( A − I ) Induction of GOMED in Atg5 KO MEFs by CBM treatment . ( C - F ) Involvement of Golgi membranes in the generation of AL - like structures . ( D , E ) Requirement of Grasp65 in the generation of AL - like structures . Atg5 KO and Grasp65 - silenced Atg5 KO MEFs were treated without ( NT ) or with CBM ( 2 mM ) for the indicated times followed by immunostaining with an anti - Lamp2 antibody . Representative images ( at 24 h ) are shown in ( D ) . Scale bars = 5 µm . A magnified image of the dashed square is shown in the inset . Scale bars = 1 µm . CBM induced large ring - like Lamp2 fluorescence . ( E ) Percentages of cells with large ring - like Lamp2 immunofluorescence ( mean ± s . e . m . , n = 4 ) . * p < 0 . 01 vs the value of Atg5 KO MEF .

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

(A−I) Induction of GOMED in Atg5 KO MEFs by CBM treatment. (C-F) Involvement of Golgi membranes in the generation of AL-like structures. (D, E) Requirement of Grasp65 in the generation of AL-like structures. Atg5 KO and Grasp65-silenced Atg5 KO MEFs were treated without (NT) or with CBM (2 mM) for the indicated times followed by immunostaining with an anti-Lamp2 antibody. Representative images (at 24 h) are shown in (D). Scale bars = 5 µm. A magnified image of the dashed square is shown in the inset. Scale bars = 1 µm. CBM induced large ring-like Lamp2 fluorescence. (E) Percentages of cells with large ring-like Lamp2 immunofluorescence (mean ± s.e.m., n = 4). *p < 0.01 vs the value of Atg5 KO MEF.


( A − I ) Induction of GOMED in Atg5 KO MEFs by CBM treatment . ( C - F ) Involvement of Golgi membranes in the generation of AL - like structures . ( F ) Colocalization of Lamp2 and GFP - syntaxin 6 in CBM - treated Atg5 KO MEFs . GFP - syntaxin 6 - expressing Atg5 KO MEFs were treated with ( CBM ) or without ( NT ) CBM ( 2 mM ) for 6 hr and immunostained with an anti - Lamp2 antibody . The ring - like Lamp2 fluorescence merged with the signal for syntaxin 6 . Scale bar = 5 µm . A magnified image of the dashed square is shown in the inset . Scale bars = 2 µm .

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

(A−I) Induction of GOMED in Atg5 KO MEFs by CBM treatment. (C-F) Involvement of Golgi membranes in the generation of AL-like structures. (F) Colocalization of Lamp2 and GFP-syntaxin 6 in CBM-treated Atg5 KO MEFs. GFP-syntaxin 6-expressing Atg5 KO MEFs were treated with (CBM) or without (NT) CBM (2 mM) for 6 hr and immunostained with an anti-Lamp2 antibody. The ring-like Lamp2 fluorescence merged with the signal for syntaxin 6. Scale bar = 5 µm. A magnified image of the dashed square is shown in the inset. Scale bars = 2 µm.


( G ) The monomeric red fluorescent protein ( mRFP ) - green fluorescent protein ( GFP ) tandem protein assay revealed the induction of GOMED . Atg5 KO MEFs stably expressing tandem mRFP - GFP were incubated without ( no treatment ) or with CBM ( 2 mM ) for 24 hr . Red signals indicate acidic compartments . Lysosomes were counterstained with an anti - Lamp2 antibody ( cyan ) . Scale bars = 5 µm . Regions of interest ( ROI ) are indicated by dashed squares . Arrowheads indicate GOMED structure . Scale bars = 2 µm .

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

(G) The monomeric red fluorescent protein (mRFP)-green fluorescent protein (GFP) tandem protein assay revealed the induction of GOMED. Atg5 KO MEFs stably expressing tandem mRFP-GFP were incubated without (no treatment) or with CBM (2 mM) for 24 hr. Red signals indicate acidic compartments. Lysosomes were counterstained with an anti-Lamp2 antibody (cyan). Scale bars = 5 µm. Regions of interest (ROI) are indicated by dashed squares. Arrowheads indicate GOMED structure. Scale bars = 2 µm.


( A − I ) Induction of GOMED in Atg5 KO MEFs by CBM treatment . ( H , I ) Degradation of GFP - fused proteins indicated the induction of GOMED . Atg5 KO MEFs stably expressing VSVG - GFP ( H ) or M6PR - GFP ( I ) were incubated with CBM ( 5 mM ) for the indicated times . Cell lysates were subjected to immunoblotting with the anti - GFP antibody .

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

(A−I) Induction of GOMED in Atg5 KO MEFs by CBM treatment. (H, I) Degradation of GFP-fused proteins indicated the induction of GOMED. Atg5 KO MEFs stably expressing VSVG-GFP (H) or M6PR-GFP (I) were incubated with CBM (5 mM) for the indicated times. Cell lysates were subjected to immunoblotting with the anti-GFP antibody.


( J − M ) Genetic induction of GOMED in Atg5 KO MEFs . Atg5 KO MEFs were treated with the indicated small interfering ( si ) RNAs for 24 hr and GOMED was assessed by EM ( J - L ) . Representative images are shown in ( J , K ) . Arrows indicate AL - like structures . Scale bars = 2 µm . ( L ) The number of AP - like and AL - like structures was calculated from the EM images of cells treated with the indicated siRNAs . Error bars indicate s . e . m . ( siControl : n = 23 , siPI4k2α + siPI4k3β : n = 20 , siArfaptin1 : n = 20 ) . * p < 0 . 01 vs the value of siControl .

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

(J−M) Genetic induction of GOMED in Atg5 KO MEFs. Atg5 KO MEFs were treated with the indicated small interfering (si)RNAs for 24 hr and GOMED was assessed by EM (J-L). Representative images are shown in (J, K). Arrows indicate AL-like structures. Scale bars = 2 µm. (L) The number of AP-like and AL-like structures was calculated from the EM images of cells treated with the indicated siRNAs. Error bars indicate s.e.m. (siControl: n = 23, siPI4k2α + siPI4k3β: n = 20, siArfaptin1: n = 20). *p < 0.01 vs the value of siControl.


( J − M ) Genetic induction of GOMED in Atg5 KO MEFs . Atg5 KO MEFs were treated with the indicated small interfering ( si ) RNAs for 24 hr and GOMED was assessed by the mRFP - GFP tandem protein assay ( M ) . ( M ) The same experiments as in ( G ) were performed using cells treated with the indicated siRNAs . Representative images are shown . Arrowheads indicate GOMED structure . Scale bars = 5 µm .

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

(J−M) Genetic induction of GOMED in Atg5 KO MEFs. Atg5 KO MEFs were treated with the indicated small interfering (si)RNAs for 24 hr and GOMED was assessed by the mRFP-GFP tandem protein assay (M). (M) The same experiments as in (G) were performed using cells treated with the indicated siRNAs. Representative images are shown. Arrowheads indicate GOMED structure. Scale bars = 5 µm.


( A ) Suppression of insulin secretion in Atg5 knockout ( KO ) MIN6 cells after glucose deprivation . Atg5 KO MIN6 cells were incubated with normal culture medium ( NT ) or glucose - depleted medium for 1 hr . The amount of secreted insulin is expressed as the percentage of that of the untreated control ( NT ) ( mean ± s . e . m , n = 3 ) . * p < 0 . 01 vs the value of NT .

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

(A) Suppression of insulin secretion in Atg5 knockout (KO) MIN6 cells after glucose deprivation. Atg5 KO MIN6 cells were incubated with normal culture medium (NT) or glucose-depleted medium for 1 hr. The amount of secreted insulin is expressed as the percentage of that of the untreated control (NT) (mean ± s.e.m, n = 3). *p < 0.01 vs the value of NT.


( B , C ) EM analysis revealed the induction of GOMED structures . Atg5 KO MIN6 cells were subjected to glucose deprivation for 1 hr . Representative images are shown . The white arrow and black arrows indicate the AP - like structure and insulinvesicles , respectively . Scale bars = 0 . 2 µm . ( D ) The number of AP - like and AL - like structures was calculated from the EM images of cells left untreated ( NT ) or subjected to glucose deprivation . Error bars indicate s . e . m . ( NT : n = 17 , glucose deprivation : n = 84 ) . * p < 0 . 01 and # p < 0 . 05 vs the value of NT .

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

(B, C) EM analysis revealed the induction of GOMED structures. Atg5 KO MIN6 cells were subjected to glucose deprivation for 1 hr. Representative images are shown. The white arrow and black arrows indicate the AP-like structure and insulinvesicles, respectively. Scale bars = 0.2 µm. (D) The number of AP-like and AL-like structures was calculated from the EM images of cells left untreated (NT) or subjected to glucose deprivation. Error bars indicate s.e.m. (NT: n = 17, glucose deprivation: n = 84). *p < 0.01 and #p < 0.05 vs the value of NT.


( E ) Involvement of Golgi membrane in the generation of GOMED . Lamp1 - GFP / mCherry - Syntaxin 6 - expressing Atg5 KO MIN6 cells were subjected to glucose deprivation , and analysed by TEM and confocal microscopy . CLEM analysis revealed that AL - like structures ( arrows ) merged with Lamp1 - GFP fluorescence and Syntaxin 6 fluorescence . Scale bars = 5 µm . A magnified image of the dashed square is shown in the inset . Scale bars = 2 µm .

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

(E) Involvement of Golgi membrane in the generation of GOMED. Lamp1-GFP/mCherry-Syntaxin 6-expressing Atg5 KO MIN6 cells were subjected to glucose deprivation, and analysed by TEM and confocal microscopy. CLEM analysis revealed that AL-like structures (arrows) merged with Lamp1-GFP fluorescence and Syntaxin 6 fluorescence. Scale bars = 5 µm. A magnified image of the dashed square is shown in the inset. Scale bars = 2 µm.


( F ) GOMED - mediated degradation of ( pro ) insulin by glucose deprivation of Atg5 KO MIN6 cells . Atg5 KO MIN6 cells were incubated in glucose - depleted medium in the absence or presence of bafilomycin A1 ( 10 nM ) for the indicated times . The cell lysates were then subjected to immunoblot analysis using an anti - ( pro ) insulin antibody .

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

(F) GOMED-mediated degradation of (pro)insulin by glucose deprivation of Atg5 KO MIN6 cells. Atg5 KO MIN6 cells were incubated in glucose-depleted medium in the absence or presence of bafilomycin A1 (10 nM) for the indicated times. The cell lysates were then subjected to immunoblot analysis using an anti-(pro)insulin antibody.


( G ) Immunofluorescence analysis of ( pro ) insulin and Lamp2 . Atg5 KO MIN6 cells were left untreated ( NT ) or subjected to glucose deprivation for 1 hr in the presence of E64d ( 25 µM ) or bafilomycin A1 ( 10 nM ) , and were stained with anti - ( pro ) insulin and anti - Lamp2 ( lysosomal marker ) antibodies and observed by fluorescence microscopy . Scale bar = 1 µm . Arrowheads indicate the colocalization of ( pro ) insulin with GOMED structures .

