 0 . 001 ) . ( e ) Percent survival of Col - 0 plants with and without ACC under HS , HS + ACC , LAT and LAT + ACC conditions . Data information : All plots represent the means of 3 biological replicates ( n = 36 , 12 plants per biological repeat ) . Error bars represent SD of three biological repeats . Asterisks indicate a statistical difference based on the Student ' s t - test ( * P ≤ 0 . 05 ; * * P ≤ 0 . 01 ; * * * P ≤ 0 . 001 ) .

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Figure 4(b,c) Fresh weight and percent survival of Col-0, hsfa2, hsfa1q and ein3-1 plants in HS, HS+187, LAT and LAT+187 treatments. Due to the dwarf size of hsfa1q mutants, LAT treatment was performed on d 18 and HS at d 20. All treatments are compared with plants upon 44°C HS. Data information: All plots represent the means of 3 biological replicates (n=36, 12 plants per biological repeat). Error bars represent SD of three biological repeats. Asterisks indicate a statistical difference based on the Student's t-test (* P≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001).(e) Percent survival of Col-0 plants with and without ACC under HS, HS+ACC, LAT and LAT+ACC conditions. Data information: All plots represent the means of 3 biological replicates (n=36, 12 plants per biological repeat). Error bars represent SD of three biological repeats. Asterisks indicate a statistical difference based on the Student's t-test (* P≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001).


Figure 5 ( c ) Relative enrichment of H3K4me3 at APX2 and HSP18 . 2 in control non - primed ( NP ) , SA187 - colonized non - primed plants ( NP + 187 ) , 37 ° C - primed ( P ) and 37 ° C - primed SA187 - colonized plants ( P + 187 ) at 24 and 72 h after priming as determined by chromatin immunoprecipitation - qPCR for the indicated regions of APX2 and HSP18 . 2 ( Fig 3F ) . Data information : For H3K4me3 enrichment analysis , amplification values were normalized to input and H3 and region 1 of non - primed ( NP ) plants . All treatments are compared with direct 44 ° C HS treatment for statistical significance . All plots represent the means of 3 biological replicates . Error bars indicate SE . Asterisks indicate a statistical difference based on the Student ' s t - test * P ≤ 0 . 05 ; * * P ≤ 0 . 01 ; * * P ≤ 0 . 001 for differences between NP in comparison to NP + 187 , P and P + 187 treatments . ( f ) Relative enrichment of H3K4me3 levels at APX2 and HSP18 . 2 memory genes in hsfa2 mutant . Data information : For H3K4me3 enrichment analysis , amplification values were normalized to input and H3 and region 1 of non - primed ( NP ) plants . All treatments are compared with direct 44 ° C HS treatment for statistical significance . All plots represent the means of 3 biological replicates . Error bars indicate SE . Asterisks indicate a statistical difference based on the Student ' s t - test * P ≤ 0 . 05 ; * * P ≤ 0 . 01 ; * * P ≤ 0 . 001 for differences between NP in comparison to NP + 187 , P and P + 187 treatments .

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Figure 5(c) Relative enrichment of H3K4me3 at APX2 and HSP18.2 in control non-primed (NP), SA187-colonized non-primed plants (NP+187), 37°C-primed (P) and 37°C-primed SA187-colonized plants (P+187) at 24 and 72 h after priming as determined by chromatin immunoprecipitation-qPCR for the indicated regions of APX2 and HSP18.2 (Fig 3F). Data information: For H3K4me3 enrichment analysis, amplification values were normalized to input and H3 and region 1 of non-primed (NP) plants. All treatments are compared with direct 44°C HS treatment for statistical significance. All plots represent the means of 3 biological replicates. Error bars indicate SE. Asterisks indicate a statistical difference based on the Student's t-test *P ≤ 0.05; **P ≤ 0.01; **P ≤ 0.001 for differences between NP in comparison to NP+187, P and P+187 treatments.(f) Relative enrichment of H3K4me3 levels at APX2 and HSP18.2 memory genes in hsfa2 mutant. Data information: For H3K4me3 enrichment analysis, amplification values were normalized to input and H3 and region 1 of non-primed (NP) plants. All treatments are compared with direct 44°C HS treatment for statistical significance. All plots represent the means of 3 biological replicates. Error bars indicate SE. Asterisks indicate a statistical difference based on the Student's t-test *P ≤ 0.05; **P ≤ 0.01; **P ≤ 0.001 for differences between NP in comparison to NP+187, P and P+187 treatments.