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

(G) Immunofluorescence analysis of (pro)insulin and Lamp2. Atg5 KO MIN6 cells were left untreated (NT) or subjected to glucose deprivation for 1 hr in the presence of E64d (25 µM) or bafilomycin A1 (10 nM), and were stained with anti-(pro)insulin and anti-Lamp2 (lysosomal marker) antibodies and observed by fluorescence microscopy. Scale bar = 1 µm. Arrowheads indicate the colocalization of (pro)insulin with GOMED structures.


( A − D ) EM analysis demonstrated the induction of GOMED . Primary Atg7 KO β - cells were subjected to glucose deprivation for 1 hr . A representative AP - like structure containing insulingranule is shown in ( A ) . An AL - like structure containing insulingranules is shown in ( B ) . Scale bars = 0 . 2 µm .

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

(A−D) EM analysis demonstrated the induction of GOMED. Primary Atg7 KO β-cells were subjected to glucose deprivation for 1 hr. A representative AP-like structure containing insulingranule is shown in (A). An AL-like structure containing insulingranules is shown in (B). Scale bars = 0.2 µm.


( A − D ) EM analysis demonstrated the induction of GOMED . Primary Atg7 KO β - cells were subjected to glucose deprivation for 1 hr . ( C , D ) GOMED structures containing Golgi vesicles . An AP - like structure containing multiple Golgi vesicles ( arrows ) ( C ) and an AL - like structure fusing with a lysosome ( arrowhead ) ( D ) are shown . Scale bar = 0 . 5 µm . A magnified image of the dashed rectangle is shown in the inset . Scale bar = 0 . 2 µm . AP - like structure contained double - membrane .

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

(A−D) EM analysis demonstrated the induction of GOMED. Primary Atg7 KO β-cells were subjected to glucose deprivation for 1 hr. (C, D) GOMED structures containing Golgi vesicles. An AP-like structure containing multiple Golgi vesicles (arrows) (C) and an AL-like structure fusing with a lysosome (arrowhead) (D) are shown. Scale bar = 0.5 µm. A magnified image of the dashed rectangle is shown in the inset. Scale bar = 0.2 µm. AP-like structure contained double-membrane.


( E ) The total number of AL - like structure was calculated from the EM images of WT and Atg7 KO β - cells after glucose deprivation . Error bars indicate s . e . m . ( n = 46 − 163 ) . * p < 0 . 01 and # p < 0 . 05 vs the value of 0 min .

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

(E) The total number of AL-like structure was calculated from the EM images of WT and Atg7 KO β-cells after glucose deprivation. Error bars indicate s.e.m. (n = 46−163). *p < 0.01 and #p < 0.05 vs the value of 0 min.


( F ) The total number of GOMED structure was calculated from the EM images of Atg7 KO β - cells after glucose deprivation in the absence or presence of bafilomycin A1 ( 10 nM ) . Error bars indicate s . e . m . ( n = 109 − 116 ) . * p < 0 . 01 vs the value of 60 min .

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

(F) The total number of GOMED structure was calculated from the EM images of Atg7 KO β-cells after glucose deprivation in the absence or presence of bafilomycin A1 (10 nM). Error bars indicate s.e.m. (n = 109−116). *p < 0.01 vs the value of 60 min.


( G − J ) EM analysis showed the induction of mitophagy . WT and Atg7 KO β - cells were subjected to glucose deprivation for 1 hr . Representative images showing mitophagy in Atg7 KO β - cells are shown in ( G ) . Arrowhead ( G ) indicates mitophagy . Scale bars = 0 . 5 µm . ( H , J ) The number of structures representative of mitophagy ( H ) per cell was calculated from the EM images of β - cells after glucose depletion . Error bars indicate s . e . m . ( n = 46 − 163 ) . * p < 0 . 01 and # p < 0 . 05 vs the value of 0 min .

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

(G−J) EM analysis showed the induction of mitophagy. WT and Atg7 KO β-cells were subjected to glucose deprivation for 1 hr. Representative images showing mitophagy in Atg7 KO β-cells are shown in (G). Arrowhead (G) indicates mitophagy. Scale bars = 0.5 µm. (H, J) The number of structures representative of mitophagy (H) per cell was calculated from the EM images of β-cells after glucose depletion. Error bars indicate s.e.m. (n = 46−163). *p < 0.01 and #p < 0.05 vs the value of 0 min.


( G − J ) EM analysis showed the induction of crinophagy / SINGD . WT and Atg7 KO β - cells were subjected to glucose deprivation for 1 hr . Representative images showing crinophagy / SINGD in WT β - cells are shown in ( I ) . White arrow ( I ) indicates crinophagy / SINGD . Arrows indicate insulingranules ( I ) . Scale bars = 0 . 5 µm . ( H , J ) The number of structures representative of crinophagy / SINGD ( J ) per cell was calculated from the EM images of β - cells after glucose depletion . Error bars indicate s . e . m . ( n = 46 − 163 ) . * p < 0 . 01 and # p < 0 . 05 vs the value of 0 min .

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

(G−J) EM analysis showed the induction of crinophagy/SINGD. WT and Atg7 KO β-cells were subjected to glucose deprivation for 1 hr. Representative images showing crinophagy/SINGD in WT β-cells are shown in (I). White arrow (I) indicates crinophagy/SINGD. Arrows indicate insulingranules (I). Scale bars = 0.5 µm. (H, J) The number of structures representative of crinophagy/SINGD (J) per cell was calculated from the EM images of β-cells after glucose depletion. Error bars indicate s.e.m. (n = 46−163). *p < 0.01 and #p < 0.05 vs the value of 0 min.


( K ) The number of ( pro ) insulingranule - containing AL - like structure was calculated from the EM images of wild - type ( WT ) and Atg7 KO β - cells after glucose deprivation . Error bars indicate s . e . m . ( n = 46 − 163 ) . * p < 0 . 01 and # p < 0 . 05 vs the value of 0 min .

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

(K) The number of (pro)insulingranule-containing AL-like structure was calculated from the EM images of wild-type (WT) and Atg7 KO β-cells after glucose deprivation. Error bars indicate s.e.m. (n = 46−163). *p < 0.01 and #p < 0.05 vs the value of 0 min.


A . Confocal images show Iba1 - immunoreactive microglia and macrophages in RPE / choroid whole mount preparations 48 hours after laser coagulation and intravitreal application of low ( 0 . 2 µg ) and high ( 3 µg ) dose polySia avDP20 or PBS - vehicle . Laser spots of vehicle - injected SIGLEC11 transgenic ( tg ) and wild type control animals revealed strong accumulation of activated microglia and macrophages , which was effectively decreased in polySia avDP20 - injected SIGLEC11 tg animals and to a lower extent in wild type controls . Representative images out of at least three independent experiments are shown . Scale bar : 50µm . B . Quantification of the average pixel intensity of Iba1 - positive area in laser spots reflects accumulation of reactive microglia / macrophages on RPE / choroid whole mounts . In comparison to the accumulation of reactive microglia / macrophages in control animals , there is a reduced pixel intensity found in polySia avDP20 - treated SIGLEC11 tg animals . Notably , treatment with a high polySia avDP20 dose ( 3 µg ) also reduced accumulation of microglia / macrophages in laser spots of wild type animals . Data show mean + / - SD . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , One - way ANOVA followed by Fisher ' s LSD . WT PBS ( n = 24 spots ) , WT 0 . 2 µg Sia ( n = 37 spots ) , WT 3 µg Sia ( n = 20 spots ) , SIGLEC11 tg PBS ( n = 25 spots ) , SIGLEC11 tg 0 . 2 µg Sia ( n = 35 spots ) , SIGLEC11 tg 3 µg Sia ( n = 31 spots ) , WT PBS vs WT 3 µg Sia p = 0 . 0147 , WT 0 . 2 µg Sia vs SIGLEC11 tg 0 . 2 µg Sia p = 0 . 0046 , SIGLEC11 tg PBS vs SIGLEC11 tg 0 . 2 µg Sia p = 0 . 0071 , SIGLEC11 tg PBS vs SIGLEC11 tg 3 µg Sia p < 0 . 0001 .

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

A. Confocal images show Iba1-immunoreactive microglia and macrophages in RPE/choroid whole mount preparations 48 hours after laser coagulation and intravitreal application of low (0.2 µg) and high (3 µg) dose polySia avDP20 or PBS-vehicle. Laser spots of vehicle-injected SIGLEC11 transgenic (tg) and wild type control animals revealed strong accumulation of activated microglia and macrophages, which was effectively decreased in polySia avDP20-injected SIGLEC11 tg animals and to a lower extent in wild type controls. Representative images out of at least three independent experiments are shown. Scale bar: 50µm.B. Quantification of the average pixel intensity of Iba1-positive area in laser spots reflects accumulation of reactive microglia/macrophages on RPE/choroid whole mounts. In comparison to the accumulation of reactive microglia/macrophages in control animals, there is a reduced pixel intensity found in polySia avDP20-treated SIGLEC11 tg animals. Notably, treatment with a high polySia avDP20 dose (3 µg) also reduced accumulation of microglia/macrophages in laser spots of wild type animals. Data show mean +/- SD. * p< 0.05, ** p< 0.01, *** p< 0.001, One-way ANOVA followed by Fisher's LSD. WT PBS (n = 24 spots), WT 0.2 µg Sia (n = 37 spots), WT 3 µg Sia (n = 20 spots), SIGLEC11 tg PBS (n = 25 spots), SIGLEC11 tg 0.2 µg Sia (n = 35 spots), SIGLEC11 tg 3 µg Sia (n = 31 spots), WT PBS vs WT 3 µg Sia p = 0.0147, WT 0.2 µg Sia vs SIGLEC11 tg 0.2 µg Sia p = 0.0046, SIGLEC11 tg PBS vs SIGLEC11 tg 0.2 µg Sia p = 0.0071, SIGLEC11 tg PBS vs SIGLEC11 tg 3 µg Sia p < 0.0001.


C . Confocal images of retinal whole mounts show Iba1 - immunoreactive microglial cells . Retinal microglia of polySia avDP20 - treated SIGLEC11 tg mice had a more ramified microglial morphology in the laser spot compared to PBS - vehicle treated mice . Interestingly , high polySia avDP20 dose ( 3 µg ) also exerted a weak therapeutic effect in wild type animals . Representative images out of at least three independent experiments are shown . Scale bar : 20 µm . D . Percentage of retina showing activated microglial cells within the laser spots was quantified . PolySia avDP20 reduced the percentage of laser spots with activated microglia in SIGLEC11 tg animals and at high dose ( 3 µg ) also in wild type controls . Data show mean + / - SEM . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , One - way ANOVA followed by Fisher ' s LSD . WT PBS ( n = 6 retinas ) , WT 0 . 2 µg Sia ( n = 8 retinas ) , WT 3 µg Sia ( n = 6 retinas ) , SIGLEC11 tg PBS ( n = 8 retinas ) , SIGLEC11 tg 0 . 2 µg Sia ( n = 7 retinas ) , SIGLEC11 tg 3 µg Sia ( n = 6 retinas ) , WT PBS vs WT 3 µg Sia p = 0 . 0052 , WT 0 . 2 µg Sia vs SIGLEC11 tg 0 . 2 µg Sia p = 0 . 0005 , SIGLEC11 tg PBS vs SIGLEC11 tg 0 . 2 µg Sia p = 0 . 0159 , SIGLEC11 tg PBS vs SIGLEC11 tg 3 µg Sia p = 0 . 001 .

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

C. Confocal images of retinal whole mounts show Iba1-immunoreactive microglial cells. Retinal microglia of polySia avDP20-treated SIGLEC11 tg mice had a more ramified microglial morphology in the laser spot compared to PBS-vehicle treated mice. Interestingly, high polySia avDP20 dose (3 µg) also exerted a weak therapeutic effect in wild type animals. Representative images out of at least three independent experiments are shown. Scale bar: 20 µm.D. Percentage of retina showing activated microglial cells within the laser spots was quantified. PolySia avDP20 reduced the percentage of laser spots with activated microglia in SIGLEC11 tg animals and at high dose (3 µg) also in wild type controls. Data show mean +/- SEM. * p< 0.05, ** p< 0.01, ***p<0.001, One-way ANOVA followed by Fisher's LSD. WT PBS (n = 6 retinas), WT 0.2 µg Sia (n = 8 retinas), WT 3 µg Sia (n = 6 retinas), SIGLEC11 tg PBS (n = 8 retinas), SIGLEC11 tg 0.2 µg Sia (n = 7 retinas), SIGLEC11 tg 3 µg Sia (n = 6 retinas),WT PBS vs WT 3 µg Sia p = 0.0052, WT 0.2 µg Sia vs SIGLEC11 tg 0.2 µg Sia p = 0.0005, SIGLEC11 tg PBS vs SIGLEC11 tg 0.2 µg Sia p = 0.0159, SIGLEC11 tg PBS vs SIGLEC11 tg 3 µg Sia p = 0.001.


A . Fundus fluorescein angiography was performed 48 hours after laser coagulation and intravitreal application of low ( 0 . 2 µg ) and high ( 3 µg ) dose polySia avDP20 or PBS - vehicle . Late stage ( 10 - 11 minutes after fluorescein injection ) fundus fluorescein angiography revealed that PBS - treated wild type controls and humanized SIGLEC11 mice showed normal levels of vessel leakage whereas polySia avDP20 - treated SIGLEC11 transgenic ( tg ) mice had lower levels of vessel leakage compared to PBS - injected wild type or SIGLEC11 tg mice . High polySia avDP20 dose reduced vascular leakage also in wild type animals . Representative images out of at least eight independent experiments are shown . B . Fundus fluorescein angiography pictures were exported from Heidelberg Eye Explorer Software and fluorescein leakage was quantified with ImageJ software ( NIH ) . Pixel intensities of 6 regions of interest per picture were quantified and background fluorescence was subtracted . PolySia avDP20 - treated SIGLEC11 tg animals showed reduced vascular leakage compared to PBS - injected wild type control and SIGLEC11 tg mice . A reduction in vascular leakage was also observed in high dose polySia avDP20 - injected wild type control mice . Data show mean + / - SD . * p < 0 . 05 , * * * p < 0 . 001 , One - way ANOVA followed by Fisher ' s LSD . WT PBS ( n = 20 eyes ) , WT 0 . 2 µg Sia ( n = 17 eyes ) , WT 3 µg Sia ( n = 9 eyes ) , SIGLEC11 tg PBS ( n = 16 eyes ) , SIGLEC11 tg 0 . 2 µg Sia ( n = 20 eyes ) , SIGLEC11 tg 3 µg Sia ( n = 15 eyes ) , WT PBS vs WT 3 µg Sia p = 0 . 0193 , WT 0 . 2 µg Sia vs SIGLEC11 tg 0 . 2 µg Sia p < 0 . 0001 , SIGLEC11 tg PBS vs SIGLEC11 tg 0 . 2 µg Sia p < 0 . 0001 , SIGLEC11 tg PBS vs SIGLEC11 tg 3 µg Sia p = 0 . 0002 .

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

A. Fundus fluorescein angiography was performed 48 hours after laser coagulation and intravitreal application of low (0.2 µg) and high (3 µg) dose polySia avDP20 or PBS-vehicle. Late stage (10-11 minutes after fluorescein injection) fundus fluorescein angiography revealed that PBS-treated wild type controls and humanized SIGLEC11 mice showed normal levels of vessel leakage whereas polySia avDP20-treated SIGLEC11 transgenic (tg) mice had lower levels of vessel leakage compared to PBS-injected wild type or SIGLEC11 tg mice. High polySia avDP20 dose reduced vascular leakage also in wild type animals. Representative images out of at least eight independent experiments are shown.B. Fundus fluorescein angiography pictures were exported from Heidelberg Eye Explorer Software and fluorescein leakage was quantified with ImageJ software (NIH). Pixel intensities of 6 regions of interest per picture were quantified and background fluorescence was subtracted. PolySia avDP20-treated SIGLEC11 tg animals showed reduced vascular leakage compared to PBS-injected wild type control and SIGLEC11 tg mice. A reduction in vascular leakage was also observed in high dose polySia avDP20-injected wild type control mice. Data show mean +/- SD. * p< 0.05, *** p< 0.001, One-way ANOVA followed by Fisher's LSD. WT PBS (n = 20 eyes), WT 0.2 µg Sia (n = 17 eyes), WT 3 µg Sia (n = 9 eyes), SIGLEC11 tg PBS (n = 16 eyes), SIGLEC11 tg 0.2 µg Sia (n = 20 eyes), SIGLEC11 tg 3 µg Sia (n = 15 eyes), WT PBS vs WT 3 µg Sia p = 0.0193, WT 0.2 µg Sia vs SIGLEC11 tg 0.2 µg Sia p < 0.0001, SIGLEC11 tg PBS vs SIGLEC11 tg 0.2 µg Sia p < 0.0001, SIGLEC11 tg PBS vs SIGLEC11 tg 3 µg Sia p = 0.0002.


C . Anti - C5b - 9 immunostaining of RPE / choroid whole mount preparations 48 hours after laser damage showed strong MAC deposition in the laser lesions of vehicle - injected controls . PolySia avDP20 treatment reduced MAC formation in a dose - dependent fashion independent of SIGLEC11 presence . Scale bar : 100 µm . D . Quantification of C5b - 9 fluorescence signal intensity in the laser lesions . In comparison to the high amount of MAC deposition in vehicle - injected controls , the pixel intensity is reduced in polySia avDP20 - treated eyes in a dose - dependent fashion and regardless of SIGLEC11 presence . Data show mean + / - SD . * * * p < 0 . 001 , One - way ANOVA followed by Fisher ' s LSD . WT PBS ( n = 12 laser spots ) , WT 0 . 2 µg Sia ( n = 12 laser spots ) , WT 3 µg Sia ( n = 7 laser spots ) , SIGLEC11 tg PBS ( n = 6 laser spots ) , SIGLEC11 tg 0 . 2 µg Sia ( n = 10 laser spots ) , SIGLEC11 tg 3 µg Sia ( n = 12 laser spots ) ; all statistical comparisons p < 0 . 0001 except SIGLEC11 tg PBS versus SIGLEC11 tg 0 . 2 µg Sia p = 0 . 0004 .

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

C. Anti-C5b-9 immunostaining of RPE/choroid whole mount preparations 48 hours after laser damage showed strong MAC deposition in the laser lesions of vehicle-injected controls. PolySia avDP20 treatment reduced MAC formation in a dose-dependent fashion independent of SIGLEC11 presence. Scale bar: 100 µm.D. Quantification of C5b-9 fluorescence signal intensity in the laser lesions. In comparison to the high amount of MAC deposition in vehicle-injected controls, the pixel intensity is reduced in polySia avDP20-treated eyes in a dose-dependent fashion and regardless of SIGLEC11 presence. Data show mean +/- SD. *** p< 0.001, One-way ANOVA followed by Fisher's LSD. WT PBS (n = 12 laser spots), WT 0.2 µg Sia (n = 12 laser spots), WT 3 µg Sia (n = 7 laser spots), SIGLEC11 tg PBS (n = 6 laser spots), SIGLEC11 tg 0.2 µg Sia (n = 10 laser spots), SIGLEC11 tg 3 µg Sia (n = 12 laser spots); all statistical comparisons p < 0.0001 except SIGLEC11 tg PBS versus SIGLEC11 tg 0.2 µg Sia p = 0.0004.


A . Analysis of relative TNFSF2 gene transcription in human control and SIGLEC11 / 16 knockout THP1 - macrophages . The levels of gene transcripts were reduced after 24 hours of co - treatment with LPS ( 1 µg / ml ) and concentrations of 0 . 15 µM and 1 . 5 µM of polySia avDP20 in the human wild type macrophages . No response to polySia avDP20 was detectable in the knockout line . Data show mean + / - SEM . * * p < 0 . 01 , ANOVA followed by Bonferroni . Statistical analysis was done in relation to the LPS control . WT : no treatment n = 7 and p < 0 . 0001 , PolySia avDP20 1 . 5 µM n = 4 and p = 0 . 0002 , LPS n = 7 , LPS / PolySia avDP20 0 . 15µM n = 3 and p = 0 . 009 , LPS / PolySia avDP20 1 . 5µM n = 5 and p = 0 . 002 . Siglec11 / 16 KO : no treatment n = 5 and p = 0 . 01 , PolySia avDP20 n = 4 and p = 0 . 122 , LPS n = 7 , LPS / PolySia avDP20 0 . 15µM n = 3 and p = 1 . 0 , LPS / PolySia avDP20 1 . 5µM n = 4 and p = 1 . 0 .

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

A. Analysis of relative TNFSF2 gene transcription in human control and SIGLEC11/16 knockout THP1-macrophages. The levels of gene transcripts were reduced after 24 hours of co-treatment with LPS (1 µg/ml) and concentrations of 0.15 µM and 1.5 µM of polySia avDP20 in the human wild type macrophages. No response to polySia avDP20 was detectable in the knockout line. Data show mean +/- SEM. ** p < 0.01, ANOVA followed by Bonferroni. Statistical analysis was done in relation to the LPS control. WT: no treatment n=7 and p< 0.0001, PolySia avDP20 1.5 µM n=4 and p=0.0002, LPS n=7, LPS/PolySia avDP20 0.15µM n=3 and p=0.009, LPS/PolySia avDP20 1.5µM n=5 and p=0.002. Siglec11/16 KO: no treatment n=5 and p=0.01, PolySia avDP20 n=4 and p=0.122, LPS n=7, LPS/PolySia avDP20 0.15µM n=3 and p=1.0, LPS/PolySia avDP20 1.5µM n=4 and p=1.0.