Figure 6 ( b ) Relative percent abundance of bacterial phyla in the soil and the wheat root endosphere without ( R - mock ) and with SA187 treatment ( R - 187 ) . Data information : Microbiome analysis was performed in triplicates .

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Figure 6(b) Relative percent abundance of bacterial phyla in the soil and the wheat root endosphere without (R-mock) and with SA187 treatment (R-187). Data information: Microbiome analysis was performed in triplicates.


Figure 1B . Western blot analysis of the dose - dependent expression of ZBP1 in HT29 / TO - ZBP1WT and HT29 / TO - ZBP1Zα1α2mut cells treated with indicated concentrations of Dox for 24 h . Blots are representative of three biological repeats . I . Western blot analysis of cells from the same wells as used in ( H ) for ZBP1 expression levels , representative of four biological replicates . L . Transwell migration of primary neutrophils towards conditioned media from HT29 / TO - ZBP1WT cells treated with DMSO or 500 ng / mL Dox for 24 hours , or HT29 chemotaxis buffer ( control buffer ) containing equal volume and amount of DMSO or Dox as the conditioned media . Data is presented as individual values with grand mean of the migrated percentage induced by control buffer containing DMSO or Dox ( n = 10 biological replicates ) , or conditioned media from DMSO - treated cells ( n = 11 biological replicates ) or Dox - treated cells ( n = 12 biological replicates ) , where the number of biological replicates is defined by the total number of independent cell cultures tested on primary neutrophils from four donors . An unpaired t - test was used to test for statistical differences between indicated conditions . * * * * p < 0 . 0001 . M . Transwell migration of primary neutrophils towards conditioned media from HT29 / TO - ZBP1WT cells treated with 0 or 500 ng / mL Dox in combination with DMSO or 20 µM zVAD + 10 μM Nec1s for 24 h . Data is presented as individual values with mean and S . E . M . ( n = 3 biological replicates of conditioned media ) . One - way ANOVA and Sidak ' s multiple comparisons test were used to test for statistical differences between indicated conditions . ns = not significant ( p = 0 . 4129 ) ; * * * p = 0 . 0004 .

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Figure 1B. Western blot analysis of the dose-dependent expression of ZBP1 in HT29 / TO-ZBP1WT and HT29 / TO-ZBP1Zα1α2mut cells treated with indicated concentrations of Dox for 24 h. Blots are representative of three biological repeats.I. Western blot analysis of cells from the same wells as used in (H) for ZBP1 expression levels, representative of four biological replicates.L. Transwell migration of primary neutrophils towards conditioned media from HT29 / TO-ZBP1WT cells treated with DMSO or 500 ng/mL Dox for 24 hours, or HT29 chemotaxis buffer (control buffer) containing equal volume and amount of DMSO or Dox as the conditioned media. Data is presented as individual values with grand mean of the migrated percentage induced by control buffer containing DMSO or Dox (n = 10 biological replicates), or conditioned media from DMSO-treated cells (n = 11 biological replicates) or Dox-treated cells (n = 12 biological replicates), where the number of biological replicates is defined by the total number of independent cell cultures tested on primary neutrophils from four donors. An unpaired t-test was used to test for statistical differences between indicated conditions. **** p < 0.0001.M. Transwell migration of primary neutrophils towards conditioned media from HT29 / TO-ZBP1WT cells treated with 0 or 500 ng/mL Dox in combination with DMSO or 20 µM zVAD + 10 μM Nec1s for 24 h. Data is presented as individual values with mean and S.E.M. (n = 3 biological replicates of conditioned media). One-way ANOVA and Sidak's multiple comparisons test were used to test for statistical differences between indicated conditions. ns = not significant (p = 0.4129); *** p = 0.0004.