B . Analysis of relative TNFSF2 protein release in human control and SIGLEC11 / 16 knockout THP1 - macrophages . The released protein levels were reduced after 24 hours of co - treatment with LPS ( 1 µg / ml ) and concentrations of 0 . 15 µM and 1 . 5 µM of polySia avDP20 in the human wild type macrophages . No response to polySia avDP20 was detectable in the knockout line . Data show mean + / - SEM . * * p < 0 . 01 , * * * p < 0 . 001 , ANOVA followed by Bonferroni . Statistical analysis was done in relation to the LPS control . WT : no treatment n = 8 and p < 0 . 0001 , PolySia avDP20 1 . 5 µM n = 5 and p < 0 . 001 , LPS n = 7 , LPS / PolySia avDP20 0 . 15µM n = 5 and p = 0 . 002 , LPS / PolySia avDP20 1 . 5µM n = 4 and p = 0 . 0003 . Siglec11 / 16 KO : no treatment n = 6 and p < 0 . 001 , PolySia avDP20 1 . 5 µM n = 5 and p < 0 . 001 , LPS n = 7 , LPS / PolySia avDP20 0 . 15µM n = 5 and p = 1 . 0 , LPS / PolySia avDP20 1 . 5µM n = 5 and p = 1 . 0 .

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

B. Analysis of relative TNFSF2 protein release in human control and SIGLEC11/16 knockout THP1-macrophages. The released protein levels were reduced after 24 hours of co-treatment with LPS (1 µg/ml) and concentrations of 0.15 µM and 1.5 µM of polySia avDP20 in the human wild type macrophages. No response to polySia avDP20 was detectable in the knockout line. Data show mean +/- SEM. ** p < 0.01, *** p < 0.001, ANOVA followed by Bonferroni. Statistical analysis was done in relation to the LPS control. WT: no treatment n=8 and p<0.0001, PolySia avDP20 1.5 µM n=5 and p<0.001, LPS n=7, LPS/PolySia avDP20 0.15µM n=5 and p=0.002, LPS/PolySia avDP20 1.5µM n=4 and p=0.0003. Siglec11/16 KO: no treatment n=6 and p<0.001, PolySia avDP20 1.5 µM n=5 and p<0.001, LPS n=7, LPS/PolySia avDP20 0.15µM n=5 and p=1.0, LPS/PolySia avDP20 1.5µM n=5 and p=1.0.


C . Analysis of relative VEGFA gene transcription in human control and SIGLEC11 / 16 knockout THP1 - macrophages . Gene transcripts were reduced after 24 hours of co - treatment with LPS ( 1 µg / ml ) and polySia avDP20 ( 0 . 15 µM and 1 . 5 µM ) in the human wild type macrophages . No response to polySia avDP20 was detectable in the knockout macrophages . Data show mean + / - SEM . * * * p < 0 . 001 , ANOVA followed by Bonferroni . Statistical analysis was done in relation to the LPS control . WT : no treatment n = 6 and p < 0 . 0001 , PolySia avDP20 1 . 5 µM n = 5 and p < 0 . 0001 , LPS n = 5 , LPS / PolySia avDP20 0 . 15µM n = 5 and p = 0 . 0002 , LPS / PolySia avDP20 1 . 5µM n = 5 and p < 0 . 0001 . Siglec11 / 16 KO : no treatment n = 5 and p = 0 . 022 , PolySia avDP20 n = 3 and p = 0 . 063 , LPS n = 4 , LPS / PolySia avDP20 0 . 15µM n = 3 and p = 1 . 0 , LPS / PolySia avDP20 1 . 5µM n = 4 and p = 1 . 0 .

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

C. Analysis of relative VEGFA gene transcription in human control and SIGLEC11/16 knockout THP1-macrophages. Gene transcripts were reduced after 24 hours of co-treatment with LPS (1 µg/ml) and polySia avDP20 (0.15 µM and 1.5 µM) in the human wild type macrophages. No response to polySia avDP20 was detectable in the knockout macrophages. Data show mean +/- SEM. *** p < 0.001, ANOVA followed by Bonferroni. Statistical analysis was done in relation to the LPS control. WT: no treatment n=6 and p<0.0001, PolySia avDP20 1.5 µM n=5 and p<0.0001, LPS n=5, LPS/PolySia avDP20 0.15µM n=5 and p=0.0002, LPS/PolySia avDP20 1.5µM n=5 and p<0.0001. Siglec11/16 KO: no treatment n=5 and p=0.022, PolySia avDP20 n=3 and p=0.063, LPS n=4, LPS/PolySia avDP20 0.15µM n=3 and p=1.0, LPS/PolySia avDP20 1.5µM n=4 and p=1.0.


D . Analysis of relative VEGFA protein release in human control and SIGLEC11 / 16 knockout THP1 - macrophages . Released protein levels were reduced after 24 hours of co - treatment with LPS ( 1 µg / ml ) and polySia avDP20 ( 0 . 15 µM and 1 . 5 µM ) in the human macrophages . Data show mean + / - SEM . * p < 0 . 05 , * * * p < 0 . 001 , ANOVA followed by Bonferroni . Statistical analysis was done in relation to the LPS control . WT : no treatment n = 9 and p < 0 . 0001 , PolySia avDP20 1 . 5 µM n = 6 and p = 0 . 009 , LPS n = 9 , LPS / PolySia avDP20 0 . 15µM n = 6 and p = 0 . 043 , LPS / PolySia avDP20 1 . 5µM n = 7 and p = 0 . 0001 . Siglec11 / 16 KO : no treatment n = 9 and p = 0 . 349 , PolySia avDP20 1 . 5 µM n = 6 and p = 0 . 249 , LPS n = 7 , LPS / PolySia avDP20 0 . 15µM n = 6 and p = 1 . 0 , LPS / PolySia avDP20 1 . 5µM n = 7 and p = 1 . 0 .

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

D. Analysis of relative VEGFA protein release in human control and SIGLEC11/16 knockout THP1-macrophages. Released protein levels were reduced after 24 hours of co-treatment with LPS (1 µg/ml) and polySia avDP20 (0.15 µM and 1.5 µM) in the human macrophages. Data show mean +/- SEM. * p < 0.05, *** p < 0.001, ANOVA followed by Bonferroni. Statistical analysis was done in relation to the LPS control. WT: no treatment n=9 and p<0.0001, PolySia avDP20 1.5 µM n=6 and p=0.009, LPS n=9, LPS/PolySia avDP20 0.15µM n=6 and p=0.043, LPS/PolySia avDP20 1.5µM n=7 and p=0.0001. Siglec11/16 KO: no treatment n=9 and p=0.349, PolySia avDP20 1.5 µM n= 6 and p=0.249, LPS n=7, LPS/PolySia avDP20 0.15µM n=6 and p=1.0, LPS/PolySia avDP20 1.5µM n=7 and p=1.0.


E . Quantification of human control and SIGLEC11 / 16 knockout THP1 - macrophages having ingested cellular debris . PolySia avDP20 ( 1 . 5 µM ) reduced the percentage of phagocytic cells having ingested drusen - like debris . No response to polySia avDP20 was detectable in the knockout macrophages . Data are presented as mean + / - SEM . n = 6 . * p ≤ 0 . 05 , * * * p ≤ 0 . 001 , ANOVA followed by Bonferroni . Debris treated WT macrophages vs debris plus polySia avDP20 treated WT macrophages p = 0 . 028 , debris plus polySia avDP20 treated WT macrophages vs KO macrophages p = 0 . 00012 .

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

E. Quantification of human control and SIGLEC11/16 knockout THP1-macrophages having ingested cellular debris. PolySia avDP20 (1.5 µM) reduced the percentage of phagocytic cells having ingested drusen-like debris. No response to polySia avDP20 was detectable in the knockout macrophages. Data are presented as mean +/- SEM. n=6. *p ≤ 0.05, *** p ≤ 0.001, ANOVA followed by Bonferroni. Debris treated WT macrophages vs debris plus polySia avDP20 treated WT macrophages p = 0.028, debris plus polySia avDP20 treated WT macrophages vs KO macrophages p = 0.00012.


F . Prevention of superoxide release in activated human control and SIGLEC11 / 16 knockout THP1 - macrophages by polySia avDP20 . Cultured human THP1 - macrophages were stimulated with RPE cell debris or co - stimulated with debris and polySia avDP20 . Addition of debris stimulated the production of superoxide . 1 . 5 µM polySia avDP20 completely prevented the release of superoxide induced by debris challenge . No response to polySia avDP20 was detectable in the knockout macrophages . Data are presented as mean + / - SEM . n = 6 . p * ≤ 0 . 05 , * * * p ≤ 0 . 001 , ANOVA followed by Bonferroni . Untreated WT macrophages vs debris treated WT macrophages p = 0 . 037 , debris treated WT macrophage vs debris plus polySia avDP20 1 . 5 µM treated WT macrophages p < 0 . 001 , debris plus polySia avDP20 WT macrophages vs KO macrophages p = 0 . 001 ,

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

F. Prevention of superoxide release in activated human control and SIGLEC11/16 knockout THP1-macrophages by polySia avDP20. Cultured human THP1-macrophages were stimulated with RPE cell debris or co-stimulated with debris and polySia avDP20. Addition of debris stimulated the production of superoxide. 1.5 µM polySia avDP20 completely prevented the release of superoxide induced by debris challenge. No response to polySia avDP20 was detectable in the knockout macrophages. Data are presented as mean +/- SEM. n=6. p* ≤ 0.05, *** p ≤ 0.001, ANOVA followed by Bonferroni. Untreated WT macrophages vs debris treated WT macrophages p = 0.037, debris treated WT macrophage vs debris plus polySia avDP20 1.5 µM treated WT macrophages p < 0.001, debris plus polySia avDP20 WT macrophages vs KO macrophages p = 0.001,


G . PolySia avDP20 ( 0 . 15 to 50 µM ) was added to normal human serum ( NHS ) to evaluate any interference with activation of the classical complement pathway by IgM . Complement activation was determined by C3b deposition . PolySia avDP20 had no effect on C3b deposition induced by activation of the classical complement pathway . Data show mean + / - SEM . n = 3 , n . s . = not significant .

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

G. PolySia avDP20 (0.15 to 50 µM) was added to normal human serum (NHS) to evaluate any interference with activation of the classical complement pathway by IgM. Complement activation was determined by C3b deposition. PolySia avDP20 had no effect on C3b deposition induced by activation of the classical complement pathway. Data show mean +/- SEM. n = 3, n.s.= not significant.


H . PolySia avDP20 inhibits the alternative complement pathway . PolySia avDP20 ( 0 . 15 to 50 µM ) was added to NHS and activation of the alternative pathway was induced by LPS . C3b deposition was monitored by ELISA . Data show mean + / - SEM ( n = 4 . * p < 0 . 05 , * * p < 0 . 01 , p * * * < 0 . 001 , ANOVA followed by Bonferroni ) .

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

H. PolySia avDP20 inhibits the alternative complement pathway. PolySia avDP20 (0.15 to 50 µM) was added to NHS and activation of the alternative pathway was induced by LPS. C3b deposition was monitored by ELISA. Data show mean +/- SEM (n = 4. * p < 0.05, ** p < 0.01, p***<0.001, ANOVA followed by Bonferroni).


Expression of Kit ( magenta ) largely overlaps with the expression of CD41 : YFP ( yellow ) in IAHC . Multistack reconstruction of confocal images . Scale bars : 30μm overview , 10μm magnification . ( C ) Snapshots of Movie EV1 . Time - lapse of embryonic organotypic slice culture from CD41 : YFP ( yellow ) : H2B - GFP ( blue ) reporter mouse shows the emergence of a single cell protruding from the endothelial layer into the aortic lumen . Scale bars : 50μm ( overview ) , 10μm ( magnification ) . Time expressed in hh : mm : ss . ( D ) Snapshots of Movie EV2 . Time - lapse of embryonic organotypic slice culture from CD41 : YFP ( yellow ) : H2B - GFP ( blue ) reporter mouse shows a single CD41 : YFP expressing cell that divides once on the aortic endothelium . Scale bars : 100μm ( overview ) , 20μm ( magnification ) . Time expressed in hh : mm : ss .

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

Expression of Kit (magenta) largely overlaps with the expression of CD41:YFP (yellow) in IAHC. Multistack reconstruction of confocal images. Scale bars: 30μm overview, 10μm magnification. (C) Snapshots of Movie EV1. Time-lapse of embryonic organotypic slice culture from CD41:YFP (yellow): H2B-GFP (blue) reporter mouse shows the emergence of a single cell protruding from the endothelial layer into the aortic lumen. Scale bars: 50μm (overview), 10μm (magnification). Time expressed in hh:mm:ss. (D) Snapshots of Movie EV2. Time-lapse of embryonic organotypic slice culture from CD41:YFP (yellow): H2B-GFP (blue) reporter mouse shows a single CD41:YFP expressing cell that divides once on the aortic endothelium. Scale bars: 100μm (overview), 20μm (magnification). Time expressed in hh:mm:ss.


BrdU incorporation in the Kit + IAHC upon cumulative administration . Multistack reconstruction of confocal images . Representative picture of a short BrdU pulse ( 1h ) . Scale bars : 50μm ( overview ) , 10μm ( magnification ) . ( F ) Quantification of the percentage of Kit + cells within IAHCs incorporating BrdU using confocal images . All cells ( Labelling Index = 100 % ) can be labelled after 8h , indicating the lack of quiescent cells inside the IAHCs . Graph represents mean ± SE .

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

BrdU incorporation in the Kit+ IAHC upon cumulative administration. Multistack reconstruction of confocal images. Representative picture of a short BrdU pulse (1h). Scale bars: 50μm (overview), 10μm (magnification). (F) Quantification of the percentage of Kit+ cells within IAHCs incorporating BrdU using confocal images. All cells (Labelling Index=100%) can be labelled after 8h, indicating the lack of quiescent cells inside the IAHCs. Graph represents mean±SE.


Quantification of recombination efficiency at E10 . 5 and E11 . 5 using confocal images . Bars represents the mean ± SD of the percentage of cells expressing single colored cassettes in CD31 + endothelial cells ( n = 2 ) .

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Quantification of recombination efficiency at E10.5 and E11.5 using confocal images. Bars represents the mean±SD of the percentage of cells expressing single colored cassettes in CD31+ endothelial cells (n=2).


Transversal section of a recombined VeCadCreER : Confetti AGM from an E10 . 5 embryo and immunostained with CD31 ( C ) ( white in image , black in label ) Multistack reconstruction of confocal images . Scale bars

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Transversal section of a recombined VeCadCreER:Confetti AGM from an E10.5 embryo and immunostained with CD31 (C) (white in image, black in label) Multistack reconstruction of confocal images. Scale bars 30μm


Transversal section of a recombined VeCadCreER : Confetti AGM from an E10 . 5 embryo and immunostained with Kit ( D ) ( white in image , black in label ) . Multistack reconstruction of confocal images . Scale bars 20

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Transversal section of a recombined VeCadCreER:Confetti AGM from an E10.5 embryo and immunostained with Kit (D) (white in image, black in label). Multistack reconstruction of confocal images. Scale bars 20 μm


Analyses of recombination in 40 clusters . Bars representing single clusters are grouped based on the number of Kit + cells . Recombination induction follows the schema in the black square . ( n = 12 embryo ) .

Analyses of recombination in 40 clusters. Bars representing single clusters are grouped based on the number of Kit+ cells. Recombination induction follows the schema in the black square. (n=12 embryo).


Representative confetti recombination on clusters of different sizes . Small cluster are monoclonal ( left panel ) , while larger clusters are polyclonal ( right panel ) . Inlet shows relevant confetti channel . Kit is shown in white in the image , in black in the label . Multistack reconstruction of confocal images . Scale bars 5 μm .

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

Representative confetti recombination on clusters of different sizes. Small cluster are monoclonal (left panel), while larger clusters are polyclonal (right panel). Inlet shows relevant confetti channel. Kit is shown in white in the image, in black in the label. Multistack reconstruction of confocal images. Scale bars 5 μm.


Detail images of Dll4 staining in Kit + cells of IAHC of different sizes . Dll4 expression inversely correlates with the size of the cluster : 2 - 5 cell IAHCs are uniformly Dll4 - positive ( upper and middle panel ) , > 5 cell IAHCs are partially Dll4 positive ( lower panel ) . Multistack reconstruction of confocal images . Scale bars : 10 μm .

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

Detail images of Dll4 staining in Kit+ cells of IAHC of different sizes. Dll4 expression inversely correlates with the size of the cluster: 2-5 cell IAHCs are uniformly Dll4-positive (upper and middle panel), >5 cell IAHCs are partially Dll4 positive (lower panel). Multistack reconstruction of confocal images. Scale bars: 10 μm.


Quantification of Kit + Dll4 + cells using confocal images . Bars represent the percentage of Dll4 expressing cells within a cluster in each category of cluster size . Mean ± SD ( total 17 clusters analysed , n = 2 ) .

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Quantification of Kit+ Dll4+ cells using confocal images. Bars represent the percentage of Dll4 expressing cells within a cluster in each category of cluster size. Mean±SD (total 17 clusters analysed, n=2).


Overview of Dll4 - ligand expression in the embryonic aorta at E10 . 5 . Dll4 is expressed in the majority of the endothelial layer . Scale bar :

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Overview of Dll4-ligand expression in the embryonic aorta at E10.5. Dll4 is expressed in the majority of the endothelial layer. Scale bar: 30μm


( A ) Whole mount images of the aorta endothelium stained for CD31 ( white in image , black in label ) and Kit ( IAHC , cyan ) upon IgG ( left ) or αDll4 antibody treatment ( right ) . Multistack reconstruction of confocal images . Red arrows indicate IAHCs on the ventral part of the aortic wall . Asterisks indicate aspecific autofluorescence . Scale bar : 50μm .

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

(A)Whole mount images of the aorta endothelium stained for CD31 (white in image, black in label) and Kit (IAHC, cyan) upon IgG (left) or αDll4 antibody treatment (right). Multistack reconstruction of confocal images. Red arrows indicate IAHCs on the ventral part of the aortic wall. Asterisks indicate aspecific autofluorescence. Scale bar: 50μm.


Representative IAHCs of IgG and αDll4 treated embryos . Kit is shown in white in the image , in black in the label . Multistack reconstruction of confocal images . Note the increased IAHC size in the right panel . Scale bars : 50μm ( overview ) , and 10μm ( magnification ) .

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

Representative IAHCs of IgG and αDll4 treated embryos. Kit is shown in white in the image, in black in the label. Multistack reconstruction of confocal images. Note the increased IAHC size in the right panel. Scale bars: 50μm (overview), and 10μm (magnification).


Frequency of IAHC categories based on the number of Kit + cells forming a cluster . Quantification of confocal images . Bars represent the mean ± SE ( n = 6 )

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

Frequency of IAHC categories based on the number of Kit+ cells forming a cluster. Quantification of confocal images. Bars represent the mean±SE (n=6)


Number of Kit + cells per IAHC increases following αDll4 antibody treatment . Each dot represents one cluster . Quantification on confocal images . Statistical analysis : Mann - Whitney U test . * * * * p < 0 . 0001 .

Number of Kit+ cells per IAHC increases following αDll4 antibody treatment. Each dot represents one cluster. Quantification on confocal images. Statistical analysis: Mann-Whitney U test. ****p< 0.0001.


Total number of IAHCs per 100μm . Quantification of confocal images . Mean ± SE . Statistical analysis : t - test . ns p > 0 . 05 ; ( n = 6 ) . ( F ) Quantification of the number of Kit + cells per 100 μm using confocal images . Bars represent the mean ± SE . Statistical analysis : t - test . ns p > 0 . 05 ; * * * p < 0 . 001 ( n = 6 ) .

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

Total number of IAHCs per 100μm. Quantification of confocal images. Mean±SE. Statistical analysis: t-test. ns p> 0.05; (n=6). (F) Quantification of the number of Kit+ cells per 100 μm using confocal images. Bars represent the mean±SE. Statistical analysis: t-test. ns p> 0.05; ***p< 0.001 (n=6).


Experimental design : E10 . 5 embryos were injected with IgG or αDll4 , incubated for 5h and then dissected . The indicated IAHC subpopulations ( kit + CD45 + ; kit + CD45 - ; kit - CD45 - CD41 + were purified and RNA was sequenced .

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

Experimental design: E10.5 embryos were injected with IgG or αDll4, incubated for 5h and then dissected. The indicated IAHC subpopulations (kit+CD45+; kit+CD45-;kit-CD45-CD41+ were purified and RNA was sequenced.


Experimental design and sorting gate for Kit + CD45 - cells for single cell RNAseq ( n = 483 cells ) .

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

Experimental design and sorting gate for Kit+CD45- cells for single cell RNAseq (n=483 cells).


Violin plots of Notch family elements expression in IgG and αDll4 treated cells ( adj p val : * < 0 . 1 and * * * p < 0 . 00001 ) .

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Violin plots of Notch family elements expression in IgG and αDll4 treated cells (adj p val: *<0.1 and *** p<0.00001).