Figure 2C . Transwell migration of primary neutrophils towards conditioned media from HT29 / TO - ZBP1WT cells treated with 0 or 500 ng / mL Dox in combination with indicated inhibitors for 24 h . Data is presented as individual values with grand mean ( n = 3 biological replicates of conditioned media from GSK ' 872 and NSA - treated wells , n = 6 biological replicates of conditioned media from other conditions ) . Two - way ANOVA and Sidak ' s multiple comparisons test were used to test for statistical differences between indicated conditions . * * * * p < 0 . 0001 ; ns = not significant ( p > 0 . 96 ) . D . CXCL8 concentration in the culture media of HT29 / TO - ZBP1WT cells transfected with siRNA targeting mismatch sequence ( MM ) or RIPK3 and treated with Dox at indicated concentrations for 24 h . Data is presented as mean with S . E . M . ( n = 4 biological replicates ) . Unpaired t - tests were used to test for statistical differences between indicated conditions . * * p = 0 . 0079 . Cell lysates from one biological replicate were analyzed by Western blotting . E . Cytokine concentration in the culture media of HT29 / TO - ZBP1WT or HT29 / RIPK1 - KO clone aA3 / TO - ZBP1WT cells treated with 0 , 50 or 500 ng / mL Dox . data is plotted as mean with S . E . M . ( n = 6 biological replicates ) . Brown - Forsythe and Welch ANOVA tests and Dunnet ' s T3 multiple comparisons test were used to test for statistical differences between indicated conditions . Cell lysates were analyzed by Western blotting for ZBP1 levels . Blots are representative of six biological replicates . ns = not significant ( p > 0 . 2 ) ; * * * p = 0 . 0003 for CXCL8 , p = 0 . 0008 for CXCL1 ; * * * * p < 0 . 0001 . G , NF - κB activity in HEK293FT cells with and without stable expression of full - length ( FL ) RIPK3 - 2xFV or RIPK3 ∆ C - 2xFV ( ∆ C ) transfected with dual luciferase reporters and ZBP1 or empty vector ( EV ) as indicated . Reporter activities were measured 24 h after transfection . Data is presented as mean with S . E . M ( n = 3 biological replicates ) . One - way ANOVA and Sidak ' s multiple comparisons test were used to test for statistical differences between indicated conditions . ns = not significant ( p > 0 . 05 ) ; * * * * p < 0 . 0001 . ( G ) Cell lysates were analyzed by Western blotting . Blots are representative of three biological replicates . Asterisk indicates background signals of the antibody .

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Figure 2C. Transwell migration of primary neutrophils towards conditioned media from HT29 / TO-ZBP1WT cells treated with 0 or 500 ng/mL Dox in combination with indicated inhibitors for 24 h. Data is presented as individual values with grand mean (n = 3 biological replicates of conditioned media from GSK'872 and NSA-treated wells, n = 6 biological replicates of conditioned media from other conditions). Two-way ANOVA and Sidak's multiple comparisons test were used to test for statistical differences between indicated conditions. **** p < 0.0001; ns = not significant (p > 0.96).D. CXCL8 concentration in the culture media of HT29 / TO-ZBP1WT cells transfected with siRNA targeting mismatch sequence (MM) or RIPK3 and treated with Dox at indicated concentrations for 24 h. Data is presented as mean with S.E.M. (n = 4 biological replicates). Unpaired t-tests were used to test for statistical differences between indicated conditions. ** p = 0.0079. Cell lysates from one biological replicate were analyzed by Western blotting.E. Cytokine concentration in the culture media of HT29 / TO-ZBP1WT or HT29 / RIPK1-KO clone aA3 / TO-ZBP1WT cells treated with 0, 50 or 500 ng/mL Dox. data is plotted as mean with S.E.M. (n = 6 biological replicates). Brown-Forsythe and Welch ANOVA tests and Dunnet's T3 multiple comparisons test were used to test for statistical differences between indicated conditions. Cell lysates were analyzed by Western blotting for ZBP1 levels. Blots are representative of six biological replicates. ns = not significant (p > 0.2); *** p = 0.0003 for CXCL8, p = 0.0008 for CXCL1; **** p < 0.0001.G, NF-κB activity in HEK293FT cells with and without stable expression of full-length (FL) RIPK3-2xFV or RIPK3∆C-2xFV (∆C) transfected with dual luciferase reporters and ZBP1 or empty vector (EV) as indicated. Reporter activities were measured 24 h after transfection. Data is presented as mean with S.E.M (n = 3 biological replicates). One-way ANOVA and Sidak's multiple comparisons test were used to test for statistical differences between indicated conditions. ns = not significant (p > 0.05); **** p<0.0001. (G) Cell lysates were analyzed by Western blotting. Blots are representative of three biological replicates. Asterisk indicates background signals of the antibody.