Percentage of cells in each cell cycle phase extracted from single cell RNA - seq analysis . Bars represent the average ± SE ( n = 483 ) .

Percentage of cells in each cell cycle phase extracted from single cell RNA-seq analysis. Bars represent the average±SE (n=483).


Bar graph represents the frequency of IAHCs containing recombined cells per embryo under the indicated conditions . Quantification of confocal images . Mean ± SE ( n = 15 for untreated , n = 7 for IgG and αDll4 treated ) .

Bar graph represents the frequency of IAHCs containing recombined cells per embryo under the indicated conditions. Quantification of confocal images. Mean±SE (n=15 for untreated, n=7 for IgG and αDll4 treated).


Representative images of recombined IAHCs following IgG or αDll4 conditions . Kit is shown in white in the image , in black in the label . Multistack reconstruction of confocal images . Scale bar : 5μm .

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Representative images of recombined IAHCs following IgG or αDll4 conditions. Kit is shown in white in the image, in black in the label. Multistack reconstruction of confocal images. Scale bar: 5μm.


Quantification and classification of IAHC containing recombined cells in TAM - induced VeCadCreER : Confetti embryos after IgG or αDll4 treatment . Embryos treated with αDll4 contain more IAHC with colored cells . Quantification of confocal images ( n = 7 embryos ) .

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

Quantification and classification of IAHC containing recombined cells in TAM-induced VeCadCreER:Confetti embryos after IgG or αDll4 treatment. Embryos treated with αDll4 contain more IAHC with colored cells. Quantification of confocal images (n=7 embryos).


Representative images of recombined endothelial cells in transversal sections of the aorta . The distribution and amount of recombined endothelial cells is similar in IgG and αDll4 conditions . CD31 is shown in white in the image , in black in the label . Multistack reconstruction of confocal images . Scale bar : 50μm . Quantification of recombined cells in the aortic endothelium using confocal images . Mean ± SE ( n = 3 ) .

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

Representative images of recombined endothelial cells in transversal sections of the aorta. The distribution and amount of recombined endothelial cells is similar in IgG and αDll4 conditions. CD31 is shown in white in the image, in black in the label. Multistack reconstruction of confocal images. Scale bar: 50μm. Quantification of recombined cells in the aortic endothelium using confocal images. Mean±SE (n=3).


Snapshots of Movie EV5 . Time Lapse of embryonic organotypic slice from a CD41 : YFP : H2B - GFP reporter mouse showing the recruitment of CD41 : YFP negative cells ( magenta central point - tracking analyses ) migrating towards the IAHC ( CD41 : YFP + cells ) . Green central point and relative tracking identify cells with non - directional movement . Scale bar : 5μm . Time expressed in hh : mm : ss .

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

Snapshots of Movie EV5. Time Lapse of embryonic organotypic slice from a CD41:YFP: H2B-GFP reporter mouse showing the recruitment of CD41:YFP negative cells (magenta central point-tracking analyses) migrating towards the IAHC (CD41:YFP+ cells). Green central point and relative tracking identify cells with non-directional movement. Scale bar: 5μm. Time expressed in hh:mm:ss.


Colony - forming assays using E10 . 5 AGMs derived from embryos treated with IgG or αDll4 .

Colony-forming assays using E10.5 AGMs derived from embryos treated with IgG or αDll4.


FACS analyses of Gfi1 + cells derived from explants treated with IgG or αDll4 for 5 hours ( C ) . Analyses of the hemogenic Gfi1 + Kit - ( D ) and the Gfi1 + Kit + ( E ) within the CD31 + CD45 - population .

FACS analyses of Gfi1+ cells derived from explants treated with IgG or αDll4 for 5 hours (C). Analyses of the hemogenic Gfi1+Kit-(D) and the Gfi1+Kit+ (E) within the CD31+CD45- population.


Percentage of reconstitution ( chimerism determined by CD45 . 1 and CD45 . 2 expression ) in peripheral blood ( PB ) . Embryos treated with IgG or αDll4 were kept in explant culture for 48hours and then transplanted into irradiated recipients . Peripheral blood was analyzed at different time points . Each dot / square represents a single transplanted mouse .

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

Percentage of reconstitution (chimerism determined by CD45.1 and CD45.2 expression) in peripheral blood (PB). Embryos treated with IgG or αDll4 were kept in explant culture for 48hours and then transplanted into irradiated recipients. Peripheral blood was analyzed at different time points. Each dot/square represents a single transplanted mouse.


Analysis of progenitors ( lin - sca1 + kit + ) and HSCs ( Lin - sca1 + kit + CD48 - CD150 + ) in the donor population in the bone marrow after 4 months of transplantation .

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Analysis of progenitors (lin-sca1+kit+) and HSCs (Lin-sca1+kit+CD48-CD150+) in the donor population in the bone marrow after 4 months of transplantation.


K . Representative immunostainings for Pax7 and MyoD in eMPs plated on Vtn and maintained in proliferation media ( PM ) after isolation . L . Representative immunostainings for Pax7 and MyoD in eMPs plated on Vtn that were first maintained in PM and then switched to differentiation media ( DM ) . hiPSC donor 1 .

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K. Representative immunostainings for Pax7 and MyoD in eMPs plated on Vtn and maintained in proliferation media (PM) after isolation. L. Representative immunostainings for Pax7 and MyoD in eMPs plated on Vtn that were first maintained in PM and then switched to differentiation media (DM). hiPSC donor 1.


M - O . Quantification of the number of Pax7 ( M ) , MyoD ( N ) and Pax7 / MyoD double - positive ( O ) cells in hskMPs and eMPs in PM and after switching to DM . hiPSC donor 1 .

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M-O. Quantification of the number of Pax7 (M), MyoD (N) and Pax7/MyoD double-positive (O) cells in hskMPs and eMPs in PM and after switching to DM. hiPSC donor 1.


P . Representative immunostaining for the muscle fiber marker myosin heavy chain ( MyHC ) of eMPs maintained in DM . Q . Quantification of the fusion index of hskMPs and eMPs in PM and DM . hiPSC donor 1 .

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P. Representative immunostaining for the muscle fiber marker myosin heavy chain (MyHC) of eMPs maintained in DM. Q. Quantification of the fusion index of hskMPs and eMPs in PM and DM. hiPSC donor 1.


U - Z , Representative immunostainings and quantification of eMPs in DM expressing Pax7 ( V ) , MyoD ( X ) , and MyoG ( Z ) , as well as the proliferation marker Ki67 . hiPSC donor 1 .

U-Z, Representative immunostainings and quantification of eMPs in DM expressing Pax7 (V), MyoD (X), and MyoG (Z), as well as the proliferation marker Ki67. hiPSC donor 1.


A . Representative immunostainings for dystrophin in cross - sections of muscles of mdx mice after transplantation of hskMPs or eMPs . Scale bars in the two left - hand side images = 300 μm . Scale bars in inserts 1 and 2 = 50 μm . B . Number of dystrophin positive fibers in mdx muscles injected with vehicle ( Ctrl ) , eMPs and hskMPs . C . Maximal distance of clusters of dystrophin positive fibers in cross - sections of mdx muscles transplanted with eMPs and hskMPs . A - C . 10 days post - transplantation . hiPSC donor 1 .

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

A. Representative immunostainings for dystrophin in cross-sections of muscles of mdx mice after transplantation of hskMPs or eMPs. Scale bars in the two left-hand side images = 300 μm. Scale bars in inserts 1 and 2 = 50 μm. B. Number of dystrophin positive fibers in mdx muscles injected with vehicle (Ctrl), eMPs and hskMPs. C. Maximal distance of clusters of dystrophin positive fibers in cross-sections of mdx muscles transplanted with eMPs and hskMPs. A-C. 10 days post-transplantation. hiPSC donor 1.


B - C Competitor donor derived Gr , B , CD4 T , and CD8 T cells in the peripheral blood . In the control group , competitor donor cells are WT HSCs . In the deficient group , competitor donor cells are uMT - / - or NSG HSCs Data information : Data are shown as percentages of the total number of white blood cells ( WBCs ) . Data were collected at month 7 post transplantation for the uMT - / - group and month 8 post - transplantation for the NSG group , and presented as mean ± SEM . n = 7 mice for the uMT - / - group and n = 6 for the NSG group . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ( two - tailed and two - sample equal variance Student " s t - test )

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

B-C Competitor donor derived Gr, B, CD4 T, and CD8 T cells in the peripheral blood. In the control group, competitor donor cells are WT HSCs. In the deficient group, competitor donor cells are uMT-/- or NSG HSCs Data information: Data are shown as percentages of the total number of white blood cells (WBCs). Data were collected at month 7 post transplantation for the uMT-/- group and month 8 post-transplantation for the NSG group, and presented as mean ± SEM. n=7 mice for the uMT-/- group and n=6 for the NSG group. *p<0.05, **p<0.01, ***p<0.001 (two-tailed and two-sample equal variance Student"s t-test)


D - E WT donor derived Gr , B , CD4 T , and CD8 T cells in the peripheral blood Data information : Data are shown as percentages of the total number of white blood cells ( WBCs ) . Data were collected at month 7 post transplantation for the uMT - / - group and month 8 post - transplantation for the NSG group , and presented as mean ± SEM . n = 7 mice for the uMT - / - group and n = 6 for the NSG group . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ( two - tailed and two - sample equal variance Student " s t - test )

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

D-E WT donor derived Gr, B, CD4 T, and CD8 T cells in the peripheral blood Data information: Data are shown as percentages of the total number of white blood cells (WBCs). Data were collected at month 7 post transplantation for the uMT-/- group and month 8 post-transplantation for the NSG group, and presented as mean ± SEM. n=7 mice for the uMT-/- group and n=6 for the NSG group. *p<0.05, **p<0.01, ***p<0.001 (two-tailed and two-sample equal variance Student"s t-test)


F - G Total production of Gr , B , CD4 T , and CD8 T cells in the peripheral blood ( derived from WT HSCs , competitor HSCs , helper whole bone marrow cells , and residual host cells ) shown as percentages of the total number of white blood cells ( WBCs ) . Data information : Data are shown as percentages of the total number of white blood cells ( WBCs ) . Data were collected at month 7 post transplantation for the uMT - / - group and month 8 post - transplantation for the NSG group , and presented as mean ± SEM . n = 7 mice for the uMT - / - group and n = 6 for the NSG group . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ( two - tailed and two - sample equal variance Student " s t - test )

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

F-G Total production of Gr, B, CD4 T, and CD8 T cells in the peripheral blood (derived from WT HSCs, competitor HSCs, helper whole bone marrow cells, and residual host cells) shown as percentages of the total number of white blood cells (WBCs). Data information: Data are shown as percentages of the total number of white blood cells (WBCs). Data were collected at month 7 post transplantation for the uMT-/- group and month 8 post-transplantation for the NSG group, and presented as mean ± SEM. n=7 mice for the uMT-/- group and n=6 for the NSG group. *p<0.05, **p<0.01, ***p<0.001 (two-tailed and two-sample equal variance Student"s t-test)


A - B Total numbers of barcoded WT clones that give rise to granulocytes ( Gr ) , B cells , CD4 T cells , and CD8 T cells Data information : Data were collected at month 7 post - transplantation for the uMT - / - group and month 8 post - transplantation for the NSG group , and presented as mean ± SEM . n = 7 mice for the uMT - / - group and n = 6 for the NSG group . * p < 0 . 05 ( two - tailed and two - sample equal variance Student " s t - test )

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

A-B Total numbers of barcoded WT clones that give rise to granulocytes (Gr), B cells, CD4 T cells, and CD8 T cells Data information: Data were collected at month 7 post-transplantation for the uMT-/- group and month 8 post-transplantation for the NSG group, and presented as mean ± SEM. n=7 mice for the uMT-/- group and n=6 for the NSG group. *p<0.05 (two-tailed and two-sample equal variance Student"s t-test)


C - J Number of HSC clones that produce different amounts of Gr , B , CD4 T , and CD8 T cells . We combined the sequencing data with the flow cytometry data to calculate clonal abundance for each clone as follows : Clonal abundance = 100 % * ( Each cell population ( Gr , B , CD4T or CD8T cells ) % WBCs ) * ( Donor % Each cell population ) * ( GFP % Donor cells ) * ( number of reads for each barcode ) / ( total reads of all barcodes Data information : Data were collected at month 7 post - transplantation for the uMT - / - group and month 8 post - transplantation for the NSG group , and presented as mean ± SEM . n = 7 mice for the uMT - / - group and n = 6 for the NSG group . * p < 0 . 05 ( two - tailed and two - sample equal variance Student " s t - test )

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

C-J Number of HSC clones that produce different amounts of Gr, B, CD4 T, and CD8 T cells. We combined the sequencing data with the flow cytometry data to calculate clonal abundance for each clone as follows: Clonal abundance =100%*(Each cell population (Gr, B, CD4T or CD8T cells) % WBCs) * (Donor % Each cell population) * (GFP % Donor cells) * (number of reads for each barcode) / (total reads of all barcodes Data information: Data were collected at month 7 post-transplantation for the uMT-/- group and month 8 post-transplantation for the NSG group, and presented as mean ± SEM. n=7 mice for the uMT-/- group and n=6 for the NSG group. *p<0.05 (two-tailed and two-sample equal variance Student"s t-test)


K - L Production of Gr , B , CD4 T , and CD8 T cells by expanding WT clones that produce more than 0 . 1 % of WBCs in each lineage . Data information : Data were collected at month 7 post - transplantation for the uMT - / - group and month 8 post - transplantation for the NSG group , and presented as mean ± SEM . n = 7 mice for the uMT - / - group and n = 6 for the NSG group . * p < 0 . 05 ( two - tailed and two - sample equal variance Student " s t - test )

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

K-L Production of Gr, B, CD4 T, and CD8 T cells by expanding WT clones that produce more than 0.1% of WBCs in each lineage. Data information: Data were collected at month 7 post-transplantation for the uMT-/- group and month 8 post-transplantation for the NSG group, and presented as mean ± SEM. n=7 mice for the uMT-/- group and n=6 for the NSG group. *p<0.05 (two-tailed and two-sample equal variance Student"s t-test)


A - B Total amount of progenitors as a percentage of ckit and Il7rα enriched bone marrow ( BM ) cells . Shown are hematopoietic stem cells ( HSCs ) , Flk2 - and Flk2 + multipotent progenitors ( MPP ) , common lymphoid progenitors ( CLP ) , common myeloid progenitors ( CMP ) , megakaryocyte - erythroid progenitors ( MEP ) , and granulocyte - monocyte progenitors ( GMP ) Data information : Data were collected at month 7 post transplantation for the uMT - / - group and month 8 post - transplantation for the NSG group , and presented as mean ± SEM . n = 7 mice for the uMT - / - group and n = 6 for the NSG group . * p < 0 . 05 and * * p < 0 . 01 ( two - tailed and two - sample equal variance Student " s t - test )

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

A-B Total amount of progenitors as a percentage of ckit and Il7rα enriched bone marrow (BM) cells. Shown are hematopoietic stem cells (HSCs), Flk2- and Flk2+ multipotent progenitors (MPP), common lymphoid progenitors (CLP), common myeloid progenitors (CMP), megakaryocyte-erythroid progenitors (MEP), and granulocyte-monocyte progenitors (GMP) Data information: Data were collected at month 7 post transplantation for the uMT-/- group and month 8 post-transplantation for the NSG group, and presented as mean ± SEM. n=7 mice for the uMT-/- group and n=6 for the NSG group. *p<0.05 and **p< 0.01 (two-tailed and two-sample equal variance Student"s t-test)


C - D WT donor derived progenitors as a percentage of ckit and Il7rα enriched BM cells . Data information : Data were collected at month 7 post transplantation for the uMT - / - group and month 8 post - transplantation for the NSG group , and presented as mean ± SEM . n = 7 mice for the uMT - / - group and n = 6 for the NSG group . * p < 0 . 05 and * * p < 0 . 01 ( two - tailed and two - sample equal variance Student " s t - test )

C-D WT donor derived progenitors as a percentage of ckit and Il7rα enriched BM cells. Data information: Data were collected at month 7 post transplantation for the uMT-/- group and month 8 post-transplantation for the NSG group, and presented as mean ± SEM. n=7 mice for the uMT-/- group and n=6 for the NSG group. *p<0.05 and **p< 0.01 (two-tailed and two-sample equal variance Student"s t-test)


( A ) survival curves of MyD88 flies injected with different doses of A . fumigatus conidia ( c / f : conidia injected per fly ; error bars represent mean ± SD of the survival of biological triplicates of 20 flies each ) .

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

(A) survival curves of MyD88 flies injected with different doses of A. fumigatus conidia (c/f: conidia injected per fly; error bars represent mean ± SD of the survival of biological triplicates of 20 flies each).


( B ) fungal loads of single MyD88 mutant and wild - type flies ( 50 conidia injected per fly ) .

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

(B) fungal loads of single MyD88 mutant and wild-type flies (50 conidia injected per fly).


( C - D ) GFP - labeled A . fumigatus ( 50 conidia per fly ) injected in wild - type ( C ) or MyD88 mutant ( D ) flies form hyphae in the thorax of the flies ( arrows ) . Scale bars 50 μm .

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

(C-D) GFP-labeled A. fumigatus (50 conidia per fly) injected in wild-type (C) or MyD88 mutant (D) flies form hyphae in the thorax of the flies (arrows). Scale bars 50 μm.


( E ) expression level of Drosomycin induced by different doses of injected A . fumigatus conidia measured by RT - qPCR ; M . luteus ( OD = 10 ) represents the positive control ( pooled data of n = 3 experiments , biological replicates ) .

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

(E) expression level of Drosomycin induced by different doses of injected A. fumigatus conidia measured by RT-qPCR; M. luteus (OD=10) represents the positive control (pooled data of n=3 experiments, biological replicates).


Hayan flies are susceptible to A . fumigatus . Survival ( F ) , of Hayan mutant flies ; Hayan 0h vs . 120h , ( error bars represent mean ± SD of the survival of biological triplicates of 20 flies each ) .

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

Hayan flies are susceptible to A. fumigatus. Survival (F), of Hayan mutant flies; Hayan 0h vs. 120h, (error bars represent mean ± SD of the survival of biological triplicates of 20 flies each).


time course of fungal loads of single Hayan mutant flies ( 500 conidia per fly ) ( G ) , and fungal load upon death ( 500 conidia per fly ) ( H ) of Hayan mutant flies ; Hayan 0h vs . 120h , ( error bars represent mean ± SD of the survival of biological triplicates of 20 flies each ) .

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

time course of fungal loads of single Hayan mutant flies (500 conidia per fly) (G), and fungal load upon death (500 conidia per fly) (H) of Hayan mutant flies; Hayan 0h vs. 120h, (error bars represent mean ± SD of the survival of biological triplicates of 20 flies each).


( A ) survival of MyD88 flies injected with ∆ pptA and wild - type ( Af ) A . fumigatus conidia ( error bars represent mean ± SD of the survival of biological triplicates of 20 flies each ) .

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

(A) survival of MyD88 flies injected with ∆pptA and wild-type (Af) A. fumigatus conidia (error bars represent mean ± SD of the survival of biological triplicates of 20 flies each).


( B ) fungal loads of single flies after the injection of 500 ∆ pptA conidia .

(B) fungal loads of single flies after the injection of 500 ∆pptA conidia.


( E ) dose response of MyD88 flies after ∆ gliP ( gliotoxin ) mutant or wild - type [ ∆ akuB ] A . fumigatus infection ; error bars represent mean ± SD of the survival of biological triplicates of 20 flies each ; wild - type flies are used as a control for the dose of 250 conidia . ( F - H ) dose response of MyD88 and wild - type flies after gliotoxin ( F ) , fumagillin ( G ) , and helvolic acid ( H ) injection at the indicated concentrations ( 20 flies per condition ) .

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

(E) dose response of MyD88 flies after ∆gliP (gliotoxin) mutant or wild-type [∆akuB] A. fumigatus infection; error bars represent mean ± SD of the survival of biological triplicates of 20 flies each; wild-type flies are used as a control for the dose of 250 conidia. (F-H) dose response of MyD88 and wild-type flies after gliotoxin (F), fumagillin (G), and helvolic acid (H) injection at the indicated concentrations (20 flies per condition).


( A ) survival of MyD88 or wild - type flies to 250 injected ∆ aspf1 ( restrictocin mutant ) or wild - type [ ∆ akuB ] A . fumigatus conidia ( 20 flies per condition ) ; MyD88 : ∆ aspf1 vs . ∆ akuB ( * * * p = 0 . 0007 ) . ( B ) survival of MyD88 flies after the injection of different concentrations of restrictocin ( R ) ( 20 flies per condition ) . ( C - D ) survival of antibiotics - treated ( C ) and axenic ( D ) MyD88 mutant flies after restrictocin injection ( 20 flies per condition ) .

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

(A) survival of MyD88 or wild-type flies to 250 injected ∆aspf1 (restrictocin mutant) or wild-type [∆akuB] A. fumigatus conidia (20 flies per condition); MyD88: ∆aspf1 vs. ∆akuB (***p=0.0007). (B) survival of MyD88 flies after the injection of different concentrations of restrictocin (R) (20 flies per condition ). (C-D) survival of antibiotics-treated (C) and axenic (D) MyD88 mutant flies after restrictocin injection (20 flies per condition).