Figure 3A . Western blot analysis of anti - FLAG ( ZBP1 ) immunoprecipitation from HT29 / TO - ZBP1WT cells treated with 0 or 500 ng / mL Dox for 16 h . Blots are representative of three biological replicates . Immunoprecipitated material was treated or not with USP21 ( 1 μM ) for deubiquitination . Input loaded was 5 % for ZBP1 and 1 % for co - immunoprecipitants . Asterisk indicates antibody heavy chain signal in IP samples . B . Enrichment of Ub - conjugates by GST - 1xUBA for analysis of ubiquitination status of RIPK1 , ZBP1 and RIPK3 in HT29 / TO - ZBP1WT cells treated with 0 or 500 ng / mL Dox for 16h . Blots are representative of at least three biological replicates . After enrichment , samples were treated or not with USP21 ( 1 μM ) for deubiquitination . Asterisk indicates unspecific bands . Arrows indicate RIPK3 signal . C . Enrichment of Ub - conjugates for analysis of the ubiquitination status of RIPK1 and ZBP1 using GST - 1xUBA or linkage - specific SUBs in HT29 / TO - ZBP1WT cells treated with 0 or 500 ng / mL Dox for 16 h . D . Enrichment of Ub - conjugates by GST - 1xUBA for analysis of ubiquitination status of RIPK1 and ZBP1 in HT29 , HT29 / TO - ZBP1WT and HT29 / RIPK1 - KO aA3 / TO - ZBP1WT cells treated with 500 ng / mL Dox for 16 h . Blots are representative of four biological replicates . Asterisk indicates antibody background signal . E . NF - κB activity in HEK293FT / RIPK3 - 2xFV cells transfected with dual luciferase reporters , ZBP1 or EV , and GFP ( Ctrl ) or GFP - tagged SUBs as indicated . Luciferase reporter activities were measured 24 h after transfection and normalized to GFP + EV - transfected condition . Data is plotted as mean with S . E . M . ( n = 4 biological replicates ) . One - way ANOVA and Sidak ' s multiple comparisons tests were used to test for statistical differences between the indicated condition and ZBP1 / GFP - transfected condition . * * * * p < 0 . 0001 . Cell lysates were analyzed by Western blotting to determine expression of ZBP1 , GFP or GFP - tagged SUBs and Actin . Blots are representative of three biological replicates . F , NF - κB activity in HEK293FT / RIPK3 - 2xFV cells transfected with dual luciferase reporters , ZBP1 or EV , and variants of OTULIN . Luciferase reporter activities were measured 24 h after transfection and normalized to ZBP1 / EV - transfected cells . Data is plotted as mean with S . E . M . Multiple Welch t - tests were used to test for statistical differences between the indicated condition and the ZBP1 / EV - transfected condition . ( F ) n = 4 biological replicates . * * p = 0 . 0012 , ns = not significant ( p = 0 . 2342 ) . Cell lysates were analyzed by Western blotting to determine expression of ZBP1 , or OTULIN , and Actin . Blots are representative of ( F ) four biological replicates . G . NF - κB activity in HEK293FT / RIPK3 - 2xFV cells transfected with dual luciferase reporters , ZBP1 or EV , and variants of CYLD Luciferase reporter activities were measured 24 h after transfection and normalized to ZBP1 / EV - transfected cells . Data is plotted as mean with S . E . M . Multiple Welch t - tests were used to test for statistical differences between the indicated condition and the ZBP1 / EV - transfected condition . ( G ) n = 3 biological replicates . * p = 0 . 0137 ; ns = not significant ( p = 0 . 8374 ) . Cell lysates were analyzed by Western blotting to determine expression of ZBP1 , CYLD and Actin . Blots are representative of ( G ) three biological replicates .