( E - F ) ribosomal RNA cleavage measurement after restrictocin or PBS injection in wild - type ( E ) and MyD88 ( F ) flies ; the arrowheads show the position of the 28S RNA - derived α - sarcin fragments whereas arrows on the right show its electrophoretic band position .

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

(E-F) ribosomal RNA cleavage measurement after restrictocin or PBS injection in wild-type (E) and MyD88 (F) flies; the arrowheads show the position of the 28S RNA-derived α-sarcin fragments whereas arrows on the right show its electrophoretic band position.


( H ) SDS - PAGE analysis of 35S - labelled translated proteins produced in a rabbit reticulocytes lysate from a m7G - capped reporter RNA containing the 5 ' UTR of β - globin followed by the Renilla luciferase coding sequence ( arrow ) , in the presence of increasing concentrations ( 0 . 125 to 25 nM ) of restrictocin .

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

(H) SDS-PAGE analysis of 35S-labelled translated proteins produced in a rabbit reticulocytes lysate from a m7G-capped reporter RNA containing the 5'UTR of β-globin followed by the Renilla luciferase coding sequence (arrow), in the presence of increasing concentrations (0.125 to 25 nM) of restrictocin.


( A - B ) survival ( A ) and fungal load ( B ) of Bom ∆ 55C ( 55C ) deficient flies compared to wild - type and MyD88 after injection of 250 conidia ( error bars represent mean ± SD of the survival of biological triplicates of 20 flies each ) ; * * * * p < 0 . 0001 . ( B ) : the fungal burden does not increase in Bom ∆ 55C - deficient flies ; wt 0h vs . 48h , * * p = 0 . 001 ; 55C 0h vs . 48h , * * p = 0 . 007 ; pooled data of n = 3 experiments , biological replicates .

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

(A-B) survival (A) and fungal load (B) of Bom∆55C (55C) deficient flies compared to wild-type and MyD88 after injection of 250 conidia (error bars represent mean ± SD of the survival of biological triplicates of 20 flies each); ****p < 0.0001. (B): the fungal burden does not increase in Bom∆55C-deficient flies; wt 0h vs. 48h, **p=0.001; 55C 0h vs. 48h, **p=0.007; pooled data of n=3 experiments, biological replicates.


( C - D ) rescue of the sensitivity of Bom ∆ 55C flies to verruculogen ( C ) or to restrictocin ( D ) by the transgenic expression of individual 55C locus genes ( caption in D also applies to ( C ) ) . 55C flies vs . BomS1 , * p = 0 . 0495 , vs . BomS6 * p = 0 . 011 for verruculogen assay ( C ) ; 55C flies vs . BomBc1 or BomS3 , * * * * p < 0 . 0001 , 55C flies vs . BomS6 , * * p = 0 . 0028 for restrictocin assay ( D ) ( 20 flies per condition ) .

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

(C-D) rescue of the sensitivity of Bom∆55C flies to verruculogen (C) or to restrictocin (D) by the transgenic expression of individual 55C locus genes (caption in D also applies to (C)). 55C flies vs. BomS1, *p=0.0495, vs. BomS6 *p=0.011 for verruculogen assay (C); 55C flies vs. BomBc1 or BomS3, ****p<0.0001, 55C flies vs. BomS6, **p=0.0028 for restrictocin assay (D) (20 flies per condition).


( E - F ) expression levels of BomBc1 , and BomS3 measured by RT - digital PCR 48 hours after challenge ; BomBc1 PBST vs . Af , * p = 0 . 015 , PBST vs . restrictocin ( R ) , * p = 0 . 015 ; BomS3 : PBST vs . Af , * p = 0 . 02 , PBST vs . R , * p = 0 . 03 ( pooled data of n = 3 experiments , biological replicates ) .

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

(E-F) expression levels of BomBc1, and BomS3 measured by RT-digital PCR 48 hours after challenge; BomBc1 PBST vs. Af, *p=0.015, PBST vs. restrictocin (R), *p=0.015; BomS3: PBST vs. Af, *p=0.02, PBST vs. R, *p=0.03 (pooled data of n=3 experiments, biological replicates).


( A - B ) tremor rate ( A ) and survival ( B ) of flies ( 20 flies per condition ) overexpressing Tl [ 10B ] in neurons compared to wild - type after injection of verruculogen . ( A ) Each dot corresponds to the tremor rate measured in a batch of 20 flies ; tremor rate wt vs . elav > UAS - Toll10B , * * p = 0 . 002 . ( C - D ) tremor rate ( C ) and survival ( D ) of flies ( 20 flies per condition ) overexpressing Tl [ 10B ] in glia compared to wild - type after injection of verruculogen . ( C ) Each dot corresponds to the tremor rate measured in a batch of 20 flies ; tremor rate wt vs . repo > UAS - Toll10B , * * p = 0 . 002 . ( D ) Survival wt V vs . repo > UAS - Toll10B V , * * p = 0 . 005 .

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

(A-B) tremor rate (A) and survival (B) of flies (20 flies per condition) overexpressing Tl[10B] in neurons compared to wild-type after injection of verruculogen. (A) Each dot corresponds to the tremor rate measured in a batch of 20 flies; tremor rate wt vs. elav> UAS-Toll10B, **p=0.002. (C-D) tremor rate (C) and survival (D) of flies (20 flies per condition) overexpressing Tl[10B] in glia compared to wild-type after injection of verruculogen. (C) Each dot corresponds to the tremor rate measured in a batch of 20 flies; tremor rate wt vs. repo> UAS-Toll10B, **p=0.002. (D) Survival wt V vs. repo> UAS-Toll10B V, **p=0.005.


( E - G ) recovery time from tremor ( E , F ) and survival ( G ) of single flies overexpressing BomS6 ubiquitously ( E , G ) or in neurons ( F - G ) ( biological replicates ) compared to wild - type after injection of verruculogen ; in ( G ) the inset represents the survival of vehicle control groups .

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

(E-G) recovery time from tremor (E,F) and survival (G) of single flies overexpressing BomS6 ubiquitously (E,G) or in neurons (F-G) (biological replicates) compared to wild-type after injection of verruculogen; in (G) the inset represents the survival of vehicle control groups.


( H - I ) recovery time from tremor ( H ) and survival ( I ) of single flies overexpressing BomS6 in glia ( pooled data from n = 3 experiments , biological replicates ) compared to wild - type after injection of verruculogen ; in ( I ) the inset represents the survival of vehicle control groups . Recovery time ( H ) repo > mCherry vs . repo > BomS6 , p = 0 . 058 , survival ( I ) repo > mCherry V vs . repo > BomS6 V , * p = 0 . 016 .

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

(H-I) recovery time from tremor (H) and survival (I) of single flies overexpressing BomS6 in glia (pooled data from n=3 experiments, biological replicates) compared to wild-type after injection of verruculogen; in (I) the inset represents the survival of vehicle control groups. Recovery time (H) repo>mCherry vs. repo>BomS6, p=0.058, survival (I) repo>mCherry V vs. repo>BomS6 V, *p=0.016.


( J - M ) expression of BomS4 ( J ) and BomS6 ( K ) in head after verruculogen powder challenge , and BomS4 ( L ) and BomS6 ( M ) in head after A . fumigatus ( Af ) , restrictocin or M . luteus injection ( pooled data from n = 3 experiments , biological replicates ) .

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(J-M) expression of BomS4 (J) and BomS6 (K) in head after verruculogen powder challenge, and BomS4 (L) and BomS6 (M) in head after A. fumigatus (Af), restrictocin or M. luteus injection (pooled data from n=3 experiments, biological replicates).


( A ) SPT activity and Western blot ( WB ) analysis of S1PR1 in S1pr1f / f and S1pr1ECKO endothelial cells after 4 - OHT ( 1μM , 72h ) treatment . ( n = 8 / group from 3 independent EC isolations / group ; 4 mice / EC isolation ) .

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(A) SPT activity and Western blot (WB) analysis of S1PR1 in S1pr1f/f and S1pr1ECKO endothelial cells after 4-OHT (1μM, 72h) treatment. (n=8/group from 3 independent EC isolations/group; 4 mice/EC isolation).


( A , B ) ( A ) WB analysis of ORMDLs , NOGO - B , SPTLC1 and SPTLC2 in HUVEC lysates in absence or presence of S1P ( 300nM , 30 ' ) and ( B ) relative ORMDLs quantification ( n = 7 biological replicates ) .

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(A,B) (A) WB analysis of ORMDLs, NOGO-B, SPTLC1 and SPTLC2 in HUVEC lysates in absence or presence of S1P (300nM, 30') and (B) relative ORMDLs quantification (n=7 biological replicates).


( C ) WB analysis of ORMDLs , SPTLC1 and SPTLC2 in HUVEC lysates in absence or presence of C16 : 0 - ceramide ( 300nM , 30 ' ) , with or without SKI II 1µM , 1h pre - treatment ) . ( D ) SPT activity in HUVEC in absence or presence of SEW2871 ( 300nM , 15 ' ) ( n = 7 biological replicates ) .

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(C) WB analysis of ORMDLs, SPTLC1 and SPTLC2 in HUVEC lysates in absence or presence of C16:0-ceramide (300nM, 30'), with or without SKI II 1µM, 1h pre-treatment). (D) SPT activity in HUVEC in absence or presence of SEW2871 (300nM, 15') (n=7 biological replicates).


( E , F ) ( E ) WB analysis of ORMDLs , SPTLC1 and SPTLC2 in HUVEC lysates in absence or presence of SEW2871 ( 300nM , 15 ' and 30 ' ) and ( F ) relative ORMDLs quantification ( n = 6 biological replicates ) .

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(E,F) (E) WB analysis of ORMDLs, SPTLC1 and SPTLC2 in HUVEC lysates in absence or presence of SEW2871 (300nM, 15' and 30') and (F) relative ORMDLs quantification (n=6 biological replicates).


( G , H ) SPT activity and WB analysis of HUVEC treated with ( G ) siCTRL and siNOGO ( 40nM , 72h ) ( n ≥ 4 / group ) , or with ( H ) siCTRL and siORMDL1 / 2 / 3 ( 40nM , 72h ) ( n = 4 biological replicates ) .

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(G,H) SPT activity and WB analysis of HUVEC treated with (G) siCTRL and siNOGO (40nM, 72h) (n≥4/group), or with (H) siCTRL and siORMDL1/2/3 (40nM, 72h) (n=4 biological replicates).


( I ) WB analysis of ORMDLs , P - p38 , p38 , P - JNK , JNK , P - ERK , ERK , P - AKT , and AKT in HUVEC lysates in absence or presence of the indicated inhibitors , with or without S1P ( 300nM , 30 ' ) .

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(I) WB analysis of ORMDLs, P-p38, p38, P-JNK, JNK, P-ERK, ERK, P-AKT, and AKT in HUVEC lysates in absence or presence of the indicated inhibitors, with or without S1P (300nM, 30').