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Figure 3A. Western blot analysis of anti-FLAG (ZBP1) immunoprecipitation from HT29 / TO-ZBP1WT cells treated with 0 or 500 ng/mL Dox for 16 h. Blots are representative of three biological replicates. Immunoprecipitated material was treated or not with USP21 (1 μM) for deubiquitination. Input loaded was 5% for ZBP1 and 1% for co-immunoprecipitants. Asterisk indicates antibody heavy chain signal in IP samples.B. Enrichment of Ub-conjugates by GST-1xUBA for analysis of ubiquitination status of RIPK1, ZBP1 and RIPK3 in HT29 / TO-ZBP1WT cells treated with 0 or 500 ng/mL Dox for 16h. Blots are representative of at least three biological replicates. After enrichment, samples were treated or not with USP21 (1 μM) for deubiquitination. Asterisk indicates unspecific bands. Arrows indicate RIPK3 signal.C. Enrichment of Ub-conjugates for analysis of the ubiquitination status of RIPK1 and ZBP1 using GST-1xUBA or linkage-specific SUBs in HT29 / TO-ZBP1WT cells treated with 0 or 500 ng/mL Dox for 16 h.D. Enrichment of Ub-conjugates by GST-1xUBA for analysis of ubiquitination status of RIPK1 and ZBP1 in HT29, HT29 / TO-ZBP1WT and HT29 / RIPK1-KO aA3 / TO-ZBP1WT cells treated with 500 ng/mL Dox for 16 h. Blots are representative of four biological replicates. Asterisk indicates antibody background signal.E. NF-κB activity in HEK293FT / RIPK3-2xFV cells transfected with dual luciferase reporters, ZBP1 or EV, and GFP (Ctrl) or GFP-tagged SUBs as indicated. Luciferase reporter activities were measured 24 h after transfection and normalized to GFP+EV-transfected condition. Data is plotted as mean with S.E.M. (n = 4 biological replicates). One-way ANOVA and Sidak's multiple comparisons tests were used to test for statistical differences between the indicated condition and ZBP1/GFP-transfected condition. **** p < 0.0001. Cell lysates were analyzed by Western blotting to determine expression of ZBP1, GFP or GFP-tagged SUBs and Actin. Blots are representative of three biological replicates.F, NF-κB activity in HEK293FT / RIPK3-2xFV cells transfected with dual luciferase reporters, ZBP1 or EV, and variants of OTULIN. Luciferase reporter activities were measured 24 h after transfection and normalized to ZBP1/EV-transfected cells. Data is plotted as mean with S.E.M. Multiple Welch t-tests were used to test for statistical differences between the indicated condition and the ZBP1/EV-transfected condition. (F) n = 4 biological replicates. ** p = 0.0012, ns = not significant (p = 0.2342). Cell lysates were analyzed by Western blotting to determine expression of ZBP1, or OTULIN, and Actin. Blots are representative of (F) four biological replicates.G. NF-κB activity in HEK293FT / RIPK3-2xFV cells transfected with dual luciferase reporters, ZBP1 or EV, and variants of CYLD Luciferase reporter activities were measured 24 h after transfection and normalized to ZBP1/EV-transfected cells. Data is plotted as mean with S.E.M. Multiple Welch t-tests were used to test for statistical differences between the indicated condition and the ZBP1/EV-transfected condition. (G) n = 3 biological replicates. * p = 0.0137; ns = not significant (p = 0.8374). Cell lysates were analyzed by Western blotting to determine expression of ZBP1, CYLD and Actin. Blots are representative of (G) three biological replicates.


Figure 4A . NF - κB activity in HEK293FT / RIPK3 - 2xFV cells transfected with siRNA targeting HOIP ( siHOIP ) or a mismatch sequence ( siMM ) , and 48 h later transfected with dual luciferase reporters and ZBP1 or GFP ( control ) . Luciferase reporter activities were measured 24 h after transfection and normalized to the siMM + ZBP1 samples . Data is plotted as mean with S . E . M . ( n = 4 biological replicates ) . A Welch ' s t - test was used to test for the statistical difference between indicated conditions . * * * * p < 0 . 0001 . HOIP knockdown and ZBP1 expression levels were analyzed by Western blotting . Blots are representative of three biological replicates . D - F . Western blot analysis of anti - FLAG ( ZBP1 ) immunoprecipitation from HT29 or HT29 / TO - ZBP1WT or HT29 / RIPK1 - KO clone aA3 / TO - ZBP1WT cells treated with 0 or 500 ng / mL Dox for 16 h . Asterisk indicates antibody heavy chain signal in IP samples . Blots are representative of at least ( D ) two and ( E - F ) three biological replicates .

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Figure 4A. NF-κB activity in HEK293FT / RIPK3-2xFV cells transfected with siRNA targeting HOIP (siHOIP) or a mismatch sequence (siMM), and 48 h later transfected with dual luciferase reporters and ZBP1 or GFP (control). Luciferase reporter activities were measured 24 h after transfection and normalized to the siMM+ZBP1 samples. Data is plotted as mean with S.E.M. (n = 4 biological replicates). A Welch's t-test was used to test for the statistical difference between indicated conditions. **** p < 0.0001. HOIP knockdown and ZBP1 expression levels were analyzed by Western blotting. Blots are representative of three biological replicates.D-F. Western blot analysis of anti-FLAG (ZBP1) immunoprecipitation from HT29 or HT29 / TO-ZBP1WT or HT29 / RIPK1-KO clone aA3 / TO-ZBP1WT cells treated with 0 or 500 ng/mL Dox for 16 h. Asterisk indicates antibody heavy chain signal in IP samples. Blots are representative of at least (D) two and (E-F) three biological replicates.